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Immature Seed Endosperm and Embryo Proteomics of the Lotus (Nelumbo Nucifera Gaertn.) by One-Dimensional Gel-Based Tandem Mass Spectrometry and a Comparison with the Mature Endosperm Proteome

1
Laboratory of Molecular Food Functionality, College of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan
2
Plant Global Educational Project, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan
3
Department of Bioinformatics, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
4
Global Research Center for Innovative Life Sciences, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan
5
Faculty of Health and Sport Sciences & Tsukuba International Academy for Sport Studies (TIAS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8574, Japan
6
Research Laboratory for Biotechnology and Biochemistry (RLABB), GPO 13265, Kathmandu 44600, Nepal
7
GRADE (Global Research Arch for Developing Education) Academy Pvt., Ltd., Adarsh Nagar-13, Birgunj 44300, Nepal
*
Authors to whom correspondence should be addressed.
Academic Editor: Jacek R. Wisniewski
Proteomes 2015, 3(3), 184-235; https://doi.org/10.3390/proteomes3030184
Received: 5 July 2015 / Accepted: 7 August 2015 / Published: 14 August 2015

Abstract

Lotus (Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed’s development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.
Keywords: 1-DGE; LC-MS/MS; lotus; seed; proteome analysis; plant proteomics 1-DGE; LC-MS/MS; lotus; seed; proteome analysis; plant proteomics

1. Introduction

Nelumbo nucifera (Gaertn.) is an aquatic perennial belonging to the family of Nelumbonaceae, whose most used common name is the lotus. The lotus typically grows in shallow ponds, with its rhizomes under the mud and its large leaves rising on stalks 1–2 m above the water surface. Flowers are white to rosy, sweet-scented, solitary, hermaphrodite and 10–25 cm in diameter, while its fruits are ovoid having nut like achenes. Seeds are black, hard and ovoid [1]. In its immature form, the lotus seed is initially of a yellowish color (early stages) and becomes green as it grows and matures. In its late immature stages, the seed is a 1.2–1.5 cm long ovoid covered in a soft green husk containing a moist and soft endosperm and the developing embryo. When the seed reaches maturity, the husk turns dark brown and hardens, and both the endosperm and embryo become considerably dry. The lotus embryo, or germ, is a small, stalk-like tissue at the core of the lotus seed. The embryo is green and yellow in color. In the mature seed, the embryo tissue is dry, and while inside an intact seed, it can remain viable for germination for more than a thousand years, making it the most durable seed known [2,3,4,5]. The immature seed, which is composed largely of the endosperm, has a water content of 77.5%, as opposed to the 13.1% water content of the mature seed. The immature seed also has lower protein and carbohydrate content, 5.9% and14.9%, respectively, compared to 19.1% and 62.6% for the mature seed [6].
The lotus seeds and rhizome are extensively consumed as food in China and Japan and regarded as a health food [7,8,9], and the plant is also utilized as a source of traditional medicine in India and China [1,10]. Furthermore, extracts from the lotus leaves, rhizomes, and seeds have been shown possess multiple health benefits and a diverse amount of secondary metabolites (more details are given in our review [11] and references therein). The genome of the lotus has only recently been sequenced [12], and a few targeted genome and transcriptome-level works have led to the identification of some functional proteins, as well as their successful cloning and transgenic expression [13,14,15,16]. Considering its documented health benefits and several desirable characteristics for nutritional, agricultural and scientific uses, such as its protein content, ability to be cultivated in flooded areas, growth and germination vigor, and extreme seed durability, the lotus plant would consist of an excellent candidate as a crop, source of recombinant genes, or even as potential model organism. However, despite these characteristics, proteome analysis of the plant is still at the initial stages of research. Figure 1 depicts the lotus fruit and seed, its importance and proteomic study goals.
Figure 1. Overview of the significance and goals of the proteomic research of the lotus. The fruit (seedpod) with seeds from a lotus plant growing in Ibaraki University pond, and the open seed with endosperm and embryo is shown.
Figure 1. Overview of the significance and goals of the proteomic research of the lotus. The fruit (seedpod) with seeds from a lotus plant growing in Ibaraki University pond, and the open seed with endosperm and embryo is shown.
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Aiming to develop a proteome catalogue of the lotus plant—starting with its seed, the nutrient rich food source—the first study by our research group has unraveled the mature endosperm proteome of the lotus seed, which included the establishment of protocols for protein extraction and analyses by one-dimensional gel electrophoresis (1-DGE) and by two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry [17]. In the present work, we advance our study of the lotus seed by further analyzing the endosperm of the lotus seed in its immature stage and the embryo, the other prominent component of the mature seed, by utilizing 1-DGE linked with tandem mass spectrometry proteomic approach. The resulting proteome from each tissue (immature endosperm and embryo) is compared with the mature endosperm proteins in hope to bring to light any notable differences in protein content between the different tissue locations and developmental stages.

2. Experimental Section

2.1. Plant Material and Tissues (Immature Endosperm and Embryo of Lotus Seed) Preparation

Lotus seeds, both mature and immature, were obtained from a small cultivation pond in the Ibaraki University’s College of Agriculture campus in Ami town, Ibaraki, Japan [17]. The immature seed endosperm was collected from seeds extracted from the lotus seedpod in their post-pollination late immature stage. At the point of collection, the seeds were approximately 1.3 cm long, and the external husk was still green and soft. The seeds were washed and stored whole at −80 °C until tissue extraction. The seeds were cut open and the soft and white core was removed whole and then cut across its length. The translucent sheet around the core, any discernible embryo tissue, as well as the central portion of the core immediately around the embryo was removed. The remaining soft endosperm fragments were ground under liquid nitrogen and the resulting powder was stored in sterile BD Falcon tubes at −80 °C until extraction of protein. For embryo tissue sample preparation, the mature seeds (stored at room temperature) were cracked open in a clean environment and the endosperm and embryo portions were cleanly separated and stored in sterile BD Falcon tubes at −80 °C. The embryo fragments were ground into a fine powder in liquid nitrogen, with a pre-chilled mortar and pestle. Resulting powder was stored in sterile 2.0 mL microfuge tubes at −80 °C until further analysis.

2.2. Extraction of the Lotus Seed Immature Endosperm and Embryo Proteins

Proteins were extracted from the powdered samples using the Tris-buffered saline (TBS) extraction method described in a previous study [17]. Briefly, a 3:1 mixture of TBS-20 buffer [10 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1% (v/v) Tween-20, plus one tablet of EDTA-free proteinase inhibitor (cOmplete Mini, Roche) per 50 mL] and SDS (sodium dodecyl sulfate) reducing buffer [62 mM Tris (pH 6.8), 10% (v/v) glycerol, 2.5% (w/v) SDS, 5% (v/v) 2-mercaptoethanol] was used to extract the powdered samples at 2 mL/100 mg. The sample/buffer mixtures were also subjected to several 30 s ultrasonic bath cycles and at 95 °C heating for 5 min to help extraction. The extract was separated by centrifugation, and its proteins precipitated and purified using the ProteoExtract kit (Calbiochem). The dry protein pellets obtained were either resolubilized in LB-TT (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 18 mM Tris-HCl (pH 8.0), 14 mM Trizma base, 0.2% (v/v) Triton X-100 and 50 mM dithiothreitol) for immediate use or stored at −80 °C. Prior to use, protein content of the resolubilized extracts was measured by Bradford assay [18].

2.3. Extraction of the Lotus Seed Immature Endosperm and Embryo Proteins

Protein samples from both tissues were subjected to 1-DGE (SDS-PAGE, 12.5%), both for visualization of protein profiles (Figure 2) using Coomassie Brilliant Blue [19] staining, and prior to analysis by 1DGE-MS.
The 1DGE-MS analyses followed the same methodology as with the previous lotus seed analyses [17]. The extracts were initially separated using SDS-PAGE. The resulting vertical protein lanes were sliced into eight pieces of equal length (regardless of apparent protein concentration) giving fraction 1: <120 kDa, fraction 2: 120–60 kDa, fraction 3: 60–40 kDa, fraction 4: 40–30 kDa, fraction 5: 30–22 kDa, fraction 6: 22–17 kDa, fraction 7: 17–14 kDa, and fraction 8: 14–10 kDa. Each fraction was digested with 1 µg of trypsin at 37 °C for 16 h [17,18,19,20]. Digested peptides were recovered twice with 20 µL of 5% (v/v) formic acid in 50% (v/v) acetonitrile. Extracted peptides were combined and then evaporated in a vacuum concentrator until liquid was dry. Dried peptides were dissolved into 20 µL of 5% acetonitrile/0.1% formic acid and then filtrated by the Ultrafree-MC Centrifugal Filters (Millipore, PVDF 0.45 µm, Darmstadt, Germany). Liquid chromatography–tandem mass spectrometry (MS/MS) analysis was performed using the LTQ-Orbitrap XL-HTC-PAL system (Thermo, Waltham, MA, USA). Trypsin digests were loaded on the column (100 µm internal diameter, 15 cm length, l-Column, CERI) using the Paradigm MS4 HPLC pump (Michrom BioResources, Auburn, AL, USA) and HTC-PAL Autosampler (CTC Analytics, Zwingen, Switzerland), and were eluted by a gradient of 5%–45% (v/v) acetonitrile in 0.1% (v/v) formic acid for 26 min. The eluted peptides were introduced directly into an LTQ-Orbitrap with a flow rate of 500 nL/min, and a spray voltage of 2.0 kV. The range of MS scan was m/z 450–1500. The top three peaks were subjected to MS/MS analysis. MS/MS spectra were analyzed by Mascot server (version 2.4.1, Matrix Science, Boston, MA, USA) in house (http://www.matrixscience.com/) and compared against proteins registered in the SwissProt (SwissProt_2012_03) database (total sequences: 428650; sequences after taxonomy filter (Viridiplantae): 27008; date: 26 July 2013). The Mascot search parameters were set as follows: threshold of the ion score cutoff, 0.05, peptide tolerance, 10 ppm, MS/MS tolerance, 0.5 Da, and peptide charge, 2+ or 3+. The search was also set to allow one missed cleavage by trypsin, a carboxymethylation modification of Cys residues, and a variable oxidation modification of Met residues. Gene ontology analysis on the data was performed using the Uniprot (www.uniprot.org) and the EMBL-EBI (www.ebi.ac.uk) databases.
Figure 2. 1D SDS-PAGE of protein extracts from lotus seed mature endosperm (MtE), immature endosperm (ImE), and embryo (Emb). SDS-PAGE, 12.5%; Coomassie brilliant blue stained. Molecular weight markers are shown on left-hand side of each gel image.
Figure 2. 1D SDS-PAGE of protein extracts from lotus seed mature endosperm (MtE), immature endosperm (ImE), and embryo (Emb). SDS-PAGE, 12.5%; Coomassie brilliant blue stained. Molecular weight markers are shown on left-hand side of each gel image.
Proteomes 03 00184 g002

3. Results and Discussion

3.1. Protein Content of the Immature Endosperm and Embryo Tissues

Protein extracts from the lotus immature seed endosperm presented very low protein yield (ca. 1.5% in the TBS method), requiring larger amounts of tissue to be extracted in order to obtain a suitable amount of protein. The reason for low protein yield lies in the high water content of the immature seed compared to its mature form. The lotus seed embryo showed a similar total protein yield to the endosperm extract [17] when extracted by the TBS/clean-up method (ca. 9%, compared to ca. 11% for the mature endosperm).
A comparison of the of the 1-D band profile on the SDS-PAGE of the embryo extract with the endosperm one showed many similarities, but also some noticeable differences, such as an absence of strongly stained bands at ca. 20 kDa and 40 kDa, and more numerous bands at low-molecular weights, under 30 and 20 kDa (see above, Figure 2). In the case of the immature endosperm, the 1-D profile is more similar to the mature endosperm than the embryo, but still was found to be different from both tissues profiles. Compared with the mature endosperm extract, the immature endosperm extract most notably does not present a high amount of protein bands around the 20 kDa range. The cluster of bands around 50 kDa is similar to that in both the endosperm and embryo, and the immature endosperms profile of bands in the 60–90 kDa range seems more similar to the mature endosperm than the embryo.

3.2. Lotus Immature Endosperm Proteins Identified by 1-DGE and MS/MS Analyses

The 1-DGE separation (SDS-PAGE) of proteins in an extract, followed by MS/MS analysis is part of the so-called “bottom-up” approach to proteomics, a methodology in which proteins are proteolytically digested into peptides prior to mass spectrometric analysis, and the ensuing peptide masses and sequences are used to identify corresponding proteins. This simple approach is a useful method for performing large-scale analyses of complex samples [21]. For the sample consisting of a purified extract of lotus immature endosperm proteins, after separation by SDS-PAGE, the sample was divided into eight fractions, analyzed by LC-MS/MS, and matched against a green plant database, as detailed in the Experimental Section. Results revealed more than 500 protein matches with at least two confirmed peptide fragment matches were identified amongst all fractions, and from these 333 unique protein matches were identified. Different database matches that were likely to refer to the same protein in the sample, such as two or more matches for the same protein but from different database organisms, were grouped together based on taxonomical proximity and similarity of identified peptide sequences. Finally, 122 non-redundant (nr) protein matches were listed, along with the number of repeated matches found for each one (Table 1), with the protein match listed being the one with the highest score amongst its group of similar proteins.

3.3. Lotus Embryo Proteins Identified by 1-DGE and MS/MS Analyses

The 1-DGE-MS analysis of the lotus embryo protein extract was performed following the same methodology, green plant database, and same parameters as for the immature endosperm extract. For the sample consisting of a purified extract of lotus embryo proteins, after separation by SDS-PAGE, the sample was divided into eight fractions, analyzed by LC-MS/MS, and matched against a green plant database, as above. From the initial results, 500+ protein matches with at least two confirmed peptide fragment matches were identified. After removing duplicate results from different gel fractions, there were 373 unique protein matches remaining. After grouping results likely to be the same protein in the sample, based on protein taxonomy and similarity of identified peptide sequences, 141 nr protein matches were listed (Table 2).
Table 1. List of top-scored non-redundant (nr) protein matches of the lotus immature endosperm 1-D shotgun mass spectroscopy results, as matched to Green Plant proteome database (SwissProt 57.0, http://www.uniprot.org/statistics/UniProtKB%2015).
Table 1. List of top-scored non-redundant (nr) protein matches of the lotus immature endosperm 1-D shotgun mass spectroscopy results, as matched to Green Plant proteome database (SwissProt 57.0, http://www.uniprot.org/statistics/UniProtKB%2015).
Fractions 1Protein AccessionProtein DescriptionSimilar 2Score 3Cover (%)PEPTIDE SequencesSig. Peptide NumberFunc. Cat. 4
6,5,8,(7,4,3,1)ENO1_HEVBREnolase 1 OS = Hevea brasiliensis11147143.4TAIAK, YNQLLR, LTSEIGEK, ACNALLLK, DGGSDYLGK, AGWGVMASHR, EKACNALLLK, MGAEVYHHLK, RAGWGVMASHR, LGANAILAVSLAVCK, VQIVGDDLLVTNPK, AAVPSGASTGIYEALELR, LAMQEFMILPVGASSFK, SGETEDTFIADLSVGLATGQIK, YGQDATNVGDEGGFAPNIQENK, KYGQDATNVGDEGGFAPNIQENK, YGQDATNVGDEGGFAPNIQENKEGLELLK17II
7,8,6,5,1,4,2G3PC_ANTMAGlyceraldehyde-3-phosphate dehydrogenase, cytosolic OS = Antirrhinum majus23124243.6AAAHLK, KATYEQIK, AAIKEESEGK, AGIALNDNFVK, DAPMFVVGVNEK, AASFNIIPSSTGAAK, VPTVDVSVVDLTVR, DAPMFVVGVNEKEYK, VPTVDVSVVDLTVRLEK, FGIVEGLMTTVHSITATQK, GILGYTEDDVVSTDFVGDSR, LTGMSFRVPTVDVSVVDLTVR, LKGILGYTEDDVVSTDFVGDSR, VINDRFGIVEGLMTTVHSITATQK14II
4,8,6,7,5HSP7D_ARATHHeat shock 70 kDa protein 4 OS = Arabidopsis thaliana1062523.8IEEVD, LSKEEIEK, ITITNDKGR, DAGVISGLNVMR, NALENYAYNMR, MVNHFVQEFKR, TTPSYVAFTDSER, IINEPTAAAIAYGLDK, ATAGDTHLGGEDFDNR, NAVVTVPAYFNDSQR, IINEPTAAAIAYGLDKK, EQIFSTYSDNQPGVLIQVYEGER12IX
4HSP7E_SPIOLChloroplast envelope membrane 70 kDa heat shock-related protein OS = Spinacia oleracea158021.7LSKEEIEK, DAGVISGLNVMR, EIAEAYLGSTVK, NALENYAYNMR, TTPSYVAFTDSER, IINEPTAAAIAYGLDK, ATAGDTHLGGEDFDNR, NAVVTVPAYFNDSQR, IINEPTAAAIAYGLDKK, EQVFSTYSDNQPGVLIQVYEGER10IX
4BIP4_TOBACLuminal-binding protein 4 OS = Nicotiana tabacum554221.6VQQLLK, NTVIPTKK, IMEYFIK, LSQEEIER, ITITNDKGR, DYFDGKEPNK, FEELNNDLFR, EAEEFAEEDKK, IVNKDGKPYIQVK, ARFEELNNDLFR, NGHVEIIANDQGNR, IINEPTAAAIAYGLDK, IINEPTAAAIAYGLDKK, IKDAVVTVPAYFNDAQR14IX
4,5,6,7,8METE_ARATH5-methyltetrahydropteroyltriglutamate—homocysteine methyltransferase OS = Arabidopsis thaliana451619.2AAAALK, VVEVNALAK, SWLAFAAQK, AVNEYKEAK, YLFAGVVDGR, SDEKLLSVFR, FALESFWDGK, GNASVPAMEMTK, YGAGIGPGVYDIHSPR, GMLTGPVTILNWSFVR10I
6,1,5,(7,8,3,2,4)EF1A_TOBACElongation factor 1-alpha OS = Nicotiana tabacum848434.7YDEIVK, GFVASNSK, QTVAVGVIK, EVSSYLKK, LPLQDVYK, ARYDEIVK, IGGIGTVPVGR, STNLDWYK, STTTGHLIYK, EHALLAFTLGVK, GFVASNSKDDPAK, YYCTVIDAPGHR, MIPTKPMVVETFSEYPPLGR, NMITGTSQADCAVLIIDSTTGGFEAGISK14V
4HSP7L_ARATHHeat shock 70 kDa protein 12 OS = Arabidopsis thaliana147916.5VQQLLK, NTVIPTKK, IMEYFIK, FDLTGVPPAPR, FEELNNDLFR, EAEEFAEEDKK, ARFEELNNDLFR, NGHVEIIANDQGNR, IINEPTAAAIAYGLDK, IINEPTAAAIAYGLDKK, IKDAVVTVPAYFNDAQR11IX
4,8,6HSP7N_ARATHHeat shock 70 kDa protein 18 OS = Arabidopsis thaliana147418.5ITITNDKGR, EIAEAYLGSSIK, MVNHFVQEFKR, TTPSYVAFTDSER, IINEPTAAAIAYGLDK, ATAGDTHLGGEDFDNR, NAVVTVPAYFNDSQR, IINEPTAAAIAYGLDKK8IX
7MDHM_CITLAMalate dehydrogenase, mitochondrial OS = Citrullus lanatus545918.2TFYAGK, LFGVTTLDVVR, TQDGGTEVVEAK, DDLFNINAGIVK, KLFGVTTLDVVR, RTQDGGTEVVEAK, VAVLGAAGGIGQPLALLMK, KVAVLGAAGGIGQPLALLMK8II
4,5HSP80_SOLLCHeat shock cognate protein 80 OS = Solanum lycopersicum142720.9AVENSPFLEK, LGIHEDSQNR, ADLVNNLGTIAR, KAVENSPFLEK, HFSVEGQLEFK, GIVDSEDLPLNISR, SLTNDWEEHLAVK, SGDEMTSLKDYVTR, KPEEITKEEYAAFYK, MKEGQNDIYYITGESK10IX
5CH62_MAIZEChaperonin CPN60-2, mitochondrial OS = Zea mays642223.3VTDALNATK, GVEELADAVK, IGGASEAEVGEK, SVAAGMNAMDLR, IGGASEAEVGEKK, NVVIEQSFGAPK, AAVEEGIVPGGGVALLYASK, TPVHTIASNAGVEGAVVVGK, QRPLLIVAEDVESEALGTLIINK9IX
7,8,6,1ACT_GOSHIActin OS = Gossypium hirsutum1941432.6AGFAGDDAPR, GYSFTTTAER, EITALAPSSMK, DAYVGDEAQSK, AVFPSIVGRPR, DAYVGDEAQSKR, SYELPDGQVITIGAER, VAPEEHPVLLTEAPLNPK, TTGIVLDSGDGVSHTVPIYEGYALPHAILR9VII
5,8,7ENO2_ARATHBifunctional enolase 2/transcriptional activator OS = Arabidopsis thaliana141134.5YNQLLR, DGGSDYLGK, ISGDALKDLYK, LGANAILAVSLAVCK, VNQIGSVTESIEAVK, TYDLNFKEENNNGSQK, SGETEDTFIADLAVGLSTGQIK, YGQDATNVGDEGGFAPNIQENK, YGQDATNVGDEGGFAPNIQENKEGLELLK9IV
4,5,6HSP83_IPONIHeat shock protein 83 OS = Ipomoea nil140523VIVTTK, VVVSDR, AILFVPK, DVDGEQLGR, APFDLFDTR, AVENSPFLER, LGIHEDSQNR, LDAQPELFIR, RAPFDLFDTR, ADLVNNLGTIAR, ELISNASDALDK, HFSVEGQLEFK, GVVDSDDLPLNISR, ELISNASDALDKIR, ITLFLKEDQLEYLEER14IX
7,6,1,2ADH1_SOLTUAlcohol dehydrogenase 1 OS = Solanum tuberosum739023.7ELELEK, SDIPSVVEK, FGVTEFVNPK, GTFFGNYKPR, THPMNLLNER, KFGVTEFVNPK, YMNKELELEK, TLKGTFFGNYKPR, GSSVAIFGLGAVGLAAAEGAR9II
4,5,7,8ENPL_CATROEndoplasmin homolog OS = Catharanthus roseus338710.5FWNEFGK, ESFKELTK, YGWSSNMER, ELISNASDALDK, IMQSQTLSDASK, GLVDSDTLPLNVSR, ELISNASDALDKIR, VFISDEFDELLPK, RVFISDEFDELLPK9IX
7,(8,6)RBL_MAIZERibulose bisphosphate carboxylase large chain OS = Zea mays5238227.5AMHAVIDR, AQAETGEIK, DTDILAAFR, DDFIEKDR, VALEACVQAR, EITLGFVDLLR, LTYYTPEYETK, MSGGDHIHSGTVVGK, YGRPLLGCTIKPK, GGLDFTKDDENVNSQPFMR10I
6SAHH_MEDSAAdenosylhomocysteinase OS = Medicago sativa336915.1ATDVMIAGK, HSLPDGLMR, ITIKPQTDR, TEFGPSQPFK, VAVVCGYGDVGK, SKFDNLYGCR, IVGVSEETTTGVK, IVGVSEETTTGVKR8I
4,(5,6)HSP82_ORYSJHeat shock protein 81-2 OS = Oryza sativa subsp. Japonica236821.5VVVSDR, IAELLR, AILFVPK, APFDLFDTR, AVENSPFLEK, RAPFDLFDTR, KAVENSPFLEK, SDLVNNLGTIAR, HFSVEGQLEFK, GIVDSEDLPLNISR, SLTNDWEEHLAVK, HSEFISYPISLWTEK, KPEEITKEEYAAFYK12IX
4,6,7HSP70_DAUCAHeat shock 70 kDa protein OS = Daucus carota133515.7IEEVD, NALENYAYNMR, NQVAMNPSNTVFDAK, NQVAMNPSNTVFDAKR, SINPDEAVAYGAAVQAAILSGEGNER, EQIFSTYSDNQPGVLIQVYEGER6IX
5CPNA1_ARATHChaperonin 60 subunit alpha 1, chloroplastic OS = Arabidopsis thaliana133116.4KVTISK, VVNDGVTIAR, NVVLDEFGSPK, VGAATETELEDR, GYISPQFVTNPEK, TNDSAGDGTTTASILAR6IX
4,5HSP82_MAIZEHeat shock protein 82 OS = Zea mays229714.4APFDLFDTR, AVENSPFLER, LGIHEDSQNR, RAPFDLFDTR, SDLVNNLGTIAR, ELISNASDALDK, HFSVEGQLEFK, GVVDSDDLPLNISR, ELISNASDALDKIR9IX
8,7ALF_CICARFructose-bisphosphate aldolase, cytoplasmic isozyme OS = Cicer arietinum228914.2ANSEATLGTYK, GILAADESTGTIGK, GILAADESTGTIGKR, YHDELIANAAYIGTPGK4II
4,(3,5)CD48A_ARATHCell division control protein 48 homolog A OS = Arabidopsis thaliana328514TLLAK, KGDLFLVR, ELVELPLR, LAEDVDLER, LAGESESNLR, GILLYGPPGSGK, IVSQLLTLMDGLK, ELVELPLRHPQLFK, NAPSIIFIDEIDSIAPK9III/IV
7,8,6,1PGKH_TOBACPhosphoglycerate kinase, chloroplastic OS = Nicotiana tabacum628415.4AAVPTIK, AHASTEGVTK, FAVGTEAIAK, VILSSHLGRPK, GVTTIIGGGDSVAAVEK, LASLADLYVNDAFGTAHR, KLASLADLYVNDAFGTAHR7II
5,(4)PGMC_POPTNPhosphoglucomutase, cytoplasmic OS = Populus tremula128112.2YLFEDGSR, FFEVPTGWK, LSGTGSEGATIR, SMPTSAALDVVAK, YDYENVDAGAAK, VETTPFGDQKPGTSGLR6II
4,(5)HS903_ARATHHeat shock protein 90-3 OS = Arabidopsis thaliana326920.2IAELLR, AILFVPK, AVENSPFLEK, LGIHEDSQNR, ADLVNNLGTIAR, KAVENSPFLEK, HFSVEGQLEFK, GIVDSEDLPLNISR, HSEFISYPISLWIEK9IX
5,6PMG2_ARATHProbable 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 2 OS = Arabidopsis thaliana125713.6VHILTDGR, ARDAILSGK, LVDLALASGK, TFACSETVK, MKALEIAEK, GWDAQVLGEAPHK, RGWDAQVLGEAPHK, AVGPIVDGDAVVTFNFR8II
5,6PMG1_ARATH2,3-bisphosphoglycerate-independent phosphoglycerate mutase 1 OS = Arabidopsis thaliana522410.2VHILTDGR, ARDAILSGK, LDQLQLLIK, GWDAQVLGEAPHK, RGWDAQVLGEAPHK, AVGPIVDGDAVVTFNFR6II
5SSG1_HORVUGranule-bound starch synthase 1, chloroplastic/amyloplastic OS = Hordeum vulgare52147.5FFHCYK, EALQAEVGLPVDR, FSLLCQAALEAPR, VAFCIHNISYQGR4I
6RL4_PRUAR60S ribosomal protein L4 OS = Prunus armeniaca320020.8AGQGAFGNMCR, AGHQTSAESWGTGR, YAVVSAIAASAVPSLVLAR, AWYQTMISDSDYTEFDNFTK4V
8H2B_GOSHIHistone H2B OS = Gossypium hirsutum520049IYIFK, LVLPGELAK, AMGIMNSFINDIFEK3VII
5RUBA_RICCORuBisCO large subunit-binding protein subunit alpha (Fragment) OS = Ricinus communis220016NVVLDEFGSPK VGAATETELEDR, GYISPQFVTNPEK, LGLLSVTSGANPVSIK4I
8H2B1_MEDTRProbable histone H2B.1 OS = Medicago truncatula219945.3IYIFK, LVLPGELAK, AMGIMNSFINDIFEK3VII
8RL182_ARATH60S ribosomal protein L18-2 OS = Arabidopsis thaliana118513.4APLGQNTVLLR, AGGECLTFDQLALR2V
5CALR_BERSTCalreticulin OS = Berberis stolonifera31759.1LAEETWGK, LLSGDVDQK, KLAEETWGK, TLVFQFSVK, LLSGDVDQKK, YVGIELWQVK6V
81433E_TOBAC14-3-3-like protein E OS = Nicotiana tabacum517226.8NVIGAR, NLLSVAYK, DSTLIMQLLR, TVDVEELTVEER, IISSIEQKEESR, SAQDIALAELAPTHPIR6VIII
8H4_ARATHHistone H4 OS = Arabidopsis thaliana116045.6TLYGFGG, IFLENVIR, DAVTYTEHAR, ISGLIYEETR, DNIQGITKPAIR5VII
6,5,8KPYC_SOYBNPyruvate kinase, cytosolic isozyme OS = Glycine max21579.2KGSDLVNVR, GDLGMEIPVEK, VENQEGVLNFDEILR3II
8RS6_ASPOF40S ribosomal protein S6 OS = Asparagus officinalis215511.6LVTPLTLQR, ISQEVSGDALGEEFK, ISQEVSGDALGEEFKGYVFK3V
5TCPA_ARATHT-complex protein 1 subunit alpha OS = Arabidopsis thaliana11536.4YFVEAGAIAVR, VLVELAELQDR, NKIHPTSIISGYR3V
1ADT1_GOSHIADP, ATP carrier protein 1, mitochondrial OS = Gossypium hirsutum11435.7SSLDAFSQILK, LLIQNQDEMIK2VI
4HSP7S_SPIOLStromal 70 kDa heat shock-related protein, chloroplastic (Fragment) OS = Spinacia oleracea21427.2QFAAEEISAQVLR, AVVTVPAYFNDSQR, IINEPTAASLAYGFEK3IX
7,8GCST_PEAAminomethyltransferase, mitochondrial OS = Pisum sativum214014.7LYFGEFR, GGAIDDSVITK, SLLALQGPLAAPVLQHLTK, TGYTGEDGFEISVPSEHGVELAK4I
3,2HSP7O_ARATHHeat shock 70 kDa protein 14 OS = Arabidopsis thaliana11397.7ILSHAFDR, AVLDAATIAGLHPLR, AVEKEFEMALQDR, RAVLDAATIAGLHPLR4IX
4HSP7F_ARATHHeat shock 70 kDa protein 6, chloroplastic OS = Arabidopsis thaliana11397.5TTPSVVAYTK, QFAAEEISAQVLR, QAVVNPENTFFSVK, LSFKDIDEVILVGGSTR4IX
8RS4_GOSHI40S ribosomal protein S4 OS = Gossypium hirsutum213520.6LSIIEEAR, LGNVFTIGK, FDVGNVVMVTGGR, LGGAFAPKPSSGPHK4V
3,4CLPA_BRANAATP-dependent Clp protease ATP-binding subunit clpA homolog, chloroplastic (Fragment) OS = Brassica napus11355.8VIGQDEAVK, TAIAEGLAQR, YRGEFEER, VLELSLEEAR4I
4,5,8EF2_BETVUElongation factor 2 OS = Beta vulgaris11315.5GGGQIIPTAR, EGALAEENMR, RVFYASQLTAKPR, LWGENFFDPATKK4V
8RL12_PRUAR60S ribosomal protein L12 OS = Prunus armeniaca112922.3VSVVPSAAALVIK, VTGGEVGAASSLAPK2V
6,(7,8)ATPBM_NICPLATP synthase subunit beta, mitochondrial OS = Nicotiana plumbaginifolia412912.5VLNTGSPITVPVGR, TVLIMELINNVAK, IPSAVGYQPTLATDLGGLQER3I
4,(7)PHSH_SOLTUAlpha-glucan phosphorylase, H isozyme OS = Solanum tuberosum61255.6AFATYTNAK, QLLNILGVIYR, HMEIIEEIDKR, TIAYTNHTVLPEALEK4II
6RL3_ORYSJ60S ribosomal protein L3 OS = Oryza sativa subsp. Japonica21246.9VIAHTQIR, HGSLGFLPR, GKGYEGVVTR3V
8TPIS_MAIZETriosephosphate isomerase, cytosolic OS = Zea mays212411.9FFVGGNWK, VAYALSQGLK, VIACVGETLEQR3VII
8LE194_HORVULate embryogenesis abundant protein B19.4 OS = Hordeum vulgare11219.2GGLSTMNESGGER, KGGLSTMNESGGER2IX
1,(2)AVP_VIGRRPyrophosphate-energized vacuolar membrane proton pump OS = Vigna radiata var. Radiata21173.3AADVGADLVGK, YIEAGASEHAR, AADVGADLVGKVER3VI
8RL6_MESCR60S ribosomal protein L6 OS = Mesembryanthemum crystallinum111510.7VDISGVNVEK, ASITPGTVLIILAGR2V
5SSG1_ARATHProbable granule-bound starch synthase 1, chloroplastic/amyloplastic OS = Arabidopsis thaliana11153.9FFHCYK, YGTVPIVASTGGLVDTVK2I
8RL10_VITRI60S ribosomal protein L10 OS = Vitis riparia111410.5VSIGQVLLSVR, ENVSSEALEAAR2V
8RS18_ARATH40S ribosomal protein S18 OS = Arabidopsis thaliana111121.7LRDDLER, VLNTNVDGK, IMFALTSIK, IPDWFLNR4V
7AATM_LUPANAspartate aminotransferase P2, mitochondrial (Fragment) OS = Lupinus angustifolius11116.8IADVIQEK, LNLGVGAYR, VATVQGLSGTGSLR3I
8GBLPA_ORYSJGuanine nucleotide-binding protein subunit beta-like protein A OS = Oryza sativa subsp. Japonica11109DGVTLLWDLAEGK, FSPNTFQPTIVSGSWDR2VIII
8H2AX_CICARHistone H2AX OS = Cicer arietinum110815.1AGLQFPVGR, GKGEIGSASQEF2VII
4VATA_GOSHIV-type proton ATPase catalytic subunit A OS = Gossypium hirsutum21085LAADTPLLTGQR, LVSQKFEDPAEGEEALVAK2VI
8PARP3_SOYBNPoly [ADP-ribose] polymerase 3 OS = Glycine max11074.2VLCSQEIYK, LEPLVANFMK, LFEEITGNEFEPWER3III
3,4,(5)CLPC1_ARATHChaperone protein ClpC1, chloroplastic OS = Arabidopsis thaliana51065.4TAIAEGLAQR, YRGEFEER, VLELSLEEAR3IX
8NDK1_ARATHNucleoside diphosphate kinase 1 OS = Arabidopsis thaliana21039.4NVIHGSDSVESAR, NVIHGSDSVESARK2I
8RL13_TOBAC60S ribosomal protein L13 OS = Nicotiana tabacum19816.3SLEGLQTNVQR, KLAPTIGIAVDHR2V
8RS5_CICAR40S ribosomal protein S5 (Fragment) OS = Cicer arietinum29515.2GSSNSYAIK, AQCPIVER, VNQAIYLLTTGAR3V
5,6PDC2_ORYSIPyruvate decarboxylase isozyme 2 OS = Oryza sativa subsp. Indica2944.5AVKPVLVGGPK, ILHHTIGLPDFSQELR2II
8HSP14_SOYBN17.5 kDa class I heat shock protein OS = Glycine max49224.7AIEISG, ADIPGLK, VLQISGER, FRLPENAK4IX
6AMPL1_ARATHLeucine aminopeptidase 1 OS = Arabidopsis thaliana2924.6GLTFDSGGYNIK, TIEVNNTDAEGR2I/IX
6ACT5_ARATHPutative actin-5 OS = Arabidopsis thaliana19215.9AGFAGDDAPR, IWHHTFYNELR2VII
8RS14_CHLRE40S ribosomal protein S14 OS = Chlamydomonas reinhardtii18715.7TPGPGAQSALR, IEDVTPIPTDSTR2V
8RS3A1_VITVI40S ribosomal protein S3a-1 OS = Vitis vinifera2866.5TTDNYTLR, LRAEDVQGR2V
7AAT3_ARATHAspartate aminotransferase, chloroplastic OS = Arabidopsis thaliana1834.9LNLGVGAYR, TEEGKPLVLNVVR2I
1COB21_ORYSJCoatomer subunit beta-1 OS = Oryza sativa subsp. Japonica1834.5HNEIQTVNIK, DTNTFASASLDR2VI
8GRDH1_ARATHGlucose and ribitol dehydrogenase homolog 1 OS = Arabidopsis thaliana3838GAIVAFTR, EGSSIINTTSVNAYK2II
8ANXD1_ARATHAnnexin D1 OS = Arabidopsis thaliana1805AQINATFNR, SKAQINATFNR2IX
7PDI21_ORYSJProtein disulfide isomerase-like 2-1 OS = Oryza sativa subsp. Japonica18010.9KLAPEYEK, YGVSGFPTLK, YGVSGYPTIQWFPK3V
8,(6)ATPAM_NICPLATP synthase subunit alpha, mitochondrial OS = Nicotiana plumbaginifolia4799.4VVSVGDGIAR, TAIAIDTILNQK2I
8,(1)CB2_PHYPAChlorophyll a-b binding protein, chloroplastic OS = Physcomitrella patens subsp. Patens1753.7ELEVIHAR, NRELEVIHAR2II
8HSP12_SOYBNClass I heat shock protein (Fragment) OS = Glycine max17518.9AIEISG, ILQISGER2IX
8BAS1_ORYSJ2-Cys peroxiredoxin BAS1, chloroplastic OS = Oryza sativa subsp. Japonica1699.6LSDYIGKK, SGGLGDLKYPLISDVTK2IX
8RLA0_LUPLU60S acidic ribosomal protein P0 OS = Lupinus luteus1697.5VGSSEAALLAK, GTVEIITPVELIK2V
7EF1G2_ORYSJElongation factor 1-gamma 2 OS = Oryza sativa subsp. Japonica1685NPLDLLPPSK, SFTSEFPHVER2V
1MDAR_SOLLCMonodehydroascorbate reductase OS = Solanum lycopersicum1689.7AYLFPEGAAR, IVGAFLESGSPEENKAIAK2IX
7RSSA_BRANA40S ribosomal protein SA OS = Brassica napus16510.3LLILTDPR, VIVAIENPQDIIVQSARPYGQR2V
6,8IF4A1_ARATHEukaryotic initiation factor 4A-1 OS = Arabidopsis thaliana1658.3ELAQQIEK, VLITTDLLAR2V
7HSP11_PEA18.1 kDa class I heat shock protein OS = Pisum sativum16414.6SIEISG, VLQISGER2IX
8RS16_FRIAG40S ribosomal protein S16 OS = Fritillaria agrestis16412.4ALVAYYQK, AFEPILLLGR2V
8RL51_ARATH60S ribosomal protein L5-1 OS = Arabidopsis thaliana1647.3KLTYEER, GALDGGLDIPHSDKR2V
4HSP7M_PHAVUHeat shock 70 kDa protein, mitochondrial OS = Phaseolus vulgaris1646.1HLNITLTR, SSGGLSEDEIEK2IX
8,7HSP12_MEDSA18.2 kDa class I heat shock protein OS = Medicago sativa16322.8TIDISG, VLQISGER, FRLPENAK3IX
8RS102_ARATH40S ribosomal protein S10-2 OS = Arabidopsis thaliana1618.9TYLNLPSEIVPATLK, TYLNLPSEIVPATLKK2V
8RS193_ARATH40S ribosomal protein S19-3 OS = Arabidopsis thaliana16015.4DVSPHEFVK, ELAPYDPDWYYIR2V
1CYF_AETCOApocytochrome f OS = Aethionema cordifolium2608.7NILVIGPVPGQK, SNNTVYNATAGGIISK2II
8UBIQP_ACECLPolyubiquitin (Fragment) OS = Acetabularia cliftonii1608.7IIFAGK, TLADYNIQK, ESTLHLVLR3V
8RL40A_ARATHUbiquitin-60S ribosomal protein L40-1 OS = Arabidopsis thaliana16037.5LIFAGK, TLADYNIQK, ESTLHLVLR3V
3UREA_CANENUrease OS = Canavalia ensiformis1593NYFLF, TIHTYHSEGAGGGHAPDIIK2I
8RS13_PEA40S ribosomal protein S13 OS = Pisum sativum15817.2DSHGIAQVK, AHGLAPEIPEDLYHLIK2V
1,8RAN_VICFAGTP-binding nuclear protein Ran/TC4 OS = Vicia faba25813.1HLTGEFEK, NLQYYEISAK2III/VI
7PYRB_ARATHAspartate carbamoyltransferase, chloroplastic OS = Arabidopsis thaliana1575.4GETLEDTIR, LGGEVLTTENAR2I
8HSP11_CHERU18.3 kDa class I heat shock protein OS = Chenopodium rubrum15218.6FRLPENAK, IDWKETPEAHVFK2IX
5CLAH1_ARATHClathrin heavy chain 1 OS = Arabidopsis thaliana1510.9ILALK, SPEQVSAAVK2VI
6PDI_RICCOProtein disulfide-isomerase OS = Ricinus communis1494.2FFNSPDAK, SEPIPEVNNEPVK2V
7PDIA6_MEDSAProbable protein disulfide-isomerase A6 OS = Medicago sativa1487.4KLAPEYEK, YGVSGYPTIQWFPK2V
4SUSY_MEDSASucrose synthase OS = Medicago sativa1462.9NITGLVEWYGK, SGFHIDPYHGDR2II
7FKB62_ARATHPeptidyl-prolyl cis-trans isomerase FKBP62 OS = Arabidopsis thaliana1424.4SDGVEFTVK, FTLGQGQVIK2V
5DLDH2_ARATHDihydrolipoyl dehydrogenase 2, mitochondrial OS = Arabidopsis thaliana1403.4AAQLGLK, SLPGITIDEK2II
7WIT2_ARATHWPP domain-interacting tail-anchored protein 2 OS = Arabidopsis thaliana1393.3ELELEK, AESGEAKIK2III
8TBA_PRUDUTubulin alpha chain OS = Prunus dulcis2377.8DVNAAVATIK, LVSQVISSLTASLR2VII
7PER1B_ARMRUPeroxidase C1B OS = Armoracia rusticana1355.7VPLGR, MGNITPLTGTQGEIR2IX
7,(8,6)YCF1_IPOPUPutative membrane protein ycf1 OS = Ipomoea purpurea3351.3ALILK, IVIEK, VIQEKER3X
6RFS_ORYSJGalactinol--sucrose galactosyltransferase OS = Oryza sativa subsp. Japonica1272.8VELAK, LMEEK2II
7Y1497_ARATHProbable receptor-like protein kinase At1g49730 OS = Arabidopsis thaliana1201.7FLLAK, NLVALK2V
1 Fraction corresponding to slice of the 1-D gel in which matches for the protein were found. Numbers in parenthesis indicate fractions where additional similar matches (see 2.) were found. 2 Number of protein matches of high taxonomical and sequence similarity grouped together with this match. (Match displayed was the top-scored one.) 3 MASCOT score. 4 I: metabolism, II: energy, III: cell growth/division, IV: transcription, V: protein synthesis/destination, VI: transporters, VII: cell structure, VIII: signal transduction, IX: disease/stress defense, and X: unclassified.
Table 2. List of top-scored non-redundant (nr) protein matches of the lotus embryo 1-D shotgun mass spectroscopy results, as matched to Green Plant proteome database (SwissProt 57.0).
Table 2. List of top-scored non-redundant (nr) protein matches of the lotus embryo 1-D shotgun mass spectroscopy results, as matched to Green Plant proteome database (SwissProt 57.0).
Fractions 1Protein AccessionProtein DescriptionSimilar 2Score 3Cover (%)Peptide sequencesSig. Peptide NumberFunc. Cat. 4
6,(5,2,1,7,3)ENO1_HEVBREnolase 1 OS = Hevea brasiliensis19112542TAIAK, YNQLLR, LTSEIGEK, DGGSDYLGK, AGWGVMASHR, MGAEVYHHLK, DGGSDYLGKGVSK, VQIVGDDLLVTNPK, VNQIGSVTESIEAVK, EAMKMGAEVYHHLK, AAVPSGASTGIYEALELR, LAMQEFMILPVGASSFK, SGETEDTFIADLSVGLATGQIK, YGQDATNVGDEGGFAPNIQENK, KYGQDATNVGDEGGFAPNIQENK, YGQDATNVGDEGGFAPNIQENKEGLELLK16II
4,(1,2,3,5)HSP7C_PETHYHeat shock cognate 70 kDa protein OS = Petunia hybrida3392233.6IEEVD, DISGNPR, NTTIPTKK, ITITNDKGR, DAGVIAGLNVMR, MVNHFVQEFK, NALENYAYNMR, MVNHFVQEFKR, TTPSYVGFTDTER, ARFEELNMDLFR, IINEPTAAAIAYGLDK, NQVAMNPINTVFDAK ATAGDTHLGGEDFDNR, NQVAMNPINTVFDAK, NAVVTVPAYFNDSQR, EQVFSTYSDNQPGVLIQVYEGER16IX
1,2,3,4,5,6,7ACT_GOSHIActin OS = Gossypium hirsutum1490350.7DLTDALMK, AGFAGDDAPR, IKVVAPPER, GYSFTTTAER, HTGVMVGMGQK, EITALAPSSMK, DAYVGDEAQSK, AVFPSIVGRPR, IWHHTFYNELR, LDLAGRDLTDALMK, GYSFTTTAEREIVR, SYELPDGQVITIGAER, VAPEEHPVLLTEAPLNPK, VAPEEHPVLLTEAPLNPK, TTGIVLDSGDGVSHTVPIYEGYALPHAILR15VII
6,(1,2,3,7)ACT12_SOLTUActin-100 (Fragment) OS = Solanum tuberosum587253.5AGFAGDDAPR, IKVVAPPER, HTGVMVGMGQK, EITALAPSSMK, DAYVGDEAQSK, AVFPSIVGRPR, DAYVGDEAQSKR, GEYDESGPSIVHR, IWHHTFYNELR, SYELPDGQVITIGAER, LAYVALDYEQELETAK, YPIEHGIVSNWDDMEK, TTGIVLDSGDGVSHTVPIYEGYALPHAILR13VII
7,(8,2)G3PC_ANTMAGlyceraldehyde-3-phosphate dehydrogenase, cytosolic OS = Antirrhinum majus2074943.6VALQR, SSIFDAK, KATYEQIK, AAIKEESEGK, AGIALNDNFVK, DAPMFVVGVNEK, AASFNIIPSSTGAAK, VPTVDVSVVDLTVR, VPTVDVSVVDLTVRLEK, FGIVEGLMTTVHSITATQK, GILGYTEDDVVSTDFVGDSR, LTGMSFRVPTVDVSVVDLTVR, LKGILGYTEDDVVSTDFVGDSR, VINDRFGIVEGLMTTVHSITATQK14II
4,(1,5,2)HSP83_IPONIHeat shock protein 83 OS = Ipomoea nil471731VIVTTK, VVVSDR, KLVSATK, AILFVPK, EMLQQNK, DVDGEQLGR, FESLTDKSK, APFDLFDTR, AVENSPFLER, LGIHEDSQNR, DIYYITGESK, LDAQPELFIR, RAPFDLFDTR, ADLVNNLGTIAR, ELISNASDALDK, KAVENSPFLER, HFSVEGQLEFK, GVVDSDDLPLNISR, ELISNASDALDKIR, SGDELTSLKDYVTR, KPEEITKEEYASFYK, HSEFISYPIYLWTEK, ITLFLKEDQLEYLEER23IX
6ATPBM_MAIZEATP synthase subunit beta, mitochondrial OS = Zea mays371231.8IGLFGGAGVGK, VVDLLAPYQR, TIAMDGTEGLVR, AHGGFSVFAGVGER, VGLTGLTVAEHFR, VLNTGSPITVPVGR, TVLIMELINNVAK, FTQANSEVSALLGR, QISELGIYPAVDPLDSTSR, EAPAFVEQATEQQILVTGIK, IPSAVGYQPTLATDLGGLQER11I
4,(5,2,1,3)HSP7E_SPIOLChloroplast envelope membrane 70 kDa heat shock-related protein OS = Spinacia oleracea563131.2NTTIPTKK, LSKEEIEK, TRDNNLLGK, DAGVISGLNVMR, EIAEAYLGSTVK, NALENYAYNMR, TTPSYVAFTDSER, IINEPTAAAIAYGLDK, ATAGDTHLGGEDFDNR, NQVAMNPINTVFDAK, NAVVTVPAYFNDSQR, EQVFSTYSDNQPGVLIQVYEGER12IX
4,(2,1,5)HSP81_ORYSIHeat shock protein 81-1 OS = Oryza sativa subsp. Indica961035.1NLVKK, VVVTTK, IAELLR, KLVSATK, EMLQQNK, FESLTDKSK, APFDLFDTR, DSSMAGYMSSK, RAPFDLFDTR, KAVENSPFLEK, SDLVNNLGTIAR, HFSVEGQLEFK, EVSHEWSLVNK, GIVDSEDLPLNISR, SLTNDWEEHLAVK, SGDELTSLKDYVTR, LDAQPELFIHIVPDK, HSEFISYPISLWTEK, KPEEITKEEYAAFYK, MKEGQNDIYYITGESK, KHSEFISYPISLWTEK21IX
6,(3,1,2)TBB_HORVUTubulin beta chain OS = Hordeum vulgare2158339.1YLTASAMFR, IREEYPDR, LAVNLIPFPR, VSEQFTAMFR, YTGTSDLQLER, MMLTFSVFPSPK, EVDEQMINVQNK, LHFFMVGFAPLTSR, AVLMDLEPGTMDSVR, LHFFMVGFAPLTSR, NSSYFVEWIPNNVK, ALTVPELTQQMWDAK, GHYTEGAELIDSVLDVVRK, TGPYGQIFRPDNFVFGQSGAGNNWAK14VII
6,(1,7,3,4,2)EF1A_TOBACElongation factor 1-alpha OS = Nicotiana tabacum1757438.5YDEIVK, GFVASNSK, EVSSYLK, QTVAVGVIK, EVSSYLKK, RGFVASNSK, LPLQDVYK, ARYDEIVK, IGGIGTVPVGR, STNLDWYK, STTTGHLIYK, EHALLAFTLGVK, GFVASNSKDDPAK, YYCTVIDAPGHR, YDEIVKEVSSYLK, YYCTVIDAPGHRDFIK, MIPTKPMVVETFSEYPPLGR, NMITGTSQADCAVLIIDSTTGGFEAGISK18V
4,(1,2,5,4)HS901_ARATHHeat shock protein 90-1 OS = Arabidopsis thaliana653926.4VVVTTKVVVTTK, VVVSDR, KLVSATK, AILFVPK, FESLTDKSK, APFDLFDTR, AVENSPFLER, LGIHEDSQNR, DSSMSGYMSSK, RAPFDLFDTR, ADLVNNLGTIAR, KAVENSPFLER, HFSVEGQLEFK, TLSIIDSGIGMTK, GVVDSDDLPLNISR, KPEEITKEEYAAFYK, HSEFISYPIYLWTEK17IX
4,(2,1,3)METE_ARATH5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase OS = Arabidopsis thaliana1253620.9AAAALK, VVEVNALAK, SWLAFAAQK, AVNEYKEAK, YLFAGVVDGR, SDEKLLSVFR, FALESFWDGK, GNASVPAMEMTK, YGAGIGPGVYDIHSPR, GMLTGPVTILNWSFVR10I
4ENPL_CATROEndoplasmin homolog OS = Catharanthus roseus347212.6NLGTIAK, FWNEFGK, YGWSSNMER, ELISNASDALDK, IMQSQTLSDASK, GLVDSDTLPLNVSR, ELISNASDALDKIR, VFISDEFDELLPK, RVFISDEFDELLPK, LMDIIINSLYSNKDIFLR10IX
4,(5,2)BIP4_TOBACLuminal-binding protein 4 OS = Nicotiana tabacum645421LIGEAAK, NTVIPTKK, IMEYFIK, LSQEEIER, ITITNDKGR, ALSSQHQVR, EAEEFAEEDKK, IVNKDGKPYIQVK, ARFEELNNDLFR, IINEPTAAAIAYGLDK, IKDAVVTVPAYFNDAQR11IX
4,(1,3,2,7)EF2_BETVUElongation factor 2 OS = Beta vulgaris645416.6DLYVK, VASDLPK, GGGQIIPTAR, MIPASDKGR, IRPVLTVNK, EGALAEENMR, NMSVIAHVDHGK, FGVDESKMMER, VFYASQLTAKPR, LWGENFFDPATK, IRPVLTVNKMDR, RVFYASQLTAKPR, GHVFEEMQRPGTPLYNIK, RGHVFEEMQRPGTPLYNIK15V
4,(1,2,3)HSP82_MAIZEHeat shock protein 82 OS = Zea mays542916.1VVVSDR, KLVSATK, APFDLFDTR, AVENSPFLER, LGIHEDSQNR, RAPFDLFDTR, SDLVNNLGTIAR, ELISNASDALDK, KAVENSPFLER, HFSVEGQLEFK, GVVDSDDLPLNISR, ELISNASDALDKIR, HSEFISYPIYLWTEK13IX
6,(2,3)IF4A1_ORYSJEukaryotic initiation factor 4A-1 OS = Oryza sativa subsp. Japonica441831.4ALGDYLGVK, ELAQQIEK, KGVAINFVTR, VLITTDLLAR, QSLRPDYIK, RDELTLEGIK, GLDVIQQAQSGTGK, GIYAYGFEKPSAIQQR, GFKDQIYDIFQLLPSK9V
3,(2,1)CAPPC_FLATRPhosphoenolpyruvate carboxylase 2 OS = Flaveria trinervia3241718GIAAGMQNTG, MNIGSRPSK, VILGDVRDK, KPSGGIESLR, LSAAWQLYK, SPEEVFDALK, RPLFGPDLPK, TPPTPQDEMR, QVSTFGLSLVR, VTIDLVEMVFAK, AGMSYFHETIWK, AIPWIFAWTQTR, VPYNAPLIQFSSWMGGDRDGNPR13I
6,(2)ENO2_ARATHBifunctional enolase 2/transcriptional activator OS = Arabidopsis thaliana239133.3YNQLLR, DGGSDYLGK, ISGDALKDLYK, DGGSDYLGKGVSK, VNQIGSVTESIEAVK, IVLPVPAFNVINGGSHAGNK, SGETEDTFIADLAVGLSTGQIK, YGQDATNVGDEGGFAPNIQENK, KYGQDATNVGDEGGFAPNIQENK, YGQDATNVGDEGGFAPNIQENKEGLELLK10IV
5,(2)CH61_CUCMAChaperonin CPN60-1, mitochondrial OS = Cucurbita maxima638733.33ISSINAVVK, VTDALNATK, VTKDGVTVAK, KISSINAVVK, IGGASEAEVGEK, IGVQIIQNALK, IGGASEAEVGEKK, GYISPYFITNQK, AAVEEGIVPGGGVALLYASK, TPVHTIASNAGVEGAVVVGK10IX
4HSP7L_ARATHHeat shock 70 kDa protein 12 OS = Arabidopsis thaliana138216.8NTVIPTKK, IMEYFIK, ALSSQHQVR, EAEEFAEEDKK, ARFEELNNDLFR, ARFEELNNDLFR, IINEPTAAAIAYGLDK, IKDAVVTVPAYFNDAQR8IX
2,(1)CLAH1_ARATHClathrin heavy chain 1 OS = Arabidopsis thaliana437410.3TVDNDLALK, SPEQVSAAVK, VANVELYYK, DPTLAVVAYR, FQELFAQTK, VEEDAVWSQVAK, GNLPGAENLVVQR, EGLVSDAIESFIR, GNMQLFSVDQQR, KNLLENWLAEDK, RGNLPGAENLVVQR, QLIDQVVSTALPESK, YKEAAELAAESPQGILR13VI
6,(2,5,1)ATPAM_PEAATP synthase subunit alpha, mitochondrial OS = Pisum sativum136429.4VVSVGDGIAR, TGSIVDVPAGK, AAELTTLLESR, VVDALGVPIDGR, TAIAIDTILNQK, KSVHEPMQTGLK, GIRPAINVGLSVSR, EAFPGDVFYLHSR, ITNFYTNFQVDEIGR, LTEVLKQPQYAPLPIEK, EVAAFAQFGSDLDAATQALLNR11I
5RUBB_PEARuBisCO large subunit-binding protein subunit beta, chloroplastic OS = Pisum sativum235723.98IAALK, VVLTK, NVVLESK, VEDALNATK, IVNDGVTVAK, KGVVTLEEGK, LADLVGVTLGPK, GYISPYFVTDSEK, EVELEDPVENIGAK, TNDLAGDGTTTSVVLAQGLIAEGVK, IVNDGVTVAKEVELEDPVENIGAK11I
5,(2)PGMC_PEAPhosphoglucomutase, cytoplasmic OS = Pisum sativum533341.16YLFEDGSR, FFEVPTGWK, LSGTGSEGATIR, SMPTSAALDVVAK, YDYENVDAGAAK5II
6,(2)SAHH_MESCRAdenosylhomocysteinase OS = Mesembryanthemum crystallinum633011.1ATDVMIAGK, HSLPDGLMR, ITIKPQTDR, TEFGPSQPFK, LVGVSEETTTGVK, TEFGPSQPFKGAK, LVGVSEETTTGVKR7I
7PGKY_TOBACPhosphoglycerate kinase, cytosolic OS = Nicotiana tabacum730221.7LAELSGK, YSLKPLVPR, YLKPAVAGFLMQK, GVSLLLPTDVVIADK, GVTTIIGGGDSVAAVEK, LASLADLYVNDAFGTAHR, KLASLADLYVNDAFGTAHR7II
4SUSY_SOYBNSucrose synthase OS = Glycine max1229513.4YLEMFYALK, VVHGIDVFDPK, NITGLVEWYGK, ELVNLVVVAGDR, LLPDAVGTTCGQR, SGFHIDPYHGDR, LGVTQCTIAHALEK7II
5CPNB3_ARATHChaperonin 60 subunit beta 3, chloroplastic OS = Arabidopsis thaliana129225.35VVLTK, NVVLESK, VEDALNATK, KGVVTLEEGK, LADLVGVTLGPK, GYISPYFVTDSEK, EVELEDPVENIGAK, TNDLAGDGTTTSVVLAQGLIAEGVK8IX
7,(8)MDHC2_ARATHMalate dehydrogenase, cytoplasmic 2 OS = Arabidopsis thaliana228823.2GAAIIK, NVSIYK, SQASALEK, EFAPSIPEK, MELVDAAFPLLK, VLVVANPANTNALILK, VLVTGAAGQIGYALVPMIAR7II
8,(1)1433E_TOBAC14-3-3-like protein E OS = Nicotiana tabacum1928429.8NVIGAR, VFYLK, YLAEFK, MKGDYHR, NLLSVAYK, IISSIEQK, TVDVEELTVEER, IISSIEQKEESR, SAQDIALAELAPTHPIR9VIII
5VATA_GOSHIV-type proton ATPase catalytic subunit A OS = Gossypium hirsutum227249.1SGDVYIPR, TVISQALSK, LAADTPLLTGQR, LAEMPADSGYPAYLAAR, LTTFEDSEKESEYGYVR, LVSQKFEDPAEGEEALVAK6VI
4,(3,2,1)CD48A_ARATHCell division control protein 48 homolog A OS = Arabidopsis thaliana326518.5TLLAK, KGDLFLVR, RSVSDADIR, DFSTAILER, LAEDVDLER, GILLYGPPGSGK, LAGESESNLRK, IVSQLLTLMDGLK, ELVELPLRHPQLFK, NAPSIIFIDEIDSIAPK10III/IV
7ALF_CICARFructose-bisphosphate aldolase, cytoplasmic isozyme OS = Cicer arietinum124511.1GILAADESTGTIGK, GILAADESTGTIGKR, YHDELIANAAYIGTPGK3II
7MDHM_CITLAMalate dehydrogenase, mitochondrial OS = Citrullus lanatus223517.6LFGVTTLDVVR, TQDGGTEVVEAK, DDLFNINAGIVK, RTQDGGTEVVEAK, VAVLGAAGGIGQPLALLMK5II
6,7ACT5_ARATHPutative actin-5 OS = Arabidopsis thaliana121320.4AGFAGDDAPR, IKVVAPPER, IWHHTFYNELR, TTGIVLDSGDGVSHTVPIYEGYALPHAILR4VII
6UGPA_MUSACUTP--glucose-1-phosphate uridylyltransferase OS = Musa acuminata319918VANFLSR, GGTLISYEGR, VLQLETAAGAAIR, FFDHAIGINVPR, LQSAVAELNQISENEK5II
8ADT1_GOSHIADP, ATP carrier protein 1, mitochondrial OS = Gossypium hirsutum21948.8SSLDAFSQILK, LLIQNQDEMIK, YFPTQALNFAFK3VI
8RAN_VICFAGTP-binding nuclear protein Ran/TC4 OS = Vicia faba119326.2NVPTWHR, HLTGEFEK, AKQVTFHR, LVIVGDGGTGK, NLQYYEISAK, SNYNFEKPFLYLAR6VIII
3CLPB1_ARATHChaperone protein ClpB1 OS = Arabidopsis thaliana219211.1TAVVEGLAQR, YRGEFEER, TKNNPVLIGEPGVGK, KVESASGDTNFQALK, VQLDSQPEEIDNLER, LIGAPPGYVGHEEGGQLTEAVR6IX
5CPNA1_ARATHChaperonin 60 subunit alpha 1, chloroplastic OS = Arabidopsis thaliana118982.82VVNDGVTIAR, NVVLDEFGSPK, VGAATETELEDR3IX
3ACOC_CUCMAAconitate hydratase, cytoplasmic OS = Cucurbita maxima61879.2NFEGR, ILLESAIR, STYESITK, DFNSYGSR, RGNDEVMAR, TSLAPGSGVVTK, ATIANMSPEYGATMGFFPVDHVTLQYLK7II
3SYA_ARATHAlanine--tRNA ligase OS = Arabidopsis thaliana11815.4LTSVLQNK, HVDTGMGFER, ESDGSLKPLPAK, AFALLSEEGIAK, AVFGEVYPDPVR5IV
6,(7)PRS6A_SOLLC26S protease regulatory subunit 6A homolog OS = Solanum lycopersicum41708.5IIKEELQR, GVLLYGPPGTGK, LAGPQLVQMFIGDGAK3I
7AATM_LUPANAspartate aminotransferase P2, mitochondrial (Fragment) OS = Lupinus angustifolius117010.8IADVIQEK, NLGLYAER, LNLGVGAYR, ISLAGLSLAK, VATVQGLSGTGSLR5I
6UGDH_SOYBNUDP-glucose 6-dehydrogenase OS = Glycine max11686.5IAILGFAFK, LAANAFLAQR, AADLTYWESAAR3II
2,8ANX4_FRAANAnnexin-like protein RJ4 OS = Fragaria ananassa11639.6VGTDEDALTR, LLVALVTAYR2IX
8RS4_GOSHI40S ribosomal protein S4 OS = Gossypium hirsutum416322.9LSIIEEAR, LGNVFTIGK, GIPYLNTYDGR, LGGAFAPKPSSGPHK, TDKTYPAGFMDVVSIPK5V
7,(8)RSSA_SOYBN40S ribosomal protein SA OS = Glycine max216119LLILTDPR, YVDIGIPANNK, HTPGTFTNQLQTSFSEPR, VIVAIENPQDIIVQSARPYGQR4V
8RAA1D_ARATHRas-related protein RABA1d OS = Arabidopsis thaliana916126.6AITSAYYR, VVLIGDSGVGK, STIGVEFATR, HSTFENVER, AQIWDTAGQER5VIII
8RS18_ARATH40S ribosomal protein S18 OS = Arabidopsis thaliana115523.7LRDDLER, VLNTNVDGK, IPDWFLNR, YSQVVSNALDMK4V
1AVP_VIGRRPyrophosphate-energized vacuolar membrane proton pump OS = Vigna radiata var. Radiata11519.5TDALDAAGNTTAAIGK, AAVIGDTIGDPLKDTSGPSLNILIK2VI
5ILV5_ARATHKetol-acid reductoisomerase, chloroplastic OS = Arabidopsis thaliana115027.45SDIVVK, SVVLAGR, QIGVIGWGSQGPAQAQNLR3I
7AATC_DAUCAAspartate aminotransferase, cytoplasmic OS = Daucus carota214910.4ISMAGLSSR, LNLGVGAYR, LIFGADSPAIQENR3I
8RL13_TOBAC60S ribosomal protein L13 OS = Nicotiana tabacum114923.3GFSLEELK, TWFNQPAR, SLEGLQTNVQR, KLAPTIGIAVDHR4V
5ACLB1_ORYSJATP-citrate synthase beta chain protein 1 OS = Oryza sativa subsp. Japonica114835.6FNNIPQVK, FGGAIDDAAR, SEVQFGHAGAK, SIGLIGHTFDQKR, VVAIIAEGVPESDTK5I
2,(3)COPA1_ARATHCoatomer subunit alpha-1 OS = Arabidopsis thaliana31474.4VWDIGALR, YVLEGHDR, AWEVDTLR, VVIFDLQQR, TLDVPIYITK, QDIIVSNSEDK6VI
6CATA2_RICCOCatalase isozyme 2 OS = Ricinus communis21469.3FSTVIHER, APGVQTPVIVR, EGNFDIVGNNFPVFFIR3IX
8RAN3_ORYSIGTP-binding nuclear protein Ran-3 OS = Oryza sativa subsp. Indica114514.2HITGEFEK, NLQYYEISAK, SNYNFEKPFLYLAR3III/VI
6MDAR_SOLLCMonodehydroascorbate reductase OS = Solanum lycopersicum113912.5AYLFPEGAAR, LSDFGVQGADSK, IVGAFLESGSPEENKAIAK3IX
6,(1)RL3_ORYSJ60S ribosomal protein L3 OS = Oryza sativa subsp. Japonica41389.3VIAHTQIR, HGSLGFLPR, LALEEIKLK, GKGYEGVVTR4V
5PMGI_RICCO2,3-bisphosphoglycerate-independent phosphoglycerate mutase OS = Ricinus communis413643.5ARDAILSGK, LVDLALASGK, LDQLQLLLK, AHGTAVGLPTEDDMGNSEVGHNALGAGR4II
5RUBA_RICCORuBisCO large subunit-binding protein subunit alpha (Fragment) OS = Ricinus communis213471.19NVVLDEFGSPK, VGAATETELEDR, LGLLSVTSGANPVSIK3I
7RL4_PRUAR60S ribosomal protein L4 OS = Prunus armeniaca113414.5AGHQTSAESWGTGR, YAVVSAIAASAVPSLVLAR2V
5G6PI_SPIOLGlucose-6-phosphate isomerase, cytosolic OS = Spinacia oleracea213127SQQPVYLK, FLANVDPIDVAK, TFTTAETMLNAR3II
8RS8_MAIZE40S ribosomal protein S8 OS = Zea mays113021.7LDTGNYSWGSEAVTR, ILDVVYNASNNELVR2V
8RL11_MEDSA60S ribosomal protein L11 OS = Medicago sativa112917.7YEGVILNK, AMQLLESGLK, VLEQLSGQTPVFSK3V
8,(7)H4_ARATHHistone H4 OS = Arabidopsis thaliana212846.6TLYGFGG, IFLENVIR, DAVTYTEHAR, ISGLIYEETR4VII
5TCPE_ARATHT-complex protein 1 subunit epsilon OS = Arabidopsis thaliana112373.41IAEGYEMASR, QQQILLATQVVK2V
5TCPA_ARATHT-complex protein 1 subunit alpha OS = Arabidopsis thaliana111971.23YFVEAGAIAVR, NKIHPTSIISGYR2V
4TKTC_SPIOLTransketolase, chloroplastic OS = Spinacia oleracea41186.1FLAIDAVEK, ALPTYTPETPGDATR, VIPGLLGGSADLASSNMTLLK3II
8TPIS_MAIZETriosephosphate isomerase, cytosolic OS = Zea mays111711.9FFVGGNWK, VAYALSQGLK, VIACVGETLEQR3VII
8PROF3_ARATHProfilin-3 OS = Arabidopsis thaliana211717.2LGDYLLEQGL, YMVIQGEPGAVIR2VII
8RS92_ARATH40S ribosomal protein S9-2 OS = Arabidopsis thaliana111519.3LVGEYGLR, ERLDAELK, RPYEKER, RLQTIVFK, IFEGEALLR5V
6VATB1_ARATHV-type proton ATPase subunit B1 OS = Arabidopsis thaliana111510.5YQEIVNIR, TVSGVAGPLVILDK, QIYPPINVLPSLSR3VI
6ERF1X_ARATHEukaryotic peptide chain release factor subunit 1-1 OS = Arabidopsis thaliana11139.9GFGGIGGILR, QSVLGAITSAQQR2V
5HSP7M_PHAVUHeat shock 70 kDa protein, mitochondrial OS = Phaseolus vulgaris311124.87HLNITLTR, VIENSEGAR, TTPSVVAFNQK, SSGGLSEDEIEK4IX
8ANXD1_ARATHAnnexin D1 OS = Arabidopsis thaliana11105AQINATFNR, SKAQINATFNR2IX
8RS16_FRIAG40S ribosomal protein S16 OS = Fritillaria agrestis310913.8ALVAYYQK, AFEPILLLGR, YKAFEPILLLGR3V
8RS5_CICAR40S ribosomal protein S5 (Fragment) OS = Cicer arietinum110815.7IGSAGVVRR, GSSNSYAIK, VNQAIYLLTTGAR3V
8ARF_VIGUNADP-ribosylation factor OS = Vigna unguiculata110428.7ILMVGLDAAGK, NISFTVWDVGGQDK2VIII
4SYGM1_ARATHGlycine--tRNA ligase 1, mitochondrial OS = Arabidopsis thaliana11035.1LFYIPSFK, VFTPSVIEPSFGIGR2IV/VI
7RGP1_ORYSJUDP-arabinopyranose mutase 1 OS = Oryza sativa subsp. Japonica11016.9ILGPK, ASNPFVNLK, ASNPFVNLKK, YVDAVMTVPK4I
3HSP7O_ARATHHeat shock 70 kDa protein 14 OS = Arabidopsis thaliana11015.1ILSHAFDR, NAVESYVYDMR, AVLDAATIAGLHPLR3IX
8RS15A_DAUCA40S ribosomal protein S15a OS = Daucus carota110029.2VSVLNDALK, HGYIGEFEYVDDHR2V
8RS61_ARATH40S ribosomal protein S6-1 OS = Arabidopsis thaliana210018LVTPLTLQR, KGENDLPGLTDTEKPR, ISQEVSGDALGEEFKGYVFK3V
6ACCC2_POPTRBiotin carboxylase 2, chloroplastic OS = Populus trichocarpa1977.8LLEEAPSPALTPELR, ALDDTVITGVPTTIDYHK2I
8RLA2_PARAR60S acidic ribosomal protein P2 OS = Parthenium argentatum19710.5DITELIASGR, GKDITELIASGR2V
8RS33_ARATH40S ribosomal protein S3-3 OS = Arabidopsis thaliana39610.9ELAEDGYSGVEVR, FKFPQDSVELYAEK2V
5KPYC_SOYBNPyruvate kinase, cytosolic isozyme OS = Glycine max19455.66KGSDLVNVR, STPLPMSPLESLASSAVR2II
7GMD1_ARATHGDP-mannose 4,6 dehydratase 1 OS = Arabidopsis thaliana2935RGENFVTR, LFLGNIQASR2II
4HSP7S_SPIOLStromal 70 kDa heat shock-related protein, chloroplastic (Fragment) OS = Spinacia oleracea2935.3HIETTLTR, IINEPTAASLAYGFEK2IX
6EF1G2_ORYSJElongation factor 1-gamma 2 OS = Oryza sativa subsp. Japonica39316.7EVAIK, LYSNTK, NPLDLLPPSK, MILDEWKR, SFTSEFPHVER5V
8RLA0_LUPLU60S acidic ribosomal protein P0 OS = Lupinus luteus1909.9EYLKDPSK, VGSSEAALLAK2V
7GCST_PEAAminomethyltransferase, mitochondrial OS = Pisum sativum1898.3GGAIDDSVITK, TGYTGEDGFEISVPSEHGVELAK2I
8APX1_ORYSJL-ascorbate peroxidase 1, cytosolic OS = Oryza sativa subsp. Japonica18915.2TGGPFGTMK, LSELGFADA, ALLSDPAFRPLVEK3IX
8RS193_ARATH40S ribosomal protein S19-3 OS = Arabidopsis thaliana18823.1AYAAHLKR, TVKDVSPHEFVK, ELAPYDPDWYYIR3V
1,(2)RPN1A_ARATH26S proteasome non-ATPase regulatory subunit 2 1A OS = Arabidopsis thaliana3875.5VGQAVDVVGQAGRPK, NLAGEIAQEYTKR2I
4,(3)PPDK_FLABRPyruvate, phosphate dikinase, chloroplastic OS = Flaveria brownii8872.9SDFEGIFR, AALIADEIAK, AMDGLPVTIR3II
8RS13_PEA40S ribosomal protein S13 OS = Pisum sativum18725.8DSHGIAQVK, GLTPSQIGVILR, KGLTPSQIGVILR, AHGLAPEIPEDLYHLIK4V
8RS14_CHLRE40S ribosomal protein S14 OS = Chlamydomonas reinhardtii18518.3TPGPGAQSALR, IEDVTPIPTDSTRR2V
6VATB2_GOSHIV-type proton ATPase subunit B 2 (Fragment) OS = Gossypium hirsutum18410.1FVTQGAYDTR, QIYPPINVLPSLSR2VI
8,(5)CYPH_MAIZEPeptidyl-prolyl cis-trans isomerase OS = Zea mays38316.3SGKPLHYK, VFFDMTVGGAPAGR2V
8NDK1_ARATHNucleoside diphosphate kinase 1 OS = Arabidopsis thaliana1829.4NVIHGSDSVESAR, NVIHGSDSVESARK2I
7GLN11_ORYSJGlutamine synthetase cytosolic isozyme 1-1 OS = Oryza sativa subsp. Japonica1797.6DIVDSHYK, HKEHISAYGEGNER2I
7SERC_SPIOLPhosphoserine aminotransferase, chloroplastic OS = Spinacia oleracea1795.3FGLIYAGAQK, NVGPSGVTIVIVR2I
7PDI21_ARATHProtein disulfide-isomerase like 2-1 OS = Arabidopsis thaliana1788.9KLAPEYEK, YGVSGFPTLK, YGVSGYPTIQWFPK3V
8RS254_ARATH40S ribosomal protein S25-4 OS = Arabidopsis thaliana27629.6LITPSILSDR, MVAAHSSQQIYTR2V
6IDHC_TOBACIsocitrate dehydrogenase [NADP] OS = Nicotiana tabacum2757.5HAFGDQYR, DLALIIHGSK, TIEAEAAHGTVTR3II
6OPD22_ARATHDihydrolipoyllysine-residue acetyltransferase component 2 of pyruvate dehydrogenase complex, mitochondrial OS = Arabidopsis thaliana1733.9ISVNDLVIK, VIDGAIGAEWLK2II
8RL40A_ARATHUbiquitin-60S ribosomal protein L40-1 OS = Arabidopsis thaliana17045.3ESTLHLVLR, TITLEVESSDTIDNVK2V
8RL24_PRUAV60S ribosomal protein L24 OS = Prunus avium1697SIVGATLEVIQK, SIVGATLEVIQKR2V
7,(5)SAPK6_ORYSJSerine/threonine-protein kinase SAPK6 OS = Oryza sativa subsp. Japonica2687.4DIGSGNFGVAR, STVGTPAYIAPEVLSR2III
6GME2_ORYSJGDP-mannose 3,5-epimerase 2 OS = Oryza sativa subsp. Japonica1677NSDNTLIKEK, ISITGAGGFIASHIAR2II
8EF1D1_ORYSJElongation factor 1-delta 1 OS = Oryza sativa subsp. Japonica2667.9LVPVGYGIK, KLDEYLLTR2V
8IF5A1_ARATHEukaryotic translation initiation factor 5A-1 OS = Arabidopsis thaliana16412VVEVSTSK, TYPQQAGTIR, TYPQQAGTIRK3V
8H2B11_ARATHHistone H2B.11 OS = Arabidopsis thaliana16230LVLPGELAK, QVHPDIGISSK, YNKKPTITSR3VII
8PSA3_ARATHProteasome subunit alpha type-3 OS = Arabidopsis thaliana1607.6VFQIEYAAK, VPDDLLEEAK2I
7AAT3_ARATHAspartate aminotransferase, chloroplastic OS = Arabidopsis thaliana1604.9LNLGVGAYR, TEEGKPLVLNVVR2I
1PDR4_ORYSJPleiotropic drug resistance protein 4 OS = Oryza sativa subsp. Japonica1601.5TTLLLALAGK, VTTGEMLVGPAR2IX
2,(8)UBQ12_ARATHPolyubiquitin 12 OS = Arabidopsis thaliana36023.9MQIFLKTLTGK, IQDKEGIPPDQQR, TITLEVESSDTIDNVK3V
2,(1)UBIQ_AVESAUbiquitin OS = Avena sativa16057.9TLADYNIQK, IQDKEGIPPDQQR, TITLEVESSDTIDNVK3V
5,(1)DIM_PEADelta(24)-sterol reductase OS = Pisum sativum25945.72NILDIDKER, SDLEAPLRPK2I
8HSP11_PEA18.1 kDa class I heat shock protein OS = Pisum sativum2568.9SIEISG, VLQISGER2IX
6MPPA_SOLTUMitochondrial-processing peptidase subunit alpha OS = Solanum tuberosum1523.4QLLTYGER, MVASEDIGR2I
8RL17_MAIZE60S ribosomal protein L17 OS = Zea mays15210.5NAESNADVK, YLEDVIAHK2V
8RL51_ARATH60S ribosomal protein L5-1 OS = Arabidopsis thaliana2529.3VFGALK, KLTYEER, GALDGGLDIPHSDKR3V
3PHSL1_SOLTUAlpha-1,4 glucan phosphorylase L-1 isozyme, chloroplastic/amyloplastic OS = Solanum tuberosum1501.8NDVSYPIK, AFATYVQAK2II
6PRS4A_ARATH26S proteasome regulatory subunit 4 homolog A OS = Arabidopsis thaliana1499.5VVGSELIQK, GVILYGEPGTGK2II
8YPTC1_CHLREGTP-binding protein YPTC1 OS = Chlamydomonas reinhardtii24917.2TITSSYYR, LLLIGDSGVGK2III/VI
4HSP7G_ARATHHeat shock 70 kDa protein 7, chloroplastic OS = Arabidopsis thaliana1479.5HIETTLTR, TTPSVVAYTK, QAVVNPENTFFSVKR3IX
8SODM_HEVBRSuperoxide dismutase [Mn], mitochondrial OS = Hevea brasiliensis14611.2HHQTYITNYNK, LVVETTANQDPLVTK2IX
5CALX2_ARATHCalnexin homolog 2 OS = Arabidopsis thaliana24530.3NPAYK, SEGHDDYGLLVSEK2V
8RS30_ARATH40S ribosomal protein S30 OS = Arabidopsis thaliana14430.6GKVHGSLAR, FVTAVVGFGK2V
8RL7A1_ARATH60S ribosomal protein L7a-1 OS = Arabidopsis thaliana1449.3TLDKNLATSLFK, LKVPPALNQFTK2V
7METK4_POPTRS-adenosylmethionine synthase 4 OS = Populus trichocarpa14212.3FVIGGPHGDAGLTGR, VLVNIEQQSPDIAQGVHGHLTK2I
8RL18A_CASSA60S ribosomal protein L18a OS = Castanea sativa1419.6ASRPNLFM, FHQYQVVGR2V
7EFTM_ARATHElongation factor Tu, mitochondrial OS = Arabidopsis thaliana1419.7QAILK, VLAEEGKAK, GITIATAHVEYETAKR3V
1CALSB_ARATHCallose synthase 11 OS = Arabidopsis thaliana1381.5ILFNEAFSR, LGEGKPENQNHALIFTR2IX
3APBLB_ARATHBeta-adaptin-like protein B OS = Arabidopsis thaliana1272EAENIVER, DSQDPNPLIR2VII
1 Fraction corresponding to slice of the 1-D gel in which matches for the protein were found. Numbers in parenthesis indicate fractions where additional similar matches (see 2) were found. 2 Number of protein matches of high taxonomical and sequence similarity grouped together with this match. (Match displayed was the top-scored one.) 3 MASCOT score. 4 I: metabolism, II: energy, III: cell growth/division, IV: transcription, V: protein synthesis/destination, VI: transporters, VII: cell structure, VIII: signal transduction, IX: disease/stress defense, and X: unclassified

3.4. Comparative Analysis of Lotus Seed (Immature Endosperm, Mature Endosperm, and Embryo) Proteins

As is to be expected, there were many proteins in common found among the immature endosperm and embryo tissues, as well as with the mature endosperm previously analyzed [17]. Amongst all three seed tissues, a total of 206 nr proteins were identified against the plant database (Figure 3). Of these, 31 (15%) were common to all three tissues, 40 (19%) were unique to the immature endosperm, and 65 (32%) were unique to the embryo; only 14 (7%) were exclusively found in the mature endosperm. To note, the larger share of embryo-only proteins is a consequence of the embryo tissue being much more involved in plant metabolism, and therefore is expected to express a larger number of functional proteins than the endosperm, which, especially in its mature phase, has nutrient storage as its primary function. The immature endosperm, as a developing tissue, also expresses a larger number of proteins than its mature form, and also shares a significant number of proteins with the embryo—35 (17%) of the identified ones. Common proteins between mature and immature endosperm only amounted to 5% of the identified ones (same as for between the mature endosperm and embryo). Although, considering that both immature endosperm and immature embryo are much softer and with a higher water content than their mature stages, there is a possibility that some of the proteins in common with the embryo identified in the immature endosperm might have originated from the embryo and diffused through the endosperm, despite the care taken to remove embryo fragments and the endosperm immediately around them in the sample preparation.
Figure 3. Venn diagram displaying distribution of non-redundant (nr) proteins amongst lotus seed immature endosperm (ImE), mature endosperm (MtE), and embryo (Emb) (a); Listing of the total and nr protein matches found for each lotus seed tissue analyzed (b); * see reference [17].
Figure 3. Venn diagram displaying distribution of non-redundant (nr) proteins amongst lotus seed immature endosperm (ImE), mature endosperm (MtE), and embryo (Emb) (a); Listing of the total and nr protein matches found for each lotus seed tissue analyzed (b); * see reference [17].
Proteomes 03 00184 g003

3.5. Functional Significance of the Identified Seed Proteins

Gene ontology data (biological processes, molecular functions and cellular localization) for all identified proteins were obtained from the UniProtKB database, using the EMBL-EBI (www.ebi.ac.uk) search tool (Table 3).
Table 3. List of all 206 non-redundant (nr) proteins found across the three tissues of the lotus seed (embryo, immature endosperm and mature endosperm).
Table 3. List of all 206 non-redundant (nr) proteins found across the three tissues of the lotus seed (embryo, immature endosperm and mature endosperm).
Protein AccessionProtein DescriptionTissues 1
1433E_TOBAC14-3-3-like protein E OS = Nicotiana tabacumM/I/E
HSP14_SOYBN17.5 kDa class I heat shock protein OS = Glycine maxI
HSP11_SOLLC17.8 kDa class I heat shock protein OS = Solanum lycopersicumM
HSP11_PEA18.1 kDa class I heat shock protein OS = Pisum sativumM/I/E
HSP12_MEDSA18.2 kDa class I heat shock protein OS = Medicago sativaM/I
HSP11_CHERU18.3 kDa class I heat shock protein OS = Chenopodium rubrumI
PMG1_ARATH2,3-bisphosphoglycerate-independent phosphoglycerate mutase 1 OS = Arabidopsis thalianaM/I/E
PRS6A_SOLLC26S protease regulatory subunit 6A homolog OS = Solanum lycopersicumE
RPN1A_ARATH26S proteasome non-ATPase regulatory subunit 2 1A OS = Arabidopsis thalianaE
PRS4A_ARATH26S proteasome regulatory subunit 4 homolog A OS = Arabidopsis thalianaE
BAS1_ORYSJ2-Cys peroxiredoxin BAS1, chloroplastic OS = Oryza sativa subsp. JaponicaI
RS102_ARATH40S ribosomal protein S10-2 OS = Arabidopsis thalianaI
RS13_PEA40S ribosomal protein S13 OS = Pisum sativumI/E
RS14_CHLRE40S ribosomal protein S14 OS = Chlamydomonas reinhardtiiI/E
RS15A_DAUCA40S ribosomal protein S15a OS = Daucus carotaE
RS16_FRIAG40S ribosomal protein S16 OS = Fritillaria agrestisI/E
RS18_ARATH40S ribosomal protein S18 OS = Arabidopsis thalianaI/E
RS193_ARATH40S ribosomal protein S19-3 OS = Arabidopsis thalianaI/E
RS254_ARATH40S ribosomal protein S25-4 OS = Arabidopsis thalianaE
RS30_ARATH40S ribosomal protein S30 OS = Arabidopsis thalianaE
RS33_ARATH40S ribosomal protein S3-3 OS = Arabidopsis thalianaE
RS3A1_VITVI40S ribosomal protein S3a-1 OS = Vitis viniferaI
RS4_GOSHI40S ribosomal protein S4 OS = Gossypium hirsutumI/E
RS5_CICAR40S ribosomal protein S5 (fragment) OS = Cicer arietinumI/E
RS6_ASPOF40S ribosomal protein S6 OS = Asparagus officinalisI
RS61_ARATH40S ribosomal protein S6-1 OS = Arabidopsis thalianaE
RS8_MAIZE40S ribosomal protein S8 OS = Zea maysE
RS91_ARATH40S ribosomal protein S9-1 OS = Arabidopsis thalianaM
RS92_ARATH40S ribosomal protein S9-2 OS = Arabidopsis thalianaE
RSSA_SOYBN40S ribosomal protein SA OS = Glycine maxI/E
METE_ARATH5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase OS = Arabidopsis thalianaM/I/E
RLA0_LUPLU60S acidic ribosomal protein P0 OS = Lupinus luteusI/E
RLA2_PARAR60S acidic ribosomal protein P2 OS = Parthenium argentatumE
RL10_VITRI60S ribosomal protein L10 OS = Vitis ripariaI
RL11_MEDSA60S ribosomal protein L11 OS = Medicago sativaE
RL12_PRUAR60S ribosomal protein L12 OS = Prunus armeniacaI
RL13_TOBAC60S ribosomal protein L13 OS = Nicotiana tabacumI/E
RL17_MAIZE60S ribosomal protein L17 OS = Zea maysE
RL182_ARATH60S ribosomal protein L18-2 OS = Arabidopsis thalianaI
RL18A_CASSA60S ribosomal protein L18a OS = Castanea sativaE
RL24_PRUAV60S ribosomal protein L24 OS = Prunus aviumE
RL3_ORYSJ60S ribosomal protein L3 OS = Oryza sativa subsp. JaponicaI/E
RL4_PRUAR60S ribosomal protein L4 OS = Prunus armeniacaI/E
RL51_ARATH60S ribosomal protein L5-1 OS = Arabidopsis thalianaI/E
RL6_MESCR60S ribosomal protein L6 OS = Mesembryanthemum crystallinumI
RL7A1_ARATH60S ribosomal protein L7a-1 OS = Arabidopsis thalianaE
ACOC_CUCMAAconitate hydratase, cytoplasmic OS = Cucurbita maximaM/E
ACT_GOSHIActin OS = Gossypium hirsutumI/E
ACT1_ORYSIActin-1 OS = Oryza sativa subsp. IndicaM
ACT12_SOLTUActin-100 (fragment) OS = Solanum tuberosumM/E
ACT1_SOLLCActin-41 (fragment) OS = Solanum lycopersicumM
ACT7_ARATHActin-7 OS = Arabidopsis thalianaM
SAHH_MEDSAAdenosylhomocysteinase OS = Medicago sativaM/I/E
ADT1_GOSHIADP, ATP carrier protein 1, mitochondrial OS = Gossypium hirsutumM/I/E
ARF_VIGUNADP-ribosylation factor OS = Vigna unguiculataE
SYA_ARATHAlanine--tRNA ligase OS = Arabidopsis thalianaE
ADH1_SOLTUAlcohol dehydrogenase 1 OS = Solanum tuberosumM/I
PHSL_IPOBAAlpha-1,4 glucan phosphorylase L isozyme, chloroplastic/amyloplastic OS = Ipomoea batatasM/E
PHSH_ARATHAlpha-glucan phosphorylase, H isozyme OS = Arabidopsis thalianaM/I
GCST_PEAAminomethyltransferase, mitochondrial OS = Pisum sativumI/E
ANXD1_ARATHAnnexin D1 OS = Arabidopsis thalianaM/I/E
ANX4_FRAANAnnexin-like protein RJ4 OS = Fragaria ananassaE
CYF_AETCOApocytochrome f OS = Aethionema cordifoliumI
AATM_LUPANAspartate aminotransferase P2, mitochondrial (fragment) OS = Lupinus angustifoliusE
AATM_LUPANAspartate aminotransferase P2, mitochondrial (fragment) OS = Lupinus angustifoliusI
AAT3_ARATHAspartate aminotransferase, chloroplastic OS = Arabidopsis thalianaI/E
AATC_DAUCAAspartate aminotransferase, cytoplasmic OS = Daucus carotaE
PYRB_ARATHAspartate carbamoyltransferase, chloroplastic OS = Arabidopsis thalianaI
ATPAM_HELANATP synthase subunit alpha, mitochondrial OS = Helianthus annuusM/I/E
ATPBM_NICPLATP synthase subunit beta, mitochondrial OS = Nicotiana plumbaginifoliaM/I/E
ACLB1_ORYSJATP-citrate synthase beta chain protein 1 OS = Oryza sativa subsp. JaponicaE
CLPA_BRANAATP-dependent Clp protease ATP-binding subunit clpA homolog, chloroplastic (fragment) OS = Brassica napusI
APBLB_ARATHBeta-adaptin-like protein B OS = Arabidopsis thalianaE
ENO2_ARATHBifunctional enolase 2/transcriptional activator OS = Arabidopsis thalianaI/E
ACCC2_POPTRBiotin carboxylase 2, chloroplastic OS = Populus trichocarpaE
CALSB_ARATHCallose synthase 11 OS = Arabidopsis thalianaE
CALX2_ARATHCalnexin homolog 2 OS = Arabidopsis thalianaE
CALR_BERSTCalreticulin OS = Berberis stoloniferaI
CATA2_RICCOCatalase isozyme 2 OS = Ricinus communisE
CD48A_ARATHCell division control protein 48 homolog A OS = Arabidopsis thalianaM/I/E
CLPB1_ARATHChaperone protein ClpB1 OS = Arabidopsis thalianaE
CLPC1_ARATHChaperone protein ClpC1, chloroplastic OS = Arabidopsis thalianaI
CPNA1_ARATHChaperonin 60 subunit alpha 1, chloroplastic OS = Arabidopsis thalianaI/E
CPNB3_ARATHChaperonin 60 subunit beta 3, chloroplastic OS = Arabidopsis thalianaE
CH60A_ARATHChaperonin CPN60, mitochondrial OS = Arabidopsis thalianaM/I/E
CB2_PHYPAChlorophyll a-b binding protein, chloroplastic OS = Physcomitrella patens subsp. patensI
HSP7E_SPIOLChloroplast envelope membrane 70 kDa heat shock-related protein OS = Spinacia oleraceaM/I/E
HSP12_SOYBNClass I heat shock protein (fragment) OS = Glycine maxI
CLAH1_ARATHClathrin heavy chain 1 OS = Arabidopsis thalianaI/E
COPA1_ARATHCoatomer subunit alpha-1 OS = Arabidopsis thalianaE
COB21_ORYSJCoatomer subunit beta-1 OS = Oryza sativa subsp. JaponicaI
RH2_ORYSJDEAD-box ATP-dependent RNA helicase 2 OS = Oryza sativa subsp. JaponicaM
DIM_PEADelta(24)-sterol reductase OS = Pisum sativumE
DLDH2_ARATHDihydrolipoyl dehydrogenase 2, mitochondrial OS = Arabidopsis thalianaI
OPD22_ARATHDihydrolipoyllysine-residue acetyltransferase component 2 of pyruvate dehydrogenase complex, mitochondrial OS = Arabidopsis thalianaE
EF1A_TOBACElongation factor 1-alpha OS = Nicotiana tabacumM/I/E
EF1D1_ORYSJElongation factor 1-delta 1 OS = Oryza sativa subsp. JaponicaE
EF1G2_ORYSJElongation factor 1-gamma 2 OS = Oryza sativa subsp. JaponicaM/I/E
EF2_BETVUElongation factor 2 OS = Beta vulgarisM/I/E
EFTM_ARATHElongation factor Tu, mitochondrial OS = Arabidopsis thalianaE
ENPL_CATROEndoplasmin homolog OS = Catharanthus roseusM/I/E
ENO1_HEVBREnolase 1 OS = Hevea brasiliensisM/I/E
IF4A1_ARATHEukaryotic initiation factor 4A-1 OS = Arabidopsis thalianaM/I/E
ERF1X_ARATHEukaryotic peptide chain release factor subunit 1-1 OS = Arabidopsis thalianaE
IF5A1_ARATHEukaryotic translation initiation factor 5A-1 OS = Arabidopsis thalianaE
ALF_CICARFructose-bisphosphate aldolase, cytoplasmic isozyme OS = Cicer arietinumM/I/E
RFS_ORYSJGalactinol--sucrose galactosyltransferase OS = Oryza sativa subsp. JaponicaI
GME2_ORYSJGDP-mannose 3,5-epimerase 2 OS = Oryza sativa subsp. JaponicaE
GMD1_ARATHGDP-mannose 4,6 dehydratase 1 OS = Arabidopsis thalianaE
GRDH1_ARATHGlucose and ribitol dehydrogenase homolog 1 OS = Arabidopsis thalianaM/I
GLGS_BETVUGlucose-1-phosphate adenylyltransferase small subunit, chloroplastic/amyloplastic (fragment) OS = Beta vulgarisM
G6PI2_CLACOGlucose-6-phosphate isomerase, cytosolic 2 OS = Clarkia concinnaM/E
GPT2_ARATHGlucose-6-phosphate/phosphate translocator 2, chloroplastic OS = Arabidopsis thalianaM
GLN11_ORYSJGlutamine synthetase cytosolic isozyme 1-1 OS = Oryza sativa subsp. JaponicaE
G3PC_ANTMAGlyceraldehyde-3-phosphate dehydrogenase, cytosolic OS = Antirrhinum majusM/I/E
SYGM1_ARATHGlycine--tRNA ligase 1, mitochondrial OS = Arabidopsis thalianaE
SSG1_HORVUGranule-bound starch synthase 1, chloroplastic/amyloplastic OS = Hordeum vulgareM/I
RAN_VICFAGTP-binding nuclear protein Ran/TC4 OS = Vicia fabaM/I/E
RAN3_ORYSIGTP-binding nuclear protein Ran-3 OS = Oryza sativa subsp. IndicaE
YPTC1_CHLREGTP-binding protein YPTC1 OS = Chlamydomonas reinhardtiiE
GBLPA_ORYSJGuanine nucleotide-binding protein subunit beta-like protein A OS = Oryza sativa subsp. JaponicaI
HSP7L_ARATHHeat shock 70 kDa protein 12 OS = Arabidopsis thalianaI/E
HSP7O_ARATHHeat shock 70 kDa protein 14 OS = Arabidopsis thalianaI/E
HSP7N_ARATHHeat shock 70 kDa protein 18 OS = Arabidopsis thalianaI
HSP7D_ARATHHeat shock 70 kDa protein 4 OS = Arabidopsis thalianaI
HSP7F_ARATHHeat shock 70 kDa protein 6, chloroplastic OS = Arabidopsis thalianaI
HSP7G_ARATHHeat shock 70 kDa protein 7, chloroplastic OS = Arabidopsis thalianaE
HSP70_DAUCAHeat shock 70 kDa protein OS = Daucus carotaM/I/E
HSP7M_PHAVUHeat shock 70 kDa protein, mitochondrial OS = Phaseolus vulgarisI/E
HSP80_SOLLCHeat shock cognate protein 80 OS = Solanum lycopersicumM/I
HS101_ARATHHeat shock protein 101 OS = Arabidopsis thalianaM
HS101_ORYSJHeat shock protein 101 OS = Oryza sativa subsp. JaponicaM
HSP81_ORYSIHeat shock protein 81-1 OS = Oryza sativa subsp. IndicaM/I/E
HSP82_TOBACHeat shock protein 82 (fragment) OS = Nicotiana tabacumM
HSP82_MAIZEHeat shock protein 82 OS = Zea maysM/I/E
HSP83_IPONIHeat shock protein 83 OS = Ipomoea nilM/I/E
HS901_ARATHHeat shock protein 90-1 OS = Arabidopsis thalianaE
HS903_ARATHHeat shock protein 90-3 OS = Arabidopsis thalianaI
H2AX_CICARHistone H2AX OS = Cicer arietinumI
H2B_GOSHIHistone H2B OS = Gossypium hirsutumI/E
H4_ARATHHistone H4 OS = Arabidopsis thalianaI/E
IDHC_TOBACIsocitrate dehydrogenase [NADP] OS = Nicotiana tabacumE
ILV5_ARATHKetol-acid reductoisomerase, chloroplastic OS = Arabidopsis thalianaE
APX1_ORYSJL-ascorbate peroxidase 1, cytosolic OS = Oryza sativa subsp. JaponicaE
LE194_HORVULate embryogenesis abundant protein B19.4 OS = Hordeum vulgareI
AMPL1_ARATHLeucine aminopeptidase 1 OS = Arabidopsis thalianaM/I
BIP4_TOBACLuminal-binding protein OS = Nicotiana tabacumM/I/E
MDHC2_ARATHMalate dehydrogenase, cytoplasmic 2 OS = Arabidopsis thalianaM/E
MDHM_CITLAMalate dehydrogenase, mitochondrial OS = Citrullus lanatusM/I/E
MPPA_SOLTUMitochondrial-processing peptidase subunit alpha OS = Solanum tuberosumE
MDAR_SOLLCMonodehydroascorbate reductase OS = Solanum lycopersicumI/E
MAOX_POPTRNADP-dependent malic enzyme OS = Populus trichocarpaM
NDK1_ARATHNucleoside diphosphate kinase 1 OS = Arabidopsis thalianaM/I/E
FKB62_ARATHPeptidyl-prolyl cis-trans isomerase FKBP62 OS = Arabidopsis thalianaI/E
PER1B_ARMRUPeroxidase C1B OS = Armoracia rusticanaI
CAPPC_FLATRPhosphoenolpyruvate carboxylase 2 OS = Flaveria trinerviaE
PGMC_PEAPhosphoglucomutase, cytoplasmic OS = Pisum sativumM/I/E
PGKH_TOBACPhosphoglycerate kinase, chloroplastic OS = Nicotiana tabacumM/I
PGKY_TOBACPhosphoglycerate kinase, cytosolic OS = Nicotiana tabacumM/E
SERC_SPIOLPhosphoserine aminotransferase, chloroplastic OS = Spinacia oleraceaE
PDR4_ORYSJPleiotropic drug resistance protein 4 OS = Oryza sativa subsp. JaponicaE
PARP3_SOYBNPoly [ADP-ribose] polymerase 3 OS = Glycine maxI
UBIQP_ACECLPolyubiquitin (fragment) OS = Acetabularia cliftoniiI
UBQ12_ARATHPolyubiquitin 12 OS = Arabidopsis thalianaE
PMG2_ARATHProbable 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 2 OS = Arabidopsis thalianaM/I
SSG1_ARATHProbable granule-bound starch synthase 1, chloroplastic/amyloplastic OS = Arabidopsis thalianaI
H2B1_MEDTRProbable histone H2B.1 OS = Medicago truncatulaI
PDIA6_MEDSAProbable protein disulfide-isomerase A6 OS = Medicago sativaI
Y1497_ARATHProbable receptor-like protein kinase At1g49730 OS = Arabidopsis thalianaI
PROF3_ARATHProfilin-3 OS = Arabidopsis thalianaE
PSA3_ARATHProteasome subunit alpha type-3 OS = Arabidopsis thalianaE
PDI21_ORYSJProtein disulfide isomerase-like 2-1 OS = Oryza sativa subsp. JaponicaI
PDI21_ARATHProtein disulfide-isomerase like 2-1 OS = Arabidopsis thalianaM/I/E
ACT5_ARATHPutative actin-5 OS = Arabidopsis thalianaI/E
YCF1_IPOPUPutative membrane protein ycf1 OS = Ipomoea purpureaM/I
AVP_VIGRRPyrophosphate-energized vacuolar membrane proton pump OS = Vigna radiata var. radiataI/E
PDC1_TOBACPyruvate decarboxylase isozyme 1 (fragment) OS = Nicotiana tabacumM/I
KPYC_SOYBNPyruvate kinase, cytosolic isozyme OS = Glycine maxM/I/E
PPDK2_ORYSJPyruvate, phosphate dikinase 2 OS = Oryza sativa subsp. JaponicaM
PPDK_FLABRPyruvate, phosphate dikinase, chloroplastic OS = Flaveria browniiM/E
RAA1D_ARATHRas-related protein RABA1d OS = Arabidopsis thalianaE
RBL_MAIZERibulose bisphosphate carboxylase large chain OS = Zea maysI
RUBA_RICCORuBisCO large subunit-binding protein subunit alpha (fragment) OS = Ricinus communisI/E
RUBB_PEARuBisCO large subunit-binding protein subunit beta, chloroplastic OS = Pisum sativumE
METK4_POPTRS-adenosylmethionine synthase 4 OS = Populus trichocarpaE
SAPK6_ORYSJSerine/threonine-protein kinase SAPK6 OS = Oryza sativa subsp. JaponicaE
HSP7S_SPIOLStromal 70 kDa heat shock-related protein, chloroplastic (fragment) OS = Spinacia oleraceaI/E
SUSY_SOYBNSucrose synthase OS = Glycine maxI/E
SODM_HEVBRSuperoxide dismutase [Mn], mitochondrial OS = Hevea brasiliensisE
TCPA_ARATHT-complex protein 1 subunit alpha OS = Arabidopsis thalianaI/E
TCPE_ARATHT-complex protein 1 subunit epsilon OS = Arabidopsis thalianaM/E
TKTC_SPIOLTransketolase, chloroplastic OS = Spinacia oleraceaE
TCTP_TOBACTranslationally-controlled tumor protein homolog OS = Nicotiana tabacumM
TPIS_MAIZETriosephosphate isomerase, cytosolic OS = Zea maysI/E
TBA_PRUDUTubulin alpha chain OS = Prunus dulcisI
TBB_HORVUTubulin beta chain OS = Hordeum vulgareE
UBIQ_ARATHUbiquitin OS = Arabidopsis thalianaM/E
RL40A_ARATHUbiquitin-60S ribosomal protein L40-1 OS = Arabidopsis thalianaI/E
RGP1_ORYSJUDP-arabinopyranose mutase 1 OS = Oryza sativa subsp. JaponicaE
UGDH_SOYBNUDP-glucose 6-dehydrogenase OS = Glycine maxE
UREA_CANENUrease OS = Canavalia ensiformisI
UGPA1_ARATHUTP--glucose-1-phosphate uridylyltransferase 1 OS = Arabidopsis thalianaM/E
VATA_GOSHIV-type proton ATPase catalytic subunit A OS = Gossypium hirsutumI/E
VATB2_GOSHIV-type proton ATPase subunit B 2 (fragment) OS = Gossypium hirsutumE
VATB1_ARATHV-type proton ATPase subunit B1 OS = Arabidopsis thalianaE
WIT2_ARATHWPP domain-interacting tail-anchored protein 2 OS = Arabidopsis thalianaI
1 M: Mature endosperm; I: Immature endosperm; E: Embryo
Figure 4. Distribution of the top gene ontology (GO) data for lotus immature endosperm proteome based on 1-DGE-MS analysis.
Figure 4. Distribution of the top gene ontology (GO) data for lotus immature endosperm proteome based on 1-DGE-MS analysis.
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Analysis of the annotations referent to the immature endosperm revealed that functions related to protein synthesis (translation, protein folding and polymerization, etc.), general metabolism (amino acid, carbon fixation) and carbohydrate metabolism (glycolysis, etc.) are all considerably represented, with the proteins in the first category being relatively more numerous (Figure 4).
Figure 5. Distribution of the top gene ontology (GO) data for lotus embryo proteome based on 1-DGE-MS analysis.
Figure 5. Distribution of the top gene ontology (GO) data for lotus embryo proteome based on 1-DGE-MS analysis.
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On the other hand, the embryo proteome shows considerable prevalence of proteins involved in protein synthesis, followed then by carbohydrate and general metabolism processes (Figure 5).

3.6. Biological Function of the Identified Seed Proteins

Furthermore, the nr protein matches were also classified according to their broader biological function [22,23], divided into 10 categories: metabolism, energy, cell growth/division, transcription, protein synthesis/destination, transporters, cell structure, signal transduction, stress response, and unclassified (Figure 6).
Figure 6. Bar charts displaying the division according to functional categories, of the non-redundant (nr) protein matches found in the lotus seed embryo, immature endosperm, and mature endosperm, as determined by 1-DGE-MS.
Figure 6. Bar charts displaying the division according to functional categories, of the non-redundant (nr) protein matches found in the lotus seed embryo, immature endosperm, and mature endosperm, as determined by 1-DGE-MS.
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A comparison of the distribution of protein functionality between the seed immature endosperm and embryo, and the previous results obtained from the mature endosperm shows that immature endosperm and embryo have a quite similar functionality profile of the mature endosperm. However, in the embryo the identified proteins related to general cell housekeeping functions (non-energy metabolism, cell growth, transcription, transport, and signaling) were slightly more apparent than in the immature endosperm. In contrast with the mature endosperm, both immature endosperm and embryo show a larger percentage of the identified proteins related to protein synthesis. This correlated well with the fact that the tissues are either in a growing phase, i.e., immature endosperm or have growth as their main function, i.e., embryo. The mature endosperm, on the other hand, having its primary function as energy and nutrient storage, has the larger share of its proteins related to energy metabolism. A common element for all the lotus seed tissues is the large presence of stress-/defense-related proteins across all samples.

3.7. Lotus Seed Proteome Compared with Other Seed Proteomes

Unlike some seeds, such as tomato, where non-germinating embryo and endosperm were shown to have very similar proteomes [24], the analysis of lotus seed proteomes showed some remarkable difference in proteins identified/function between the non-germinating embryo and mature endosperm. Contrary to other seed proteomes like Jatropha curcas [23] and sugarbeet [25], the lotus embryo in its pre-germination stage did not seem to have a considerably higher expression of metabolism- and energy-related proteins compared to the mature endosperm. Structural proteins, however, did seem to be at least slightly more represented in the endosperm, as in the case of J. curcas. Compared with other embryo proteomes, such as Brassica campestri [26], and sugarbeet, the lotus embryo appears to have a larger percentage of proteins related to protein synthesis in comparison to primary and energy metabolism, as well as a much greater presence of defense related proteins. We further discuss below the key proteins identified in this study.

3.8. Key Proteins of the Lotus Immature Seed Endosperm

Contrary to the mature endosperm, the key functional proteins identified in the lotus immature endosperm mostly consisted of proteins related to plant growth and development (Figure 7).
Figure 7. Key functional proteins identified in the lotus seed immature endosperm, and subdivided according to their role in plant metabolism.
Figure 7. Key functional proteins identified in the lotus seed immature endosperm, and subdivided according to their role in plant metabolism.
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Amongst the identified proteins were several transcription proteins (cell division control and transcription factors), translation (ribosomal) proteins, post-translational modification proteins (elongation factors and ubiquitins) and nutrient production proteins (RuBisCO subunits and sucrose synthase). Many stress response- and plant defense-related proteins were also present in the immature endosperm. Of these, the largest subgroup is the heat shock response proteins (high- and low-molecular weight heat shock proteins (HSPs), as well as chaperone and annexin proteins). Anti-oxidative stress (peroxidases, endoplasmin, and monodehydroascorbate reductase) are also present, more so than in the mature endosperm (see below section). Proteins related to carbohydrate metabolism are also present in the immature endosperm, but in a smaller number.

3.9. Key Proteins Previously Identified in the Lotus Mature Seed Endosperm

In the case of mature endosperm proteome [17], the two most significant groups of proteins identified were related to energy/carbohydrate metabolism, and stress response and plant protection (Figure 8). In the first group, several proteins that are part of glycolysis, gluconeogenesis, citric acid cycle and starch metabolism including other carbohydrate metabolism proteins, were identified. Of the stress response proteins, HSPs, along with other heat response proteins (chaperones, annexin), constituted the most numerous category. Anti-oxidative stress proteins were not greatly represented. Of note is the identification of storage proteins (such as globulins, castanins) for the mature endosperm by 2-D MS and N-terminal sequencing, but not by 1-D MS [17,27], which might indicate a possible detection gap of this technique.
Figure 8. Key functional proteins identified in the lotus seed mature endosperm, and subdivided according to their role in plant metabolism. * for original protein lists, see reference [17].
Figure 8. Key functional proteins identified in the lotus seed mature endosperm, and subdivided according to their role in plant metabolism. * for original protein lists, see reference [17].
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3.10. Proteome Changes between Mature and Immature Stages of the Endosperm

Despite constituting the endosperm tissue samples, protein extracts from the mature and immature seed presented a notably different proteome composition (Figure 9).
Figure 9. Depiction of the changes in biological function, nutrient content and functional proteome composition from the immature (left) to the mature (right) endosperm in the lotus seed.
Figure 9. Depiction of the changes in biological function, nutrient content and functional proteome composition from the immature (left) to the mature (right) endosperm in the lotus seed.
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This reflects the changes the endosperm undergoes during the maturation process, where it develops from a soft wet tissue to a dry one with a large amount per weight of both carbohydrates and proteins [6]. The endosperm’s main role in the seed is as a nutrient storage tissue, so it is expected that during the maturation phase, these nutrients are going to be produced for later storage, hence the larger number of functional proteins related to the protein and carbohydrate synthesis categories. In the mature endosperm, a large percentage of the total protein content is expected to be seed storage proteins (SSPs). Although not many SSPs were identified by MS analysis of the mature endosperm, several possible matches were found by N-terminal sequencing analysis [27]. The prevalence of carbohydrate metabolism proteins amongst the identified functional proteins in the mature endosperm could be a result of production in the late maturation stage, with such proteins playing a quasi-dormant role in managing the nutrient content of the seed before and during germination.

3.11. Key Proteins of the Lotus Seed Embryo

In the case of the embryo proteins identified by database matching, the distribution of key proteins was similar to that of the immature endosperm, in that they can be divided in the same main groups: proteins related to plant growth, and proteins responsible for plant protection and germination vigor (Figure 10). Of the first group, those also include the same subgroups of transcription, translation, and post-translation proteins as well as nutrient production proteins. In the case of stress/defense-related proteins, the embryo was also found to possess the largest number heat shock response proteins (12 HSPs, mostly of high-molecular weight, five chaperone proteins and two annexins). However, the embryo also contained a larger number of anti-oxidative stress proteins, including l-ascorbate peroxidase, catalase, monodehydroascorbate reductase, superoxide dismutase [Mn], and endoplasmin. S-adenosylmethionine synthase and adenosylhomocysteinase (also found in the endosperm tissues), and two proteins from the active methyl cycle, which is of great importance to plant metabolism as well as their nutritional value [28], were also identified in the lotus embryo.
Figure 10. Key functional proteins identified in the lotus seed embryo, and subdivided according to their role in plant metabolism.
Figure 10. Key functional proteins identified in the lotus seed embryo, and subdivided according to their role in plant metabolism.
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4. Conclusions

Analysis of protein extracts from the lotus seed embryo and immature seed endosperm was performed following 1-DGE separation in conjunction with LC-MS/MS analysis. This “bottom-up” proteomics analysis, represented by the SDS-PAGE technique, has been shown to be a good approach for identifying the lotus seed proteins [17]. For both tissues, a great number of proteins were identified by database matching. A total of 141 nr protein matches were identified in the embryo, and 122 in the immature endosperm. Together with the 66 proteins previously identified for the mature endosperm, a total of 206 nr proteins have been identified to date.
Combined datasets are a resource in itself towards complete proteomics analysis of lotus seeds and plants. By producing more extensive datasets, these results help toward forming a complete proteomic picture of the lotus seeds. The analysis of protein makeup and functionality across different tissues within the seed also permits a comparison of metabolic functions across different tissues and developmental stages of the lotus seed, as well as allowing for the comparison with similar tissues from other plants. Furthermore, the identification of proteins of interest—such as key proteins in the metabolism, proteins that confer resistance against stress or germination vigor—opens up several possibilities for more specific studies on these proteins and their possible use in producing transgenic varieties of interest.
Future work will both strive to expand the lotus proteome to other developmentally important tissues, such as seedling and rhizome, as well as to isolate and characterize functional proteins of interest in the seed proteome. Moreover, 2-DGE-MS analysis of individual proteins, especially by de novo proteome analysis techniques, coupled with genome comparison, can help obtain more detailed sequences of lotus-specific proteins, since the high taxonomical distance of the lotus in relation to other modern plants hinders the achievement of higher homology values when database-matching proteins.

Acknowledgments

CFM greatly appreciates and acknowledges the financial support of the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) in the conducting this research. Authors appreciate the International Plant Proteomics Organization (INPPO) initiative (www.inppo.com) for connecting plant proteomic researchers between Brazil and Japan, and continuing collaborations between INPPO-India-Nepal chapter and INPPO-Japan.

Author Contributions

C.F.M., R.R., G.K.A., S.S., Y.K. and M.Y. were responsible for the conception of the study; C.F.M. and J.S. performed sample preparation and gel analysis; C.F.M. and Y.F. were responsible for mass spectrometry analyses; C.F.M. and R.R. performed the data analysis; C.F.M., R.R., G.K.A. and M.Y. wrote or contributed to the manuscript; figures and tables were prepared by C.F.M. and R.R. All authors read and approved the final version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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