The seed proteome of pea (Pisum sativum L.) lines different in their response to beneficial microorganisms highlights the different strategies for seed production in plants

The analysis of the seed proteome of pea lines contrasting in their responsiveness to inoculation with soil microorganisms revealed the mechanisms underlying the extension of the seed filling phase in highly responsive line K-8274.

Characterization of Seed Proteome Profile of Wild and Cultivated Chickpeas of India

Background: Chickpea is a widely grown legume in India, Australia, Canada, and Mediterranean regions. Seeds of chickpea are good source of protein for both human and animals. Wild relatives of chickpea (Cicer arietinum) are the potential gene pool for crop improvement; however, very little information is available on the seed proteome of these wild chickpeas. Objective: We aimed to analyze the seed proteome profiles of three wild relatives of chickpea, Cicer bijugum, Cicer judaicum and Cicer microphyllum along with two cultivated varieties JG11 and DCP 92/3. Method: Total seed proteins were extracted using various extraction buffers for 2-D gel electrophoresis. Protein separated in a 2-D gels were subjected to image analyses, differentially expressed proteins were extracted from the gels and identified by the MALDI TOF/TOF. Seed protease inhibitors were analysed biochemically. Results: We have standardized the 2-D gel electrophoresis method and separated seed proteins using the modified method. We identified a large number (400) of protein proteins which were differentially expressed in cultivated and wild type species of chickpea. A comparative analysis between C. bijugum and JG 11 revealed the presence of 9 over-expressed and 22 under-expressed proteins, while the comparison between C. bijugum with DCP 92/3 showed 8 over-expressed and 18 under-expressed proteins. Similarly, comparative analysis between C. microphyllum with DCP 92/3 showed 8 over-expressed proteins along with 22 under-expressed proteins, while the comparative study of C. microphyllum with JG11 displayed 9 over-expressed and 24 under-expressed proteins. We also compared C. judaicum with DCP 92/3 which revealed 15 overexpressed and 11 under-expressed proteins. On the other hand, the comparative analysis of C. judaicum with JG11 showed 10 over-expressed proteins, while the numbers of under-expressed proteins were 14. Among the differentially expressed protein proteins, 19 proteins were analyzed by the MS/MS, and peptides were identified using the MASCOT search engine. In teh wild relatives the differtially expressed proteins are phosphatidylinositol 4-phosphate 5- kinase, β-1-6 galactosyltransferase, RNA helicase, phenyl alanine ammonia lyase 2, flavone 3’-0-methyl transferase, Argonaute 2, Myb related protein, Tubulin beta-2 chain and others. The most important one was legumin having α- amylase inhibition activity which was up regulated in C. bijugum. We also studied the activity of protease inhibitor (trypsin and α- amylase inhibitors) in these seed lines which showed differential activity of protease inhibitors. The highest trypsin and α- amylase inhibition was observed in C. judaicum and C. bijugum, respectively. Conclusion: The differentially expressed proteins of wild relatives of chickpea appeared to be involved in various metabolic pathways. The study provides us information about the differences in the seed proteome of these wild species and cultivated varieties for the first time.

In-Depth Investigation of Low-Abundance Proteins in Matured and Filling Stages Seeds of Glycine max Employing a Combination of Protamine Sulfate Precipitation and TMT-Based Quantitative Proteomic Analysis

Despite the significant technical advancements in mass spectrometry-based proteomics and bioinformatics resources, dynamic resolution of soybean seed proteome is still limited because of the high abundance of seed storage proteins (SSPs). These SSPs occupy a large proportion of the total seed protein and hinder the identification of low-abundance proteins. Here, we report a TMT-based quantitative proteome analysis of matured and filling stages seeds of high-protein (Saedanbaek) and low-protein (Daewon) soybean cultivars by application of a two-way pre-fractionation both at the levels of proteins (by PS) and peptides (by basic pH reverse phase chromatography). Interestingly, this approach led to the identification of more than 5900 proteins which is the highest number of proteins reported to date from soybean seeds. Comparative protein profiles of Saedanbaek and Daewon led to the identification of 2200 and 924 differential proteins in mature and filling stages seeds, respectively. Functional annotation of the differential proteins revealed enrichment of proteins related to major metabolism including amino acid, major carbohydrate, and lipid metabolism. In parallel, analysis of free amino acids and fatty acids in the filling stages showed higher contents of all the amino acids in the Saedanbaek while the fatty acids contents were found to be higher in the Daewon. Taken together, these results provide new insights into proteome changes during filling stages in soybean seeds. Moreover, results reported here also provide a framework for systemic and large-scale dissection of seed proteome for the seeds rich in SSPs by two-way pre-fractionation combined with TMT-based quantitative proteome analysis.

Proteomics reveals commitment to germination in barley seeds is marked by loss of stress response proteins and mobilisation of nutrient reservoirs

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used DIA/SWATH-MS proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We validated our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or breeding to enhance germination or control dormancy.

Eggplant Germination is Promoted by Hydrogen Peroxide and Temperature in an Independent but Overlapping Manner

Hydrogen peroxide promotes seed germination, but the molecular mechanisms underlying this process are unclear. This study presents the results of eggplant (Solanum melongena) germination analyses conducted at two different temperatures and follows the effect of hydrogen peroxide treatment on seed germination and the seed proteome. Hydrogen peroxide was found to promote eggplant germination in a way not dissimilar to that of increased temperature stimuli. LC–MS profiling detected 729 protein families, 77 of which responded to a temperature increase or hydrogen peroxide treatment. These differentially abundant proteins were found to be involved in a number of processes, including protein and amino acid metabolism, carbohydrate metabolism, and the glyoxylate cycle. There was a very low overlap between hydrogen peroxide and temperature-responsive proteins, highlighting the differences behind the seemingly similar outcomes. Furthermore, the observed changes from the seed proteome indicate that hydrogen peroxide treatment diminished the seed endogenous hydrogen peroxide pool and that a part of manifested positive hydrogen peroxide effect might be related to altered sensitivity to abscisic acid.