Dynamic proteomic changes in soft wheat seeds during accelerated ageing

Wheat seeds and artificial ageing treatment

The ‘Yangmai 15’ soft cultivars used in this study were purchased from the Henan Academy of Agricultural Science. The original germination rate (Gr) was 97.0% (designated as YM97), and the average seed moisture content was 12.19%. A 400 g sample of seeds with a similar size and weight was stored in a 5 L plastic bottle at 45 ± 1 °C and 50% relative humidity in a constant temperature- and humidity-controlled cabinet (Binder KMF720; Binder, Tuttingen, Germany) according to our previous research (Lv et al., 2016). Measurement of CO₂ from wheat respiration and calculation of Gr were performed as previously described (Dong et al., 2015; Zhang et al., 2014). Gr and the CO₂ concentration were determined regularly and seed samples with three lots of biological replicates were collected for further proteomic analysis when Gr was 97%, 45%, 28% and 6% (designated as YM97, YM45, YM28 and YM6, respectively).

Protein extraction, SDS-PAGE analysis, trypsin digestion, TMT labelling and high-pressure liquid chromatography (HPLC) fractionation

Protein extraction, trypsin digestion and HPLC fractionation were performed as reported previously (Lv et al., 2016). Extracted proteins were boiled in loading buffer (100 mM Tris-HCl buffer (pH 8.0) containing 2% SDS, 5% glycerol, and 1% β-mercaptoethanol) for 5 min. They were centrifuged at 12,000× g for 5 min, and supernatants were subjected to 12% SDS-PAGE in a vertical slab gel apparatus (LIUYI, Beijing China). After trypsin digestion, peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. Peptides were reconstituted in 0.5 M triethylammonium bicarbonate and processed according to the manufacturer’s protocol for the TMT kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile, and peptides from YM97, YM45, YM28, and YM6 (labelled with 126, 127, 130, and 131, respectively) were then incubated with TMT reagent for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation. Labeled samples combined for further analysis.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and database searches

LC-MS/MS and database searches were performed as described previously (Lv et al., 2016). Briefly, peptides were dissolved in 0.1% formic acid (solvent A) and separated using a reversed-phase analytical column (Acclaim PepMap RSLC; Thermo Fisher Scientific, Waltham, MA, USA). The gradient comprised an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, an increase to 80% in 3 min, and holding at 80% for 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed and subjected to a nanospray ion source followed by tandem mass spectrometry (MS/MS) in a Q Exactive™ instrument (Thermo Fisher Scientific, Waltham, MA, USA) coupled online to an ultra-performance LC module. Peptides were selected for MS/MS using NCE setting of 28, and fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were used as queries against the Uniprot_Triticum_aestivum database (136,892 sequences) concatenated with the reverse decoy database. The false discovery rate was adjusted to <1%, and the minimum score for peptides was set at >40. Proteins displaying a 1.2-fold change between artificially aged and normal seeds (YM45 vs. YM97, YM28 vs. YM97 and YM6 vs. YM97) were considered as DEPs if p < 0.05.

Protein annotation and functional enrichment analysis

Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed as previously described ( Wu et al., 2006). Additionally, domain annotation and subcellular localization were performed using InterProScan on InterPro (http://www.ebi.ac.uk/interpro/) and WoLFPSORT systems (PSORT/PSORT version II; http://psort.hgc.jp/), respectively (Horton et al., 2007; Zdobnov & Apweiler, 2001). Functional-enrichment-based clustering for different protein groups (GO, Domain, Pathway) was performed and cluster membership was visualized using a heat map generated with the “heatmap.2” function in the “gplots” program within the R package as previous reported (Lv et al., 2016).

Seed respiration and germination rate are altered during accelerated ageing

To characterize physiological changes occurring during artificial ageing, respiration and Gr were investigated (Fig. 1). The Gr of seeds following artificial ageing treatment displayed decreased germinability from 97% to 87% (30 days), 70% (60 days), 45% (90days), 28% (120 days), 15% (150 days), and 6% (180 days), while the amount of CO₂ produced increased from 0% to 0.25% (30 days), 0.30% (60 days), 0.34% (90 days), 0.37% (120 days), 0.39% (150 days), and 0.40% (180 days). These results showed that Gr and respiration changed rapidly from 30 d to 90 d indicating drastic seed vigor and metabolic alterations.

Overview of quantitative proteomics analysis

To characterize the wheat seed proteome, a TMT-based gel-free quantitative proteomics analysis of seeds with three biological replicates was performed during artificial ageing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins from wheat seeds is shown in Fig. S1A. Each lane was loaded with 30 µg of protein, and proteins of different molecular weight were distributed in each lane, indicating that the protein was not degraded and hence suitable for subsequent analysis. For LC-MS/MS analysis, distribution of peptide length (8–16), mass error (<0.02 Da), and repeatability of relative protein quantitation evaluation (characterized with pair-wise Pearson’s correlation coefficients) indicated that MS data accuracy, sample preparation, and reproducibility were acceptable (Figs. S1B–S1D). In this study, 6,271 proteins were identified, among which 4,509 were quantified. Of the quantified proteins, 1,010 displayed significant changes, of which 519 were upregulated (ratio > 1.2; p < 0.05) and 491 were downregulated (ratio < 1/1.2; p < 0.05) in the artificial aged seeds compared with untreated (control) seeds (Table S1). Of the upregulated proteins, there were 29 in YM45 vs. YM97, 46 in YM28 vs. YM97, and 444 in YM6 vs. YM97; of the downregulated proteins, there were 36 in YM45 vs. YM97, 28 in YM28 vs. YM97, and 427 in YM6 vs. YM97. The above results showed that most of the DEPs distributed in YM6 (Gr was 6%) compared with YM97, indicating that the proteomic profiles were significantly changed during accelerated ageing. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaíno et al., 2015) partner repository with the dataset identifier hyperlink to PXD009156.

Protein annotation and functional enrichment analysis of DEPs

To determine the functions of the quantified DEPs, annotation and functional enrichment analyses were performed including subcellular localization, GO, domain, and pathway analysis (Table S1). To better understand the dynamics of DEPs during accelerated ageing, a hierarchical clustering analysis was carried out to obtain dynamic expression patterns (Figs. 2 and 3). The biological process category of GO analysis indicated that upregulated proteins involved in resistance (wheatwin and defensin-like proteins), killing of cells of other organisms, and disruption of cells of other organisms were enriched in YM28. Additionally, upregulated proteins participating in macromolecular metabolic processes were also enriched in YM6, indicating the degradation of storage nutrients (Fig. 2A). Meanwhile, downregulated proteins were mainly involved in regulation of carbon, nitrogen and macromolecular metabolic processes (alpha-amylase/trypsin inhibitor and transcriptional factors) when the Gr was 6%. For the cellular component subclass, upregulated proteins involved in proteasome, endoplasmic reticulum membrane, and extracellular region were enriched in YM6. At the beginning of storage, enriched downregulated proteins were mainly associated with membrane component (YM45), but with an extended artificial ageing time (YM28), enriched downregulated proteins were mainly linked to nucleosome, chromatin and chromosome, and downregulated proteins in YM6 were mainly enriched in respiratory chain, ribosome and ribonucleoprotein complex categories (Fig. 2B). Molecular function analysis indicated that upregulated proteins participated in amylase activity, hydrolase activity, cellulase activity and transporter activity, while down-regulated proteins were involved in peptidase regulatory activity, nutrient reservoir activity, structural constituents of ribosome, enzyme regulator activity and protein dimerization/heterodimerization (Fig. 2C). Protein domain analysis demonstrated that many upregulated proteins contained domains involved in pathogenesis-related proteins, alpha-amylase and glycosyl hydrolase when Gr was 28%, whereas when Gr was 6%, upregulated proteins were mainly associated with cytochrome c, glycoside hydrolase, and 14-3-3 protein. Additionally, upregulated proteins with a mitochondrial carrier domain were enriched in YM45, proteins with histone fold and histone H2 were abundant in YM28, and proteins with 50S ribosomal protein, proteinase inhibitor, non-specific lipid-transfer protein and bifunctional trypsin/alpha-amylase inhibitor/seed storage domains were enriched in YM6 (Fig. 3A). KEGG analysis indicated that upregulated proteins involved in MAPK signaling pathway, signal transduction and plant pathogen interactions were mainly enriched when Gr was 28% and 6%, and upregulated proteins associated with glycan, amino acids, glutathione metabolism, pentose phosphate pathway and starch and sucrose metabolism were enriched when Gr was 6%. Downregulated proteins related to protein processing in the endoplasmic reticulum were enriched when Gr was 45%, and proteins linked to oxidative phosphorylation and DNA modification were downregulated following extended storage time (Fig. 3B).

Classification of DEPs during artificial ageing

To better understand the functions of proteins, representative DEPs were classified into six groups involving enzyme activity and regulation, nutrient reservoir activity, electron transport and signal transduction, energy and metabolism, defense/stress responses and chromatin and ribosome (Table 1).

Enzymes involved in starch hydrolytic activity and proteolysis such as alpha/beta-amylase, beta-glucosidase, and proteasome subunit were upregulated, while alpha-amylase/trypsin inhibitor and proteinase inhibitor subtilisin-chymotrypsin were downregulated, which was inconsistent with the increased enzyme activities. Accompanying the increased levels of hydrolytic enzymes was the reduced abundance of storage proteins avenin, gliadin, gamma-hordein and oleosin. Additionally, proteins involved in electron transport and signal transduction including cytochrome c oxidase, and calcium-dependent protein kinase and 14-3-3 protein were upregulated. These results also indicated that proteins in energy and metabolism were differently regulated. Two downregulated ATP synthase submits were observed and glyceraldehyde-3-phosphate dehydrogenase that is involved in glycolysis was downregulated, whereas glucose-6-phosphate isomerase, 6-phosphofructokinase, acyl-coenzyme A oxidase, and pyruvate kinase were upregulated. Malate dehydrogenase and succinate dehydrogenase that are involved in the tricarboxylic acid cycle were downregulated, whereas citrate synthase, and isocitrate dehydrogenase were upregulated. Additionally, glucose-6-phosphate 1-dehydrogenase that is involved in the pentose phosphate pathway was upregulated. Among the proteins involved in defense/stress responses, peroxidases and superoxide dismutases, protein disulfide-isomerases, non-specific lipid-transfer proteins, puroindoline-B, and L-ascorbate peroxidase were downregulated, whereas glutathione S-transferase, glutathione synthetase, pathogenesis-related protein, defensin-like protein, wheatwin-2, and heat stress-associated DnaJ were upregulated. During accelerated ageing, proteins participating in chromatin and ribosome organization such as protein H2A, histone H2B, and the 40S and 60S ribosomal proteins were downregulated.