Teyssier, C Alexandre Morel

Caroline ; Teyssier

Jean-François Trontin

Kateřina Eliášová

Bedřich Pešek

Martine Beaufour

Domenico Morabito

Nathalie Boizot

Claire Le Metté

Leila Belal-Bessai

HARVENGT Isabelle ; Fcba

Luc ; Fcba

Isabelle Reymond

Luc Harvengt

Martine Cadene

Françoise Corbineau

Martin Vágner

Label

Marie-Anne Lelu-Walter
email: marie-anne.lelu-walter@orleans.inra.fr

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses

Keywords: somatic embryo development, germin-like protein, glycolysis, protein phosphatase 2C, ubiquitin-protein ligase, embryonal masses; SE, somatic embryo

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

Introduction 1

Maritime pine, Pinus pinaster Ait., is industrially important in South-Western Europe, especially in 2 France, Portugal and Spain. Maintenance of plantation productivity, together with increasing forest 3 resilience to major biotic and abiotic risks, are thus major objectives for this species. In order to 4 bypass the current limitations of classical methods for accelerating tree domestication, technological 5 innovation is required, and this may result from any of a number of biotechnological inputs into 6 improvement programs (Harfouche et al. 2012).

7

Biotechnology offers new opportunities for improving selection efficiency (balancing genetic gain and 8 genetic diversity), conservation and deployment of new varieties for reforestation. As a high-9 performance, clonal propagation system, somatic embryogenesis is a powerful tool for scaling up 10 production of genetically improved varieties (Park, 2002). During the last decade, methodology for 11 somatic embryogenesis in conifers has undergone rapid and significant progress (see review Lelu-12 Walter et al. 2013). However, somatic embryo (SE) maturation a challenging step (Stasolla et al. 13 2002) towards rapid production of uniform and consistent emblings quality up to the accepted standard 14 by foresters for conventional seedlings in commercial deployment of new varieties. Since the early 15 protocols were developed for spruce species (Attree and Fowke, 1993), it was assumed that they 16 would be applicable to pines; however, it subsequently became apparent that pines were less 17 responsive to these approaches. When effective, somatic embryogenesis could also result in qualitative 18 and quantitative differences in gene expression between somatic and reference zygotic embryos (Lara-19 Chavez et al. 2012;Pérez Rodríguez et al. 2006). Such distinctive features raised the important 20 question of achieving in pine similar SE development to high-quality seedlings.

21

With regard to zygotic embryogenesis, the genus Pinus differs markedly from Picea, the main 22 difference in Pinus being the type of cleavage that takes place at the late embryogeny stage (Dogra, 23 1978). In pine somatic embryogenesis, recovery of cotyledonary SE occurs only if the availability of 24 water to the embryonal masses is reduced (Klimaszewska and Smith, 1997). This is achieved by 25 increasing the gel strength of the medium, by using a high gellan gum concentration (Klimaszewska et 26 al. 2000). In maritime pine, SE maturation, which is a protracted process (requiring 12 weeks to reach 27 the cotyledonary stage), is obtained only on high gellan gum concentration, i.e. 9-10 g L -1 28 (Ramarosandratana et al. 2001;Lelu-Walter et al. 2006). In contrast, under low gellan gum 29 concentrations, e.g. 4 g L -1 as used for spruce species, embryonal masses undergo excessive 30 proliferation which interferes with embryo maturation.

31

In both angiosperms (Black, 1991) and gymnosperms (Kong et al. 1997), abscisic acid (ABA) is 32 involved in embryogenesis. Apart from certain species such as Pinus taeda (Kapik et al. 1995), ABA 33 content increases during seed maturation, reaches a maximum and then declines as the seed dehydrates 34 (reviewed by King, 1982). In Picea glauca the main source of ABA for the developing zygotic 35 embryo is from the surrounding tissue, the megagametophyte (Kong et al. 1997). Interestingly, for 36 conifers exogenous ABA promotes SE maturation through accumulation of storage products such as 37

Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu (Gutmann et al. 1996). ABA can regulate the switch from embryonal mass proliferation to 1 embryo development (Stasolla et al. 2002).

2

Many stress treatments, such as water stress, are known to induce de novo synthesis of ABA and 3 therefore to enhance endogenous ABA levels. These increases generally occur during the first days of 4 the treatment. Desiccation of hybrid larch SE was found to result in reduced water content associated 5 with a transient increase in ABA (Dronne et al. 1997). ABA signaling involves complex transduction 6 mechanisms in which there is cross-talk between serine/threonine kinase (SRK2) and protein 7 phosphatase 2C (PP2C) during ontogenesis (Umezawa et al. 2009).

8

In addition to water availability and ABA, carbohydrate supply is of great importance for pine somatic 9 embryogenesis. In Pinus pinaster, maturation of SE is enhanced by raising the sucrose concentration 10 to 0.2M (Lelu-Walter et al. 2006). Clearly, endogenous carbohydrate status varies throughout somatic 11 embryogenesis (see review by Lipavská and Konrádová, 2004). In general, during the proliferation 12 phase embryonal masses have a high hexose content and almost no sucrose, resulting in a high 13 hexose/sucrose ratio. On the other hand, maturation results in a decrease in hexose content whereas a 14 gradual increase in sucrose content is observed in the SE (Lipavská et al. 2000). Starch, accumulates 15 mainly during the first weeks of the maturation process.

16

In addition to these physiological studies, efforts have recently been made to describe SE development

32

Carbohydrate and starch assay. Soluble sugars were extracted as described by Black et al. (1996).

33

EMs (approximately 40 mg FW) were ground in 1 mL 80% aqueous ethanol containing melezitose as 34 an internal standard and lipids were removed using chloroform (1:1, v/v 12 Results, which were expressed as µg g -1 DW, corresponded to the mean of 3 (carbohydrates), 5 (total 13 protein) or 6 (starch) replicates ± confidence interval (CI) at the 5% error level.

15

Transcriptomic analysis

16

Maturation was carried out as described above. EMs were collected after 1 week of maturation on 4G 17 and 9G, frozen in liquid nitrogen, and ground for 5 min with a mortar and pestle to a fine powder.

18

Frozen powder samples were divided into two equal portions (each about 400 mg FW) for 19 transcriptomic and proteomic analyses. For transcriptomic analysis, three biological replicates were 20 sampled for each maturation conditions, producing a total of 6 samples.

21

Sequencing. Library construction and sequencing was done by a commercial service provider (GATC

22

Histological and biological characteristics of maritime pine EMs

23

EMs from proliferation medium consisted of single early SEs each being composed of several 24 meristematic cells and a few voluminous suspensor cells with starch grains present (Fig. 1a). After 25 transfer onto 4G maturation medium, embryos started to develop; however, after 2 weeks dead cells 26 were detected in meristematic centres (Fig. 1b). In the third week, dying or dead cells were observed 27 in the majority of suspensors (Fig. 1c). On 9G, meristematic centres gradually enlarged, and 28 suspensors became well organized, comprising a number of long narrow cells after 3 weeks (Fig 1d).

29

Vital staining showed that dead cells were mainly confined to very distal parts of suspensors. A 30 difference in starch grain distribution was clear after 2 weeks of maturation. On 4G, starch grains 31 spread throughout the suspensors and there were a few in basal parts of meristems (Fig. 1e). On 9G,

32

starch grains accumulated to a noticeably higher extent in suspensors and parts of meristems (Fig. 1f).

33

After 12 weeks, EMs on 4G showed overgrowth all over the filters but no embryos (Fig. 2a), whereas Compared with EMs matured on 4G, those transferred onto 9G showed no FW increase (Fig. 3A), but 1 there was a significant increase in their DW (P< 0.05, Fig. 3B) and decrease in their water content (P< 2 0.05, Fig. 3C). After 4 weeks, EMs matured on 9G had a significantly higher DW content (10.1% FW) 3 and lower water content (9.0 g H 2 O g -1 DW) than those on 4G (5.1% FW, and 19.0 g H 2 O g -1 DW; 4 Fig. 3B, 3C respectively). Availability of water from the maturation medium was significantly lower 5 on 9G (0.580 g H 2 O ± 0.009, mean ± CI at the 5% error level.) than on 4G (0.635 g H 2 O ± 0.008).

7

ABA content 8

At the beginning of the maturation period, the ABA content of EMs was low (2.1 nmol g -1 DW). EMs 9 subsequently showed a significant increase in endogenous ABA (Fig. 4) whereas ABA glucose ester 10 conjugate (ABA-GE) remained undetectable whatever the conditions tested (data not shown). On 9G,

11

ABA content increased slowly during the 3 first weeks of maturation but peaked at 21.1 nmol g -1 DW 12 after 4 weeks. A similar initial increase in ABA content was observed in EMs matured on 4G, but after 13 4 weeks of maturation, the ABA content was less than half (9.0 nmol g -1 DW) that on 9G.

15

Carbohydrate and total protein contents 16

At 1 week of maturation, similar amounts of total protein were observed for EMs developed on 4G or 17 9G (Table 1). In contrast, significant differences in carbohydrates contents were observed. EMs 18 matured on 4G had a greater amount of fructose (Fru, 27.4 µg g -1 DW) and glucose (Glu, 26.6 µg g -1 19 DW) but a similar sucrose content (Su, 6.88 µg g -1 DW) to that of EMs matured on 9G. In 20 consequence, the ratio (Fru+Glu)/Su was significantly higher for EMs matured on 4G than for those 21 matured on 9G (7.9 and 4.4, respectively). Other carbohydrates such as stachyose, raffinose, maltose 22 and mannose were not detected whatever the conditions. Finally, EMs matured on 9G contained a 23 significantly larger amount of starch (46.30 µg g -1 DW) than those matured on 4G (32.70 µg g -1 DW).

25

Transcriptomic analysis 26 1103 transcripts were considered to be differentially expressed, 632 for EMs matured on 9G and 471

27

for EMs matured on 4G (Appendix S1 in Supporting Information).

28

Data annotation by enrichment analysis and functional classification. Using the Blast2GO program, 29 significant transcripts were annotated against the Plants/Arabidopsis thaliana section of the protein 30 sequences database. Next, the annotated significant transcripts were mapped against the GO database 31 to retrieve the relevant GO terms. In total 4865 GO terms were assigned to the 529 mapped transcripts 32 with an average of five GO terms assigned to each transcript. Of these GO terms, the majority were 33 assigned to the Biological Process category (3118, 64%

16

Transcript expression levels. 17

Among the 471 transcripts overexpressed in EMs grown on 4G, the most prominent were 9 encoding 18 "pyruvate decarboxylase" with a mean of 12.0 FPKM, 7 encoding "alcohol dehydrogenase" (8.3 19 FPKM), 7 annotated as "bifunctional enolase 2" and related to a glucose catabolic process (15.2 20 FPKM), 1 annotated as "6-phosphofructokinase 2" involved in glycolysis (7.5 FPKM) and 1 annotated 21 as "sucrose synthase 3" responsive to oxygen levels (9.8 FPKM).

22

For EMs grown on 9G, the most interesting transcripts among the 632 that were overexpressed are 20 23 annotated as either "SUMO-conjugating enzyme" or "ubiquitin-conjugating enzyme" (25.6 FPKM)

24

with an ontology related to endosperm development, 5 encoding "Naringenin,2-oxoglutarate 3-25 dioxygenase", also known as flavanone 3-hydroxylase (F3H) and associated with a flavonoid 26 biosynthetic process (8.1 FPKM), 3 encoding "chitinase" (18.6 FPKM), 2 transcript for "protein 27 phosphatase 2C 16" which is putatively involved in negative regulation of the abscisic acid mediated 28 signaling pathway (28.3 FPKM), and 3 "serine/threonine-protein kinases" transcripts reported as being 29 involved in the positive regulation of abscisic acid-mediated signaling pathway, SRK2E (55.2 FPKM) 30 and SRK2H (18.3 FPKM). In addition, one transcript annotated as "pollen Ole e 1 allergen and 31 extensin family protein", but with a weak similarity of 47%, was the most highly over-expressed 32 9

Microscopic observations (Fig. 1) showed that EMs grown on 4G produced less structured immature 10 embryos and more undifferentiated tissues through cell proliferation (Fig. 2), an observation 11 corroborated by the increase in FW due mainly to water uptake throughout the culture period (Fig. 3).

12

In general, for pine species maturation in the presence of low concentrations of gelling agent sustains

6

This result again suggests that 4G may be a stressful culture condition for both cell proliferation and 7

(to an even greater extent) differentiation.

8

Our proteomic results revealed a high level of protein synthesis after 1 week of maturation on 4G. This 9 is probably a prerequisite for cell division. We identified many transcripts (cell division control 10 protein 48-e, growth-regulating factor 5 and mitogen-activated protein kinase 6) and proteins (DNA 11 replication licensing factor MCM3 homolog 3 and cell division protein FtsQ) involved in both DNA 12 replication and cell division on 4G. Because of the increase in FW and DW and the histological results

13

(Fig. 1), we propose that these genes and their products may be involved in cell proliferation. One of 14 the most significantly overexpressed proteins on 4G is related to a 26S proteasome regulatory subunit 15 which is involved in selective breakdown of proteins (Vierstra, 2003) but also in many other aspects of 16 cell regulation including embryogenesis and cell senescence. This is an interesting point, since we 17 observed necrotic cells in meristematic centers as early as 2-3 weeks after the start of maturation (Fig.

23

Which components appear during maturation of somatic embryos?

24

ABA is synthesized at a low level during the three first weeks of culture (Fig. 4), but peaks at week 4 25 when SE are differentiating, while the water content remains relatively high. On 9G the EMs are not 26 much dehydrated, even though water availability is relatively limited compared to that on 4G. The 27 major increase in ABA at week 4 suggests that there is an ontogenetic signal for SE development at

13 14Fresh2

 13 Weight (FW), Dry Weight (DW), and water content of EMs 15 Measurements were performed after 0, 1, 2, 3 and 4 weeks of maturation on both media. Each week, 16 each filter paper disc with dispersed EMs was weighed and replaced on the maturation medium. FW 17 of EMs was obtained by subtracting the mass of the filter paper disc from the total FW. DW was 18 determined after oven-drying at 70°C for 24 h and calculated by multiplying the DW to FW ratio by 19 100 (Teyssier et al. 2011). Water content was calculated as (FW-DW)/DW and expressed as g H 2 O g -1 20 DW (Dronne et al. 1997). Finally, water availability from the maturation media was determined by 21 measuring the amount of water absorbed by the filter paper disc (Teyssier et al. 2011). Each assay 22 (FW, DW, water content, water availability) was carried out on ten biological replicates for after 0, 1, 2 and 3 weeks of maturation on both media. EM viability was 27 determined by double staining with fluorescein diacetate (FDA) (Sigma-Aldrich) and propidium 28 iodide (PI) (Sigma-Aldrich) according to modified protocols (Jones and Senft, 1985; Petřek et al. 29 2005; Vondráková et al. 2010). A LSM5 Duo confocal laser scanning microscope (Zeiss, Jena, 30 Germany) equipped with an Argon/2 laser (FDA excitation at 488 nm, emission filter-set BP 505-550) 31 and a DPSS laser (PI excitation at 561 nm, emission filter-set LP 650) was used to visualize stained 32 EMs. Viable cells exhibit bright green fluorescence, non-viable or dead cells show bright red 33 fluorescence. Starch grains in squashed fresh EMs were stained with iodine/potassium iodide and 34 observed with a Jenaval transmission light microscope (Zeiss, Jena, Germany) equipped with a DS-355M digital camera (Nikon, Tokyo, Japan). All images were processed using the computer image 36 analysis system NIS Elements, version 3.2 (Laboratory Imaging, Prague, Czech Republic).Sample preparation and determination of ABA and ABA-GE. EMs were collected after 0, 1, 2, 3 and 4 3 weeks of maturation. Samples (each approximately 0.5 g FW) were ground to powder in liquid 4 nitrogen using a pestle and mortar, and each sample was mixed with 3 mL of modified Bieleski 5 solution; a further 2 x 3 mL of the solution was used to rinse the mortar and the three extracts were 6 combined. ABA and ABA-GE labeled with stable isotopes were added to the extraction mixture as 7 internal standards and the mixture was left overnight to extract. The extract was centrifuged and 8 applied on C18 SPE column (Strata C18-T, Grace, U.S.A.) to eliminate non-polar compounds. The 9 eluate was partly evaporated by the rotary vacuum evaporator (RVE) to approx. 3 mL and acidified by 10 adding of 1 mL of 1 M formic acid. The eluate was applied on the MCX SPE column (Oasis MCX, 11 Waters, U.S.A.), cleaned by 2 mL of 1 M formic acid, then eluted by 5 mL of 100 % methanol and 12 evaporated on the rotary vacuum concentrator to dryness. Dried samples were diluted in 100 µl of 13 15% (v/v acetonitrile:water) solution and filtered on Nylon 0.2 µm Micro-Spin (Grace, USA) filters. 14 The filtrates were divided into two parts, one for determination of ABA, the second for determination 15 of ABA-GE. 16 ABA determination by GC-MS. ABA was analyzed by GC-MS/MS (Vandenbusche et al., 2010). The 17 MS detector (ion-trap) was operated in MS/MS mode: MS/MS precursor 190.2, product full scan 65-18 200 amu for ABA; MS/MS precursor 194.2, product full scan 70-200 amu for labeled ABA. At the 19 first stage, MS full scan 50-300 amu was added to confirm the identity of the substance. A calibration 20 graph for each substance was constructed to cover the range 1-1000 ng mL-1 of final extract. 21 ABA-GE determination by LC-MS. A 5 µL sample of filtrate was injected into an LC-MS system 22 consisting of an HTS PAL autosampler (CTC Analytics, Switzerland), Rheos 2200 gradient HPLC 23 pump (Flux, Switzerland) and Quantum Ultra AM triple-quadrupole mass spectrometer (Thermo 24 Instruments, USA) equipped with an ESI interface. Separation was performed on an HPLC column, 50 25x 2.1 mm Kinetex C18 (Phenomenex, USA) using gradient elution with a mobile phase consisting of 26 acetonitrile, water and 1% acetic acid. The mass spectrometer was set to record SRM transitions 27 425→153 for ABA-GE and, as internal standard, 430→157 for D5-ABA-GE. A calibration graph was 28 constructed to cover the concentration range 1-100 ng mL-1 of extract. as described above and EMs were collected after 1 week.

  34 those on 9G yielded cotyledonary SE (Fig 2b), the embryogenic potential being estimated at 191 ± 23 35 (mean ± CI at the 5% error level). 36 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Physiologia Plantarum, 2014, 152(1), 184-201 http://dx.doi.org/10.1111/ppl.12158

  High 26S proteasome subunit activity suggests both active regulation of cell proliferation and 19 senescence on 4G. No related transcripts could be detected in the same samples, but transcript 20 abundance is not always well correlated with levels of the final products (Dembinsky et al. 2007; Pan 21 et al. 2012).

  28this stage, as has been observed in larch(Label and Lelu, 2000). For instance, many ubiquitin-protein 29 ligase transcripts are detected in 9G. More recent findings indicate that this enzyme is associated with 30 the activation of the small ubiquitin-related modifier (SUMO), leading to specific post-transcriptional 31 modifications of chromatin and thereby influencing gene transcription (Cubenas-Potts and Matunis, 32 2013). Our results suggest that in differentiated EMs, gene expression at week 1 may be undergoing 33 large-scale reorganization to promote SE development. In addition to the ubiquitin-protein ligase 34 transcripts, two proteasome subunit beta type-1 proteins were identified among the proteins over-35 expressed on 9G. The ubiquitin/proteasome pathway is reported to be a major factor in controlled 36 proteolysis, in which ubiquitinized proteins are recognized and catabolized by the 26S proteasome 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014

  18 developing and maturing wheat embryo in relation to its desiccation tolerance. J Exp Bot 47Harvengt L, Trontin J-F, Bouvet A, Favre J-M (2005) High subculture frequency, maltose-21 based and hormone-free medium sustained early development of somatic embryos in maritime pine. 22 In Vitro Cell Dev Biol Plant 41: 494-504 23 Caliskan M, Turet M, Cuming A (2004) Formation of wheat (Triticum aestivum L.) embryogenic 24 callus involves peroxide-generating germin-like oxalate oxidase. Planta 219: 132-140 25 Cairney J, Pullman GS (2007) The cellular and molecular biology of conifer embryogenesis. Wyse R, Hein M, Brenner ML (1984) Abscisic acid metabolism by source and sink tissues of 35 sugar beet. Plant Physiol74: 810-814 36 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014

3

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Electrophoresis 20: 1109-1119 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Physiologia Plantarum, 2014, 152(1), 184-201 http://dx.doi.org/10.1111/ppl.12158 1998) Mutational analysis of protein phosphatase 2C involved in abscisic acid signal 1 transduction in higher plants. Proceedings National Acad Sci 95: 975-980 2 Stasolla C, Kong L, Yeung EC, Thorpe T (2002) Maturation of somatic embryos in conifers: 3 morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol Plant 38Zoglauer K, Milhinhos A, Miguel C, Oliveira M (2007) Zygotic and somatic embryo 11 morphogenesis in Pinus pinaster: comparative histological and histochemical study. Tree Physiol 27Grondin C, Bonhomme L, Lomenech A-M, Vallance M, Morabito D, Label P, Lelu-14 Walter M-A (2011) Increased gelling agent concentration promotes somatic embryo maturation in 15 hybrid larch (Larix x eurolepis): a 2-DE proteomic analysis. Physiol Plant 141: 152-165 16 Teyssier C, Maury S, Beaufour M, Grondin C, Delaunay A, Le Metté C, Ader K, Cadene M, Label P, 17 Lelu-Walter M-A (2013) In search of markers for somatic embryo maturation in hybrid larch (Larix 18 x eurolepis): global DNA methylation and proteomic analyses. Physiol Plant (in press) doi: F, Shealy RT, Khanna A, Vodkin LO (2003) Clustering of microarray data reveals 21 transcript patterns associated with somatic embryogenesis in soybean. Plant Physiol 132: 118-136 22 Trontin J-F, Harvengt L, Garin E, Vernaza ML, Arancio L, Hoebeke J, Canlet F, Paques M (2002) 23 Towards genetic engineering of maritime pine (Pinus pinaster Ait.). Annals of Forest Science 59Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama 26 Y, Hirayama T, Shinozaki K (2009) Type 2C protein phosphatases directly regulate abscisic acid-27 activated protein kinases in Arabidopsis. Proceedings National Acad Sci 106: 17588-17593 28 Vandenbussche F, Petrasek J, Zadniková P, Hoyerová K, Pesek B, Raz V, Swarup R, Bennett M, 29 Zazimalova E, Benkova E, Van Der Straeten D (2010) The auxin influxcarriers AUX1 and LAX3 30 are involved in auxin-ethylene interactions during apical hook development in Arabidopsis thaliana 31 seedlings. Development 137: 597-606 32 Vierstra RD (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of 33 many plant proteins. Trends Plant Sci 8: 135-142 34 Vernoud V, Horton AC, Yang Z, Nielsen E (2003) Analysis of the small GTPase gene superfamily of 35 arabidopsis. Plant Physiol 131:1191-1208 36 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014

Fig. 1 .

 1 Figure legends:

Fig. 2 .

 2 Fig. 2. Morphological appearance of embryonal masses of maritime pine matured for 12weeks on either 4 g L -1 (a) or 9 g L -1 (b) gellan gum. Note embryonal mass proliferation on 4G (a1, a2) and presence of cotyledonary somatic embryos on 9G (b1, b2). Scale bar = 1cm (a1, b1), 1mm (a2, b2).

Fig. 3 .

 3 Fig.3. Biological characteristics of immature somatic embryos of maritime pine during maturation in the presence of either low (4 g L -1 , ) or high (9 g L -1 , ) gellan gum. A, Fresh weight (FW); B, Dry weight (DW); C, Water content. Bars represent confidence interval at the 5% error level. Letters represent statistical groups defined by the Multiple Comparisons of Means method (P<0.05, N = 10).

Fig. 4 .

 4 Fig.4. Endogenous abscisic acid (ABA) content of immature somatic embryos of maritime pine during maturation in the presence of either low (4 g L -1 , ) or high (9 g L -1 , ) gellan

Fig. 5 .Figure 3 .Figure 4

 534 Fig.5. Gene ontology (Biological Process categories at level 2) terms found to be significantly enriched after 1 week of maturation with either low (4 g L -1 , ) or high (9 g L -1 , ) gellan gum. Only GO categories for which >5 genes were represented are included. To test whether Gene Ontology terms were enriched in a test group, when compared to a reference group, Fisher's Exact Test with Multiple Testing Correction of FDR (FDR<0.05, Benjamini and Hochberg, 1995) was used.Numbers of sequences: Numbers of sequences involved in each biological process.

EMs when somatic embryo maturation is hindered?

		Physiologia Plantarum, 2014, 152(1), 184-201 Physiologia Plantarum, 2014, 152(1), 184-201	http://dx.doi.org/10.1111/ppl.12158 http://dx.doi.org/10.1111/ppl.12158
	1 1	Appendix S3 in the Supporting Information). The gellan gum concentration had no significant effect embryogenesis in white pine (Klimaszewska et al. 2000), and Scots pine (Lelu-Walter et al. 2008),
	2 2	(t-test, P<0.05), on the normalized volumes of most spots. Significant differences were detected for where high gellan gum concentrations were linked to lower water availability and enhanced SE
	3 3	only 83 spots, 35 with greater abundance on 4G and 48 on 9G. Of the significant spots, 58 were picked development. Low water and carbon source availability also had a major impact on SE maturation and
	4 4	for analysis by mass spectrometry. Protein identification was successful for 56 spots, 27 of which were germination in carrot (Lee et al. 2001), suggesting that the induction of embryo development by
	5 5	overproduced on 4G and 29 on 9G (Table 2). Detailed information on the statistical parameters used temporary growth starvation observed in natural conditions can be mimicked in vitro in a broad range
	6 6	for protein identification is given in Appendix S4 in the Supporting Information. All identified of species. Our results indicate that gellan gum concentration and associated modulation of water
	7 7	proteins were grouped into functional categories within the Biological Process ontology (level 2) using availability in the medium is critical in controlling SE development in maritime pine.
	8 8	Blast2GO (Appendix S5 in Supporting Information). A striking result of this classification was that
	9	around 50% of all spots identified were placed in only three categories: metabolic process, cellular What happens in
	10	process and response to stimulus.
	11	F o F o
		r r
		P P
		e e r e e r
	26 27	R e v i e w pair that we obtained mapped specifically to one reference transcript, and the results indicate some R e v i e w
	28	diversity in the range of transcripts deriving from different gene families; further characterization of
	29	these families at the genome level is still necessary. We decided to limit data analysis to the genes that
	30	were most differentially expressed according to FPKM ratio statistics. These genes were grouped on
	31	the basis of GO annotation at the Biological Process level. From this data analysis, it appears that EMs
	32	expressed markedly different set of genes according to the maturation medium used.
	33
		Does gellan gum concentration control maturation of somatic embryos?
		12 13

transcript, at 230.4 FPKM. 33 34 2-D gel proteomic analysis 35 Changes in EM protein profiles were investigated using 2D gel electrophoresis. Separation with high 36 resolution was achieved, allowing detection of a total of 1,428 spots (representative gel shown in 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Discussion 12 We determined key molecular, physiological and biological parameters in order to develop a 13 comprehensive picture of SE maturation in maritime pine. In addition to SE growth and water content, 14 we measured endogenous ABA, total protein, soluble carbohydrate and starch contents. We also 15 tentatively identified as many protein and transcript levels as possible in order to gain information 16 about the cellular environment as a whole. For this study, we used a culture medium parameter 17 specifically appropriate for pine species, namely gellan gum concentration, to trigger the maturation 18 process. Transcriptomic and proteomic analyses were performed at week 1 after initiation of cultures, 19 whereas other parameters were monitored weekly. This sampling strategy was designed to focus 20 mainly on early molecular mechanisms (as reflected in transcript and protein profiles) that could be 21 involved in subsequent physiological modifications promoting SE development. 22 Since the Pinus pinaster genome has yet not been completely sequenced, we are aware that it is not 23 currently possible to infer numbers of genes per gene family. 'Hence, we cannot estimate whether 24 variant transcripts detected in this study represent alternative transcripts from individual members of 25 gene families or are products of paralogs. 'However, we can state with confidence that each short-read 34 When compared to the lower concentration, 4G, it appears that on 9G, maritime pine SE differentiated 35 better, based on macroscopical and histological observations (Fig. 1, 2), and on measurements of DW 36 and water content (Fig. 3). Our results reinforce findings previously reported for somatic 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in :

  Version définitive du manuscrit publié dans / Final version of the manuscript published in :

		Physiologia Plantarum, 2014, 152(1), 184-201	http://dx.doi.org/10.1111/ppl.12158
	1	glycolysis pathway genes) that could result in the rapid onset (within 1 week) of alcoholic
	2	fermentation (expression of anoxia-related genes) and cell senescence.
	3	Additionally, in EMs grown on 4G we observed over-expression of several bifunctional enolase-2
	4	transcripts which may be related to abiotic stress-regulated transcriptional networks (Chinnusamy et
	5	al. 2007). Bifunctional enolase 2, also known as low expression of osmotically responsive genes 2
		(LOS2), appears to be a negative regulator of transcription factors involved in abiotic stress responses.
		F o
		r
		P
		e e r
	23 24 25 26 27	2004). Transcriptionally, our results show an increased level of expression of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) genes in 4G-grown EMs, along with over-expression of sucrose synthase. At the proteomic level, ADH, phosphoglycerate kinase and PDC isozyme 2 are also R e v i e w
	28	significantly up-regulated. The two datasets are consistent, and support possible stimulation of the
	29	glycolytic pathway, as also suggested by the results of carbohydrate analysis. However a recent paper
	30	on Arabidopsis reported that ADH and PDC enzymes and their corresponding transcripts could be
	31	modulated to overcome unfavorable conditions such as anoxia brought about by submergence in
	32	culture medium (Mithran et al. 2013). In addition, utilization of sucrose, provided in our study by the
	33	culture medium, is important in anoxia tolerance mediated by alcoholic fermentation (Loreti et al.
	34	2005). These findings, taken together with our results, suggest that for EMs, 4G is i) a condition
		14

13

proliferation of immature SE but is detrimental to SE development

(Klimaszewska et al. 2000)

.

14

In 4G-grown EMs, endogenous synthesis of ABA is limited and there is no detectable ABA 15 conjugation, at least via ABAGE synthesis. We avoided exogenous application of ABA at levels high 16 enough to obscure endogenous ABA synthesis by several orders of magnitude because of the 17 occurrence of high levels of ABA conjugation (Daie et al. 1984) as reported for larch (Label and Lelu, 18 2000). 19 Carbohydrate contents in EMs cultured on 4G is characterized by a high hexose:sucrose ratio, and 20 fairly low starch accumulation which is in accordance with microscopic observations (Fig. 1e). These 21 results suggest stimulation of the glycolytic pathway, possibly related to cell proliferation (Fig.2a). A 22 high hexose:sucrose ratio has been observed in proliferating conifer EMs (Lipavská and Konrádová, unfavorable for SE differentiation and maturation, possibly due to an excess of water at this low gellan 35 gum concentration and ii) a suboptimal condition for proliferation (indicated by the expression of 36 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : This pathway supplies amino acids for the biosynthesis of new proteins(Vierstra, 2003). A 1 recent paper (Rode et al. 2012) links high levels of enzymes involved in the proteasome-dependent 2 proteolysis pathway and the switch from callus to globular embryos in cyclamen.Version définitive du manuscrit publié dans / Final version of the manuscript published in :Version définitive du manuscrit publié dans / Final version of the manuscript published in :

			Physiologia Plantarum	Page 18 of 37
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	embryo stage (Zhen et al. 2012). It has been suggested that the ability of embryos to develop an efficient redox homeostasis system may be key for successful regulation and maintenance of cell phosphatase 2C (PP2C) may be also a suitable marker for monitoring adaptive molecular responses to favourable maturing conditions that may have a beneficial impact on regeneration capacity of cultures. contract 09012579-045), and the French Ministry of Foreign Affairs and the French Ministry of Higher Education and Research through the France/Czech Republic Science Cooperation Bibliography Andème-Ondzighi C, Christopher DA, Cho EJ, Chang S-C, Staehelin LA (2008) Arabidopsis protein complex. 3 1 2 1 2 1 2 1 2 differentiation. 3 The identification of relevant biomarkers at the molecular level needs validation experiments but has 3 BARRANDE Programme. Data analysis was made possible through the involvement of INRA's 3 disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed 3
	4 4 4 4 4	During somatic embryogenesis in gymnosperms, chitinases are reportedly excreted into the culture Our results also show that many transcripts related to F3H, as well as several other transcripts related clear practical implications for implementation of somatic embryogenesis in both conifer fundamental GenoToul bioinformatics platform in Toulouse (France). cell death of the endothelium in developing seeds. Plant Cell 20: 2205-2220
	5 5 5 5 5	medium. These enzymes are involved in the regulation of the EM-to-SE transition and massive to flavonol metabolism, are over-expressed in 9G-grown EMs. F3H catalyses hydroxylation of research and commercial applications. Functional genomics based on reverse genetics in maritime pine Attree SM, Fowke LC (1993) Embryogeny of gymnosperms: advances in synthetic seed technology of
	6 6 6 6 6	programmed cell death (Wiweger et al. 2003; Vestman et al. 2011). Our results showed over-flavanones to dihydroflavonols. Flavonoids are ubiquitous in plants, and some of these compounds is a long process requiring transgenic plant regeneration through somatic embryogenesis and conifers. Plant Cell Tiss Org 35: 1-35
	7 7 7 7	expression of transcripts for chitinase in EMs grown on 9G. In Norway spruce, programmed cell death play specific roles in seed development (Lepiniec et al. 2006). Among these roles, stress resistance is a cryopreservation of selected transgenic lines (Trontin et al. 2002). Labor intensiveness would be Beaufour M, Godin F, Vallée B, Cadene M, Bénédetti H (2012) Interaction proteomics suggests a new
	8 8 8 8	is responsible for the degradation of EMs when early SEs differentiate (Vestman et al. 2011). The key biological function which leads to the synthesis of several secondary compounds all related to significantly decreased if the regeneration capacity of transgenic lines could be predicted from early role for the Tfs1 protein in yeast. J Proteome Res 11: 3211-3218
	9 9 9 9	timing of programmed cell death can also be modulated by disulfide isomerase, which was up-flavanones hydroxylation and subsequent metabolism down to condensed tannins (Winkel-Shirley, molecular monitoring of rapid maturation tests (1 week). Furthermore untargeted (genome-wide) or Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: a practical and powerful
	10 10 10 10	regulated on 9G. This enzyme seems to be required for proper embryo development in Arabidopsis 2002). Our results suggest that EMs cultivated on 9G may overcome constraints imposed by culture targeted transcriptomic and proteomic profiling (candidate genes and proteins markers) may be approach to multiple testing. J Royal Stat Society, Series B 57: 289-300
	11 12 13 11 12 13 11 12 13 11 12 13	F o seeds (Andème-Ondzighi et al. 2008). Expansin S2 and putative expansin-B14 are known to play roles in embryogenesis. In Arabidopsis, they are involved in the cell cycle, as key regulators of wall and cell expansion (Thibaud-Nissen et al. 2003; Spencer et al. 2007). The proteins tubulin beta-2 chain and F o conditions by synthesizing protective secondary metabolites. This does not seem to occur in EMs grown on 4G, where many stress response-related transcripts were identified as discussed above. At the proteomic level, two enzymes (glucose-1-phosphate adenylyltransferase AGPP; 4-alpha-F o invaluable for qualitative and quantitative selection of elite embryogenic lines that are receptive to maturing conditions and productive enough for breeding application (clonal and varietal tests) and new variety deployment in the frame of multivarietal forestry. F o Bernier F, Berna A (2001) Germins and germin-like proteins: Plant do-all proteins. But what do they do exactly? Plant Physiol Bioch 39: 545-554 F o Bergmeyer HU, Bernt E, Schmidt F, Stork H (1974) D-glucose. In: Bergmeyer HU (ed) Methods of
	14 14 14 14	r GTP-binding nuclear protein Ran-A1 participate in cytoskeletal process such as nucleocytoplasmic r glucanotransferase DPE2, disproportionating enzyme) reportedly involved in starch biosynthesis r r r enzymatic analysis, vol III. Verlag Chemie, Weinheim, pp 1196-1201
	15 16 15 16 15	P transport (Pan et al. 2009; Vernoud et al. 2003). All these proteins, which are up-regulated in 9G, may direct cell division towards cell remodeling for embryogenesis, a hypothesis consistent with our P P Author contributions P P Black M (1991) Involvement of ABA in the physiology of developing and mature seeds. In: Davies
	19	histological observations.	e e r e e r e e r e e r e e r
	21 22 23 24 25 26 27 21 22 23 24 25 26 27 20 21 22 23 24 25 26 27	R e v i e w Endogenous ABA synthesis was significantly enhanced on 9G, i.e when water availability was reduced. In addition, the detection of highly over-expressed protein phosphatase 2C (PP2C) transcripts in 9G grown EMs reinforces the likelihood of such a -possibly adaptive -molecular response (Sheen, 1998). PP2Cs are reported to be key protein-protein interactors mediating ABA responses in plants (Yoshida et al. 2006). Our results suggest that immature SE respond to reduced availability of water in the maturation medium through ABA-mediated molecular mechanism. R e v i e w Conclusions This is the first study on the early molecular mechanisms involved in somatic embryogenesis of pine following an increase in gellan gum concentration in the medium, and it is also the first report combining transcriptomic and proteomic data on somatic embryogenesis in conifers. Our transcriptomic and proteomic results indicate that glycolysis is enhanced under conditions R e v i e w embryogenesis and drafted the manuscript. KE carried out histological and microscopical analysis and drafted the manuscript. BP carried out ABA and ABA GE analyses. MB carried out Gel spot processing and protein identification by database sequence assignment. DM participated in the design of the study and helped to draft the manuscript. NB carried out carbohydrate analyses and helped to draft the manuscript. CLM carried out somatic embryogenesis and collected the material. LB-B participated in 2-D gel proteomic analyses. IR carried out somatic embryogenesis and collected the material. LH participated in the design of the study and helped to draft the manuscript. MC participated in gel spot processing and protein identification by database sequence assignment and R e v i e w R e v i e w
	28 28 28	Two superoxide dismutases are overexpressed at the proteomic level, as is one germin-like protein. unfavourable for SE maturation (4G) which lead to EM proliferation. Under favourable conditions helped to draft the manuscript. FC participated in study design, participated in carbohydrate analysis
	29 29 29	This latter is also detected at the transcriptomic level, suggesting an ongoing process of antioxidant (9G), we observed adaptive, ABA-mediated molecular and physiological responses to reduced water and drafted the manuscript. MV participated in the design of the study and drafted the manuscript. PL
	30 30 30	protein production through mRNA synthesis EMs growing on 9G. The role of germin-like proteins in availability resulting in early transition of EMs from proliferation to the SE developmental pathway conceived in the design of the study, carried out transcriptomic analysis, performed the statistical
	31 31 31	plant development and defense is well documented (Bernier and Berna, 2001). Their involvement as (indicated by active protein synthesis, and overexpression of proteins involved in cell division, analysis and drafted the manuscript. MALW conceived in the design of the study and its coordination,
	32 32 32	markers of early stages of somatic embryogenesis in both conifers (Neutelings et al. 1998; Mathieu et embryogenesis and starch synthesis). Specific pathways (synthesis of protective secondary and drafted the manuscript. All authors read and approved the final manuscript.
	33 33 33	al. 2006) and angiosperms (Caliskan et al. 2004; Kim and Triplett, 2004) makes them especially metabolites, regulation of oxidative stress) are concomitantly activated, apparently to overcome
	34 34	valuable in this context. Superoxide dismutase is involved in the regulation of oxidative stress. It constraints due to culture conditions.
		15 16 17 18 19

). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water 17 Several serine/threonine kinase (SRK2) transcripts are highly over-expressed in EMs growing on.

18

SRK2s are plant homologs of AMPK/SNF1 genes, which in other eukaryotes are considered to be 19 crucial in linking stress and metabolic responses

(Kulik et al. 2011)

. They are reported to be key 20 players in osmotic stress responses and ABA-dependent plant development

(Fujii et al. 2011)

.

destroys free radicals which are normally produced within the cells and are toxic to biological systems.

35

In a proteomic study on the early stages of seed development in Pinus massoniana, a correlation was 36 established between the level of this protein and the transition from the pro-embryo to the columnar 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water (Keeling and Myers, 2010) were found to be overexpressed on 9G. This is consistent with the high 15 starch content of immature SE cultivated in these favorable conditions (Fig. 1, Table 1). It is 16 interesting to note that glucose and fructose contents were correlated with the difference in starch grain 17 accumulation on 4G and 9G (Fig. 1e, f; Table 1). In maritime pine, a low abundance of starch granules 18 was reported in proliferating EMs (Breton et al. 2005) whereas starch was found to accumulate 19 throughout developing embryos during early maturation (Tereso et al. 2007). A similar picture was 20 reported during larch SE maturation (Gutmann et al. 1998).

34 Based on both our transcriptomic and proteomic data and related information available in the 35 literature, we proposed the germin-like protein and ubiquitin-protein ligase as potential marker genes 36 and proteins for early evidence of effective SE development on maturation medium. Protein-37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water AM participated in the acquisition of the data, carried out all types of analyses and drafted the 17 manuscript. CT conceived in the design of the study, participated in 2-D gel proteomic analysis and 18 drafted the manuscript. JFT conceived in the design of the study, participated in somatic Acknowledgments 35 This work was supported by grants from the French "Conseil Régional de la Région Centre" 36 (EMBRYOME project, contract 33639), the "Conseil Régional de la Région Aquitaine" (Embryo2011, 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water WJ, jones HG (eds) Abscisic acid: physiology and biochemistry. Oxford: BIOS Scientific Publisher 16 pp 99-124 17 Black M, Corbineau F, Grzesik M, Guy P, Côme D (1996) Carbohydrate metabolism in the

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). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining 16 with fluorescein diacetate-propidium iodide. J Histoch Cytoch 33: 77-79 17 Kapik RH, Dinus RJ, Dean, JFD (1995) Abscisic acid and zygotic embryogenesis in Pinus taeda. regulators of plant response to abiotic stresses. Omics: a journal of integrative biology 15: 859-872 35 Label P, Lelu MA (2000) Exogenous abscisic acid fate during maturation of hybrid larch (Larix x 36 leptoeuropaea) somatic embryos. Physiol Plant 109: 456-462 37 Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water

Table 1 .

 1 ). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in : Carbohydrate and protein content in maritime pine immature somatic embryos of 2 maritime pine matured for one week in the presence of either 4 or 9 g L -1 gellan gum. Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Version définitive du manuscrit publié dans / Final version of the manuscript published in :

	Physiologia Plantarum, 2014, 152(1), 184-201	http://dx.doi.org/10.1111/ppl.12158

Table 2 :

 2 Characteristics of differentially expressed proteins in immature somatic embryos of maritime pine matured for 1 week on either 4 or 9 g L -1 gellan gum. Excised spot number refers to the spot marked in Fig.S2. The protein assignments and accession numbers were retrieved from the GenoToul database (http://genotoul-contigbrowser.toulouse.inra.fr:9092/Pinus_pinaster2/index.html) by the PEAKS identification engine.The most homologous protein in the UniProt database (http://www.uniprot.org/) and its species affiliation are given. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Version définitive du manuscrit publié dans / Final version of the manuscript published in : Version définitive du manuscrit publié dans / Final version of the manuscript published in : Version définitive du manuscrit publié dans / Final version of the manuscript published in : Physiologia Plantarum, 2014, 152(1), 184-201 http://dx.doi.org/10.1111/ppl.12158

	Gellan gum: protein relative abundance (9>4 : protein over-expressed on 9G, 4>9 : protein over-expressed on 4G).
	th: theoretical; exp : expected
	Spot	Accession number	Gellan	Protein name	UniProt	Organism	MW	pHi
	number		gum		accession		th./exp.	th./exp.
	5 SC_RP12A_ARATH.1.6	4>9 26S proteasome regulatory particle	Q0GEA1 Camellia sinensis	31 / 26.3	4.95 / 4.96
				subunit
	56 SC_STAD_RICCO.1.4	4>9 Acyl-[acyl-carrier-protein] desaturase	A9NVD4 Picea sitchensis	46.9 / 44.3	5.65 / 5.5
	36 SC_00001024.1.1	4>9 Alanine aminotransferase	Q84UX4 Oryza sativa	53.6 / 52.3	8.01 / 5.9
	30 SC_ADH3_SOLTU.1.15	4>9 Alcohol dehydrogenase	Q43025	Pinus banksiana	40.5 / 44.6	6.12 / 6.02
	34 SC_ADH_FRAAN.16.30	4>9 Alcohol dehydrogenase	Q43025	Pinus banksiana	40.5 / 46.4	6.12 / 5.86
	32 SC_00002407.1.1	4>9 At5g54100 (Similarity to stomatin like	Q9LVW0 A. thaliana	43.6 / 47.2	9.11 / 5.96
				protein)
	59 SC_00001003.1.1	4>9 BAHD family acyltransferase, clade V	D8T9Y4	S. moellendorffii	49.4 / 55.4	5.91 / 5.5
	10 SC_RRAA3_ARATH.2.3	4>9 Cell division protein FtsQ	A3TJS8	Oryza sativa	18 / 9.8	5.6 / 5.7
	55 SC_CH62_CUCMA.1.9	4>9 Chaperonin CPN60-2, mito.	Q05046	Cucurbita maxima	57.6 / 52.7	5.35 / 5.67
	62 SC_CH62_CUCMA.2.9	4>9 Chaperonin CPN60-2, mito.	Q05046	Cucurbita maxima	57.6 / 61.5	5.35 / 5.47

Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water Comment citer ce document : Morel, A., Teyssier, C., Trontin, J.-F., Eliášová, K., Pešek, B., Beaufour, M., Morabito, D., Boizot, N., Le Mette, C., Belal-Bessai, L., Reymond, I., Harvengt, L., Cadene, M., Corbineau, F., Vágner, M., Label, P., Lelu-Walter, M.-A. (Auteur de correspondance) (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water

Physiologia Plantarum

Supporting information

1 Additional Supporting Information may be found in the online version of this article: 2 3 Appendix S1 Identification of differentially expressed transcripts in immature somatic embryos of 4 maritime pine matured for 1 week on either 4 or 9 g L -1 gellan gum.