Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression
Edited by Roland Douce, Université de Grenoble, Grenoble, France, and approved April 11, 2008
Abstract
Proteomic analysis of mature sugarbeet seeds led to the identification of 759 proteins and their specific tissue expression in root, cotyledons, and perisperm. In particular, the proteome of the perispermic storage tissue found in many seeds of the Caryophyllales is described here. The data allowed us to reconstruct in detail the metabolism of the seeds toward recapitulating facets of seed development and provided insights into complex behaviors such as germination. The seed appears to be well prepared to mobilize the major classes of reserves (the proteins, triglycerides, phytate, and starch) during germination, indicating that the preparation of the seed for germination is mainly achieved during its maturation on the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic trait defined as the potential to produce vigorous seedlings, such as glycine betaine accumulation in seeds. This study also identified several proteins that, to our knowledge, have not previously been described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by a cap-independent process. The study of the tissue specificity of the seed proteome demonstrated a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, indicating a division of metabolic tasks between the various tissues. Furthermore, the perisperm, although it is known as a dead tissue, appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo.
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Sugarbeet (Beta vulgaris L.) is a dicotyledonous plant of the Amaranthaceae family that has a high economic importance because it is one of the two main sources of sucrose, the other being sugarcane. Furthermore, there is growing interest in the use of this crop to produce bioethanol. The quality of seed germination has a direct impact on the final yield of the culture and is conditioned by the number of plants issued from successful germinations and by the vigor of the seedlings, i.e., the potential to produce vigorous seedlings.
The seed is the main form of dissemination of plants. It results 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 compounds necessary for the embryo's nutrition during germination, and seed coats to ensure the seed's protection against biotic and abiotic stress. A specific feature of sugarbeet is that the maternal nucellus is not fully digested during maturation and gives rise to the central perisperm, in which starch reserves accumulate (1–2). For most plant species growing in temperate climates, seed development ends with a phase of intense desiccation, then the embryo enters a dormant state, allowing its survival for many years. Two phytohormones, abscisic acid (ABA) and gibberellins (GA), play key roles in seed formation, dormancy, and germination. The first inhibits germination and is involved in the development of the embryo and maintenance of dormancy, and the second stimulates germination (3).
The use of metabolic inhibitors (α-amanitin and cycloheximide) showed that transcription is not required for the completion of germination in Arabidopsis, implying that the potential of germination is largely programmed during seed maturation on the mother plant (4). Therefore, in this work, we have characterized sugarbeet seed¶ vigor by proteomics. This was challenging, however, because there are virtually no genomics data presently available on this plant that could be used for protein identification, but recent successes illustrated the ability of mass spectrometry to identify and quantify thousands protein profiles from diverse species (5–6). By using this approach, we have also determined the tissue specificity of the accumulation of the seed proteins, allowing us to described the proteome of the perisperm storage tissue.
Results and Discussion
Proteome-Wide Analysis Allows Metabolic Network Reconstruction in Sugarbeet Seeds.
Of 784 protein spots submitted to proteomic analysis, we identified 759 proteins [Fig. 1, supporting information (SI) Figs. S1–S3, Table S1, and Table S2], of which the majority is associated with unique proteins. Seventy spots gave two identifications, and 14 spots gave three identifications (Table S3). This corresponds to an overall success rate of ≈80%.
Fig. 1.
Metabolic network reconstruction is a fundamental task in systems biology with an ultimate goal of full-scale in silico simulations (7). Based on ontological classification (8) and established features of metabolism, notably in plants (refs. 9 and 10 and http://metacyc.org), the metabolism of the sugarbeet seed can be reconstructed by 121 biochemical functions, covering 561 of the 759 proteins presently identified (Table S4) and showing a tight organization (Fig. 2). Several metabolic modules have been identified in a complete manner, such as glycolysis, fatty acid β-oxidation, glyoxylate cycle, protein degradation, or starch metabolism, allowing unveiling major metabolic features of seed development. It is clear that the mature seed is well prepared to mobilize the major classes of reserves (proteins, triglycerides, starch, and phytate) during germination. Here, we discuss some of the salient features revealed by the present study.
Fig. 2.
A Complete Glyoxylate Cycle Is Present in Sugarbeet Seeds.
The occurrence of a complete glyoxylate cycle in the sugarbeet seeds is in excellent agreement with the work of Elamrani et al. (11), showing that the sugarbeet embryo stores many lipid reserves as an initial energy source during germination and seedling establishment. Furthermore, the differential activity of the glyoxylate cycle has been shown to be a physiological marker that distinguishes between high- and low-vigor sugarbeet cultivars (12).
Caleosin, which is present in sugarbeet seeds (Table S2), is one of the two proteins associated with oil bodies, the other being oleosin. Its role is to dock oil bodies to glyoxysomes during germination to foster the mobilization of lipid reserves (13). In agreement with this, caleosin-deficient mutants in Arabidopsis display altered rates of degradation of oil bodies during seed germination (14). Taken together, these results confirm the important role of the glyoxylate cycle in germination and vigor (12, 15).
Two Pathways for Synthesis of Isopentenyl Diphosphate Are Present in Sugarbeet Seeds.
Plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidial nonmevalonate [methylerythritol phosphate (MEP)] pathway. The MVA pathway leads to the biosynthesis of sterols, sesquiterpens, and triterpenoids, whereas the alternative MEP route is required for the synthesis of plastid isoprenoids, phytol, and plastoquinones (16). Both pathways are presently described here in seeds (arrows 91 and 92 in Fig. 2; see Table S2). Note that GAs, which play an essential role in germination (3), are presumably synthesized through the MEP pathway (17).
Seeds from Chenopodiaceae Specifically Accumulate Glycine Betaine.
Sugarbeet is a halophyte plant, able to adapt to environments of high salinity or high osmotic pressure because of its ability to accumulate a specific molecule, glycine betaine, which in plants is synthesized from choline via the action of choline monoxygenase and betaine aldehyde dehydrogenase (arrows 99 and 100 in Fig. 2) (18). However, these enzymes have not, to our knowledge, been described previously in seeds. That we detected them suggests that sugarbeet seed germination is tolerant to salt or water stress. This hypothesis was experimentally verified by measuring glycine betaine contents of seeds from sugarbeet, spinach, quinoa, tomato, Arabidopsis, and carrot. Only the seeds from sugarbeet, spinach, and quinoa, which belong to the Chenopodiaceae (now included in the Amaranthaceae family) contain glycine betaine (Table S5). Because sugarbeet seed germination proves fully resistant to 200 mM NaCl, or to 300 mM mannitol, whereas, under the same conditions, Arabidopsis seed germination is reduced two and five times, respectively (Fig. S4), the combined results reveal new insights as to the role of glycine betaine in sugarbeet seed vigor.
The synthesis of glycine betaine requires S-adenosyl-l-methionine, which derives from methionine (Fig. 2), as a methyl donor. However, mature seeds do not seem to store important stocks of methionine as judged by strong impediment of germination in the presence of propargylglycine, a methionine biosynthesis inhibitor (19). The detection of an almost complete methyl cycle (20) in sugarbeet seeds thus confirms its importance in germination.
Sugarbeet Seeds Display an Impressive Set of Stress-Defense Mechanisms.
Besides >60 chaperones and HSPs, the sugarbeet seeds possess different enzymes to cope with reactive oxygen species (Table S2). During germination, massive quantities of H2O2 are produced in the peroxisomes as a byproduct of fatty acid β-oxidation (21). We identified several catalases (arrow 90 in Fig. 2) and several components of the ascorbate-dependent peroxisomal electron transfer system (22), e.g., ascorbate peroxidase (arrow 73 in Fig. 2), monodehydroascorbate reductase (MDAR) (arrow 74 in Fig. 2), and glutathione-dependent dehydroascorbate reductase (arrow 72 in Fig. 2). An investigation of the sugar-dependent2 Arabidopsis mutant that is deficient in the peroxisomal isoform of MDAR showed that the ascorbate-dependent electron transfer system is necessary to detoxify the H2O2 escaping from the peroxisomes. This function is critical to protect oil bodies against oxidative damage (21). Aconitase (arrow 37 in Fig. 2; see Table S2), which participates in the glyoxylate cycle, is also very sensitive to H2O2 and must be protected (23).
NO can serve as a signal molecule against oxidative stress (24) or during developmental processes, such as the breaking of seed dormancy (25). In addition, protein S-nitrosylation is a biologically important role of NO (26). In sugarbeet seeds, three glutathione-dependent formaldehyde dehydrogenases, also called GSNO reductases (GSNOR) (arrow 77 in Fig. 2), and two [Cu-Zn] superoxide dismutases (arrow 78 in Fig. 2) have been identified (Table S2). GSNOR is the crucial enzyme for the degradation of GSNO, thereby protecting cells against nitrosylation stress (27). The [Cu-Zn] superoxide dismutase also catalyzes the decomposition of S-nitrosothiols (28). The present detection of these enzymes implies an important role of NO and/or of protein nitrosylation in sugarbeet seed physiology.
In conclusion, the sugarbeet seeds exhibit an impressive set of defense mechanisms that could be useful to overcome oxidative stress due to the resumption of metabolic activity during germination and seedling establishment.
Amino Acid Biosynthesis Pathways Are Omnipresent in Sugarbeet Seeds.
Impressively, enzymes involved in the biosynthesis of 17 of the 20 amino acids entering in protein composition are identified in the sugarbeet seed (Table S2 and Table S4). This finding contrasts with the long-standing view that storage proteins are the major sources of nitrogen and carbon in germinating seeds and clearly indicates, in agreement with metabolite profiling in germinating Arabidopsis seeds (29), that de novo synthesis of amino acids is required to support germination.
The proteome of the sugarbeet seed reveals many enzymes involved in the metabolism of branched chain amino acids (BCAA) (Val, Leu, Ile) [see Fig. 2; Thr synthase (arrow 101), acetolactate synthase (arrow 102), acetohydroxyacid isomeroreductase (arrow 103), and dihydroxyacid dehydratase (arrow 104); see Table S4]. Studies on the mode of action of commercial herbicides targeting acetolactate synthase demonstrated the existence of a relationship between synthesis of BCAA and functionality of the cell cycle (30). In Arabidopsis, the cell cycle is activated before testa rupture during seed germination (31). In sugarbeet seeds, several cell cycle proteins are evidenced, such as the proliferating cell nuclear antigen and the Cdc48p/p97 cyclins (Table S2). We also identified components of the cytoskeleton, including five tubulin subunits and five actin isoforms (Table S2). An accumulation of tubulin subunits occurs during imbibition of seeds from different species (32–33) in conjunction with the resumption of cell cycle activity (34). The role of actin in germination is essential because Arabidopsis mutants of the act7 isoform display altered germination, seedling growth, and root development (35). Thus, the present results support the importance of the BCCA pathway for both cell cycle activation and initiation of protein synthesis (see below) in preparation to germination.
Sugarbeet Seeds Use Distinct Mechanisms for Translation Initiation.
In eukaryotes, proteins are synthesized through cap-dependent and cap-independent translation initiation mechanisms. For the cap-dependent mechanism, mRNAs are recruited by the elF-4F complex composed of the eIF-4E and eIF-4G subunits (36). eIF-4E is involved in mRNA cap recognition, and eIF-4G interacts with the polyA tail recognition protein and several factors, including eIF-4A and eIF-3. In sugarbeet seeds, we identified the initiation factors eIF-3 and eIF-4A (Table S2).
Although the cap-independent process was originally described for the translation of viral RNAs, this mechanism is now surmised to occur for the translation of many cell mRNAs, which may represent up to 10% of cellular RNA, particularly during stress (37). It proceeds by the direct recruitment of ribosomal subunits on RNA sequences defined as internal ribosome entry sites (IRESs). Recruitment of canonical translation initiation factors is accompanied by that of stimulating proteins named IRES-specific cellular transacting factors (ITAFs), of which one, ITAF45, is better known as ErbB3-binding protein 1 (EBP1) (38). Four EPB1 proteins are detected in the sugarbeet seeds (Table S2). This is the first description of EBP1 in seeds, and there is only a single recent report describing these proteins in plants (39). Thus, our data unveil a complex mechanism of translation initiation in seeds. Because two different mRNA pools exist in germinating seeds, the stored mRNAs, and the de novo-synthesized mRNAs (4, 40), it is possible that these different initiation systems allow their respective recruitment during germination. Consistent with this, maize embryonic axes were shown to contain stored mRNAs, some of which are efficiently translated via a cap-independent mechanism during germination (41).
Sugarbeet Seeds Are Equipped with Components for Protein Metabolism and Import.
Besides 19 protease spots, we identified 20 proteins of the 26S proteasome (Table S2). Furthermore, three proteins correspond to the E1 class of ubiquitin activation enzymes (Table S2). Thus, the 26S proteasome/ubiquitin system (43) is well represented in the sugarbeet seed proteome. It is noted that several systems playing a role in seed germination depend on the activity of the proteasome, such as GA signaling via the degradation of DELLA proteins, which are negative regulators of GA action and repress germination (44).
Proteomics-based approaches recently revealed the role of the regulatory disulfide protein thioredoxin in seeds, notably during germination, where it catalyzes the reduction of disulfide bridges in storage proteins to increase their solubility and favor their mobilization (45). The occurrence of the cytosolic thioredoxin h in sugarbeet seeds (Table S2) suggests the ubiquity of this redox control of protein conformation in seeds.
Two spots correspond to the stress inducible STI1 protein (Table S2). In Saccharomyces cerevisiae, this protein is a cochaperone involved in the mediation of the response of HSP70s to heat shock (46). In plants, STI1 domains have been identified in translocon in outer (TOC) and inner (TIC) envelopes of chloroplast complexes, which are located in plastid membranes, where they realize the import of proteins encoded by the nuclear genome (47).
The mitochondrial-processing peptidase has also been identified in the sugarbeet seeds (Table S2). This peptidase specifically recognizes mitochondrial preproteins encoded by the nuclear genome and removes their N-terminal signal prepeptides during protein import in mitochondria (48). Therefore, its activity ensures the functionality of these organelles. The translocase of outer mitochondrial membrane (TOM)20 translocase has also been identified (49) (Table S2), which is one of the central components of the protein import machinery into mitochondria. In potato and Arabidopsis, the crucial role of the TOM complex is to discriminate between mitochondrial and plastidial transit peptides (50). The occurrence of these proteins in the proteome of the sugarbeet seeds reinforces the key role of mitochondria in seed metabolism, because it is known that the quality of seed mitochondria is strongly related to germination vigor (51, 52).
Our identification of the main components of the plastid and mitochondrial protein addressing systems provides a means to uncover one of the major mechanisms governing seed vigor.
Sugarbeet Seed Proteome Exhibits Tissue-Specific Features.
Quantitative image analysis of the 2D gels reveals that the root and cotyledon proteomes are fairly similar. However, there are quantitative differences in the accumulation level of several proteins between the two tissues: 76 proteins are more abundant in the root proteome, whereas 157 proteins are more abundant in the cotyledon proteome (Figs. S5 and S6 and Table S6). In contrast, the perisperm proteome is markedly different (Fig. 1). Proteins differentially accumulated in the different tissues are involved in various biological functions (Fig. 1). In the root proteome, the major functional categories represented are the storage proteins, protein folding and turnover, protein synthesis, and components of the cell structure. For the cotyledons, the main functional categories represented are neoglucogenesis that includes the glyoxylate cycle, the TCA cycle, lipid metabolism, sterols, amino acid metabolism, defense reaction, and secondary metabolism. In the case of the perisperm, the major functional category revealed is sugar and polysaccharide metabolism. Each tissue is therefore assigned to specific metabolic functions.
In addition to the many proteins involved in sugar metabolism, the terminal enzyme in the biosynthesis of ascorbic acid (AsA), l-galactono-1,4-lactone dehydrogenase (arrow 75 in Fig. 2), is present in the perisperm (Table S6), suggesting that the synthesis of vitamin C occurs at least in part in this tissue. We found that oxalate, which derives from AsA, is present in root [4.4 mg per gram of fresh weight (FW)], cotyledons (0.5 mg per gram of FW) and perisperm (0.3 mg per gram of FW). Because an oxalate oxidase (arrow 89 in Fig. 2) is also located in the perisperm (Table S6), H2O2 can be produced in this tissue, which can help sustaining oxalate production from ascorbate and provide CO2 (53). This CO2 can then be transformed into bicarbonate by carbonic anhydrase, an enzyme exclusively located in the perisperm (arrow 49 in Fig. 2; see Table S6). Bicarbonate is the cosubstrate for two important enzymes, phosphoenolpyruvate carboxylase (arrow 48 in Fig. 2) and acetyl-CoA carboxylase (arrow 62 in Fig. 2), both of which are detectable in the proteome of the sugarbeet embryo (Table S6). It appears that bicarbonate is produced by carbonic anhydrase in the perisperm and distributed to the embryo. Oxalate oxidase behaves as a marker of germination vigor in sugarbeet (54). Note also that formate dehydrogenase (arrow 86 in Fig. 2), which produces CO2 from formate, is detectable in the perisperm (Table S6).
In the perisperm, we identified four very abundant spots as purple acid phosphatase (arrow 122 in Fig. 2; see Table S6 and Fig. S2). In seeds, this enzyme corresponds to phytase that hydrolyses phytate during germination, the main storage form of phosphorus (55–56). We found that phytate is present in roots (33 mg per gram of FW) and cotyledons (6 mg per gram of FW) but is undetectable in the perisperm, in agreement with myo-inositol-1-phosphate synthase, the first committed enzyme in phytate biosynthesis, being detected only in roots and cotyledons (arrow 121 in Fig. 2; see Table S6). From these results, the exclusive detection of phytase in the perisperm is intriguing. These results raise the possibility that a compartmentalization of phytate and phytase activity might preserve the integrity of the phytate reserves up to germination. In turn, this hypothesis raises the question of the involvement of a selective transport system of phytase from the perisperm (a dead tissue) to the embryo (a living tissue). Note that myo-inositol, the product of phytase, is a substrate for AsA biosynthesis (55, 56), lending further support to the existence of cross-talk between the perisperm and the embryo, notably concerning AsA/oxalate/CO2 metabolism.
Conclusion
The present study identified >750 proteins and allowed us to reconstruct in detail the metabolism of sugarbeet seeds, providing a proteome-wide fingerprint of their metabolic activity during development. The mature seed appears to be well prepared to mobilize its major reserve compounds during germination. Interestingly, our study also identified a number of proteins that, to our knowledge, have not previously been described in seeds. For example, we discovered that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by an alternative cap-independent process.
Strikingly, this study reveals a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, which indicates a division of metabolic tasks between the various tissues and supports the results of Gallardo et al. (57), which showed that, in developing Medicago truncatula seeds, sulfur metabolism is partitioned between seed coats and the embryo, and the results of Koller et al. (58), which documented the existence of divergent regulatory mechanisms in starch biosynthesis and degradation in different rice tissues.
We also established the perisperm proteome. Unexpectedly, besides its role in starch metabolism, this tissue is involved in the metabolism of phytate, AsA, and oxalate. Even though it is known as a dead tissue, the perisperm appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo.
The reference maps presently established provide important new information about mechanisms controlling germination and suggest new ways to improve sugarbeet seed quality and vigor and aid further progress in crop yield. Also, this comprehensive proteomic analysis will facilitate annotation of the sugarbeet genome of which sequencing is in preparation.
Materials and Methods
For detailed materials and methods, see SI Appendix.
Preparation of Protein Extracts.
For each condition assayed, protein extracts were prepared from 100 sugarbeet seeds (KWS). After grinding of the seeds in liquid nitrogen with a mortar and pestle, soluble proteins were extracted as described in ref. 59, using an extraction buffer composed of 50 mM Hepes (pH 8.0), 1 mM EDTA (pH 9.0). For tissue proteomics, whole seeds were dissected under a binocular microscope (ref. 2 and SI Appendix). Each part (root, stem, cotyledons, and perisperm) was immediately frozen in liquid nitrogen and protein extractions were carried out as above.
2D Gel Electrophoresis (2DE) and Protein Quantification.
2D gel electrophoresis was carried out as described in ref. 59, except that isoelectric focusing was run, using 24-cm immobilized pH gradient (3–10 nonlinear) immobilized pH gradient strips (GE Healthcare). For each condition, analyzed 2D gels were made at least in triplicate and for a minimum of three independent extractions. After silver-nitrate staining of the 2D gels, quantification of spots and comparative analysis were performed with the Image Master 2D Elite software (Amersham Biosciences) (59).
Protein Identification by Mass Spectrometry.
Proteins were submitted to in-gel 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 software as described in ref. 60. Individual peptide MS/MS spectra were checked manually (Fig. S3 and SI Appendix). Criteria used for protein identifications followed the general guidelines for reporting proteomic experiments (MIAPE; www.psidev.info). Peaks software (BSI; Bioinformatics Solutions) was used to obtain sequence tag from MS/MS data to realize sequence alignments with MSBlast.
Determination of Glycine Betaine, Oxalate, and Phytate.
Acknowledgments.
We thank Juliane Meinhard, Andeas Menze, Uwe Fischer (KWS), Elena Pestsova, Peter Westhoff (University of Düsseldorf, Düsseldorf, Germany), Karine Gallardo (Institut National de la Recherche Agronomique, Dijon, France), and Maya Belghazi (Centre National de la Recherche Scientifique, Marseilles, France) for helpful discussions. This work was supported in part by Génoplante-Genomanalyse im Biologischen System Pflanze, a French–German joint program in plant genomics.
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Cover image: The parasitic wasp Trichogramma brassicae preys on eggs of the cabbage butterfly Pieris brassicae. P. brassicae, in turn, is a parasite of Brussels sprout plants. Nina E. Fatouros et al. find that a compound in male butterfly ejaculate triggers the plant to secrete phytochemicals that recruit the parasitic wasp to kill butterfly larvae. See the article on pages 10033–10038. Image courtesy of Nina E. Fatouros.
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© 2008 by The National Academy of Sciences of the USA. Freely available online through the PNAS open access option.
Submission history
Received: January 19, 2008
Published online: July 22, 2008
Published in issue: July 22, 2008
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Acknowledgments
We thank Juliane Meinhard, Andeas Menze, Uwe Fischer (KWS), Elena Pestsova, Peter Westhoff (University of Düsseldorf, Düsseldorf, Germany), Karine Gallardo (Institut National de la Recherche Agronomique, Dijon, France), and Maya Belghazi (Centre National de la Recherche Scientifique, Marseilles, France) for helpful discussions. This work was supported in part by Génoplante-Genomanalyse im Biologischen System Pflanze, a French–German joint program in plant genomics.
Notes
This article is a PNAS Direct Submission.
¶
Throughout the article, “sugarbeet seed” refers to the botanically true seed, which includes the embryo, the perisperm, the remnants of the endosperm, and the testa (seed coat), surrounded by a thick pericarp (see refs. 1 and 2 and SI Appendix).
This article contains supporting information online at www.pnas.org/cgi/content/full/0800585105/DCSupplemental.
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Competing Interests
The authors declare no conflict of interest.
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