Julie Oustric

Yann Quilichini

Raphaël Morillon
email: raphael.morillon@cirad.fr

Stéphane Herbette

Luro François

Jean Giannettini
email: gianetti@univ-corse.fr

Liliane Berti
email: berti@univ-corse.fr

Jérémie Santini
email: jeremie.santini@univ-corse.fr

François Luro
email: luro@corse.inra.fr

Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones

Keywords: Citrus, Nutrient deficiency, Oxidative stress, Photosynthesis, Polyploidy, Ultrastructure Abbreviations 2x: diploid, 4x: doubled-diploid, APX: ascorbate peroxidase, CAT: catalase, CM2x: diploid Citrumelo 4475, CM4x: doubled-diploid Citrumelo 4475, F0: minimal chlorophyll a fluorescence, Fm: maximal fluorescence, Fv: variable fluorescence, Fv/Fm: maximum quantum efficiency of PSII, Gs: stomatal conductance, MDA: malondialdehyde, Pnet: Leaf net photosynthetic rate, ROS: reactive oxygen species, SOD: superoxide dismutase, VK2x: diploid Volkameriana, VK4x: doubled-diploid Volkameriana

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Polyploidy is a common feature and a major facilitator of adaptive evolution in plants [START_REF] Wendel | Genome evolution in polyploids[END_REF][START_REF] Chen | Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids[END_REF]. It arises spontaneously from unreduced gametes or duplicated chromosomes. Two types of polyploidy are generally recognized: autopolyploidy, which is considered extremely rare in nature (Stebbins 1950;[START_REF] Grant | Plant speciation[END_REF], and allopolyploidy.

Autopolyploid genomes are composed of identical sets of chromosomes, while allotetraploid genomes are formed through interspecific hybridization (Soltis and Soltis, 2009). Genome variations in polyploidy have induced changes in anatomical and morphological characteristics, physiological functions and gene expression [START_REF] Leitch | Genomic plasticity and the diversity of polyploid plants[END_REF]. Polyploid plants frequently occur in adverse environmental conditions, like high altitude with high radiation, more polar latitude, deserts, and nutrient-poor soil [START_REF] Ehrendorfer | Polyploidy and distribution[END_REF]Thompson and Lumaret, 1992;Soltis and Soltis 2000). Their potential to improve tolerance has prompted a number of investigations into diploids (2x) and doubled-diploid (4x) genotypes under several environmentally stressful conditions, and some studies have found a tolerance improvement in 4x under heat, drought and salt stress than in their related 2x (Saleh et al., 2008;[START_REF] Li | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF][START_REF] Zhang | Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance[END_REF].

A common consequence of environmental stresses is the overproduction of free radicals and other reactive oxygen species (ROS) that can deteriorate the ultrastructure of plant components (e.g. photosynthetic pigments) and cell membranes (Foyer et al. 1994, Taylor andCraig 1971). The major sources of ROS in plants cells are chloroplasts, mitochondria and peroxisomes. Plants possess two antioxidant systems-one enzymatic (mainly superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and dehydroascorbate reductase (DHAR)), the other non-enzymatic (mainly ascorbate, glutathione and proline). However, stress response may change depending on genotype, intensity of stress, and ploidy level (Santini et al., 2013;[START_REF] Liu | In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance[END_REF].

Nutrient deficiency is one of the major environmental factors that severely limit crop development and production worldwide [START_REF] Foyer | Hydrogen peroxide-and glutathione-associated mechanisms of acclimatory stress tolerance and signalling[END_REF]. Essential nutrients are mainly N, P and K, which have a direct impact on plant growth. A deficiency in these elements would disturb the vegetative or reproductive lifecycle of the plant. Mineral-deficient plants consequently show various visual symptoms, such as necrosis, chlorosis, dark green foliage, or stunted growth [START_REF] Ericsson | Growth and shoot: root ratio of seedlings in relation to nutrient availability[END_REF]. These minerals have various functions: (i) as essential Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. (2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 4 metabolites (e.g. proteins, enzymes and coenzymes, cell walls and chlorophyll), (ii) as enzyme activators or regulators of enzyme-associated processes, and (iii) as non-structural factors in physiological processes (i.e. membrane integrity, photosynthesis, stomatal movement and environmental signalling) [START_REF] Lal | Encyclopedia of soil science[END_REF]. Deficiency in one or more mineral elements causes a degradation of the chloroplasts and thus disruption of their photosynthetic capacity [START_REF] Kalaji | Identification of nutrient deficiency in maize and tomato plants by in vivo chlorophyll a fluorescence measurements[END_REF].

The citrus industry has long used rootstocks to enhance tolerance to biotic and abiotic stresses and to improve fruit output and quality. The majority of the rootstocks used are 2x, with 2n = 2x = 18 chromosomes [START_REF] Krug | Chromosome numbers in the subfamily Aurantioideae with special reference to the genus Citrus[END_REF]. However, 4x resulting from incomplete mitosis of somatic embryos frequently occur in seedlings of 2x apomictic genotypes [START_REF] Cameron | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF]. Tetraploid citrus are characterized by various phenotypic traits including greater flower number, greener (high chlorophyll production), thicker and larger leaves and fruits, and reduced growth compared to the related 2x (Stebbins,1950;Ollitrault and Jacquemond, 1994;Romero-Aranda et al., 1997). Leaves of 4x generally have larger cells, guard cells, stomata and chlorophyll, and a higher number of chloroplasts [START_REF] Beck | Evaluation of induced polyploidy in Acacia mearnsii through stomatal counts and guard cell measurements[END_REF][START_REF] Jellings | Anatomical variation in first leaves of nine Tritucum genotypes, and its relationship to photosynthetic capacity[END_REF][START_REF] Zhang | Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance[END_REF]. The greater genetic variability of 4x could be used in citrus to improve abiotic stress tolerance [START_REF] Allario | Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production[END_REF]Oustric et al., 2017;[START_REF] Balal | Kinnow mandarin plants grafted on tetraploid rootstocks are more tolerant to Cr-toxicity than those grafted on its diploids one[END_REF].

Fertilization is a big economic and ecological issue in the citrus industry, making it essential to better understand plant responses to nutrient deficiency and urgent to develop new rootstocks that require less fertilizer. New tetraploid rootstocks offer a promising way forward to lower use of fertilizers.

Here we compared the impact of nutrient deficiency in two rootstock genotypes that are used worldwide, i. The objective of this study was to differentiate these 4 genotypes by investigating the impact of nutrient deficiency on leaf structure and ultrastructure associated with physiological and biochemical parameters. Increased tolerance to nutritional stress in 4x (lower structure and ultrastructure damage associated with higher photosynthetic capacities and more efficient antioxidant system) would confirm the value of using polyploids when exposed to environmentally harsh conditions.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 5

Materials and Methods

Plant material and experimental design

Plants of 2x Citrumelo 4475 (CM2x) and Volkamer lemon (VK2x) genotypes and their related doubled-diploids (CM4x and VK4x, respectively) were grown from seedling stages (8 weeks old). Seedlings of each genotype were selected after checking their ploidy status by flow cytometry (Partec I, Germany) according to [START_REF] Froelicher | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF]. Clonal propagation by nucellar embryogenesis was checked by genotyping with SSR markers as described in Vieira et al. (2016).

The 6 seedlings of each genotype, giving a total of 24 plants, were grown under identical conditions in vermiculite with ferti-irrigation and water (1L/h) during 4 years in a tunnel greenhouse at the AREFLEC experimental station in San Giuliano, Corsica (41°47'27''N and 09°23'40''E). The ferti-irrigation solutions were prepared and applied with a metering pump.

The stock solution used for irrigation included: N (nitrogen), P (phosphorus) and K (potassium) (20-5-10 fertilizer unit) + 2MgO (magnesium oxide) + trace elements. The fertilization rate supplied was in accordance with the French department of agriculture recommendations.

After 4 years and before starting the experiment, the vermiculite was washed with distilled water for 48 hours in order to eliminate any nutritional reserves in the pot. The 4-yearsold trees were then divided into 2 blocks: one with reference ferti-irrigation (control plants) and the other with irrigation water (without nutrient inputs). In each block, a total of 3 trees of each genotype was randomized, giving a total of 12 trees per block.

Experiments were carried out in December 2016 after 210 days of nutrient deficiency.

Measurements and sampling were performed on homogeneous plants composed of 4 branches with fully-expanded leaves. For physiological measurements, 9 fully expanded leaves were analyzed, i.e. 3 per tree (9 replicates), between 7 AM and 11 AM. For biochemical analyses, 3 samples were collected for each genotype, i.e. 1 per tree, and each sample is obtained by pooling 8 fully-expanded leaves. All leaves were immediately frozen in liquid nitrogen and stored at -80°C, and each leaf sample was ground to a fine powder prior performing biochemical analyses. For the mineral measurements, 3 samples were collected for each genotype, i.e. one per tree, and each sample is obtained by pooling 8 fully-expanded leaves (3 replicates).

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 6 At the same time of measurements and sampling, five 1 cm² pieces for scanning electron microscopy and five 1-2 mm² pieces for transmission electron microscopy were cut with a razor blade from mid-laminar areas of both sets of leaves (5 replicates for each genotype).

2.2.Measurements of gas exchange

Leaf net photosynthetic rate (Pnet) and stomatal conductance (Gs) were measured using a LI-6400 portable photosynthesis system (Li-COR, Lincoln, NE, USA) with the LI6400-40 Leaf Chamber. This experiment was conducted at photosynthetic photon flux density (PPFD) of 1400 μmol photon•m -2 •s -1 with a Li-COR red-blue light source (6400-02B no. SI-710, Li-Cor, Lincoln, NE), a constant carbon dioxide concentration (CO2) of 380 μmol•mol -1 , air flow rate of 500 μmol•s -1 , and leaf temperature of 25ºC.

Measurements of chlorophyll a fluorescence

Chlorophyll a fluorescence of dark-acclimated leaves was measured using a Handy-PEA portable fluorometer (Hansatech Instruments, Ltd, Norfolk, UK). Leaves were firstly darkacclimated with a "leafclip" for 30 min. Then, leaves were illuminated with a light intensity of 3000 μmol photon•m -2 •s -1 , by an array of three light-emitting diodes (650 nm) for a duration of 1 s. Among the measured parameters, F0 represents the minimal values of chlorophyll a fluorescence, Fm represents the maximum fluorescence, and Fv is variable fluorescence, which is obtained by subtracting Fo from Fm. The Fv/Fm ratio corresponds to the maximum quantum efficiency of photosystem II (PSII) [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF].

Foliar mineral analysis

All ground samples were dried at 65 ± 10° C in an oven overnight, transferred into a desiccator until cooling and sent to a CIRAD laboratory (Montpellier, France) for analysis of macro-and micro-nutrients.

Total P, K, Ca, Mg, Na, B, Cu, Fe, Zn and Mn in leaves were determined using an Agilent 720 simultaneous ICP-OES after double calcination including silica removal by adding hydrofluoric acid.

N content in leaves was measured by combustion using a Leco TruMac N determinator.

Scanning electron microscopy (SEM)

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. (2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 7 Leaf pieces (typically 1 cm²) were cut with a razor blade from mid-laminar areas of leaves and fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, dehydrated through a graded ethanol series (30%, 50%, 75%, 90% and 100%), and dried with CO2 in an Emitech K850 critical point dryer (Quorum Technologies Ltd, Ashford, U.K.). Specimens were mounted on aluminum stubs with carbon double-sided adhesive disks, coated with gold/palladium in a SC7640 sputter coater (Quorum Technologies Ltd, Newhaven, U.K.), and examined under a S-3400N scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 5 at the University of Corsica (Corte, France). SEM measurements were realized using the software of the microscope. 30 independent measurements on 5 different leaves of each variety and each genotype were realized in order to estimate length and width of stomata and ostioles. 5 independent measurements on 5 different leaves of each variety and each genotype were analyzed using Adobe Photoshop software in order to estimate the stomata density. The number of stomata was counted on photographs and taken into account the stomata cut only on two sides of the photograph.

Transmission electron microscopy (TEM)

Leaf pieces (typically 1 mm²) were cut with a razor blade from mid-laminar areas of leaves and fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M cacodylate buffer at pH 7.2, post-fixed in cold (4°C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated through a graded ethanol series (70% and 100%) and propylene oxide, embedded in Spurr, and polymerized at 60°C for 24 h. Ultra-thin sections (60-90 nm) were cut using an Power tome PC ultramicrotome (RMC Boeckeler, Tuscon, U.S.A.).

The sections were placed on 200-and 300-mesh copper grids and stained with UranyLess (Delta Micoscopies, France) and lead citrate. Sections were examined on a Hitachi H-7650 (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 80 kV at the University of Corsica (Corte, France). TEM measurements were realized using the software of the microscope. 10 cells of each variety, each genotype and each mesophyll type were analysed in order to estimate length, width and thickness of cells and length, width and number of chloroplasts, starches, plastoglobuli and mitochondria. 30 independent measurements of each variety, each genotype and each mesophyll type were analysed in order to estimate length Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 8 and width of chloroplasts, starches, plastoglobuli, mitochondria, grana, number of grana per cell section, and number of thylakoids per granum.

Assay of oxidation markers

Malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents and antioxidant enzyme activities (superoxide dismutase, catalase and ascorbate peroxidase) were determined using the method described by Santini et al. (2013). A V-630 spectrophotometer (Jasco Inc., Tokyo, Japan) was used to measure the absorption spectra of the samples.

Statistical analyses

The experimental was set up as a split-plot design with genotype as main plot and ploidy and treatment as subplot. R statistical software (http://www.R-project.org) was used to analyze all the data. Multi-way ANOVA followed by least significant difference (LSD) test at P < 0.05 was used for evaluating the influence of genotypes, ploidy and treatments. Heat map was made to determine the differences between genotypes and treatments for abaxial epidermis and ultrastructural palisadic and spongic mesophyll characters.

Results and Discussion

Anatomical properties of doubled-diploid versus diploid genotypes under control conditions

Regardless of seedling genotype and ploidy level, stomata were observed only on leaf abaxial surface and were surrounded by ordinary epidermal cells (anomocytic organization).

The epidermal carpet architecture showed no appendages (trichome) (Fig. 1). Similar results were found by [START_REF] Inyama | Comparative morphology of the leaf epidermis in six Citrus species and its biosystematic importance[END_REF] in other Citrus species. In agreement with other studies, stomatal density was lower in 4x genotypes than in their related 2x, and correlated with an increase in size (Fig. 1; Table 1; Sup. Table 1) [START_REF] Aryavand | Stomatal frequency and size differentiate ploidy levels in Aegilops neglecta[END_REF][START_REF] Li | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF][START_REF] Allario | Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression[END_REF][START_REF] Meng | Comparison of photosynthesis and leaf ultrastructure on two black locust (Robinia pseudoacacia L.)[END_REF]. At ultrastructure level, whether in the palisade or spongy mesophylls, cells were larger with a thicker wall in 4x genotypes than in their related 2x (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3), as reported in Romero-Aranda et al. (1997). Doubled-diploid genotypes had more chloroplasts but their size remained broadly similar to their 2x counterparts (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3), in line with previous reports [START_REF] Jellings | Anatomical variation in first leaves of nine Tritucum genotypes, and its relationship to photosynthetic capacity[END_REF][START_REF] Mathura | Comparative study of chlorophyll content in diploid and tetraploid black wattle (Acacia mearnsii)[END_REF]. Conversely, ploidy level did Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 9 not influence number and size of starches and plastoglobuli or number of grana per chloroplasts, which were approximately identical between the different ploidy levels and whatever the parenchyma (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3). This result was also observed in 4x black locust (Robinia pseudoacacia L.) [START_REF] Meng | Comparison of photosynthesis and leaf ultrastructure on two black locust (Robinia pseudoacacia L.)[END_REF].

Impact of tetraploidization on photosynthetic properties under nutrient deficiency

Deficiency of one or more essential minerals is known to affect the photosynthetic process [START_REF] Lima | Leaf gas exchange and chlorophyll fluorescence parameters in Phaseolus vulgaris as affected by nitrogen and phosphorus deficiency[END_REF][START_REF] Huang | Effects of nitrogen deficiency on gas exchange, chlorophyll fluorescence, and antioxidant enzymes in leaves of rice plants[END_REF][START_REF] Boussadia | Effects of nitrogen deficiency on leaf photosynthesis, carbohydrate status and biomass production in two olive cultivars 'Meski' and 'Koroneiki[END_REF]. The decrease in Pnet was associated with a decrease in Gs in all genotypes (Fig. 4A andB; Sup. Table 4), suggesting a stomatal limitation [START_REF] Zhao | Nitrogen deficiency effects on plant growth, leaf photosynthesis, and hyperspectral reflectance properties of sorghum[END_REF] that would lead to down-regulation of the CO2 accumulation [START_REF] Flexas | Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency[END_REF].

Pnet was higher in 4x honeysuckle (Lonica japonica Thunb.) [START_REF] Li | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF], spathe flower (Spathiphyllum wallisii) (Van Laere et al., 2011) and in Carrizo citrange citrus seedlings (Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf) (Oliveira et al, 2017) than their 2x counterparts under drought stress, due to higher maintained degree of stomatal opening.

However, nutrient deficiency caused less pronounced inhibition of photosynthesis in 4x seedlings than in related 2x (Fig. 4C; Sup. Table 4) possibly due to an upregulation of nonstomatal factors (biochemical) [START_REF] Klamkowski | Response to drought stress of three strawberry cultivars grown under greenhouse conditions[END_REF]Shahid et al., 2011) or a decrease in protective non-radiative energy dissipation, photodamage of the PSII centers, or both (Osmond, 1994). Photoinhibitory impacts on PSII occurred proportionally more in 2x honeysuckle (Lonicera japonica Thunb.) under salt stress [START_REF] Yan | Contrasting photosynthesis and photoinhibition in tetraploid and its autodiploid honeysuckle (Lonicera japonica Thunb.) under salt stress[END_REF] and heat stress [START_REF] Li | Chromosome doubling can increase heat tolerance in Lonicera japonica as indicated by chlorophyll fluorescence imaging[END_REF] than in 4x counterparts. The lower decrease of photosynthetic rate in 4x genotypes under nutrient deficiency could be due to ultrastructural changes in leaves (Parisod et al., 2010;[START_REF] Xiong | Performance of wheat crops with different chromosome ploidy: root-sourced signals, drought tolerance, and yield performance[END_REF].

The higher photosynthetic rates of doubled-diploid genotypes under nutrient stress could be explained by their leaf and cell anatomy.

In each genotype, nutrient deficiency decreased the total content of all minerals except for K (Fig. 6) and was associated with leaf alteration (Fig. 5). Despite a similar decrease in minerals content (Fig. 6), leaves in 2x genotypes had a greater proportion of chlorotic leaves than in their related 4x (Fig. 5), suggesting a substantial loss of chlorophyll pigments (Zhang et al., 2016). However, taking in account the long-term nutrient deficiency, there was no large Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 10 change in stomata size and density in either 2x or 4x genotypes (Fig. 1; Table 1; Sup. Table 1), suggesting that stomata do not play a major role in response to nutritional stress. Furthermore, all genotypes presented ultrastructural damages (chloroplasts, starches, mitochondria and plastogobuli) under nutrient deficiency. In spongy and palisadic mesophyll cells, number of chloroplasts and mitochondria decreased and granal thylakoids were distorted in all genotypes (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3). Doubled-diploid genotypes were less impacted by these changes than their related 2x. Loss of chloroplast integrity and functioning (photosynthesis, potential quantum yield of photosystem II) would be characteristic of senescence. Chloroplast degradation plays an important role in leaf senescence for nutrient recycling from reproductive organs or younger leaves [START_REF] Makino | Effects of nitrogen nutrition on nitrogen partitioning between chloroplasts and mitochondria in pea and wheat[END_REF][START_REF] Hortensteiner | Nitrogen metabolism and remobilization during senescence[END_REF]. Moreover, thylakoid membranes and their associated PSII are considered as targets of abiotic stress [START_REF] Ding | Effects of nitrogen deficiency on photosynthetic traits of maize hybrids released in different years[END_REF].

The lower deterioration of thylakoids in 4x genotypes could explain their better Fv/Fm ratio and, in turn, the higher photosynthetic activity maintained in 4x genotypes than their respective 2x (Fig. 4C; Sup. Table 4). Interestingly, lower chloroplast damage associated with higher photosynthetic activity was also observed in 4x black locust (Robinia pseudoacacia L.) under salt stress [START_REF] Wang | Physiological and proteomic responses of diploid and tetraploid black locust (Robinia pseudoacacia L.) subjected to salt stress[END_REF] than in their related 2x.

Plastoglobuli containing products of membrane degradation showed an abnormal increase in size (spongy and palisadic mesophyll cells) in all genotypes except VK4x (Fig. 2 and 3; Tables 2 and3; Sup. Tables 2 and3), which may indicate a link between chloroplast disorganization and plastoglobuli [START_REF] Dou | Growth and chloroplast ultrastructure of two citrus rootstock seedlings in response to ammonium and nitrate nutrition[END_REF]. In the chloroplast stroma, nutrient deficiency led to a decrease in the number as well as the length and width of starch granules in both spongy and palisadic mesophyll cells in all genotypes except VK4x (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3). VK4x showed an increase in starch length and width (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3), as reported in other studies under nutrient deficiency [START_REF] Bondada | Leaf chlorophyll, net gas exchange and chloroplast ultrastructure in citrus leaves of different nitrogen status[END_REF][START_REF] Zhao | Effect of nitrogen and phosphorus deficiency on transcriptional regulation of genes encoding key enzymes of starch metabolism in duckweed (Landoltia punctata)[END_REF]. The swelling of starch granules could be induced by an up-regulation of transcripts encoding key enzymes involved in starch biosynthesis and a down-regulation of transcripts encoding enzymes involved in starch consumption (Tao et al., 2013). In CM4x, VK2x and CM2x, the decline in length and width of transitory starch (Fig. 2 and3; Tables 2 and3; Sup. Tables 2 and3) would suggest a high mobilization and translocation of starch leading, in turn, to a more advanced senescence process than in VK4x [START_REF] Lindroth | Effects of genotype and nutrient availability on phytochemistry of trembling aspen (Populus tremuloides Michx.) during leaf senescence[END_REF]. In leaves, stimulation of the degradation of starch granules Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 11 would supply the carbon and energy needed for the stress response, especially when photosynthesis becomes too weak, and protect cellular structures by increasing soluble sugars as osmoprotectants [START_REF] Kaplan | β-Amylase induction and the protective role of maltose during temperature shock[END_REF]Rizhsky et al., 2004;[START_REF] Naeem | Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones[END_REF].

The greater tolerance to nutrient stress of 4x genotypes is associated with better redox status

Mesenchymal cells, especially chloroplasts, endure ultrastructure damage from abiotic stresses such as salinity [START_REF] Hernandez | Salt-induced oxidative stress in chloroplasts of pea plants[END_REF][START_REF] Fidalgo | Effects of long-term salt stress on antioxidant defence systems, leaf water relations and chloroplast ultrastructure of potato plants[END_REF], drought and heat [START_REF] Grigorova | Combined drought and heat stress in wheat: changes in some heat shock proteins[END_REF], chilling stress (Kratsh and Wise, 2000), nutrient excess (Papadakis et al., 2004) and nutrient deficiency [START_REF] Bondada | Leaf chlorophyll, net gas exchange and chloroplast ultrastructure in citrus leaves of different nitrogen status[END_REF]Papadakis et al., 2007).

Reduction of chloroplast numbers leads to a decrease in photosynthetic activity under nutrient deficiency. This decrease is probably due to oxidative stress, i.e. an imbalance between ROS accumulation and antioxidant system capacity [START_REF] Djanaguiraman | Ethylene production under high temperature stress causes premature leaf senescence in soybean[END_REF]. Previous studies have reported that nutrient deficiency enhances oxidative metabolism [START_REF] Cakmak | Activity of ascorbate-dependent H2O2-scavenging enzymes and leaf chlorosis are enhanced in magnesium-and potassium-deficient leaves, but not in phosphorus-deficient leaves[END_REF]Tewari et al., 2006). Here, SOD and APX activities differed between genotypes, but CAT activity decreased with nutrient deficiency in all genotypes (Fig. 7A, B andC; Sup. Table 4).

CAT activity decreases in several species under numerous environmental stresses such as salt stress (Tanaka et al., 1999), shading stress [START_REF] Causin | Down-regulation of catalase activity contributes to senescence induction in wheat leaves exposed to shading stress[END_REF] and nutrient deficiency [START_REF] Huang | Effects of nitrogen deficiency on gas exchange, chlorophyll fluorescence, and antioxidant enzymes in leaves of rice plants[END_REF]Tewari et al, 2004). The increase in APX only seemed to compensate for the lower CAT activity in CM4x, which limited accumulation of H2O2 (Fig. 7D; Sup. Table 4). The increase in MDA content in CM2x and CM4x (Fig. 7E; Sup. Table 4) could result directly from the transformation of the H2O2 in OH° via Fenton or Haber-Weiss reactions, but also from a direct conversion of O2 to OH • from an addition of three electrons. The lower MDA and H2O2 accumulation in CM4x was associated with greater SOD and APX activities. This result could explain that CM4x had less ultrastructural damage and decreased less in photosynthetic activity than CM2x. Higher antioxidant capacity was associated to better ultrastructural cell integrity for better protection under drought stress in a tolerant apple rootstock compared to a sensitive one [START_REF] Wang | Influence of drought stress on the cellular ultrastructure and antioxidant system in leaves of drought-tolerant and droughtsensitive apple rootstocks[END_REF]. In agreement with our results, MDA and H2O2 were also less accumulated in 4x Ignam (Dioscorea) under heat stress [START_REF] Zhang | Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance[END_REF] and in black locust (Robinia pseudoacacia L.) under salt stress [START_REF] Meng | Tetraploid black locust (Robinia pseudoacacia L.) increased salt tolerance by activation of the antioxidant system[END_REF] than in their related 2x.

In VK2x and VK4x, the increase in SOD activity and the lower or similar APX and CAT activities could explain their higher accumulation of H2O2. These results imply that factors Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 12 other than redox status could explain the stronger ultrastructural degradations in VK2x than VK4x, i.e. transcriptional regulations, phytohormones and nutrient signalling [START_REF] Woo | Plant leaf senescence and death-regulation by multiple layers of control and implications for aging in general[END_REF][START_REF] Liu | The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89)[END_REF].

Conclusion

Polyploidization lends citrus genotypes greater tolerance to a prolonged nutrient deficiency, as indicated by better photosynthetic activity for doubled-diploid genotypes under stress conditions. This greater tolerance and photosynthetic activity were associated with limited ultrastructural changes of leaf cells, especially chloroplasts and starch granules, in doubled-diploid genotypes. These limited structural changes argue for a long-lasting tolerance of doubled-diploid genotypes under nutrient deficiency, suggesting that these genotypes may be good candidates for citrus cultivation as part of the effort to reduce fertiliser use. However, better protection against nutrient deficiency is not still related to a better antioxidant system for citrus.

The next step of this study will be to test these genotypes as rootstocks and to graft them with an economically productive variety.

Contributions

J. Oustric is a student and main author. She collected test data, performed statistical analyses, interpreted the results and drafted the manuscript. J. Santini was involved in the write-up, planning and discussion of results.

Y. Quilichini performed microscopic analysis.

L. Berti helped in paper discussion and was the leader of the team. R. Morillon, S. Herbette, F. Luro and J. Giannettini helped in research trial and to draft the manuscript.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 13 Gilles Paolacci and Paul Martin for installing and monitoring the ferti-irrigation system in the greenhouse.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 17 57, 84-92. Oliveira, T.M., Yahmed, J.B., Dutra, J., Maserti, B.E., Talon, M., Navarro, L., Ollitraut, P., Gesteira, A. da S., Morillon, R., 2017. Better tolerance to water deficit in doubled diploid 'Carrizo citrange' compared to diploid seedlings is associated with more limited water consumption. Acta Physiol. Plant. 39, 204. Santini, J., Giannettini, J., Pailly, O., Herbette, S., Ollitrault, P., Berti, L., Luro, F., 2013.

Comparison of photosynthesis and antioxidant performance of several Citrus and

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 18 Fortunella species (Rutaceae) under natural chilling stress. Trees 27, 71-83. Shahid, M.A., Pervez, M.A., Balal, R.M., Ahmad, R., Ayyub, C.M., Abbas, T., Akhtar, N., 2011. Salt stress effects on some morphological and physiological characteristics of okra (Abelmoschus esculentus L.). Soil Environ. 30. Plant Physiol. 47, 719-725. Tanaka, Y., Hibino, T., Hayashi, Y., Tanaka, A., Kishitani, S., Takabe, T., Yokota, S., 1999. Salt tolerance of transgenic rice overexpressing yeast mitochondrial Mn-SOD in chloroplasts. Plant Sci. 148, 131-138. Tao, X., Fang, Y., Xiao, Y., Jin, Y., Ma, X., Zhao, Y., He, K., Zhao, H., Wang, H., 2013. Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation. Biotechnol. Biofuels 6, 72. Tewari, R.K., Kumar, P., Sharma, P.N., 2006. Magnesium deficiency induced oxidative stress and antioxidant responses in mulberry plants. Sci. Hortic. 108, 7-14. Tewari, R.K., Kumar, P., Tewari, N., Srivastava, S., Sharma, P.N., 2004. Macronutrient deficiencies and differential antioxidant responses-influence on the activity and expression of superoxide dismutase in maize. Plant Sci. 166, 687-694. Thompson, J.D., Lumaret, R., 1992. The evolutionary dynamics of polyploid plants: origins, establishment and persistence. Trends Ecol. Evol. 7, 302-307.

Van Laere, K., França, S.C., Vansteenkiste, H., Van Huylenbroeck, J., Steppe, K., Van Labeke, M.-C., 2011. Influence of ploidy level on morphology, growth and drought susceptibility in Spathiphyllum wallisii. Acta Physiol. Plant. 33, 1149-1156. Vieira, D.D.S.S., Emiliani, G., Michelozzi, M., Centritto, M., Luro, F., Morillon, R., Loreto, F., Gesteira, A., Maserti, B., 2016. Polyploidization alters constitutive content of volatile organic compounds (VOC) and improves membrane stability under water deficit in Volkamer lemon (Citrus limonia Osb.) leaves. Environ. Exp. Bot. 126, 1-9.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 20 Table 1. Abaxial epidermis study of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days.

Values are means (± standard error) of 30 independent measurements on 5 different leaves (n = 150) for length and width of stomata and ostioles, and of 5 independent measurements on 5 different leaves (n = 25) for stomatal density. The heat map shows the differences between genotypes and treatments for each measured characteristic. Values are associated with color ranging from white (low) to dark green (high).

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 21 Table 2. Ultrastructure of palisade mesophyll cells of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days.

Values are means (± standard error) of independent measurements on five different cells section (n = 5) for length, width and thickness of cells and for number of chloroplasts, starches, plastoglobuli and mitochondria and of 30 independent measurements on different cells section (n = 30) for length and width of chloroplasts, starches, plastoglobuli, mitochondria, grana, number of grana per cells section and number of thylakoids per granum. The heat map shows the differences between genotypes and treatments for each measured characteristic. Values are associated with color ranging from white (low) to dark green (high).

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 22 Table 3. Ultrastructure of spongy mesophyll cells of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days.

Values are mean (± standard error) of independent measurements on 10 different cells section (n = 10) for length, width and thickness of cells, and for number of chloroplasts, starches, plastoglobuli and mitochondria and of 30 independent measurements on different cells section (n = 30) for length and width of chloroplasts, starches, plastoglobuli, mitochondria, grana, number of grana per cell section, and number of thylakoids per granum. The heat map shows the differences between genotypes and treatments for each measured characteristic. Values are associated with color ranging from white (low) to dark green (high).

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 23 CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubleddiploid (VK4x) Volkamer lemon. The white circles correspond to the control (100%) and the black circles to the plant without nutrition (0%). Values are mean (± standard error) of 9 independent measurements from 3 different trees for each genotype (n = 9). Ploidy and treatment effects were analyzed using ANOVA and Fisher LSD tests (P < 0.05). Distinct letters indicate significant differences between all genotypes and treatments.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 24 Volkamer lemon. The white circles correspond to the control (100%) and the black circles to the plant without nutrition (0%). Values are mean (± standard error) of 3 independent measurements from 3 samples for each genotype, i.e. one per tree, obtained by pooling 8 fullyexpanded leaves (n = 3). Ploidy and treatment effects were analyzed using ANOVA and Fisher LSD tests (P < 0.05). Distinct letters indicate significant differences between all genotypes and treatments.

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020

Figures

Fig. 1 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 26 Fig. 2 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 27 Fig. 3 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 28 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 29 Fig. 5 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 30 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 32 Supplementary data Table 1. Probability values produced by analysis of variance (ANOVA) of abaxial epidermis datasets of experimentation conditions. Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 33 Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 34 Table 3. Probability values produced by analysis of variance (ANOVA) on spongic mesophyll cells ultrastructural datasets of experimentation conditions. Initial data were subjected to a two-way analysis of variance. The qualitative factors studied were genotype, ploidy, treatment and their interaction. The analyses were performed on a group of three trees. The p values showing the level of significance of each qualitative factor are presented. Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 35 Table 4. Probability values produced by analysis of variance (ANOVA) on physiological and biochemical datasets of experimentation conditions. Initial data were subjected to a two-way analysis of variance. The qualitative factors studied were genotype, ploidy, treatment and their interaction. The analyses were performed on a group of three trees. The p values showing the level of significance of each qualitative factor are presented.

Cells

Pnet

Gs Fv/Fm SOD CAT APX H2O2 MDA A (genotype) 0.654 0.243 0.531 10 -4 0.005 0.07 0.000 0.022 B (ploidy) 0.288 0.992 0.256 0.305 0.845 0.13 0.195 0.148 C (treatment) 10 -7 10 -4 10 -4 0.082 0.002 0.04 0.078 0.002 D (A x B x C) 10 -6 0.008 10 -4 10 -6 10 -5 10 -6 0.000 10 -5

Comment citer ce document : Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J. ( 2019). Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiology and Biochemistry, 135, 372-384. , DOI : 10.1016/j.plaphy.2018.12.020 36 Table 5. Probability values produced by analysis of variance (ANOVA) on minerals datasets of experimentation conditions. Initial data were subjected to a two-way analysis of variance. The qualitative factors studied were genotype, ploidy, treatment and their interaction. The analyses were performed on a group of three trees. The p values showing the level of significance of each qualitative factor are presented.

N%

P% K% Mg% Ca% Na% Fe mg.kg -1 Mn mg.kg -1 Cu mg.kg -1 Zn mg.kg -1 B mg.kg

  e. Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf. (CM2x)) and Volkamer lemon (Citrus limonia Osb. (VK2x)), in both 2x and their respective 4x types (CM4x and VK4x, respectively).

  Ollitrault, P., Jacquemond, C., 1994. Facultative apomixis, spontaneous polyploidization and inbreeding in Citrus volkameriana seedlings.Fruits 49, 479-480. Osmond, C.B., 1994. What is photoinhibition? Some insights from comparisons of shade and sun plants. Photoinhibition of photosynthesis from molecular mechanisms to the field.In: Baker, N.R., Boyer, J.R., (eds), Photoinhibition of Photosynthesis: From Molecular Mechanisms to the Field. Bios Scientific Publishers, Lancaster, pp. 1-24. Oustric, J., Morillon, R., Luro, F., Herbette, S., Lourkisti, R., Giannettini, J., Berti, L., Santini, J., 2017. Tetraploid Carrizo citrange rootstock (Citrus sinensis Osb. x Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (Citrus clementina Hort. ex Tan). J. Plant Physiol. 214, 108-115. Papadakis, I.E., Dimassi, K.N., Bosabalidis, A.M., Therios, I.N., Patakas, A., Giannakoula, A., 2004. Effects of B excess on some physiological and anatomical parameters of 'Navelina' orange plants grafted on two rootstocks. Environ. Exp. Bot. 51, 247-257. Papadakis, I.E., Bosabalidis, A.M., Sotiropoulos, T.E., Therios, I.N., 2007. Leaf anatomy and chloroplast ultrastructure of Mn-deficient orange plants. Acta Physiol. Plant. 29, 297-301. Parisod, C., Holderegger, R., Brochmann, C., 2010. Evolutionary consequences of autopolyploidy. New Phytol. 186, 5-17. Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., Davletova, S., Mittler, R., 2004. When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol. 134, 1683-1696.Romero-Aranda, R., Bondada, B.R., Syvertsen, J.P., Grosser, J.W., 1997. Leaf characteristics and net gas exchange of diploid and autotetraploid citrus. Ann.Bot. 79, 153-160. Saleh, B., Allario, T., Dambier, D., Ollitrault, P., Morillon, R., 2008. Tetraploid citrus rootstocks are more tolerant to salt stress than diploid. C. R.Biol. 331,[703][704][705][706][707][708][709][710]

  Soltis, P.S., Soltis, D.E., 2000. The role of genetic and genomic attributes in the success of polyploids. Proc. Natl. Acad. Sci. 97, 7051-7057. Soltis, P.S., Soltis, D.E., 2009. The role of hybridization in plant speciation. Ann. Rev. Plant Biol. 60, 561-588. Stebbins, G. L. (1950). Variation and evolution in plants. Oxford University Press, London, UK. Taylor, A.O., Craig, A.S., 1971. Plants under climatic stress II. Low temperature, high light effects on chloroplast ultrastructure.

Figure captions Figure 1 .

 captions1 Figure captions Figure 1. Scanning electron micrographs of abaxial epidermis (1A, 1B, 1E, 1F, 1I, 1J, 1M, 1N; scale bar: 50 µm) and stomata (1C, 1D, 1G, 1H, 1K, 1L, 1O, 1P; scale bar: 10 µm) in diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days. ST: ostiole (stoma); EC: epithelial cells; GC: guard cells.

Figure 2 .

 2 Figure 2. Transmission electron migrographs of palisade mesophyll cells (3A, 3B, 3C, 3D, 3I, 3J, 3K, 3L; scale bar: 5 µm) and their respective chloroplasts (3E, 3F, 3G, 3H, 3M, 3N, 3O, 3P; scale bar: 1 µm) in diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days. St: starch; Pg: plastoglobule; Mt: mitochondria; Gr: granum.

Figure 3 .

 3 Figure 3. Transmission electron migrographs of spongy mesophyll cells (3A, 3B, 3C, 3D, 3I, 3J, 3K, 3L; scale bar: 5 µm) and their respective chloroplasts (3E, 3F, 3G, 3H, 3M, 3N, 3O, 3P; scale bar: 1 µm) in diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) for 210 days. St: starch; Pg: plastoglobule; Mt: mitochondria; Gr: granum.

Figure 4 .

 4 Figure 4. Determination of (A) net photosynthetic rate (Pnet), (B) stomatal conductance (Gs), and (C) chlorophyll a fluorescence (Fv/Fm) after 210 days of nutrient deficiency in leaves of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-

Figure 5 .

 5 Figure 5. Leaf damages after 210 days of total nutrient deficiency.

Figure 6 .

 6 Figure 6. Total content of nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe), manganese (Mn), copper, zinc (Zn) and boron (B) in leaves of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x) Volkamer lemon grown in nutrient reference solution (100%) and without nutrient solution (0%) after 210 days. The white circles correspond to the control (100%) and the black circles to the plant without nutrition (0%). Values are mean (± standard error) of 3 independent measurements from 3 samples for each genotype, i.e. one per tree, obtained by pooling 8 fully-expanded leaves (n = 3). Data were analysed using ANOVA and Fisher LSD tests (P < 0.05). Different letters indicate significant differences between genotypes and treatments.

Figure 7 .

 7 Figure 7. Determination of specific activities of (A) superoxide dismutase (SOD), (B) catalase (CAT) and (C) ascorbate peroxidase (APX) and (D) hydrogen peroxide (H202) and (E) malondialdehyde (MDA) after 210 days of nutrient deficiency in leaves of diploid (CM2x) and doubled-diploid (CM4x) Citrumelo 4475 and diploid (VK2x) and doubled-diploid (VK4x)

  Fig. 4

  Fig 6.

  Fig. 7

  Initial data were subjected to a two-way analysis of variance. The qualitative factors studied were genotype, ploidy, treatment and their interaction. The analyses were performed on a group of three trees. The p values showing the level of significance of each qualitative factor are presented.

  subjected to a two-way analysis of variance. The qualitative factors studied were genotype, ploidy, treatment and their interaction. The analyses were performed on a group of three trees. The p values showing the level of significance of each qualitative factor are presented.

Table 2 .

 2 Probability values produced by analysis of variance (ANOVA) on palisadic mesophyll cells ultrastructural datasets of experimentation conditions.

			Cells	Chloroplasts	Starches		Plastoglobuli		Mitochondria		Grana
		Length (μm)	Width (μm)	Thickness (μm)	Number	Length (μm)	Width (μm)	Number	Length (μm)	Width (μm)	Number	Length (μm)	Width (μm)	Number	Length (μm)	Width (μm)	Number of grana per cell	Length (μm)	Width (μm)	Number of thylakoids per grana
	A (genotype) 0.367 0.013	10 -4	0.149 10 -12 10 -15	10 -3	0.145 10 -10	10 -4	0.141 0.573 10 -3	0.032 0.800	10 -4	0.054 10 -6	0.010
	B (ploidy)	10 -8	10 -3	10 -5	0.002 0.005 0.505 0.048 0.242 10 -6	0.91 0.876 0.849 0.573 0.535 0.330	10 -3	0.389 0.810	0.642
	C (treatment) 0.093 0.008 0.048	10 -8	0.962 0.878 10-8 0.529 0.013 0.714 10 -4	10	-8	10 -4	0.088 0.211	NA	NA	NA	NA
	D (A x B x C) 10

Acknowledgements

Julie Oustric received grant support from the "Collectivité Territoriale de Corse" (CTC). This work was funded by the "Collectivité Territoriale de Corse" as part of the research project "Sélection de porte-greffes pour le développement d'une agrumiculture durable en Corse". We thank Jean-Claude Ribaut for hosting this study at the AREFLEC experimental station, and