Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.

BackgroundChilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments.MethodsRecombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength.ResultsLarval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested.ConclusionsOf the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum.General significanceIngestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.