A comparative proteomic analysis of responses to high temperature stress in hypocotyl of Canola (Brassica napus L.).

High temperature stress, especially on the early season of plant growth stages, is an agricultural problem in many areas in the world. A temporary or continually high temperature leads to a set of morphological, biochemical and physiological changes in plants, which consequently reduces the plant growth and development and finally may cause a severe reduction in economic yield. The main goal of this study was to assess plant response to high temperature stress (HTS) in early seedling of canola. This study is the first experiment on the effect of heat stress on proteome of canola. In the present research, a proteomics approach was used to evaluate the effects of high temperature stress, including 45 °C day/34 °C night for 2, 6 and 12 hour, on early seedling stage (2-day old) of canola. Proteins were isolated from hypocotyl and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 381 protein spots, 28 and 34 proteins were significantly down- and up-regulated, respectively. The trend of mRNA expression for sucrose binding protein, a scorbate peroxidase and triosephosphateisomerase, was in accordance with their trend at translation level. Results of this study suggest that the up-regulation of proteins involved in cellular traffic, energy and metabolism, and down-regulation of some proteins involved in disease and defense, protein synthesis and signal transduction could be the main reason of physiological and morphological responses to high temperature stress. The observed increases in the level of ascorbate peroxidase protein and mRNA expression in canola hypocotyl in response to HTS suggests that ascorbate peroxidase is a short term high temperature stress response protein and is thus a candidate for gene modification strategies aimed at producing high temperature canola varieties. These results also suggest that the up regulation of protein involved in energy and metabolism in response to the heat stress can use most of nutritive reserves in seedling of canola and might explain the reduced growth of canola in heat stress conditions.

Proteomic insights into intra- and intercellular plant-bacteria symbiotic association during root nodule formation.

Over the last several decades, there have been a large number of studies done on the all aspects of legumes and bacteria which participate in nitrogen-fixing symbiosis. The analysis of legume-bacteria interaction is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. Bacteria regulate their quorum-sensing genes to enhance their ability to induce conjugation of plasmids and symbiotic islands, and various protein secretion mechanisms; that can stimulate a collection of chain reactions including species-specific combinations of plant-secretion isoflavonoids, complicated calcium signaling pathways and autoregulation of nodulation mechanisms. Quorum-sensing systems are introduced by the intra- and intercellular organization of gene products lead to protein-protein interactions or targeting of proteins to specific cellular structures. In this study, an attempt has been made to review significant contributions related to nodule formation and development and their impacts on cell proteome for better understanding of plant-bacterium interaction mechanism at protein level. This review would not only provide new insights into the plant-bacteria symbiosis response mechanisms but would also highlights the importance of studying changes in protein abundance inside and outside of cells in response to symbiosis. Furthermore, the application to agriculture program of plant-bacteria interaction will be discussed.

Analysis of the proteome of common bean (Phaseolus vulgaris L.) roots after inoculation with Rhizobium etli.

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.

A comparative proteomics analysis in roots of soybean to compatible symbiotic bacteria under flooding stress.

A proteomics approach was used to evaluate the effects of flooding stress on early symbiotic interaction between soybean roots and soil bacteria, Bradyrhizobium japonicum. Three-day-old soybean was inoculated with B. japonicum followed by flooding. The number of root hairs in seedlings, without or with flooding stress, was increased after 3 days of inoculation. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 219 protein spots, 14 and 8 proteins were increased and decreased, respectively, by inoculation under flooding compared with without flooding. These proteins were compared in untreated and flooded seedlings. Increased level of 6 proteins in flooded seedlings compared with untreated seedlings was suppressed by inoculation in seedlings under flooding. They were related to disease/defense, protein synthesis, energy, and metabolism. Differential abundance of glucan endo-1,3-beta-glucosidase, phosphoglycerate kinase, and triosephosphate isomerase, based on their localization in middle and tip of root, indicated that they might be related to increase in number of root hairs. These results suggest that disease/defense, energy, and metabolism-related proteins may be particularly subjected to regulation in flooded soybean seedlings, when inoculated with B. japonicum and that this regulation may lead to increase in number of root hair during early symbiotic differentiation.

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding.

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.

A comparative proteomic analysis of the early response to compatible symbiotic bacteria in the roots of a supernodulating soybean variety.

To reveal the processes involved in the early stages of symbiosis between soybean plants and root nodule bacteria, we conducted a proteomic analysis of the response to bacterial inoculation in the roots of supernodulating (En-b0-1) and non-nodulating (En1282) varieties, and their parental normal-nodulating variety (Enrei). A total of 56 proteins were identified from 48 differentially expressed protein spots in normal-nodulating variety after bacterial inoculation. Among 56 proteins, metabolism- and energy production-related proteins were upregulated in supernodulating and downregulated in non-nodulating varieties compared to normal-nodulating variety. The supernodulating and non-nodulating varieties responded oppositely to bacterial inoculation with respect to the expression of 11 proteins. Seven proteins of these proteins was downregulated in supernodulating varieties compared to non-nodulating variety, but expression of proteasome subunit alpha type 6, gamma glutamyl hydrolase, glucan endo-1,3-beta glucosidase, and nodulin 35 was upregulated. The expression of seven proteins mirrored the degree of nodule formation. At the transcript level, expression of stem 31kDa glycoprotein, leucine aminopeptidase, phosphoglucomutase, and peroxidase was downregulated in the supernodulating variety compared to the non-nodulating variety, and their expression in the normal-nodulating variety was intermediate. These results suggest that suppression of the autoregulatory mechanism in the supernodulating variety might be due to negative regulation of defense and signal transduction-related processes.