8. The way forward

are by-products as a result of disruption of normal homeostasis and those involved in signal transduction in response to the stresses [85]. Due to involvement of metabolites in important life processes, the field of metabolic profiling could contribute significantly to the study of stress biology in plants. Both primary and secondary metabolites have been shown to play important roles in responses of plants to drought and salinity stresses. Primary metabolites such as sugars, amino acids and intermediates of Krebs cycle were found with important roles in photosynthetic dysfunction and osmotic readjustment. While, the secondary metabolites such as antioxidant scavengers, coenzymes and regulatory molecules responded to specific stress conditions. Both qualitative and quantitative studies of metabolites in response to abiotic stress are helpful in not only determining the phenotypic response of the plant and screening for stress tolerant lines but also reveal the genetic and biochemical mechanisms underlying the

Drought and salt stresses affect the process of photosynthesis, affecting CO2 diffusion leading to photorespiration and hydrogen peroxide production, causing cell damage [87]. Most recently, Rabara et al. [88] analyzed the metabolomics profile of tobacco and soybean roots and leaves facing dehydration stress. The study revealed highest tissue specific accumulation of 4-hydroxy-2-oxoglutaric acid in tobacco roots and coumestrol in soybean roots; indicating 4-hydroxy-2 oxoglutaric acid and coumestrol can be used as markers for drought stress. Metabolomic analysis of intense drought-stressed grapevine leaves was conducted to reveal induction of several metabolites [89]. Metabolomic profiling of Arabidopsis exposed to drought and heat stresses in combination revealed accumulation of sucrose, maltose and glucose [90]. In tolerant and sensitive thyme facing water stress, metabolomics analysis revealed differential changes in carbohydrates, amino acids, fatty acids and organic acids profiles [91]. Metabolites related to the mechanisms of osmotic adjustment, ROS scavenging, cellular components protection and membrane lipid showed significant changes. Metabolomic and proteomic analysis of xylem sap in maize under drought stress revealed a higher abundance of cationic peroxidases, which with the increase in phenylpropanoids may lead to a reduction in lignin biosynthesis in the xylem vessels and could induce cell wall stiffening [92]. Catola et al. [93] reported that trans-2-hexenal showed a significant increase in water-stressed and recovered leaves respect to the well-watered ones in pomegranate plants. This indicated a possible role of the oxylipin pathway in the response to water stress. Metabolites changes in rice grains during water-stressed and recovery indicated involvement in stress signaling pathways such as gamma-amino butyric acid (GABA) biosynthesis, sucrose metabolism and antioxidant defense [94]. Zhang et al. [95] reported that myo-inositol and proline had striking regulatory profiles in Medicago indicating involvement in drought tolerance. Metabolite profiling of hybrid poplar genotypes revealed that amino acids, the antioxidant phenolic compounds catechin and kaempferol, as well as the osmolytes raffinose and galactinol exhibited increased abundance under drought stress, whereas metabolites involved in photorespiration, redox regulation and carbon fixation showed decreased abundance under drought stress [96]. Concentrations of flavonoids, glycosides of kaempferol, quer-

cetin and cyanidin were found in Arabidopsis during drought stress [97].

Salinity stress has been investigated at metabolite level to reveal the response mechanism. In salinity-stressed barley plants, cell division and root elongation was found associated with accumulation of amino acids, sugars and organic acids [98]. Chen and Hoehenwarter [99] reported that sucrose, fructose, glycolysis intermediates and amino acids levels were altered

stress condition [86].

86 Transgenic Crops - Emerging Trends and Future Perspectives

RNA-Seq and genome sequencing and proteomic techniques/technologies (2D, iTRAQ, MALDI, gel-free, label-free, LC-MS/MS-based technologies) have widened the dimensions of analyzing plant responses to abiotic stresses such as drought and salinity. Recent advances in the omics technologies have contributed considerably to our understanding of the plant abiotic stress-responsive mechanisms. In addition to advancing research in other related areas, emphasis has been on the proteomic analysis specific to whole plants, individual organs, tissues and cells [55]. These technologies are helping to characterize individual proteins specific to different organs, tissues and cells subjected to various abiotic stresses. Advanced proteomic information, coupled with other omics approaches would further strengthen the efforts to develop breeding programs based on identification of novel proteins/genes and their integration through marker-assisted selection. However, further efforts are required to focus on individual target points associated with "Omics" technologies and their application to dissect stress-responsive mechanisms. Research needs to be focused on several fronts such as more studies that target post translational modifications (PTMs), cell type-specific proteome analysis, advanced mapping populations in crop plants and comparative proteomic studies. PTMs of proteins may change their stability, subcellular localization, interactions with other proteins and ultimately proteins functioning. A number of studies revealed the important role of PTMs in protein functioning. Studies have been conducted to analyses protein phosphorylation in maize [108, 109], phosphorylation and ubiquitination in Arabidopsis [110, 111] and glycosylation in soybean [112] under various abiotic stresses. In addition to improved methodologies, identification of more PTMs would unravel functional characterization of important proteins involved in stress-responsive mechanisms and plant adaptation to various abiotic stresses.

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Individual proteins characterization and quantification is essential to fully explore the stressresponsive mechanisms in organs, tissues and cells. However, problems may arise due to the conventional methodologies such as protein detection on 2-DE gels [55]. Improved extraction methodologies may overcome such problems. Poor proteome coverage may be the result while detecting leaf proteome with abundance of RuBisCO that constitutes almost half of the total leaf proteins. However, proteome coverage may be improved with the recently adopted fractionation of crude protein extract. Similarly, quantification of stress responsive low abundance target proteins may be improved through selected reaction monitoring (SRM) technique [113, 114]. Such improved techniques would also help unravel commonly expressed proteins in different organs under multiple abiotic stresses. These advanced techniques coupled with improved bioinformatics approaches may help shed further light on plant responses to abiotic stresses. Recently, transgenic plants conferring abiotic stress tolerance have entered vigorous evaluations under greenhouse and filed conditions. Comparative proteomic studies of these transgenic plants may be helpful to characterize key stress-responsive factors among large number of commonly expressed proteins. Identification of major stress-responsive proteins coupled with advances in transcriptomics, metabolomics and bioinformatics tools would help unravel the complex interactions among stress-responsive signaling pathways. Moreover, omics approaches such as proteomics can be extremely helpful in analyzing post-stress recovery responses in the plants, revealing the key proteins/genes involved in the recovery stage [115].
