Proteomics of seed development, desiccation tolerance, germination and vigor.

Plant Physiology and Biochemistry 86 (2015) 1e15

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Review

Proteomics of seed development, desiccation tolerance, germination and vigor Wei-Qing Wang a, Shu-Jun Liu a, Song-Quan Song a, **, Ian Max Møller b, * a b

Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China Department of Molecular Biology and Genetics, Aarhus University, Flakkebjerg, DK-4200 Slagelse, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2014 Accepted 3 November 2014 Available online 4 November 2014

Proteomics, the large-scale study of the total complement of proteins in a given sample, has been applied to all aspects of seed biology mainly using model species such as Arabidopsis or important agricultural crops such as corn and rice. Proteins extracted from the sample have typically been separated and quantified by 2-dimensional polyacrylamide gel electrophoresis followed by liquid chromatography and mass spectrometry to identify the proteins in the gel spots. In this way, qualitative and quantitative changes in the proteome during seed development, desiccation tolerance, germination, dormancy release, vigor alteration and responses to environmental factors have all been studied. Many proteins or biological processes potentially important for each seed process have been highlighted by these studies, which greatly expands our knowledge of seed biology. Proteins that have been identified to be particularly important for at least two of the seed processes are involved in detoxification of reactive oxygen species, the cytoskeleton, glycolysis, protein biosynthesis, post-translational modifications, methionine metabolism, and late embryogenesis-abundant (LEA) proteins. It will be useful for molecular biologists and molecular plant breeders to identify and study genes encoding particularly interesting target proteins with the aim to improve the yield, stress tolerance or other critical properties of our crop species. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Proteome Seed development Desiccation tolerance Seed germination Seed vigor

1. Introduction Almost all plant cultivation in agriculture and horticulture is based on seeds. The vast majority of our domesticated plants are propagated via seeds and seeds provide most of our caloric intake either directly as food or indirectly as feed for our domestic animals. To feed the ever-growing human population we need to increase the productivity of our crop species by exploiting their full genetic

Abbreviations: ABA, abscisic acid; ADH, aldehyde dehydrogenase; APX, ascorbate peroxidase; CAT, catalases; Cys, cysteine; DAF, days after flowering; DAP, days after pollination; GA, gibberellic acid; GC, green carbohydrate abundant; GO, green oil abundant; GPX, glutathione peroxidase; HSP, heat shock proteins; LEA, late embryogenesis abundant; LHC, light harvesting complex; MDHR, monodehydroascorbate reductase; Met, methionine; MV, methylviologen; NGO, nongreen oil abundant; PDC, pyruvate dehydrogenase complex; PEG, polyethylene glycol; Prx, peroxiredoxin; PSII, photosystem II; PTM, post-translational modification; ROS, reactive oxygen species; Rubisco, ribulose bisphosphate carboxylase/ oxygenase; SAM, S-adenosylmethionine; Ser, serine; SOD, superoxide dismutase; TCA, tricarboxylic acid; TPX, thioredoxin peroxidase; Tyr, tyrosine. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S.-Q. Song), [email protected] (I.M. Møller). http://dx.doi.org/10.1016/j.plaphy.2014.11.003 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

potential in plant breeding. This requires that we understand the fundamental processes taking place in the seeds during their development, storage, germination and growth. An excellent overview of all aspects of seed biology is found in Bewley et al. (2013). Seed development. The seed develops from a single fertilized zygote into an embryo and endosperm in association with the surrounding maternal tissues. Most seeds contain large quantities of nutrient reserves, mainly carbohydrates, oils, and/or proteins, which are biosynthesized and deposited during seed development. These reserves are not only important for seed germination and seedling growth, but are also vital components of human and animal diets. Their production in crops is the basis of agriculture. Before reaching maturity, the seed develops other important properties, including desiccation tolerance, germination/dormancy and vigor (Bewley et al., 2013). Seed desiccation tolerance. Considered only in terms of tolerance of, or sensitivity to, desiccation, seeds can be categorized as orthodox or recalcitrant (Berjak and Pammenter, 2008). The orthodox seed acquires desiccation tolerance during seed development approximately halfway through development. This trait ensures that the seeds passes unharmed through maturation drying and

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W.-Q. Wang et al. / Plant Physiology and Biochemistry 86 (2015) 1e15

retains viability in the dry state for long periods of time (up to hundreds of years in some case) under natural or artificial conditions (Kermode and Finch-Savage, 2002). In contrast, the recalcitrant seed is desiccation sensitive and can not survive drying during ex situ conservation (Berjak and Pammenter, 2008). This creates a serious problem in the conservation of recalcitrant species. Many species with recalcitrant seeds are economically important, such as avocado, mango, lychee, cocoa, coffee, citrus, and rubber. Seed germination. Seed germination is the most critical phase in the seed plant life cycle. It determines when the plant enters natural or agricultural ecosystems. Cultivation of most crop species is dependent on seed germination. Seeds of most species acquire the ability to germinate during development. This is important for crop production, because it ensures that the untreated seed quickly germinates after sowing. However, in a few species, such as maize, wheat and rice, it can result in precocious germination, which typically occurs when developing seeds with a low degree of dormancy experience rainfall or humid conditions (Bewley et al., 2013). Precocious germination can decrease the grain quality and cause great economic losses. In some species, seeds are dormant at the end of development. Seed dormancy is defined as the failure of an intact viable seed to complete germination under favorable conditions (Bewley, 1997). It is an adaptive strategy for seed to survive under adverse natural conditions, but it also creates an obstacle for agricultural production, where rapid germination and growth are required. Seed vigor. Seed deterioration occurs always during storage of orthodox seeds, resulting in the gradually loss of vigor and even death. For crop species, preventing or minimizing the loss of vigor during storage is critical for the production in the subsequent seasons. Seed longevity is dependent on storage temperature and moisture (Walters et al., 2005). Seed priming, imbibing seeds in water or chemicals, such as PEG, for a period of times followed by dehydration, is utilized commercially to increase seed vigor (Heydecker et al. 1973; McDonald, 2000). Seed proteomics. The great biological and economic importance of seeds has led to a vast number of studies of all the above aspects of seed biology. One type of study is proteomics, the study of all the expressed proteins. Since proteins are responsible for most metabolic processes in the seed, in addition to being important structural components in the cytoskeleton, membranes, the cell wall, etc., it makes excellent sense to describe the proteome of a seed, a seed tissue, a specific cell type or a subcellular compartment. However, proteomics are also a powerful tool for detecting changes in the protein composition in response to developmental or environmental stimuli, so-called differential proteomics. In other words, proteomics can be used analytically rather than descriptively to identify proteins associated with, and therefore probably important for, specific processes and specific responses. This will be the focus in the present review e qualitative and quantitative changes in the seed proteome during the life cycle of the seed. We will start by giving a very brief overview of the methods used to study seed proteomics. We will then review the proteomics of seed development, desiccation tolerance, germination and dormancy release and vigor. Finally, we will attempt to give some perspectives. 1.1. Proteomic methods The seed proteome can be analyzed like any other proteome using the standard general procedure of protein extraction, separation and identification. The sampling is, as always, the basis for obtaining meaningful results. As discussed by Miernyk (2014) this is not a trivial point: The seed part to be analyzed needs to be considered carefully in relation to the question asked. It is also

essential to ensure the physiological (developmental stage) and genetic uniformity of the seeds sampled. The dynamic range for protein amount may be as high as 1010e1012 whereas the dynamic range for the analytical methods are much lower perhaps only 103e104 (Hortin and Sviridov, 2010; Miernyk, 2014). It can therefore be an advantage to remove the superabundant storage proteins during protein extraction or during the early stages of separation in order to improve the chances of detecting lower-abundance proteins (Miernyk and Hajduch, 2011). Gel-based methods for separation e particularly twodimensional polyacrylamide gel electrophoresis (2D-PAGE) e have dominated and will probably continue to dominate because, in addition to being reasonably quantitative, they provide a lot of information about the proteins not provided by the gel-free shotgun methods, such as changes in protein size, pI, and posttranslational modifications (PTMs) (Rogowska-Wrzesinska et al., 2013). This is always useful information, and particularly so for species where the genome has not yet been fully sequenced. Mass spectrometer-based methods for protein identification have dominated completely in recent years also in seed proteomics, but the methods have been extensively reviewed recently (e.g. Pan et al., 2009; Walther and Mann, 2010; Bantscheff et al., 2012; Rogers and Overall, 2013) and since they are essentially independent of species (except for the question of access to a full genome sequence), they will not be reviewed here. 1.2. Pitfalls in the use of 2D-PAGE and other quantitative proteomic methods Although 2D-PAGE is reasonably quantitative, a note of caution is in order concerning the way it is routinely used. The standard procedure is to extract all proteins from a series of samples, for instance a time series during seed development. The same fixed amount of protein (typically 100e500 mg protein) from each sample is then separated by 2D-PAGE and, when the gels are stained typically using Coomassie Brilliant Blue or silver nitrate, the total staining on all the gels is very similar. A total of 500e1000 discrete spots are typically discernable, where each spots normally represents one dominant unique protein. When a change in the number of pixels in a given spot is observed between different samples, it is then concluded that the amount of that protein has changed. Contrariwise, if no significant change in the number of pixels in a given spot is observed between different samples, it is concluded that the amount of that protein has not changed. Both of these conclusions can actually be wrong! The problem is that the standard procedure for sampling and loading outlined above ensures that the number of pixels in a spot represents not the absolute amount of protein in the sample, but the fraction of the sample protein contributed by that protein. To appreciate this point, let us consider two extreme cases in seed biology: (1) The composition of the proteome changes strongly, e.g. due to biosynthesis of storage proteins. This happens during the reserve deposition stage. Let us assume that the storage proteins go from constituting 10% of the total protein in the seed to 40% between two samples, while all the rest of the proteins in the seed are present in unchanged amounts. This means that the non-storage proteins will make up 90% in the first sample, but only 60% in the second. As a result all their spots will therefore decrease in size to 60%/90% ¼ 0.67 of their original size (a decrease by a factor 1.5). The relative amounts of the non-storage proteins have changed, in spite of the fact that there was no change in the absolute amounts of those proteins!


(2) The total amount of protein per seed changes strongly between samples, e.g. due to general growth and protein biosynthesis. This might happen during the early stages of seed development. In this case the amount of many of the proteins increase similarly, but on the gel their spot size will remain unchanged. Their relative amounts are constant, but their absolute amounts are not. The way to avoid such misinterpretations is to quantify the total amount of protein extracted per unit sample (per seed, per leaf, etc) and to take major shifts in protein pattern into account when interpreting spot volume changes. Similar words of caution are also relevant for non-gel-based proteomic methods such as iTRAQ, where several samples are labeled separately and then mixed before MS analysis. If the same amount of protein from each sample is mixed, it corresponds precisely to comparing several 2D-gels with the same loading.

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2. Proteomic studies of seed development Seed development can be divided into three sequential, temporal phases: histodifferentiation, reserve deposition and maturation drying (Kermode and Finch-Savage, 2002; Black et al., 2006; Bewley et al., 2013). During the histodifferentiation stage, the fertilized egg cells undergoes rapid cell division and develops into different seed tissues; thereafter, little cell division occurs and cell expansion and deposition of seed reserves (normally proteins, along with lipids or carbohydrates) occurs primarily in the storage tissue (i.e., cotyledons or endosperm); finally, seed development ends by a maturation drying, which slows down and stops the dry matter accumulation and leads the seed into a metabolically quiescent state (Kermode and Finch-Savage, 2002; Black et al., 2006; Bewley et al., 2013). Seed development has been studied by proteomics in many species, such as Medicago truncatula (Gallardo et al., 2003, 2007;

Table 1 Recent proteomic studies on seed development. Species

Arabidopsis thaliana Brassica campestri Brassica napus (rapeseed) Cunninghamia lanceolata Glycine max (soybean)

Development stage Reserve deposition

5,7 DAFb

9,11,13 DAF

Whole seed

Hajduch et al., 2010; Meyer et al., 2012

10, 16, 20, 25 DAF

35 DAF

Whole seed

Li et al., 2012

2 WAFc

3, 4, 5 WAF

Whole seed

Hajduch et al., 2006; Meyer et al., 2012

Whole seed

Shi et al., 2010

Whole seed

Hajduch et al., 2005; Agrawal et al., 2008; Meyer et al., 2012 Miernyk and Johnston, 2013 Finnie et al., 2002

Cleavage polyembryony, dominant embryo, columnar embryo, and early cotyledonary 2, 3 WAF

Medicago truncatula

Oryza sativa (rice) 5, 7 DAF 6, 8 DAF

Oryza sativa (rice, othersa) Pinus massoniana Cleavage polyembryony, dominant embryo, columnar embryo, and early cotyledonary Ricinus communis 2, 3 WAF (castor) Stage III, IV Stage IV Triticum aestivum Stage I, II (wheat) 35, 88, 125, 195, 227 and 280 -days 10, 36 DAF Zea mays (maize) 4, 7, 10 DAF

Maturation drying

6 WAF

4, 5, 6 WAF S4, S6 Stage 80, 82

S8, S9 Coat Stage 85, 86, Whole seed 87 20, 25, 30 DAF Endosperm 16e25 DAF 43 DAF Whole seed 19, 22, 31 DAF Whole seed and pod 12, 14, 16, 18, 20 Whole seed DAF 12, 14, 16, 20, Embryo, 24, 36 DAF endosperm, seed coat 13 DAF 21, 30 DAF Embryo 10, 12, 14, 16, Whole seed 18, 20 DAF 10, 20 DAF 30, 40 DAF Whole seed 12, 15, 18 DAF Endosperm

4, 5, 6 WAF

Stages VI Stage III

17, 22, 25, 28 DAF 14, 21, 30 DAF

Stages X Stage IV,V

40 DAF 40, 65 AF

28 DAF b c

Reference

Histodifferentiation

S2 Hordeum vulgare (barley) Jatropha curcas 5, 10, 15 DAF Lotus japonicus 7, 13 DAF

a

Tissue

See Deng et al. (2013). DAF: days after flowering. WAF: weeks after flowering.

52 AF

Liu et al., 2013 Dam et al., 2009 Nautrup-Pedersen et al., 2010 Gallardo et al., 2003 Gallardo et al., 2007

Xu et al., 2012 Xu et al., 2008 Lee and Koh, 2011; Sano et al., 2013a,b Xu et al., 2010 Deng et al., 2013

Whole seed

Zhen et al., 2012

Whole seed

Houston et al., 2009

Nucellus whole seed Whole seed

Nogueira et al., 2012 Nogueira et al., 2013 Guo et al., 2012

Whole seed Endosperm Embryo and endosperm Whole kernel or endosperm Embryo and endosperm Embryo

Nadaud et al., 2010 Vensel et al., 2005 Jin et al., 2013 chin et al., 2007 Me Wang et al., 2014 Huang et al., 2012

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^telain et al., 2012), soybean (Hajduch et al., 2005; Agrawal et al., Cha 2008; Miernyk and Johnston, 2013), rice (Xu et al., 2008, 2012; Lee and Koh, 2011; Sano et al., 2013a, 2013b), Arabidopsis (Hajduch et al., 2010), and castor bean (Houston et al., 2009; Nogueira et al., 2012, 2013) (Table 1). The proteomic studies on rice seed development were reviewed recently (Deng et al., 2013). Most of the proteomic studies of seed development have used whole seeds as the experimental material and only in a few cases has the proteome of embryo, endosperm and/or seed coat been studied separately (Table 1). Therefore, this section will focus mainly on proteomic changes occurring in the whole seeds during seed development. Seed filling has been the main developmental stage to be investigated in most proteomic studies. This stage starts during the late histodifferentiation and continues during reserve deposition. Some important events occurring during maturation drying are also covered in the present review although this process has only been investigated in a few species (Table 1). 2.1. Protein synthesis Cell division and enlargement occurring during seed development require proteins involved in processes such as DNA replication, transcription, cytoskeleton and cell wall formation. In many species, such as castor (Houston et al., 2009; Nogueira et al., 2013), soybean (Hajduch et al., 2005; Agrawal et al., 2008) and rice (Xu et al., 2008), most of the proteins involved in protein synthesis including ribosomal proteins, translation factors and tRNA synthases, in protein folding and stability, such as chaperones and heat shock proteins, and in biosynthesis of amino acids accumulated most abundantly at the stage of histodifferentiation and/or the early stage of reserve deposition. Their accumulation would help produce the various proteins used for cell division and expansion during these stages. During reserve deposition when cell division ceases, protein synthesis slows down, and the above proteins therefore decrease in abundance. However, storage proteins and proteins involved in cell defense and rescue are synthesized at high rates during this stage. The accumulation of storage proteins is important for nutrition during seed germination and seedling growth (Rajjou et al., 2012; Tan-Wilson and Wilson, 2012), while the proteins involved in cell defence and rescue are associated with ^telain et al., increased seed desiccation tolerance and vigor (Cha 2012; Rajjou et al., 2012; Wang et al., 2014). In Arabidopsis, some proteins related to protein synthesis, such as cytosolic ribosomal protein S15, 60S acidic ribosomal protein P2 and elongation factor EF-2 increased in abundance during reserve deposition (Hajduch et al., 2010). In addition, proteins related to protein folding and stability in some species, such as protein disulfide isomerase in developing seeds of Arabidopsis (Hajduch et al., 2010), M. truncatula (Gallardo et al., 2003) and rice (Xu et al., 2008) and BiP proteins in soybean (Hajduch et al., 2005) and rice (Xu et al., 2008) showed a high accumulation at this stage. Protein disulfide isomerase, an abundant 57-kDa protein, is a chaperone in eukaryotes, including yeast, mammals, and plants and it may participate in the synthesis and deposition of storage proteins (Kim et al., 2012). BiP, an ER-localized member of the heat shock 70 family, has been proposed to play a role in protein body assembly and retention of zeins within the ER (Boston et al., 1991; Vitale and Ceriotti, 2004). Therefore, it is not surprising to find that these proteins have an accumulation profile similar to storage proteins. High proteolytic activity occurs simultaneously with protein biosynthesis during histodifferentiation. Most of the proteolytic proteins such as proteasome family proteins, ubiquitin protease family proteins, and amino acid peptidases showed a high accumulation at the stage of histodifferentiation, and then decreased gradually in abundance during the following stage. These results

suggest that protein turnover and rearrangements during or at the end of histodifferentiation is important. Protein degradation would allow a reallocation of amino acids to pathways involved in synthesis of storage products. Proteolysis will occur across all the development stages. Some proteolytic proteins such as the ubiquitin carboxyl-terminal hydrolase-related in rapeseed (Hajduch et al., 2006), putative aminopeptidase N in rice (Xu et al., 2008), 20S proteasome a subunit E2 and 20S proteasome b subunit C1 in Arabidopsis (Hajduch et al., 2010) were found to accumulate abundantly during the reserve deposition, while others, such as 26S protease regulatory subunit proteins in rice (Xu et al., 2008) and cystatin in maize (Wang et al., 2014), had high accumulation at the stage of maturation drying. The proteases accumulating during seed development have been proposed to play a key role in nitrogen remobilization by releasing the free amino acids in the endosperm and seed coat for storage protein synthesis in the embryo (Gallardo et al., 2007). In addition, some of proteases may act as storage proteins in mature dry seed and function in the degradation of other storage proteins during seed germination (Tan-Wilson and Wilson, 2012). 2.2. Reserve accumulation Most mature seeds contain two or more reserve compounds including carbohydrates, oils and proteins, and to a large extent they are synthesized during seed development, especially during the stage of reserve deposition. Sucrose and amino acids, imported from the parent plant, are the major carbon and nitrogen sources for the synthesis of reserve compounds (Bewley et al., 2013). Enzymes involved in carbohydrate synthesis showed the highest abundance at the early stage of reserve deposition. For instance, UDP-glucose pyrophosphorylase 2 reached its peak abundance at 11 days after flowering (DAF) in Arabidopsis (Hajduch et al., 2010); in rice, starch synthase showed the highest abundance at 14 DAF (Xu et al., 2012). In developing maize seed, sorbitol dehydrogenase had reached its maximal abundance at 25 DAF and it was suggested that sorbitol dehydrogenase is an important regulator of maize grain filling, especially for hybrid Zhengdan 958 (Jin et al., 2013). Proteins involved in oil synthesis showed different accumulation profiles in non-oilseed and oilseed species. Enoyl-CoAhydratase reached the peak level at the stage of histodifferentiation in the non-oilseed species rice (Xu et al., 2008). In oilseed species, these proteins can be divided into two groups according to the trend of change. One group including ATP-citrate lyase B-1 in Arabidopsis (Hajduch et al., 2010) and rapeseed (Hajduch et al., 2006), has a similar accumulation pattern as in non-oilseed plants; the other group shows the maximal accumulation at the middle and/or late stage of reserve deposition. Change in abundance of pyruvate dehydrogenase E1 b subunit, enoyl-ACP reductase and malonyl-CoA ACP transacylase in Arabidopsis (Hajduch et al., 2010), b-ketoacyl-ACP synthetase 1, enoyl acyl carrier protein reductase and pyruvate dehydrogenase E1 a subunit in rapeseed (Hajduch et al., 2006) belongs to the latter group. 2.3. Energy production The central metabolism (glycolysis and tricarboxylic acid (TCA) cycle) provides most of the energy for processes in the seed. In developing seeds, most of the glycolytic enzymes in the cytosol and a few in plastids have been identified in many species by proteomics (Fig. 1). In castor seeds nearly all the glycolytic enzymes in both cytosol and plastids, with the exception of plastidic phosphoglyceromutase, were identified and shown to change in abundance during seed development (Nogueira et al., 2013). These data gave what appeared to be a relatively clear picture of the


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Fig. 1. Changes in the amounts of identified proteins involved in glycolysis and the TCA cycle during seed development. The figure was plotted using data reported for soybean (Glycine max, Gm) (Hajduch et al., 2006), rapeseed (Brassica napus, Bn) (Agrawal et al., 2008), rice (Oryza sative, Os) (Xu et al., 2008; Lee and Koh, 2011), castor (Ricinus communis, Rc) (Houston et al., 2009) and Arabidopsis (Arabidopsis thaliana, At) (Hajduch et al., 2010) and modified from Houston et al. (2009). The relative abundance of each enzyme was extracted for each species and plotted against the developmental time (horizontal axis) to show the trend of the change. Only the plastidial glycolytic pathway enzymes identified in these species are shown. In rice seed, the TCA cycle, PDC, CST and ODC were identified by Lee and Koh (2011), while MDH was identified by Xu et al. (2008). In each part figure, the left gray part shows the developmental stage of histodifferentiation and the following white part is the reserve deposition. In some part figures, maturation drying is depicted by the light gray area on the right. Abbreviations: CST, citrate synthase; FBA, fructose bisphosphate adolase; FK, fructose kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PDC, pyruvate dehydrogenase complex; PFK, phosphofructose kinase; PGM, phosphoglucomutase; PGI, phosphoglucose isomerase; PK, pyruvate kinase; PGK, phosphoglycerate kinase; PGAM, phosphoglycerate mutase; ODC, 2-oxoglutarate dehydrogenase complex; SuSy, Sucrose synthase; SCST, succinyl-CoA synthetase; SDH, succinate dehydrogenase; TPI, triose phosphate isomerase; UDP, UDP-glucose pyrophosphorylase.

involvement of glycolysis during seed development. However, when similar studies were performed in other species, it became clear that the identified glycolytic enzymes vary greatly not only in number, but also in accumulation pattern, among different species (Fig. 1). For example, using the same 2-D PAGE proteomic approach, a total of 63 protein spots involved in glycolysis were identified in developing castor seeds (Houston et al., 2009), while this number was only 19 in Arabidopsis seeds (Hajduch et al., 2010). In castor seeds, the identified glycolytic proteins in both cytosol and plastids

accumulated most abundantly at the stage of histodifferentiation and decreased gradually during reserve deposition, while in Arabidopsis, various trends of change were observed (Fig. 1). A variation in the number and accumulation pattern was also observed for the enzymes of the TCA cycle (Fig. 1). The above results imply that the participation of glycolysis and the TCA cycle in seed development is not entirely straightforward and possibly regulated by different mechanisms in different plant species. In addition to providing energy in the form of ATP,

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glycolysis and the TCA cycle also provide many intermediates for the biosynthesis of storage reserves, secondary metabolites, nucleotides, etc. (Møller et al., 2014), where the need differs among different plant species. Therefore, the various accumulation patterns of proteins involved in glycolysis and the TCA cycle may reflect different requirement for glycolytic and/or TCA cycle intermediates in biosynthesis. In many species, it was interesting to find that proteins of the pyruvate dehydrogenase complex (PDC) had a consistent accumulation pattern during seed development. They accumulated abundantly at the stage of histodifferentiation and decreased during reserve deposition (Fig. 1). The PDC catalyzing the conversion of pyruvate to acetyl-CoA is a step linking the glycolytic pathway to the TCA cycle. When the accumulation of PDC decreased, the carbon flux through the TCA cycle would be expected to decline. The time at which the PDC declines coincides with the time that storage reserves start to be deposed (Fig. 1). Therefore, it is possible that the decrease in PDC plays a role in switching carbon flux through the energy production to the deposition of storage reserve. In addition, a decrease in the flux through the TCA cycle would decrease the production of NADH and therefore limit the oxygen consumption. Because the internal oxygen level is low inside developing seeds (Geigenberger, 2003; Borisjuk and Rolletschek, 2009), this will limit the occurrence of anoxia inside the seeds, which is a damaging to the cells (Geigenberger, 2003). Proteins involved in photosynthesis accumulated during seed development in almost all the species, but the pattern differed between green oil abundant (GO) seeds and nongreen oil abundant (NGO) and green carbohydrate abundant (GC) seeds. In general, photosynthetic proteins in NGO, e.g. castor (Nogueira chin et al., 2007) seeds and GC seeds, et al., 2013) and maize (Me such as barley (Finnie et al., 2002), rice (Xu et al., 2008) and wheat (Guo et al., 2012) seeds, accumulated to high abundance at the stage of histodifferentiation and/or the early stage of reserve deposition and then decreased during the following development, while in GO seeds, such as soybean (Agrawal et al., 2008), M. truncatula (Gallardo et al., 2003, 2007), Arabidopsis (Hajduch et al., 2010) and rape (Hajduch et al., 2006) seeds, those proteins accumulated most abundantly at the stage of reserve deposition. In GO seeds, light is known to provide the energy for oil biosynthesis (Ruuska and Ohlrogge, 2004; Goffman et al., 2005; Allen et al., 2009; Borisjuk et al., 2013), and a number of studies have shown that the accumulation of photosystem components coincides with oil accumulation in GO seeds. The components were photosystem (PS) II oxygen-evolving complex 1, PSII subunit P-1 and 2, chlorophyll a/b binding protein/light harvesting complex (LHC) II type I and PSI LHC gene 3 in Arabidopsis (Hajduch et al., 2010), chlorophyll a/b binding protein and oxygen-evolving enhancer protein in M. truncatula (Gallardo et al., 2003, 2007), oxygen-evolving enhancer protein and water-soluble chlorophyll protein in rape (Hajduch et al., 2006), and chlorophyll a/b binding protein in soybean (Agrawal et al., 2008) seeds. Photosystem components have also been identified in GC seeds, such as wheat (Guo et al., 2012) and rice (Xu et al., 2008) seeds. This indicates that photosynthesis occurs also in the GC seeds where it may support starch biosynthesis. In barley seeds, pericarp photosynthesis supplied oxygen to the growing lateral and peripheral regions of the endosperm, and the accumulation of oxygen correlated well with the accumulation of ATP and starch (Rolletschek et al., 2004). Ribulose bisphosphate carboxylase/oxygenase (Rubisco) has been found in the above seed types. In GO seeds, Rubisco may play also an important role in the biosynthesis of oil. It has been reported that carbon flux through Rubisco without the Calvin cycle improved the efficiency of carbon use in the developing green seeds

during biosynthesis of oil (Schwender et al., 2004; Allen et al., 2009). In developing rape seeds, Rubisco participated in an alternative pathway in conversion of carbohydrate to oil that provided 20% more acetyl-CoA for oil synthesis and resulted in 40% smaller loss of carbon as CO2 (Schwender et al., 2004). The role of Rubisco in NGO and GO seeds is still unclear. 2.4. Formation of cytoskeleton and cell wall Cell division and expansion during seed development is expected to rely largely on the biosynthesis of proteins involved in the formation of the cytoskeleton and the cell wall. The microtubules and actin filaments, assembled from tubulin and actin molecules, respectively, are the two key components of the cytoskeleton (Mayer and Jürgens, 2002). Proteomic analysis of seed development revealed that tubulins accumulated at high abundance during histodifferentiation, and then decreased during the following developmental stage. In most species, actin also accumulated abundantly during histodifferentiation, but decreased later than the tubulins, normally at the mid or late stage of reserve deposition. These results imply that tubulins may play more roles in cell division, while actin may be essential for both cell division and expansion during seed development. The actin cytoskeleton seems to be a dynamic structure, which undergoes dramatic reorganization during seed development. Profilin in rice (Xu et al., 2008) and castor (Nogueira et al., 2013) seeds and actin depolymerizing factor in Arabidopsis (Hajduch et al., 2010), rice (Xu et al., 2008) and castor (Houston et al., 2009) seeds accumulated to high levels at the stage of histodifferentiation and early stage of reserve deposition and then decreased in abundance during the following stage. These two proteins are both thought to play a role in reorganizing the actin cytoskeleton (Staiger et al., 1997; Henty-Ridilla et al., 2013). The polysaccharides, cellulose, hemicelluloses and pectin, are the main components of the plant cell wall. Proteomic studies identified many proteins involved in the biosynthesis of these polysaccharides in developing seeds. The identified proteins vary greatly among different plant species and are involved in various processes of cell wall formation. The a-1,4-glucan-protein synthase, identified in developing M. truncatula (Gallardo et al., 2007) and castor (Houston et al., 2009; Nogueira et al., 2013) and cellulose synthase catalytic subunit in rape (Hajduch et al., 2006) seeds are involved in biosynthesis of cellulose. Rhamnose synthase found in Arabidopsis (Hajduch et al., 2010) and UDP-D-apiose/xylose synthase in Jatropha curcas (Liu et al., 2013) seeds are related to the biosynthesis of pectin. Pectin methylesterase, involved in cell wall modification, was found in rice (Xu et al., 2008) and castor (Nogueira et al., 2013) seeds. Reversibly glycosylated polypeptide, possibly participating in the synthesis of xyloglucan, one of the hemicelluloses (Dhugga et al., 1997) differentially accumulated in M. truncatula (Gallardo et al., 2003) and Arabidopsis (Hajduch et al., 2010) seeds. In addition, glycoside hydrolases, such as b-D-xylosidase, b-galactosidase and xyloglucan endotransglycosylases/hydrolases, involved in the biosynthesis and remodeling of glycans (Minic, 2008), were found in developing seeds of many species. The dynamic changes in the cytoskeleton and cell wall would assist cell expansion and reserve nutrition deposition. The accumulation of the cell wall-related proteins fall into two groups: (1) Proteins whose abundance increases transiently during histodifferentiation, e.g. rhamnose synthase in Arabidopsis, a-1,4glucan-protein synthase in M. truncatula, rice and castor, cellulose synthase catalytic subunit in rape and xyloglucan endotransglycosylases/hydrolases in soybean (Agrawal et al., 2008) seeds; (2) Proteins whose abundance increases strongly during reserve deposition, e.g. reversibly glycosylated polypeptide in rice and M. truncatula, UDP-D-apiose/xylose synthase in J. curcas (Liu


et al., 2013) and b-D-xylosidase in rice (Xu et al., 2008) and wheat (Guo et al., 2012). These results suggest that cell wall formation differs between species and even within species between different developmental stages. 2.5. Removal of reactive oxygen species The production of reactive oxygen species (ROS), such as superoxide radical (O 2 ) and hydrogen peroxide (H2O2), is an unavoidable consequence of aerobic metabolism (Møller et al., 2001). ROS can act as signal molecules to regulate biological processes, but can also damage cellular components (Møller, 2001; Møller et al., 2007). The amount of ROS must be tightly controlled in the cell. At the stage of histodifferentiation, production of ROS is high due to the high metabolic and respiratory activity in the developing seeds (Bailly, 2004; Bailly et al., 2008). It appears that the enzymes of ascorbateeglutathione cycle play a major role in removal of ROS at this stage. In Arabidopsis, six of ten proteins accumulating abundantly during histodifferentiation belonged to antioxidant enzymes of the ascorbateeglutathione cycle. They were three ascorbate peroxidases (APX) and three monodehydroascorbate reductases (MDHR) as well as one glutathione synthetase (Hajduch et al., 2010). In addition, two catalases (CAT) accumulated in high abundance during this stage (Hajduch et al., 2010). In rice, five antioxidant enzymes of the ascorbateeglutathione cycle, including three APXs, one MDHR and one superoxide dismutase (SOD) and one protein involved in the detoxification of aldehydes, lactoylglutathione lyase accumulated abundantly during histodifferentiation (Xu et al., 2008). The antioxidant enzymes of the ascorbateeglutathione cycle were also observed to accumulate abundantly during histodifferentiation in many other species, such as M. truncatula (Gallardo et al., 2003, 2007), soybean (Agrawal et al., 2008), castor (Houston et al., 2009; Nogueira et al., 2013), J. curcas (Liu et al., 2013), and wheat (Guo et al., 2012). As the metabolic and respiratory activity decreases during reserve deposition and maturation drying, the production of ROS will be reduced. However, the internal hypoxic condition of the developing seeds (Borisjuk and Rolletschek, 2009) and the metabolic imbalance resulting from the desiccation at these stages could well cause ROS production to increase (Bailly, 2004; Bailly et al., 2008). The antioxidant enzymes of the ascorbateeglutathione cycle were constant or increased at the early stage of reserve deposition, but decreased during later development. However, many thiol-dependent antioxidant enzymes increased in abundance during reserve deposition and even during maturation drying. For example, in castor seeds, glutaredoxin, glutathione peroxidase (GPX), glutathione reductase, thioredoxin and one peroxiredoxin (Prx), increased in abundance during reserve deposition (Nogueira et al., 2013). In rice seeds, one proteomic study observed that thioredoxin peroxidase (TPX) and GPX increased in abundance during reserve deposition (Xu et al., 2008), while another study found that glutathione S-transferase accumulated abundantly not only during reserve deposition, but also in the fully mature seeds (Lee and Koh, 2011). This would suggest that thiol-dependent antioxidant proteins play a major role in the removal of ROS during reserve deposition and maturation drying. It appears that detoxification of aldehydes is also a necessary process for seed development, especially in the late stages. During rice seed development, it was striking to observe that glyoxalase was the most abundant protein accounting for more than 1.6% of the total protein (Xu et al., 2008). This protein not only increased in abundance at the stage of reserve deposition, but also during maturation drying (Lee and Koh, 2011). Glyoxalase was also observed to increase in abundance during reserve deposition in M. truncatula (Gallardo et al., 2007) and during maturation drying

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in maize (Wang et al., 2014) seeds. This enzyme is involved in the detoxification of methylglyoxal, a highly reactive aldehyde derived from glycolysis (Thornalley, 2003). The important role of aldehyde detoxification for seed developments is underlined by the observation that aldehyde dehydrogenase (ADH) accumulated highly during reserve deposition but also during maturation drying in several species, such as rice (Xu et al., 2008; Lee and Koh, 2011) and maize seeds (Wang et al., 2014). 2.6. Protein modifications Few studies have looked for protein modifications during seed development. Meyer et al. (2012) analyzed the phosphoproteome of Arabidopsis, rapeseed and soybean at five sequential stages during seed development. During seed development, 459, 325 and 172 phosphoproteins accumulated differentially in soybean, rapeseed and Arabidopsis, respectively. In soybean and Arabidopsis seeds, protein phosphorylation occurs mainly during the stage of histodifferentiation and early stage of reserve deposition; while in rapeseed, a majority of the phosphoproteins were identified during maturation drying (Meyer et al. 2012). Protein phosphorylation varied greatly among species and only few phosphopeptides and phosphoproteins were identified in all three species (Meyer et al. 2012). Tyrosine (Tyr) phosphorylation was thought to be more prevalent in crop oilseeds, since the occurrence rate of Tyr phosphorylation was 7.1, 7.2 and 3.5% for soybean, rapeseed and Arabidopsis, respectively. This should be compared to an overall frequency of 5.5% phospho-Tyr out of a reported 100,000 phosphorylation sites in all proteins and all species, but only 1.3% in Arabidopsis (out of 1057 sites) (Rao and Møller, 2012). Two protein phosphorylation motifs found by Meyer et al. (2012), the Protargeted motif E-X-X-X-X-S-P and the Thr motif X-T-D-X, may turn out to be unique for the developing seed. The former can be recognized by protein kinases that are involved in the regulation of transcription (Meyer et al., 2012). 3. Desiccation tolerance Desiccation tolerance refers to the ability of an organism to endure loss of all or almost all of its cellular water without irreversible damage (Leprince and Buitink, 2010). This phenomenon is widely observed in the plant kingdom, including ferns, mosses, pollen and seeds of higher plants and resurrection plants (Hoekstra et al., 2001; Oliver et al., 2005). In higher plant, only orthodox seeds are desiccation tolerant. This property gives the orthodox seeds the ability to survive under extreme environmental conditions. In the dry state, the orthodox seeds can be stored for long periods of time, depending on species and storage temperature and humidity (Walters et al., 2005; Berjak and Pammenter, 2008). A number of protective mechanisms have been proposed for seed desiccation tolerance, including metabolic ‘switch off’, structural stabilization, accumulation of protective molecules and removal of ROS (Pammenter and Berjak, 1999; Berjak and Pammenter, 2008). Details of some of these protective mechanisms have been identified by proteomic studies as outlined in the following sections. 3.1. Experimental approaches Orthodox seeds acquire desiccation tolerance during development, and lose it during germination (Kermode and Finch-Savage, 2002). Thus, comparing the dehydration response of seeds during development and/or germination is a common approach for the study of desiccation tolerance. In proteomic studies, this approach has been applied to rice (Sano et al., 2013a, 2013b) and maize (Huang et al., 2012) seeds. However, it is not easy to differentiate

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the events related to seed desiccation tolerance from those of seed development or germination (Leprince and Buitink, 2010). Some physiological models have been developed to overcome this difficulty. Re-establishment of desiccation tolerance in germinating seeds by application of a mild osmotic stress with polyethylene glycol (PEG) solution is one such model (Bruggink and van der Toorn, 1995) and has been used in the proteomic analysis of M. truncatula seed desiccation tolerance (Boudet et al., 2006). In pea, application of the chemical reagents, CaCl2 and methylviologen (MV), can increase and decrease desiccation tolerance of germinated seeds, respectively (Wang et al., 2012a). In this way, seed desiccation tolerance and germination are uncoupled. This approach was used to identify the potential proteins related to desiccation tolerance in pea (Wang et al., 2012b). It is difficult to establish a comparable model in recalcitrant seeds during their development and germination. However, their tolerance to desiccation can be altered by application of chemical reagents, like NO (Bai et al., 2011), and H2O2 scavengers (Chen et al., 2011). The mechanism of the effect of NO on seed desiccation tolerance was investigated by the proteomic analysis of recalcitrant Antiaris toxicaria seeds (Bai et al., 2011). In view of the contrasting dehydration properties between orthodox and recalcitrant seeds, a comparison between the seeds of these two types of species, especially the closely related ones is a useful model to identify the mechanisms related to desiccation tolerance (Kermode, 1997; Oliver et al., 2011). This has been applied in the proteomic study of seeds of two Papilionaceae species with different desiccation tolerance (Delahaie et al., 2013). 3.2. Accumulation of LEA proteins Late embryogenesis abundant (LEA) proteins are well characterized as protective molecules against desiccation stress (Cuming, 1999). They are thought to act by replacing water, sequestering ions, removing ROS and/or stabilizing protein and membrane structure (Cuming, 1999; Tunnacliffe and Wise, 2007; Battaglia et al., 2008). LEA proteins are commonly found in proteomic analyses of desiccation tolerance. Boudet et al. (2006) investigated the change of the heat-stable proteome during germination and after re-establishment of desiccation tolerance in M. truncatula seeds. Six LEA proteins from four gene groups, including Em6, MP2, an isoform of PM18, six isoforms of SBP65, PM25, and one isoform of DHN3 were identified to be associated with desiccation tolerance (Boudet et al., 2006). In this species, LEA proteins of Em6, PM18 and PM25 were also observed to accumulate abundantly during late seed development when the seed acquired desiccation tolerance by a similar proteo^telain et al., 2012). mic analysis (Cha Recently, Delahaie et al. (2013) compared the heat-stable proteome between recalcitrant Castanospermum australe and orthodox M. truncatula seeds, both of which belong to the Papilionaceae subfamily. They found that, out of 12 LEA proteins, six (EM1, EM6, MP2, PM25, LEAm and SBP65) accumulated only at low levels and six (PM1, D113.I, two D34 members, PM10 and PM18) were undetectable in the C. austral seed proteome. Nine of these proteins (EM1, EM6, MP2, PM25, SBP65, D113.I, D34.I, PM10, and PM18) were also absent from or accumulated only to low levels in the desiccation-sensitive seeds of M. truncatula mutants of ABI3 (Delahaie et al., 2013). This further validates a correlation between the absence of LEA protein accumulation and seed desiccation sensitivity. This study also revealed that most of the desiccation tolerance-related LEA proteins were positively regulated by ABI3. Analysis of the proteome changes during pea seed germination revealed that the abundance of SBP65 (belonging to the group 3 of LEA proteins) continuously decreased after 18 h of germination,

coinciding with the loss of desiccation tolerance (Wang et al., 2012b). At the same imbibition time, seeds imbibed in CaCl2 were more tolerant to dehydration than seeds imbibed in distilled water, while the SBP65 protein accumulated to a higher level in the former seeds (Wang et al., 2012b). In rice, a proteomic investigation of the change in the amount of stress-related proteins during seed development showed that several LEA proteins accumulated after maturation drying and remained at high levels in the mature seeds (Sano et al., 2013a). In maize seeds, one LEA protein, EMB564 accumulated abundantly in desiccation-tolerant seeds and decreased in amount during germination when the seed lost its desiccation tolerance (Huang et al., 2012). 3.3. Removal of ROS Dehydration will disrupt the metabolism of seeds and lead to production of ROS, such as H2O2, O2, singlet oxygen (1O2) and the hydroxyl radical (HO ) (Bailly, 2004; Kranner and Birtic, 2005; Berjak and Pammenter, 2008). At lower concentration, ROS can act as a messenger to regulate biological process, while they can damage cellular components, like lipids, proteins and DNA at higher concentration (Møller, 2001; Bailly, 2004; Møller et al., 2007). It is also possible that breakdown products, e.g. oxidized peptides deriving from oxidized proteins, can act as signals (Møller and Sweetlove, 2010). Thus, ROS and the various oxidation products must be strictly controlled in seeds during dehydration (Bailly, 2004; Kranner and Birtic, 2005; Berjak and Pammenter, 2008). In the rice seed proteome, Prx and a putative aldose reductase were observed to accumulate at the beginning of the dehydration phase, and continuously increased their abundance with seed dehydration, while glyoxalase I accumulated during the later stages of dehydration (Sano et al., 2013a). These proteins may play a role in protecting seeds against ROS damage during desiccation (Sano et al., 2013a). Protein carbonylation is one of the most commonly occurring protein modifications by ROS (Møller et al., 2007). Analysis of protein carbonylation in recalcitrant A. toxicaria seeds using 2-D PAGE showed that the number of carbonylated proteins increased during dehydration, but was reduced by pretreating the seeds with NO, which decreased the desiccation sensitivity of A. toxicaria seeds (Bai et al., 2011). It has been proposed that NO promotes seed desiccation tolerance by decreasing and increasing carbonylation and S-nitrosylation of antioxidant enzymes, respectively, which increases the ability of antioxidant enzymes to remove ROS (Bai et al., 2011). 3.4. Stabilization of structure In the pea seed proteome, the amounts of two proteins related to structural stabilization, the TCP-1/cpn60 chaperonin family protein and tubulin a-1 chain decreased during seed germination. Pea seeds imbibed in CaCl2 and MV were more and less tolerant to dehydration, respectively, compared to seeds imbibed in distilled water. In agreement, the chaperonin TCP-1/cpn60 showed lower accumulation in seeds imbibed in MV than in seeds imbibed in distilled water, while tubulin a-1 chain had higher accumulation in seeds imbibed in CaCl2 (Wang et al., 2012b). Members of the TCP-1 chaperonin family act as molecular chaperones for the cytoskeleton proteins, tubulin and actin and probably also for other proteins (Yaffe et al., 1992; Vinh and Drubin, 1994), while tubulin is an important cytoskeleton component. 3.5. Other mechanisms Other mechanisms for desiccation tolerance highlighted by seed proteomic studies include pathogen resistance (Huang et al.,


2012; Wang et al., 2012b), removal of unneeded or damaged proteins (Huang et al., 2012), and/or stabilization of long-lived mRNAs (Sano et al., 2013b). Accumulation of vicilin-like antimicrobial peptides 2e3 in pea seeds, NBS-LRR resistance-like protein RGC456 and major allergen Bet v 1.01C in maize seeds and some storage proteins in both pea and maize seeds all correlated well with the loss or acquisition of desiccation tolerance (Huang et al., 2012; Wang et al., 2012b). The proteasome is a multi-subunit proteinase complex that is involved in ATP/ubiquitin-dependent proteolytic pathways (Sassa et al., 2000), which removes redundant or damaged proteins (Grune et al., 1997; Li et al., 2010). In maize seeds, the change in the amount of proteasome subunit a type l correlated with the change in desiccation tolerance during seed development and germination (Huang et al., 2012). It has been proposed that the synthesis of proteins, but not mRNAs, is a prerequisite for seed germination (Rajjou et al., 2004; Sano et al., 2012). Dry seeds contain a large amount of stored long-lived mRNA (Nakabayashi et al., 2005; Howell et al., 2009), which may support protein biosynthesis during the early stages of seed germination. Sano et al. (2013b) suggested that the accumulation of glycine-rich RBP 1A during seed development may play a role in stabilizing these long-lived mRNA during dehydration and in the dry seeds. 4. Seed germination and dormancy release 4.1. Seed germination versus seedling growth Germination begins with water uptake by the seed (imbibition) and ends with the emergence of the embryonic axis, usually the

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radicle, through the structures surrounding it (Bewley et al., 2013). This is known as “germination sensu stricto” (simply called “germination” in the following) and does not include seedling growth. During germination and seedling growth, the fresh weight follows a tri-phasic curve (Fig. 2). In phase I there is a rapid increase in fresh weight caused by water uptake. During the slower phase II metabolism gets underway initially supported by mRNA present in the mature seed. After emergence of the radicle, signaling the end to germination, a rapid growth phase follows (phase III) (Bewley et al., 2013). Note that the term “seed vigor” discussed in the following section includes both germination and growth. 4.2. Proteomic changes during seed germination The ability of the seeds to germinate rapidly and vigorously under suitable environmental conditions is an important trait for any plant species and no less so for agricultural species. The mature seed is an easily accessible, compact and well-defined object, which is relatively simple to study under a variety of conditions. As a result, in more than 50 papers proteomics have been used to study changes in the seed proteome during germination as affected by a range of physical, chemical and biological conditions. In this section we will describe the way the proteome changes during normal germination while proteomic changes associated with processes like dormancy release and responses to environmental stress will be treated in the following sections. Because of the limited space available, we have been quite selective in our coverage of the literature in this section. The discussion will also focus more on the protein groups involved and less on the individual proteins than in

Fig. 2. Major biological processes identified by proteomic studies during germination sensu stricto (phases I and II). The figure was modified from Nonogaki et al. (2010) using data reported for barley (Hordeum vulgare, Hv) (Bønsager et al., 2007), cress (Lepidium sativum, Ls) (Müller et al., 2010), alfalfa (Medicago sativa, Ms) (Yacoubi et al., 2011), pea (Pisum sativum, Ps) (Wang et al., 2012), Arabidopsis (At) (Galland et al., 2014) and rice (Os) (Han et al., 2014a,b). Phase I of germination has only been investigated by proteomics for barley and Arabidopsis seeds and the biological processes identified in both species are shown in the figure. Proteins involved in amino acid metabolism, translation, reserve mobilization, energy production and detoxification increase in abundance during phase II of germination in at least three species. Only these biological processes are shown in the figure. APX, Ascorbate peroxidase; ASL, O-acetylserine (thiol) lyase; ASP, aspartate aminotransferase; CAT, catalase; Cys, cysteine; DHAR, dehydroascorbate reductase; GR, glutathione reductase; HSPs, heat shock proteins; LEA, Late embryogenesis abundant protein; LGL, lactoylglutathione lyase; MDAR, monodehydroascorbate reductase; Met, methionine; PDI, protein disulfide isomerase; PER, peroxiredoxin; ROC, rotamase cyclophilin; SAM, S-adenosylmethionine synthetase; SHM, serine hydroxymethyltransferase; SOD, superoxide dismutase. Abbreviations of proteins involved in glycolysis are same to Fig. 1. Note that different translation elongation factors, ribosomal proteins, proteasomes, proteases, chaperonins, HSPs, LEA and PDI were identified in different species.

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the other sections. Several recent reviews have been published on proteomic changes associated with various aspects of seed germination (Arc et al., 2011; Rajjou et al., 2012; He and Yang, 2013; Tan et al., 2013). The processes taking place during germination, identified mainly by physiological and genomic methods, are well illustrated in the tri-phasic germination model (Bewley, 1997; Nonogaki et al., 2010; Weitbrecht et al., 2011; Bewley et al., 2013). Some of the processes occurring during germination, especially during germination sensu stricto (phase I and II) have also been identified by many proteomic studies (Fig. 2) e translation, reserve mobilization and detoxification occurs during both phase I and II of germination (Fig. 2). In many species, amino acid metabolism and energy production are mainly activated during phase II of germination (Fig. 2). The mature seed contains all the proteins and mRNA needed to get metabolism started. During the early stages of seed imbibition de novo protein biosynthesis is at least partly dependent on these stored mRNAs (Nonogaki et al., 2010; Rajjou et al., 2012). The general changes in the seed proteome during germination will be exemplified by two studies, one on Arabidopsis (Galland et al., 2014) and one on rice (Yang et al., 2007). In Arabidopsis seeds, Galland et al. (2014) carried out a very interesting study where not only differential protein expression, but also de novo protein synthesis measured as 35S-methionine (Met) incorporation, was followed during different germination phases. A total of 202 proteins spots (representing 158 nonredundant proteins) were radioactively labeled and therefore at least partially de novo synthesized. Out of the 273 spots not radioactively labeled (representing 140 non-redundant proteins), 123 spots showed increased abundance in spite of the fact that they were not de novo synthesized. This demonstrates the extent to which PTMs such as phosphorylation or protease degradation, modify the proteome composition as viewed by 2D-PAGE (see also later). When the frequency distribution of functional categories was compared for de novo synthesized protein spots and the unlabeled protein spots also increasing in abundance, some marked differences were observed. The categories energy, metabolism (including enzymes involved in Met metabolism e see later) and protein fate included >53% of the de novo synthesized proteins, but only 34% for unlabeled protein spots, the difference being the 35% unlabeled storage proteins, which was not unexpected. However, the de novo synthesized proteins included 9% “cell components” and “cell cycle” proteins, which were almost entirely absent in unlabeled spots. Thus, these latter proteins were not present in the mature seeds, but were synthesized during germination. In rice seeds, the abundance of 63 proteins was observed to decrease during germination, while the abundance of 69 proteins increased (including 20 induced proteins) (Yang et al., 2007). The decreasing proteins were mainly storage proteins (globulins), proteins associated with seed maturation (e.g. LEA proteins) and proteins related to dehydration. Among the increasing proteins, enzymes involved in starch degradation and glycolysis dominated, but also breakdown products of storage proteins increased in abundance. Most of these changes are quite predictable. Methionine-related enzymes have been observed to change in abundance during germination of seeds from a range of different species e Arabidopsis (Gallardo et al., 2001, 2002; Galland et al., 2014), pea (Wang et al., 2012b), wheat (Fercha et al., 2013), and rice (Liu et al., 2014) as well as beech, Norway maple and sycamore (Pawłowski, 2010). In addition to the obvious importance of Met itself in protein biosynthesis, the role of Met in the biosynthesis of polyamines, ethylene and biotin (Roje, 2006), all via S-adenosylmethionine (SAM), is probably also significant.

4.3. Proteomic changes associated with seed dormancy Dormancy is the temporary failure of a seed to complete germination under favorable conditions. This means that they cannot complete phase II (Bewley et al., 2013). “According to the hormone balance theory, the relative actions of abscisic acid (ABA) (inhibitory) and gibberellic acid (GA) (promotive) are the primary determinants of seed dormancy and germination” (Bewley et al., 2013). However, a proteomic study in Arabidopsis showed that the proteomic profile of dormant seeds was quite different from that of non-dormant seeds treated with exogenous ABA to make them dormant. This indicates that the mechanism of dormancy induction also differed (Chibani et al., 2006). Seed dormancy can be broken by a number of treatments where low temperature for longer periods of time (cold stratification) is probably the best studied. However, nitrate or nitrite treatment can also break certain types of dormancy (e.g. Arc et al., 2012). It is likely that this is connected to the production of NO from nitrite in the mitochondria under the hypoxic conditions of the seed interior (Hebelstrup and Møller, in press), since NO by itself can also break dormancy. In Arabidopsis, dormancy breaking by stratification and by exogenous nitrate gave very similar proteome adjustments (Arc et al., 2012). About 35 proteins belonging to the groups of storage proteins and stress response and detoxification were more abundant in the dormant seeds, while proteins belonging to the groups of energy (20), amino acid metabolism, folding and stability, proteolysis and mRNA metabolism and protein synthesis (about 10 each) were more abundant in the non-dormant seeds. 4.4. Changes in post-translational modifications during germination PTMs in proteins can be regulatory, e.g. phosphorylation, they can be part of protein degradation pathways, e.g. ubiquitination, or they can perhaps be both as proposed for carbonylation (Arc et al., 2011). Protein carbonylation occurs as a result of metal-catalyzed oxidation involving ROS and it is probably the most common irreversible protein oxidation PTM (Møller et al., 2011). Han et al. (2014a,b) identified more than 800 phosphoproteins in rice seeds, out of which 149 changed in amount during germination. Protein destination-related proteins were the largest functional category and half of these proteins were protein kinases and phosphatases. This demonstrates that protein phosphorylation is heavily involved in reprogramming cellular metabolism during seed germination and provides a catalog of potential regulatory proteins. Job et al. (2005) investigated the occurrence of protein carbonylation during germination of Arabidopsis seeds. They found that the carbonylation of a number of important metabolic proteins, e.g. glycolytic enzymes, mitochondrial ATP synthase and Rubisco, increased during germination without any apparent ill effects on the seeds, which germinated at high rates and grew vigorously. They therefore suggested that this could be “a means to adapt embryo metabolism to the oxidative conditions encountered during germination” (Job et al., 2005). We need to get a more precise idea of the meaning of this metabolic adaption. However, since the tissues inside a mature seed are hypoxic (e.g. Rolletschek et al., 2004), the sudden influx of oxygenated water into the seed during phase I will expose the seed to a serious oxidative challenge. Another type of protein oxidation involving Met sidechains, the first step of which to Met-SO is reversible, is involved in protecting more sensitive sites from oxidative damage (Levine et al., 1996) and in regulating metabolic processes (Rao et al., 2014). Whether irreversible carbonylation, which is usually thought to lead to protein degradation, can act in similar ways is still an open question.


4.5. Proteomic changes in response to abiotic and biotic stress Tan et al. (2013) summarized the results from 13 studies in which proteomics had been used to study the effect of a range of environmental conditions on the germinating seed proteome. A total of 561 proteins changed their abundance level in response to the treatments. The largest protein groups affected were glycolysis (43 proteins), storage protein mobilization (117), protein processing (55), osmotic homeostasis (37) and ROS scavenging (42). However, the very diverse nature of the stresses studied e copper and cadmium ions, drought, low temperature, hormones (GA, ABA, salicylic acid) and a-amanitin (inhibitor of RNA polymerase II), as well as Fusarium graminearum infection e in combination with eight different plant species meant that a consistent response could not be seen for any protein group. There was, for instance, no indication that an increase in the abundance of ROS scavenging enzymes was a general stress response (Tan et al., 2013 and references therein). 5. Seed vigor The definition of seed vigor adopted by the Association of Seed Analysts (AOSA) states: “Seed vigor comprises those seed properties that determine the potential for rapid, uniform emergence, and development of normal seedlings under a wide range of field conditions” (Black et al., 2006). Seed vigor is therefore central to the successful propagation of agricultural crops. However, this very complex property obviously depends on a wide range of biochemical and molecular variables making it difficult to characterize (Rajjou et al., 2012). Seeds with different vigor resulting from aging and priming, where aging decreases and priming increases seed vigor, have been studied by proteomics (Gallardo et al., 2001; Rajjou et al., 2008; Catusse et al., 2011; Wu et al., 2011; Xin et al., ^telain et al., 2011; Yacoubi et al., 2011, 2013; Chu et al., 2012; Cha 2012; Wang et al., 2012). These studies have led to the identification of many proteins and metabolic processes potentially important for seed vigor. 5.1. Methionine metabolism Met is essential in all organisms because it functions not only as a building block for protein synthesis but also as the precursor of SAM, the methyl group donor for the biosynthesis of polyamines, ethylene and biotin (Roje, 2006; Rajjou et al., 2012). Met synthase catalyzes the formation of Met by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in Met biosynthesis, and also serves to regenerate the methyl group of SAM (Eichel et al., 1995). Met synthase decreased in abundance in untreated control alfalfa seeds in the presence of NaCl during germination (Yacoubi et al., 2013). This decrease did not occur in osmoprimed seeds imbibed under salinity or control (water) conditions, which paralleled an increased seed vigor afforded by the osmopriming treatment (Yacoubi et al., 2013). SAM synthetase is a key enzyme converting the Met to SAM. For sugarbeet seeds, priming treatment promoted germination and increased the accumulation of SAM synthetase, while aging treatment caused a delay and decrease in seed germination and decreased the accumulation of this protein (Catusse et al., 2011). Cysteine (Cys) is synthesized from serine (Ser), and serves as the sulfur donor for Met. The amounts of several proteins responsible for Cys and Ser biosynthesis also exhibit a positive correlation with seed vigor. Cys synthase (O-acetyl-serine (thiol) lyase) is responsible for the formation of Cys from O-acetyl-serine and hydrogen sulfide (Rolland et al., 1996). In sugarbeet, this enzyme increased in abundance in primed and aged-primed seeds, but decreased in aged seeds (Catusse et al., 2011). Aging treatment also decreased

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the accumulation of this protein during imbibition. The abundance of Cys synthase decreased in untreated control seeds, but was constant in osmoprimed seeds during germination in the presence of NaCl (Yacoubi et al., 2013). The de novo synthesis of cysteine synthase was also inhibited in artificially aged seeds during germination (Rajjou et al., 2008). In plants, Ser is biosynthesized by two different pathways: a phosphorylated pathway via 3phosphoglycerate and a photorespiratory pathway via glycine (Ho et al., 1998). Catusse et al. (2011) observed that phosphoserine aminotransferase and serine hydroxymethyltransferase increased in abundance in primed and aged-primed seeds, and decreased in aged sugarbeet seeds. The conclusion from these observations is that proteins involved in Met metabolism or the methyl cycle are very important in seed vigor. 5.2. Protein synthesis and destination Proteins involved in protein synthesis. Seed aging has a strong impact on the translation process. Many translation factors, including translation initiation factors eIf4A, eIf3 and EBP1 and translation elongation factor eIF-1a, eIF-1b, eIF-1 g and eEF-2 decreased in abundance in sugarbeet seeds during aging (Catusse et al., 2011). In Arabidopsis seeds, several proteins related to translation, such as initiation factor 4A-1, elongation factor 1-g2, elongation factor1B-g, and ribosomal protein 60S were less abundant in aged seeds than in control seeds during germination (Rajjou et al., 2008). Artificial aging of pre-harvest soybean seeds decreased significantly the accumulation of translation elongation factor Tu1, Tu2 and 1-a (Wang et al., 2012). Priming treatment enhances seed vigor and increases the accumulation of the proteins involved in translation. In sugarbeet seeds, accumulation of eIF-1a, IF-1 g and eEF-2 was reversed when aged seeds were subjected to a priming treatment (Catusse et al., 2011). All these results indicate that translation initiation and elongation factors are important for a high degree of seed vigor. Proteins involved in protein folding. The amounts of heat shock proteins, chaperones and LEA proteins all correlate closely with seed vigor. In maize, HSP18, 17.2 and 16.9 were more abundant in high-vigor seeds (Wu et al., 2011). Osmopriming treatment increased the abundance of HSP 70, 20 and 17.7, class I HSP 18.2 and 17.4 in Arabidopsis (Gallardo et al., 2001) and alfalfa (Yacoubi et al., 2011) seeds and GroEL, a complex oligomeric protein and a molecular chaperone (Ryabova et al., 2013) in alfalfa (Yacoubi et al., 2011) seeds. In sugarbeet seeds, HSP17 decreased in abundance during aging, but increased during priming for both control and aged seeds (Catusse et al., 2011). De novo synthesis of HSP 101, 70 and 17.6 and T-complex protein 1 q-subunit (TCP-1-q), a molecular chaperone, was inhibited during germination in artificially aged sugarbeet seeds compared to control seeds (Rajjou et al., 2008). LEA proteins are presumed to be involved in binding or replacement of water, in sequestering ions that will be concentrated under dehydrated conditions, or in maintaining protein and membrane structure (Cuming, 1999; Tunnacliffe and Wise, 2007; Battaglia et al., 2008). EMB564 (group 1 LEA family) and LEA-3 accumulated to a relatively high abundance in high vigor maize seeds (Wu et al., 2011). De novo synthesis of Em-like protein GEA1 was inhibited during germination of sugarbeet seeds after aging (Rajjou et al., 2008). In M. truncatula, the abundance of four LEA proteins, EM (LEA_5), D113.I/II (LEA_1), D-34.III (SMP) and CapLEA I/II (LEA_4) and two chaperone-related proteins, glycine-rich RNAbinding protein RPN-1 and sHSP20, increased in parallel to acqui^telain et al., 2012). sition of longevity during seed development (Cha Protein involved in protein repair. One of the spontaneous changes occurring in seed proteins during aging is the formation of

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isoaspartyl residues, which destabilizes the secondary protein structure. The enzyme L-isoaspartyl methyltransferase recognizes such isoaspartyl residues and catalyzes the methylation of its free acid group using S-adenosyl-L-methionine as the methyl donor. This is the first step towards the conversion back to an aspartyl residue. This repair enzyme has been shown to be important for et al., 2008). both seed vigor and longevity in Arabidopsis (Oge Met is very susceptible to ROS-mediated oxidation, but the first step is reversible as mentioned in Section 4.4. The enzyme responsible for this, methionine sulfoxide reductase, has been demonstrated to be important for seed longevity by repairing proteins oxidized on Met groups during aging (Ch^ atelain et al., 2013).

(Rajjou et al., 2008). These results suggest that tubulin and actin might be a seed vigor markers. Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids (Mortimer et al., 2008; Laohavisit and Davies, 2011). One annexin increased significantly in abundance in sacred lotus (Nelumbo nucifera) seeds during heat stress. Transgenic Arabidopsis seeds ectopically expressing this annexin exhibited improved resistance to the accelerated aging treatment that is used for assessing seed vigor (Chu et al., 2012). Annexins could be a potential seed vigor marker, which displayed decreased and increased abundance in untreated control and osmoprimed alfalfa seeds, respectively (Yacoubi et al., 2013).

5.3. Glycolysis

5.6. ROS detoxification

In central metabolism, the glycolytic pathway appears to be affected much more by seed vigor than other pathways. Accumulation of glycolytic enzymes in many species, such as fructose-1,6bisphosphatase in maize (Wu et al., 2011) and Arabidopsis (Rajjou et al., 2008), phosphoglucomutase, 3-phosphoglycerate kinases and 3-phosphoglycerate kinases (cytosolic) in maize (Xin et al., 2011) and Arabidopsis (Rajjou et al., 2008), glyceraldehyde-3phosphate dehydrogenase in sugarbeet (Catusse et al., 2011) and Arabidopsis (Rajjou et al., 2008) seeds all correlated positively with seed vigor.

Production of ROS may be one of the main causes of loss of seed vigor. Rajjou et al. (2008) revealed that protein oxidation (carbonylation) increased strongly in aged seeds. The amounts of enzymes involved in ROS removal correlate with seed vigor. SOD increased in abundance in high-vigor maize seeds (Wu et al., 2011) and in osmoprimed alfalfa seeds (Yacoubi et al., 2011), while CAT 2 increased in hydro-primed Arabidopsis seeds (Gallardo et al., 2001). In addition, CAT and reduced glutathione-dependent dehydroascorbate reductase decreased in abundance in artificially aged Arabidopsis seeds during germination (Rajjou et al., 2008). The abundance of a number of thiol-dependent antioxidant proteins and enzymes have also been observed to relate to seed vigor. In maize seeds, 2-Cys Prx BAS1, TPX and glutathione S-transferase accumulated abundantly in high vigor seeds (Wu et al., 2011). Osmopriming treatment of alfalfa seeds increased the abundance of alkyl hydroperoxide reductase (thiol-specific antioxidant/Mal allergen), thioredoxin, 1Cys Prx, GST and mitochondrial Prx (Yacoubi et al., 2011). During aging, GST part A decreased significantly in abundance in maize seed (Xin et al., 2011). Detoxification of aldehydes is also a necessary process for acquisition of seed vigor. Glyoxalase, involved in the detoxification of methylglyoxal, a highly reactive aldehyde derived from glycolysis (Thornalley, 2003), increased in abundance in high-vigor maize seeds (Wu et al., 2011). In maize seeds, aging decreased the accumulation level of glyoxalase and ADH (Xin et al., 2011). ADH, catalyzing the irreversible oxidation of a wide range of reactive aldehydes to their corresponding carboxylic acids, has been suggested to play a pivotal role in detoxifying the aldehydes generated by environmental stress (Perozich et al., 1999).

5.4. Signal transduction Phosphorylation and dephosphorylation of key regulatory proteins serve as an “oneoff” switch in the control of cellular activities in eukaryotic cells. Serine/threonine protein phosphatases are ubiquitous enzymes in all eukaryotes. Protein phosphatase 2A (PP2A), a subfamily of serine/threonine protein phosphatases, has been proposed to play positive and dynamic roles in stress signaling (País et al., 2009). The abundance of this protein decreased during aging, but increased during priming in sugarbeet seeds (Catusse et al., 2011). Plant 14-3-3 proteins function by binding to phosphorylated client proteins to modulate their function. Through the regulation of a diverse range of proteins including kinases, transcription factors, structural proteins, ion channels and pathogen defense-related proteins, they are being implicated in many physiological functions in plants (Denison et al., 2011). In sugarbeet, 14-3-3 proteins decreased in abundance in aged seeds and increased in primed and aged-primed seeds (Catusse et al., 2011).

6. Perspectives 5.5. Cell structure Microtubule arrays play critical roles in intracellular organization and cell division in all eukaryotes. The a- and b-tubulin subunits of microtubule heterodimerize in a head-to-tail fashion, giving rise to polarity that plays a crucial role in the function of the microtubule array (Eckardt, 2006). In Arabidopsis, the tubulin b2b3 chain decreased in abundance not only during the aging process, but also during imbibition of aged seed (Rajjou et al., 2008). Hydropriming increased the abundance of both tubulin a-chain and tubulin b-2, while osmo-priming only promoted the accumulation of tubulin b-2 (Gallardo et al., 2001). In eukaryotic cells, a large number of proteins are targeted after translation to specific organelles by a process called intracellular trafficking. Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells (Augustine et al., 2008; Ketelaar, 2013). Actin 7 was observed to accumulate less abundantly in artificially aged Arabidopsis seeds than in control seeds during germination

In the proteomic studies discussed in the previous sections it has generally been assumed that, when an increase in the abundance of a given protein correlates with e.g. an increase in vigor or some other beneficial trait, it is an indication that the process in which the protein is involved is important for that process; and vice versa for proteins decreasing in abundance. Although that need not be the case, the proteomic studies have identified many proteins that are potentially important for seed development, seed vigor and/or seed germination or for more specific seed traits such as resistance to fungal pathogens. Proteins that have been identified to be particularly important for at least two of these seed processes are involved in ROS detoxification, the cytoskeleton, glycolysis, protein biosynthesis, post-translational modifications, methionine metabolism, and late embryogenesis-abundant (LEA) proteins. This type of data cannot be obtained by transcriptomics as there is generally a poor correlation between mRNA amounts and protein amounts. However, even when we know which proteins are


particularly important for a specific process, we still need information about the extent to which each individual protein is posttranslationally modified, e.g. by oxidation, phosphorylation or by proteases. These modifications can strongly affect the function of the protein and often give strong hints about metabolic regulation or metabolic adjustments to environmental factors. To study these PTMs often requires special enrichment steps because the PTMs are sub-stoichiometric and because they in some cases (e.g. protein phosphorylation) make the modified peptide more difficult to detect by mass spectrometry. To study PTMs, gel-free systems and shot-gun proteomics using some type of quantification tagging will be increasingly useful. The highlighting of potentially important proteins by proteomics provides researchers and plant breeders with starting points for further studies where the next step will often be to look at the expression and regulation of the gene encoding the protein of interest in their favorite crop plant. The ultimate aim is to incorporate genes encoding particularly promising proteins into the breeding programs. Contributions Abstract, Introduction, Seed germination and dormancy and Perspectives were written by IMM, Seed development was written by WQW and SJL, Seed desiccation tolerance was written by WQW, Seed vigor was written by SQS, while IMM, SQS and WQW revised the manuscript. Acknowledgments This work was supported by the National Science and Technology Support Program (2012BAC01B05) and by National Science Foundation of China (31171624). IMM was supported by a Chinese Academy of Sciences Visiting Professorship for senior international scientists. References Agrawal, G.K., Hajduch, M., Graham, K., Thelen, J.J., 2008. In-depth investigation of the soybean seed filling proteome and comparison with a parallel study of rapeseed. Plant Physiol. 148, 504e518. Allen, D.K., Ohlrogge, J.B., Shachar-Hill, Y., 2009. The role of light in soybean seed filling metabolism. Plant J. 58, 220e234. Arc, E., Galland, M., Cueff, G., Godin, B., Lounifi, I., Job, D., Rajjou, L., 2011. Reboot the system thanks to protein post-translational modifications and proteome diversity: how quiescent seeds restart their metabolism to prepare seedling establishment. Proteomics 22, 1606e1618. Arc, E., Chibani, K., Grappin, P., Jullien, M., Godin, B., Cueff, G., Valot, B., Balliau, T., Job, D., Rajjou, L., 2012. Cold stratification and exogenous nitrates entail similar functional proteome adjustments during Arabidopsis seed dormancy release. J. Proteome Res. 11, 5418e5432. Augustine, R.C., Vidali, L., Kleinman, K.P., Bezanilla, M., 2008. Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth. Plant J. 54, 863e875. Bai, X.G., Yang, L.M., Tian, M.H., Chen, J.H., Shi, J.S., Yang, Y.P., Hu, X.Y., 2011. Nitric oxide enhances desiccation tolerance of recalcitrant Antiaris toxicaria seeds via protein S-nitrosylation and carbonylation. PLoS One 6 (6), e20714. Bailly, C., 2004. Active oxygen species and antioxidants in seed biology. Seed Sci. Res. 14, 93e107. Bailly, C., El-Maarouf-Bouteau, H., Corbineau, F., 2008. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. CR Biol. 331, 806e814. Bantscheff, M., Lemeer, S., Savitski, M.M., Kuster, B., 2012. Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal. Bioanal. Chem. 404, 939e965. Battaglia, M., Olvera-Carrillo, Y., Garciarrubio, A., Campos, F., Covarrubias, A.A., 2008. The enigmatic LEA proteins and other hydrophilins. Plant Physiol. 148, 6e24. Berjak, P., Pammenter, N.W., 2008. From avicennia to zizania: seed recalcitrance in perspective. Ann. Bot. 101, 213e228. Bewley, 1997. Seed germination and dormancy. Plant Cell 9, 1055e1066. Bewley, J.D., Bradford, K.J., Hilborst, H.W.M., Nonogaki, H., 2013. Seeds e Physiology of Development, Germination and Dormancy, third ed. Springer, New York.

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