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. METHODS: 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 the wild relatives the differentially expressed proteins are phosphatidylinositol 4-phosphate 5- kinase, ß-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.

Defense Response in Chickpea Pod Wall due to Simulated Herbivory Unfolds Differential Proteome Profile.

The pod wall of legumes is known to protect the developing seeds from pests and pathogens. However, the mechanism of conferring defense against insects has not yet been deciphered. Here, we have utilized 2-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS/MS) to identify over expressed proteins in the pod wall of two different cultivars (commercial cultivar: JG 11 and tolerant cultivar: ICC 506-EB) of chickpea after 12 h of application of Helicoverpa armigera oral secretions (simulated herbivory). The assays were performed with a view that larvae are a voracious feeder and cause substantial damage to the pod within 12 h. A total of 600 reproducible protein spots were detected on gels, and the comparative analysis helped identify 35 (12 up-regulated, 23 down-regulated) and 20 (10 up-regulated, 10 down-regulated) differentially expressed proteins in JG 11 and ICC 506-EB, respectively. Functional classification of protein spots of each cultivar after MS/MS indicated that the differentially expressed proteins were associated with various metabolic activities. Also, stress-related proteins such as mannitol dehydrogenase (MADH), disease resistance-like protein-CSA1, serine/threonine kinase (D6PKL2), endoglucanase-19 etc. were up-regulated due to simulated herbivory. The proteins identified with a possible role in defense were further analyzed using the STRING database to advance our knowledge on their interacting partners. It decoded the involvement of several reactive oxygen species (ROS) scavengers and other proteins involved in cell wall reinforcement. The biochemical analysis also confirmed the active role of ROS scavengers during simulated herbivory. Thus, our study provides valuable new insights on chickpea-H.armigera interactions at the protein level.