A proteomic insight into the MSP1 and flg22 induced signaling in Oryza sativa leaves.

Previously, we reported a novel Magnaporthe oryzae- secreted protein MSP1, which triggers cell death and pathogen-associated molecular pattern (PAMP)-triggered immune (PTI) responses in rice. To investigate the MSP1 induced defense response in rice at the protein level, we employed a label-free quantitative proteomic approach, in parallel with flg22 treatment, which is a well-known elicitor. Exogenous application of MSP1 to rice leaves induced an oxidative burst, MAPK3/6 activation, and activation of pathogenesis-related genes (DUF26, PBZ, and PR-10). MaxQuant based label free proteome analysis led to the identification of 4167 protein groups of which 433 showed significant differences in response to MSP1 and/or flg22 treatment. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins associated mainly with the stress response, post-translational modification and signaling were increased in abundance. Moreover, several peroxidases and receptor kinases were induced by both the elicitors, highlighting their involvement in MSP1 and flg22 induced signaling in rice. Taken together, the results reported here contribute to our understanding of MSP1 and flg22 triggered immune responses at the proteome level, thereby increasing our overall understanding of PTI signaling in rice. BIOLOGICAL SIGNIFICANCE: MSP1 is a M. oryzae secreted protein, which triggers defense responses in rice. Previous reports have shown that MSP1 is required for the pathogenicity of rice blast fungus, however, the exact mechanism of its action and its downstream targets in rice are currently unknown. Identification of the downstream targets is required in order to understand the MSP1 induced signaling in rice. Moreover, key proteins identified could also serve as potential candidates for the generation of disease resistance crops by modulating stress signaling pathways. Therefore, here we employed, for the first time, a label-free quantitative proteomic approach to investigate the MSP1 induced signaling in rice together with flg22. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins related to the defense response, signaling and ROS detoxification were majorly increased. Thus, as an elicitor, recombinant MSP1 proteins could be utilized to inducing broad pathogen resistance in crops by priming the local immune responses.

Comparative Biochemical and Proteomic Analyses of Soybean Seed Cultivars Differing in Protein and Oil Content.

This study develops differential protein profiles of soybean (Glycine max) seeds (cv. Saedanbaek and Daewon) varying in protein (47.9 and 39.2%) and oil (16.3 and 19.7%) content using protamine sulfate (PS) precipitation method coupled with a 2D gel electrophoresis (2DGE) approach. Of 71 detected differential spots between Daewon and Saedanbaek, 48 were successfully identified by MALDI-TOF/TOF. Gene ontology analysis revealed that up-regulated proteins in Saedanbaek were largely associated with nutrient reservoir activity (42.6%), which included mainly seed-storage proteins (SSPs; subunits of glycinin and ß-conglycinin). Similar results were also obtained in two cultivars of wild soybean (G. soja cv. WS22 and WS15) differing in protein content. Western blots confirmed higher accumulation of SSPs in protein-rich Saedanbaek. Findings presented and discussed in this study highlight a possible involvement of the urea cycle for increased accumulation of SSPs and hence the higher protein content in soybean seeds.

Protamine sulfate precipitation method depletes abundant plant seed-storage proteins: A case study on legume plants.

Depletion of abundant proteins is one of the effective ways to improve detection and identification of low-abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable for their in-depth proteome investigation. In this study, we provide evidence that the PSP method can also be effectively used for depletion of abundant seed-storage proteins (SSPs) from the total seed proteins of diverse legume plants including soybean, broad bean, pea, wild soybean, and peanut. The 0.05% protamine sulfate (PS) was sufficient to deplete major SSPs from all legumes tested except for peanut where 0.1% PS was required. SDS-PAGE, Western blotting and 2DE analyses of PS-treated soybean and peanut seed proteins showed enriched spots in PS-supernatant than total proteins. Coefficient of variation percentage (%CV) and principal component analysis of 2DE spots support the reproducibility, suitability, and efficacy of the PSP method for quantitative and comparative seed proteome analysis. MALDI-TOF-TOF successfully identified some protein spots from soybean and peanut. Hence, this simple, reproducible, economical PSP method has a broader application in depleting plant abundant proteins including SSPs in addition to RuBisCO, allowing discussion for comprehensive proteome establishment and parallel comparative studies in plants.

Comparative investigation of seed coats of brown- versus yellow-colored soybean seeds using an integrated proteomics and metabolomics approach.

Seed coat color is an important attribute determining consumption of soybean seeds. Soybean cultivar Mallikong (M) has yellow seed coat while its naturally mutated cultivar Mallikong mutant (MM), has brown colored seed coat. We used integrated proteomics and metabolomics approach to investigate the differences between seed coats of M and MM during different stages of seed development (4, 5, and 6 weeks after flowering). 2DE profiling of total seed coat proteins from three stages showed 178 differentially expressed spots between M and MM of which 172 were identified by MALDI-TOF/TOF. Of these, 62 were upregulated and 105 were downregulated in MM compared with M, while five spots were detected only in MM. Proteins involved in primary metabolism showed downregulation in MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via secondary metabolic pathways that leads to the development of brown seed coat color. Besides, downregulation of two isoforms of isoflavone reductase indicated reduced isoflavones in seed coat of MM that was confirmed by quantitative estimation of total and individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer a high substrate for proanthocyanidin production that results in the development of brown seed coat in MM.