By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.

Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

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Untimely rains in wheat fields during harvest season can cause pre-harvest sprouting (PHS), which deteriorates the yield and quality of wheat crop. Metabolic homeostasis of the embryo plays a role in seed dormancy, determining the status of the maturing grains either as dormant (PHS-tolerant) or non-dormant (PHS-susceptible). Very little is known for direct measurements of global metabolites in embryonic tissues of dormant and non-dormant wheat seeds. In this study, physiologically matured and freshly harvested wheat seeds of PHS-tolerant (cv. Sukang, dormant) and PHS-susceptible (cv. Baegjoong, non-dormant) cultivars were water-imbibed, and the isolated embryos were subjected to high-throughput, global non-targeted metabolomic profiling. A careful comparison of identified metabolites between Sukang and Baegjoong embryos at 0 and 48 h after imbibition revealed that several key metabolic pathways [such as: lipids, fatty acids, oxalate, hormones, the raffinose family of oligosaccharides (RFOs), and amino acids] and phytochemicals were differentially regulated between dormant and non-dormant varieties. Most of the membrane lipids were highly reduced in Baegjoong compared to Sukang, which indicates that the cell membrane instability in response to imbibition could also be a key factor in non-dormant wheat varieties for their untimely germination. This study revealed that several key marker metabolites (e.g., RFOs: glucose, fructose, maltose, and verbascose), were highly expressed in Baegjoong after imbibition. Furthermore, the data showed that the key secondary metabolites and phytochemicals (vitexin, chrysoeriol, ferulate, salidroside and gentisic acid), with known antioxidant properties, were comparatively low at basal levels in PHS-susceptible, non-dormant cultivar, Baegjoong. In conclusion, the results of this investigation revealed that after imbibition the metabolic homeostasis of dormant wheat is significantly less affected compared to non-dormant wheat. The inferences from this study combined with proteomic and transcriptomic studies will advance the molecular understanding of the pathways and enzyme regulations during PHS.

Nitrogen fertilization is essential for increasing rice production to meet the food demands of increasing world's population. We established an in vivo hydroponic rice seedling culture system to investigate physio-biochemical/molecular responses of various rice japonica and indica cultivars to low nitrogen (N). Three-week-old seedlings grown in Yoshida's nutrient solution manifested stable and reproducible symptoms, such as reduced shoot growth and length under low N. Out of 12 genetically selected cultivars, 11 cultivars showed varied degrees of growth reduction response to applied N (4 and 40 ppm N for treatment and control, respectively), whereas one cultivar (no. 12) showed similar growth as the control though its leaf width was smaller than control. Leaves of a representative low N-responsive cultivar (BG90-2) were sampled for revealing protein profiles between low and normal (control) N application by two-dimensional gel electrophoresis (2-DGE). Forty-one proteins were identified with MALDI-TOF-MS and nESI-LC-MS/MS. Assignment of proteins into major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealed many novel low N-responsive proteins, including those having energy/photosynthesis-and defense/stress-and iron homeostasis-related functions. Results suggest the usefulness of proteomics in identifying novel N-responsive proteins and may provide potential markers for rice response to low N.

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Silicon (Si) is a micronutrient. Its amount has been found to vary from plant to plant. Grasses contain much higher Si than Arabidopsis. Interestingly, Si in plants has been shown to enhance their tolerance against various abiotic and biotic stresses. Silicon induced resistance in rice against pathogenic fungi Magnaporthe grisea and Rhizoctonia solini have been well demonstrated. In addition, Si also plays an important role in providing tolerance to heavy metal toxicity and water stress. Systematic identification and characterization of Si-responsive genes responsive genes and proteins will help in better understanding the underlying mechanism of Si-induced tolerance in plants. High-throughput technologies, such as transcriptomics and proteomics, have tremendous potential in establishing the Si-responsive genes and proteins network in order to design next generation crop plants. Here, we will focus on the role of Si in conferring tolerance in plants against various environmental stressors. We highlight the importance of genomics and potential of proteomics and metabolomics in investigating Si responses in plants and discuss its suitability in crop improvement.

Rice is one of the most important food and cereal crop plants in the world. Rice proteomics began in the 1990s. Since then, considerable progress has been made in establishing protocols from isolation of rice proteins from different tissues, organs, and organelles, to separation of complex proteins and to their identification by mass spectrometry. Since the year 2000, global proteomics studies have been performed during growth and development under numerous biotic and abiotic environmental conditions. Two-dimensional (2-D) gel-based proteomics platform coupled with mass spectrometry has been retained as the workhorse for proteomics of a variety of rice samples. In this chapter, we describe in detail the different protocols used for isolation of rice proteins, their separation, detection, and identification using gel-based proteomics and mass spectrometry approaches.