Proteome-Wide Characterization of Sugarbeet Seed Vigor and Its Tissue Specific Expression

Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression

Julie Catusse*†, Jean-Marc Strub†‡, Claudette Job*, Alain Van Dorsselaer†, and Dominique Job*§

*Centre National de la Recherche Scientifique-UniversiteĆlaude Bernard Lyon 1, Institut National des Sciences Appliqueés–Bayer CropScience Joint Laboratory, Unite´Mixte de Recherche 5240, Bayer CropScience, 14-20 rue Pierre Baizet, F69263 Lyon Cedex 9, France; and ‡Laboratoire de Spectrome´trie de Masse Bio-Organique, De´partement des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien, Unite´Mixte de Recherche 7178, Centre National de la Recherche Scientifique-Universite´Louis Pasteur, Ecole Europeénne de Chimie, Mate´riaux et Polyme`res, 25 rue Becquerel, F67087 Strasbourg Cedex 2, France

Edited by Roland Douce, Universite´de Grenoble, Grenoble, France, and approved April 11, 2008 (received for review January 19, 2008) Proteomic analysis of mature sugarbeet seeds led to the identifi- The use of metabolic inhibitors (␣-amanitin and cyclohexi- cation of 759 proteins and their specific tissue expression in root, mide) showed that transcription is not required for the comple- cotyledons, and perisperm. In particular, the proteome of the tion of germination in Arabidopsis, implying that the potential of perispermic storage tissue found in many seeds of the Caryophyl- germination is largely programmed during seed maturation on lales is described here. The data allowed us to reconstruct in detail the mother plant (4). Therefore, in this work, we have charac- the metabolism of the seeds toward recapitulating facets of seed terized sugarbeet seed¶ vigor by proteomics. This was challeng- development and provided insights into complex behaviors such as ing, however, because there are virtually no genomics data germination. The seed appears to be well prepared to mobilize the presently available on this plant that could be used for protein major classes of reserves (the proteins, triglycerides, phytate, and identification, but recent successes illustrated the ability of mass starch) during germination, indicating that the preparation of the spectrometry to identify and quantify thousands protein profiles seed for germination is mainly achieved during its maturation on from diverse species (5–6). By using this approach, we have also the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic determined the tissue specificity of the accumulation of the seed trait defined as the potential to produce vigorous seedlings, such proteins, allowing us to described the proteome of the perisperm as glycine betaine accumulation in seeds. This study also identified storage tissue. several proteins that, to our knowledge, have not previously been Results and Discussion described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional Proteome-Wide Analysis Allows Metabolic Network Reconstruction in cap-dependent mechanism or by a cap-independent process. The Sugarbeet Seeds. Of 784 protein spots submitted to proteomic study of the tissue specificity of the seed proteome demonstrated analysis, we identified 759 proteins [Fig. 1, supporting informa- a compartmentalization of metabolic activity between the roots, tion (SI) Figs. S1–S3, Table S1, and Table S2], of which the cotyledons, and perisperm, indicating a division of metabolic tasks majority is associated with unique proteins. Seventy spots gave between the various tissues. Furthermore, the perisperm, although two identifications, and 14 spots gave three identifications (Table it is known as a dead tissue, appears to be very active biochemi- S3). This corresponds to an overall success rate of Ϸ80%. cally, playing multiple roles in distributing sugars and various Metabolic network reconstruction is a fundamental task in metabolites to other tissues of the embryo. systems biology with an ultimate goal of full-scale in silico simulations (7). Based on ontological classification (8) and proteomics ͉ germination ͉ perisperm ͉ Amaranthaceae established features of metabolism, notably in plants (refs. 9 and 10 and http://metacyc.org), the metabolism of the sugarbeet seed ugarbeet (Beta vulgaris L.) is a dicotyledonous plant of the can be reconstructed by 121 biochemical functions, covering 561 SAmaranthaceae family that has a high economic importance of the 759 proteins presently identified (Table S4) and showing because it is one of the two main sources of sucrose, the other a tight organization (Fig. 2). Several metabolic modules have being sugarcane. Furthermore, there is growing interest in the been identified in a complete manner, such as glycolysis, fatty use of this crop to produce bioethanol. The quality of seed acid ␤-oxidation, glyoxylate cycle, protein degradation, or starch germination has a direct impact on the final yield of the culture metabolism, allowing unveiling major metabolic features of seed and is conditioned by the number of plants issued from success- development. It is clear that the mature seed is well prepared to ful germinations and by the vigor of the seedlings, i.e., the mobilize the major classes of reserves (proteins, triglycerides, potential to produce vigorous seedlings. starch, and phytate) during germination. Here, we discuss some The seed is the main form of dissemination of plants. It results of the salient features revealed by the present study. from the conversion of a fertilized egg and contains a zygotic embryo (the future plant), one or more storage tissues [a triploid albumen, cotyledon(s), and perisperm] that accumulate the Author contributions: D.J. designed research; J.C., J.-M.S., and C.J. performed research; J.C., compounds necessary for the embryo’s nutrition during germi- J.-M.S., C.J., A.V.D., and D.J. analyzed data; and D.J. wrote the paper. nation, and seed coats to ensure the seed’s protection against The authors declare no conflict of interest. biotic and abiotic stress. A specific feature of sugarbeet is that the This article is a PNAS Direct Submission. maternal nucellus is not fully digested during maturation and Freely available online through the PNAS open access option. gives rise to the central perisperm, in which starch reserves †J.C. and J.-M.S. contributed equally to this work. accumulate (1–2). For most plant species growing in temperate §To whom correspondence should be addressed. E-mail: dominique.job@bayercropscience. climates, seed development ends with a phase of intense desic- com. cation, then the embryo enters a dormant state, allowing its ¶Throughout the article, ‘‘sugarbeet seed’’ refers to the botanically true seed, which survival for many years. Two phytohormones, abscisic acid includes the embryo, the perisperm, the remnants of the endosperm, and the testa (seed (ABA) and gibberellins (GA), play key roles in seed formation, coat), surrounded by a thick pericarp (see refs. 1 and 2 and SI Appendix). dormancy, and germination. The first inhibits germination and This article contains supporting information online at www.pnas.org/cgi/content/full/ is involved in the development of the embryo and maintenance 0800585105/DCSupplemental. of dormancy, and the second stimulates germination (3). © 2008 by The National Academy of Sciences of the USA

10262–10267 ͉ PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800585105 Downloaded by guest on September 24, 2021 pI MM 76 157 110 A (kDa) 4.3 4.8 4.9 5.1 5.4 6.25.9 6.5 6.6 7.6 8.0 9.2 B E

116 97 84 66

36 29 24

12 RPC e C 4.3 4.8 4.9 5.1 5.4 6.25.9 6.5 6.6 7.6 8.0 9.2 D F Legend 116 97 20 Secondary metabolism 02.16 Fermentation 84 13 Unclassified 02.13 Respiration 66 12 Unclear classification 02.10 TCA pathway 55 11 Disease/defense 02.07 Pentose phosphate 10 Signal transduction 02.02 Gluconeogenesis 45 09 Cell structure 02.01 Glycolysis 08 Intracellular traffic 01.07 Cofactors 36 07 Transporters 01.06 Lipid and sterol 29 06 Protein destination and storage 01.05 Sugars and polysaccharides 24 05 Protein synthesis 01.04 Phosphate 04 Transcription 01.03 Nucleotides 03 Cell growth/division 01.02 Nitrogen and sulfur 17 02.30 Photosynthesis 01.01 Amino acid 02.20 E-transport 12

Fig. 1. Proteome of the sugarbeet seed. (A) 2D electrophoresis analysis of total soluble proteins (100 ␮g) from whole seeds (759 proteins identified) (Table S2). (B) Ontological classification (8) of total soluble sugarbeet seed proteins. (C) 2DE analysis of total soluble perisperm proteins (100 ␮g) from sugarbeet seeds (172 proteins identified) (Table S6). (D) Ontological classification of total soluble proteins from the perisperm of sugarbeet seeds. (E) Ontological signatures of proteomes specifically expressed in root (R; 76 proteins) (Table S6), cotyledons (C; 157 proteins) (Table S6) and perisperm (Pe; 110 proteins) (Table S6) from sugarbeet seeds. (F) Ontological classes (8).

A Complete Glyoxylate Cycle Is Present in Sugarbeet Seeds. The accumulate a specific molecule, glycine betaine, which in plants occurrence of a complete glyoxylate cycle in the sugarbeet seeds is synthesized from choline via the action of choline monoxyge- is in excellent agreement with the work of Elamrani et al. (11), nase and betaine aldehyde dehydrogenase (arrows 99 and 100 in showing that the sugarbeet embryo stores many lipid reserves as Fig. 2) (18). However, these enzymes have not, to our knowledge, an initial energy source during germination and seedling estab- been described previously in seeds. That we detected them lishment. Furthermore, the differential activity of the glyoxylate suggests that sugarbeet seed germination is tolerant to salt or cycle has been shown to be a physiological marker that distin- water stress. This hypothesis was experimentally verified by guishes between high- and low-vigor sugarbeet cultivars (12). measuring glycine betaine contents of seeds from sugarbeet, Caleosin, which is present in sugarbeet seeds (Table S2), is one spinach, quinoa, tomato, Arabidopsis, and carrot. Only the seeds of the two proteins associated with oil bodies, the other being from sugarbeet, spinach, and quinoa, which belong to the oleosin. Its role is to dock oil bodies to glyoxysomes during Chenopodiaceae (now included in the Amaranthaceae family) germination to foster the mobilization of lipid reserves (13). In contain glycine betaine (Table S5). Because sugarbeet seed agreement with this, caleosin-deficient mutants in Arabidopsis germination proves fully resistant to 200 mM NaCl, or to 300 mM display altered rates of degradation of oil bodies during seed mannitol, whereas, under the same conditions, Arabidopsis seed germination (14). Taken together, these results confirm the germination is reduced two and five times, respectively (Fig. S4), important role of the glyoxylate cycle in germination and vigor the combined results reveal new insights as to the role of glycine

(12, 15). PLANT BIOLOGY betaine in sugarbeet seed vigor. The synthesis of glycine betaine requires S-adenosyl-L- Two Pathways for Synthesis of Isopentenyl Diphosphate Are Present in Sugarbeet Seeds. Plants have two metabolic pathways for methionine, which derives from methionine (Fig. 2), as a methyl isoprenoid biosynthesis: the cytosolic mevalonate (MVA) path- donor. However, mature seeds do not seem to store important way and the plastidial nonmevalonate [methylerythritol phos- stocks of methionine as judged by strong impediment of germi- phate (MEP)] pathway. The MVA pathway leads to the biosyn- nation in the presence of propargylglycine, a methionine bio- thesis of sterols, sesquiterpens, and triterpenoids, whereas the synthesis inhibitor (19). The detection of an almost complete alternative MEP route is required for the synthesis of plastid methyl cycle (20) in sugarbeet seeds thus confirms its importance isoprenoids, phytol, and plastoquinones (16). Both pathways are in germination. presently described here in seeds (arrows 91 and 92 in Fig. 2; see Table S2). Note that GAs, which play an essential role in Sugarbeet Seeds Display an Impressive Set of Stress-Defense Mech- germination (3), are presumably synthesized through the MEP anisms. Besides Ͼ60 chaperones and HSPs, the sugarbeet seeds pathway (17). possess different enzymes to cope with reactive oxygen species (Table S2). During germination, massive quantities of H2O2 are Seeds from Chenopodiaceae Specifically Accumulate Glycine Betaine. produced in the peroxisomes as a byproduct of fatty acid Sugarbeet is a halophyte plant, able to adapt to environments of ␤-oxidation (21). We identified several catalases (arrow 90 in high salinity or high osmotic pressure because of its ability to Fig. 2) and several components of the ascorbate-dependent

Catusse et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10263 Downloaded by guest on September 24, 2021 7 sucrose-6P maltose Starch Seed proteins glycosyl- ubiquitin UDP 6 protein 9 8 7 8 ATP 2 Sucrose lipids 1 15 α-dextrins protein Fatty acids gluconate-6P gluconolactone-6P Glc-6P 1011 9 18 12 UDPGlc Glc amino acids 3 UDP- glycerol acyl-Coa 57 ribose-5P sedoheptulose-7P 22 ADPGlc glucuronate 14 fatty acids 33 55 56 19 17 polypeptides amino acyl tRNA 13 UDP- 16 ribulose-5P Fru-6P enoyl-Coa 4 rhamnose Glc-1P UDP- Fru 34 ribosomal subunits xilulose-5P glyceraldehyde-3P erythrose-4P 5 apiose 21 121 translation factors 20 Glc-6P 3-L-hydroxyacyl-Coa phytate 55 34 glyceraldehyde-3P Fru-6P 22 122 protein 24 β-ketoacyl-Coa 23 Fru-1,6P2 myo-inositol 35 phosphate 25 acyl-Coa phospho enolpyruvate glyceraldehyde-3P DHAP acetyl-Coa CO 2 + 2 NH 3 glutamate 27 26 SAH 58 acetyl-CoA 1,3-bisphophoglycerate 60 28 shikimate 61 urea 29 citrate SAM 3-phosphoglycerate acetyl-CoA 37 50 118 IPP arginine ornithine 58 49 30 36 malate CO HCO 2-phosphoglycerate oxaloacetate chorismate 42 2 3 glutamic fumarate 43 48 31 aconitate oxaloacetate phospho enolpyruvate γ-semialdehyde 37 41 36 32 tryptophane tyrosine proline pyruvate 43 acetaldehyde isocitrate citrate 51 phenylalanine succinate 46 52 37 47 ethanol 44 40 acetyl-CoA malate aconitate succinate succinyl-CoA 37 glyoxylate O 2 + H 2O 39 38 45 α-keto isocitrate glutarate glutamate acetyl-CoA 49 90 79 γ-hydroxybutyrate succinate 86 HCO 3 CO 2 methanol CO 2 + H 2O 2 O 2 62 Fru-6P 117 52 89 77 myo-inositol formaldehyde formate oxalate protein GABA malonyl-CoA 76 54 ascorbate NO alanine 63 radical protein-SNO formyl-CoA 75 78 glutamate 74 73 DHAP heme GSSG 66 chlorophylls 64 oxalyl-CoA ascorbate 53 65 77 GSNO glutamyl-tRNA 88 72 protein-SH glutamine dehydroascorbate 67 formate GSH methylglyoxal 85 protein fatty acids 70 protein 71 10-formyl-THF NAD 87 5-amino-levulinate lactoyl-GSH seryl-tRNA GSR 84 5-methyl-THF 5,10-methylene-THF 3 protein* acetoacetyl-CoA serine 69 lactate cysteine aspartate MVAPP glutamine 105 91 83 82 sulfide IPP carbamoyl-P 3-phosphoglycerate glycine 68 92 APS 101 80 sulfate glyceraldehyde-3P homocysteine 120 3-phospho-hydroxypyruvate 3 93 threonine pyruvate pyruvate 81 95 linalool 108 glycyl-tRNA adenosine methyl-THF 102 3-phosphoserine 96 methionine 102 FGAM thiamine 106 AMP 119 94 103 103 111 S S 109 -adenosylhomocysteine -adenosylmethionine UMP serine 104 104 ferulolyl 97 F0F1- AIR protein -CoA caffeoyl ATP synthase serine -CoA ACC isoleucine valine UDP phosphoryl- ATP 107 115 choline 98 ethylene 113 114 ethanolamine coniferyl- complex III 116 ADP IMP UTP CoA IMP GMP choline spermidine complex II 99 41 110 phosphoryl AMP complex I fumarate ethanolamine glycine betaine adenine GTP GDP succinate betaine aldehyde 112 96 107 100 NAD+ adenosine NADH

Fig. 2. Proteomics-based metabolic pathway reconstruction for the mature sugarbeet seed. Red arrows and numbers correspond to biochemical functions identiﬁed by functional gene ontology annotation (8) from the proteome of mature sugarbeet seed (Table S2 and Table S4). Colored blocks represent known metabolic modules (10). Black arrows represent individual functions undetected in the metabolic modules. Dotted black lines represent several consecutive functions undetected in these modules. Gray arrows materialize transfers of metabolites between different modules.

peroxisomal electron transfer system (22), e.g., ascorbate per- detection of these enzymes implies an important role of NO oxidase (arrow 73 in Fig. 2), monodehydroascorbate reductase and/or of protein nitrosylation in sugarbeet seed physiology. (MDAR) (arrow 74 in Fig. 2), and glutathione-dependent In conclusion, the sugarbeet seeds exhibit an impressive set of dehydroascorbate reductase (arrow 72 in Fig. 2). An investiga- defense mechanisms that could be useful to overcome oxidative tion of the sugar-dependent2 Arabidopsis mutant that is deficient stress due to the resumption of metabolic activity during ger- in the peroxisomal isoform of MDAR showed that the ascor- mination and seedling establishment. bate-dependent electron transfer system is necessary to detoxify the H2O2 escaping from the peroxisomes. This function is critical Amino Acid Biosynthesis Pathways Are Omnipresent in Sugarbeet to protect oil bodies against oxidative damage (21). Aconitase Seeds. Impressively, enzymes involved in the biosynthesis of 17 of (arrow 37 in Fig. 2; see Table S2), which participates in the the 20 amino acids entering in protein composition are identified glyoxylate cycle, is also very sensitive to H2O2 and must be in the sugarbeet seed (Table S2 and Table S4). This finding protected (23). contrasts with the long-standing view that storage proteins are NO can serve as a signal molecule against oxidative stress (24) the major sources of nitrogen and carbon in germinating seeds or during developmental processes, such as the breaking of seed and clearly indicates, in agreement with metabolite profiling in dormancy (25). In addition, protein S-nitrosylation is a biolog- germinating Arabidopsis seeds (29), that de novo synthesis of ically important role of NO (26). In sugarbeet seeds, three amino acids is required to support germination. glutathione-dependent formaldehyde dehydrogenases, also The proteome of the sugarbeet seed reveals many enzymes called GSNO reductases (GSNOR) (arrow 77 in Fig. 2), and two involved in the metabolism of branched chain amino acids [Cu-Zn] superoxide dismutases (arrow 78 in Fig. 2) have been (BCAA) (Val, Leu, Ile) [see Fig. 2; Thr synthase (arrow 101), identified (Table S2). GSNOR is the crucial enzyme for the acetolactate synthase (arrow 102), acetohydroxyacid isomerore- degradation of GSNO, thereby protecting cells against nitrosy- ductase (arrow 103), and dihydroxyacid dehydratase (arrow lation stress (27). The [Cu-Zn] superoxide dismutase also cat- 104); see Table S4]. Studies on the mode of action of commercial alyzes the decomposition of S-nitrosothiols (28). The present herbicides targeting acetolactate synthase demonstrated the

10264 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800585105 Catusse et al. Downloaded by guest on September 24, 2021 existence of a relationship between synthesis of BCAA and in sugarbeet seeds (Table S2) suggests the ubiquity of this redox functionality of the cell cycle (30). In Arabidopsis, the cell cycle control of protein conformation in seeds. is activated before testa rupture during seed germination (31). In Two spots correspond to the stress inducible STI1 protein sugarbeet seeds, several cell cycle proteins are evidenced, such (Table S2). In Saccharomyces cerevisiae, this protein is a cochap- as the proliferating cell nuclear antigen and the Cdc48p/p97 erone involved in the mediation of the response of HSP70s to cyclins (Table S2). We also identified components of the cy- heat shock (46). In plants, STI1 domains have been identified in toskeleton, including five tubulin subunits and five actin isoforms translocon in outer (TOC) and inner (TIC) envelopes of chlo- (Table S2). An accumulation of tubulin subunits occurs during roplast complexes, which are located in plastid membranes, imbibition of seeds from different species (32–33) in conjunction where they realize the import of proteins encoded by the nuclear with the resumption of cell cycle activity (34). The role of actin genome (47). in germination is essential because Arabidopsis mutants of the The mitochondrial-processing peptidase has also been iden- act7 isoform display altered germination, seedling growth, and tified in the sugarbeet seeds (Table S2). This peptidase specif- root development (35). Thus, the present results support the ically recognizes mitochondrial preproteins encoded by the importance of the BCCA pathway for both cell cycle activation nuclear genome and removes their N-terminal signal prepeptides and initiation of protein synthesis (see below) in preparation to during protein import in mitochondria (48). Therefore, its germination. activity ensures the functionality of these organelles. The translocase of outer mitochondrial membrane (TOM)20 translocase Sugarbeet Seeds Use Distinct Mechanisms for Translation Initiation. In has also been identified (49) (Table S2), which is one of the eukaryotes, proteins are synthesized through cap-dependent and central components of the protein import machinery into mito- cap-independent translation initiation mechanisms. For the cap- chondria. In potato and Arabidopsis, the crucial role of the TOM dependent mechanism, mRNAs are recruited by the elF-4F complex is to discriminate between mitochondrial and plastidial complex composed of the eIF-4E and eIF-4G subunits (36). transit peptides (50). The occurrence of these proteins in the eIF-4E is involved in mRNA cap recognition, and eIF-4G proteome of the sugarbeet seeds reinforces the key role of interacts with the polyA tail recognition protein and several mitochondria in seed metabolism, because it is known that the factors, including eIF-4A and eIF-3. In sugarbeet seeds, we quality of seed mitochondria is strongly related to germination identified the initiation factors eIF-3 and eIF-4A (Table S2). vigor (51, 52). Although the cap-independent process was originally de- Our identification of the main components of the plastid and scribed for the translation of viral RNAs, this mechanism is now mitochondrial protein addressing systems provides a means to surmised to occur for the translation of many cell mRNAs, which uncover one of the major mechanisms governing seed vigor. may represent up to 10% of cellular RNA, particularly during stress (37). It proceeds by the direct recruitment of ribosomal Sugarbeet Seed Proteome Exhibits Tissue-Specific Features. Quanti- subunits on RNA sequences defined as internal ribosome entry tative image analysis of the 2D gels reveals that the root and sites (IRESs). Recruitment of canonical translation initiation cotyledon proteomes are fairly similar. However, there are factors is accompanied by that of stimulating proteins named quantitative differences in the accumulation level of several IRES-specific cellular transacting factors (ITAFs), of which one, proteins between the two tissues: 76 proteins are more abundant ITAF45, is better known as ErbB3-binding protein 1 (EBP1) in the root proteome, whereas 157 proteins are more abundant (38). Four EPB1 proteins are detected in the sugarbeet seeds in the cotyledon proteome (Figs. S5 and S6 and Table S6). In (Table S2). This is the first description of EBP1 in seeds, and contrast, the perisperm proteome is markedly different (Fig. 1). there is only a single recent report describing these proteins in Proteins differentially accumulated in the different tissues are plants (39). Thus, our data unveil a complex mechanism of involved in various biological functions (Fig. 1). In the root translation initiation in seeds. Because two different mRNA proteome, the major functional categories represented are the pools exist in germinating seeds, the stored mRNAs, and the de storage proteins, protein folding and turnover, protein synthesis, novo-synthesized mRNAs (4, 40), it is possible that these dif- and components of the cell structure. For the cotyledons, the ferent initiation systems allow their respective recruitment dur- main functional categories represented are neoglucogenesis that ing germination. Consistent with this, maize embryonic axes includes the glyoxylate cycle, the TCA cycle, lipid metabolism, were shown to contain stored mRNAs, some of which are sterols, amino acid metabolism, defense reaction, and secondary efficiently translated via a cap-independent mechanism during metabolism. In the case of the perisperm, the major functional germination (41). category revealed is sugar and polysaccharide metabolism. Each Several translation elongation factors are also evidenced in the tissue is therefore assigned to specific metabolic functions. sugarbeet seeds (Table S2), such as eEF-1␣, eEF-1␤␥, and In addition to the many proteins involved in sugar metabolism, eEF-2. Taken together, these results demonstrate the impor- the terminal enzyme in the biosynthesis of ascorbic acid (AsA), PLANT BIOLOGY tance of protein translation in seed vigor (42). L-galactono-1,4-lactone dehydrogenase (arrow 75 in Fig. 2), is present in the perisperm (Table S6), suggesting that the synthesis Sugarbeet Seeds Are Equipped with Components for Protein Metab- of vitamin C occurs at least in part in this tissue. We found that olism and Import. Besides 19 protease spots, we identified 20 oxalate, which derives from AsA, is present in root [4.4 mg per proteins of the 26S proteasome (Table S2). Furthermore, three gram of fresh weight (FW)], cotyledons (0.5 mg per gram of FW) proteins correspond to the E1 class of ubiquitin activation and perisperm (0.3 mg per gram of FW). Because an oxalate enzymes (Table S2). Thus, the 26S proteasome/ubiquitin system oxidase (arrow 89 in Fig. 2) is also located in the perisperm (43) is well represented in the sugarbeet seed proteome. It is (Table S6), H2O2 can be produced in this tissue, which can help noted that several systems playing a role in seed germination sustaining oxalate production from ascorbate and provide CO2 depend on the activity of the proteasome, such as GA signaling (53). This CO2 can then be transformed into bicarbonate by via the degradation of DELLA proteins, which are negative carbonic anhydrase, an enzyme exclusively located in the regulators of GA action and repress germination (44). perisperm (arrow 49 in Fig. 2; see Table S6). Bicarbonate is the Proteomics-based approaches recently revealed the role of the cosubstrate for two important enzymes, phosphoenolpyruvate regulatory disulfide protein thioredoxin in seeds, notably during carboxylase (arrow 48 in Fig. 2) and acetyl-CoA carboxylase germination, where it catalyzes the reduction of disulfide bridges (arrow 62 in Fig. 2), both of which are detectable in the proteome in storage proteins to increase their solubility and favor their of the sugarbeet embryo (Table S6). It appears that bicarbonate mobilization (45). The occurrence of the cytosolic thioredoxin h is produced by carbonic anhydrase in the perisperm and distrib-

Catusse et al. PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10265 Downloaded by guest on September 24, 2021 uted to the embryo. Oxalate oxidase behaves as a marker of known as a dead tissue, the perisperm appears to be very active germination vigor in sugarbeet (54). Note also that formate biochemically, playing multiple roles in distributing sugars and dehydrogenase (arrow 86 in Fig. 2), which produces CO2 from various metabolites to other tissues of the embryo. formate, is detectable in the perisperm (Table S6). The reference maps presently established provide important In the perisperm, we identified four very abundant spots as new information about mechanisms controlling germination and purple acid phosphatase (arrow 122 in Fig. 2; see Table S6 and suggest new ways to improve sugarbeet seed quality and vigor Fig. S2). In seeds, this enzyme corresponds to phytase that and aid further progress in crop yield. Also, this comprehensive hydrolyses phytate during germination, the main storage form proteomic analysis will facilitate annotation of the sugarbeet of phosphorus (55–56). We found that phytate is present in genome of which sequencing is in preparation. roots (33 mg per gram of FW) and cotyledons (6 mg per gram of FW) but is undetectable in the perisperm, in agreement with Materials and Methods myo-inositol-1-phosphate synthase, the first committed en- For detailed materials and methods, see SI Appendix. zyme in phytate biosynthesis, being detected only in roots and cotyledons (arrow 121 in Fig. 2; see Table S6). From these Preparation of Protein Extracts. For each condition assayed, protein extracts were prepared from 100 sugarbeet seeds (KWS). After grinding of the seeds results, the exclusive detection of phytase in the perisperm is in liquid nitrogen with a mortar and pestle, soluble proteins were extracted as intriguing. These results raise the possibility that a compart- described in ref. 59, using an extraction buffer composed of 50 mM Hepes (pH mentalization of phytate and phytase activity might preserve 8.0), 1 mM EDTA (pH 9.0). For tissue proteomics, whole seeds were dissected the integrity of the phytate reserves up to germination. In turn, under a binocular microscope (ref. 2 and SI Appendix). Each part (root, stem, this hypothesis raises the question of the involvement of a cotyledons, and perisperm) was immediately frozen in liquid nitrogen and selective transport system of phytase from the perisperm (a protein extractions were carried out as above. dead tissue) to the embryo (a living tissue). Note that myo- inositol, the product of phytase, is a substrate for AsA 2D Gel Electrophoresis (2DE) and Protein Quantification. 2D gel electrophoresis was carried out as described in ref. 59, except that isoelectric focusing was run, biosynthesis (55, 56), lending further support to the existence using 24-cm immobilized pH gradient (3–10 nonlinear) immobilized pH gra- of cross-talk between the perisperm and the embryo, notably dient strips (GE Healthcare). For each condition, analyzed 2D gels were made concerning AsA/oxalate/CO2 metabolism. at least in triplicate and for a minimum of three independent extractions. After silver-nitrate staining of the 2D gels, quantification of spots and com- Conclusion parative analysis were performed with the Image Master 2D Elite software The present study identified Ͼ750 proteins and allowed us to (Amersham Biosciences) (59). reconstruct in detail the metabolism of sugarbeet seeds, provid- ing a proteome-wide fingerprint of their metabolic activity Protein Identification by Mass Spectrometry. Proteins were submitted to in-gel during development. The mature seed appears to be well pre- digestion by trypsin. Extracted peptides were analyzed by nano-LC-MS-MS on a Q-TOF2 mass spectrometer (Micromass) and identified by using the Mascot pared to mobilize its major reserve compounds during germination. software as described in ref. 60. Individual peptide MS/MS spectra were Interestingly, our study also identified a number of proteins that, to checked manually (Fig. S3 and SI Appendix). Criteria used for protein identi- our knowledge, have not previously been described in seeds. For fications followed the general guidelines for reporting proteomic experi- example, we discovered that the sugarbeet seed can initiate trans- ments (MIAPE; www.psidev.info). Peaks software (BSI; Bioinformatics Solu- lation either through the traditional cap-dependent mechanism or tions) was used to obtain sequence tag from MS/MS data to realize sequence by an alternative cap-independent process. alignments with MSBlast. Strikingly, this study reveals a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, Determination of Glycine Betaine, Oxalate, and Phytate. Glycine betaine was which indicates a division of metabolic tasks between the various determined in whole seeds by the method of Bessieres et al. (61). Oxalate was determined in isolated seed tissues, using a commercial assay (Boehringer tissues and supports the results of Gallardo et al. (57), which Manhein/R-Biopharm). Phytate content was determined in isolated seed tis- showed that, in developing Medicago truncatula seeds, sulfur sues as described in ref. 62. metabolism is partitioned between seed coats and the embryo, and the results of Koller et al. (58), which documented the ACKNOWLEDGMENTS. We thank Juliane Meinhard, Andeas Menze, Uwe existence of divergent regulatory mechanisms in starch biosyn- Fischer (KWS), Elena Pestsova, Peter Westhoff (University of Du¨sseldorf, Du¨s- thesis and degradation in different rice tissues. seldorf, Germany), Karine Gallardo (Institut National de la Recherche We also established the perisperm proteome. Unexpectedly, Agronomique, Dijon, France), and Maya Belghazi (Centre National de la Recherche Scientifique, Marseilles, France) for helpful discussions. This work besides its role in starch metabolism, this tissue is involved in the was supported in part by Geńoplante-Genomanalyse im Biologischen System metabolism of phytate, AsA, and oxalate. Even though it is Pflanze, a French–German joint program in plant genomics.

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