Spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process.

Grain filling and grain development are essential biological processes in the plant's life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In-gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule-bound and soluble starch synthase, pyruvate phosphate dikinase, 14-3-3 proteins as well as sugar precursors undergo a major tissue-dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue-specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes.

Proteomic analysis of drought stress response mechanism in soybean (Glycine max L.) leaves.

Knowledge of the physiological and molecular mechanisms of drought responses is fundamental for developing genetically drought tolerant and high yielding crops. To understand molecular mechanism of drought tolerance of soybean (Glycine max L.), we compared leaf proteome patterns of in two genotypes GN-3074 (drought tolerant) and GN-2032 (drought-sensitive) under drought stress during vegetative stage. Proteins were extracted from leaves of well-watered and drought-treated plants by using the trichloroacetic acid (TCA)-acetone precipitation method and analyzed by two-dimensional polyacrylamide gel electrophoresis. Out 488 reproducibly detected and analyzed on two-dimensional electrophoresis gels, 26 proteins showed significant changes in at least one genotype. The identification of 20 differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in cellular metabolisms including photosynthesis, oxidative stress defense, respiration, metabolism process, signal transduction, phosphorus transduction, and methyl transduction which enable plant to cope with drought conditions. The most identified proteins involved in photosynthesis and oxidative stress defense system. The up-regulation of several photosynthetic proteins and also high abundance of oxidative stress defense proteins in GN-3074 genotypes as compare to GN-2032 genotypes might reflect the fact that drought tolerance of GN-3074 is due to effective photosynthetic machinery and more defense against oxidative stress. Our results suggest that soybean plant might response to drought stress by applying efficiently stay-green mechanism through coordinated gene expression during vegetative stage.

Physiological and Proteomic Signatures Reveal Mechanisms of Superior Drought Resilience in Pearl Millet Compared to Wheat.

Presently, pearl millet and wheat are belonging to highly important cereal crops. Pearl millet, however, is an under-utilized crop, despite its superior resilience to drought and heat stress in contrast to wheat. To investigate this in more detail, we performed comparative physiological screening and large scale proteomics of drought stress responses in drought-tolerant and susceptible genotypes of pearl millet and wheat. These chosen genotypes are widely used in breeding and farming practices. The physiological responses demonstrated large differences in the regulation of root morphology and photosynthetic machinery, revealing a stay-green phenotype in pearl millet. Subsequent tissue-specific proteome analysis of leaves, roots and seeds led to the identification of 12,558 proteins in pearl millet and wheat under well-watered and stress conditions. To allow for this comparative proteome analysis and to provide a platform for future functional proteomics studies we performed a systematic phylogenetic analysis of all orthologues in pearl millet, wheat, foxtail millet, sorghum, barley, brachypodium, rice, maize, Arabidopsis, and soybean. In summary, we define (i) a stay-green proteome signature in the drought-tolerant pearl millet phenotype and (ii) differential senescence proteome signatures in contrasting wheat phenotypes not capable of coping with similar drought stress. These different responses have a significant effect on yield and grain filling processes reflected by the harvest index. Proteome signatures related to root morphology and seed yield demonstrated the unexpected intra- and interspecies-specific biochemical plasticity for stress adaptation for both pearl millet and wheat genotypes. These quantitative reference data provide tissue- and phenotype-specific marker proteins of stress defense mechanisms which are not predictable from the genome sequence itself and have potential value for marker-assisted breeding beyond genome assisted breeding.

Proteomic analysis of rice anthers under salt stress.

Salinity is a major factor that limits rice production worldwide. Rice is considered generally to be sensitive to salt stress during the reproductive stage. To determine the molecular mechanisms of salt tolerance at the reproductive stage, anther proteomic patterns for two contrasting rice genotypes IR64 (salt sensitive) and Cheriviruppu (salt tolerant) under salt stress were compared. Plants were grown in a greenhouse and salt stress (100 mM NaCl) was imposed at the booting stage. Anther samples were collected from control and salt-treated plants at the anthesis stage. The Na(+)/K(+) ratio in IR64 anthers under salt stress was >1.7 times greater than that under control conditions, whereas no significant change was observed in Cheriviruppu. We also observed an 83% reduction in IR64 pollen viability, whereas this reduction was only 23% in Cheriviruppu. Of 454 protein spots detected reproducibly on two-dimensional electrophoresis gels, 38 showed significant changes in at least one genotype in response to stress. Using Mass spectrometry (MALDI TOF/TOF) analysis, we identified 18 protein spots that were involved in several processes that might increase plant adaptation to salt stress, such as carbohydrate/energy metabolism, anther wall remodelling and metabolism, and protein synthesis and assembly. Three isoforms of fructokinase-2 were upregulated only in Cheriviruppu under salt stress. This upregulation might result in increased starch content in pollen, which would support pollen growth and development under salt stress. The results also suggested that anther and pollen wall remodelling/metabolism proteins contribute to the tolerance of rice to salt stress.

A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat.

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.