J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 3 8 9 – 4 0 0

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Review

Seed proteomics

Ján A. Miernyka, Martin Hajduchb,⁎aUSDA, Agricultural Research Service, Plant Genetics Research Unit, Department of Biochemistry, Interdisciplinary Plant Group,University of Missouri, Columbia, MO 65211, USAbInstitute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovak Republic

A R T I C L E I N F O

Abbreviations: CM, central metabolism; CShormones and signalling; LC–MS/MS, liqudesorption/ionization peptide mass fingerprprotein folding; PS, protein synthesis; PSV, pproteins of unknown function; SSP, seed sto⁎ Corresponding author. Institute of Plant Gen

Nitra, Slovak Republic. Tel.: +421 37 6943346;E-mail address: hajduch@savba.sk (M. Ha

1874-3919/$ – see front matter © 2010 Elsevidoi:10.1016/j.jprot.2010.12.004

A B S T R A C T

Article history:Received 27 October 2010Accepted 10 December 2010Available online 21 December 2010

Seeds comprise a protective covering, a small embryonic plant, and a nutrient-storageorgan. Seeds are protein-rich, and have been the subject of many mass spectrometry-basedanalyses. Seed storage proteins (SSP), which are transient depots for reduced nitrogen, havebeen studied for decades by cell biologists, and many of the complicated aspects of theirprocessing, assembly, and compartmentation are now well understood. Unfortunately, theabundance and complexity of the SSP requires that they be avoided or removed prior to gel-based analysis of non-SSP. While much of the extant data from MS-based proteomicanalysis of seeds is descriptive, it has nevertheless provided a preliminarymetabolic pictureexplainingmuch of their biology. Contemporary studies aremovingmore toward analysis ofprotein interactions and posttranslational modifications, and functions of metabolicnetworks. Many aspects of the biology of seeds make then an attractive platform forheterologous protein expression. Herein we present a broad review of the results from theproteomic studies of seeds, and speculate on a potential future research directions.

© 2010 Elsevier B.V. All rights reserved.

Keywords:ElectrophoresisMass spectrometryProteinsProteomicsSeedsSeed storage proteins

Contents

1. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3902. Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3903. Seed storage proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

3.1. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3903.2. Seed storage protein synthesis and processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3913.3. Seed storage proteins; the dynamic range problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

4. Seed development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3924.1. Endosperm development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3934.2. Embryo development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

, cellular structures; DIGE, difference in gel electrophoresis; ER, endoplasmic reticulum; HS,id chromatography coupled with tandem-MS; MALDI-TOF PMF, matrix-assisted laserinting; MT, membrane transport; MS, mass spectrometry; NA, nucleic acid metabolism; PF,rotein storage vacuoles; PT, protein targeting; PTM, posttranslational modification (s); PUF,rage proteins; SR, stress responseetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P. O. Box 39A, 950 07fax: +421 37 7336660.jduch).

er B.V. All rights reserved.

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5. Seed germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3945.1. Endosperm-dominant seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3945.2. Cotyledon-dominant seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

6. Seeds as a biotechnology platform. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3957. The future of seed proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

“Animals are something invented by plants to move seedsaround. An extremely yang solution to a peculiar problemwhich they faced.” – Terence McKenna

1. Proteomics

As a discipline, proteomics is a broad, instrument-intensiveresearch area that has progressed rapidly since its genesisalmost twenty years ago. While protein isolation and separa-tion methods have improved during this period [1–3], it hasbeen improved instrumentation that has driven expansion ofthe field [4–6]. Significant improvements in sensitivity, massaccuracy, and fragmentation in recent years have led towidespread adoption of proteomic strategies [7]. As the fieldof proteomics matures there have been an increasing numberof very specialized studies; organism-specific [e.g., [8,9]], cellor organelle-specific [e.g. ][10,11], and analyses that targetnarrowphysiological [12] or developmental [13] targets. Hereinwe address the burgeoning field of seed proteomics.

2. Seeds

The most prominent case of plant domestication by man wasduring the Neolithic agricultural revolution [14]. Humans wereseed-gathering foragers during most of prehistory, but beganto cultivate plants starting in the Epipaleolithic/NeolithicPeriod. Cultivation and selection ultimately gave rise toseeds with increased levels of protein and oil, characteristicswhich stimulated both human evolution and, more recently,plant biology. Seeds can be protein-dense based upon arelatively small number of genes encoding seed storageproteins (SSP) [15–17], and this relationship between humandiet and seed protein density has encouraged the early studiesin plant proteomics [18–20].

Seeds comprise the three basic parts of embryonic plants;the embryo proper [21], a tissue containing nutrients stored foruse by the embryo prior to achieving autotrophy, and the seed-coat protective covering [22]. Formation of the seed completesa reproductive cycle that begins with development of flowers,and pollination [13]. The embryo develops from the zygoteformed by the fusion of an egg and a sperm cell, and the seedcoat develops from the integuments of the ovule [23]. Thenutrient-storage tissue can be the cotyledon(s), the endo-sperm, or the megagametophyte in gymnosperms. Theendosperm, derived from the parent plant via double fertil-

ization, is usually triploid, and is rich in either oil and proteinor starch and protein [24]. In some species, the embryo isembedded in the endospermwhile in others the endosperm isabsorbed by the embryo as the latter grows within thedeveloping seed, and the cotyledons of the embryo becomefilledwith the stored nutrients. Thusmature seedsmight haveno remaining endosperm (embryo dominant) or theymight bemostly endosperm (endosperm dominant) [13]. The embryo-dominant seeds include all of the agriculturally importantlegumes while endosperm-dominant seeds include all of thegrains plus a few exceptional dicots (e.g. Brazil nut (Bertholletiaexcelsa HBK)) and castor oil seeds (Ricinus communis L.) [13].

3. Seed storage proteins

3.1. Terminology

The terminology of SSP is complex and can be dauntinglyconfusing to the non-expert. The first systematic classifica-tion system was developed by Osborne [25], and grouped SSPon the basis of solubility in H2O (albumins), dilute saline(globulins), alcohol: water mixtures (prolamins), and dilutealkali or acid (glutelins). The majority of agriculturallyimportant SSP are albumins, globulins, or prolamins[16,17,26]. While albumins are found in all seeds, prolaminsand glutelins are most abundant in monocotyledon seeds andglobulins are prevalent in dicotyledon seeds [17,27].

The globulin SSP have additionally been grouped basedupon sedimentation, 7S and 11S [26]. While this systemappears simple and straightforward, some authors haveeschewed clarity for specific scientific detail. As a result, it isnot uncommon to additionally find reference to 3S or 12Sglobulins. Taking this to the extreme, there are also literaturereports of 2.2S and 11.3S globulins [28].

Furthermore, there are a plethora of trivial names assignedto SSP. Initially, the cereal prolamins were named based ontheir Latin generic names; the zeins from maize (Zea mays)[29], hordeins from barley (Hordeum vulgare) [30], secalins fromrye (Secale cereale) [31], etc. There are exceptions to even thisnomenclature however, and the wheat (Triticum aestivum)prolamins were named gliadins [32,33]. The 7S globulins arecalled vicilins [34,35], while the 11S globulins are calledlegumins [36]. A third group of globulins, the convicilinshave been found only among members of the legume tribeVicieae [37]. The convicilins arose from one or more insertionswithin the N-terminal region of vicilin-like progenitor, and arenot in any way related to the jackbean (Canavalia ensiformis)seed lectin, concanavalin A. Finally, the trivial name cactin

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has been used to describe the 2S albumin SSP from seeds ofCereus jamacaru [38].

Finally, in the postgenomics era it has been well estab-lished that SSP are encoded by multigene families of varyingsizes [33,39–42]. Unfortunately, even here this is no consis-tency in nomenclature as; α, α', A, B, 1, 2, etc. are all variouslyused, sometimes in confusing hybrid configurations (e.g., 2S1).

3.2. Seed storage protein synthesis and processing

The SSP are synthesized on the rough-endoplasmic reticulum(ER), and targeted to the ER lumen by an N-terminal signalsequence which is co-translocationally removed by signalpeptidase [43]. The SSP are then sorted from bulk proteintraffic through the secretory pathway to their site of deposi-tion, the protein storage vacuole (PSV) [44]. There are twotargeting pathways for SSP, one for prolamins and a second forthe non-prolamin SSP [44]. The prolamin-containing PSV ariseby direct vesiculation from the ER, and the prolamin SSP donot traverse the classical secretory pathway. Non-prolaminSSP do pass through the secretory pathway, and are sortedfrom a post-Golgi compartment to the PSV [45].

Fig. 1 – A schematic model for the synthesis, processing, and assprepro-polyprotein in the cytoplasm has a MW of 70,302 Da andco-translocational import into the lumen of the rough-ER, the sigand two high-mannose glycans are added to the primary sequonMW is increased to 71,736 Da by glycan addition. After transfer tomodified (Man removal; Xyl and Fuc addition). The slight decreatransport to the protein storage vacuole (PSV) the pro-sequencemodified glycan) is removed by a Kexin-type protease [155]. Withtwice within specific linker regions (blue) [156]. The internal fragand the two remaining fragments are joined by formation of a dglycolytic processing yield multiple products, each with a uniqueCys; ○, Man; □, GlcNAc; Δ, Fuc; ◊, Xyl.

The prolamin-PSV can have a complex internal architec-ture, which is thought to be the result of different rates ofsynthesis of the various subunits [46]. In contrast, non-prolamin PSV have a uniformly granular appearance [47]. Anadditional morphological characteristic of prolamin-PSV is,since they are directly derived from the rough-ER, that theyare studdedwith ribosomes [48]. Rice (Oryza sativa) endospermis unusual in containing both prolamin- and non-prolaminPSV [49].

The 2S, 7S, and 11S SSP are synthesized as large precursors(now known to be prepro-polyproteins) (Fig. 1) which undergoinitial processing and assembly while still within the ER [50–55]. In addition to the N-terminal signal sequence, thecanonical SSP-precursor primary sequence includes a pro-sequence containing the PSV-targeting information [56], plusat least two linker/protease cleavage sites [57,58]. It is notuncommon for the SSP pro-proteins to display the canonicalAsn-X-Ser/Thr glycosylation sequon, and become N-glycosy-lated during passage through the ER [59]. In some instancesthe N-glycosylation sequons are within the pro-sequence orlinker regions, and are subsequently removed during proteo-lytic processing (Fig. 1) [60]. In other instances, however, they

embly of a hypothetical 2S/7S/11S seed storage protein. Thewould be cleaved into 44 peptides by tryptic digestion. Uponnal sequence (green) would be removed by signal peptidases Asn-X-Thr/Ser. Despite removal of the signal sequence, thethe Golgi apparatus, the glycan located in the pro-sequence isse in MW (71,041) reflects removal of six Man residues. Upon(red) that contained the PSV sorting information (plus thein the PSV the primary sequence of the model SSP is cleavedment (grey) is degraded by non-specific vacuolar peptidases,isulfide bond. The multiple steps of both proteolytic andMW and pI value, and pattern of tryptic peptides. N, Asn; C,

Fig. 2 – Depletion of the abundant storage proteins as aprelude to proteomic analysis of seed proteins. Lane A,fractional solubility of the maize endosperm prolamins. A1,total proteins; A2, dilute-buffer soluble proteins, A3,extraction of the zein SSP with 35% (v/v) ethanol. Lane B,“salting out” of soybean globulin SSP [73]. B1, total proteinsfrom mature seeds, B2, globulin-depleted proteins; B3,proteins precipitated by incubation with 10 mM CaCl2.Lane C, reduction of the beta-conglycinins from a totalprotein fraction isolated from developing soybean seeds byimmobilized-lectin chromatography [74]. Lane D,immunoremoval of the agglutinin proteins from maturecastor seed endosperm preparations. D1, total endospermproteins, D2, ricin-depleted endosperm proteins, D3,removed castor SSP [75]. The positions of size markerproteins are indicated to the left and right of the Y-axes.

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persist and are present in the final fully-processed SSP (Fig. 1)[61]. Proteolytic processing and assembly continue duringtransit through the secretory pathway, and are completedwithin the PSV [56,62,63].

After proteolytic processing has been completed, the finalpolypeptide associations are stabilized by formation of at leastone disulfide bond [64,65]. The final stable SSP structures canbe as simple as a “αβ-heterodimer” or as complex as a α3β3-heterohexamer [55,66]. It is noteworthy that the incisive SSP-based research of a generation of plant cell biologists has beenmechanistically verified and extended by recent results basedupon availability of genome sequence information andapplication of tandem mass spectrometry (MS/MS) [39,66,67].It is equally important to note that the understanding of SSPprocessing can greatly simplify interpretation of MS results(Fig. 1). This is especially true of gel-based results, where itcould be difficult to interpret the position of a protein that isvery different from the MW and pI of the primary translationproduct unless one has prior knowledge of both proteolyticand glycolytic processing events (Fig. 1).

3.3. Seed storage proteins; the dynamic range problem

This brings us to an important point in the review. Even if theseeds of a hypothetical plant have only, for example, a 2Salbumin and an 11S globulin as SSP, the complexity in 2D gelspot-patterns can be nearly overwhelming because of thecontributions of extended multigene families [26,27,33,41,42]plus heterogeneity in both proteolytic [68] and glycolytic[60,61,69] processing!

The abundance of the SSP can be a great benefit……if youare studying SSP. If not, especially if a gel-based strategy isemployed, then the SSP can substantially interfere withanalysis of total proteins (Fig. 2). In some instances, priorknowledge of the amino acid composition of the SSP can beexploited to allow their avoidance. An excellent example ofthis addresses the SSP of castor endosperm which containrelatively low levels of Lys [70]. By using a Lys-specific N-hydroxysuccinimide-activated Cy-dye to stain the gels, thereappear to be far fewer SSP spots than would have beenvisualized after staining with Coomassie Blue, Sypro, etc.Using this approach, itwas possible to visualize castor proteinsat the same pI and Mr values as the SSP [71].

Otherwise, SSP-depletion can be an important componentof sample preparation [56]. With the exception of theprolamins (Fig. 2A), fractional solubility has not proved to begenerally useful in SSP-depletion. However, if a “total protein”fraction has been initially isolated, then fractional solubility ofthe globulins can be exploited by selective precipitation [72,73](Fig. 2B). If the interfering SSP are N-glycosylated, it is possibleto use immobilized-lectin affinity chromatography for theirremoval. This strategy was useful in removing the acidicsubunit of beta-conglycinin from total soybean (Glycine max (L.)Merr.) seed proteins [74] (Fig. 2C). If suitable antibodies areavailable [75], then immunoremoval can be a useful strategyfor the depletion of SSP (Fig. 2D). Application of any of thesemethods will need to be individually refined. For example,antibodies and lectins might need to be chemically cross-linked in order to be useful if SSP are prepared underdenaturing conditions [e.g., 74].

4. Seed development

Specific characteristics of seeds simplify their study. Firstly, allcell division is completed within a few days after fertilization.This marks the line of demarcation between embryogenesisand seed development. Subsequent cellular specializationtakes place in the absence of cell division [13,76]. Secondly,nearly all biosynthetic activity during seed development isdirected toward the accumulation of storage polymers (oils,polysaccharides, and proteins) [77,78]. These compounds, andthe subcellular structures that contain them, are inert depotsawaiting the activities responsible for mobilizing them toprovide biosynthetic intermediates necessary until the devel-oping seedling becomes autotrophic [79,80]. Thirdly, seeds arerelatively simple anatomically; comprising the embryonicaxes, storage tissue(s), and the protective seed-coat.

During typical analyses, seed-coats are removed anddiscarded, while embryos are usually either removed or,because of their relatively small size, ignored. Thus, “seedproteomics” usually means proteomic analysis of the storagetissues/organs. While SSP are very abundant, accounting foras much as 60% of total seed protein, they are generallyconsidered inert. If they were not biochemically removed priortoMS analysis, then they have beenmanually subtracted fromthe analyses described herein.

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4.1. Endosperm development

The endosperm is composed of specialized cells that producelarge quantities of storage polymers [81,82]. The amount ofendosperm in a mature seed is variable. For example, itcomprises the majority of the whole seed mass in cereals,such as rice, maize, and wheat, and it is prominent in theseeds of a few dicots, such as castor and Brazil nut. In otherspecies, for examplemouse-ear cress (Arabidopsis thaliana), theendosperm is almost completely absent in the mature seed[22,83]. Regardless of the pattern of endosperm persistence, itis vital for embryo development in much the same way theplacenta is essential for mammalian embryos [22].

Similar patterns of identified protein distribution wereseen among the endosperm-dominant starch-rich seeds ofwheat, barley, rice, andmaize. Using a 2-DE plus eitherMALDI-TOF PMF [81,84,85] or LC–MS/MS [86–89] experimental design,a total of 1496 proteins were identified. When the proteins areseparated into 10 functional classes [13]; modified from [90],the distribution is: Central Metabolism (CM), 34%; CellularStructures (CS), 12; Stress Response (SR), 5; Nucleic Acidmetabolism (NA), 2; Protein Synthesis (PS), 2; Protein Folding(PF), 5; Protein Targeting (PT), 7; Hormones and Signalling (HS),2; Membrane Transport (MT), 2; and Proteins of UnknownFunction (PUF), 29 (Fig. 3). It is noteworthy that conditions thatmight be considered as stress for the non-seed components ofa plant are part of the normal developmental programof seeds

Fig. 3 – The functional annotation of MS-identified seed proteinsStructure (CS), Stress Response (SR), Nucleic Acid metabolism (NTargeting (PT), Hormones and Signalling (HS), Membrane Transponot included in the analysis. The starch-rich endosperm categorymaize [81,84–89]. The oil-rich endosperm category includes 437cotyledon-dominant seeds of A. thaliana [105] and B. napus [102,proteins were identified from the starch-rich cotyledon-dominanManual subtraction of the SSP reduced this total to 64. A total ofmegagametophyte of Cunninghamia lanceolata (Lamb) Hook seed

[91]. Water “stress” is a good example. With the possibleexception of recalcitrant seeds, all seeds become dehydratedas they approach maturity and prepare for quiescence [91].

The results from separate proteomic analyses of develop-ingwheat [81] and rice [88] endosperm, andwheat [89] and riceembryos [92,93] highlight the differences in these two seedorgans. The CM proteins were the most abundant in allinstances; however members of the PF category were relative-ly more abundant in endosperm, while proteins included inthe SR andHS categories weremore abundant in embryos. Thehigher proportion of proteins involved in signalling mightreflect the role(s) of the embryo in controlling metabolism inthe endosperm [c.f., [94]]. Proportionally, there are far morePUF proteins in endosperm than in embryos irrespective of theavailability of whole genome sequence data.

Castor is an unusual example of an oil-rich endosperm-dominant seed; most accumulate starch rather than oil. Usinga 2-DE plus LC–MS/MS strategy, Houston et al., [71] identified522 proteins from developing castor endosperm. Discountingthe SSP, themost abundant castor proteinswere involvedwithCM, PF, SR, and CS (Fig. 3).

Amyloplasts are non-green plastids specialized for thesynthesis and accumulation of starch [95]. Balmer et al. [96]described isolation of amyloplasts from developing wheatendosperm, and used a 2-DE LC–MS/MS strategy to identify289 proteins. In addition to all of the enzymes necessary forstarch biosynthesis, they were also able to identify many

. The 10 categories [13] are; Central Metabolism (CM), CellularA), Protein Synthesis (PS), Protein Folding (PF), Proteinrt (MT), and Proteins of Unknown Function (PUF). The SSP arecomprises 1496 proteins identified from rice,wheat, barley, orproteins identified from castor [71]. Analysis of the oil-rich106] yielded 1049 identified proteins, while a total of 278t seeds of pea (P. sativum) [109], and lentil (L. culinaris) [110].71 non-SSP proteins were identified from the oil-richs [134].

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proteins involved with nitrogen and sulfur assimilation, andamino acid and lipid biosynthesis. The composition of thewheat endosperm amyloplast proteome is more similar tothat of castor endosperm leucoplasts [97] or even greenplastids [98,99] than the wheat endosperm cell cytoplasm.

4.2. Embryo development

Typically embryo-dominant seeds contain starch plus SSP asthe storage polymers (peas, beans, lentils), or oil plus SSP(mouse-eared cress, canola (Brassica napus L.)), soybean/barrelmedic (Medicago truncatula Gaertn.)/lotus (Lotus japonicus L.). Todate, the cotyledons of legume seeds have received the mostattention from “omics biologists,” in part due to theiragronomic importance [13,15,79,80,100].

There have been extensive proteomic studies of developingG. max [101,102], M. truncatula [77,103], and L. japonicus [104]seeds. Experimental strategies included 2-DE plusMALDI-TOF,2-DE plus LC–MS/MS, or GeLC–MS (Proteins are separatedusing 1-D SDS-gels which are subsequently divided intoequally-sized segments. Proteins in each segment are re-duced, alkylated, and digested in-gel. An LC-system then usedto separate and supply the peptides to the mass detector)analysis of tryptic peptides. Seed proteome data for G. max,M.truncatula, and L. japonicus have been collected, analyzed, andcan be retrieved from: http://bioinfoserver.rsbs.anu.edu.au/utils/PathExpress/pathexpress4legumes.php. A total of 1723proteins were identified. Manual subtraction of 316 SSP left1407 identified proteins from the three legume species. Whenthese proteins were categorized, the percent distribution was:CM, 49%; CS, 19; NA, 2; PS, 3; PF, 5; PT, 3; HS, 3; MT, 4; SR, 3; and,PUF, 10. In all three species a relatively large number of lateembryo abundant proteins were identified.

Similar studies of A. thaliana [105] and B. napus [102,106]seeds yielded similar results. A total of 1290 proteins wereidentified either by 2-DE plus LC–MS/MS or MudPIT. Aftermanual removal of 241 SSP entries, 1049 identified proteinsremained. Once again, the CM category was by far the mostpopulous (36%), followed by CS (19), and PUF (17).

Agrawal and Thelen [107] used the phospho-proteinspecific fluorescent dye ProQ Diamond as a probe for analysisof developing B. napus seeds. They were able to detect 234phospho-protein spots, 103 of which were identified by LC–MS/MS. Not surprisingly, most of the identified proteins werein the CM and HS categories. It was, however, somewhatsurprising that many of the cruciferin SSP subunits gave apositive phospho-protein stain since these proteins had notbeen previously described as phospho-proteins.

Jain et al. [108] used MudPIT to identify 80 proteins fromplastids purified from developing B. napus embryos. Theplastid proteins complement was enriched in enzymes ofthe CM grouping, and was mid-way between the non-greenplastids from wheat and castor endosperm [96,97] and thechloroplasts isolated from green organs [98,99].

In contrast with these oil plus SSP embryo-dominant seeds,the main starch plus SSP seeds that have been characterizedare pea (Pisum sativum) [109], and lentil (Lens culinaris) [110]. Acombination of MALDI-TOF PMF and LC–MS/MS was used toidentify 122 proteins from mature L. culinaris seeds. Manualsubtraction of the SSP reduced this number to 25. Of these 6

were grouped in CM, 4 each in CS and PT, and 3 each in NA andSR. Similar results were obtained when Bourgeois et al. [109]usedMALDI-TOF PMF to identify 156 proteins frommature peaseeds. Manual subtraction of the SSP left 39, distributedamong CM (16), PUF (8), CS (7), and PF (4). A persistent problemwith analysis of the starch plus SSP legume seeds is the lack ofgood genomic or EST resources.

5. Seed germination

Mature quiescent seeds are dispersed at low (5–15%) moisturecontent, andwithmetabolic activity at a standstill [91,111]. Forgermination, quiescent seeds need only be hydrated at asuitable temperature in the presence of O2. Germinationbegins with water uptake by the seed (imbibition), continuesthrough the elongation the embryonic axis inside the seed,and is visiblymanifest by protrusion of the radicle through theseed coat [112]. This sequence continues until the transition ofthe heterotrophic seedling to an autotrophic plant. Germina-tion involvesmany cellular andmetabolic events, coordinatedby complex regulatory networks. Seed dormancy, the intrinsicability to temporarily block radicle elongation in order tooptimize the timing of germination, will be treated herein as aspecialized variant of quiescence [113–115].

Dormancy is a process whereby germination is delayed inorder to avoid conditions adverse for seedling survival.Release from dormancy is controlled by perception of acombination of environmental signals [91,112,113,115,116].Temperature, light quality, and hormonal balance (abscisicacid and gibberellins) all play key roles in release fromdormancy.

The period between germination and assumption ofautotrophy is broadly referred to as postgerminative growth.It is during this period that the seed storage polymers (oil,polysaccharides, SSP) are mobilized to provide biosyntheticintermediates. In general, the enzymes necessary for polymerdegradation are synthesized de novo during postgerminativegrowth. Thus, SSP proteases (endo- and exo-), lipases and theglyoxylate cycle enzymes, and starch-degrading enzymes canserve as markers to define the period of postgerminativegrowth [114].

5.1. Endosperm-dominant seeds

Total endosperm proteins from germinated castor seeds wereanalyzed using a 2-DE plus MALDI-TOF/TOF MS strategy, andnearly 400 distinct proteins were identified [97]. Essentially allof these proteins are the same as were previously describedfrom analysis of developing castor endosperm [71]. Withouttheir removal in this study, the extremely high “background”of SSP and CM proteins precluded identification of anyproteins that could be specifically attributed to germination/postgerminative growth.

Castor endosperm plastidial and mitochondrial fractionswere also prepared and subjected to proteomic analysis [97].The proteins identified are the same as have been previouslydescribed for these organelles from other plant species andorgans [98,117,118]. Maltman et al. [119] used 2-DE, DIGE,MALDI-TOF PMF, and LC–MS/MS to show that more than 100

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proteins are differentially abundant when comparing the ERfrom developing castor endospermwith that from germinatedseeds. With the exception of a few contaminants from otherorganelles (RuBisCO, plastids; malate synthase, glyoxysomes),and PUF proteins, those identified were all either SSP ormembers of the PF and PT groups.

A proteomics strategy employing 2-DE and MALDI-TOFPMF analysis was used to identify nearly 200 proteins fromgerminating barley seeds [120]. Nearly all of the proteinsidentified are included in the CM, PF, CS, and SR categories. Bythree days after imbibitions there were large increases in theenzymes involved with starch breakdown and mobilization,such as the α- and β-amylases.

Because barley is used extensively in both the food andbrewing industries, there have been detailed analyses of a fewspecific proteins; α-amylase, peroxidases, and thioredoxins[85]. Hynek et al. [121] used LC–MS/MS to analyze a plasmamembrane-enriched fraction isolated from germinating bar-ley. Many of the proteins identified appear to be contaminantsfromother subcellular organelles, demonstrating howdifficultit is to obtain purified subcellular components. Despite thedifficulties, they also identified several bona fide plasmamembrane proteins including a H+-ATPase, a pyrophospha-tase, and a voltage-dependent anion channel [104].

Analysis of germinating rice endosperm using a 2-DE plusMALDI-TOF MS strategy allowed Yang et al. [122] to studyproteins that changed in abundance. They found that the SSP,along with members of the CM and CS groups decreased inabundance during germination, while different members ofthe CM group increased, some of which are involved withstarch breakdown. Results similar to those found withgerminating rice and barley endosperm proteins were alsofound during studies of wheat and maize [81,86].

Evidence is accumulating that indicates the cellularenvironment is increasingly oxidizing as seeds dehydrateand approach quiescence. Formation of disulfide bonds is onemanifestation of the changing redox environment. Subse-quently, in response to imbibition, the thioredoxin (NADPH,thioredoxin h, and NADP-thioredoxin reductase) and ascor-bate/glutathione systems are activated [123,124]. These sys-tems reduce disulfide bonds, and in doing so increases proteinsolubility, the rate of proteolysis, and ultimately the extent ofnitrogen and carbonmobilization [125]. The endosperm-basedthioredoxin paradigm was subsequently extended to includedicot seeds [126].

5.2. Cotyledon-dominant seeds

Essentially all reported proteomic analyses of “germinatingseeds” are actually from studies of postgerminative growth.Inevitably, 2-DE-based LC–MS/MS analyses of the postgermi-native growth of cotyledon-dominant seeds yield datasetsidentical to those of mature seeds and are dominated by thepresence of SSP [127–132]. If proteins were quantified, then themajor theme is the decrease in levels of SSP. In some instancesthis overall decrease is accompanied by transient accumula-tion of SSP degradation intermediates.

In contrast to most “shotgun” proteomic analyses of seeds,Müller et al. [133] reported the results from analysis of aspecific tissue, the endosperm cap, during germination of

cotyledon-dominant Lepisium sativum seeds. The cap is aspecific part of the endosperm surrounding the embryoradicle. Using a 2-DE plus LC–MS/MS strategy, 140 proteinswere identified. The largest group of proteins was the CMcluster, followed by SR, and PF. The endosperm cap proteomewas both qualitatively and quantitatively different from therest of the endosperm, but no proteins were identified thatmight be responsible for modification of the endospermstructure in response to radicle protrusion.

“Logically,” one might reasonably have expected the suiteof proteins identified in oil-rich seeds [12] to differ from thoseof starch-rich seeds, or for endosperm-dominant seeds todiffer from cotyledon-dominant seeds. It was surprising thenthat the patterns seen with these four groups are quite similar(Fig. 3). The implication, that a seed proteome is more similarto that of another seed than it is to another organ of even thesame plant [13], requires more attention. In Fig. 3, theapparently different pattern of proteins seen in the oil-richmegagametophyte must be considered in the context of arelatively small number of proteins identified from a singlespecies of gymnosperm (C. lanceolata) [134].

6. Seeds as a biotechnology platform

Because seeds have a high intrinsic capacity for proteinsynthesis, packaging, and storage, they should be an excellentplatform for production of transgenic products [135]. Proteinsynthesis can be driven by the strong, tissue-specific SSPpromoters [e.g., [136]]. Successful early targets have includedexpression of both industrial [137] and biomedical [138]proteins. There is relatively little information availableaddressing potential pleiotropic effects of heterologous pro-tein expression on expression of native seed proteins[139,140]. Should this become problematic, Schmidt andHerman [141] have developed a unique strategy for rebalan-cing the seed proteome in order to increase expression ofheterologous non-seed proteins.

7. The future of seed proteomics

One goal of this review was to identify areas that needincreased attention from proteomics-based researchers. Oneclear need is for more comparative analyses, especially interms of gymnosperm seeds, although in many cases this willrequire parallel development of better genomic/EST resourcesto facilitate protein identification. Proteomics researchers areincreasingly moving from gel-based to gel-free approaches,which will contribute to identification of more proteins and isamenable to use with automated, high-throughput platforms[142]. At the same time, gel-free analyses require applicationof more robust standards of statistical analysis [143]. In theforeseeable future there will remain applications for gel-basedanalyses, especially in targeted-proteomic studies such asthose involving DIGE. A combination of gel-based and gel-freemethods will likely comprise the state-of-the-art for years tocome [c.f., [144]].

There can be no debate about the need to move fromqualitative to quantitative proteomic analyses, and there are

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proponents of both protein labeling and of label-free methodsto achieve this end [142,145]. While the debate will likelycontinue into the future, an increasing number of researchersare using a label-free method termed spectral counting [c.f., ][146–148]. Regardless of the method finally adopted, it is clearthat future proteomics-based studies of seeds will benefitfrom employing quantitative approaches.

One aspect of biology that cannot be addressed in anyother large-scale survey (e.g., transcript profiling) is theposttranslational modification (PTM) of proteins. Much ofthe elaboration of the proteome is the result of PTM [149]. Inaddition to the nature of the PTM, it is also important todetermine which residue is modified and the result of themodification. Do the PTM affect protein turnover, activity,interactions? In addition to using bottom up analysisstrategies, it is important that top down proteomic analysesare included in analysis of PTM [150]. Thus far only a handfulof studies of seed proteins have included systematic analysisof PTM, and these have addressed only phosphorylation [107].In addition to identification of the nature of PTM, it isimportant to determine their stoichiometry in the proteome.Changes in PTM stoichiometry likely indicate a specificfunctional change, while increase in protein amount with aparallel increase in the extent of PTM means that thestoichiometry has not changed and there is unlikely to beany functional consequence.

The othermajor aspect of biology that cannot be addressedin any sort of high-throughput or computational context isthat of protein interactions (e.g., the interactome). There areno extant publications addressing MS-based analysis ofprotein interactions as a component of seed biology. Butthere should be! The next wave of understanding of howproteins function will involve analysis of protein interactionsin a cellular or subcellular context [151,152].

Finally, an exciting and relatively new application of MS inproteomics studies is at the tissue- and sometimes evencellular level of analysis; imaging MS or MALDI-imaging[153,154]. By gating the detector to a specified mass (orrange), it is possible to detect the location of a protein inwhole tissue/organ mounts. Especially in conjunction withhigh-sensitivity instruments, this method could providesignificant insight into the cellular and even molecularorganization of seeds and their component tissues.

Acknowledgements

This investigation was supported by the Seventh FrameworkProgram of the European Union – International ReintegrationGrant (MIRG-CT-2007-200165). The graphics were created byM.L. Johnston. Professor J.J. Thelen provided the antibodiesused in Fig. 2D.

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