Keywords: oil palm, potassium, metabolomics, proteomic, leaflet

Oil palm (Elaeis guineensis Jacq.) is one of the most productive oil crop in the world, with yield values of about 4 t oil ha-1. Potassium (K) is of considerable importance for oil palm production. In fact, K availability is crucial for fruit development by increasing bunch weight and number. Unfortunately, such positive effects of K fertilization remain rather difficult to predict because of the lack of knowledge of underlying metabolic mechanisms. In effect, understanding these mechanisms would allow agronomists to better anticipate oil palm responses in terms of photosynthetic sugar production, respiratory loss, or lipid biosynthesis.

Résumé

Le palmier à huile (Elaeis guineensis Jacq.) est l'un des oléagineux les plus productifs au monde, avec des rendements d'environ 4 t huile ha-1. Le potassium (K) a une importance considérable pour la production. En effet, l'apport en K est crucial pour le développement des fruits, augmentant non seulement le nombre mais aussi le poids des régimes. Malheureusement, les effets bénéfiques de K sont peu prédictibles car on ignore encore les mécanismes métaboliques sous-jacents. De fait, comprendre ces derniers permettrait aux agronomes de mieux anticiper les effets potassiques sur la production photosynthétique des sucres, les pertes respiratoires et la biosynthèse des lipides.

L'objectif de cette thèse était précisément de regarder les effets de la disponibilité en K sur les voies métaboliques et de voir s'il pouvait y avoir une relation entre les modifications métaboliques et la production d'huile. Pour cela, nous avons réalisé une étude sur le terrain à Sumatra (Indonésie) en utilisant des niveaux de K réalistes évitant l'excès ou la déficience, sur deux croisements largement utilisées commercialement (Deli x La Mé, Deli x Yangambi). Nous avons combiné une étude des traits fonctionnels (biomasse, phénologie, analyse des régimes, échanges gazeux), des teneurs élémentaires, et de génomique fonctionnelle (protéomique et métabolomique) dans les folioles et les fruits en cours de maturation. De plus, pour mieux comprendre les effets de K sur l'allocation des photosynthats des feuilles sources aux organes puits et juger si un marquage isotopique sur le terrain est possible, nous avons fait des manipulations préliminaires de marquages avec du 13CO2.

Outre les effets attendus sur des traits végétatifs ou les régimes (nombre et/ou poids), nos résultats montrent que l'apport en K impacte le métabolisme primaire du carbone et de l'azote aussi bien dans les folioles que dans les fruits. Dans les feuilles, à K élevé, il y a une capacité photosynthétique accrue que reflètent des teneurs en enzymes-clef (ex. sucrose phosphatase) ou en sucres (disaccharides notamment), et ce de façon croisement-dépendante. De plus, le K élevé reprogramme le catabolisme, avec une augmentation de la respiration, et de la teneur en enzymes du cycle de Krebs et de protéines de la chaîne de transport d'électrons mitochondriale. Dans les fruits, à K élevé, il y a une accélération de la cinétique de dépôt des lipides (synthèse et élongation des acides gras) et des voies associées, comme la synthèse transitoire des sucres et de l'amidon, la remobilisation des acides aminés ou l'activité du cycle des pentoses phosphate. Pourtant, la composition finale des lipides du mésocarpe reste inchangée.

Cette thèse présente ainsi, pour la première fois, une étude détaillée du métabolisme du palmier à huile au champ, et montre que certains traits métaboliques (métabolites ou enzymes) sont liés à la disponibilité en K, mettant en abyme une potentielle utilisation de biomarqueurs foliaires pour piloter la nutrition minérale du palmier.

The objective of this thesis was precisely to assess the effect of K availability on oil palm metabolic pathways and determine if metabolic changes could be related to oil production. To do so, we conducted our research work in situ, that is, in plantations in Sumatra (Indonesia), using realistic K levels far from excess or deficiency, with two contrasted oil palm crosses (Deli x La Mé and Deli x Yangambi) that are widely used in commercial planting. We combined analysis of functional traits (vegetative biomass, phenology, production and fruit analysis, gas exchanges), elemental analyses, functional genomics (proteomics and metabolomics) in leaflets and fruits during maturation. Furthermore, in order to gain knowledge on the effect of K on the redistribution of photosynthetic products from source to sink organs and show the feasibility of isotopic tracing in the field, a preliminary 13CO2 labelling experiment was carried out.

In addition to expected effects of potassium on vegetative traits and bunches (number of fruits and/or total fruit biomass), our results show that K availability affected carbon and nitrogen primary metabolism in both leaflets and developing fruits. In leaves, high K conditions led to an increased photosynthetic capacity reflected by higher content in several enzymes (e.g. sucrose phosphatase) and sugar content (in particular disaccharides), in a cross-dependent manner. In addition, high K reconfigured catabolism, with an increase in respiration, Krebs cycle enzyme content and proteins of the mitochondrial electron transfer chain. In fruits, high K accelerated the kinetics of lipid production (fatty acid synthesis and elongation) and associated pathways, including transitory sugar and transitory starch storage and utilization, amino acid remobilization or activity of the oxidative pentose phosphate cycle. However, final mesocarp lipid composition at maturity appeared to be unchanged by K availability. This thesis presents, for the first time, a detailed metabolic exploration of oil palm in the field and shows that some metabolic traits (metabolites or enzymes) are linked to K availability, thereby opening avenues for the use of leaf biochemical markers to monitor oil palm mineral nutrition.

I would like to warmly thank all the people who contributed in many different ways to this Ph.D. work, and more particularly my supervisors: Emmanuelle Lamade, Guillaume Tcherkez and Jaleh Ghashghaie. They contributed widely to the elaboration, the comprehension and the writing of this Ph.D. thesis. I just want to say thank you for your time, your patience and for sharing your knowledge and your skills with me during this 3 years.

I also thank all the Ph.D. board members for their patience during the steering committee meetings and their numerous comments and advice: Jean Ollivier, Edward Gerardeaux and Arthur Gessler. I thank all colleagues of my research teams UPR34 -Système de pérennes, who welcomed me at CIRAD in Montpellier, and I thank also the team Ecophysiologie Végétal, who welcomed me at Ecology Systematic and Evolution (ESE), and also the doctoral school Science du végétale.

I also thank all members of research teams who welcomed me as a visitor, for trainings or field work:

• the team of PSBB, Socfindo in Indonesia, including Indra, Dadang, Deni, Chandra, the field workers and the students, who widely helped me on the field,

• the team of the platform PAPPSO, INRA, Le Moulon, with Dr. Michel Zivy and Mr. Thierry Balliau,

• the team of the platform Metabolism -Metabolome at IPS2, with Dr. Françoise Gilard, Mme. Florence Guérard and Pr. Bertrand Gakière,

• the team of the Research School of Biology at Australian National University, including Jing and Illa,

• and the team SMS in INRA at Angers with Pr. Anis Limami and Jean-Baptiste. I would like to thank Chantal Fresneau from ESE for helping me in the lab with starch extractions and Clément Piel from the ecotron, CNRS at Montpellier, who had the kindness to lend me all the materials necessary for my labelling experiment. I thank also the greenhouse workers Mr. Christian Chaine and Mr. Semi Melliti at CIRAD in Montpellier who helped me built and organize my pre-labelling experiment in the greenhouse. III.1. Correlation analysis between oil content and mesocarp metabolome ................. Table IV.1. Correlation analysis between leaflet K content and oil palm functional traits. .. Table IV.3 S1. List of proteins significant for the cross effect in 2018 ......................................... Table S2. List of proteins significant for the cross effect in 2017 ......................................... Table S3. List of proteins with best loading values (first decile) in the proteome-metabolome correlation ..

List of tables

Context of the study

Oil palm is one of the most productive vegetable oil crop in the world with an oil yield of about 4 t ha-1. Palm oil is the leading vegetable oil in terms of production, volume and consumption (Statista, 2019). Oil palm represents only 5.5% of agricultural land surface area planted with oil crops, but accounts for 35% of global consumption in oils and fats, a number larger than of any other crops (Statista, 2019). In 2017 and 2018, worldwide palm oil production represented roughly 70.5 million metric tons.

Oil palm grows in Asia, Africa and Latin America. Indonesia and Malaysia are the main palm oil producers, followed by Thailand, Colombia, and Nigeria (FIG. 1). According to FAOSTAT, Asia accounts for around 89% of global production of palm oil in 2013. Palm oil production is also of importance in India (province of Goa) and Brazil (province of Para) [START_REF] Sparks | Advances in agronomy[END_REF]. The global demand in palm oil is expected to rise in the coming years, primarily for food consumption, due to population growth and rising living standards. As such, by 2027, an increase of about 25 million tons of palm oil per year is anticipated (www.oilworld.biz, 2019).

Oil palm is cultivated mainly on tropical soils (and humid tropical climate) with low-toaverage level of fertility (i.e., with limited cation exchange capacity) like ultisols (red clay soil) and oxisols (e.g. ferralitic soils) (FIG. 2) and therefore requires fertilization, in particular with potassium. In fact, higher potassium application rates are found in Asia on ultisol soils and in Latin America on oxisol and ultisol soils (FIG. 3).That is, the particularly high potassium requirement in oil palm comes not only from the dependency of bunch production on nutrient availability, loss via leaching and cultural practices (potassium removal from bunches harvesting and leaf pruning) but also from nutrient scarcity in oil palm cultivation areas [START_REF] Omoti | Leaching losses of nutrients in oil palm plantations determined by tension lysimeters[END_REF]Foong, 1991;[START_REF] Chang | Leaching losses of nutrients from a mature oil palm catchment in Malaysia[END_REF]. Potassium is known to sustain both vegetative growth and fruit production. As a result, there is an increase in potassium consumption in oil palm cultivating countries [START_REF] Ollivier | Genetic variation in nutrient uptake and nutrient use efficiency of oil palm[END_REF]. It represents up to 114 kg of potash per hectare and thus an annual cost of about US$ 1b at the global scale. Fertilization management thus represents a crucial aspect of oil palm agriculture and economy. Considerable advances have been made in understanding nutrient requirements in oil palm over the past few decades [START_REF] Woittiez | Yield gaps in oil palm: a quantitative review of contributing factors[END_REF]. However, because of the complexity of oil palm agrosystems (considering soil properties, climatic conditions, nutrient sources and oil palm varieties), fertilization management is still an intense field of research and unsurprisingly therefore, a better understanding of physiological aspects of potassium fertilization related to yield is needed. The oil palm tree 1. Overview Oil palm Elaeis guineensis Jacq. was named by Jacquin in 1763 using specimens collected in Martinique, and originates from the tropical rain forest of West Africa (Henson, 2012). Along with coconuts, oil palm is a perennial C3 monocotyledonous plant of the Arecaceae family.

Characterized as a ripicole (riparian) species, oil palm grows naturally in forest fringes, river banks, and swampy areas at low altitudes forming natural palm grove galleries [START_REF] Zeven | The semi-wild oil palm and its industry in Africa: Pudoc[END_REF].

Within E. guineensis, several genetically distinct forms are recognized based on the structure and coloration of fruits. The major structural character is the thickness of the shell or endocarp that surrounds the seed or kernel. Three main forms are referred to as dura, tenera, and pisifera, (more details in paragraph II. A.3). The most producing oil palm trees (tenera type) derives from dura x pisifera crosses and dura × tenera crosses (illustrated below in FIG. I.1) [START_REF] Billotte | Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.)[END_REF][START_REF] Cochard | Geographic and genetic structure of African oil palm diversity suggests new approaches to breeding[END_REF]. Amongst tenera forms, the crosses Deli x La Mé and Deli x Yangambi are the most widely commercialized families. They have been obtained

by crossing the Asian dura line Deli with the tenera African lines La Mé (Ivory Coast) or

Yangambi (Democratic Republic of Congo). In general, these crosses show differences in morphology, yield and oil composition. Usually, Deli lines have a small number of large bunches and La Mé and Yangambi have a large number of smaller bunches [START_REF] Meunier | Le schéma général d'amélioration du palmier à huile à l'IRHO[END_REF]. ‰

Oil palm is cultivated mainly as a source of oil but there are side utilizations such as production of palm wine. Two types of oil can be extracted: [START_REF]A Brief History of the Oil Palm[END_REF] crude palm oil extracted from the mesocarp of the fruit, and (2) the palmist oil extracted from the kernel. Palm oil is a multipurpose oil since it has several useful properties. It can be used as cooking oil, to manufacture margarine, bakery and confectionery fats (substitute for cocoa butter), or to produce oleochemicals for cosmetics, domestic and industrial products or biofuels.

While oil palm was widely present in West Africa, Central America and Asia, the use of palm oil in the international market expanded significantly as a result of the British Industrial Revolution and the expansion of overseas trade. Major importers of palm oil worldwide in 2018

were India (import of USD 6916.13 million), followed by Pakistan, Netherlands and USA (FIG.

4) (Statista 2019

). Import volume of palm oil has increased worldwide over the past few years.

According to the World Bank, in 2017, the average price for palm oil was about 649 nominal U.S. dollars per metric ton. It is expected to rise to about 744 nominal U.S. dollars per metric ton by 2025. While there is an obvious increase in global palm oil need, oil palm production has to face strong criticism in recent years, mostly due to the negative impact of oil palm cultivation on environment. In particular, the development of oil palm plantations at the expense of tropical forest in Indonesia (also in Papua) is believed to cause biodiversity loss, with near-extinction of emblematic species like the orangutan, pygmy elephant and the Sumatran rhino. As a result, public and private efforts led by both consumer and producer countries have been devoted to improve standards of palm oil production. This process is at the origin of the Roundtable of Sustainable Palm Oil (RSPO). RSPO is a non-profit organization that unites stakeholders from several sectors of the palm oil industry in order to develop and implement global standards and produce Certified Sustainable Palm Oil (CSPO) (www. rspo.org).

Oil palm ecophysiology

Oil palm is characterized by a simple architecture and indefinite growth which produces successive leaves ("palms") on an unique cylindrical trunk [START_REF] Jacquemard | Le palmier à huile: Quae[END_REF][START_REF] Corley | The oil palm[END_REF]. At adult stage, the palm tree has a crown of 30 to 45 leaves (5 to 9 m long). In the first year after planting, the number of new leaves produced each year increases progressively, reaches 40 leaves yr-1 2 years after planting and then declines with age [START_REF] Jacquemard | Jacq.)[oil palm, Ivory Coast[END_REF][START_REF] Gerritsma | An analysis of the growth of leaf area of oil palms in Indonesia[END_REF]. After 8-12 years, leaf emission is thus about 20-24 leaves yr-1 while leaf area and dry weight increase steadily with palm age. Leaf area reaches a plateau at 8-10 years after planting, but petiole cross section and presumably leaf dry weight continue to increase slowly [START_REF] Corley | The oil palm[END_REF]. At the adult stage, a new leaf is therefore emitted approximatively every two weeks (17 days in fact) with a divergence (phyllotaxic) angle of about 137.5°. The crown is organized in eight spirals where leaves are numbered (as ranks) following emergence sequence, from the youngest (rank 1) to the oldest (for example, rank 42). That is, leaf no. 1 is the starting point of the spiral "one". By convention, leaf rank no. 1 is the youngest leaf presenting 75% of leaflets opened and is considered to be the first functional (i.e. autotrophic) leaf by planters (FIG. I.2). At adult stage, the leaf emission rate also depends considerably on water availability (a dry period causes an accumulation of spear leaves), temperature (low temperature decreases growth rate) and also probably on carbon reserves of the tree. By contrast, leaf area and specific leaf weight are not very sensitive to external factors but may show significant responses to fertilizers [START_REF] Corley | The oil palm[END_REF].

Oil palm trees can reach 20-25 m height at adult stage, with a trunk diameter varying between 60 and 90 cm, depending on varieties and carbon reserve accumulation. The growth of trunk diameter is limited and usually stops when the tree reaches 4 years. Annual height increment varies from 0.3 to 0.6 m yr-1. It depends on environment and crosses but also on leaf production rate and the internode length (Hartley, 1988a;[START_REF] Corley | The oil palm[END_REF]. The roots system originates from the trunk base and form a fasciculate rootstock, which spreads around the tree, reaching neighbor trees with a potential length at soil surface larger than 9 m. Furthermore, roots can grow up to 6 m depth when grown on sandy soil. The root system is made of four-fold branched roots (the primary roots (I): responsible for sap circulation and anchorage; the secondary roots (II): which are devoted to resources prospections; the tertiary and quaternary roots (III+IV): which form the absorption system) (Jourdan & Rey, 1997a).

Oil palm is a C3 plant with a high leaf surface area (from 6 to 12 m2 leaf-1, depending on crosses), and a large carbon storage mostly in the form of starch and glucose located in the trunk [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]Legros et al., 2009c). Oil palm leaves have high photosynthesis rates, with a maximum for leaves at rank 8 to 10 (from 23 up to 30 μmol m-2 s-1) [START_REF] Dufrene | Gas exchange of oil palm in relation to light, vapour pressure deficit, temperature and leaf age[END_REF][START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF] even in trees that are 4-5 years old only. Concerning photosynthesis activity, a mid-day depression is generally observed due to stomatal closure when the VPD (Vapor Pressure Deficit) reaches 1.7 kPa. In young palms, the rate of photosynthesis goes up with developmental stage but also appears to be controlled by the presence of developing bunches, acting as sinks for assimilates [START_REF] Gerritsma | Simulation of oil palm yield[END_REF]. This feedback control is believed to be mediated by carbohydrate accumulation in leaflets during the day [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF]. In fact, [START_REF] Henson | Photosynthesis and source-sink relationships in oil palm (Elaeis guineensis)[END_REF] found higher leaf non-structural carbohydrate levels in non-fruiting than fruiting palms. However, on mature oil palm under drought condition, no negative feedback has been observed with strong sink limitation on light-saturated leaf CO2 assimilation rate (Amax) (Legros et al., 2009b). In such conditions, strong sink limitation, has led to non-soluble carbohydrates (NSC) accumulation in the trunk (mainly as starch), reaching up to 50% (dw:dw) in trunk top. Leaf physiological parameters also vary with leaf ageing. The study of [START_REF] Suresh | Variations in photosynthetic rate and associated parameters with age of oil palm leaves under irrigation[END_REF] has shown that in leaf number 9, net photosynthesis, transpiration, and stomatal conductance, for an adult palm tree, are higher than in younger or older leaves, meaning that leaf gas exchange progressively declines with leaf age (TABLE I.1). In addition, photosynthetic rate has been found to be positively correlated to stomatal conductance and negatively correlated to leaf mass per area [START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF].

Dark respiration rates are also lower in older and shaded leaves compared to young and unshaded leaves [START_REF] Henson | Adaptation to light environment by leaves of oil palm (Elaeis guineensis)[END_REF]. In oil palm leaflets, the dark respiration rate is about 1.5-2.5 µmol m-2 s-1 [START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF][START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]. At the whole oil palm tree level, about 60 to 80 % of gross assimilation is thought to be respired not only by leaf respiration but also by other organs [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF][START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]. [START_REF] Marcelis | Sink strength as a determinant of dry matter partitioning in the whole plant[END_REF][START_REF] Heuvelink | Effect of fruit load on dry matter partitioning in tomato[END_REF]. [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF] and [START_REF] Dufrene | Gas exchange of oil palm in relation to light, vapour pressure deficit, temperature and leaf age[END_REF] have estimated that oil palm photosynthates are used in priority for vegetative growth and maintenance. Also, Henson [START_REF] Henson | Modelling vegetative dry matter production of oil palm[END_REF][START_REF] Henson | Modelling the effects of physiological and morphological characters on oil palm growth and productivity[END_REF] reported that vegetative growth and development does not vary much and has priority for photosynthate allocation perhaps due to the low architectural plasticity, whereas fruit production and thus fruit sink strength can adjust to available resources (FIG. I.3). In fact, severe leaf pruning reduces vegetative dry matter (VDM) production by 4% only and reduces fruit yield by 65%; conversely, removing bunches increases VDM with an increase in leaf production, leaf dry weight, trunk height and diameter, as well as leaf area [START_REF] Corley | Fruiting activity, growth and yield of oil palm. I. Effects of fruit removal[END_REF]. Therefore, fruit development seems to occur only when fixed carbon (gross production) is larger than the demand of vegetative development, and thus is very sensitive to photosynthetic conditions. In fact, it has been shown that intercepted radiation per tree and photosynthesis were linearly related to bunch yield [START_REF] Squire | A physiological analysis for oil palm trials[END_REF][START_REF] Squire | Oil palm[END_REF] suggesting that bunch yield is source limited, that is, limited by the supply of carbohydrates from photosynthesis. Moreover, in the fruit, the kernels had a higher priority for carbohydrate supply for their development than the mesocarp or shell [START_REF] Harun | Fruit set and oil palm bunch components[END_REF]. It is worth noting that according to [START_REF] Corley | The oil palm[END_REF], roots may have, like leaves, priority over bunches for photosynthates utilization. The physiology of roots is far less documented than leaves and thus this suggestion awaits further evidence. Still, root biomass certainly differs with oil palm age, between crosses, soils and environments and this add to the complexity of physiological determinants of fruit production. Root turnover represents about 9-11.5t ha-1 year-1, made of 15% primary roots, 31% secondary and 57% fine roots [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF]Lamade et al., 1996;Jourdan & Rey, 1997a;Jourdan & Rey, 1997b). The lines are fitted regressions (both with slopes significantly different from zero) and indicate that 91% of all additional dry matter, above a threshold of 77 kg palm-1 year-1, goes into bunches. Bunch dry matter is adjusted for the energy content of the oil. Data are from [START_REF] Corley | Effects of plant density on growth and yield of oil palm[END_REF] for 6-7 year-old palms in a density trial, plotted as in [START_REF] Squire | The physiology of tropical crop production: CAB International[END_REF].

To our knowledge, the precise photosynthate allocation pattern in oil palm has never been examined with isotopic tracers (13C or 14C labelling), likely due to the scale of experiments required (tree-scale) and thus the induced cost. Nevertheless, a recent study [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]) used 13C at natural abundance (δ13C values) to compute the most probable allocation pattern (FIG. I.4). It suggests that fruits and roots represent the largest carbon sinks (20-30% each) while heterotrophic leaves represent about 11-15% [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF].

Ecophysiological modelling and biomass measurements have suggested that in mature trees, fruit bunches and roots are responsible for a respiratory loss of 18 and 14% of gross primary production, respectively and that carbon partitioning to fruits and roots stands for 30 and 10% of gross carbon fixation, respectively [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF][START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF]. However, δ13C values suggest that fruit development represents by far the largest respiratory loss (40% of tree respiratory loss) while leaves and roots represent only 12 and 5-10% of respiratory loss [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]. Although more data are certainly needed to provide a more precise picture of allocation and respiratory losses in oil palm, a high respiratory loss in fruits would be consistent with the low metabolic carbon use efficiency of oil (fatty acid) production. In adult oil palm trees under favorable conditions, non-structural carbohydrates (NSC)

represents about 20% of vegetative plant dry matter (DM) including roots, with 65% located in the trunk [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]. In general, it is believed that perennials plants accumulate NSC during periods of excess production of photoassimilates and use them when the demand exceeds production [START_REF] Kozlowski | Carbohydrate sources and sinks in woody plants[END_REF]. In oil palm, reserves are predominantly located in the trunk, however, other organs like petioles or leaves may also play a role of a transitory storage. [START_REF] Zahari | Renewable sugars from oil palm frond juice as an alternative novel fermentation feedstock for value-added products[END_REF] observed also substantial carbon reserves in the form of glucose in petioles. The chemical nature of NSC reserves varies among perennial species [START_REF] Kozlowski | Carbohydrate sources and sinks in woody plants[END_REF] but in adult oil palm, glucose is an important reserve sugar (50% of NSC), followed by starch and sucrose (20% each) and a small amount of fructose [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]. In other words, oil palm is one of the rare species using glucose as a transitory reserve sugar. However, starch is the main metabolic "buffer" compensating for source and sink variations, whereas glucose dynamics remain poorly explained (Legros et al., 2009d). The same authors suggested that the glucose content might be directly driven by environmental variables. Still, sucrose is by far the major carbon transport form in the tree from source leaves to sinks (fruits) [START_REF] Houngbossa | The nature of organic nutrients exported from the leaf of oil palm (Elaeis guineensis): effect of ethylenediaminetetraacetic acid on exudation[END_REF][START_REF] Obahiagbon | Assessment of the nutritional constituents of Elaeis guineensis Jacq. exudates from different states of Nigeria[END_REF]. In fact, sucrose is the prevalent sugar in oil palm phloem sap extracted from leaves, and furthermore, it is the major 14C-compound in sap upon 14C-labelling [START_REF] Houngbossa | The nature of organic nutrients exported from the leaf of oil palm (Elaeis guineensis): effect of ethylenediaminetetraacetic acid on exudation[END_REF][START_REF] Obahiagbon | Assessment of the nutritional constituents of Elaeis guineensis Jacq. exudates from different states of Nigeria[END_REF]. Thus presumably, sucrose is the carbon source for lipid and starch biosynthesis in fruits (starch being then used as a carbon source for lipid synthesis) [START_REF] Dussert | Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition[END_REF]. In fact, genomics analyses have suggested that fruit development is primarily sustained by sucrose, since fruit tissues have a high expression level of sucrose synthase (Susy; which catalyzes sucrose cleavage) during maturation [START_REF] Wong | Differential gene expression at different stages of mesocarp development in high-and low-yielding oil palm[END_REF].

Oil production and composition in fruits

Oil palm is a monoecious species, with successive male and female inflorescences that develop separately on the same plant. Pollination, which must be crossed between two individual palm trees at least, is usually anemophilous but can done either by insects such as Elaeidobius kamerunicus (Coleoptera Curculionidae), or manually. After pollination, fruits start maturation, which takes 5 to 6 months until ripe. Generally, bunches are harvested from the third to fourth year after planting. Fresh fruit bunch yield increases for 10 years, then reaches a maximum and declines progressively [START_REF] Corley | oil palm research[END_REF] or stay stable (see https://www.palmelit.com, Catalogue-PalmElit-Oil-Palm-Seeds.pdf)

The oil palm fruit is a sessile drupe composed of five different parts: (1) the exocarp (outer skin); (2) mesocarp (oily fibrous layer containing palm oil): (3) the endocarp (kernel shell); and (4) the kernel containing palmist oil (Teh et al., 2013b). As already mentioned page 10, there are three forms of oil palm fruit: a thick-shelled form (2-8 mm) with a thin mesocarp (35-65 % mesocarp/fruit) called dura, a shell-less form, usually female sterile called pisifera and a thin shelled form (0.5-4 mm) with a high mesocarp content (55-96 %) called tenera (FIG.

I.5).

The pisifera is not well spread as a commercial planting material as it is mostly infertile.

Oil represents up to 90% of mesocarp dry weight, and this is the highest lipid content in oil-producing crops. The bunch has a main stalk, spikelet stalks, spines (transformed bracts), fruits and parthenocarpic (seedless) fruits, which are developed in case of poor pollination conditions. The parthenocarpic fruits in the inner bunch increased with fruit set and contain very little oil due to decreased mean fruit size [START_REF] Harun | Fruit set and oil palm bunch components[END_REF]. Oil palm fruit development and oil deposition has been well-described by [START_REF] Thomas | Fruit ripening in the oil-palm Elaeis guineensis[END_REF] and Hartley (1988a). After anthesis, the mesocarp is visible and composed of 92% of water. At this stage, the kernel is still liquid and the endocarp is thin. Twenty days after anthesis, the endocarp becomes thicker and the kernel hardens. By 11 weeks after anthesis (WAA), depending on varieties, the kernel completely fills the seed cavity and the endocarp starts dehydration while sclerifying to isolate and protect the kernel (Hermine [START_REF] Bille | Morphogenesis of oil palm fruit (Elaeis guineensis Jacq.) in mesocarp and endocarp development[END_REF]. By 15 WAA, the mesocarp begins a continuous dehydration by transpiration which is associated with active lipid biosynthesis through fatty acids (FA) synthesis until maturation (around 20-21 WAA) [START_REF] Jeje | Transpiration and oil accumulation rates for developing oil palm fruits Elaeis guineensis Jacq[END_REF]Teh et al., 2013a). Reciprocally, fruit sugar content tends to decrease to very low level by 18 WAA [START_REF] Neoh | Profiling of metabolites in oil palm mesocarp at different stages of oil biosynthesis[END_REF]Teh et al., 2013a).

Fatty acids (FA) are mostly produced in plastids, and are then assembled into triglycerides and other lipids in the cytoplasm (oil bodies). In fact, [START_REF] Bourgis | Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning[END_REF] From [START_REF] Guschina | Studies on the regulation of lipid biosynthesis in plants: application of control analysis to soybean[END_REF].

Changes in lipid class and fatty acid composition during development of the oil palm mesocarp are well reviewed in [START_REF] Sambanthamurthi | Chemistry and biochemistry of palm oil[END_REF]. Before oil deposition, phospholipids are the major lipid class, accounting for about 60% of the total lipids at 8-12 WAA (with 51% of phosphatidylcholine (PC), followed by phosphatidylinositol (PI) and lysophosphatidyl-choline (LPC)). At maturity, however, glycolipids form the major component of the polar lipids [START_REF] Bafor | Changes in lipid class and fatty acid composition during maturation of mesocarp of oil palm (Elaeis guineensis) variety dura[END_REF]. Triacylglycerols (TAG) increase rapidly from 16

WAA and reaches a maximum at 20 WAA [START_REF] Sambanthamurthi | Chemistry and biochemistry of palm oil[END_REF]. Palmitoleic and linolenic acids are present in significant amounts in the early stages of lipid synthesis. These fatty acids (FA) are typical constituent of chloroplast and membrane, suggesting a high ratio of chloroplast and cellular synthesis to storage lipid synthesis at this stage. These fatty acids, however, are undetectable after 16 WAA, probably because of a dilution effect by the accumulation of storage lipids [START_REF] Sambanthamurthi | Chemistry and biochemistry of palm oil[END_REF]. In addition, immature mesocarp contains large amounts of chlorophyll, which decline by about 17 WAA, accompanied by a massive accumulation of carotenes as the fruit ripens [START_REF] Ikemefuna | Chlorophyll and carotenoid changes in ripening palm fruit, Elaeis guineënsis[END_REF]. Also immature mesocarp tissue is characterized by high amounts of sterols. As the fruit matures, sterols decrease as a consequence of dilution by the huge amount of TAG synthesized [START_REF] Ikemefuna | Chlorophyll and carotenoid changes in ripening palm fruit, Elaeis guineënsis[END_REF].

When ripe, fruits (including kernels) not only contain lipids but also structural constituents (lignin, hemicellulose, cellulose and pectic substances) at 18-20%, soluble proteins and sugars accounting for 2-3% of mesocarp weight. On average, crude palm oil contains 44% palmitic acid (C16:0), 5% stearic acid (C18:0) and traces of myristic acid (C14:0), which together constitute half of fatty acids found in triacylglycerols (TAG) in fruit mesocarp [START_REF] Sambanthamurthi | Chemistry and biochemistry of palm oil[END_REF]. TAG with unsaturated fatty acids are represented by 40% oleic acid (C18:1), 10% linoleic acid (C18:2) and traces of linolenic acid (C18:3) ( [START_REF] Barcelos | Oil palm natural diversity and the potential for yield improvement[END_REF]. This right balance between saturated and unsaturated fatty acids makes palm oil suitable for a number of food applications.

In oil palm agronomy, the term "production" refers to fresh fruit bunch (FFB), completed by bunch number, bunch weight and oil-to-bunch ratio. The number of bunches results from leaf emission rate, sex ratio, early and late abortion rate. The weight of a mature bunch depends on the number of spikelets, the number of flowers per spikelet, the fruit set (percentage of flowers that develop into fruit or pollination efficiency), the mean weight per fruit and the weight of the stalk [START_REF] Corley | The oil palm[END_REF]. The oil-to-bunch ratio is defined as the amount of oil recovered in a bunch (i.e. the oil extraction rate) [START_REF] Rahim | An economic perspective of oil extraction rate in the oil palm industry of Malaysia[END_REF]. The oil extraction rate (OER) is calculated as:

F B× M F×OFM OER 0.855 10, 000 = 
where F/B, M/F and OFM stand for the percentage of fruits biomass in bunch total biomass, the percentage of mesocarp biomass in total fruit biomass, and the percentage of oil in mesocarp biomass, respectively. The correction factor of 0.855 is used to account for losses in factory oil extraction. Of course, the OER is influenced by oil palm crosses, environmental conditions or the addition of fertilizers [START_REF] Menon | Factors affecting oil and kernel extraction rates[END_REF]. At maturity, bunch weight is in average 23-27 kg and OER is about 23% of fresh weight (TABLE I.2). [START_REF] Schroeder | Structure and weathering of potassium containing minerals[END_REF]. However, potassium is not readily accessible in soils since its availability for plants depends on exchange capacity and most of soil potassium is fixed in minerals or non-exchangeable and embedded in silicates. In addition, due to intensive agricultural production systems leading to coarse-textured or organic soils, and specific soil composition (sandy, waterlogged, saline, or acidic), K can be limiting for plant growth [START_REF] Goulding | The classification and mapping of potassium reserves in soils of England and Wales[END_REF]. This is the case in the tropical region where oil palm is cultivated, where strong weathering leads to clay-rich or ferralitic soils with limited cation exchange capacity and low K bioavailability. Many sources of K are used as fertilizers, including potassium chloride (KCl, the most used by far), potassium nitrate (KNO3), potassium sulfate (K2SO4), potassium carbonate (K2CO3) and potassium-malate (C4H4O5K2). Of course, the efficiency of K+ fertilization depends on soil texture, propensity for leaching, soil moisture content, pH, etc.

In some cases such as sandy soil or waterlogged conditions, K is applied as a foliar spray. In fact, [START_REF] Ashraf | Alleviation of waterlogging stress in upland cotton (Gossypium hirsutum L.) by exogenous application of potassium in soil and as a foliar spray[END_REF] reported that foliar spray application of potassium alleviated the adverse effects of waterlogging on cotton plants. In olive tree, potassium nitrate (KNO3) or mono potassium phosphate (MPK) enhanced leaf mineral status, yield and fruit quality in a sandy soil [START_REF] Sarrwy | Effect of foliar sprays with potassium nitrate and mono-potassium phosphate on leaf mineral contents, fruit set, yield and fruit quality of picual olive trees grown under sandy soil conditions[END_REF]. However, while K+ is highly soluble in water and diffuses relatively easily through the cuticle and the free space of the cell wall, K foliar spray application can only partially compensate for insufficient uptake by roots [START_REF] Mengel | Alternative or complementary role of foliar supply in mineral nutrition International Society for Horticultural Science[END_REF].

Potassium absorption

Plants uptake of K as a cation (K+) occurs via root hairs and then cortical roots cells and once in the stele, K+ is transported to the shoot and distributed to the leaves and others organs via the xylem. K+ can be stored in the vacuole, from which it can be remobilized to keep cytoplasmic K+ concentration constant. In fact, the concentration of K+ in the cytoplasm is kept relatively constant at about 50-150 mM, while the concentration in the vacuole varies substantially depending on supply status. In the soil, potassium concentration is between 0.1 and 1 mM [START_REF] Britto | Cellular mechanisms of potassium transport in plants[END_REF]White & Karley, 2010). Therefore, K+ enters root cells against the concentration gradient but follows the transmembrane potential, which favors the entry of cations. The cornerstone of cation and charge distribution across the plasma membrane is the activity of H+-ATPases that pumps H+ out of the cell by consuming ATP.

Potassium uptake is facilitated by different transporters or channels allowing transport of K+ between the outer environment and the cytoplasm or between different cellular compartments. Two transport systems are involved in K+ uptake based on their affinity. The high-affinity system operates at low external concentrations. Electrophysiological evidence indicates that this pathway involves a H+:K+ symporter coupled to the activity of the plasma membrane H+-ATPase [START_REF] Maathuis | Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana[END_REF]. The low affinity pathway, which operates at higher concentrations, usually more than 0.3 mM external K+, has the characteristics of a channel-mediated transport. Channels of the Shaker family have been implicated in K+ permeation through the plasma membrane. Other channels have been also identified in plants (FIG. I.8). In Arabidopsis thaliana, the voltage-gated K+-selective channel protein K+ Transporter 1 (AKT1) has been shown to be essential for K+ uptake by roots [START_REF] Spalding | Potassium uptake supporting plant growth in the absence of AKT1 channel activity: inhibition by ammonium and stimulation by sodium[END_REF][START_REF] Xu | A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis[END_REF]. In Elaeis guineensis, the transporter EgKUP8 has been shown to be transcriptionally activated in roots under K+-deficiency, indicating its importance for high affinity K+ uptake in K+-limited environment [START_REF] Husri | Importance of KUP8 for K+ uptake in rooted plantlets of Elaeis guineensis under K+ sufficient conditions[END_REF]. Stelar K+ Outward Rectifier (SKOR) is structurally similar to AKT1 but mediates K+ efflux in root stellar cells to facilitate K+ loading into the xylem [START_REF] Gaymard | Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap[END_REF]. In Arabidopsis guard cells, where K+ fluxes mediate stomatal movements, stomatal opening driven by K+ entry occurs mainly through K+ channel in Arabidopsis thaliana 1 (KAT1) and KAT2, whereas stomatal closing is caused by K+ efflux through Gated Outwardly Rectifying K+, a channel activated by membrane depolarization [START_REF] Ache | GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+selective, K+-sensing ion channel[END_REF][START_REF] Hosy | The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration[END_REF][START_REF] Lebaudy | Preferential KAT1-KAT2 heteromerization determines inward K+ current properties in Arabidopsis guard cells[END_REF].

Potassium distribution in the plant has been reviewed recently [START_REF] Ahmad | Cellular and tissue distribution of potassium: Physiological relevance, mechanisms and regulation[END_REF][START_REF] Wigoda | Is the leaf bundle sheath a "smart flux valve" for K+ nutrition?[END_REF]. K is allocated to growing and metabolically active tissues, often at the cost of older and less crucial cell types. Sensing of and response to K+ appears to be mediated by several cellular events. These include cell membrane potential (depolarization), reactive oxygen species (ROS), Ca2+, hormones such as ethylene, jasmonic acid or cytokinins and direct sensing of the environmental K+ concentrations by K+ channels.

Physiological roles of potassium a) Cells turgor and guard cells

As stated above, plant cells contain two major pools of K+, one in the vacuole and one in the cytosol (while mitochondria represent a smaller pool but of physiologic ,al importance). K+ plays the role of an osmoticum in the vacuole and as such, is involved in turgor and cell enlargement. Similarly, K+ plays a role in the opening and closing of stomata, which regulate leaf conductance for water vapor and CO2. Thus K nutrition has been suggested to be essential for water use efficiency [START_REF] Mengel | Der Einfluß der Kaliumkonzentration der 'Bodenlösung'auf den Ertrag, den Wasserverbrauch und die K-Aufnahmearten von Zuckerrüben (Beta vulgaris ssp. esculenta var. altissima)[END_REF]. In general, K-deficient plants have a low stomatal conductance, and a low water use efficiency because of the concurrent decrease in the synthesis of the photosynthetic machinery component (for a recent example in oil palm, see Cui et al. (2019b)).

Stomatal opening starts with membrane hyper-or re-polarization caused by H+-ATPases, leading to K+ uptake through inward-rectifier K+ channels [START_REF] Ache | GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+selective, K+-sensing ion channel[END_REF]. The influx of K+, Cl-, and NO3-and the synthesis of malate (accumulated in the vacuole) increases the concentration in osmoticum and thus a water influx thereby ensuring turgor of the guard cells and stomatal opening. The rapid loss of K+ from guard cells during stomatal closure stems from anion efflux, which causes plasma membrane depolarization and the opening of outward K+ channels that leads to K+ efflux from the vacuole and across the plasma membrane. Among ions released from guard cells, it is believed that >90% originate from vacuoles.

b) Cation-anion balance

Potassium has also a function in the transport of water and nutrients throughout the plant in the xylem and participate therefore to the cation-anion balance. Although, evidence has been provided that when K availability is low, translocation of nitrates, phosphates, calcium (Ca), magnesium (Mg), and amino acids is depressed [START_REF] Schwartzkopf | Potassium, calcium, magnesium-How they relate to plant growth[END_REF], increasing amount of potassium in all plant tissues and decreasing amounts of sodium, calcium, magnesium, iron and copper in certain tissues were found in potassium fertilization conditions (from complete deficiency to luxury consumption levels) [START_REF] Ward | Potassium in plant metabolism: II. Effect of potassium upon the carbohydrate and mineral composition of potato plants[END_REF]Cui et al., 2019a). In fact, there is a well-known antagonistic effect between potassium and calcium and between potassium and magnesium in the leaf and stem [START_REF] Ward | Potassium in plant metabolism: II. Effect of potassium upon the carbohydrate and mineral composition of potato plants[END_REF].

Previous research has indicated a close relationship between K+ and NO3-uptake by roots [START_REF] Marschner | Nutrient availability in soils. Marschner's mineral nutrition of higher plants[END_REF], while evidence has been provided that the nitrate reduction rate does not decrease in the short and mid-term with low K [START_REF] Agüera | Nitrate and nitrite uptake and reduction by intact sunflower plants[END_REF]. That is, K+ represents the major counter cation for nitrate and thus is transported together with NO3-in the xylem [START_REF] Wang | The critical role of potassium in plant stress response[END_REF][START_REF] Coskun | The nitrogen-potassium intersection: membranes, metabolism, and mechanism[END_REF].

In addition, NH4+ has also a marked inhibitory effect on the high-affinity K+ uptake system [START_REF] Coskun | Regulation and mechanism of potassium release from barley roots: an in planta 42K analysis[END_REF][START_REF] Coskun | Capacity and plasticity of potassium channels and high-affinity transporters in roots of barley and Arabidopsis[END_REF]. This inhibition appears to be reciprocal, resulting in an 'antagonism' between the two ions, with both competitive and non-competitive components.

Potassium and metabolism

Although K+ itself is not metabolized by plants, it plays a vital role in many aspects of plant metabolism. Potassium is involved in the activity of some 46 enzymes [START_REF] Leigh | A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell[END_REF][START_REF] Britto | Cellular mechanisms of potassium transport in plants[END_REF][START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF], it acts as a cofactor of pyruvate kinase (PK), starch synthase, Rubisco or nitrate reductase (NR) amongst other enzymes [START_REF] Sorger | Effects of univalent cations on the immunoelectrophoretic behavior of pyruvic kinase[END_REF][START_REF] Evans | Role of mineral elements with emphasis on the univalent cations[END_REF][START_REF] Nitsos | Effects of univalent cations on the inductive formation of nitrate reductase[END_REF][START_REF] Beevers | Nitrate reduction in higher plants[END_REF][START_REF] Peoples | Role of potassium in carbon dioxide assimilation in Medicago sativa L[END_REF] a) Carbohydrate metabolism

• Photosynthesis and sugar synthesis

As mentioned above, potassium is essential for stomatal movement and therefore photosynthetic gas exchange. The effect of K on photosynthesis has been investigated in a wide range of plants including: maize [START_REF] Du | Effects of potassium deficiency on photosynthesis, chloroplast ultrastructure, ROS, and antioxidant activities in maize (Zea mays L.)[END_REF]), bean (O'Toole et al., 1980), cotton [START_REF] Zhao | Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants[END_REF], eucalyptus (Battie- [START_REF] Battie-Laclau | Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment[END_REF], sugar beet [START_REF] Terry | Effects of potassium deficiency on the photosynthesis and respiration of leaves of sugar beet[END_REF] and Brassica napus L. (Lu et al., 2016b). In all cases, K deficiency significantly decreased the rate of photosynthesis. The role of K on photosynthesis has been associated to stomatal and chloroplast functions but also to non-stomatic pathway.

Since leaf chlorosis appeared on K deficient plant, a direct effect of K on chlorophyll content has been suggested (Bolle- [START_REF] Notton | The relative proportions of the chloroplast pigments as influenced by different levels of iron and potassium supply[END_REF]. In fact, in potatoes, high potash evidently suppressed chloroplast pigment formation [START_REF] Schertz | The effect of potassium, nitrogen and phosphorus fertilizing upon the chloroplast pigments, upon the mineral content of the leaves, and upon production in crop plants[END_REF]. Moreover, evidences

showed in maize at seedling stage that Chlorophyll a, b and (a + b) were significantly decreased under different K deficiency treatments [START_REF] Zhao | Effects of different potassium stress on leaf photosynthesis and chlorophyll fluorescence in maize (Zea Mays L.) at seedling stage[END_REF]. However, such effect has not been observed in rice [START_REF] Watanabe | Effects of nitrogen, phosphorus, and potassium on photophosphorylation in rice in relation to the photosynthetic rate of single leaves[END_REF] and maize [START_REF] Baszynski | Some biochemical characteristics of chloroplasts from mineral-deficient maize[END_REF].

Several studies have showed that insufficient leaf K+ content generally leads to decreased stomatal conductance (gs), decreased mesophyll conductance (gm) and a decreased content in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), thereby inhibiting photosynthesis per unit leaf area [START_REF] Zhao | Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants[END_REF][START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF]. However, results from [START_REF] Jin | Effects of potassium supply on limitations of photosynthesis by mesophyll diffusion conductance in Carya cathayensis[END_REF] in hickory seedlings indicate that the photosynthetic rate is primarily limited by the biochemical processes of photosynthesis such as the maximum carboxylation rate of Rubisco (Vc,max) and the maximum rate of electron transport (Jmax), rather than by gm and gs in K-deficient plants. The mesophyll conductance limitation contributed to more than one-half of photosynthesis decline (Lu et al., 2016b). Additionally, gm was closely correlated with gs and the leaf dry mass per unit area (SLW) in hickory seedlings, which indicates that decreased gm and gs may be a consequence of leaf anatomical adaptation.

In fact, potassium level affects photosynthesis through changes in leaf morphology and anatomy, as the SLW as well as the leaf thickness and density, stomatal size and intercellular air spaces in bean [START_REF] O'toole | Effect of potassium nutrition on leaf anatomy and net photosynthesis of Phaseolvs vulgaris L[END_REF] and cotton [START_REF] Zhao | Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants[END_REF]. In fact, under long term K deficiency, the total stomatal pore area decreases, resulting in lower stomatal conductance in eucalyptus (Battie- [START_REF] Battie-Laclau | Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment[END_REF], and in higher mesophyll resistance to CO2 in sugar beet [START_REF] Terry | Effects of potassium deficiency on the photosynthesis and respiration of leaves of sugar beet[END_REF]. At the opposite, sufficient K availability increases the volume fraction of intercellular air space and gas-phase internal conductance in eucalyptus (Battie- [START_REF] Battie-Laclau | Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment[END_REF], enhances the chloroplast surface area exposed to intercellular air space per leaf area and reduces the gas-phase resistance of CO2 in the cytoplasm in Brassica napus L. (Lu et al., 2016b). Therefore, total CO2 conductance from the sub-stomatal cavity to the site of carboxylation in the chloroplast is enhanced at high K, which also stimulates total Rubisco enzyme activity [START_REF] Galmés | Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration under water stress[END_REF], although, previous studies have showed that K excess does not stimulate CO2 transport ability [START_REF] O'toole | Effect of potassium nutrition on leaf anatomy and net photosynthesis of Phaseolvs vulgaris L[END_REF][START_REF] Jin | Effects of potassium supply on limitations of photosynthesis by mesophyll diffusion conductance in Carya cathayensis[END_REF]Lu et al., 2016a) and Rubisco activity [START_REF] Weng | Characteristics of photosynthesis and functions of the water-water cycle in rice (Oryza sativa) leaves in response to potassium deficiency[END_REF].

Potassium can also modulate photosynthesis by non-stomatic pathways. K plays a role in the conversion of light energy into chemical energy by pumping proton out of the stroma into the cytosol and maintains therefore the balance of electric charges in chloroplasts. In fact, the two-pore potassium (K+) channel TPK3, a component of the thylakoid membrane, modulates the size and the composition of the light-induced proton motive force (pmf) across the thylakoid membrane of chloroplasts through ion counterbalancing. A study of [START_REF] Carraretto | A thylakoid-located two-pore K+ channel controls photosynthetic light utilization in plants[END_REF], in Arabidopsis, has showed that silencing TPK3 led to reduced growth and altered thylakoid membrane organization, resulting in reduced CO2 assimilation and deficient nonphotochemical dissipation of excess absorbed light. Hence, K improves the transfer of radiation energy into primary chemical energy in the form of ATP (photophosphorylation) [START_REF] Pfluger | The photochemical activity of chloroplasts from various plants with different potassium nutrition[END_REF][START_REF] Tester | Direct measurement of K channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers[END_REF] and NADPH [START_REF] Wallingford | Function of potassium in plants[END_REF] which are produced both in photosynthesis and transpiration processes.

Through its action on photosynthesis, K affects sugar synthesis. The most consistent observation is an accumulation of soluble sugars in K-deficient plants both in leaves ( [START_REF] Cakmak | Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants[END_REF] (bean), [START_REF] Pettigrew | Potassium deficiency increases specific leaf weights and leaf glucose levels in field-grown cotton[END_REF] (cotton), [START_REF] Ward | Potassium in plant metabolism: II. Effect of potassium upon the carbohydrate and mineral composition of potato plants[END_REF]) (potato)) and in roots ( [START_REF] Volenec | Physiological control of alfalfa growth and yield[END_REF] (alfalfa), [START_REF] Farley | Growth and yield of sugar beet in relation to potassium and sodium supply[END_REF]) (sugar beet)). Nevertheless, in another study opposite effect of K on sugar contents was found in cabbage (Freeman & Mossadeghi, 1970). The observed accumulation of soluble sugars in K-deficient plants can be explained with Kdependence of both starch synthase and pyruvate kinase as they determine the rate of incorporation of C into starch or organic/amino acids respectively [START_REF] Amtmann | The effect of potassium nutrition on pest and disease resistance in plants[END_REF]. In fact, evidence has been provided that the activity of starch synthase, which is involved in starch synthesis in the chloroplast, depends on K concentration [START_REF] Läuchli | Potassium transport through plant cell membranes and metabolic role of potassium in plants[END_REF]. Thus, with insufficient K supply, the level of starch declines in general. In potato leaves, [START_REF] Ward | Potassium in plant metabolism: II. Effect of potassium upon the carbohydrate and mineral composition of potato plants[END_REF] has showed that the amount of starch was a direct function of the amount of potassium applied.

However, [START_REF] Pettigrew | Potassium deficiency increases specific leaf weights and leaf glucose levels in field-grown cotton[END_REF] has revealed that in cotton, only glucose content was consistently altered by the K deficiency in leaflet while K deficiency increased root tissue concentrations of starch, glucose and fructose. K is also necessary for ribulose bisphosphate carboxylase activity in alfalfa plants [START_REF] Peoples | Role of potassium in carbon dioxide assimilation in Medicago sativa L[END_REF] and sucrose synthesis (SuSy) is highly sensitive to K deficiency in cotton [START_REF] Hu | Effects of potassium deficiency on the enzymatic changes in developing cotton fibers[END_REF].

• Sugar transport

As the major cation in phloem sap, K+ availability has dramatic effect on phloem functions such as sugar transport. Sugar produced during photosynthesis are transported through the phloem to others organs to insure their growth or storage. The energy necessary for the transport of sugars is provided by an H+/pumping ATPase which establishes a proton gradient and a transmembrane potential regulated by potassium channels of the AKT2/3 type [START_REF] Lemoine | Sourceto-sink transport of sugar and regulation by environmental factors[END_REF]. Evidences showed that loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis [START_REF] Deeken | Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis[END_REF]. As mentioned above, K deficiency in plant leads therefore to accumulation of sugars in leaves (sucrose as in cotton see [START_REF] Gerardeaux | Effect of carbon assimilation on dry weight production and partitioning during vegetative growth[END_REF]). However, according to [START_REF] Huber | Biochemical basis for effects of K-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves[END_REF] decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.

• Respiration

Respiration is also affected by K availability in plants. Low K progressively decreased the photorespiratory evolution of CO2 into CO2-free air, but steadily increased the rate of CO2 flux in darkness [START_REF] Terry | Effects of potassium deficiency on the photosynthesis and respiration of leaves of sugar beet[END_REF]. [START_REF] Okamoto | The respiration in leaf disks from younger taro plants under a moderate potassium deficiency[END_REF] suggested that at low K, respiratory CO2 production may be uncoupled from oxidative phosphorylation in leaves. High respiration rates under K deficiency are believed to be due, in part, to increased activity of the tricarboxylic acid cycle [START_REF] Okamoto | The respiration in leaf disks from younger taro plants under a moderate potassium deficiency[END_REF]. Recently, (Cui et al., 2019a) observed that leaf respiration rate correlated to putrescine and citramalate, two metabolites also associated with low K, and anticorrelated to succinate, malate, and fumarate. Potassium is required for maximal activity (in the forward direction) of succinate thiokinase [START_REF] Bush | Succinyl CoA synthetase of tobacco[END_REF][START_REF] Besford | Effect of potassium nutrition on some enzymes of the tomato plant[END_REF], which catalyzes the reversible reaction of succinyl-CoA conversion to succinate [START_REF] Yamashita | Respiration and organis acid metabolism in potassium deficient rice plant[END_REF], but also of pyruvate kinase. In fact, low K+ impedes pyruvate formation from phosphoenolpyruvate [START_REF] Evans | Effect of potassium & other univalent cations on activity of pyruvate kinase in Pisum sativum[END_REF][START_REF] Nowak | Nuclear magnetic resonance studies of the function of potassium in the mechanism of pyruvate kinase[END_REF][START_REF] Besford | Effect of potassium nutrition on some enzymes of the tomato plant[END_REF].

Therefore, K shortage must in principle lead to a drastic change in mitochondrial metabolism.

b) Nitrogen assimilation and protein synthesis

Potassium is involved directly and indirectly in plant protein metabolism not only for its role in enzyme activation but also for ribosome synthesis and mRNA turnover [START_REF] Evans | Potassium and its role in enzyme activation[END_REF][START_REF] Blevins | Role of potassium in protein metabolism in plants[END_REF][START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF] and K dependency of nitrate and amino acid transport [START_REF] Amtmann | Potassium in plants. e LS[END_REF]. Generally, K supply promotes the incorporation of N into proteins leading to higher levels of proteins and lower concentrations of amino acids in K-sufficient plants (in cotton (Hu et al., 2016b), in tobacco [START_REF] Koch | The influence of the level of potassium supply to young tobacco plants (Nicotiana tabacum L.) on short-term uptake and utilisation of nitrate nitrogen (15N)[END_REF], in rice [START_REF] Mengel | Effect of potassium on uptake and incorporation of ammonium-nitrogen of rice plants[END_REF] and in barley [START_REF] Helal | Nitrogen metabolism of young barley plants as affected by NaClsalinity and potassium[END_REF]). However, an increase of both protein and amino acid levels with increased K supply was found for tea leaves [START_REF] Ruan | Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensisL)[END_REF] and cucumber [START_REF] Ruiz | Relationship between potassium fertilisation and nitrate assimilation in leaves and fruits of cucumber (Cucumis sativus) plants[END_REF], while in several other studies, no effect of K on protein and/or amino acid levels was detected [START_REF] Amtmann | Potassium in plants. e LS[END_REF]. Moreover, K deficiency increases the content in several free amino acids in oil palm [START_REF] Ollagnier | Interaction entre l'azote et le potassium dans la nutrition des oléagineux tropicaux[END_REF] as well as Arabidopsis [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF].

In fact, potassium is further involved in the partitioning of NR activity between the root and the shoot (Blevins et al., 1978b;[START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF], which also depends on plant species, external nitrate supply, temperature and light intensity [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF][START_REF] Smirnoff | Nitrate assimilation and translocation by higher plants: comparative physiology and ecological consequences[END_REF][START_REF] Andrews | The partitioning of nitrate assimilation between root and shoot of higher plants[END_REF]. As mentioned earlier, K promotes long-distance transport of NO3-from roots to the leaves and thus improves NO3-contents and nitrate reductase activity in leaves, facilitating N use efficiency and metabolism in tobacco [START_REF] Zioni | Nitrate uptake by roots as regulated by nitrate reduction products of the shoot[END_REF], barley (Blevins et al., 1978b) and soybean [START_REF] Touraine | Charge balance in NO3--fed soybean: estimation of K+ and carboxylate recirculation[END_REF], while less N assimilation occurs in barley (Blevins et al., 1978b) or corn [START_REF] Rufty | Nitrate reduction in roots as affected by the presence of potassium and by flux of nitrate through the roots[END_REF] roots. At the opposite, K deficiency leads to lower translocation of NO3-and then a higher N assimilation in corn roots [START_REF] Rufty | Nitrate reduction in roots as affected by the presence of potassium and by flux of nitrate through the roots[END_REF]. Moreover, K was found to be involved in ammonium assimilation by up-regulating glutamine synthetase (GS), ferredoxin-glutamine-2-oxoglutarate aminotransferase (Fd-GOGAT) and glutamate dehydrogenase (GDH) [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]Hu et al., 2016b), suggesting a K effect as a compensatory response to maintain C flux through the TCA cycle and into amino acids and proteins. It is also important to note that individual amino acids show different responses to K. For example, in rice seedlings, K deficiency causes an increase in glutamine but a decrease in glutamate and aspartate [START_REF] Yamashita | Metabolism of acetate-1-14C in excised leaves from potassium deficient rice seedlings[END_REF].

c) C/N metabolism

According to [START_REF] Yamada | Effect of potassium nutrition on current photosynthesized carbon distribution to carbon and nitrogen compounds among rice, soybean, and sunflower[END_REF], K deficiency decreased carbon flux into the TCA cycle and amino acids and restricted the distribution of carbon from serine into other amino acids in sunflower, while rice and soybean were less affected. In fact, metabolite analysis in Arabidopsis roots revealed a decrease in nitrate, glycolytic intermediates (pyruvate), organic acids (malate, 2-oxoglutarate (2-OG), isocitrate), and negatively charged amino acids (glutamate, aspartate)

and an increase in the levels of soluble carbohydrates (sucrose, glucose, fructose) and many amino acids, notably those with high N-carbon (C) ratio and/or a positive charge (glutamine, glycine, arginine) in K-deficient plants [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]. FIGURE I.9 exemplifies the type of information that was obtained in this study by mapping the changes of transcript levels, enzyme activities and metabolites into a given branch of the metabolic network. In this study, most metabolite changes were at least partially reversed within 24 hours of K re-supply, a notable exception being sucrose, glucose and fructose in the shoots. In roots, pyruvate levels were replenished within minutes, whereas malate and 2-oxoglutarate levels recovered more slowly [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]. According to [START_REF] Amtmann | Potassium in plants. e LS[END_REF], the reversible change in the metabolite profile was accompanied by a reversible change in the activity of enzymes, including those involved in sugar metabolism, glycolysis, TCA cycle and nitrogen assimilation. [START_REF] Amtmann | Potassium in plants. e LS[END_REF] suggested that regulation of the enzymes is posttranslational rather than transcriptional and that the changes in enzyme activities were the result of negative feedback control through the reaction products. Moreover, [START_REF] Amtmann | Potassium in plants. e LS[END_REF] suggested that pyruvate kinase can be viewed as a central integrator of C and N metabolism since it is the primary enzymatic target of K deficiency. The genome microarray study by [START_REF] Armengaud | The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling[END_REF] showed that several enzymes in glycolysis and anaplerosis are regulated during K stress; in particular, the upregulation of two genes for malic enzyme, which catalyses an alternative pathway for pyruvate synthesis, was observed [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]. Transcript changes in response to K-deficiency and re-supply are shown above and below the enzymes respectively. Individual boxes represent individual genes encoding different enzyme isoforms. For details see [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]. From [START_REF] Amtmann | Potassium in plants. e LS[END_REF].

d) K role in plant growth, morphology and yield

Considering the multiple roles of potassium in physiology, it is unsurprising that potassium fertilization has a positive effect on plant growth and increases plant biomass [START_REF] Ebelhar | Effects of nitrogen and potassium on growth and cation composition of corn genotypes differing in average ear number 1[END_REF][START_REF] Mullins | In-row subsoiling and potassium placement effects on root growth and potassium content of cotton[END_REF][START_REF] Pettigrew | Dry matter production, nutrient uptake, and growth of cotton as affected by potassium fertilization[END_REF]. In the case of potassium deficiency, plant biomass is reduced and accompanied also by a reduction in leaf area mainly because of a slower rate of leaf appearance and a reduced final size of individual leaves [START_REF] Kimbrough | Potassium effects on regrowth of alfalfa (Medicago sativa L.)[END_REF][START_REF] Pettigrew | Dry matter production, nutrient uptake, and growth of cotton as affected by potassium fertilization[END_REF][START_REF] Jordan-Meille | Leaf area establishment of a maize (Zea mays L.) field crop under potassium deficiency[END_REF]. In fact, for eucalyptus, the leaf area reduction can come from a reduction in the number of leaves produced, a reduction in the size of individual leaves or both [START_REF] Laclau | Influence of nitrogen and potassium fertilization on leaf lifespan and allocation of above-ground growth in Eucalyptus plantations[END_REF].

Taken as a whole, potassium plays also a crucial role in crop yield [START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF]. In soybean plants, K affects positively the yield by increasing yield component such as the number of pods per plant [START_REF] Nelson | Fruit development, seed quality, chemical composition, and yield of soybeans as affected by potassium and magnesium 1[END_REF][START_REF] Jones | Effects of phosphorus and potassium on soybean nodules and seed yield 1[END_REF][START_REF] Bharati | Soybean response to tillage and nitrogen, phosphorus, and potassium fertilization[END_REF] and the weight of individual seeds [START_REF] Bharati | Soybean response to tillage and nitrogen, phosphorus, and potassium fertilization[END_REF] or the number of seed per pods [START_REF] Coale | Root distribution and shoot development in no-till full-season and double-crop soybean[END_REF].

In wheat, K increases yield by increasing in the number of heads per unit area and the number of kernels per head [START_REF] Haeder | Influence of potassium nutrition and water stress on the content of abscisic acid in grains and flag leaves of wheat during grain development[END_REF], but in other study, this trend was not observed [START_REF] Sweeney | Phosphorus, potassium, chloride, and fungicide effects on wheat yield and leaf rust severity[END_REF].

Fruit crop quality is also enhanced by K through its influence on size, appearance, color, soluble solids, acidity, vitamin contents, disease resistance, and shelf-life of fruits. K improves quality of the fruits in several species by maintaining desirable sugar-to-acid ratio, ripening of fruit and many other processes [START_REF] Kumar | Role of potassium in fruit crops-a review[END_REF]. In oil crop, effect of potassium on oil composition is still unclear. In some studies, K was found to increase seed linoleic acid content and decrease oleic acid content [START_REF] Gaydou | Effects of phosphorus, potassium, dolomite, and nitrogen fertilization on the quality of soybean. Yields, proteins, and lipids[END_REF], while other studies did not find consistent results from K+ fertilization on seed oil and protein content [START_REF] Haq | Response of soybean grain oil and protein concentrations to foliar and soil fertilization[END_REF][START_REF] Seguin | Potassium, phosphorus, sulfur, and boron fertilization effects on soybean isoflavone content and other seed characteristics[END_REF]. Similarly, effect of K on isoflavones soybean seeds is controversial [START_REF] Yin | Critical leaf potassium concentrations for yield and seed quality of conservation-till soybean[END_REF][START_REF] Seguin | Potassium, phosphorus, sulfur, and boron fertilization effects on soybean isoflavone content and other seed characteristics[END_REF].

C. Oil palm mineral nutrition 1. K needs in oil palm

a) Fertilization recommendation

Oil palm is amongst C3 crops that has a very high fruit production rate. However, as mentioned above, oil palm growth requires high fertilization rates since physiological requirements (to get optimal yield) are high in this species, and furthermore, soils on which oil palm is grown is generally K-poor. Potassium chloride (KCl) is the most widely used fertilizer in oil palm plantations and also the most expensive. As mentioned above, other forms of potassium fertilizers are also used, such as potash sulphate (K2SO4), (potassium and magnesium sulfate, K2Mg2(SO4)3), compound fertilizers (mix of two or three nutrients of N, P, K) or palm leaf and trunk residues.

According to [START_REF] Corley | The oil palm[END_REF], the total K demand is assumed to have two components: "growth" demand and "deficiency" demand. The "growth" demand is determined by the balance between initial content of mineral nutrient, enlargement of plant organs and production of parts that are removed such as leaves, bunches and inflorescences. The growth demand can therefore be calculated as the K demand for the increasing dimensions of the palm trunk, the production of new organs as bunches (or inflorescences) and leaves, less any return of K nutrient. Conversely, when the oil palm is not in a satisfactory nutrient balance, a deficiency situation can take place. In case of K deficiency, a supply is therefore necessary in order to replenish oil palm K mineralomass but also potassium soil content. The "deficiency" demand is thus defined as the amount of K required to fulfill optimum yield and growth.

In mature oil palm, K elemental content differs greatly between organs. Most of the K applied can be stored in the trunk and represents a useful reserve [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF]. Teoh and Chew (1988a) estimated that trunk reserve can be sufficient to support growth and yield for 2 to 6 years. Under standard cultivation conditions in Sumatra, K content is between 0.6 and 1.2 % of dry matter in leaflet and 1.3-1.5% in rachis. In fruit bunches, potassium is by far the most abundant macroelement. The highest K content can be found in stalks (5-8% of dry matter), which account for up to 26% of total bunch K content. Due to its high biomass, the trunk alone represents 34 to 50% of the total palm K [START_REF] Teoh | Use of rachis analysis as an indicator of K nutrient status in oil palm[END_REF]Teoh & Chew, 1988a).

According to [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF], most of the increase in K in the plant is accounted for in vegetative parts. The partitioning of absorbed K by the oil palm to its vegetative parts seemed to be related to its nutritional status and K requirement. It is worth noting that within a crown, leaflet K content varies with palm age [START_REF] Tinker | Cation relationships and magnesium deficiency in the oil palm[END_REF] and leaf rank. Samples taken from leaf 9, 17, 25, and 33 show decreasing in K content in leaflet with increasing palm age [START_REF] Fairhurst | Management of nutrients for efficient use in small holder oil palm plantations[END_REF]. Ng et al. (1999) have proposed that on tropical soils of poor fertility, the total demand in potassium of a mature plantation producing 20 tons fresh fruit bunches (FFB) per year is about 205 kg K ha-1. The annual nutrient demand of oil palm, depending on tree age, has been characterized further in several studies (TABLE I.3). Table I.3 summarizes data from several sources on the annual nutrient demand of oil palm of various ages based on the need of different plant parts. In practice, K is usually applied once to twice a year. Increasing the frequency of fertilizer application up to 6 times a year does not improve the yield but rather the risk of loss by leaching.

b) K nutrition monitoring

In oil palm plantations, potassium deficiency is mainly due to removing bunches (up to 248 kg K ha-1 y-1 is abstracted from oil palm agrosystems during bunch harvesting [START_REF] Heffer | Assessment of fertilizer use by crop at the global level[END_REF]) and losses by leaching (up to 30% of potassium applied [START_REF] Omoti | Leaching losses of nutrients in oil palm plantations determined by tension lysimeters[END_REF]Foong, 1991;[START_REF] Chang | Leaching losses of nutrients from a mature oil palm catchment in Malaysia[END_REF]). K deficiency is also common on peat soils, sandy soils with low pH derived from sandstone and granite and acid soils with low cation exchange capacity. To characterize or predict the deficiency demand, and to monitor oil palm K requirements, there are different agronomical tools such as fertilization trials, leaf diagnosis, observation of leaf symptoms and soil analysis.

• Fertilization trials are used to determine the response curve of yield with respect to K input, and therefore to provide recommendations for fertilization to achieve optimum production. This will be illustrated further below in Section 2.

• According to [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF], K deficiency symptoms are rare in established plantations. Leaf diagnosis (LD) is therefore widely used to detect sub-optimal nutrition as leaf nutrient status is directly correlated with FFB yield (See Chapter I.2), and nutrient imbalance.

Leaf diagnosis is based on the potential relationship between the nutrient content in leaflet tissue and in other organs. Usually, palm rank 17 is used as the standard sample for analysis. Main mineral contents are measured and compared to known critical values. Potassium deficiency may happen when leaflet K elemental content is lower than 0.7% in mature oil palm [START_REF] Jacquemard | Le palmier à huile: Quae[END_REF]. However, leaf nutrient concentration is also influenced by different factors such as leaf age, leaflet rank, leaf number, tree age, sampling time along the fruiting cycle, planting material (cross), palm density, fertilizer treatment, rainfall and soil properties [START_REF] Fairhurst | Management of nutrients for efficient use in small holder oil palm plantations[END_REF], but also ratio to other nutrient concentrations (See Chapter I.3). Therefore, all these parameters have to be taken into account for potassium fertilization. But quite often, it is assumed that leaflet K content reflects the K status of the tree [START_REF] Chapman | Leaf analysis and the nutrition of the oil palm (Elaeis guineensis Jacq.)[END_REF].

However, in general, leaf K content does not directly reflect K availability depending on soil properties or oil palm varieties [START_REF] Ollivier | Genetic variation in nutrient uptake and nutrient use efficiency of oil palm[END_REF][START_REF] Dubos | High soil calcium saturation limits use of leaf potassium diagnosis when KCl is applied in oil palm plantations[END_REF][START_REF] Dubos | High soil calcium saturation limits use of leaf potassium diagnosis when KCl is applied in oil palm plantations[END_REF]. In fact, on soils of volcanic origin, rachis potassium concentration has been found to be better correlated to soil potassium than was leaf potassium content but not to the yield [START_REF] Ollivier | Genetic variation in nutrient uptake and nutrient use efficiency of oil palm[END_REF] and then rachis cannot be used as a better proxy than leaflet for K as recommended by Foster (2002).

• Observation of leaf symptoms are also used to detect deficiency. In case of potassium deficiency, pale green spot becoming gradually colored with yellow or orange appear on leaflets. Symptoms expand across and between veins, until necrotic spots appear, with possible fungus invasion giving thus a 'bronzed' appearance to the palm. However, leaf symptoms as an indicator of potassium deficiency are not very reliable, since sometimes potassium deficiency is not accompanied by visible leaf symptoms. In general, symptoms appear on older leaves as K is translocated to young fronds, thereby exaggerating K deficiency in old leaves [START_REF] Rankine | Management of phosphorus, potassium and magnesium in mature oil palm[END_REF].

• In oil palm, soil analysis is used either as a diagnostic tool to group the soil types and to approximate the soil nutrient supply to oil palm, or as a prognostic tool to predict the yield response curve of oil palm to fertilizer rates (Foster, 1985;Foster et al., 1985). Both methods are briefly reviewed in [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF]. In both cases, the soil physical, chemical and mineralogical properties are determined. The diagnostic tool is based on a general classification table for soil nutrients. For example, potassium deficiency may occur when the concentration in exchangeable K in soil is less than 0.2 cmol kg-1. Also, soil exchangeable Mg/K has to be above two to avoid magnesium deficiency on acid soils in West Africa [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF]. In addition, a soil-based system to predict the optimum N and K rates for oil palm in West Malaysia has been described in [START_REF] Fairhurst | Oil palm: management for large and sustainable yields. Oil palm: management for large and sustainable yields[END_REF]. This system was developed by

Foster and his associates, using around 50 factorial fertilizer experiments in West Malaysia.

The system, which was statistical in nature, attempted to reconstruct the yield response curve to N and K fertilizer inputs based on site characteristics. The system essentially had three steps (Foster, 1985;Foster et al., 1985): ( 1) predict yield without N and/or K (starting point of the system) (2) predict yield response to N at non-limiting K and vice-versa and ( 3) predict yield at any combination of N and K fertilizers. However, this system depended on statistical relations, and not on a basic understanding of the underlying mechanisms for plant nutrient uptake, growth and yield [START_REF] Goh | Fertilizer recommendation systems for oil palm: estimating the fertilizer rates[END_REF]. In fact, soil fertility is affected not only by soil nutrient content but also texture, structure, consistency, terrain, moisture status and mineralogy.

Moreover, soil nutrient variation is extremely high between soil types and within the palm area, and error in sampling a fertilized field is too large, making interpretation difficult and probably unreliable.

• Metabolic signature : as suggested by (Corley et al., 1976), some metabolites could be affected by K content and thus their analysis could be helpful to monitor oil palm K status. For example, putrescine accumulates in several species under potassium deficiency, more or less specifically [START_REF] Hoffman | Free amine content in fruit tree organs as an indicator of the nutritional status with respect to potassium[END_REF]Cui et al., 2019b). However, this point has never been used in practice in the field so far.

Oil palm growth and yield responses to K availability

In oil palm, the positive effects of K fertilization on growth and yield are well-established [START_REF] Ochs | Contribution à l'étude de la fumure potassique du palmier à huile[END_REF]Hartley, 1988a;[START_REF] Corley | The oil palm[END_REF]. In immature oil palm (aged 1 to 3 years), potassium fertilization affects significantly plant morphology, and increases plant height, trunk diameter, leaf number, leaf length, and leaf area of the reference palm rank no. 17 [START_REF] Purwanto | Roles and optimisation rate of potassium fertiliser for immature oil palm (Elaeis guineensis Jacq.) on an Ultisol soil in Indonesia[END_REF]. An increase in leaf area has also been found in mature oil palm under potassium fertilization, partly due to an increase in petiole cross section [START_REF] Ruer | Premières observations sur les caractéristiques du pétiole de la feuille de palmier à huile[END_REF]Corley, R & Mok, CK, 1972).

The beneficial effects of potassium fertilization on oil palm growth probably come from an increased carbon metabolism. Evidences showed that potassium application significantly increases carbon assimilation via higher chlorophyll and Rubisco contents, and stomatal density and conductance in plant [START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF][START_REF] Battie-Laclau | Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment[END_REF]. Reciprocally, in oil palm saplings cultivated in the greenhouse, K deficiency leads to a considerable increase in respiratory CO2 production and a decline in the biosynthesis of proteins involved in photosynthetic machinery (Cui et al., 2019b). In addition, high K availability stimulates nitrogen metabolism while K deficiency (or K excess) leads to oxidative stress (Cui et al., 2019b). However, higher K availability seems to decrease specific leaf weight (g DW m-2), perhaps because it changes non-structural carbohydrate metabolism and thus leaf starch and glucose contents. For example, potassium is essential for phloem sugar movement [START_REF] Deeken | Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis[END_REF], and thus the remobilization of soluble sugars from vegetative parts like trunk base and leaves (incl. petioles) to sustain fruit development [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF]. Therefore, high K availability tends to accelerate sugar movement and decrease sugar content in leaflets.

However, excess of K may lead to an inhibition of the transport of assimilates from source leaves to sink organs and consequently to an inhibition of the photosynthesis [START_REF] Senbayram | Role of magnesium fertilisers in agriculture: plant-soil continuum[END_REF].

The response curve of fresh fruit bunch production to K fertilization is well known (Foster, 2002;[START_REF] Corley | The oil palm[END_REF]. FIG. I.9 shows the relationship between leaflet potassium content and yield. Fresh fruit bunch production increases with K fertilization level up to an optimum at 2.0 kg K tree-1 yr-1 (TABLE I.4) simultaneously with K leaflets and rachis contents. Above this amount of K, there is no significant increase in fresh fruit bunch production (TABLE I.4) [START_REF] Imogie | Effect of potassium (K) source on oil palm yield at Okomu oil palm Plc, Ovia North East LGA of Edo State[END_REF]. Increasing yield using K fertilization involves a change in bunch components. Bunch number, bunch weight and fresh fruit bunch production are indeed significantly higher with increasing K fertilization [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF][START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b). However, in some studies, potassium has been shown to decrease the oil-to-bunch ratio significantly on inland soils while this ratio increased on coastal soils, but in both cases potassium fertilization led to a significant increase in oil yield [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]. While the effect of K on bunch number is well -established, its effect on lipid accumulation in mesocarp (oleosynthesis) is unclear. There are specific dynamics of K content in fruits that suggest a role in fruit metabolism. In fact, in fruit mesocarp, the K content has been shown to increase progressively for 15 weeks after anthesis (WAA) and then decreases during last developmental stages where lipid biosynthesis occurs [START_REF] Desassis | Sur les modalités de formation des matières grasses dans le fruit d'Elaeis guineensis Jacq[END_REF]. Several studies have shown that higher K availability tends to increase average mesocarp oil content (as well as the proportion of oleate in oil fatty acid composition) [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]Ollagnier & Olivin, 1984b) while others found a negative effect [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]. It has been suggested that this occasional negative effect may come from an increased tissue chloride (Cl-) concentration (which inevitably comes along with KCl-based fertilization), resulting in lower mesocarp-to-kernel biomass ratio in fruits [START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b). Deli x Yangambi trees subjected to different forms of chloride fertilization (either as NaCl or KCl) have been shown to form heavier bunches with more and bigger fruits, and higher proportion of kernel biomass, with no change in OER (Ollagnier & Olivin, 1984a), and this effect might also be related to Cl-. The positive effect of potassium fertilization on lipid synthesis seems to partially come from its effect on moisture content. In other words, if increasing potassium is associated to a lower fruit water content, higher lipid accumulation is observed by mass-balance. Interestingly, fruit transpiration rate (water loss) has been found to be positively related to lipid biosynthesis [START_REF] Jeje | Transpiration and oil accumulation rates for developing oil palm fruits Elaeis guineensis Jacq[END_REF]Teh et al., 2013a).

Interaction of K and others nutrients

In oil palm, optimum leaf nutrient ranges varied from 2.24 to 2.97 %, 0.08 to 0.14 % and 0.78 to 0.91 % for N, P and K, respectively, from 0.74 to 1.53 %, 0.25 to 0.98 % and 0.72 to 1.09 % for Ca, Mg and sulfur (S), respectively, and from 5.71 to 31.0 mg kg-1, 7.42 to 12.9 mg kg-1, 33.6 to 58.6 mg kg-1, 82.5 to 681 mg kg-1 and 82.8 to 936 mg kg-1 for boron (B), copper (Cu), zinc (Zn), manganese (Mn) and iron (Fe), respectively [START_REF] Behera | Soil nutrient status and leaf nutrient norms in oil palm (Elaeis guineensis Jacq.) plantations grown on southern plateau of India[END_REF]. As expected, there is an interaction between K fertilization and others nutrients, with an antagonistic response that has been documented recently in oil palm saplings, between K, Na, Ca and Mg (Cui et al., 2019b). The antagonism simply comes from (i) competition for exchange sites in soil and (ii) competition for transporters in roots. In some cases, tissues K concentration may be low due to uptake competition such as between K and Mg, while soil K is sufficient [START_REF] Pushparajah | Leaf analysis and soil testing for plantation tree crops: ASPAC & FFTC[END_REF].

This antagonistic effect is particularly visible for K and Mg fertilization so that an excess of one may interfere with the availability of the other [START_REF] Senbayram | Role of magnesium fertilisers in agriculture: plant-soil continuum[END_REF]. Studies have shown that optimum K in leaflet depends strongly on soil Mg content [START_REF] Ochs | Contribution à l'étude de la fumure potassique du palmier à huile[END_REF][START_REF] Corley | The oil palm[END_REF]. Particularly, in sandy soils, application of high rates of K fertilizer often enhances the risk of Mg deficiency, however, at a higher Mg concentration in the soil solution, generally K uptake is not disturbed [START_REF] Senbayram | Role of magnesium fertilisers in agriculture: plant-soil continuum[END_REF]. This is due to the fact that in the root, the specific K transporters cannot be blocked by others nutrients, whereas Mg transporters are non-specific and can be used by other cations such as K. Therefore, when K concentration in the soil-root interface is high, plant ability to take up sufficient Mg is limited. It is therefore useful to consider (Ca, Na, Mg)/K ratio under K deficiency for a better K fertilization [START_REF] Fallavier | Etude expérimentale de la dynamique du potassium dans deux sols tropicaux utilisés pour la culture du palmier à huile[END_REF][START_REF] Corley | The oil palm[END_REF]. However, as mentioned above, in the oil palm such correlations may disappear when the sum K + Ca + Mg is lower than 2.0% [START_REF] Fairhurst | Oil palm: management for large and sustainable yields. Oil palm: management for large and sustainable yields[END_REF].

At the opposite, there is a synergy between K and N nutrition. For example, leaf analysis may indicate both N and K deficiencies, but a parallel soil analysis may reveal that soil K is adequate and only N needs to be added, allowing significant cost savings [START_REF] Pushparajah | Leaf analysis and soil testing for plantation tree crops: ASPAC & FFTC[END_REF].

According to [START_REF] Teoh | Investigations on areas and frequencies of fertiliser application in mature oil palms[END_REF], in mature oil palm, N and K fertilizers are usually applied once or twice a year on coastal marine, nutrient-rich clay soils, and at least twice per year on alluvial, nutrient-poor inland soils. Both nutrients are normally applied together to avoid imbalances and allow optimum yield. In fact, N increased yield by 49% in the presence of high K level rate and there was a 25% positive yield response to K when high N rate was applied [START_REF] Harun | Fruit set and oil palm bunch components[END_REF]. By contrast, in the absence of N, increasing K application rates depressed yield but had no effect on growth (Foster & Mohammed, 1988). Positive effects on oil-to-bunch ratio have been observed too (Foster & Mohammed, 1988).

Moreover, as N has a linear relationship with phosphorus (P) content in leaflet [START_REF] Tampubolon | Réponses du palmier à huile aux fumures azotée et phosphorée à Sumatra[END_REF]Foster, 2002), potassium interacts indirectly with P. In particular, K has an effect on rachis P content and modifies therefore P allocation at the plant level. Also, because of the synergism between N and P uptake, leaf P concentration must be assessed in relation to leaf N concentration during field monitoring [START_REF] Ollagnier | Management of mineral nutrition in industrial oil palm plantations. Fertilizer savings[END_REF]. This is due to the constant N/P ratio in protein compounds found in plant tissue [START_REF] Fairhurst | Oil palm: management for large and sustainable yields. Oil palm: management for large and sustainable yields[END_REF].

D. Specific objectives of the thesis

The positive effect of K fertilization in oil palm yield has been well known for several decades. agrosystem husbandry practices, is the investigation of the impact of K on this metabolic aspect.

Then the following research questions are central to the present thesis; they all relate to defining the effect of K availability on metabolic pathways and metabolic changes related to yield:

(A) What is the impact of K fertilization on proteomic and metabolomic responses of leaflets on two genetically contrasted material, presenting different leaflet K mineral signature? (B) What is the impact of K fertilization on omics responses of the oil palm fruit mesocarp during maturation, on two genetically contrasted material? (C) Can a 13CO2 labelling help identifying sugars produced and C allocation and is all this can be affected by K availability?

In this PhD thesis, we used realistic K conditions in the field in Sumatra, K0 to K3 (that is, no KCl applied for 3 yr before and also during the experiment (K0), 1.5 kg KCl tree-1 yr-1 (K1), 3 kg KCl tree-1 yr-1 (K2), and 4.5 kg KCl tree-1 yr-1 (K3)), far from excess or deficient potassium conditions, with two contrasted oil palm crosses (Deli x La Mé and Deli x Yangambi).

We took advantage of several methods, combining functional traits (production and fruit analysis), elemental analyses, functional genomics (proteomics and metabolomics) in (i) leaflets and (ii) fruits during maturation. In addition, a preliminary 13CO2 labelling experiment has been carried out on a palm as a perspective to study carbon flux from source to sink organs and to appreciate the feasibility of isotopic tracing in oil palm in the field.

A mid-term objective of this overall study "K/omics" for oil palm is to identify molecular biomarkers susceptible to precise the K nutritional status of trees studied and then allow the monitoring of K fertilization with higher accuracy than with LD which takes into account only elemental analyses.

In order to answer our first question, which is "What is the impact of K fertilization on proteomic and metabolomic responses of leaflets on two genetically contrasted material, presenting different leaflet K mineral signature?", the following chapter explores key leaf metabolic pathways under K fertilization treatments, linked to functional traits and yield. This chapter is in the form of a research article that has been submitted to Environmental and Experimental Botany.

A. Introduction

Oil palm (Elaeis guineensis Jacq., Arecaceae) is the most widely cultivated oil crop in the world. Because of both its economic efficiency as a high-yielding source of edible and technical oils, the global demand in palm oil for food industry, already very high, will probably increase in the next decade. Presently, palm oil represents the largest oil production globally (about 75

Mt y-1, FAOSTAT). Oil palm plantations are widespread in tropical countries such as Indonesia and Malaysia. In the past decades, intense efforts have been devoted to improve oil palm yield, and this includes not only the generation of better cultivars (crosses) but also the identification of optimal mineral nutrition. Nitrogen (N), phosphorus (P) or potassium (K) fertilization are used, sometimes intensively, to increase fruit development and thus palm oil production.

Typical K fertilization consists, on average, of 180 kg K ha-1 y-1 (≈1.5 kg K tree-1 y-1) [START_REF] Heffer | Assessment of fertilizer use by crop at the global level[END_REF], which represents an annual cost of about US$ 1b at the global scale.

In fact, potassium supply (generally in the form of potassium chloride, KCl) is by far the most important input in oil palm agrosystems. In general, oil palm biomass contains more K than N, and K elemental content can reach very high values, up to 8% in rachis bunch (about 1% in leaf organic matter) [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF][START_REF] Corley | The oil palm[END_REF]. In addition, fruit bunch harvesting removes substantial amounts of K and this has to be compensated for by K fertilization [START_REF] Heffer | Assessment of fertilizer use by crop at the global level[END_REF]. The positive effects of K fertilization on tree development are now well established: K fertilization increases total leaf biomass by increasing total leaf area (by increasing rachis length), and depending on the cross, increases bunch weight and/or bunch number [START_REF] Ochs | Contribution à l'étude de la fumure potassique du palmier à huile[END_REF]Hartley, 1988a;[START_REF] Corley | The oil palm[END_REF][START_REF] Mirande-Ney | Effects of potassium fertilization on oil palm fruit metabolism and mesocarp lipid accumulation[END_REF].

However, potassium requirements in oil palm depend on soil quality (such as the Ca or Mg content), other nutrients (N and P fertilization) as well as other environmental conditions (such as water availability) [START_REF] Corley | The oil palm[END_REF]. Therefore, K fertilization as KCl commonly carried out in the field can be inadequate. This may have side effects, such as Cl-excess in tissues (causing a decline in fruit quality) [START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b), oxidative stress, or potassium and chloride losses by leaching [START_REF] Omoti | Leaching losses of nutrients in oil palm plantations determined by tension lysimeters[END_REF]Foong, 1991;[START_REF] Chang | Leaching losses of nutrients from a mature oil palm catchment in Malaysia[END_REF].

It is only recently that physiological mechanisms of the response to K availability have been examined in oil palm. In oil palm trees cultivated in North Sumatra (Indonesia) in an agronomic trial with contrasted K fertilization levels, it has been shown that elemental K content and glucose concentration generally correlate positively, with substantial differences in K content between organs [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF]. Since fruits are fed with sugars (glucose, sucrose) from other organs to sustain oleosynthesis [START_REF] Dussert | Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition[END_REF][START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF][START_REF] Wong | Differential gene expression at different stages of mesocarp development in high-and low-yielding oil palm[END_REF], this correlation may explain why K availability is related to yield. In oil palm saplings cultivated in the greenhouse, leaf metabolism and proteins have been found to respond strongly to K availability, with a considerable increase in respiratory CO2 and a decline in the biosynthesis of photosynthetic machinery under K deficiency; in addition, changes in K availability were associated with modifications in metabolism, such as polyamine synthesis under K deficiency and oxidative stress under both K deficiency and K excess [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF][START_REF] Hussain | Polyamines: natural and engineered abiotic and biotic stress tolerance in plants[END_REF][START_REF] Ahmad | Cellular and tissue distribution of potassium: Physiological relevance, mechanisms and regulation[END_REF]Cui et al., 2019b). In species other than oil palm, K availability is well known to play an important role in several biochemical processes such as protein synthesis (translation), carbohydrate metabolism, phloem transport, and enzyme activation [START_REF] Corley | Effects of nitrogen, phosphorus, potassium and magnesium on growth of the oil palm[END_REF][START_REF] Mengel | Effect of potassium on the assimilate conduction to storage tissue[END_REF][START_REF] Marschner | 8 -Functions of mineral nutrients: macronutrients[END_REF][START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF][START_REF] Zörb | Potassium in agriculturestatus and perspectives[END_REF][START_REF] Hu | Potassium application affects carbohydrate metabolism in the leaf subtending the cotton (Gossypium hirsutum L.) boll and its relationship with boll biomass[END_REF]Hu et al., 2016a;Hu et al., 2016b;[START_REF] Zahoor | Potassium fertilizer improves drought stress alleviation potential in cotton by enhancing photosynthesis and carbohydrate metabolism[END_REF]. In particular, K deficiency leads to a strong decrease in key enzymatic activities such as pyruvate kinase, thereby impeding glycolysis (Cui et al., 2019b). Other enzymes are also K-dependent (such as starch synthase, succinate thiokinase, etc.) and this biochemical effect probably contributes to changes in metabolite contents (Cui et al., 2019b). Also, many studies have shown that K deficiency affects nitrogen metabolism, with lower nitrate assimilation, higher putrescine biosynthesis from glutamate, and changes in some amino acids due to the restriction of protein synthesis and pyruvate synthesis [START_REF] Mengel | Effect of potassium on the assimilate conduction to storage tissue[END_REF][START_REF] Blevins | Role of potassium in protein metabolism in plants[END_REF]Hu et al., 2016b). At the leaf level, K deficiency lowers stomatal conductance, and thus causes a decline in the photosynthesis rate [START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF][START_REF] Favreau | Distinct leaf transcriptomic response of water deficient Eucalyptus grandis submitted to potassium and sodium fertilization[END_REF]. Conversely, K fertilization leads to an increase in stomatal and mesophyll conductances and in the biosynthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) leading thus to a larger photosynthesis rate [START_REF] Hu | Potassium application affects carbohydrate metabolism in the leaf subtending the cotton (Gossypium hirsutum L.) boll and its relationship with boll biomass[END_REF]Hu et al., 2016a). Taken as a whole, K availability may have primarily an impact on oil palm tree photosynthesis (via total leaf surface area, photosynthate export or synthesis of photosynthetic machinery) and leaf primary C and N metabolism and thus on the efficiency of leaves to sustain fruit development.

However, this question has never been examined precisely, in particular in oil palm grown in the field. This lack of knowledge is problematic not only because K availability is an important determinant of palm oil production (see above) but also because most studies looking at the effect of K availability on metabolism take advantage of extreme situations (deficiency, or excess) that are unlikely to reflect realistic field conditions. In other words, the actual physiological effect of changes in K availability is poorly known, in particular before symptoms become visible. In the case of oil palm, a tree crop, the question as to whether changes in tree K provision (fertilization) might have rapid effects on leaf metabolism is of particular significance since in principle, low K availability could be compensated for by K remobilization from other K-rich organs (such as the trunk) to sustain leaf photosynthesis and fruit development instead of a lower yield or a deficiency. A better knowledge of leaf metabolism under varying K availability would also be of interest to monitor K requirements and thus adjust K fertilization before consequences on fruit production and yield. Presently, K fertilization decisions are based on leaflet K content (about 10 mg g-1 dry weight (DW) under K-sufficient conditions) using the so-called LD (leaf diagnosis) method [START_REF] Chapman | Leaf analysis and the nutrition of the oil palm (Elaeis guineensis Jacq.)[END_REF], but in some agronomic trials, LD has been found not to reflect perfectly K requirements (Foster, 2002).

As an aid in clarifying physiological mechanisms involved in early leaf response to K availability, we conducted here an analysis of leaf metabolism (proteome and metabolome) coupled to tree phenology and elemental analyses, using oil palm trees of two crosses, Deli x La Mé (referred to as DL) and Deli x Yangambi (referred to as DY), grown in the field in North Sumatra (Indonesia). Here, the objective of the study was not to describe the agronomical effects of K availability but rather, to elucidate biochemical ("omics") leaf responses to K availability in the field and thus compare with greenhouse conditions that have been documented previously (Cui et al., 2019b). We used different K fertilization regimes maintained for two years, with conditions far from deficiency and excess, such that final leaf K varied within a rather narrow range (18-38 mmol m-²), similar to what is usually observed in the field. Our results show that K availability altered N metabolism, photosynthesis and reorchestrated mitochondrial metabolism in leaves, with changes not only in respiratory efflux, but also in mitochondrial metabolites and proteins involved in catabolism.

B.

Material and methods Then in 2015, the same amount of fertilizers was applied to all trees (1.75 kg of urea and 2 kg of KCl per tree) so as to sustain growth and avoid extreme differences between K0 and K3 treatments that would not be close enough to realistic field conditions. K fertilization treatments began in May 2016 with 2 kg of urea tree-1 and 0, 1.5, 3 or 4.5 kg of KCl tree-1 y-1.

Trees were fertilized with nitrogen using 2 kg urea tree-1 y-1. There was no K deficiency under K0 conditions, since natural K in the soil under K0 treatment represents about 0.2 meq exchangeable K per 100 g soil, and some K remained from previous agronomic trials conducted before ours on the same location (information from soil analysis). As such, oil palm tree tissues were not under very low or very high K conditions and K content was always in the 18-38 mmol The number of leaves and bunches (males and females) present on trees at the time of sampling was determined by visual inspection of trees. Specific leaf weight (SLW) was measured in 2017, as dry weight per leaf surface area (g DW m-2) by weighing precisely 30 leaflet samples of 10 cm2 on a representative palm of the crown. Leaf area was determined in 2017 on the leaf rank number 17 following the method of [START_REF] Tailliez | A method for measuring oil palm leaf area[END_REF][START_REF] Tailliez | A method for measuring oil palm leaf area[END_REF][START_REF] Tailliez | A method for measuring oil palm leaf area[END_REF].

Overall foliar emission rate was calculated as the number of leaves present on the tree plus

Climatic conditions

Climatic conditions (solar radiation, rainfall, temperature and relative humidity) were recorded near the fertilizer trial at Bangun Bandar (Nord Sumatra, Indonesia) using a minimeteorological station (Watchdog, Spectrum, France). The

Elemental analysis

Mineral content analysis was done on leaf and rachis by PSBB (Pusat Seleksi Bangun Bandar) at the SOCFINDO laboratory (Bangun Bandar, Indonesia). Leaflet samples were first cleaned with deionized water, then oven-dried and ground into fine powder. Analyses (N, P, K, Ca, Mg and Cl elemental contents) were carried out either by titration (N), spectrophotometry (P and Mg) or by flame photometry (K, Ca, Cl) following methods from [START_REF] Van Ranst | Manual for the soil chemistry and fertility laboratory: analytical methods for soils and plants equipment, and management of consumables[END_REF].

Leaflet nitrate was analyzed using the LAQUA Twin Nitrate Tester® (Spectrum Technologies) using 3-fold diluted leaf extracts (3 mg leaf powder extracted with 4 mL deionized water).

Sampling for omics

For proteomics and metabolomics, leaflets were sampled between 8 and 10 am. Samples were rapidly cleaned with deionized water, frozen in liquid nitrogen then freeze dried (lyophilized)

for metabolomics, and kept frozen prior to total protein extraction (for proteomics). Proteomics and metabolomics analyses were performed on leaflets only.

Metabolomics analysis

After lyophilization, leaflets were ground into fine powder and 10 mg of powder of each sample was extracted with 1 mL cold (-20°C) water-acetonitrile-isopropanol mixture (2:3:3, v:v:v) containing 4 pg mL-1 ribitol as an internal standard. Samples were placed for 10 min at 4°C and 1,400 rpm shaking in a thermomixer (Eppendorf). After centrifugation (10 min, 4°C, 13,500 rpm), 10 µL d27-myristic acid (30 pg mL-1; internal standard for retention time calculation) was added to 75 µL of supernatant. The solution was spin-dried under vacuum for 4 h and stored at -80°C until further analysis. Extracts were derivatized with methoxylamine and N-methyl-N-

Proteomics analysis

Proteomic analysis was performed using a method adapted from Cui et al. (2019b). Proteins were extracted using TCA-acetone, digested and after desalting on solid phase extraction, peptides were purified using the column Pipette Tip ZipTipSCX according to the protocol of the supplier (Millipore). (http://www.merckmillipore.com, document no. 201306-4805).

Column Pipette Tips ZipTipSCX were first equilibrated with 310µL washing solution (0.1% TFA, 30% methanol in MilliQ water), then the sample was loaded, washed with 510 µL washing solution and eluted with 10 µL 5% ammonium hydroxide, 30% methanol in MilliQ water. Eluted peptides were speed-vac dried and re-suspended in a solution containing 2% acetonitrile, 0.05% TFA and 0.05 % formic acid. Samples were then analyzed by LC-MS/MS (nano-HPLC coupled to mass spectrometry via a nano-electrospray interface). Except for the separation parts of the chromatographic run that is achieved in 110 min against 75 min, acquisition parameters are the same as in Cui et al. (2019b). Protein identification, filtering and grouping were carried out using the X!Tandem pipeline, after [START_REF] Langella | X!TandemPipeline: a tool to manage sequence redundancy for protein inference and phosphosite identification[END_REF] by querying the MS/MS data against the Elaeis guineensis sequenced genome (GCF_000442705.1_EG5, 41887 entries from https://bigd.big.ac.cn/ [START_REF] Singh | Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds[END_REF]) together with a custom contaminant database (trypsin, keratins, 58 entries). The false discovery rates (FDRs) at the peptide and protein levels were 0.02% and 0.15%, respectively. Relative peptide quantification by peak-area integration on eXtracted ion chromatogram (XICs) was performed using the MassChroQ software (version 2.2.16; pappso.inra.fr/bioinfo/masschroq/) [START_REF] Valot | MassChroQ: a versatile tool for mass spectrometry quantification[END_REF].

Relative protein abundance was calculated and defined as the sum of peptide intensities considering only (1) reproducible peptides, (2) unique peptides, and (3) correlated peptides belonging to the same protein, which correspond to peptides-mz whose intensity profile do not deviates from the average profile of the peptides-mz from the same protein.

Statistical and hierarchical clustering analysis

For all analyses, 3 to 5 replicates were taken for all conditions each year. Univariate and multivariate analyses of omics data were conducted using an ANOVA (MeV version 4.9) and orthogonal projection on latent structures (OPLS, SIMCA version 14.0, Umetrics), respectively. Phenological data and elemental content were analyzed separately in each sampling year using a 2-way ANOVA (with a threshold P-value of 0.05) with crosses and K as factors, followed by a post-hoc Tukey test. For each sampling year, we used also a 2-way ANOVA with a threshold P-value of 0.05 (metabolomics) or Benjamini-Hochberg-adjusted Pvalue to keep a false discovery rate < 0.05 (proteomics), with cross and K as factors. Omics results were analyzed using a heatmap associated with a hierarchical clustering analysis (Pearson correlation) for significant metabolites. The OPLS analysis used the K level (quantitative) and cross (qualitative) as predicted Y variables and metabolites or proteins as predicting X variables. The goodness of the OPLS model was assessed using the determination coefficient R2 and the predictive power was quantified by the cross-validated determination coefficient, Q2. Best discriminating metabolites were identified using volcano plots whereby the logarithm of the P-value obtained in univariate analysis (ANOVA) was plotted against the rescaled loading (pcorr) obtained in the OPLS. In such a representation, best discriminating metabolites have both maximal -log(P) and pcorr values. Also, to investigate correlations between leaf proteome and metabolome, a multivariate analysis was performed with proteomics data as X variables and metabolomics data as Y variables (presented in Supplementary Material).

C. Results

Functional traits

Elemental contents (nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and chlorine (Cl)) in leaflet and rachis were analyzed in two different conditions of K availability (K0, K3) and two crosses (DL, DY) for both sampling years (2017,2018). Using a two-way ANOVA, only the nitrogen content in leaflet was found to be significant for the K effect (P<0.05) in 2017, with no significant effect of the cross nor the interaction cross  K, regardless of the element. In 2018, N and P contents in leaflet and K content in rachis were significantly different between the two crosses, while leaflet N P, K contents were significant for the K effect (P<0.05) with no significant effect of the interaction. In other words, in 2017, despite the significant effect of K fertilization treatments on total leaflet N, there was no significant effect on leaflet K content while K fertilization had a significant effect in rachis K In terms of phenology, there was a strong effect of the cross on female and male inflorescences in 2018 and on leaf area in 2017 (P<0.05). As expected, there was a change in total leaf number between the two years (with more leaves in 2018), and a slight change in the male/female bunch number ratio (FIG. II.4A). There were significant differences between crosses, with DY having more apparent leaves with lower leaf surface and less apparent female Amongst proteins that increased with K availability (in one cross only or both crosses) were proteins related to (FIG. II.7A-B): photosynthesis and sucrose synthesis (UDP-sugar pyrophosphorylase, phosphofructokinase/fructose-2,6-bisphosphatase, HHL1 protein, zeaxanthin epoxidase, NADPH-quinone oxidoreductase, chloroplastic aldolase), mitochondrial metabolism (ubiquinone-NADH dehydrogenase, mitochondrial dicarboxylate/tricarboxylate transporter, γ-aminobutyrate transaminase), redox homeostasis (superoxide dismutase, ascorbate peroxidase), protein turn-over (ribosomal proteins, peptidases, signal recognition particle protein and chloroplastic translocase, chloroplastic ribosome binding factor, histidinyl-tRNA synthetase) as well as other proteins involved in secondary metabolism and housekeeping functions. Interestingly, a K+ channel of the nuclear envelope was also found to increase with K availability (DUF 1012 domain-containing ion channel POLLUX). Quite similarly, proteins decreasing with K availability were associated with photosynthesis and photorespiration (NDH subunit, protein H of the glycine decarboxylase complex), mitochondrial metabolism (aconitase, cytochrome c oxidase, 3-isopropylmalate dehydratase), redox homeostasis (peroxidase), protein turn-over (ribosomal proteins, nascent polypeptide associated complex subunit, ubiquitin-domain containing protein), and well as other proteins involved in secondary metabolism (e.g., alkenal reductase) and housekeeping functions (e.g. actin). Taken as a whole, rather than a general protein increase or decrease, changes in K availability led to a reconfiguration of photosynthetic, mitochondrial or protein synthesis machinery. However, the OPLS model was not significant in 2017 (PCV-ANOVA = 1 [K effect] and 0.96 [cross effect]), and was significant for the cross effect in 2018 (PCV-ANOVA = 0.67 [K effect] and 0.036 [cross effect]). It explained most of total variance (R2 = 0.908 and 0.899) and was predictive in 2018 but not in 2017 (Q2 = 0.583 and < 0, respectively). Unsurprisingly, the two crosses exhibited significant differences in many metabolites (FIG. S1). DY had more hexoses (glucose, fructose), Krebs cycle intermediates (citrate, fumarate, isocitrate), amino acids (glycine, isoleucine, serine, threonine) than DL. Conversely, DL had more lipids (such as stearate, digalactosylglycerol, arachidate). Also, crosses differed in their secondary metabolite composition such as phenylpropanoids (blue rectangle, FIG. S1): DY had more cis-caffeate, ferulate and vanillin, while DL had more trans-caffeate, catechin and sinapate. In univariate analysis, 12 analytes were significant for the K effect and 10 for the interaction K  cross in 9E-F). The importance of metabolites in sample discrimination associated with K availability was easily visible in the volcano plot which combined univariate and multivariate analyses (-log(P) obtained in the two-way ANOVA was plotted against the loading (pcorr) along axis, Fig. 5a-b). In both sampling years, higher K availability led to an increase in amino acids (methionine, glutamate, serine, aspartate and methyl-aspartate), sugars and their derivatives (sucrose, gluconate, and a disaccharide), polyamines (spermidine and cadaverine), organic acids (3-methylglutarate, dehydroascorbate, succinate, and citrate), a polyol (galactitol) and an alkaloid (dopamine).

Correlation between proteome and metabolome

A bidimensional statistical multivariate analysis (O2PLS) was conducted to investigate the potential linkage between proteome and metabolome. In such an analysis, it is not necessary to give classes (K0/K3, DY/DL) as an input to discriminate samples, since samples should be discriminated from their metabolomics pattern (Y variable). Interestingly, samples appeared to be naturally discriminated following crosses (axis 1) and K availability (axis 2), demonstrating that the proteome-metabolome association was K-and cross-specific (FIG. II.11A). However, the O2PLS model was not significant, the minimal individual PCV-ANOVA value being obtained for caffeate (P = 0.102). Caffeate correlated to various proteins involved in metabolism (adenosine kinase, cinnamoyl CoA reductase, cytosolic phosphoglycerate kinase, phosphopyruvate hydratase, linoleate lipoxygenase, tryptophan synthase, V type proton ATPase) and anti-correlated to some proteins involved in photorespiration and photosynthesis (glutamate glyoxylate aminotransferase, rubisco activase, sucrose phosphatase) (P-value of correlations < 0.01). The loading bi-plot of the O2PLS analysis further showed that metabolite and protein families were not randomly distributed (FIG. II.11B). In fact, disaccharides (green dots) and some proteins of photosynthesis (sucrose-phosphatase, glycerate dehydrogenase, carbohydrate kinase domain-containing protein, Rubisco activase, transketolase) co-localized (i.e. correlated) on the DL side (left). Moreover, most intermediates of the Krebs cycle (red arrows) co-localized with proteins involved in catabolism (phosphoenolpyruvate carboxylase, phosphopyruvate hydratase, etc.) on the DY side (right), and appeared to be anti-correlated to mitochondrial malate dehydrogenase (Table S4). Most hexoses and their derivatives (yellow dots) appeared to correlate with several chloroplastic proteins involved in photosynthesis (NADH dehydrogenase subunit, PSII subunit PsbP, PSI chlorophyll a/b-binding protein) in the lower part of the plot, i.e., under low K availability.

Taken as a whole, the O2PLS analysis suggests that there were differences in both photosynthetic capacity and mitochondrial metabolism between crosses. In particular, it suggests that the abundance of organic acids of the Krebs cycle was dictated by the balance between phosphoenolpyruvate carboxylase and malate dehydrogenase. Also, K availability impacted on photosynthesis, with changes in both the protein composition of the photosynthetic machinery and an increase in free hexoses.

D. Discussion

1.

Time lag between K treatment and visible effects

The present study shows that unsurprisingly, the effect of K fertilization on leaf K content (and proteome) was minimal after one year (in 2017) plus three years preconditioning (FIGS. III.1 AND III.9). That is, it took two years to see a significant effect of K fertilization on leaf K content (FIG. II.1F). This is consistent with past agronomic trials that show potassium fertilization affects leaf K pools about four years after the treatment onset (Corley et al., 1976).

Also, it is worth noting that in 2017, based on the weighted tree-average elemental K content (1.1%), considering that tree total standing biomass is about 295 kg DW, tree K mineralomass was about 2.6 kg K tree-1. Since, on average, 1.8 leaves (FIG. II.4) are produced each month and 23 bunches each year [START_REF] Mirande-Ney | Effects of potassium fertilization on oil palm fruit metabolism and mesocarp lipid accumulation[END_REF], this represents about 107 kg y-1 DW produced, and a K demand of about 1.1 kg K tree-1 y-1. If we also account for K represented by leaves cut when bunches are harvested, the overall K requirement was about 43% of total K mineralomass of trees (note this value is slightly underestimated due to the K demand by trunk growth). Without any supply of potassium (no fertilization), standing K mineralomass may be sufficient to feed growth explaining the lack of effects in 2017. In addition, during preconditioning, all trees received 2 kg KCl tree-1, which compensate for the K0 treatment in 2016 onwards. The lack of significant effect of K fertilization on leaf K content in 2017 could have also come from changes in allocation, with more inflorescences and less leaves compared to 2018 (FIGS. III.1 AND III.4). That is, some K could have been remobilized from leaves or trunk to fruits to cover the K demand of bunch maturation, thereby dampening changes in leaf K despite the K fertilization treatment. Parenthetically, with 4.5 kg KCl tree-1 y-1 (i.e. 2.3 kg K tree-1 y-1), fertilization in the K3 conditions represented therefore an excess, i.e. twice as much the K demand.

However, despite the lack of effect of fertilization on leaf K content and proteome in 2017, several metabolites appeared to be significant (FIG. II.9), suggesting that there were some changes in, e.g., K+ distribution amongst leaf tissues or cellular compartments. In fact, we found that there was more succinate, spermidine and methionine at high K (K3 conditions), suggesting a higher activity associated with two well-known K+-dependent enzymes, succinyl-CoA thiokinase (Krebs cycle enzyme which forms succinate) and S-adenosylmethionine synthase (required for spermidine synthesis and the methionine salvage pathway). That is, it is probable that even in 2017, K0/K3 conditions were associated with subtle changes in cytosolic and mitochondrial K+ concentration.

K availability interacts with N and P

Both elemental and omics analyses suggested that K availability impacted on nitrogen metabolism. Our results show that potassium fertilization increased N content in leaflets of both crosses (FIG. II.1). This agrees with previous agronomical observations that increasing K stimulates N use efficiency and eventually, increases %N in leaflets [START_REF] Ollagnier | Interaction entre l'azote et le potassium dans la nutrition des oléagineux tropicaux[END_REF].

However, despite a tendency to increase leaf nitrate (NO3-) in DY (FIG. II.1A,E), no significant effect of K was found on nitrate overall, in contrast to what has been observed elsewhere in other species [START_REF] Blevins | Role of potassium in protein metabolism in plants[END_REF]Hu et al., 2016b). In fact, it is believed that K plays a key role in nitrate absorption and circulation, as a counter-cation (Blevins et al., 1978a;Hu et al., 2016b;[START_REF] Coskun | The nitrogen-potassium intersection: membranes, metabolism, and mechanism[END_REF][START_REF] Wang | Regulation of potassium transport and signaling in plants[END_REF]). An effect of K availability on both leaf nitrate content and natural 15N abundance (δ15N) has also been found in sunflower, showing a change in nitrate influx-to-reduction ratio in leaves (Cui et al., 2019a). Nitrate reductase isoforms were not detected in our proteomics analyses. However, leaf glutamate synthase (Fd-GOGAT) content increased with K availability (P=0.008) (not shown) and we also found an increase in glutamate (FIG. II.9), suggesting a stimulation of N assimilation by K availability (FIG. II.12). That said, it is worth noting that, in accordance with usual agronomical practices, N fertilization was here done with urea, using 1 kg tree-1 y-1. The mineralization rate in tropical soils like those found in oil palm fields in Indonesia is in principle sufficient to mineralize such an amount of urea to nitrate within 1 year [START_REF] Allen | Soil nitrogen-cycling responses to conversion of lowland forests to oil palm and rubber plantations in Sumatra, Indonesia[END_REF] but it is likely that at least for a brief period after urea fertilization, available soil nitrogen was made of a mixture of urea and nitrate. It has been found

in maize that while urea can be assimilated directly via urease, urea potentializes N assimilation, in particular the expression of genes encoding GOGAT and glutamine synthetase [START_REF] Zanin | Measurement of net high-affinity urea uptake in maize plants[END_REF]. This effect probably explains why the effect of K on leaf nitrogen was more pronounced on glutamate metabolism (and thus on many amino acid contents) than on nitrate content.

Several amino acids increased with K availability, such as methionine, serine, leucine or valine as a likely consequence of both augmented synthesis and proteolysis (protein-turnover). In fact, amongst significant proteins were isopropyl malate dehydratase (involved in leucine biosynthesis), and peptidases (FIGS. III.7, III.9 AND III.12). However, the effect of K availability appeared to depend on the cross. DY was generally enriched in amino acids compared to DL (FIG. S1). K availability had also an effect on protein synthesis machinery, in particular ribosomal proteins (FIG. II.7). K+ is also required for translational activity [START_REF] Blevins | Role of potassium in protein metabolism in plants[END_REF] and it is thus likely that protein synthesis was higher at high K availability. In fact, total leaf protein content was larger under K3 conditions (FIG. II.5). Previous study showed that in cotton, tea and oil palm, K fertilization improved leaf protein content and changed free amino acids concentration, depending on the species and variety [START_REF] Ollagnier | Interaction entre l'azote et le potassium dans la nutrition des oléagineux tropicaux[END_REF][START_REF] Ruan | Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensisL)[END_REF]Hu et al., 2016b) In addition to its effect on nitrogen, K fertilization impacted on phosphorus content and metabolism (FIGS. II.1 AND II.9). This may have in turn had an effect on protein synthesis and leaf N content [START_REF] Marschner | 8 -Functions of mineral nutrients: macronutrients[END_REF]. The concurrent effect of K on N and P metabolism comes as no surprise since in oil palm, P and N contents have been found to be linearly correlated in leaflets [START_REF] Tampubolon | Réponses du palmier à huile aux fumures azotée et phosphorée à Sumatra[END_REF]. Transcriptomics analysis of Eucalyptus leaves have shown that K supply influenced the expression of genes involved in phosphate homeostasis and the response to phosphate starvation [START_REF] Favreau | Distinct leaf transcriptomic response of water deficient Eucalyptus grandis submitted to potassium and sodium fertilization[END_REF]. Here, we show that leaf free phosphate (Pi), fructose 6-phosphate, glycerol 1-phosphate and 3-phosphoglycerate decreased at high K (FIG. II.9), in contrast to elemental P content, which was found to increase slightly in leaflets (FIG. II.1) and in rachis elsewhere (Cui et al., 2019b). The decrease in Pi was further confirmed by 31P-NMR (data not shown). Therefore, it is likely that higher K availability was associated with an increase in P-containing compounds that could not be analysed here, such as phospholipids, nucleotides or non-soluble phosphates (such Ca/Mg phytate and phosphate).

More generally, K fertilization affected the cation balance, with, in 2017, a lower content in Ca and Mg in leaflets (but higher content in rachis). We also found an increase in the content of the ion channel POLLUX, which is a Ca-gated, potassium channel of the nuclear envelope.

Evidence has been provided that POLLUX modulates nuclear envelope membrane potential and therefore opening of nuclear calcium channels or conversely, and compensate for calcium efflux by playing a role of a nuclear K+ inward rectifier [START_REF] Charpentier | Lotus japonicus CASTOR and POLLUX are ion channels essential for perinuclear calcium spiking in legume root endosymbiosis[END_REF][START_REF] Chen | Antiquity and function of CASTOR and POLLUX, the twin ion channel-encoding genes key to the evolution of root symbioses in plants[END_REF][START_REF] Checchetto | Physiology of intracellular potassium channels: a unifying role as mediators of counterion fluxes?[END_REF]. Thus, our data suggests that K availability directly impacted cellular K+ and Ca2+ pools. Of course, our present analysis does not cover all aspects related to channels since the protocol used for protein extraction was generic. A targeted analysis on membrane-associated proteins would provide more insight on cellular ion balance. At the transcript level, it has also been shown in Eucalyptus that K availability impacted on the expression of genes involved in photosynthesis [START_REF] Favreau | Distinct leaf transcriptomic response of water deficient Eucalyptus grandis submitted to potassium and sodium fertilization[END_REF]. The net effect of such changes is likely to be an increase in photosynthetic CO2 assimilation rate, as shown in oil palm saplings grown under different K conditions (Cui et al., 2019b) and here, using gas exchange in a separate agronomic trial (FIG. II.13). This effect is also associated with a higher content in sugars, here disaccharides such as sucrose (FIGS. II.9 AND II.10). It is interesting to note that leaf sucrose accumulation can also been found in K deficient plants not due to an increase in photosynthetic rate but rather, because of inhibited phloem loading [START_REF] Cakmak | Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants[END_REF][START_REF] Zhao | Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants[END_REF][START_REF] Gerardeaux | Changes in plant morphology and dry matter partitioning caused by potassium deficiency in Gossypium hirsutum (L.)[END_REF].

Figure thiokinase activity). We nevertheless recognize that the increase in succinate content could have also been due to the increase in γ-aminobutyrate (GABA) transaminase, which can generate succinate semialdehyde in the GABA shunt. There was also an increase in other mitochondrial proteins (cytochrome c oxidase subunit 5b-2, NADH dehydrogenase, di/tricarboxylate transporter, etc.). Altogether, these changes point to an increase flux in organic acid metabolism and thus an increase in respiratory activity. In fact, we found nearly a 2-fold increase in dark respiration (CO2 evolution) using gas-exchange measurements (FIG. II.13). The rationale of such an important stimulation of mitochondrial metabolism at higher K availability is presently unclear. In fact, it might have been driven by ion balance (generation of organic acids carrying a negative charge to compensate for changes in relative K+ excess, Fig. 1), the demand in carbon skeletons for nitrogen assimilation (discussed above), or the increase in growth rate and sucrose export. In that regard, it is very different from the stimulation of respiration observed under K deficiency, which is linked to a loss in ATP generation efficiency and the onset of CO2producing alternative pathways (Cui et al., 2019b).

E. Conclusions and perspectives

Taken as a whole, K availability had important effects on several metabolic pathways in leaflets of oil palm trees grown in the field (summarized in FIG. II.12). These effects were visible one to two years only after the onset of K fertilization regime, that is, before significant changes could be found on bunch number (FIG. II.4) and fresh fruit bunches weight (Mirande-Ney et al., 2019). As such, our results obtained at the leaf scale suggest that it could be of interest to conduct experiments at the plantation scale (agronomical trials) to assess the potential of the metabolic signature of leaflets as a technique for K fertilization monitoring. In addition, the simultaneous effect of K availability on photosynthesis and respiration suggests that the carbon use efficiency could have also been impacted. Recent studies have shown that in oil palm saplings, K deficiency leads to ≈10% loss in carbon use efficiency only (Cui et al., 2019b) and under field conditions similar to ours, the effect of K availability on total fruit and oil production was rather small [START_REF] Hartley | The oil palm (Elaeis guineensis Jacq[END_REF]. That is, despite significant changes in photosynthetic capacity and respiratory efflux, K availability has, perhaps, a limited effect on tree-scale carbon use efficiency. Of course, measuring the overall effect of K availability on tree carbon balance would require further work, e.g. to monitor biomass and respiration of all organs and estimate total tree photosynthesis. This will be addressed in a future study.

F. Acknowledgements

C. Mirande-Ney is grateful to CIRAD for the PhD fellowship funding and for financial support of the experiments and divers analyses, and also to IDEEV for financial support. The authors would like to thank the company SOCFINDO for their material and technical support during field experiment, as well as Bertrand Gakiere from the facility Metabolism-Metabolome (IPS2).

We also thank Jean Ollivier for providing agronomical data.

Proteomics analyses were performed on the PAPPSO platform (http://pappso.inra.fr) which is supported by INRA (http://www.inra.fr), the Ile-de-France regional council (https://www.iledefrance.fr/education-recherche ), IBiSA (https://www.ibisa.net), CNRS (http://www.cnrs.fr ) and SPS (https://www6.inra.fr/saclay-plant-sciences).

In Chapter III, fruit mesocarp metabolome was explored during five steps of maturation from pollination to harvesting, and the quality and quantity of mesocarp oil was assessed. This

Chapter is now published as a research article in Journal of Agricultural and Food Chemistry.

A. Introduction

Oil palm (Elaeis guineensis Jacq.) is a major oil crop with a global production of about 77 Mt y-1 in 2018. Oil palm is also the world highest yielding oil crop species, with a productivity (production per hectare) up to 10 times larger than other leading vegetable oil crops like canola.

Due to its unique chemical composition (relatively rich in saturated fatty acids), palm oil has a long shelf-life. In addition, it offers a number of nutritional benefits since it is cholesterol-free and rich in carotenoids, representing a natural source of vitamin A.

Oil palm growth is highly dependent on potassium (K) provision, and the elemental K content in total oil palm tissues is often larger than that of nitrogen. As such, K fertilization is common practice in the field, with potassium chloride (KCl) or potassium sulphate (K2SO4), representing an annual cost of about US$ 1b at the global scale. Typical oil palm K fertilization consists, on average, of 180 kg K ha-1 y-1 (≈2.5 kg KCl tree-1 y-1), while N and P represent about 95 and 11 kg ha-1 y-1, respectively [START_REF] Heffer | Assessment of fertilizer use by crop at the global level[END_REF]. The particularly high K requirement in oil palm comes not only from the dependency of bunch production to K availability, but also from K scarcity in soils of oil palm cultivation areas, and the considerable loss of K applied, via leaching (up to 30% [START_REF] Omoti | Leaching losses of nutrients in oil palm plantations determined by tension lysimeters[END_REF]Foong, 1991;[START_REF] Chang | Leaching losses of nutrients from a mature oil palm catchment in Malaysia[END_REF]). Elemental bunch analyses have shown that K is by far the most abundant macroelement in fruit bunches. The highest K content can be found in stalks (5-8% of dry matter), which account for up to 26% of total bunch K content [START_REF] Teoh | Use of rachis analysis as an indicator of K nutrient status in oil palm[END_REF]Teoh & Chew, 1988a). Also, it has been estimated that up to 248 kg K ha-1 y-1 is abstracted from oil palm agrosystems during bunch harvesting [START_REF] Heffer | Assessment of fertilizer use by crop at the global level[END_REF], and therefore K efflux (harvesting) can exceed K influx (fertilization). K fertilization is thus crucial to allow proper development and avoid progressive K depletion in soils and trees.

Furthermore, high K availability is beneficial to fruit development, bunch biomass and bunch number and thus increases yield [START_REF] Hartley | The oil palm (Elaeis guineensis Jacq[END_REF][START_REF] Corley | The oil palm[END_REF]. In fruit mesocarp, the K content has been shown to increase progressively for 15 weeks after anthesis (WAA) and then decreases during last developmental stages where lipid biosynthesis occurs [START_REF] Desassis | Sur les modalités de formation des matières grasses dans le fruit d'Elaeis guineensis Jacq[END_REF]. K+ has also been suggested to drive phloem sugar movement, in particular for the remobilization of soluble sugars from the trunk base (and petioles) to sustain fruit development [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF]. However, to our knowledge, there is considerable uncertainty as to whether K fertilization affects mesocarp oil content and composition. Some studies have shown that on average, higher K availability tends to increase mesocarp oil content (as well as the proportion of oleate in oil fatty acid composition) [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]Ollagnier & Olivin, 1984b) but a negative effect of K on oil content has been found elsewhere [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]. It has been suggested that this negative effect may stem from an increased tissue chloride (Cl-) concentration (which inevitably comes along with KCl-based fertilization), resulting in lower mesocarp-to-kernel biomass ratio in fruits [START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b). On average, crude palm oil triacylglycerols (TAG) contain unsaturated and saturated fatty acid chains in near-equal proportions, which makes palm oil suitable for a number of food, medicinal or biofuel applications. Saturated fatty acid chains are formed by 44% palmitic acid (C16:0), 5% stearic acid (C18:0) and traces of myristic acid (C14:0) [START_REF] Sambanthamurthi | Chemistry and biochemistry of palm oil[END_REF]. Unsaturated fatty acid chains are formed by 40% oleic acid (C18:1), 10% linoleic acid (C18:2) and traces of linolenic acid (C18:3) ( [START_REF] Barcelos | Oil palm natural diversity and the potential for yield improvement[END_REF]. In other species such as olive tree, it has been shown that K affects oil composition by increasing the proportion of mono-unsaturated (C20:1)

and long saturated (C22:0) fatty acids [START_REF] Dag | Olive oil composition as a function of nitrogen, phosphorus and potassium plant nutrition[END_REF]. Similarly, high K availability leads to increased fatty acid unsaturation in linseed, sunflower and sesame oil [START_REF] Seo | The effects of fertilization level on the growth and oil quality in sesame (Sesamum indicum L.)[END_REF][START_REF] Salama | Yield and oil quality of sunflower as affected by fertilization and growthregulating hormones[END_REF][START_REF] Froment | Effect of nutrition on growth and oil quality in linseed[END_REF]. K availability has a well-known effect on metabolism, via its action on several enzymatic activities such as pyruvate kinase, the last step of glycolysis that generates pyruvate from phosphoenolpyruvate. In oil palm cultivated under low K conditions in the greenhouse, a general reorchestration of carbon primary metabolism has been shown in leaves, with drastic changes in organic acid content and the involvement of an alternative pathway (parapyruvate aldolase) to synthesize pyruvate (Cui et al., 2019b). Since pyruvate is the key metabolite that links glycolysis to fatty acid synthesis (via acetyl-CoA production), an impact of K availability on oil production and composition can be anticipated. However, amongst metabolomics analyses conducted in oil palm fruits [START_REF] Neoh | Profiling of metabolites in oil palm mesocarp at different stages of oil biosynthesis[END_REF]Teh et al., 2013a), none has looked at the effect of K availability on fruit development and oil biosynthesis.

This lack of knowledge impedes our current understanding of how K availability regulates lipid biosynthesis and thus oil palm productivity. In particular, since the impact of K fertilization on oil composition and final oil content in mature fruits has never been assessed precisely, K fertilization strategies that are best adapted to fruit metabolic efficiency are unknown. To address this issue, we examined fruit metabolism during maturation, using oil palm cultivated in the field under different K availability conditions. Bunch analysis, mesocarp metabolomics and 1H-NMR oil analysis were carried out using two oil palm crosses (Deli x La Mé (DL) and Deli x Yangambi (DY)) grown under different K fertilization treatments (from 0 to 4.5 kg KCl tree-1 y-1) in a plantation located in North Sumatra (Indonesia). Fruit metabolism and bunch analysis were monitored at five developmental maturation stages (depicted in Fig. S1 and explained in Supplemental Text S1), and metabolomics and lipid analyses were carried out.

B. Material and Methods

Field location, fertilization and sampling

The experimental field was located at SOCFINDO plantation (Bangun Bandar Estate, North Sumatra, Indonesia; 3°18'19.60"N, 99°3'24.33"E) and sampling was carried out in Oct-Nov KCl tree-1 y-1 (K3). In an effort to examine the influence of K under realistic conditions, we used K availability levels that are far from extreme situations (i.e., K deficiency or K excess).

In fact, residual K in soil under the K0 treatment represents about 0.2 meq exchangeable K per 100 g soil, showing that there was no K deficiency during the experiment. Trees were also fertilized with nitrogen using 2 kg urea tree-1 y-1. Leaflets used for mineral analysis were sampled on trees fertilized under treatments K0, K2 and K3 on leaf rank number 9, at point B and a distance of two third of the leaflet length from the rachis, using leaflets on both sides of the leaf [START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]. Leaf samples were cleaned with deionized water, frozen in liquid nitrogen and then oven-dried prior to analysis. Fruit sampling for metabolomics analysis was performed with treatments K0 and K3 at five different developmental maturation stages (30 d between them) numbered from 1 (1 month after anthesis) to 5 (at ripening) so as to follow fruit formation and maturation described by [START_REF] Thomas | Fruit ripening in the oil-palm Elaeis guineensis[END_REF]) (stages are further described in

FIG. III.1 and SUPPLEMENTAL TEXT S1).

Five fruits (per stage) were randomly collected on each respective bunch the same day for all maturation stages and all trees studied. Sampled fruits were rapidly cleaned with deionized water, cut in small pieces, frozen in liquid nitrogen then frozen-dried (lyophilized). Once dried, fruit mesocarp was ground and stored at -20°C.

Leaf samples were ground in a fine powder and mineral analyses (N and K elemental content)

were carried out by ICP-OES by SOCFINDO.

Starch and metabolites extraction

Starch was extracted following the procedure described in [START_REF] Duranceau | δ13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought[END_REF]. Briefly, 50 mg of ground mesocarp was extracted with 1 mL MilliQ water, centrifuged and the supernatant was discarded. The pellet was rinsed with absolute ethanol and starch was extracted using solubilization with 6 M HCl and subsequent flocculation in methanol at 5°C. For metabolomics, 10 mg of ground dried mesocarp was extracted with 1 mL cold (-20°C) water-acetonitrileisopropanol mixture (2:3:3, v:v:v) containing 4 pg mL-1 ribitol as an internal standard, using 2 stainless steel 3-mm balls for tissue lysing (1.5 min,30 Hz). Samples were placed for 10 min WAA at 4°C and 1,400 rpm shaking in a thermomixer (Eppendorf). After centrifugation (10 min, 4°C, 13,500 rpm), 10 µL d27-myristic acid (30 pg mL-1; internal standard for retention time locking (RTL) using the Agilent software [START_REF] Tavares | 1,5-Anhydroglucitol predicts CKD progression in macroalbuminuric diabetic kidney disease: results from non-targeted metabolomics[END_REF][START_REF] Fu | Metabolomic profiling of zebrafish (Danio rerio) embryos exposed to the antibacterial agent triclosan[END_REF]) was added to 50 µL of supernatant. The solution was spin-dried under vacuum for 4 h and stored at -80°C until further analysis.

Deli x La Mé Deli x Yangambi

3.

Metabolomics by gas chromatography-mass spectrometry (GC-MS)

GC-MS analyses were carried out as in [START_REF] Fiehn | Metabolite profiling in Arabidopsis[END_REF]; Fiehn et al. (2008). Frozen samples were warmed up for 15 min and vacuum-dried for 1.5 h at 35°C. Extracts were derivatized with 10 µL methoxyamine in pyridine (20 mg mL-1) for 90 min at 30°C with continuous shaking then 90 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide (Regis Technologies) was added and the reaction ran for 30 min at 37°C. After cooling to ambient temperature, 100 µL was transferred to a glass vial. Four hours after derivatization, 1 µL was injected in split-less mode into the GC-MS (GC Agilent 7890B coupled to MS Agilent 5977A). The GC column contained a standard low-polarity phase with 1,4-bis(dimethylsiloxy)phenylene dimethyl polysiloxane (RXI-5SIL MS, 30 m with 10 m integra-guard column, Restek). Injection in split mode (ratio 1:30) was also done for saturating (highly concentrated) compounds quantification. Oven temperature was 60°C for 1 min then ramped at 10°C min-1 up to 325°C for 10 min. Helium was used as vector gas under a constant flow at 1.1 mL min-1. Other temperature values were as follows: injector 250°C, transfer line 290°C, source 230°C, and quadrupole 150°C. The solvent delay was 5.9 min, and the scan window during acquisition was 50-600 amu. Retention indexes were calculated against the internal standard (d27-myristic acid) using the RTL system provided in Masshunter® (Agilent) in order to reduce run-to-run variations. In addition, a fatty acid methyl ester mix (from C8 to C30) was injected each 8 samples for external retention index calibration. Samples were randomized amongst treatments and crosses to avoid statistical bias.

Raw data files were analyzed with AMDIS (http://chemdata.nist.gov/mass-spc/amdis), and the Fiehn GC-MS Metabolomics RTL Library (version June 2008) was employed for metabolite identification, which was also checked using the NIST library and authentic standards injected separately. Peak areas were determined with Masshunter® (quantitative analysis version, Agilent). Because automated peak integration was occasionally erroneous, integration was verified manually for each compound in all analyses. Resulting areas were compiled in one single Excel file for comparison. Peak areas were normalized to ribitol (internal standard) and dry weight. In addition to GC-MS metabolomics, we also conducted the analysis of total lipids by NMR in order to measure saturated and unsaturated fatty acid contents and the iodine index (SUPPLEMENTAL TEXT S2).

Bunch analysis

Components of oil extraction rate were determined by SOCFINDO using standard procedures adapted from [START_REF] Blaak | Breeding and inheritance in the oil palm (Elaeis guineensis Jacq.). Part II. Methods of bunch quality analysis[END_REF]. Bunch recording was performed from the onset of the productive period of the trial. One mature bunch (5 to 6 months after pollination) per tree was sampled for analysis: bunch, stalk and spikelet fresh biomass was measured and fruit biomass, mesocarp-to-fruit (M/F), oil-to-mesocarp (OFM) and kernel-to-fruit (K/F) ratios (expressed in %) were computed using a set of 30 fruits. Mesocarp relative water content (in %) was calculated as the difference between fresh and dry mesocarp biomass divided by fresh mesocarp biomass. The oil extraction rate (OER) was calculated as:

F B× M F×OFM OER 0.855 10, 000 = 
where F/B, M/F and OFM stand for the percentage of fruit biomass in bunch total biomass, the percentage of mesocarp biomass in total fruit biomass, and the percentage of oil in mesocarp biomass, respectively. The correction factor of 0.855 is used to account for losses in factory oil extraction. The proportion of kernels in bunch biomass was computed as:

kernel F K 0.91 B F 100 p =  
where, K/F stands for the percentage of kernel biomass in fruit biomass and 0.91 represent the fraction of non-parthenocarpic fruits.

Statistical and hierarchical clustering analysis

For OER components, elemental content and metabolomics, 4 to 5 replicates were taken for all conditions. Univariate and multivariate analyses of metabolomics data were conducted using an ANOVA (MeV version 4.9) and orthogonal projection on latent structures (OPLS, SIMCA version 14.0, Umetrics), respectively. For each cross, we used a 2-way ANOVA (threshold Pvalue for significance 0.01) with developmental stage and K as factors and results are presented as a heatmap associated with a hierarchical clustering analysis (Pearson correlation) for significant metabolites. The OPLS analysis used the K level (qualitative) and maturation stage (quantitative) as predicted Y variables and metabolites as predicting X variables. The goodness of the OPLS model was assessed using the determination coefficient R2 and the predictive power was quantified by the cross-validated determination coefficient, Q2. Best discriminating metabolites were identified using volcano plots whereby the logarithm of the P-value obtained in univariate analysis (ANOVA) was plotted against the rescaled loading (pcorr) obtained in the OPLS. In such a representation, best discriminating metabolites have both maximal -log(P) and pcorr values. Metabolic pathway enrichment was analyzed with the tool MetaboAnalyst (Xia & Wishart, 2016) using as input metabolites significant for the K effect (P<0.01) for DL and DY mixed and the plant metabolite database (Arabidopsis thaliana; the database for oil palm is not currently available). A Pearson correlation analysis was also performed on metabolomics data with R (version 3.6.0, R Development Core Team 2005) in order to identify correlations between metabolites and agronomical parameters such as OER and OFM. OER components, elemental content and lipid content (1H-NMR) were analyzed separately using a 2-way ANOVA (with a threshold P-value of 0.05) with developmental stage and K as factors, followed by a post-hoc Tukey test.

C. Results and discussion

1.

Differential effect of K on bunch composition

As expected, K fertilization conditions were reflected in leaf K elemental content while N content remained constant. In fact, there was about 0.75% K under K0 conditions and significantly more (up to 1.15%) at K2 and/or K3 (FIG. III.2). on oil content (OFM) in DY fruits could have come from excess chloride in tissues, which is also known to increase the kernel-to-mesocarp ratio, as observed here [START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]. In fact, DY trees subjected to different forms of chloride fertilization (either as NaCl or KCl)

have been shown to form heavier bunches with more and bigger fruits, and higher proportion

(f) (b) (g) (e) (d) (c) (a)
of kernel biomass, with no change in OER (Ollagnier & Olivin, 1984a). We also note that leaf K elemental content was more impacted by fertilization in DY than in DL (FIG. III.2), suggesting that KCl absorption was more efficient in DY trees. In other oil-producing species such as castor bean, higher K availability is accompanied by lower Na+ and/or Ca2+ and less chloride in phloem sap and tissues, suggesting that electroneutrality at high K requires less (not more) anions, and is achieved with anion species other than chloride [START_REF] Peuke | Flows of elements, ions and abscisic acid in Ricinus communis and site of nitrate reduction under potassium limitation[END_REF]. That is, ion balance leads to an antagonistic relationship between K and Cl and therefore explains the effect (or the lack thereof) of high KCl fertilization in which Cl-inevitably comes along with K+.

Metabolic profiling during fruit development

The metabolome of fruit mesocarp was analysed at five different developmental stages, across two K conditions (K0, K3) and two crosses (DL, DY). Overall, 131 analytes were quantified and identified and amongst them, about 100 (103 in DL, 97 in DY) were found to be significant for the developmental stage effect (P<0.01, FIG. S2). As expected, carbohydrates, organic acids and amino acids decreased between stage 2 and 3 and conversely, there was a clear increase from stage 3 to 5 not only in free fatty acids (FAs) but also in glycerol, glycerol 1-phosphate, monostearin, and oleoylglycerol, reflecting increased triglycerides synthesis. At maturity, mesocarp metabolome distribution was made of 72% FAs, 27% carbohydrates and less than 1% other metabolites across all conditions (as estimated from GC-MS signals; FIG. III.4A).

Thus, fruit development was associated with a general change in tissue composition and metabolic content, along with a typical accumulation of lipids (FIGS. III.4A AND S2) as described elsewhere [START_REF] Hartley | The oil palm[END_REF][START_REF] Thomas | Fruit ripening in the oil-palm Elaeis guineensis[END_REF][START_REF] Neoh | Profiling of metabolites in oil palm mesocarp at different stages of oil biosynthesis[END_REF]Teh et al., 2013a). Interestingly, sucrose decreased from stage 1, glucose and fructose increased until stage 2 and then decreased, while starch peaked at stage 3 and reached a minimal level at stage 5 (FIGS.

S2, III.5 AND III.6).

It strongly suggests that sucrose was used first as the carbon source for lipid and starch biosynthesis, and then starch was used as a carbon source for lipid synthesis [START_REF] Dussert | Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition[END_REF]. Accordingly, sucrose is the prevalent sugar in oil palm phloem sap [START_REF] Houngbossa | The nature of organic nutrients exported from the leaf of oil palm (Elaeis guineensis): effect of ethylenediaminetetraacetic acid on exudation[END_REF][START_REF] Obahiagbon | Assessment of the nutritional constituents of Elaeis guineensis Jacq. exudates from different states of Nigeria[END_REF]. Also, genomics analyses have suggested that fruit development is primarily sustained by sucrose, which is hydrolysed by sucrose synthase (Susy) in the mesocarp [START_REF] Wong | Differential gene expression at different stages of mesocarp development in high-and low-yielding oil palm[END_REF]. The involvement of starch metabolism is also demonstrated here by the occurrence of maltose (non-phosphorolytic starch degradation product), which increased in both crosses during stages 4 and 5 (FIG. III.4). Lipid accumulation was also accompanied by an increase in glucarate and gluconolactone, which are both associated with the oxidative pentose phosphate pathway.

Maturation was associated with a general depletion in fumarate, citrate or succinate, probably reflecting the progressive increase in energy generation and thus the consumption of Krebs cycle intermediates. The importance of respiration and oxidative phosphorylation in oil palm fruit development has also been demonstrated by protein content analysis [START_REF] Loei | Proteomic analysis of the oil palm fruit mesocarp reveals elevated oxidative phosphorylation activity is critical for increased storage oil production[END_REF]. It is worth noting that other compounds were found to accumulate during fruit maturation, such as caffeate, suggesting changes in secondary metabolism as fruits mature. In addition to changes during developmental stage, our results also show some differences between crosses. In fact, there were more carbohydrates, amino acids and organic acids in DY compared with DL (FIG.

III.4). Furthermore, we found an increase in putrescine in DL fruits during maturation, as found elsewhere [START_REF] Teh | Hormones, polyamines, and cell wall metabolism during oil palm fruit mesocarp development and ripening[END_REF]. Polyamines such as putrescine have been shown to be involved as signaling metabolites in fruit enlargement, maturation, and ripening [START_REF] Serrano | 7 -Polyamines as an ecofriendly postharvest tool to maintain fruit quality[END_REF][START_REF] Guo | Polyamines regulate strawberry fruit ripening by abscisic acid, auxin, and ethylene[END_REF]. Sugars have a signaling role in lipid synthesis. In particular, hexokinase acts as a glucose sensor and generates glucose 6-phosphate (which will then be used by the oxidative pentose phosphate pathway) and is essential for fatty acid synthesis (Aguilera-Alvarado & Sánchez-Nieto, 2017; [START_REF] Zhai | Sugar potentiation of fatty acid and triacylglycerol accumulation[END_REF]. Overall, the kinetics of lipid biosynthesis and sugar remobilization seem to be faster in DL, maybe linked to higher catabolism, or differences in metabolic signaling. This reflected the increase in glucose catabolism via the oxidative pentose phosphate pathway (which generates NADPH for fatty acid synthesis), and the redistribution of nitrogen to specific compounds (methylpiperidine, amino acids). Interestingly, pipecolate (product of lysine catabolism) was found to decrease at high K in DL and lysine found to increase in DY, suggesting that in both crosses, high K availability likely slowed down lysine catabolism.

Similarly, phenylalanine was more abundant in DY at high K and benzoate (product of phenylalanine catabolism) was less abundant in DL, suggesting that phenylalanine catabolism was also slowed down. This K effect on amino acid catabolism was mostly visible at early stages [START_REF]A Brief History of the Oil Palm[END_REF](2)[START_REF]Plateforme Métabolisme-Métabolome[END_REF] , where at least one metabolite is significantly impacted by K fertilization with P<0.01, Fig. 3). The asterisk (*) indicates when pathways are not significantly affected as per PEA analysis (P>0.05).

Differential effect of potassium on mesocarp lipid composition

Despite metabolic changes during fruit development (see above), the relative quantity in fatty acids in mesocarp and final oil composition at maturity were not significantly modified by K availability (FIGS. III.4C AND III.9). Accordingly, oil NMR profiles were extremely similar (FIG. III.10). However, at high K (K3), GC-MS showed an increase in average oleate and a decrease in linoleate content in both crosses, although it was found to be insignificant in each cross taken separately (FIG. III.9A). Consistently, NMR analyses showed a slight increase in both average mono-unsaturated fatty acids and iodine index in both crosses (FIG. III.9B). A similar effect of K has been suggested previously in oil palm [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b) and other species [START_REF] Seo | The effects of fertilization level on the growth and oil quality in sesame (Sesamum indicum L.)[END_REF][START_REF] Salama | Yield and oil quality of sunflower as affected by fertilization and growthregulating hormones[END_REF][START_REF] Froment | Effect of nutrition on growth and oil quality in linseed[END_REF][START_REF] Dag | Olive oil composition as a function of nitrogen, phosphorus and potassium plant nutrition[END_REF]. Here, we show that its effect is quantitatively small (≈1-2% change in mono-unsaturated FA content) but found in both crosses Interestingly, the biochemical response to K availability differed slightly in minor oil components between the two crosses. In fact, linoleate and palmitoleate correlated positively

Key determinants of oil production

Oil palm production not only depends on the number and the biomass of bunches formed by trees each year but also on the amount of oil that can be recovered from bunches. In other words, the oil content in fresh mesocarp (OFM) and oil extraction rate (OER) are two critical parameters to assess lipid synthesis capability of trees. Both OFM and OER depend on K availability and crosses [START_REF] Menon | Factors affecting oil and kernel extraction rates[END_REF] (FIG. III.3). Here, we took advantage of the variance in OFM and OER values and statistical methods to find potential biomarkers of oil production rate. Univariate Pearson correlation and OPLS multivariate analysis (where OFM or OER were used as predicted Y variable) were computed. Since our metabolomics analyses showed that kinetics of lipid production may vary, we used relative changes in metabolite content from stage 3 to 5 as predictive X variables rather than metabolite contents at individual maturation stages.

OPLS models were insignificant (PCV-ANOVA>0.05) demonstrating that the variance in metabolic composition and oil production was too small to generate a very strong statistical model. This result is unsurprising since the window of OER or OFM values across all conditions was narrow Univariate Pearson correlation analysis showed that OER was strongly correlated not only to morphological parameters (fruit-to-bunch ratio and bunch biomass) but also to 2hydroxypyridine (TABLE III.1). The correlation with morphological parameters comes as no surprise since it reflects the importance of fruit set rather than fruit size per se (FIG.

III.3).

OFM appeared to be positively correlated to hexoses (fructose, glucose) as well as pipecolate, aminoisobutyrate and dopamine (which are products of lysine, leucine and tyrosine degradation, respectively) and β-alanine and triethanolamine (TABLE III.1). In contrast, OFM was negatively correlated to caprate, glycerol 1-phosphate and a compound possibly identified as lauryl salicylate. This shows that final oil content in mesocarp is related to (i) the velocity of lipid production (and thus anticorrelates to pools of intermediates of lipid synthesis), (ii) the availability of carbon source (sugars) and (iii) the ability to break down amino acids and thus metabolic recycling to other N-containing metabolites (β-alanine, triethanolamine, in addition to putrescine discussed above in DL). agronomical factors and metabolites significantly correlated to (a) oil extraction rate (OER) and (b) oil content in fresh mesocarp (OFM). The correlation coefficient (which is equal to pcorr in the OPLS analysis) and the P-value (significance of the correlation with univariate analysis) are listed for all features with a P-value lower than 0.05.

Taken as a whole, our study shows that K availability had a significant effect on mesocarp metabolism but composition of fruit oil at maturity was not significantly affected.

This demonstrates that even though K changed metabolic fluxes in lipid synthesis and source sugar utilization and recycling, it had little effect on oil production.

D.

E. Associated content:

Analysis of leaf mineral content and metabolites involved in oil palm mesocarp maturation and oil biosynthesis. This material is available free of charge via the Internet at http://pubs.acs.org.

Preamble

In Chapters II and III of this thesis, we have identified main metabolic pathways impacted by potassium fertilization in leaflets and fruits. In order to better understand metabolic dynamics and the flux of carbon from leaflet assimilation to fruits, further physiological studies are necessary. The idea of this last part is to offer a new perspective for analysing carbon fluxes in adult trees upon different conditions of potassium fertilization using stable isotope of carbon, 12C and 13C. More precisely, our intention was to know how K reshaped sugar production and allocation from source organ (leaflets) to sink organs.

The experiment was performed in 2017, where rather small effects of potassium were found due to previous fertilization and therefore, there was little modification of leaflet metabolism by K treatments (despite significant changes in some metabolites, see Chapter II). Nevertheless, this study represents an innovative work using, for the first time, 13CO2 labelling in oil palm in the field. This was a challenging part of the thesis, because of leaf size and field conditions, which both made gas-exchange coupled to 13C tracing rather difficult. In addition, a purposebuilt chamber was designed and constructed to enclose leaves for labelling them with 13CO2. It should also be noted that this experiment was expensive (typically due to the cost of travel, 13CO2 and subsequent isotopic analyses) and could not be repeated in 2018. In what follows, the results are structured using the general form of an article draft that has not been submitted

for publication to a journal yet.

Chapter IV. A 13CO2 labelling experiment to study carbohydrates production and transport from leaves to sink organs under different potassium conditions in oil palm tree.

Cathleen Mirande-Ney1,4*, Guillaume Tcherkez2, Jaleh Ghashghaie3, Emmanuelle Lamade1 1Unité PERSYST, UPR34, Système de pérennes, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, F-34398 Montpellier, France.

2Research School of Biology, Australian National University, Canberra 2601, ACT, Australia.

3Ecologie Systématique Evolution, Université Paris-Sud, CNRS, AgroParisTech, Université Paris-Saclay, 91400, Orsay, France.

A. Introduction

The oil palm (Elaeis guineensis Jacq.) is one of the most productive oil crops, with a potential yield reaching 40 t fresh fruit biomass ha-1 yr-1 due to its high photosynthetic capacity (from 23 to 30 µmol m-2 s-1) for a C3 plant and an important carbon allocation to fruits [START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]. Among the ten major oil seeds, oil palm accounted for only 5.5% of agricultural land surface area planted with oil crops, and oil palm represents 35% of global consumption in oils and fats, a number that is larger than for any other crop (Statista, 2019). Moreover, with growing demand for food and decreasingly available arable land, improving yield becomes central in oil palm producer interests. According to FAO (2012), the global demand for palm oil will double by 2020, and triple by 2050. The positive effect of potassium on fresh fruit yield is well known.

While oil composition at maturity did not significantly change under low K conditions, we found clear alterations in fruit metabolism associated with lipid production during maturation with K fertilization [START_REF] Mirande-Ney | Effects of potassium fertilization on oil palm fruit metabolism and mesocarp lipid accumulation[END_REF]. Some (eco)physiological studies allowed to get a better understanding of oil palm physiology, with the ultimate objective to improve fruit bunch production. They concerned (i) distribution of assimilated carbon [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]; (ii) reserve carbon pools under source constraints such as pruning (Legros et al., 2009b); (iii) plant response to variable environmental conditions (Legros et al., 2009a); (iv) functional relationship and dependency between autotrophic source organs and heterotrophic sink tissues [START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]; and (v) the carbon transfer and allocation pathways at the tree scale [START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF][START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]. In particular, in a recent study, [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF] have computed plausible carbon fluxes (allocation patterns) using natural 13C abundance (δ13C values) and a steady-state model of 13C-distribution. However, the effect of potassium fertilization on oil palm carbon fluxes related to yield has been not investigated yet.

Stable isotopes are sensitive and multipurpose tools to study various climatic and environmental effects on plant physiology since their natural abundance responds to key parameters such as CO2 mole fraction, humidity, etc. [START_REF] Saurer | The impact of an inverse climate-isotope relationship in soil water on the oxygen-isotope composition of Larix gmelinii in Siberia[END_REF]. In particular, the 13C natural abundance (isotope composition, δ13C) in plant organic matter gives information on photosynthesis. In fact, photosynthates of C3 plants are 13C-depleted by about 20‰ compared to atmospheric carbon dioxide because of the isotope discrimination during photosynthesis.

Importantly, the δ13C value of net assimilated carbon (photosynthates) not only depends on the isotope composition of the inorganic source (atmospheric CO2) but also on isotope fractionation processes associated with transport, diffusion, phase transition, and enzyme reactions in the plant. Here, the term "fractionation" refers to the change in the δ13C between substrate and product due to different velocity of isotopic molecules (isotopologues) (i.e., the ratio of rates 12v/13v differs from unity). In practice, the δ13C value of organic plant material can be modelled as (neglecting the denominator, and neglecting internal resistance):

δ13Cleaf ≈ δ13Catm -a -(b -a)•ci/ca (1)
where δ13Catm is the carbon isotope ratio of atmospheric CO2 (about -8‰), a is the fractionation caused by diffusion (4.4‰), b is the apparent biochemical fractionation associated with carbon dioxide fixation by primary carboxylating enzymes, mainly Rubisco (here, 27‰), and ci/ca is the intercellular-to-ambient ratio of CO2 mole fraction (Francey & Farquhar, 1982). It is clear in Eq (1) that the δ13C value of a given plant tissue reflects both the carbon source and fractionations along metabolism, thereby providing physiological information [START_REF] Offermann | The long way down-are carbon and oxygen isotope signals in the tree ring uncoupled from canopy physiological processes?[END_REF]. At the whole plant scale, the carbon isotopic signal is primarily determined by photosynthesis, that is, by the δ13C value of photosynthates. Sugars exported by leaves via the phloem to sink tissues (like fruits) are used to synthesize organic matter and thus sink organs are isotopically close to leaves, but with some further fractionation steps referred to as "postphotosynthetic" fractionation may take place [START_REF] Gessler | Tracing carbon and oxygen isotope signals from newly assimilated sugars in the leaves to the tree-ring archive[END_REF]. As a result, sink organs are generally 13C-enriched compared to leaves, by a few ‰.

Here we took advantage of 13C labelling and quantified how the isotope composition in leaf matter and respired CO2 deviated from natural abundance after photosynthesis in a 13CO2containing atmosphere. Labelling of plants with isotopically enriched CO2 has been used for decades (mostly with 14C up to the 80s) to study the fate of carbon into photosynthetic products in leaves. Pulse-labelling has already been used to examine where photosynthetically fixed carbon is allocated and how fast it is transferred in trees [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF]. In this study, we performed for the first time a 13CO2 pulse-labelling experiment on oil palm leaves, submitted to two potassium fertilization conditions: K0 (no KCl applied) and K3 (with 4.5 kg KCl tree-1 yr -1), and two crosses (Deli x La Mé (DL) and Deli x Yangambi (DY)), in the field. Our primary objectives were to (i) determine the nature of the prevalent sugar exported by leaves (sucrose or glucose); (ii) follow the export of carbon from leaves to other organs; (iii) understand carbon dynamics in leaves after photosynthesis (in particular, utilization by respiration); and (iv) know whether all these processes are affected by K availability. Amongst these objectives, mainly (iii) is addressed here. Objective (i) is still in progress (samples collected in this experiment

were not of sufficient quality to allow reliable NMR analyses) and will not be reported here.

Objective (ii) has been partly addressed and will be discussed at the end. As stated above in the Preamble, objective (iv) is superficially dealt with here and awaits further experiments when the K treatment has more tangible effects on photosynthesis and allocation.

B.

Material and methods

Field location and fertilization

The field was located at the SOCFINDO station (North Sumatra, Indonesia; 3°18'19.60"N, 99°3'24.33"E). The experiment was carried out in July 2017 on oil palm trees planted in August 2013 and organized inside a K factorial agronomic trial. They belonged to two crosses: Deli x La Mé (DL) and Deli x Yangambi (DY). DL and DY have been chosen here since they are two very common crosses used in oil palm agroforestry and presented, at nursery stage, contrasting K foliar signature with DL "K--"and DY K++. Two levels of potassium fertilization were studied: (K0), no KCl added and (K3) with 4.5 kg KCl tree-1 yr-1.Trees were fertilized with nitrogen using 2 kg urea tree-1 yr-1. There was no K deficiency under K0 conditions, since K content in soil under the K0 treatment represents about 0.2 meq exchangeable K per 100 g soil, coming from some K remained from previous agronomic trials conducted before on the same location (information from soil analysis). As such, oil palm tree tissues were not under very low or very high K conditions and K content was always in the 18-38 mmol m-² range.

Climatic conditions

Climatic conditions (solar radiation, rainfall, temperature and relative humidity) were recorded near the fertilizer trial at Bangun Bandar (Nord Sumatra, Indonesia) using a minimeteorological station (Watchdog, Spectrum, France). between May and July 2017. Nevertheless, no sign of water stress could be detected in the field in July 2017. In fact, each tree studied were watered on the night before the labelling to make sure that stomates were fully opened at the beginning of the experiment in the following morning at 7 am.

Functional traits

Specific leaf weight (SLW) was measured, as dry weight per leaf surface area (kg DW m-2) by weighing precisely 30 leaflets samples of 10 cm2 of each on a representative palm of the crown.

Leaf area was determined in 2017 on the leaf rank number 17 following the method of [START_REF] Tailliez | A method for measuring oil palm leaf area[END_REF]. Overall foliar emission rate was calculated as the number of leaves present on the tree plus petiolar bases (corresponding to cut leaves) divided by tree age. Trunk heights (from the limit of the trunk basal bud to the petiole insertion of leaf rank number 33) were determined using a meter, and diameters were measured (at 1 m high from trunk base) with a specific caliper, respecting CIRAD standard procedure. Functional traits were determined on 5 replicates per condition for a better representation of the treatment effect.

Experimental design and sampling

Overview. The 13CO2 pulse-labelling experiment was carried out on 12 oil palm trees (3 replicates per condition) using a closed chamber system where CO2 is injected to maintain its mole fraction. For this purpose, a palm portion of 1m of rachis length, centered at point B of potential leaf rank number for gas exchanges (usually 10 but mainly varied until 14). In some cases, when palm 10 was absent or in bad shape, other palms (11, 12 or 14) which have approximatively the same age were used instead. See PICTURES S1-S7 illustrating the whole experiment.

The labelling chamber. For this labelling, we did a purpose-built transparent chamber with a rectangular form and a volume of 0.38 m3 (1.9 m x 1 m x 0.2 m). It was made of stainless steel half-plates covered with a polypropylene film with rectangular openings to allow the entrance of the rachis (FIG. IV.1). Air tightness was obtained with mastic (Terobon RB VII) and adhesive tape. Six axial fans (12 V) were placed in the labelling chamber to ensure good air mixing and a cold water bath (located outside) was used to help to cool air tube (connected to the pump) for maintaining air temperature inside the labelling chamber. Temperature, humidity and solar radiation were recorded inside the labelling chamber using a small meteorological station. 13CO2 circulation was obtained through the chamber and tubing (BEV A LINE IV 3.2

x 6.4 mm, Fischer Scientific, Illkirsh, France) using a pump (IP00-T 230V50HZ N828KNE, KNF, Village-Neuf, France). Silica gel was used to decrease humidity generated by high leaf transpiration rates after the closing of chamber. The labelling chamber is held by a scaffold made of wood. In the field, electricity was provided using a generator. Because the chamber was installed in the canopy, upper leaves were folded away to avoid shading.

The 13CO2 -labelling experiment. The central B leaf region (composed of around 20 leaflets)

was enclosed with care in the chamber and both entrances were then sealed with mastic (See

FIG. IV.2).

During all this time and after closing the chamber, the CO2 concentration inside the labelling chamber was continuously monitored with a gas analyzer (WALZ GFS 3000, Germany)

that was designed and calibrated for 12CO2 and exhibited low sensitivity to 13CO2. When the chamber was closed, the CO2 mole fraction decreased linearly from ambient (≈400 ppm) with an average of 0.37 ppm s-1 due to CO2 uptake by photosynthesis. When the CO2 mole fraction reached 200 ppm, 13CO2 was added to reach again 400 ppm inside the chamber (which equals external conditions). Pure 13CO2 (99.3%, Euriso-top, Saint-Aubin, France) was then constantly injected at a flow rate that compensated for photosynthetic consumption, using a mass flow controller (EL-FLOW Prestige, Bronkhorst, Montigny-Les-Cormeilles, France). Because of the low sensitivity of the gas analyzer to 13CO2, the slope of the CO2 decline in the beginning of the experiment was used to calculate net CO2 uptake and thus adjust immediately the flow rate of the 13CO2 injector. The observed flow rate for all trees was 6-10 mL min-1. The labelling was stopped when 1 L of 13CO2 had been injected in the chamber. The time required to do so was 2-5 hours, depending on individual CO2 palm absorption and leaf surface area. At the end, the chamber was rapidly opened and removed and leaflets were sampled. Labelling started at about 8:00 AM local time and ended no later than 3:00 PM. (99.3%) with a CO2 and mass flow regulator allowing a constant flow that compensated for photosynthetic consumption. The flow is also maintained using a pump, silica gel was used to decrease humidity generated by high leaf transpiration rates after the closing of chamber, CO2 level was controlled with a gas analyzer (WALZ GFS 3000, Germany), a cold water bath was used to help to cool air tube, air inside the chamber was homogenized with fans and, temperature, humidity and solar radiation (PAR) were monitored with sensors. Small arrows indicates the sens of the flow. Leaflet were sampled at: T0, before the labelling, T1, just after opening after labelling, and then at T2-T8, 1h, 2h, 3h, 5 h and 1, 2 and 7 days later. Dark respiration analysis started 8 to 10h after the labelling depending on the tree and was performed during 2h. CO2 sampling inside the chamber took place at: S0, before the installation of the chamber (control with no respired CO2) and S1-S5, 5 min, 10 min, 30 min, 1h and 2h later using a 25 mL syringe.

Samples preparation and isotope analysis. Leaflets were collected just before placing the leaf in the labelling chamber and just after opening after labelling, and then 1h, 2h, 3h, 5h and 1, 2 and 7 days later (FIG. IV.3). One leaflet was sampled per time point randomly on both sides of the rachis at the point B of the palm labelled. Seven days after the experiment, leaflets from the palm rank 1, 9, 17, 25, 33, 41 and 49 were also sampled (at point B) from the labelled trees.

Leaflet samples were cleaned with deionized water, frozen in liquid nitrogen, stored at -20 °C, and finally oven-dried and ground. Leaflet organic matter (OM) samples were weighed in tin capsules and analyzed for C isotope composition and total C using an elemental analyzer (Carlo-Erba) coupled to an isotope ratio mass spectrometer (Isochrom, Elementar) run in continuous flow at the Isotope Facility of the Australian National University.

Calculations

13C excess. The 13C atom fraction (percentage), denoted as x(13C), was calculated from the isotope composition (δ13C) using the isotope ratio (RV-PDB) of V-PDB (Vienna Pee Dee Belemnite standard, 0.0112372):

x(13C) = 13C/(12C+13C) = (δ13C+1)×RV-PDB/((δ13C+1)×RV-PDB + 1) (2)

While the isotope composition (δ13C) at natural abundance levels is often expressed as a ratio relative to V-PDB, the isotope composition of an enriched compartment can be better expressed as percent atom excess because in denominators, the isotope ratio is not negligible compared to unity [START_REF] Dawson | Stable isotopes in plant ecology[END_REF]. This value is defined using percentages, as the relative 13C abundance in a labelled sample exceeding the natural isotope abundance in an unlabelled sample. Percent atom excess 13C, denoted as xE(13C), was calculated therefore after accounting for the background 13C atom fraction measured on the same tree before labelling, x(13CUN):

xE(13C) = x(13C) -x(13CUN) (3)
xE(13C) in leaflets was calculated for each tree at each sampling time.

Turnover of assimilated C. In order to assess differences in C turnover, we consider two general analysis. The analysis was carried out by the platform GISMO (Bourgogne, France) using a GC-C-IRMS (Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry). The isotope composition of respired CO2 was obtained by linear regression using a Keeling plot.

Statistical analysis

For all analyses, 3 replicates were taken for all conditions. Univariate analyses were conducted using a two-way ANOVA (SigmaPlot version 11.0) (Fisher test with a threshold P-value of 0.05) with crosses and K as factors, followed by a post-hoc Tukey test. Also, to investigate correlations between leaflet K content or carbon isotopic signature and functional traits, a

Pearson correlation was assessed. MRT was determined using a fitted exponential decay model with SigmaPlot.

C. Results and discussion

1.

Functional traits and leaflet starch content of oil palms studied Foliar emission (FE), leaf area (LA), SLW, trunk height and diameter were determined for each K and cross conditions. Fisher tests showed, as expected, that except for trunk diameter significantly affected by both K level (P=0.01) and crosses (P=0.02), potassium and crosses did not affect significantly other traits such as FE, LA, SLW or trunk height (FIG. IV.4), as observed elsewhere [START_REF] Ruer | Premières observations sur les caractéristiques du pétiole de la feuille de palmier à huile[END_REF]Corley, R & Mok, CK, 1972;[START_REF] Purwanto | Roles and optimisation rate of potassium fertiliser for immature oil palm (Elaeis guineensis Jacq.) on an Ultisol soil in Indonesia[END_REF]. Trees emitted approximatively two new leaves by month with a leaf area of 5 m² in average with a specific weigh of about 0.102 kg DW m-². In 2017, trees were 4 -years old, with a trunk height of about 87 cm and a diameter of 76 cm. They produced between 18 and 25 bunches per year [START_REF] Mirande-Ney | Effects of potassium fertilization on oil palm fruit metabolism and mesocarp lipid accumulation[END_REF]. Trunk diameter increased slightly by 5% at high K (K3) and was higher in DL compared to DY. At this stage, K3 had a small positive effect on bunch production in DY only, but not in DL. Although K fertilization had small effects on tree morphology and phenology, leaf K content (documented further in Chapter II) was found to be correlated to crosses (R = 0.82, Pvalue = 0.01), but also (i.e. with positive R) to photosynthesis, leaflet chlorine content and oilto-bunch ratio, while it appeared to be insignificant (TABLE IV.1). These results are not surprising since K was added as KCl thereby providing chlorine. Also the positive effect of K on photosynthesis is well known (see Introduction of the thesis). However, univariate analysis showed no significant effect of potassium fertilization conditions or cross on photosynthesis (FIG. IV.5). In Chapter II, we have seen effectively that there was no significant effect of potassium fertilization or cross on leaflet K content in 2017. It seems therefore that, although leaflet mineral composition does not reflect yet fertilization condition, oil palm reacts to small variations of leaflet K content to modulate its photosynthesis. Under our labelling conditions, photosynthesis was on average about 6 µmol m-² s-1 in DL and 5.3 µmol m-² s-1 in DY, far from average usually admitted for oil palm, around 11 µmol m-2 s-1 [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF] for the same ecology and age. This was perhaps due to poor air conditioning of the chamber while under tropical conditions in Sumatra, incident radiation was high, with PAR values about 2000 µmol m-2 s-1 and high air temperature was also high, above 30 °C. Moreover, when we looked at starch content variations in leaflets under different K fertilization conditions and crosses, no significant effect of K or cross was observed while high K caused a decrease in leaflet starch content in DL and an increase in DY (FIG. IV.6). This is surprising considering that via its action on starch synthase, potassium is known to be positively correlated to leaflet starch content [START_REF] Ward | Potassium in plant metabolism: II. Effect of potassium upon the carbohydrate and mineral composition of potato plants[END_REF][START_REF] Läuchli | Potassium transport through plant cell membranes and metabolic role of potassium in plants[END_REF]. The effect of K on leaflet starch content seemed to be therefore cross-dependent. Starch content varied between 15 and 35 kg g-1 DW.

Interestingly, leaflet K content was also anti-correlated to palm length, bunch biomass, leaflet N and C content and average diameter (TABLE IV.1), while these parameters are also cross-dependent. It is possible that K has an effect on carbon partitioning but further investigations are required to differentiate the effect of K from that of the cross (genotype)

itself. Having said that, an effect of K on carbon partitioning would not be surprising because of its action on sugar synthesis and transport. In another trial with the same conditions (reported in Chapter II), potassium also had a positive effect on photosynthesis and respiration. Dark respiration was on average 0.47 µmol m-² s-1 in DY, similar to the values observed in the other trial. 2. 13C natural abundance in leaflet total organic matter (OM)

At natural abundance, on average, 13C (OM) was about -30.3 ‰ in DL (K0, -30.5 ‰ and K3, -30.67 ‰) and -29.9 ‰ in DY (K0, -30.44 ‰ and K3, -29.41 ‰) close to the 13C value observed in leaflet by [START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF]; [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]; [START_REF] Muhammad | Variations of δ13C and δ15N in oil palm tree organs: an insight into C and N distribution[END_REF].

Univariate analysis on 13C value of OM between conditions showed no significant effect of K or cross. Since K availability is known to affect photosynthesis and carbon allocation in oil palm [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF], an impact on leaflet δ13C could have been expected. Here, the effect of K fertilization was too small to affect significantly ci/ca and thus the photosynthetic fractionation.

In general, leaf 13C values at natural abundance depends on many factors such as climatic conditions but also leaflet functional traits [START_REF] Saurer | The impact of an inverse climate-isotope relationship in soil water on the oxygen-isotope composition of Larix gmelinii in Siberia[END_REF]. In our study, 13C

values at T0 (i.e. at natural abundance) were correlated to average air relative humidity in the cultivation field and leaflet starch content determined just after photosynthesis in the chamber, but anti-correlated to average temperature and leaf length ( [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]Legros et al., 2009b;[START_REF] Battie-Laclau | Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment[END_REF]. When we looked at leaflet δ13C just after labelling (T1), we could observe a strong variability between replicates in the same conditions (TABLE IV.4). Although leaflet 13C at T1 (denoted as 13C(T1)) was not significantly affected by K, cross or replicates conditions, pearson correlation analyses revealed that 13C(T1) was strongly correlated to photosynthesis, and leaflet starch content during the night. Considering the fact that leaflets were not in optimal conditions of photosynthesis during the labelling due to high temperature and RH, these correlations showed that leaflet 13C was in fact very sensitive to photosynthetic processes [START_REF] Saurer | The impact of an inverse climate-isotope relationship in soil water on the oxygen-isotope composition of Larix gmelinii in Siberia[END_REF] and may give insight on starch leaflet composition during the night thus carbon remobilization, thus respiration. [START_REF] Epron | In situ 13CO2 pulse labelling of field-grown eucalypt trees revealed the effects of potassium nutrition and throughfall exclusion on phloem transport of photosynthetic carbon[END_REF] have shown that K fertilization had a positive effect on crown CO2 uptake and the amount of 13C in excess recovered in leaves just after labelling. According to [START_REF] Dannoura | In situ assessment of the velocity of carbon transfer by tracing 13C in trunk CO2 efflux after pulse labelling: variations among tree species and seasons[END_REF], the shape of the curve given by the time course of excess 13C is affected by the duration of the labelling, thus the photosynthesis, but also to seasonal conditions. We found in fact that, 13C(T1) was anticorrelated to temperature and leaflet %C.

The time course of excess 13C, xE(13C) in leaflet of labelled palms (FIG. IV.8) was fitted according to Eq. ( 4). This is based on the assumption there are two pools of 13C in leaflets:

representing a "stabilized" (i.e. fixed constant) pool (CS) and a labile pool (CL) that is turnedover exponentially [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF][START_REF] Subke | Fast assimilate turnover revealed by in situ 13CO2 pulse-labelling in Subarctic tundra[END_REF]. CS accounted for 33% of total 13C (CS+CL) in DL (35.5% in K0 and 30.6% in K3) and for 32.5% of total 13C in DY (30.4% in K0 and 33.8% in K3) (TABLE IV.4). The 13C pool associated with CL decreased quite slowly, with a mean residence time (1/τ) ranging from 2h to 130h that was longer in DL than in DY trees (but this difference was insignificant because of large variations within condition) (TABLE IV.4). 3). Red straight lines represent fitted simple exponential decay (Eq. ( 4)).

In Betula nana [START_REF] Subke | Fast assimilate turnover revealed by in situ 13CO2 pulse-labelling in Subarctic tundra[END_REF] or eucalypts [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF], mean residence time of pulse-derived 13C are about 1.1 days or 21-31h respectively. There were also no difference between K conditions (P=0.3). In eucalypts, [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF] has observed that 1/τ tended to be shorter in K fertilized trees compared to non-fertilized trees while the difference was found to be insignificant. The presence of the 2 pools was also represented after graphed the probability density Because past studies have shown that vegetative tissues were priority sinks in carbon partitioning [START_REF] Henson | Modelling vegetative dry matter production of oil palm[END_REF][START_REF] Henson | Modelling the effects of physiological and morphological characters on oil palm growth and productivity[END_REF], we were expecting that some 13C labelling could be found in other leaves and specially young ones like leaf number 1 and 9. However, we are conscious that at this developmental stage, oil palm represents a huge biomass. We estimated that in 2017, trees had a total standing biomass of about 295 kg DW per tree, and 13C effectively assimilated by a tree represented only about 1.2 mg 13C /g C.

Natural abundance.

As observed by [START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF], there was a clear difference in 13C natural abundance between leaf ranks, suggesting changes in the origin of the carbon source used for leaf growth. In fact, it is found here that 13C values decreased with increasing rank number. Thus, leaf number 1 was more enriched than leaf number 49, regardless of the cross or K fertilization, ranging from -29.5‰ to -31.5‰ (FIG. IV.10). The δ13C difference between leaf ranks may be due to differences in the ratio of starch/soluble sugars. In young leaves, starch content (which present a less negative δ13C value, about -26 ‰) is more important than soluble sugars (which have a more negative δ13C, about -29 ‰ for glucose and fructose) [START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]. When the leaf is becoming autotrophic after rank 1, there is a change in the carbon pool participating to leaf biomass elaboration, with a higher proportion of photosynthates that are more negative. In addition, while insignificant, we observed that K tended to increase the

D. Advantages and disadvantages of the present experiment

In this study, we performed for the first time a 13CO2 pulse-labelling experiment on oil palm leaves, submitted to two potassium fertilization conditions and two crosses in the field. Clearly, 13CO2 injected into the chamber was fixed into leaflet organic matter (OM), showing that the labelling experiment was successful.

However, the efficacy of the experiment was much less than expected. In reason of uncontrolled conditions in the field, such as high temperature or air relative humidity, leading to water condensation (PICT. S4) and the large biomass that a young oil palm tree represents, it was less than 27% of 13CO2 added inside the chamber that were assimilated by the leaflets. In fact, according to [START_REF] Dufrene | Gas exchange of oil palm in relation to light, vapour pressure deficit, temperature and leaf age[END_REF] photosynthesis is very low when air temperature is over 36°C. In our experiment, air temperature inside the chamber could reach 49°C.

Moreover, because of the high water condensation inside the chamber, we had to add silica gel before the entrance of the analyzer to avoid water entry that could damage the analyzer, which made impossible the measure of water absorbed, transpiration rate and thus the calculation of stomatal conductance and water use efficiency.

Therefore, since 13C is very sensitive to climatic variations such as temperature and RH, there was a strong variability between replicate that made impossible the interpretation of the data relative to the effect of K on carbon metabolism in the leaflet. However, we could observe some tendency for K to enrich starch or 13C median of the 2 pools. In addition, 13C

was correlated to leaflet Cl content that could reflect an effect of K on trees experienced more K treatments as K applied as KCl.

Finally, this study sheds light on the basal physiology of the oil palm inside the leaflet that is the existence of 2 pools of C: a bigger pool representing the leaf biomass, and a smaller labile pools. The labile pool had a MRT reaching up to 130 h that is relatively slow comparing to Betula nana [START_REF] Subke | Fast assimilate turnover revealed by in situ 13CO2 pulse-labelling in Subarctic tundra[END_REF] or eucalypts [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF] trees, suggesting a slow exportation rate of C. It also gave us insight on the proportion of CO2 lost by respiration, although more 13C analysis of leaflets OM during respiration could have informed us on the metabolism of respiration and explained higher mitochondrial activity with K fertilization.

To conclude, this experiment has a strong potential, it can brings insight on the effect of potassium on carbon dynamic and flux related to the yield, which is relatively important for the adjustment of K fertilization in the field. Another experiment of 13CO2 labelling is therefore necessary with trees experiencing more K treatments and with controlled climatic conditions to answer the followings objectives: (i) determine the nature of the prevalent sugar exported by leaves (sucrose or glucose); (ii) follow the export of carbon from leaves to other organs; (iii) understand carbon dynamics in leaves after photosynthesis (in particular, utilization by respiration); and (iv) know whether all these processes are affected by K availability. More specifically, compound specific analysis (by LC-c-IRMS or GC-c-IRMS) of labelled leaflets following the labelling are necessary to answer (i). Moreover, as the trees at 4 years old represent a huge amount of carbon biomass, it may be difficult to answer to objective (ii)

without performing several analysis for the different organs or labelling during a long time with pure 13CO2, which represent both high cost. A solution may be to do a labelling with 13Cdepleted CO2 (i.e., near-natural abundance labelling) during a long period (see [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF]) or practice a pruning to reduce sink organs before the labelling (see Chapter IA2). In order to answer to objective (iii), analysis of CO2 respired and compound specific analysis of labelled leaflets during dark respiration should be performed in addition to gas exchange measurements.

Chapter V. General discussion and perspectives

Oil palm is characterized by a simple architecture and indefinite growth which produces successive leaves on an unique cylindrical trunk [START_REF] Jacquemard | Le palmier à huile: Quae[END_REF][START_REF] Corley | The oil palm[END_REF].

It is a C3 plant with a high leaf surface area and a generally high photosynthesis rate [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]Legros et al., 2009c). To sustain growth and get optimal yield, high fertilization is required. Potassium chloride (KCl) is the most widely used fertilizer in oil palm plantations. K has many roles in plant physiology and metabolism related to cation-anion balance, osmoregulation, water movement, phloem transport and energy transfer, and takes part in protein synthesis, carbohydrate metabolism and enzyme activation (see Introduction).

However, in oil palm, the effects of K on carbon metabolism related to the yield are not well understood and in some cases, potassium is applied excessively. Nowadays, different agronomical practices and tools are used to monitor K nutrition (fertilization trial, leaf diagnosis, leaf symptoms and soil analysis) for a better prediction and adjustment of K fertilization. However, there is still some possible improvement (and even a slight improvement of a few % in KCl management could save hundreds of k$).

The goal of this study was to assess the effect of K availability on oil palm metabolic pathways and determine if metabolic changes were related to yield. More precisely: (A) What is the impact of K fertilization on proteomic and metabolomic responses of leaflets on two genetically contrasted materials, presenting different leaflet K mineral signature? (B) What is the impact of K fertilization on omics responses of the oil palm fruit mesocarp during maturation, on two genetically contrasted materials? (C) Can a 13CO2 labelling help identifying sugars produced and C allocation, with these affected by K availability?

A.

Effect of K on oil palm growth and yield

Taken as a whole, we observed that K fertilization, in the first 3-4 years (respecting standard agronomical procedures) had small effects on functional traits. Two years were necessary to see a significant effect of K fertilization on leaf K content. Even so, while potassium fertilization increased N, P and K contents in leaflets of both crosses, no general effect of K was found on bunch production nor vegetative growth 2 years after the treatment, as observed elsewhere [START_REF] Ochs | Contribution à l'étude de la fumure potassique du palmier à huile[END_REF]Hartley, 1988a;[START_REF] Corley | The oil palm[END_REF]. On average, individual bunch production of oil palm studied was up to 25 bunch tree-1 yr-1 and bunch weight was about 9 kg, with no significant effect of K, which is coherent to the fact that it takes 3 year for a bunch to develop [START_REF] Corley | The oil palm[END_REF]. However, high K availability increased significantly specific leaf weight, trunk diameter and the number of fruits as well as mesocarp and kernel biomass proportion in bunches, and tended to increase trunk height. This suggests that possible changes in yield and therefore on leaflet and fruit metabolism can be anticipated in the next few years.

Moreover, the fact that K increased trunk diameter and tended to increase trunk height without increasing foliar emission rate, may reflect an effect of K on trunk internode length, and therefore on growth rate, carbon transport and storage in the trunk, which would be consistent to literature [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]Legros et al., 2009c;[START_REF] Corley | The oil palm[END_REF].

Our study also shows that while the effect of K fertilization on leaflet K content (and leaflet proteome) was minimal after one year (in 2017) plus three years preconditioning, several metabolites appeared to be significant, suggesting that there were some changes in, e.g., K+ distribution amongst leaf tissues or cellular compartments. That is, it is probable that even in 2017, K0/K3 conditions were associated with subtle changes in cytosolic and mitochondrial K+ concentration. In fact, we have observed in Chapter IV that in spite of the absence of significant effect of K fertilization on photosynthesis, leaflet K content was slightly correlated to photosynthesis, while not significant (R=0.52, P=0.19). It is worth noting that both crosses have contrasted leaflet K content and effect of leaflet K content on photosynthesis may be attributed to the cross effect. In fact, both crosses differed from their metabolism and particularly, the O2PLS analysis from the Chapter II suggests that there were differences in both photosynthetic capacity and mitochondrial metabolism between crosses.

We estimated that the lack of effect of K on vegetative growth and yield in 2017 may be due to K remobilization from leaves or trunk to fruits to cover the K demand of bunch maturation, thereby dampening changes in leaf K despite the K fertilization treatment. Also, in 2017, K3 fertilization (4.5 kg KCl tree-1 yr-1) represented twice as much the K demand and may reflect an excess. In a previous study [START_REF] Imogie | Effect of potassium (K) source on oil palm yield at Okomu oil palm Plc, Ovia North East LGA of Edo State[END_REF], it has been observed that even 1 year after the onset of the K treatment, fresh fruit bunch production increased with K fertilization up to an optimum at 2.0 kg K tree-1 yr-1 and above this amount of K, there was no significant increase in fresh fruit bunch production (See Chapter I.C.2), reflecting the non-linear relation between K and bunch production thus oil palm metabolism. Here, we could observe a slight depressive effect of K on bunch production in DY.

We observed that higher K availability caused an increase in kernel biomass proportion in bunches and mesocarp water content (MWC) in DY, thus leading to lower OFM, while in DL, MWC clearly decreased with K availability and accordingly, there was an increase in OFM and OER. Previous studies showed a correlation between fruit transpiration (water loss) and lipid biosynthesis [START_REF] Jeje | Transpiration and oil accumulation rates for developing oil palm fruits Elaeis guineensis Jacq[END_REF]Teh et al., 2013a). It seemed therefore that high K availability had an effect on fruit water loss, and this effect can be positive or negative depending on the cross. Leaflet elemental content analysis showed that DY (supposed to be leaflet K++) was more sensitive to K fertilization than DL (supposed to be leaflet K--) (See Chapter III). In fact, according to [START_REF] Rengel | Crops and genotypes differ in efficiency of potassium uptake and use[END_REF], genotypic differences in capacity to utilize K have been attributed to (1) differences in partitioning and redistribution of K at cellular and whole plant levels, (2) the substitution of K by other ions e g Na in the vacuole particularly important under salinity, and (3) the partitioning of resources into the economic product. However, the negative effect of high K (K3 conditions) on oil content (OFM) in DY fruits could not be assigned to an excess of K, because the same trend was observed with K1 and K2 in DY (FIG. III.3). The change in OFM may have come from excess chloride in tissues, which is also known to increase the kernel-to-mesocarp ratio, as observed in our study [START_REF] Breure | Factors affecting yield and growth of oil palm tenera in West New Britain[END_REF]Ollagnier & Olivin, 1984a). Moreover, increasing OFM in DL with K without increasing mesocarp proportion in a fruit, suggested therefore that increasing OFM may be associated to increasing lipid accumulation and/or FA synthesis (see Chapter III).

Finally, although no big changes were observed on yield or vegetative growth 2 years after the onset of the treatment, our data indicate that leaflet metabolism is sensitive to small variation of leaflet K content suggesting that possibly, important changes on yield and therefore on leaflet and fruit metabolism will take place in the next few years. It is important to realize that a direct effect of K nutrition on enzyme activity thus on leaflet metabolism requires a considerable change in cytoplasmic K concentration. However, according to [START_REF] Amtmann | Potassium in plants. e LS[END_REF], efficient usage of the vacuole as a reversible K store usually prevents large fluctuations of cytoplasmic K concentration. A considerable increase of cytoplasmic K concentrations would only be expected after prolonged K-supply.

B. Effect of K on leaflet metabolism

In order to understand the possible impact of K fertilization on leaflet metabolism, both proteome and metabolome were analyzed. First, our results showed that higher K availability increased N assimilation. Stimulation of N assimilation encompassed an increase in glutamate metabolism leading to higher amino acid content such as methionine, serine, leucine or valine, due to both augmented synthesis and proteolysis (protein-turn-over). In fact K availability had an effect on protein synthesis machinery, in particular the abundance of ribosomal proteins.

This agrees with previous agronomical observations that increasing K stimulates N use efficiency and eventually, increases %N in leaflets [START_REF] Ollagnier | Interaction entre l'azote et le potassium dans la nutrition des oléagineux tropicaux[END_REF], perhaps through enzyme activation but also ribosome synthesis and mRNA turnover [START_REF] Evans | Potassium and its role in enzyme activation[END_REF][START_REF] Blevins | Role of potassium in protein metabolism in plants[END_REF][START_REF] Pettigrew | Potassium influences on yield and quality production for maize, wheat, soybean and cotton[END_REF]). K fertilization has been found to be beneficial to ammonium assimilation by up-regulating glutamine synthetase (GS), ferredoxin-glutamine-2-oxoglutarate aminotransferase (Fd-GOGAT) and glutamate dehydrogenase (GDH) in Arabidopsis [START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]Hu et al., 2016b). This might imply that K fertilization leads to a high C flux through the TCA cycle so as to sustain amino acids and protein synthesis [START_REF] Ollagnier | Interaction entre l'azote et le potassium dans la nutrition des oléagineux tropicaux[END_REF][START_REF] Ruan | Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensisL)[END_REF][START_REF] Amtmann | Potassium in plants. e LS[END_REF][START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]Hu et al., 2016b) (See FIG. IV.1).

Moreover, higher K availability led to higher proteins contents, which may explain the increase in SLW. We are well aware, that an increase in SLW could have also come from an increase in leaflet structural components (such as lipids, cellulose, lignins, etc.); however, they have not been characterized in this study. Despite a tendency of K to increase leaflet starch content in DL and to decrease it in DY (Chapter IV), high variability between samples did not allow a consistent interpretation of the effect of K on starch content and carbon storage.

However, difference in leaflet starch content between both crosses may be consistent with the fact that DY was more sensitive to K fertilization suggesting a higher carbon assimilation and translocation resulting in lower starch content at higher K compared to DL. (see Chapter II). In fact, according to [START_REF] Yang | Potassium internal use efficiency relative to growth vigor, potassium distribution, and carbohydrate allocation in rice genotypes[END_REF], the higher K concentrations in the rice leaves (especially the lower leaves) of the K-efficient genotypes were associated with higher RuBP carboxylase activities and net photosynthetic rates allowing the leaves to maintain a higher photosynthetic capacity during grain filling. Nevertheless, at high K, the increase in leaflet total phosphorus content concurrent with a decrease in leaflet free phosphate (Pi), fructose 6-phosphate, glycerol 1-phosphate and 3-phosphoglycerate, suggests an increase in Pcontaining compounds such as phospholipids, which could participate in increasing SLW.

Moreover, we found that K availability had a significant effect on several proteins involved in photosynthesis and photorespiration, including sucrose metabolism (UDP-sugar pyrophosphorylase) that were consistent to the well-known beneficial effects of K on oil palm photosynthesis via stomatal regulation, photosynthetic capacity and photosynthate export [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF]Cui et al., 2019b). Nevertheless, in our study, we found that this effect was also associated with a higher content in sugars such as sucrose, which is not in agreement with other findings [START_REF] Cakmak | Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants[END_REF][START_REF] Zhao | Influence of potassium deficiency on photosynthesis, chlorophyll content, and chloroplast ultrastructure of cotton plants[END_REF][START_REF] Gerardeaux | Changes in plant morphology and dry matter partitioning caused by potassium deficiency in Gossypium hirsutum (L.)[END_REF] where sugar accumulation was observed at low K because of the inhibition of phloem loading. It is worth noting that in our study case, no K deficiency was observed, explaining therefore why the effect of K on sugar content in our study was different with other findings dealing with K deficiency. Further studies are necessary to evaluate effect of K on sugar transport. Still, in our study, preliminary results of 13CO2-labelling experiment (Chapter IV) indicates that leaflet carbon transport rate was relatively slow (high MRT) [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF][START_REF] Subke | Fast assimilate turnover revealed by in situ 13CO2 pulse-labelling in Subarctic tundra[END_REF]. [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF] has effectively demonstrated that carbon assimilation (at saturating light) and SLW in oil palm leaflets are correlated and suggested that assimilate transport is very slow during the day. However, increasing in SLW with photosynthesis may be also due to the fact that up to 20% of carbon are stored as starch [START_REF] Legros | Carbohydrates reserves in 9 years old oil palm: nature, distribution and seasonal changes[END_REF]. Also, under our conditions, we observed a link between sugars and photosynthetic capacity. In fact, bi-dimensional multivariate analysis showed that disaccharides did correlate to key proteins involved in photosynthesis such as sucrose phosphatase or Rubisco activase.

In addition, potassium fertilization had an effect on mitochondrial metabolism, with changes in the abundance of some Krebs cycle enzymes and associated changes in Krebs cycle intermediates: an increase in citrate (due to lower aconitase activity) and succinate (due to both larger IDH and succinate thiokinase activity). There was also an increase in other mitochondrial proteins (cytochrome c oxidase subunit 5b-2, NADH dehydrogenase, di/tricarboxylate transporter, etc.). Altogether, these changes point to an increased flux in organic acid metabolism and thus an increase in respiratory activity. Our results have effectively shown that potassium tended to increase dark respiration rate in DY (Chapters II and IV). However, such an important stimulation of mitochondrial metabolism at higher K availability is slightly surprising. In fact, recent studies have shown that in oil palm saplings, K deficiency rather than high K leads to an increase in respiration rate and ≈10% loss in carbon use efficiency only (Cui et al., 2019b). We have suggested that such increase might have been driven by the demand in carbon skeletons for nitrogen assimilation (discussed above), or the increase in growth rate and sucrose export, or the ion balance (generation of organic acids carrying a negative charge to compensate for changes in the relative K+ excess). Indeed, we also found an increase in the content of the Ca2+-sensitive ion channel POLLUX suggesting that K availability directly impacted on cellular K+ and Ca2+ pools. Moreover, as shown in (Cui et al., 2019b), effect of K in leaflet metabolism is non-linear, thus explaining similar results observed at K deficiency.

Thus, the simultaneous effect of K availability on photosynthesis and respiration suggests that the carbon use efficiency could have also been impacted. Future measures of biomass, gas exchange and thus carbon use efficiency are necessary to better understand the effect of K on leaflet metabolism.

C. Carbon utilization by fruits

Assimilates produced in the leaflets are redistributed mainly as sucrose to developing leaves, inflorescences (and bunches), trunk and roots, depending on sink strength [START_REF] Houngbossa | The nature of organic nutrients exported from the leaf of oil palm (Elaeis guineensis): effect of ethylenediaminetetraacetic acid on exudation[END_REF][START_REF] Obahiagbon | Assessment of the nutritional constituents of Elaeis guineensis Jacq. exudates from different states of Nigeria[END_REF]. In fact, in the fruit mesocarp, our data (Mirande- et al., 2019) suggested that sucrose is the carbon source for lipid and starch biosynthesis (starch being stored transiently and then used as a carbon source for lipid synthesis). This is consistent with the findings of [START_REF] Dussert | Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition[END_REF]. According to [START_REF] Bourgis | Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning[END_REF] and [START_REF] Voelker | Secrets of palm oil biosynthesis revealed[END_REF], in the mesocarp, imported sucrose is cleaved into hexoses, and intermediates are transported to the plastids, where glycolysis and FA synthesis occurs. FA are then assembled into triglycerides and other lipids in the cytoplasm. In addition, we found that lipid accumulation was associated with metabolites of the oxidative pentose phosphate pathway (generating NADPH) and a general depletion in fumarate, citrate or succinate, probably reflecting the progressive increase in energy generation and thus the consumption of Krebs cycle intermediates. In general, we observed that FA biosynthesis was accompanied by a decrease in sugar, amino acids and organic acids to very low levels at maturity (around 20-21 WAA at stage 5) as observed elsewhere [START_REF] Hartley | The oil palm[END_REF][START_REF] Thomas | Fruit ripening in the oil-palm Elaeis guineensis[END_REF][START_REF] Neoh | Profiling of metabolites in oil palm mesocarp at different stages of oil biosynthesis[END_REF]Teh et al., 2013a). Final oil content in mesocarp was found to be related to (i) the velocity of lipid production (and thus anticorrelated to pools of intermediates of lipid synthesis), (ii) the availability of carbon source (sugars) and (iii) the ability to break down amino acids and thus metabolic recycling to other N-containing metabolites (β-alanine, triethanolamine and putrescine). At maturity, the fruit mesocarp was mostly composed of 71.5% FAs (including palmitate (53-67%), oleate (23-35%), linoleate (2.5-5.3%) and stearate (3-8%)), and 27.4%

Ney
carbohydrates depending on the cross.

D. Effect of K on fruit mesocarp metabolism during maturation

Despite multiple metabolic changes during fruit development, the relative quantity in fatty acids in mesocarp and final oil composition at maturity were not modified significantly by K availability. However, K availability had an effect on fruit metabolism during maturation, with lower carbohydrate and organic acid content at high K. At high K, glucose, fructose and sucrose were lower (by 40-70%) in particular at stage 1, suggesting therefore (1) that sugar transport to the fruit could be a limiting factor to lipid accumulation as suggested earlier and/or (2) that sugars were rapidly used for FA synthesis under K fertilization.

Our data showed that high K simply led to an increase in glucose catabolism, including via the oxidative pentose phosphate pathway (which generates NADPH for fatty acid synthesis), and in redistribution of carbon skeletons to specific nitrogenous compounds (methylpiperidine, amino acids). More precisely, K slowed down lysine and phenylalanine catabolism during early stages of fruit maturation (stages 1-3). Moreover, in DY, high K was associated with more intermediates of lipid synthesis (glycerate, monostearin and azelaate) and However, it is worth noting that, (1) in young palms, there was a strong competition between vegetative growth and bunch production with a priority for growth and maintenance [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF][START_REF] Dufrene | Gas exchange of oil palm in relation to light, vapour pressure deficit, temperature and leaf age[END_REF] and (2) K increased fruit number and thus sink without increasing leaf area nor foliar emission rate, both leading to a decrease therefore in sugars allowed to the mesocarp for oleosynthesis. In addition, this may be accentuated by the fact that high K increased the leaflet respiration rate, thus the carbon loss. Previous studies have effectively shown that fruit bunches are responsible for a respiratory loss of 18-40% of gross primary production [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF][START_REF] Lamade | Variation in maximum photosynthesis of oil palm in Indonesia: comparison of three morphologically contrasting clones[END_REF][START_REF] Lamade | Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern[END_REF]. Although more data are certainly needed to provide a more precise picture of allocation and respiratory losses in oil palm, a high respiratory loss in fruits would be consistent with the low metabolic carbon use efficiency of oil (fatty acid) production.

Moreover, bunch analyses showed that K increased OFM without modifying significantly mesocarp FA content. Therefore, it seems that K slow down sugar transport to the fruit during the day as suggested by [START_REF] Dufrene | Photosynthèse, consommation en eau et modélisation de la production chez le palmier à huile (Elaeis guineensis Jacq[END_REF], but used rapidly the sugar allocated to the fruit for FA elongation. More studies are nevertheless necessary to confirm this hypothesis.

Proteomic analysis of oil palm mesocarp at different maturation stages for example could inform us on enzymes activity associated to FA biosynthesis.

Furthermore, although GC-MS showed that there was an increase in average oleate and a decrease in linoleate content in both crosses with K, consistently with NMR analysis and others findings [START_REF] Ochs | Effect of fertilizers on composition of lipids produced by perennial tropical oil plants and on their yield[END_REF]Ollagnier & Olivin, 1984a;Ollagnier & Olivin, 1984b;[START_REF] Seo | The effects of fertilization level on the growth and oil quality in sesame (Sesamum indicum L.)[END_REF][START_REF] Salama | Yield and oil quality of sunflower as affected by fertilization and growthregulating hormones[END_REF][START_REF] Froment | Effect of nutrition on growth and oil quality in linseed[END_REF][START_REF] Dag | Olive oil composition as a function of nitrogen, phosphorus and potassium plant nutrition[END_REF], it is important to keep in mind that GC-MS is not the best technique to characterize FAs and lipids (a proper lipidomics analysis with LC-MS would be necessary). We choose GC-MS here as the best compromise to study fruit mesocarp metabolism and get broad information on mesocarp metabolome, since it allows the simultaneous identification and quantitation of sugars, amino acids, organic acids, etc.

E. Conclusion

Taken as a whole, this study allowed us to assess the effect of K availability on oil palm metabolic pathways. It gives us the opportunity to identify key metabolic pathway impacted by K into leaflet but also in fruit mesocarp. Metabolomics and proteomics analyses gave us insight on photosynthetic and respiration activity, and metabolomics analyses gave information on mesocarp lipid accumulation. Finally, our results are thus of potential interest to envisage new techniques for K fertilization monitoring using the metabolic signature of leaflets. Further studies with others K fertilization conditions and oil palm tree ages are nevertheless necessary.

Also, studies on trunk composition and carbon dynamics in the phloem must be assessed, in particular, analysis of specific sugar in the phloem after a leaf 13CO2 labelling could help understanding kinetic of production, utilization and transport of specific sugars related to the yield.

Importantly, our study allowed us to identify metabolic pathways modified in a crossspecific manner and thus key components that are specific to genotypes. As such, this study may be of interest for breeding since it show how integrative analyses (omics) are implementable in oil palm and thus could be used for the selection of genotypes more sensitive (responsive) to K. This perspective would be useful to reduce K fertilization for sustainable agrosystem. More generally, omics-based selection could be interesting for selecting genotypes associated with other traits, such as higher resistance to climatic changes such as increasing air temperature, waterlogging events or decreasing solar radiation induced by fires in oil palm plantations.

Figure 1 .

 1 Figure 1. Palm oil production by country in 1000 MT in 2019 ............................................... Figure 2. Global soil regions types ........................................................................................... Figure 3. Potassium application rate (in kilograms of total nutrient per hectare of cropland) by region in 2014 ........................................................................................................................... Figure I.1. Leading importers of palm oil worldwide in 2018 ................................................. Figure I.2. Oil palm leaf organization ...................................................................................... Figure I.3. Dry matter partitioning between vegetative tissues and fruit bunches ................... Figure I.4. Allocation pattern in oil palm computed from δ13C values .................................... Figure I.5. Oil palm fruits forms of dura, pisifera and tenera ................................................. Figure I.6. The basic Kennedy pathway for glycerolipid biosynthesis .................................... Figure I.7. Simplified scheme of TAG biosynthesis in plants ................................................. Figure I.8. Overview of transport processes and proteins that are involved in K+ uptake, efflux and distribution ......................................................................................................................... Figure I.9. Example of data obtained in multi-level analysis of the effect of K deficiency and K re-supply on plant primary metabolism ................................................................................ Figure I.9. Comparison of the relationship between oil palm yield and K .............................. Figure II.1. Leaflet and rachis mineral composition ................................................................ Figure II.2. Leaf sampling design. ........................................................................................... Figure II.3. Climatic conditions in the field ............................................................................. Figure II.4. Oil palm phenology ............................................................................................... Figure II.5. Differential effect of potassium nutrition on total leaf protein content ................. Figure II.6. Output of the multivariate analysis of proteome ................................................... Figure II.7. Proteomics pattern of oil palm leaves under two K fertilization treatments ......... Figure II.8. Output of the multivariate analysis of metabolome .............................................. Figure II.9. Metabolomics pattern of oil palm leaves under two K fertilization treatments .... Figure II.10. Differential effect of potassium nutrition on leaf sugar content ......................... Figure II.11. Bidimensional multivariate analysis of metabolome and proteome ................... Figure II.12. Tentative summary of the effect of K on leaf metabolism .................................. Figure II.13. Differential effect of K fertilization on leaf gas-exchange parameters ............... Figure III.1. Fruit developmental maturation stages ................................................................ Figure III.2. Leaf nitrogen and potassium elemental contents ................................................. Figure III.3. Effect of potassium availability on bunch production ......................................... Figure III.4. Overview of mesocarp metabolism ..................................................................... Figure III.5. Effect of K availability on oil palm mesocarp lipid biosynthesis ...................... Figure III.6. Effect of K availability on FAs synthesis .......................................................... Figure III.7. Metabolomics response of mesocarp to K fertilization during fruit maturation Figure III.8. Pathway enrichment analysis (PEA) associated with the effect of potassium fertilization ............................................................................................................................. Figure III.9. Differential effect of potassium nutrition on mesocarp oil quality .................... Figure III.10. Overview of NMR spectra ............................................................................... Figure III.11. Effect of potassium availability on mesocarp carotenoids content .................. Figure III.12. Robustness of the relationship between oil content and mesocarp metabolome ................................................................................................................................................ Figure IV.1. Scheme of the 13CO2 labelling ......................................................................... Figure IV.2. Photographs of (a) the whole 13CO2 labeling experiment installation in the field and (b) external view of an oil palm leaf enclosed in the labeling chamber .......................... Figure IV.3. Temporal design of the experiment. .................................................................. Figure IV.4. Overview of functional traits of oil palm in DL and DY under K0 and K3

  ............................................................................................................................. Table I.3. Annual K demand of oil palm of various ages ........................................................ Table I.4. Effect of K application rate on Fresh Fruit Bunch (FFB) production at Okomu Oil Palm plc, from 1999 to 2008 .................................................................................................... Table II.1. Fertilization design ................................................................................................. Table

  . Summary of 13C expected and effectively found in leaflet, according to the different conditions of K and crosses. .................................................................................... Table IV.4. Model parameters describing the decrease in xE(13C), the 13C in excess, with time after labelling in leaflet........................................................................................................... Table

  .............................................................................................................................

Figure 1 .

 1 Figure 1. Palm oil production by country in 1000 MT in 2019 -Source: United States Department of Agriculture, www.indexmundi.com

Figure 3 .

 3 Figure 3. Potassium application rate (in kilograms of total nutrient per hectare of cropland) by region in 2014. Source FAO, from www.ourworldindata.org

Figure I. 1 .

 1 Figure I.1. Leading importers of palm oil worldwide in 2018 (in million U.S. dollars).

Figure I. 2 .

 2 Figure I.2. Oil palm leaf organization. (a) Photography of an oil palm tree. (b) Photography of an oil palm tree crown with associated leaf rank number on spiral one (c) Diagram of an oil palm crown with leaf rank number as a function of their chronological emergence, spiral one in red[START_REF] Lamade | Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy[END_REF].

Figure I. 3 .

 3 Figure I.3. Dry matter partitioning between vegetative tissues and fruit bunches. Dry matter (DM) per palm incorporated into vegetative tissues and bunches, compared with total dry matter production.The lines are fitted regressions (both with slopes significantly different from zero) and indicate that 91% of all additional dry matter, above a threshold of 77 kg palm-1 year-1, goes into bunches. Bunch dry matter is adjusted for the energy content of the oil. Data are from[START_REF] Corley | Effects of plant density on growth and yield of oil palm[END_REF] for 6-7 year-old palms in a density trial, plotted as in[START_REF] Squire | The physiology of tropical crop production: CAB International[END_REF].

Figure I. 4 .

 4 Figure I.4. Allocation pattern in oil palm computed from δ13C values, using a model based on sucrose redistribution directly from leaves. Numbers are in percent that is, expressed relative to photosynthetic C fixation fixed at 100. Values in black and grey were obtained at stages 0 and 6 of fruit maturation, respectively. δ13C values shown for respired CO2 were obtained by mass balance with e=+1‰ (dark) or -1‰ (grey), where e is the isotope fractionation associated with respiratory CO2 evolution in the light, with respect to net fixed CO2. Asterisks indicate that the values were fixed as a model constraint: leaf respiration (12% of fixed CO2), total respiration (fixed CUE of 0.38) and trunk growth [in (B)]. Resp, respiration; St.s., starch synthesis. (See (Lamade et al., 2016) for further details).

Figure I. 5 .

 5 Figure I.5. Oil palm fruits forms of dura, pisifera and tenera. Tenera forms are the products of dura x pisifera crosses or dura x tenera crosses (or tenera x tenera).

  has established a precise oil biosynthesis pathway for oil palm. Briefly, imported sucrose is cleaved to hexoses, and intermediates are transported to the plastids, where glycolysis and FA synthesis are massively up-regulated. The large flux of de novo FA synthesis is then channeled to the endoplasmic reticulum, where "housekeeping" levels of enzymes of the Kennedy pathway (FIG. I.6) and associated enzymes are sufficient to assemble triacylglycerols (TAG), which simply accumulate as oil droplets in the cytoplasm (FIG. I.7). Also,[START_REF] Bourgis | Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning[END_REF] found that (i) plastid glycolysis is up-regulated in oil palm temporally during ripening, and (ii) glucose-6-phosphate transporter (GPT2) and phosphoenolpyruvate (PEP) transporter (PPT) provide glycolytic substrates (hexose P) and intermediates (triose phosphate, PEP) to the plastid by transport from the cytosol, a trend that is accentuated during ripening. This implies a strong funneling of carbon towards pyruvate in the plastids of oil palm. Thus, increased fatty acid synthesis, together with plastid carbon supply, is crucial for the accumulation of oil in the mesocarp.

Figure I. 6 .

 6 Figure I.6. The basic Kennedy pathway for glycerolipid biosynthesis. From[START_REF] Guschina | Studies on the regulation of lipid biosynthesis in plants: application of control analysis to soybean[END_REF]

Figure I. 8 .

 8 Figure I.8. Overview of transport processes and proteins that are involved in K+ uptake, efflux and distribution. At the external soil:root interface transport functions are shown for passive [AKT1 and CNGC (cyclic nucleotide gated channel)] and energized (KUP/HAK) K+ uptake and channel mediated K+ release (guard cell outward rectifying K+ channel; GORK); Xylem loading mainly happens through K+ selective (SKOR) and non-selective (NCC) cation channels though energized systems may also play a role; Phloem loading of K+ for recycling and/or sucrose loading may involve the AKT2 channel; K+ flux to the seed is phloem mediated but K+ is unloaded into the seed apoplast (a) at the junction between maternal (m) and filial (f) tissues; vacuolar K+ accumulation is primarily driven by H+-coupled antiporters such as NHX while vacuolar K+ release is either passive through TPK1 type channels or, in K+ starvation conditions, active through H+-coupled KUP/HAK transporters. From[START_REF] Ahmad | Cellular and tissue distribution of potassium: Physiological relevance, mechanisms and regulation[END_REF].

Figure I. 9 .

 9 Figure I.9. Example of data obtained in multi-level analysis of the effect of K deficiency and K resupply on plant primary metabolism. A subset of reactions occurring in root cell cytoplasm, mitochondria and plastids of A. thaliana plants were quantified with respect to changes in metabolite concentrations (grey-scaled bar graphs), transcript levels (blue for increase, red for decrease) and enzyme activities (green bar graphs) of important enzymes (purple). Bar graphs show data for plants grown in control conditions (left), K-deficiency for 14 days (centre) and K re-supply for 24 h (right). Transcript changes in response to K-deficiency and re-supply are shown above and below the enzymes respectively. Individual boxes represent individual genes encoding different enzyme isoforms. For details see[START_REF] Armengaud | Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots[END_REF]. From[START_REF] Amtmann | Potassium in plants. e LS[END_REF].

Figure I. 9 .

 9 Figure I.9. Comparison of the relationship between oil palm yield and K concentration in leaflets (a) and rachis (b) tissue in a fertilizer experiment on volcanic soil in North Sumatra (Foster & Prabowo,1996). From Foster (2002).

  m-² range (see FIG. II.1).

Figure II. 1 .

 1 Figure II.1. Leaflet and rachis mineral composition: (a,e) Leaf total nitrogen (N) and nitrate (NO3-) content, (b, f) leaf phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and chlorine (Cl) content, (c, g) rachis K, Ca and Mg content and (d, h) potassium charge excess in 2017 (top) and 2018 (bottom) without (K0, light shade) or with (K3, dark shade) potassium treatments in Deli x La Mé (DL, pink) and Deli x Yangambi (DY, yellow) crosses. Potassium charge excess corresponds to the charge difference between K and Ca + Mg, i.e. K-2(Ca+Mg). Elemental data are given in millimoles per surface area in leaf and millimoles per kilogram in rachis. Mean ± SE (n = 5). Letters stand for statistical classes (two-way ANOVA, P<0.05).

  petiolar bases (corresponding to cut leaves) divided by tree age. Leaflets and rachises used for analyses were sampled on leaves number 8 and 9, at leaf B point (FIG. II.2D). Leaflet samples were taken on the blade at a distance of two third of total leaflet length from the base (attachment zone region to the rachis), using leaflets on both sides of the leaf. For memory, the palm crown is composed of 8 spirals which grow indefinitely, with leaves emerging from the apical meristem with a specific phyllotaxic angle (FIG. II.2C). Leaves were numbered following tree age, leaf #1 being the youngest (FIG. II.2B).

Figure II. 2 .

 2 Figure II.2. Leaf sampling design. (a) Photograph of an oil palm tree. (b) Photograph of an oil palm tree crown to show leaf rank number. (c) Diagram of an oil palm crown with leaf rank number as a function of chronological emergence, after Lamade et al. (2009). (d) Picture of an oil palm leaf showing the sampling location (red rectangles) at point B of the leaf. (e) Timeline showing the life cycle of leaves sampled in our study (July 2017 and October 2018).

  local weather was characterized by a rainy season in September-December for both years with low solar radiation and temperature and high relative humidity (FIG. II.3). Solar radiation varied between 428 MJ/m² and 603 MJ/m², with a maximum in June 2017 and a minimum in December 2017. Temperature varied from 21.6 to 35.6 °C. Rainfall peaked at 682 mm in October 2018.

Figure II. 3 .

 3 Figure II.3. Climatic conditions in the field. Monthly solar radiation and rainfall precipitation, monthly average temperature and relative humidity in 2017 and 2018 during the agronomical trial BBCP07 at Bangun Bandar (North Sumatra, Indonesia). Red narrow indicates sampling dates.

(

  trimethylsilyl) trifluoroacetamide (MSTFA) in pyridine and metabolomics analyses were carried out by gas chromatography coupled to mass spectrometry (GC-MS) as in[START_REF] Mirande-Ney | Effects of potassium fertilization on oil palm fruit metabolism and mesocarp lipid accumulation[END_REF]. Peak integration and identification was done using Metabolome Express as inCui et al. (2019b) and a verification was manually done for each peak using NIST database. Peak areas were normalized to ribitol (internal standard) and sample dry weight, and the sum of metabolites by sample. Similar results were found with both normalization methods. Here we choose to show results normalized with the sum.

  bunches, reflecting the higher production in DL comparing to DY (about 2 leaves are cut by bunch harvested). K availability did not change significantly the apparent leaf number, bunch number, leaf area nor foliar emission rate (FIG. II.4). By contrast, an increase in specific leaf weight was observed in both K2 and K3 conditions, in both crosses (FIG. II.4C), consistent with higher N content at higher K availability (FIG. II.1).

Figure II. 4 .

 4 Figure II.4. Oil palm phenology. (a) Total leaf and inflorescence composition of oil palm crown, (b) foliar emission rate, and (c) specific leaf weight and total leaf area of oil palm trees in 2017, under different potassium treatments (K0, K1, K2 and K3) in Deli x La Mé (DL) and Deli x Yangambi (DY) crosses. Total leaf area was determined on leaf no. 17. Mean ± SE (n = 5). Letters stand for statistical classes (two-way ANOVA, P<0.05).

Figure II. 5 .

 5 Figure II.5. Differential effect of potassium nutrition on total leaf protein content in Deli x La Mé (pink) and Deli x Yangambi (yellow) crosses sampled in 2017 (left) and 2018 (right). Protein concentration is given in milligrams per surface area. Mean ± SE (n = 5). Letters stand for statistical classes (two-way ANOVA, P<0.05).

Figure II. 7 .

 7 Figure II.7. Proteomics pattern of oil palm leaves under two K fertilization treatments, for Deli x La Mé (pink) and Deli x Yangambi (yellow), in 2018. (a,c) Heatmaps of proteins that were significant for the K effect (a) and the interaction effect K  cross (c). Scale from blue (lowest) to red (highest) to show the relative content (mean-centered). (b) Volcano plots (-log(P) from two-way ANOVA versus O2PLS loading, pcorr) associated with the effect of K availability. Abbreviations: RPS5, Ribosomal protein S5/Elongation factor G/III/V family protein (CLO); Oct./Phox/Bem1p, Octicosapeptide/Phox/Bem1p family protein; UDPSP, UDP-sugar pyrophosphorylase; 3IPMD, 3isopropylmalate dehydratase protein ; 3DHQS, 3-dehydroquinate synthase ; HHL1, Protein HHL1, chloroplastic isoform X1; FLKHD, Flowering locus K homology domain; KIN-14B, kinesin-like protein KIN-14B; APX4, Probable L-ascorbate peroxidase 4; PNDH B3, Photosynthetic NDH subunit of subcomplex B 3, chloroplastic; PSRP1, Ribosome-binding factor PSRP1, chloroplastic; SECA1, Protein translocase subunit SECA1, chloroplastic; DUF1118, Wiskott-aldrich syndrome family protein, putative (DUF1118); NAC, Nascent polypeptide-associated complex (NAC) subunit alpha-like protein 1; ARCP2, Ankyrin repeat-containing protein 2; MDTC, Mitochondrial dicarboxylate/tricarboxylate transporter DTC; PSAIX2, Puromycin-sensitive aminopeptidase isoform X2; GLP5-1, Germin-like protein 5-1; PSA, 40s ribosomal protein SA.

3 .

 3 Leaf metabolomeOverall, 208 and 181 analytes were quantified and identified in 2017 and 2018, respectively.Multivariate analysis conducted separately on the two sampling years showed that the cross and K availability could well be discriminated along axis 1 and 2 of the OPLS score plot, respectively (FIG. II.8A-B).

Figure

  Figure II.8. Output of the multivariate analysis of metabolome: score plots of the O2PLS analyses using K and cross as predicted Y variables in 2017 (a) and 2018 (b) analysis. Samples are discriminated along the y axis (K) and x axis (cross).

  2017 (FIG. II.9B-C), and 21 were significant for the K effect and 23 for the interaction K  cross in 2018 (FIG. II.

Figure II. 9 .

 9 Figure II.9. Metabolomics pattern of oil palm leaves under two K fertilization treatments, in Deli x La Mé (pink) and Deli x Yangambi (yellow) crosses, in 2017 (top) and 2018 (bottom). (a,d) Volcano plots (-log(P) from two-way ANOVA versus O2PLS loading, pcorr) associated with the effect of K availability, in 2017 (a) and 2018 (d). Heatmaps of metabolites significant for the K effect (P<0.05) (b,e) and the interaction effect K  cross (P<0.05) (c,f). Scale from blue (lowest) to red (highest) to show the relative content (mean-centered). Hierarchical clustering (Pearson correlation) is shown on left. Abbreviation 1, 2 or 3 indicate different isomers of the same compound. Abbreviations: F6P, fructose-6-phosphate; D.Asc 1, dehydroascorbate 1; Ukn D1, unknown disaccharide from the family of turanose; G1P, glycerol-1-phosphate; G3P, glycerate-3-phosphate; M.Asp.dv., methyl aspartate derivative.

Figure

  Figure II.10. Differential effect of potassium nutrition on leaf sugar content in Deli x La Mé (pink) and Deli x Yangambi (yellow) crosses sampled in 2017 (a) and 2018 (b). Sucrose, glucose and fructose leaf content were determined by 1H-NMR and are given in micromoles per square meter of leaf. Mean ± SE (n = 5). Letters stand for statistical classes (two-way ANOVA, P<0.05).

Figure

  Figure II.11. Bidimensional multivariate analysis of metabolome and proteome: (a) Score plots of the O2PLS analysis using proteomic and metabolomic data as predicting (X) and predicted (Y) variables, respectively. Samples are mostly discriminated along the y axis (K) and x axis (cross). (b) Loading plot showing the first two components (loading pq(corr)[1] plotted against pq(corr)[2]). Metabolistes are colored by classes to facilitate reading and red arrows indicate metabolites from the tricarboxylic acid cycle pathway (Krebs cycle).

Figure

  Figure II.13. Differential effect of K fertilization on leaf gas-exchange parameters for Deli x Lamé (pink) and Deli x Yangambi (yellow) under K0 and K3 treatments from a similar fertilization trial in a SOCFINDO plantation at Aek Loba, North Sumatra (Indonesia). (a) Carbon assimilation under saturated light (Amax), carbon assimilation at standard light (A) and dark respiration (Rdark), measured with the WALZ GFS 3000 (Germany), in µmol CO2 per square meter per second. (b) Stomatal conductance and intercellular-to-external CO2 mole fraction ratio (ci/ca). Mean ± SE (n = 5). Letters stand for statistical classes (two-way ANOVA, P<0.05).

  2018. Average monthly temperature was 26°C, ranging from 18 to 42°C. In this geographical region, there is a soft rainy season from Sept to Dec, and a precipitation peak (460 mm) has been observed in Dec 2017. Total annual precipitation is about 2,500 mm. Overall, 40 oil palm trees planted in Aug 2013 were organized in a K-factorial agronomic trial. They belonged to two crosses: Deli x La Mé (DL) and Deli x Yangambi (DY). DL and DY have been chosen here since they are two very common crosses used in oil palm agroforestry. The agronomic trial comprised four levels of potassium fertilization with KCl: no KCl applied for 3 y before and also during the experiment (K0), 1.5 kg KCl tree-1 y-1 (K1), 3 kg KCl tree-1 y-1 (K2), and 4.5 kg

Figure III. 1 .

 1 Figure III.1. Fruit developmental maturation stages. Fruit morphology (top) and longitudinal sections (bottom) of (a) Deli x La Mé (DL) and (b) Deli x Yangambi (DY) oil palm trees at 5 different developmental maturation stages, along with associated time-windows (in week after anthesis, WAA). These time-windows are averages and depend on crosses (maturation takes about 5.8 months in DL and 5.5 months in DY).

Figure III. 2 .

 2 Figure III.2. Leaf nitrogen and potassium elemental contents in the two crosses Deli x La Mé (DL) and Deli x Yangambi (DY) under three K treatments (K0, K2 and K3). The elemental content is given in percentage of dry weight. Mean ± SE (n = 4). Letters stand for statistical classes (P<0.05) using a Tukey test

Figure III. 3 .

 3 Figure III.3. Effect of potassium availability on bunch production: (a) Production at 4 years, (b) Bunch, stalk and spikelet fresh biomass and proportion of fruits in bunch, (c) Proportion of mesocarp and kernel in bunch biomass, (d) Fruit biomass, (e) Proportion of mesocarp and kernel in fruit biomass, (f) Mesocarp water content and (g) Oil content in mesocarp (OFM) or in bunch (OER), under different K fertilization treatments (K0 to K3) and two contrasting oil palm crosses Deli x La Mé (DL) and Deli x Yangambi (DY). Production at 4 years corresponds to the number of bunches harvested along the 4th year. Fresh biomass is given in kilograms (b) or grams (d). Proportions are given in percentage and were defined as the ratio of fruits, mesocarp or kernel biomass to that of the bunch or fruit. Abbreviations: OFM, oil content in fresh mesocarp; OER, oil extraction rate (proportion of oil in mesocarp). Mean ± SE (n = 5). Letters stand for statistical classes (Tukey test, P<0.05).

Figure III. 4 .

 4 Figure III.4. Overview of mesocarp metabolism: (a) Average mesocarp composition (across K conditions and crosses) during fruit development (relative content in % of total GC-MS signal). (b) Average content (in arbitrary unit (AU) relative to internal standard, normalized to dry weight (DW)) in carbohydrates, organic acids, amino acids, fatty acids and others metabolites across developmental stages in Deli x La Mé (DL) and Deli x Yangambi (DY). (c) Average K effect (K0 vs. K3) on metabolite classes across developmental stages and crosses. Mean ± SE (n = 10). Letters stand for statistical classes (Tukey test, P<0.05). When non-significant, the P-value is written between parentheses.

Figure III. 5 .

 5 Figure III.5. Effect of K availability on oil palm mesocarp lipid biosynthesis. Blue arrow indicates metabolites which decrease with K fertilization and red arrow indicates those which increase. Abbreviation: ADP-glucose: adenosine diphosphate glucose, Susy: Sucrose synthase

3 .

 3 Potassium availability impacts on kinetics of lipid synthesis K availability had an effect on fruit metabolism during maturation, with lower carbohydrate and organic acid content at high K (FIG. III.4C, pathways summarized in FIG. III.5). At high K, glucose, fructose and sucrose were lower (by 40-70%) compared with K0 in particular at stage 1 (FIG. III.6). Multivariate analysis conducted separately on the two crosses showed that both the developmental stage and K availability could be well discriminated along axis 1 and 2, respectively (FIG. III.7A-B). The OPLS model was significant in both DL (PCV-ANOVA = 3•10-4 [K effect] and 7•10-11 [stage]) and DY (PCV-ANOVA = 4.9•10-2 [K effect] and 6•10-13 [stage]), explained most of total variance (R2 = 0.892 and 0.739, respectively) and was predictive (Q2 = 0.69 and 0.54, respectively). The importance of metabolites in sample discrimination associated with K0 and K3 conditions was assessed using the combination of univariate and multivariate analysis whereby -log(P) obtained in the two-way ANOVA (univariate) was plotted against the loading value (pcorr) along axis 2 (volcano plot; FIG. III.7C-D).In DL, high K availability led to an increase in methylpiperidine and glucose derivatives 1,6-anhydroglucose (levoglucosan) and 6-deoxyglucose (quinovose) and in DY, there was an increase in amino acids (leucine, phenylalanine, lysine) as well as glucose derivatives (xylulose, lyxose, ribose).

Figure III. 7 .

 7 Figure III.7. Metabolomics response of mesocarp to K fertilization during fruit maturation in Deli x La Mé and Deli x Yangambi crosses. (a-b) Score plots of the O2PLS analysis using K and developmental stage as predicted Y variables in DL (a) and DY (b). Samples are discriminated along the y axis (K) and x axis (stage). (c-d) Volcano plots (-log(P) from two-way ANOVA versus O2PLS loading pcorr) showing best discriminating metabolites associated with K availability, in DL (c) and DY(d). (e-f) Heatmap of metabolites significant for the K effect (P<0.01; the threshold of 0.01 is shown with the green dotted line) in DL (e) and DY (f). Scale from blue (lowest) to red (highest) to show the relative content (mean-centered). To facilitate reading, a magnified version of this figure is available as Supplementary FigureS3.

  of fruit development since lysine and phenylalanine contents were very low at late stages (4-5) (FIG. III.7E-F). In DY, high K was associated with more intermediates of lipid synthesis (glycerate, monostearin and azelaate) and in DL, less short-chain fatty acids (caprate, caprylate), showing that K stimulated fatty acid chain elongation and triglyceride synthesis. These suggested changes in metabolic pathways as affected by K availability were further supported by pathway enrichment analysis (FIG. III.8).

Figure III. 8 .

 8 Figure III.8. Pathway enrichment analysis (PEA) associated with the effect of potassium fertilization in both crosses (DL, DY). PEA was performed with MetaboAnalyst and shows metabolic pathways affected by the K treatment in DL and DY (i.e., where at least one metabolite is significantly impacted by K fertilization with P<0.01, Fig.3). The asterisk (*) indicates when pathways are not significantly affected as per PEA analysis (P>0.05).

  even though they had very different lipid compositions (nearly 10% difference in monounsaturated FAs, FIG. III.9B). Furthermore, in the present study the difference in leaf tissue K content was modest (0.3-0.7% of dry weight (DW)) suggesting that very large changes in oil composition are to be anticipated under K deficiency or very high K fertilization.

Figure III. 9 .

 9 Figure III.9. Differential effect of potassium nutrition on mesocarp oil quality: (a) Average mesocarp free fatty acid (FA) composition of both crosses (Deli x La Mé, DL, and Deli x Yangambi, DY) under K0 and K3 conditions, with FA contents given in percentage of total FAs as measured by GC-MS. (b) Mesocarp content in linoleate, mono-unsaturated and saturated fatty acids, and iodine index, measured using 1H-NMR, in both crosses under K0 and K3 conditions. FA contents are given in percentage of total FAs and iodine index in (g iodine/100 g FA). Mean ± SE (n = 3). Letters stand for statistical classes (Tukey test, P<0.05).

(

  positive loading value) with K in DY whereas they anti-correlated (negative loading value) with K in DL (FIG. III.7C-D). Accordingly, linoleate decreased significantly at high K at stage 5 and so did palmitoleate in DL (FIGS. S2 AND III.5). Also, in terms of kinetics of lipid production, high K decreased significantly the pool of short-chain fatty acids (caprate, (a) (b) caprylate), showing the stimulation of fatty acid chain elongation in DL, while both a shortchain fatty acid (azelaate) and esterified stearate (monostearin) increased in DY, suggesting a general increase in lipid synthesis (FIGS. III.7 AND III.8). In addition, high K increased the average content in carotenoids in mesocarp oil, and this effect was easily visible from the color of chloroform oil extracts (FIG. III.11). Estimating carotenoids quantity with NMR showed an increase of the average content by 25% in DL and almost 100% in DY (FIG. III.11) at high K but this was not significant due to substantial variability between individuals. Such a variability was also due to the fact that carotenoids formed small peaks in the NMR spectrum (FIG. III.10)and this was detrimental to quantitation precision.

Figure III. 10 .

 10 Figure III.10. Overview of NMR spectra in DL and DY chloroform-extracted oil, under different K fertilization treatment (K0 and K3). Spectra were obtained by 1H-NMR analysis (see Materials and Methods) and are presented as the relative signal at the same scale, with the chemical shift (relative to TMS) on the x-axis.

  %, FIG. III.3). However, OPLS models explained nearly all of the variance (R2=0.999 and 0.922 for OER and OFM, respectively) and were robust for OER (Q2= 0.511) (FIG. III.12).

Figure III. 12 .

 12 Figure III.12. Robustness of the relationship between oil content and mesocarp metabolome in the OPLS statistical models with OER or OFM as a quantitative predicted Y variable. This figure shows the linear relationship between observed (y-axis) and predicted (x-axis) values of (a) OER and (b) OFM

Figure IV. 1 .

 1 Figure IV.1. Scheme of the 13CO2 labelling. The closed system used is alimented by pure 13CO2(99.3%) with a CO2 and mass flow regulator allowing a constant flow that compensated for photosynthetic consumption. The flow is also maintained using a pump, silica gel was used to decrease humidity generated by high leaf transpiration rates after the closing of chamber, CO2 level was controlled with a gas analyzer (WALZ GFS 3000, Germany), a cold water bath was used to help to cool air tube, air inside the chamber was homogenized with fans and, temperature, humidity and solar radiation (PAR) were monitored with sensors. Small arrows indicates the sens of the flow.

Figure IV. 2 .

 2 Figure IV.2. Photographs of (a) the whole 13CO2 labeling experiment installation in the field and (b) external view of an oil palm leaf enclosed in the labeling chamber.

Figure IV. 3 .

 3 Figure IV.3. Temporal design of the experiment.The labelling duration was about 2-5h depending on the CO2 consumption of each tree. The chamber was opened 1h after the stop of the flux. Leaflet were sampled at: T0, before the labelling, T1, just after opening after labelling, and then at T2-T8, 1h, 2h, 3h, 5 h and 1, 2 and 7 days later. Dark respiration analysis started 8 to 10h after the labelling depending on the tree and was performed during 2h. CO2 sampling inside the chamber took place at: S0, before the installation of the chamber (control with no respired CO2) and S1-S5, 5 min, 10 min, 30 min, 1h and 2h later using a 25 mL syringe.

  pools of C turnover within leaflet as in[START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF];[START_REF] Subke | Fast assimilate turnover revealed by in situ 13CO2 pulse-labelling in Subarctic tundra[END_REF]:[START_REF]A Brief History of the Oil Palm[END_REF] a labile C pool (photosynthates), and (2) leaf biomass C. All assimilated C initially forms part of the labile, non-structural C pool (Pool 1), which includes all water-soluble forms of carbohydrates (i.e. also sugars stored in cell vacuoles). As this labile pool turns over, a small fraction of it becomes incorporated into leaf biomass (Pool 2), whilst the rest is either exported to other plant parts via the phloem, or respired by the leaf.In order to calculate the export rate of labile C, we fitted the following exponential decay function in Eq. (4) to the excess 13C and xE (13C) leaflet:xE(13C)=Cs+CL*exp(-t) (4)where t is the time since pulse-labelling (in hours), and CS, CL and  are fitted parameters. In doing so, we assume that the overall pool sizes do not change over the observation period, i.e.that the reduction in 13C abundance in Pool 1 is caused by respiratory loss, phloem export or incorporation into Pool 2 and not by dilution due to an increase in unlabelled C subsequent to the 13C pulse. We recognize that a double exponential decay would be more appropriate to model 13C dynamics, in particular because it is unlikely that only one labile pool is representative of metabolic partitioning. However, we chose to use a simple exponential decay because it fitted satisfactorily our data and our time resolution would be insufficient to fit a 123 double exponential function reliably. We estimated the mean residence time (MRT) of labile C with Eq. (5) as the reciprocal of the decay constant (the exponential decay function (CS) indicates the amount of labelled C not affected by the exponential decay, i.e. the amount of C that would remain in the leaf once the labile pool of labelled C (CL) is completely turned over. Dark respiration. The night following the labelling experiment, dark respiration was quantified by analyzing CO2 respired by the same palm portion labelled in the morning using the same chamber. Experiment started at about 8:00 PM and lasted for 2h. Immediately after closing the chamber, the CO2 mole fraction inside the chamber was continuously monitored with the gas analyzer. The CO2 mole fraction increased linearly after the chamber was closed because of CO2 release by respiration and the slope was computed. The CO2 respired was sampled just before the installation of the chamber (control with no respired CO2) and directly after the closing, 5 min, 10 min, 30 min, 1h and 2h later (FIG. IV.3) using a 25 mL syringe, collected inside 5 mL air tight glass vials and sealed with parafilm. Glass vials were kept at -20°C until

  FFB was higher in DL compared to DY (see Mirande-Ney et al. (2019)).

Figure IV. 4 .

 4 Figure IV.4. Overview of functional traits of oil palm in DL and DY under K0 and K3 treatments. (a) Foliar emission (FE), specific leaf weight (SLW), leaf area (LA), (b) trunk height and trunk diameter variations with K0 and K3 and in DL and DY. FE stands for the number of leaf emitted per month and SLW, LA and trunk height and diameter are given in kg m-2, m2 and cm. Mean ± SE (n = 5). Letters stand for statistical classes (Tukey test, P<0.05). When non-significant, the P-value is written.

  [START_REF] Lamade | Occurrence of potassium location in oil palm tissues with reserve sugars: consequences for oil palm K status determination[END_REF] have shown in fact a cooccurrence of K+ and hexoses (glucose) in heterotrophic tissues (trunk, rachis, rachis bunch), suggesting an effect of K on sugar redistribution. While insignificant, it seems also that potassium addition tends to increase dark respiration rate in DY (FIG. IV.5).

Figure IV. 5 .

 5 Figure IV.5. Photosynthesis and respiration rate variation upon K fertilization for both crosses DL and DY. Photosynthesis and respiration rate are given in micromoles per square meter per second. Respiration rate was calculated only in DY. Mean ± SE (n = 3).

Figure IV. 6 .

 6 Figure IV.6. Variations of leaflet starch content upon K fertilization in (a) DL and (b) DY over different time periods after the 13CO2 labelling. Leaflet starch content are given in milligrams per gram of dry weight. They were measured on labelled leaflet until 7 days after labelling. T0 correspond to the time before the labelling and T1-T8 correspond to 1h, 2h, 3h, 5 h and 1, 2 and 7 days after the labelling. Mean ± SE (n = 3).

Figure IV. 7 .

 7 Figure IV.7. Correlation between 13C values of respired CO2 and leaflet organic matter after labelling. Just after (T1) or 1h after (T2) a 13CO2 pulse-labelling, leaflets OM of labelled palms were analyzed for C isotope composition using an elemental analyzer coupled to an isotope ratio mass spectrometer. In addition, the night following the labelling experiment CO2 respired by labelled leaflets were sampled and 13C of respired CO2 was determined for each sample using a GC-C-IRMS at S3, 30 min after closing the chamber.  13C values are given in part per million. Each point corresponds to  13C values of leaflet OM and CO2 respired of K0 and K3 from DL trees (n=3). Dotted lines corresponds to linear regressions.

Figure IV. 8 .

 8 Figure IV.8. Time courses of xE(13C), the 13C in excess, after pulse labelling in leaflets in DL and DY under K0 and K3 treatments for each replicate. xE(13C) was calculated according to Eq. (3). Red straight lines represent fitted simple exponential decay (Eq. (4)).

  of the data according to different conditions (FIG. IV.9). We can see in Fig. IV.9 that the data follow a bimodal distribution before and after the labelling, suggesting the occurrence of too carbon pools within a condition. Taken as a whole (FIG. IV.9 A), we can see that the first peak representing the biggest pool has a median carbon isotope signature of -30 ‰ which is slightly impacted by 13C labelling (FIG. IV.9 E) and the smallest has a median carbon isotope signature of -29.4‰, which is more impacted by 13C labelling (400 ‰ just after labelling). When the probability density of the different conditions taken separately (FIG. IV.9 H), we could observe a slight positive shift in the median carbon isotope composition between K0 and K3, suggesting an enrichment of the pool with 13C labelled upon K fertilization, as observed in Epron et al. (2016).

Figure IV. 9 .

 9 Figure IV.9. Probability density of 13C values before and after the labelling according to K and cross conditions. (a,c) global probability density with all the data and (b,d) probability density grouped by crosses and treatments.

13C

  value in leaflets, regardless of leaf rank (FIG. IV.10), suggesting that K either modified leaflet carbohydrate composition with for example an increase in sucrose vs. starch (see Chapter II and FIG. IV.6), or was associated with an increase in ci/ca.

Figure IV. 10 .

 10 Figure IV.10. Variation of the carbon isotope composition (13C) of organic matter (OM) across leaf ranks. Mean ± SE (n = 3).

Figure V. 1 .

 1 Figure V.1. Tentative summary of the effect of K on leaf metabolism. Blue arrows () indicate metabolites/enzymes that decrease with K fertilization while red arrows () indicate an increase. Black dots (•) indicates metabolites/enzymes that differ significantly between crosses.Enzymes are written in red italics. Abbreviation: 3DHQS, 3-dehydroquinate synthase; 3IPMD, 3-isopropylmalate dehydratase protein; 3PGA, 3-phosphoglycerate; AA, amino acid; AAA, aromatic amino acid; ADP, adenosine diphosphate; Asp, aspartate; ATP, adenosine triphosphate; Aldolase TIM, aldolase-type tim barrel family protein; CcOS5b-2, cytochrome c oxidase subunit 5b-2; CoA, coenzyme A; CTE, chain transport electron; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBA, fructose-bisphosphate aldolase 1; FBP, fructose biphosphate; G6P, glucose-6-phosphate; GCSH3, glycine cleavage system H protein 3; Gln, glutamine; Glu, glutamate; glycerol-1P, glycerol-1-phosphate; Gly, glycine; GOGAT, ferredoxin-dependent glutamate synthase; HHL1, Protein HHL1, chloroplastic isoform X1; MDTC, mitochondrial dicarboxylate/tricarboxylate transporter DTC; Met, methionine; N, nitrogen; NADH dhg, NADH dehydrogenase; NADH/NAD, nicotinamide adenine dinucleotide; NADPH/NADP, nicotinamide adenine dinucleotide phosphate; NAD(P)bRF, NAD(P)-binding Rossmann-fold superfamily protein; NDUFB11; Putative NDUFB11 Superfamily domain; Leu, leucine; Lys, lysine; PEP, phosphoenolpyruvate; PNDH B3, Photosynthetic NDH subunit of subcomplex B 3; Pyr, pyruvate; RubP, ribulose 1,5-bisphosphate; SECA1, Protein translocase subunit SECA1; Ser, serine; TCA; tricarboxylic acid cyle; TP, triose-phosphate; Thr, threonine; UDPSP, UDP-sugar pyrophosphorylase; Val, valine.

  in DL, less short-chain fatty acids (caprate, caprylate), showing that K stimulated fatty acid chain elongation and triglyceride synthesis. (See FIG. IV.2), balancing therefore toward the hypothesis (2).

Figure V. 2 .

 2 Figure V.2. Effect of K availability on oil palm mesocarp lipid biosynthesis. Blue arrow indicates metabolites which decrease with K fertilization and red arrow indicates those which increase. Abbreviation: ADP-glucose: adenosine diphosphate glucose, Susy: Sucrose synthase

Figure S2 .

 S2 Figure S2. Metabolomics response of mesocarp during fruit maturation in Deli x La Mé and Deli x Yangambi crosses. (a-b) Heatmap of metabolites significant for the maturation stage (P<0.01) in DL (a) and DY (b). Scale from blue (lowest) to red (highest) to show the relative content (mean-centered). Red frames (labelled A) show metabolites that increased during maturation and blue frames (labelled B) show metabolites that decreased with maturation. Abbreviations: DHB, 3,4dihydroxybenzoate; HB, 4-hydroxybenzoate; DHS, 3-dehydroshikimate; HC, 4-hydroxycinnamate; GABA, γ-aminobutyrate; HMB, 4-hydroxy-3-methoxybenzoate (vanillate); NADM, N-acetyl-D-mannosamine. On bottom right, two metabolites (caprylate, putrescine) that are did not vary significantly during maturation in DY but did vary significantly in DL are shown to facilitate comparison.

Table I .

 I 1. Physiological parameters variations with leaf age ................................................... Table I.2. Oil palm fruit bunch composition under ideal climatic conditions and good management.

Table I .1. Physiological parameters variations with leaf age

 I

	Leaf n°	PN	gs	E	LA LDM LMA
	1	2.94 e 0.01 a 0.72 c 2.15 b 1.80 c 0.85 ab
	9	9.18 a 0.03 a 2.26 a 3.38 a 2.61 b 0.78 ab
	17	6.16 b 0.03 a 1.58 b 3.28 a 3.08 a 0.94 a
	25	5.23 c 0.02 a 1.32 b 3.26 a 2.59 b 0.81 ab
	33	4.38 d 0.02 a 1.27 b 2.96 a 2.37 b 0.75 b
	LSD (p=0.05) 0.64 0.05 0.36 0.43 0.46	0.19
	Assimilates produced during photosynthesis are redistributed to developing leaves,

. Variation in net photosynthetic rate (PN in µmol m-2 s-1), stomatal conductance for CO2 (gs in mol m-2 s-1), transpiration rate (E in mmol m-2 s-1), leaf area (LA in m²), leaf dry mass (LDM in kg) and and leaf mass per area (LMA kg m-²). Values with the same letters do not differ significantly from each other. Adapted from

[START_REF] Corley | The oil palm[END_REF]

inflorescences (and bunches), trunk and roots, depending on sink strength. Assimilate partitioning among growing organs is generally a function of active sinks (and organ growth rates) in plants

Table I .2. Oil palm fruit bunch composition under ideal climatic conditions and good management.

 I Data are given in kilograms and percentage of fresh weight. Source: www.fao.org.

			Bunch	Standard
			component	range
			Bunch weight	23-27 kg
			Fruit/bunch	60-65 %
			Oil/bunch	21-23 %
			Kernel/bunch	5-7 %
			Mesocarp/bunch 44-46 %
			Mesocarp/fruit	71-76 %
			Kernel/fruit	21-22 %
			Shell/fruit	10-11 %
	B.	Potassium nutrition in plants: overview
	Potassium (K) is one of the most important macronutrients, along with nitrogen (N) and
	phosphorous (P), required for plant growth and development. It is also the most abundant (in
	terms of elemental composition) macronutrient in many species (Leigh & Wyn Jones, 1984),
	such as oil palm. Potassium plays a significant role in several plant physiological processes
	related to cation-anion balance, osmoregulation, water movement, phloem transport and energy
	transfer, and takes part in protein synthesis, carbohydrate metabolism, enzyme activation and
	stress resistance.
		1.	Potassium sources and fertilization
	Potassium geological reserves are generally large and represent about 2.1 to 2.3 % of Earth's
	crust

Table I .3. Annual K demand of oil palm of various ages

 I

	Palm age	Plant part	K	Reference
	0-3	Whole palm excluding roots	55	Tan (1977) citing Tan (1976)
	3-9	inflorescences Palm including prune fronds and male	387 Tan (1977)
	3-9	inflorescences Palm excluding prune fronds & male	287 Tan (1977)
	9-12	Trunk + root + FFB	167 Tarmizi and Mohd Tayeb (2006)
	15-19	FFB + Trunk only	199 Prabowo et al. (2006)
	15-19	FFB + Trunk + Frond + male inflorescences	368 Prabowo et al. (2006)

(kg ha-1)

. (Adapted from

[START_REF] Tiemann | Feeding the palm[END_REF]

).

Table I .4. Effect of K application rate on Fresh Fruit Bunch (FFB) production at Okomu Oil Palm plc, from 1999 to 2008

 I

	Rate of K application		Mean bunch Number / palm / year
	kg/palm/year 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
	0.0	8.9c	7.5b 8.01c 9.2c	10.5c 11.0c 11.3d 8.7d	9.5	10.5d
	1.0	10.4a 11.9a 12.0b 13.0b 12.0b 14.0b 14.0c 10.5c 12.0 12.5c
	1.5	9.5b 11.7a 13.0a 13.5b 13.1a 15.5a 15.0b 12.3b 14.5 15.0b
	2.0	11.6a 12.0a 14.0a 15.0a 13.6a 16.5a 17.5a 14.5a 16.0 17.5a
	2.5	10.5a 11.7a 13.5a 14.0a 13.4a 16.1a 16.5a 13.1b 15.6 17.0a

. Oil palm plantation was established in 1993 with NIFOR EWS Seedlings G99 at Okomu. The trial commenced in 1998 when the palms were about 5 years old. K was applied as muriate of potash in addition to 0.5 kg urea, 0.5 kg single super phosphate (SSP) and 0.5 kg dolomite / palm tree yr-1. Means with the same alphabet in the same column are not significantly different from each other at 5% level of probability by the New Duncan's Multiple Range Test (DMRT). Adapted from

[START_REF] Imogie | Effect of potassium (K) source on oil palm yield at Okomu oil palm Plc, Ovia North East LGA of Edo State[END_REF]

.

Table II .1. Fertilization design.

 II planted in Aug 2013 and organized inside a K factorial agronomic trial. They belonged to two crosses: Deli x La Mé (DL) and Deli x Yangambi (DY). DL and DY have been chosen here since they are two very common crosses used in oil palm agroforestry. The agronomic trial comprised four levels of potassium fertilization with KCl applied proportionally Trees were planted in August 2013. From 2013 to 2015, trees were "pre-conditioned", that is, received urea and KCl in accordance with growth demand from 2013 to 2015.

	with growth until 2015 (TABLE II.1). At the beginning of the production stage (starting in
	2016) fertilization levels were: no KCl added (K0), 1.5 kg KCl tree-1 y-1 (K1), 3 kg KCl tree-1
	y-1 (K2), and 4.5 kg KCl tree-1 y-1 (K3). Therefore, at the first sampling date, in 2017, trees had
	just experienced 1 year of K treatment plus 3 years of preconditioning, and in 2018, 2+3 years
	(TABLE II.1).
				Date	Urea (g/tree)		KCl (g/tree)
					N1	K0	K1	K2	K3
	Pre-	conditioning	2013, October 2014, February 2015 1st application: April 2nd application: September	450 750 1 000	25 300 1 000 1 000	50 600 1 000 1 000	75 900 1 000 1 000	100 1 200 1 000 1 000
			2016
			1st application: May	1 000	0	750	1 500	2 000
	treatment K	2nd application: September 2017 1st application: March 2nd application: September 2018	1 000 1 000 1 000	0 0 0	750 750 750	1 500 1 500 1 500	2 000 2 250 2 250
			1st application: April	1 000	0	750	1 500	2 250
			2nd application: September	1 000	0	750	1 500	2 250
			1.	Field location, fertilization and leaf traits

The field was located at the SOCFINDO station (North Sumatra, Indonesia; 3°18'19.60"N, 99°3'24.33"E) and sampling was carried out in July 2017 and November 2018. Overall, 40 oil palm trees were

  By contrast, in 2018, there was a significant effect on both leaflet K and N (but no significant effect on leaflet nitrate content), and leaflet K content was higher in DY comparing to DL (FIG. II.3E-F). As expected, the increase in leaflet K under K3 conditions was associated with a concurrent increase in average Cl content, but this appeared to be insignificant. It is worth noting that K availability did not affect significantly the Ca or Mg content in leaflets and caused an increase in Ca in rachis in 2017 (FIG.

in DL only (FIG. II.1). II.3C). The balance between cations was examined here by calculating the "potassium charge excess" (PCE), which is the difference between K and the double of Ca + Mg. Interestingly, there was a significant effect of K availability on leaflet PCE in 2018 in both crosses, showing that the increase in K+ was accompanied by a relative decline in divalent cations (FIG. II.3D,H). This was probably compensated for by an increase in Na+ (not analyzed here). In rachis, as opposed to leaflets, PCE was positive due to the very large K content (about 800 mmol kg-1 DW, i.e. 3.2%). Leaflet P content also increased very slightly but significantly with K availability (FIG. II.3B,F).

Table III .1. Correlation analysis between oil content and mesocarp metabolome. List of

 III

  The local weather was characterized by a rainy season in September-December for both years (2017-2018) with low solar radiation and temperature and high relative humidity. Solar radiation varied between 428 MJ/m² and 603 MJ/m², with a maximum in June 2017 and a minimum in December 2017. Temperature varied from 21.6 to 35.6 °C. Rainfall peaked at 460.3 mm in December 2017, with almost no rain

Table IV .1. Correlation analysis between leaflet K content and oil palm functional traits.

 IV

	Leaflet K content	R	Pvalue
	Palm 17 length	-0.70	0.05
	Bunch biomass (kg DW)	-0.68	0.07
	Leaflet N content	-0.66	0.08
	%C T0	-0.65	0.08
	Average diameter	-0.65	0.08
	Labelling time	-0.57	0.14
	Trunk biomass	-0.54	0.93
	Trunk volume	-0.54	0.17
	Leaf area	-0.51	0.20
	Bunch number (2017)	-0.51	0.20
	Photosynthesis	0.52	0.19
	Leaflet Cl content	0.54	0.73
	Oil-to-mesocarp ratio	0.62	0.10
	Cross	0.82	0.01 **

TABLE

  IV.2). These relationships may indicate a link between the natural abundance and photosynthetic capacity since at high carboxylation velocity for a fixed stomatal conductance, ci/ca is low. Although insignificant, leaflet δ13C was also correlated to fruit starch content at stage 3 as reported in Mirande-Ney et al. (2019), crosses, leaflet chlorine (Cl) content and replicates, and anti-correlated to leaf area, bunch biomass, bunch weight and bunch number. This tendency might be driven by K considering its known effects on bunch production, leaf area or carbohydrate metabolism

Table IV .2. Correlation analysis between leaflet carbon isotopic signatures at natural abundance (13C (T0)) and just after the labelling (13C (T1)), and oil palm functional traits.

 IV Significant correlation are written in red. 13CO2 injected into the chamber was fixed into leaflet organic matter (OM), showing that the labelling experiment was successful. However, when we compared the amount of 13C found in the leaflet just after labelling to the amount of 13C supposedly assimilated during the labelling time based on photosynthesis rate, it represented sometimes less than 10% (TABLEIV.3). The maximum of 13C assimilated by leaflet was about 2623 µg 13C/g C, representing only 27% of efficacy. Even though the export of photosynthates can explain a smaller-thanexpected 13C content, we expected to find at least 50%, which is in general the carbon partitioning to starch. Therefore, we can suppose that either there was a leak in the chamber leading to a loss of 13CO2, or photosynthesis decreased/changed during the experiment maybe due to an insufficient temperature control. There could also be a problem at the end of the labelling when there was no 13CO2 left (1 L 13CO2 was consumed) but the leaf continued assimilation for some time. In fact, at this stage, leaflet experienced about 1 h without any supply of 13CO2, and therefore CO2 mole fraction should have decreased down to the compensation point.

	3.	Dynamics of 13C in leaflets after labelling
	Clearly,
	 13 C(T0)	R	P value	 13 C(T1)	R	P value
	Palm 17 length	-0.71	0.05	Labelling time	-0.65	0.02
	Leaf area	-0.67	0.07	SLW (g/m²)	-0.60	0.12
	Bunch biomass (kg DW)	-0.67	0.07	Leaflet starch content (day)	-0.55	0.16
	Bunch weight	-0.64	0.09	%C T0	-0.51	0.20
	Average T(°C) Bunch number (2017) Rep	-0.60 -0.56 0.54	0.04 0.15 0.17	Leaflet starch content (T1) Fruit-to-bunch ratio	0.52 0.53	0.08 0.17
	Leaflet starch content (T1) RH avg Leaflet Cl content Cross Fruits starch content (Stage 3)	0.60 0.62 0.65 0.67 0.69	0.04 0.03 0.13 0.07 0.06	RH max Leaflet Cl content Photosynthesis (µmol/m²/s) Leaflet starch content (night)	0.55 0.58 0.69 0.75	0.07 0.03 0.01 0.03

Table IV .3. Summary of 13C expected and effectively found in leaflet, according to the different conditions of K and crosses. Cross K Treatment Rep Expected leaflet 13C content assimilated (µg 13C/g C) Leaflet 13C content assimilated (µg 13C/g C) Proportion of 13C effectively incorporated

 IV

		1	9558	1882	19.7%
	K0	2	10813	2584	23.9%
	DL	3 1	9153 9096	213 985	2.3% 10.8%
	K3	2	10111	1480	14.6%
		3	9722	2623	27.0%
		1	9939	1779	17.9%
	K0	2	9294	114	1.2%
	DY	3 1	9649 9098	310 103	3.2% 1.1%
	K3	2	9639	617	6.4%
		3	9327	2189	23.5%

  The decrease in leaflet 13C excess with time could be explained by sugar transport to sink organs or losses by dark respiration. As for natural abundance, no significant effect of K or cross was found on excess 13C after labeling. However, in a 13CO2 labelling study on Eucalypt trees under K nutrition,

	after having closed the chamber the night following the labelling experiment. It represents about
	0.53 of 13C excess, that is about 64% of the mean of 13C labile pool (CL) (See TABLE IV.4)
	(maximum values of 13C of CO2 respired were observed 30 min after having closed the
	chamber).
	Unsurprisingly, 13C excess in leaflet total organic matter was maximal just after
	labelling and decreased thereafter (FIG. IV.8).
	the		700	y = 0.3566x + 18.837	y = 0.2293x + 19.15
	closing		600	R² = 0.9781		R² = 0.9665
	2 respired 30min after	chamber (‰)	200 300 400 500			T1 T2
	CO		100
	 13 C		0
		-100		100	300	500	700
		-100
				 13 C leaflet OM just after labeling (T1) or 1h after
				labeling (T2) (‰)

In fact, as shown in FIG. IV.7, 13C in leaflet OM after labelling was linearly correlated to 13C of respired CO2 (r²=0.98, P=0.001). Respired CO2 had a 13C of about 164 ‰ 30min

Table IV .4. Model parameters describing the decrease in xE(13C), the 13C in excess, with time after labelling in leaflet

 IV (Eq. 4). CL is the labile pool of xE(13C) at time 0 and CS is the asymptotic remaining of xE (13C) in the leaflet. MRT is the mean residence time of labile C in leaflet and as the inverse of the decay constant ().

		CS	CL		r²	MRT (h)
	DL K0 1	0.45	0.83	0.01	0.93	82.64
	DL K0 2	0.61	0.75	0.04	0.98	27.86
	DL K0 3	0.16	0.43	0.23	0.90	4.30
	DL K3 1	0.14	0.93	0.01	0.77	130.65
	DL K3 2	0.29	0.91	0.02	0.93	59.88
	DL K3 3	NA	NA	NA	NA	NA
	DY K0 1	0.27	1.17	0.08	0.91	11.93
	DY K0 2	0.28	0.38	0.02	0.39	45.25
	DY K0 3	NA	NA	NA	NA	NA
	DY K3 1	0.13	0.54	0.03	0.55	35.21
	DY K3 2	0.44	0.58	0.46	0.48	2.16
	DY K3 3	0.52	0.80	0.04	0.99	26.18

  Black dots (•) indicates metabolites/enzymes that differ significantly between crosses. Enzymes are written in red italics. Abbreviation: 3DHQS, 3-dehydroquinate synthase; 3IPMD, 3-isopropylmalate dehydratase protein; 3PGA, 3-phosphoglycerate; AA, amino acid; AAA, aromatic amino acid; ADP, adenosine diphosphate; Asp, aspartate; ATP, adenosine triphosphate; Aldolase TIM, aldolase-type tim barrel family protein; CcOS5b-2, cytochrome c oxidase subunit 5b-2; CoA, coenzyme A; CTE, chain transport electron; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBA, fructose-bisphosphate aldolase 1; FBP, fructose biphosphate; G6P, glucose-6-phosphate; GCSH3, glycine cleavage system H protein 3; Gln, glutamine; Glu, glutamate; glycerol-1P, glycerol-1-phosphate; Gly, glycine; GOGAT, ferredoxin-dependent glutamate synthase; HHL1, Protein HHL1, chloroplastic isoform X1; MDTC, mitochondrial dicarboxylate/tricarboxylate transporter DTC; Met, methionine; N, nitrogen; NADH dhg, NADH dehydrogenase; NADH/NAD, nicotinamide adenine dinucleotide; NADPH/NADP, nicotinamide adenine dinucleotide phosphate; NAD(P)bRF, NAD(P)-binding Rossmann-fold superfamily protein; NDUFB11; Putative NDUFB11 Superfamily domain; Leu, leucine; Lys, lysine; PEP, phosphoenolpyruvate; PNDH B3, Photosynthetic NDH subunit of subcomplex B 3; Pyr, pyruvate; RubP, ribulose 1,5-bisphosphate; SECA1, Protein translocase subunit SECA1; Ser, serine; TCA; tricarboxylic acid cyle; TP, triose-phosphate; Thr, threonine; UDPSP, UDP-sugar pyrophosphorylase; Val, valine.In fact, proteins involved in catabolism (phosphoenolpyruvate carboxylase, phosphopyruvate hydratase, etc.) were up-regulated in DY, whereas disaccharides and some proteins of photosynthesis (sucrose-phosphatase, glycerate dehydrogenase, carbohydrate kinase domain-containing protein, Rubisco activase, transketolase) were up-regulated in DL

	De novo AA synthesis and derivatives -Lys -Asp -Thr -Met -Leu -Val -3-pyridinol AAA and derivatives 3DHQS Lipids -Myristate -Oleate	3IPMD Acetyl-CoA	Cadaverine Pyr PEP	TCA derivatives and others ⁻ Methylaspartate ⁻ Aminoisobutyrate ⁻ Butanediol ⁻ Beta-Alanine ⁻ Methylglutarate ⁻ Pyroglutamate Aconitase Citrate Acetyl-CoA Oxaloacetate Pyruvate dehydrogenase MDTC TP derivatives and Oxaloacetate MDTC Trans-aconitate Malate
	-Dopamine -Benzoate -Hydroxytyrosol O 2	Aldolase TIM	Shikimate pathway CO 2	PEP	3PGA		3PGA	phosphate -Glycerol -1P -Glyceraldehyde -Phosphate	Fumarate Succinate	2-Oxoglutarate Isocitrate Isocitrate dehydrogenase
	Glycolate CTE	RubP ATP NADPH ADP Calvin cycle E4P NADP		DHAP (TP) FBP F6P		DHAP (TP) FBP UDPSP G6P F6P FBA FBPase	-Xylulose -Xylose Hexose and derivatives -Desoxyribose -Galactose -Tagatose -Sorbose	Ser	GCSH3	Gly	NAD CTE -NADH dhg NADH -NAD(P)bRF -CcOS5b-2 -NDUFB11
	-PNDH B3	-NAD(P)bRF						-Galactitol
	-HHL1	-SECA1		Chloroplast Glycerate			Sucrose -Gluconate	MDTC	Mitochondrion
	Glycolate					Photorespiration cycle
		-Spermidine Glutamate derivatives		Gln	GOGAT	Glu
						N assimilation		Cytoplasm
										Export

Acknowledgments Acknowledgements

C. Mirande-Ney is grateful to CIRAD for the PhD fellowship funding and for financial support of the experiments and divers analyses. The authors would like to thank the company SOCFINDO for their material and technical support during field experiment, as well as Bertrand Gakiere from the facility Metabolism-Metabolome (IPS2). We also thank Jean Ollivier for providing agronomical data.

Arabidopsis Protocols. Totowa, NJ: Humana Press, 439-447. Fiehn O, Wohlgemuth G, Scholz M, Kind T, Lee DY, Lu Y, Moon S, Nikolau B. 2008. Quality control for plant metabolomics: reporting MSI-compliant studies. The Plant Journal 53(4): 691-704. Foong SF 1991. Potential evapotranspiration, potential yield and leaching losses of oil palm. PORIM International Palm Oil Conference. Kuala Lumpur, Malaysia: Palm Oil Research Institute of Malaysia, Ministry of Primary Industries. 105-119. Foster HL. 1985. Oil palm yield responses to N and K fertilizers in different environments in peninsular malaysia: Palm Oil Research Institute of Malaysia, Ministry of Primary Industries, Malaysia. Foster HL 2002. Assessment of oil palm requirements. In: Fairhurst TH, Härdter R eds. The oil palm, management for large and sustainable yields. . Potash and Phosphate Institute of Canada (ESEAP). Singapore. Foster HL, Dolmat MTH, Zakaria ZZ. 1985. Oil palm yields in the absence of N and K fertilizers in different environments in peninsular Malaysia: Palm Oil Research Institute of Malaysia, Ministry of Primary Industries, Malaysia. Foster HL, Mohammed AT. 1988. Variation in the fertilizer requirements of oil palm in peninsular Malaysia. I. Within the same soil series. PORIM Bulletin, Palm Oil Research Institute, Malaysia(116): 1-9. Foster HL, Prabowo NE. 1996. Variation in the potassium fertiliser requirements of oil palm in North Sumatra: Palm Oil Research Institute of Malaysia. Francey RJ, Farquhar GD. 1982. An explanation of 13C/12C variations in tree rings. Nature 297(5861): 28-31. Freeman GG, Mossadeghi N. 1970. Studies on potassium nutrition of plants IV.-Effect of potassium nutrition on the composition of leaves of cabbage seedlings (Brassica

availability tended to decrease the number of bunches per tree (FIG. III.3A) but increased significantly the number of fruits (FIG. III.3B) as well as mesocarp and kernel biomass proportion in bunches (FIG. III.3C). Interestingly, while previous studies have shown that K availability ameliorates yield by increasing bunch biomass and number (Corley & Mok, 2008), we find here that the increase in bunch number is visible only under K2 conditions, with even a slight decrease at high K (K3) in DY (FIG. III.3A). In DL, MWC clearly decreased with K availability (FIG. III.3F) and accordingly, there was an increase in OFM and OER (FIG.

III.3G).

In contrast, higher K availability caused an increase in kernel biomass proportion in bunches and MWC in DY, thus leading to lower OFM. Our results are in agreement with previous studies that showed a correlation between fruit transpiration (water loss) and lipid biosynthesis [START_REF] Jeje | Transpiration and oil accumulation rates for developing oil palm fruits Elaeis guineensis Jacq[END_REF] Teh et al., 2013a). The negative effect of high K (K3 conditions)

List of figures

Chapter II. Metabolic leaf responses to potassium availability in oil palm (Elaeis guineensis Jacq.) trees grown in the field Cathleen Mirande-Ney1,5*, Guillaume Tcherkez2, Thierry Balliau3, Michel Zivy3, Françoise Gilard4, Jing Cui2, Jaleh Ghashghaie5, Emmanuelle Lamade1 1Unité PERSYST, UPR34, Système de pérennes, Centre de Coopération Internationale en

Recherche Agronomique pour le Développement, F-34398 Montpellier, France.

2Research School of Biology, Australian National University, Canberra 2601, ACT, Australia. When tested via a ² test against a random model (averagerandom error), the OPLS model was insignificant for the K effect and highly significant for the cross effect (PCV-ANOVA = 0.14 [K effect] and 0.0008 [cross effect]) in 2018. However, it explained most of total variance (R2 = 0.977) and was very predictive (Q2 = 0.833). Similarly, the OPLS model was insignificant for the K effect and highly significant for the cross effect (PCV-ANOVA = 0.99 [K effect] and 2.81•10-5 [cross effect]) in 2017. It explained most of total variance (R2 = 0.98) and was predictive (Q2 = 0.591). Using the Benjamini-Hochberg correction in univariate analysis to identify significant proteins, 51 proteins appeared to be significantly affected by K availability, 477 by the cross and 20 by the interaction K  cross in 2018 (FIG. II.7A-C; the full list of proteins significant for the cross effect is given in TABLE S1). In 2017, no protein was significantly affected neither by K availability nor the interaction K  cross, and 301 were significant for the cross effect (full list given in TABLE S2). The 51 proteins that were significant for the K effect in 2018 could be gathered in 4 groups using hierarchical clustering.

The two first groups comprised proteins that decreased with K availability, in a slightly more pronounced manner in DL (group 1) or DY (group 2). Group 3 and 4 comprised proteins that Abstract Potassium fertilization is common practice in oil palm (E. guineensis) plantation to increase yield. However, its effect on fruit oil content and composition are not well documented. Here, we conducted bunch, metabolomics and oil composition analyses in two contrasting crosses (Deli x La Mé and Deli x Yangambi) grown under different K fertilization conditions. K availability impacted on bunch oil content, due to lower water content and higher oil proportion in fruit mesocarp in Deli x La Mé only, showing differential responses of crosses to K. Oil composition at maturity did not significantly change under low K conditions despite clear alterations in fruit metabolism associated with lipid production during maturation, demonstrating the resilience of oil biosynthetic metabolism. However, the analysis of variance in oil content (across K treatments and crosses) demonstrates that sugar availability, lipid synthesis rate and metabolic recycling are all important in determining the oil content.

Keywords: oil palm, potassium, metabolomics, oil, fruit, mesocarp

Abstract

In this study, we performed for the first time a 13CO2 pulse-labelling experiment on oil palm leaves, submitted to two potassium fertilization conditions and two crosses in the field. The goal of this study was a better understanding of metabolic dynamics and the flux of carbon from leaflet assimilation to fruits of oil palm. Preliminary results showed that 13CO2 injected into the chamber was effectively fixed into leaflet organic matter (OM), demonstrating that the labelling experiment was successful. However, the efficacy of labelling was much less than expected.

For technical reasons, such as high temperature or air relative humidity, less than 27% of 13CO2 added to air composition in the chamber was assimilated by leaflets. Also, 13C values after labelling were found to be sensitive to climatic variations such as temperature and RH, there was a strong variability between replicate that made difficult the interpretation of the data in terms of effect of K on carbon metabolism in the leaflet. However, we could observe some tendency for K to increase leaflet starch content, and the kinetics of the decline in 13C after labelling suggests there were 2 pools of C: a bigger pool representing (or feeding) leaf biomass, and a smaller "labile" (exportable) pool. The labile pool had a mean turn-over time of up to 130 h suggesting that photosynthate export rate is low.

Journal of Plant Physiology 171(9): 656-669.

Appendix

A.

Supplementary texts

Supplemental text S1: Oil palm fruit developmental stages and oil deposition kinetics

Here, we use oil palm fruit development and oil deposition kinetics that are well described by previous studies [START_REF] Hartley | The oil palm[END_REF][START_REF] Thomas | Fruit ripening in the oil-palm Elaeis guineensis[END_REF]:

-Stage 1 | 0-5 weeks after anthesis (WAA): the mesocarp is visible and composed of up to 92% of water. At this stage, the kernel is still liquid and the endocarp is rather thin.

Supplemental text S2: Total lipids analysis with 1H-NMR analyses

15 mg of ground mesocarp samples (fruits at maturity) were extracted with 500 µL KCl (0.88 %, w:w), 1 mL d-chloroform (Sigma-Aldrich), and 20 µL tetramethylsilane (TMS, 0.03% in dchloroform) in 4-mL glass vials (Supelco). Samples were vortexed, kept on ice for 10 min and then re-vortexed. After centrifugation (3 min, 2,500 rpm, 16°C), 600 µL of the organic phase was collected and transferred to 5-mm NMR tubes (Z107373, Bruker). NMR analyses were performed on a 700 MHz Advance spectrometer (Bruker) at 25°C with a standard pulse program (zg30) over 244 scans. Chemical shifts were referenced against TMS. Outre les effets attendus sur des traits végétatifs ou les régimes, nos résultats montrent que l'apport en K impacte le métabolisme primaire du carbone et de l'azote aussi bien dans les folioles que dans les fruits. Cette thèse présente ainsi, pour la première fois, une étude détaillée du métabolisme du palmier à huile au champ, et montre que certains traits métaboliques (métabolites ou enzymes) sont liés à la disponibilité en K, mettant en abyme une potentielle utilisation de biomarqueurs foliaires pour piloter la nutrition minérale du palmier. Title : C/K interactomic in oil palm metabolism and allocation related to yield Keywords : oil palm, K nutrition, metabolomic, proteomic, 13C labelling, yield Abstract: Oil palm (Elaeis guineensis Jacq.) is one of the most productive oil crop in the world. Unfortunately, positive effect of K fertilization on fruit development with increasing in bunch weight and number, remain rather difficult to predict because of the lack of knowledge of underlying metabolic mechanisms. The objective of this thesis was precisely to assess the effect of K availability on oil palm metabolic pathways and determine if metabolic changes could be related to oil production.

B. Supplementary figures

In addition to expected effects of potassium on vegetative traits and bunches, our results show that K availability affected carbon and nitrogen primary metabolism in both leaflets and developing fruits. This thesis presents, for the first time, a detailed metabolic exploration of oil palm in the field and shows that some metabolic traits (metabolites or enzymes) are linked to K availability, thereby opening avenues for the use of leaf biochemical markers to monitor oil palm mineral nutrition.