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-Universite´Claude Bernard Lyon 1, Institut National des Sciences Applique´es–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 Europe´enne 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 path- ways 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 sug- arbeet 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 55 45 36 29 24 17 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.