1.2.1 Organic and inorganic solutes

Specific types of proteins work as the essential component of cell membranes, which is porous for the ions. The membranes act as the channels to bind the ions to those proteins through diffusion and form electrochemical potential gradient. This process requires energy, which is used in the form of stored adenosine triphosphate (ATP) or ATP and pyrophosphate. This gradient across the membrane will produce differences in pH and electric potential. Therefore, the modification in electric potential forces the inward movement of cations through channels and the variation in pH regulates the movement of ions through carriers, which leads to binding of the protons and ions (Flowers and Flowers, 2005). Therefore, the efficiency of cell membranes to control the rate of ion movement in and out of the plant cell is used as an indicator of damage to a great range of tissues (Farkhondeh et al., 2012).

Ion toxicity results after the increased accumulation of salts into the crop through growing medium. This mostly includes increased levels of Na⁺ and Cl⁻, which form ionic effects and whose level is different in various crop species depending upon the severity and duration of the stress (Abrol et al., 1988). This leads to surpassing the capability of the cell to compartmentalize ions into the vacuole, or they may pass onto the cell wall and dehydrate the cell, which may result in interruption of the water relations and effective use of essential nutrients (Lacerda et al., 2003; Munns, 2005). The inclusion of Na⁺ into the plant cell taken up through growing medium will result in cytoplasmic toxicity. Therefore, uptake, accumulation, and long-distance transport of Na⁺ throughout the plant system is a critical step for plants to be affected by ion toxicity (Munns et al., 2000; Ashraf, 2004). There are reports that higher accumulation of Cl⁻ causes leaf injury in different plant species (Greenway and Munns, 1980; Flowers and Hajibagheri, 2001; Mansour et al., 2005). The toxicity of Cl⁻ causes necrosis and finally leaf senescence (Nawaz et al., 2010). The roots absorb the Cl⁻ and move through the xylem to shoots and leaves where its accumulation beyond the toxic level causes cell injury. Higher concentrations of salt increase the deposition of Na⁺ in the growing region of the root and hence decrease the selectivity for K⁺ (Ashraf et al., 2012). Therefore, proper uptake through roots and justified cellular movement, translocation, and compartmentalization in the cell are necessary for proper functioning of the plant metabolism in saline, dehydrated, and higher temperature environments (Akram et al., 2007).

Higher accumulation of Na⁺ causes a higher proportion of Na⁺/K⁺ and Na⁺/Ca⁺ in stress growth medium/conditions. The concentration of K⁺ is indirectly proportional to the Na⁺ in the cell, i.e., if Na⁺ is increasing then K⁺ and Ca²⁺ are decreasing in maize and Limonium perezii as reported by Suarez and Grieve (1988) and Carter et al. (2005), respectively, while K⁺ and Ca²⁺ both take part in the maintenance for integration and proper functioning of cell membranes, i.e., cell wall stabilization, triggering enzymes, and directing ion translocation (Carden et al., 2003; Wenxue et al., 2003).

Altered or lower levels of calcium sodium ratios in a saline environment will inhibit plant development and uptake of various nutrients in Atriplex griffithii (Khan et al., 2000), cotton (Higbie et al., 2010), and Agrostitis stolonifera (Majeed et al., 2010), and significantly change the morphological and anatomical structure (Cramer, 1992). Various nutrient deficiency with higher NaCl stress has also been reported in different crops, as can be found with K⁺ in spinach (Chow et al., 1990), artichoke (Graifenberg et al., 1995), and tomato (Lopez and Satti, 1996), and with nitrogen deficiency in cucumber (Cerda and Martinez, 1988) and lettuce and cabbage (Feigin et al., 1991), whereas phosphorus and potassium are deficient in tomato (Adams, 1991) and cucumber (Sonneveld and de Kreiji, 1999). However, artificial or foliar application of potassium in the form of KH₂PO₄, KCl, KOH, K₂CO₃, KNO₃, and K₂SO₄ improved the plant growth in wheat, sunflower, and cotton for various physiological parameters (Sherchand and Paulsen, 1985; Akram et al., 2007, 2009a,b; Jabeen and Ahmad, 2009).

The organic solutes protect the plant metabolism and play an important role in adjusting the osmotic potential. Osmotic adjustment in a plant is altered due to inorganic ion or organic solute accumulation in the cell. Net increase in the organic solutes in a cell is due to the decrease in the water potential. Besides inorganic ion accumulation, the role of the accumulation of organic solutes is also important. Cell membrane is important in maintaining the proper concentration of these solutes in the cell. Proline, trehalose, sucrose, polyols, glycinebetaine, prolinebetaine, alaninebetaine, hydroxylprolinebetaine, pipecolatebetaine, and choline O-sulfate are among the common organic solutes that play an important role for osmotic adjustment in plants under salt stress, dehydration, and some other abiotic stresses (Rhodes and Hanson, 1993; Hasegawa et al., 2000). Therefore, higher concentrations of these solutes under salt stress are favorable to plants as they participate in ROS scavenging activity, act as a ¹O₂ quencher, and maintain the OP (Marcelo-Pedrosa and Queila-Souza, 2013).

1.2.2 Role of abscisic acid under abiotic stress

Under salinity, dehydration, extreme temperatures, and other abiotic stresses, plants respond in relation to metabolic and progressive variations. Different tissues exposed to variant stresses behave in a coordinated manner to implement these responses. Salts would not be accumulated in higher concentrations in actively dividing and growing cells. Plants’ responses under stress are initiated by some indicators such as primary osmotic stress and/or secondary metabolites (Chaves et al., 2003). Secondary metabolites signals comprise growth hormones like abscisic acid, ethylene, and cytokinins, and reactive oxygen species and intracellular phospholipids or sugars. It was observed that the ability of isopentenyl transferase (IPT) to increase cytokinin biosynthesis, hence delaying senescence and the ability of CBL-interacting protein kinase to sense and respond to the calcium concentrations, has been used to make cotton plants more robust under drought conditions (Kuppu et al., 2013; He et al., 2013). Abscisic acid (ABA) is initiated to produce in roots, translocates to shoots through xylem, and signals the stomata to close, which in the long run limits the cellular division and expansion and reduction in stomatal conductance in response to stress (Aldesuquy and Ibrahim, 2001). Munns et al. (2000) conducted experiments on shoot water relations and verified that the osmotic effects under salt stress induce the hormonal signals outside the roots to regulate cell expansion. Artificial application of ABA to common bean plants induced higher K⁺/Na⁺ ratios and so restricted the translocation of sodium to shoots (Khadri et al., 2007; Chaves et al., 2009). It is evident that along with root cells, ABA may also be synthesized in leaf cells and distributed from there throughout the whole plant (Wilkinson and Davies, 2002). Flexas et al. (2006) also proved that stomatal conductance is reduced when the leaves are cut off the plants, but the same has been recovered very quickly after the foliar application of ABA to the plants. They also reported the response of stomatal conductance to alterations in photoperiod, temperature, and availability of CO₂ (Flexas et al., 2008).

The role of other growth hormones like GA₃ has been reported to compensate for the adverse effect of stress on different plant tissues (Naqvi, 1999; Chakraborti and Mukherji, 2003). Shah (2007) reported on the foliar application of GA₃ under salt-stressed plants and observed the improvements in the dry mass, leaf area expansion, photosynthetic rate, and stomatal conductance. The accumulation of salts under dehydrated conditions induces the production of ABA to close the stomata and regulate the plants’ growth under stress conditions which accounted for the restricted production of ABA during the process of conjugation. The reduced stomatal conductance and rescuing of productivity was aggravated by the foliar application of GA₃ as these results were inconsistent with those of Afroz et al. (2005). Similarly, Guo et al. (2011) guessed that GhWRKY3—a transcription factor from Gossypium sp.—will possibly play a role towards ABA- and GA₃-intermediated pathways that signal and regulate plant growth and development and generate defense responses against pathogens.

Late embryogenesis abundant (LEA) genes have been identified that are responsive to ABA, and a number of ABA responsive elements (ABREs) are present on the promoters that work together with other nuclear protein factors in different crops. These LEA genes are mostly present in mature embryos and some other vegetative tissues under abiotic stresses (Luo et al., 2008). LEA proteins are known for their role in protecting cells under osmotic stress due to hydrophilic characteristics and as they accumulate under the abiotic stress in plants. Advances in this research indicate that the pH of xylem and leaf apoplast affects ABA production and subsequently translocates to the stomata. Under salt stress or more alkaline pH, exclusion of ABA from xylem and leaf apoplast may be decreased and hence more ABA will reach the guard cells, which will facilitate the variation of stomatal aperture (Jia and Davies, 2007).

1.2.3 Reactive oxygen species

Higher amounts of salts taken up from the soil by plants at higher temperatures restrict the availability of water and cause drought stress as well. Consequently, plants close the stomata to retain water and this will also limit the entrance of carbon dioxide into the leaves. This will decrease the process of photosynthesis, or if the salts are at too high a concentration or at toxic levels, then photosynthesis will be inhibited directly. In this situation, plants suffer from oxidative stress, which is an additional significant feature of this stressful condition.

The formation of reactive oxygen species (ROS) is the outcome of sunlight absorption by plants. This mainly takes place in the chloroplast through superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet excited oxygen as a routine function of plants’ aerobic respiratory system. ROS are the reason to why cell membranes, nucleic acids degradation, and proteins in the cell are damaged because they are extremely reactive. Photorespiration, oxidation of fatty acids, and mitochondrial and chloroplast electron transport systems (PSI and PSII) are the various categories of functions performed in the cell that generate ROS (Hernàndez et al., 2001; Foyer and Noctor, 2003). A number of reports are available for the effects of abiotic stress comprising ROS (Bor et al., 2003; Stepien and Klobus, 2005), but the limit of these processes stimulated under specific conditions as well as the magnitude of the damage or the differences in their capability acquired by the plants under stress has not been well understood. Irregular concentrations of antioxidants in chloroplast and changes to enzymatic activities may regulate the ROS to damage the plant systems (Asada, 2000), but transgenic plant development is another way to control the damage (Apse et al., 1999; Kumar et al., 2012).

1.5.1 Advances in phenology assist the genomic studies of abiotic stress tolerance

An abiotic stress mechanism is complex to understand at the molecular level, but the advances in phenotypic studies have helped to quantify the components contributing to tolerate the abiotic stress at the genetic level. Therefore, phenomic developments along with genomic methods in the crops with more complex genomes are becoming increasingly amenable because the genomic evidences in non-model crops are now available (Roy et al., 2011).

Plant morphological or phenotypic studies are commonly difficult, time consuming, and destructive as most require removing the whole plant biomass. But now there are imaging tools that are non-destructive and are used to take several images of the same plant at different time intervals and wavelengths. These advances have made it possible to offer non-destructive approaches to obtain computable data in a number of crops at different growth stages under different types of abiotic stresses like drought, salt, cold, and heat (Morison et al., 2008; Rajendran et al., 2009; Sirault et al., 2009; Berger et al., 2010). Most of the phenotypic studies have concentrated upon the shoots or traits related to the stems or leaves and have ignored the roots. But in spite of the contribution of the roots to tolerate abiotic stress, the studies related to the roots are fewer. This might be because it is difficult to phenotype the roots without damaging or donating the whole plant (Richards et al., 2010; Fleury et al., 2010; Zhu et al., 2011b). Some of these reports are known for the important root traits that tolerate abiotic stresses, like metal toxicity (Jefferies et al., 1999; Ma et al., 2005) and nutrient deficiency (Laperche et al., 2006; Walk et al., 2006; Zhu et al., 2011a). Therefore, for further explanations of abiotic stress tolerance through roots, it is desirable to make improvements to the methods developed for phenotyping of roots, and environmental factors affecting plants growth may also be considered (Berger et al., 2010). Comparison of the crops growing under controlled conditions, i.e., in a greenhouse and in the field conditions, is important to elucidate the complexity of the crops’ tolerance mechanism for superimposed environmental variations or the naturally occurring climatic disorders.

With the introduction of high-throughput studies, a number of phenomics assays are now accessible online to speed up the gene identification and molecular marker-assisted genomics studies related through phenomics. One of these assays is rapid elemental analysis of plant tissue, which is helpful to estimate the ionic accumulation in different plant tissues under particular abiotic stress conditions (Baxter et al., 2007). A number of developed countries have plant growth chambers/facilities furnished with mechanized systems that bring the plants for experimental analyses to, for example, irrigation, imaging, and/or weighing stations. Data obtained are analyzed with computational software that includes: The Plant Accelerator1 (http://www.plantaccelerator.org.au) in Australia, Crop Design (http://www.cropdesign.com) in Belgium, and the Leibniz Institute of Plant Genetics and Crop Plant Research in Germany (http://www.ipk-gatersleben.de/Internet). Results of these analyses in combination with the genomics approaches will help to discover the genes contributing to the known abiotic stress tolerance mechanism in crop plants. To minimize the experimental errors, the plant populations tested in greenhouses are required to be compared/tested under different field conditions several times and the abiotic stress-related components must be studied carefully.

The latest advances in phenomics for measurement of shoot-related parameters without damaging the plant aerial parts are digital RGB images and infrared thermography (Jones et al., 2009; Berger et al., 2010; Munns et al., 2010). Similarly, minirhizotrons, ground penetrating radar, and electrical resistivity imaging have been introduced to analyze the roots non-invasively (Zhu et al., 2011b). Genomics studies for plants grown in field conditions became more accurate with high-resolution EM38 mapping for characterization of defined growth conditions (Furbank, 2009; Robinson et al., 2009). Thus, genetic approaches in combination with phenotypic studies to expose the molecular mechanism of abiotic stress tolerance in crops are becoming more easily attainable.

1.5.2 Molecular mapping

Correlation of molecular markers with phenotypic studies of plants is important to know the exact point of qualitative and quantitative genetic variation harboring the dynamics manipulating the plant’s response under abiotic stress. Yield or associated secondary traits are main components for crop improvement, and identification of quantitative trait loci (QTLs) for these traits is very strategic to improve stress tolerance in plants through marker-assisted selection. The loci for variation may be identified at structural or expression level and further tested in other germplasms, or alleles may be discovered to be used for breeding and selection into high yielding and elite cultivars of agriculturally important crops (Langridge et al., 2006). To date, QTL mapping is an exciting option, rather than using previously listed DNA markers such as RFLP, AFLP, RAPD, SSR, and SNP (Ashraf et al., 2008). So, gene pyramiding at two or more loci is possible through molecular marker-based selection (Asins, 2002). The selection procedure is mainly dependent on specific environmental conditions, which are major limitations encountered in the conventional breeding of the traits affected by abiotic stresses. Once a marker-related featured link has been marked undoubtedly, then selection could be minimized to a great extent (Humphreys and Humphreys, 2005; Tuberosa and Salvi, 2006).

Several mapping studies have identified numerous QTLs associated with salinity, drought, extreme temperatures, and other abiotic stress tolerance loci relevant to performance in less yielding areas in a number of crop species (such as that found at http://www.gramene.org/qtl/). The major crops mapped for different traits through QTL-related abiotic stresses are for cold, heat, salinity, mineral toxicities, nutrient deficiencies, and drought in cotton (Saranga et al., 2001), wheat (Quarrie et al., 1994), maize (Feng-Ling et al., 2008), soybean (Cornelious et al., 2005), barley (Teulat et al., 1997), sorghum (Sanchez et al., 2002), and rice (Lafitte et al., 2004; Mizoi and Yamaguchi-Shinozaki, 2013), among others (Table 1.1). Although the application and achievements through marker-assisted selection seems to be simple and easy-to-induce stress tolerance, the constraint is the precise and accurate identification of QTL and the proficient capability relevant to this research application. Broad diversity has been identified in the complexity of stress tolerance through the mapping program, and its association with breeding has expanded the mapping studies. Most of these studies are based on the field evaluation of crops for abiotic stress tolerance (Ahmed et al., 2013). This identified diversity in the stress tolerance mechanism may prove a key source to validate the candidate genes and will be a helpful mechanism to communicate the findings of genomics to an efficient/practical plant breeding program (Ismail et al., 2007).

Since the abiotic stress tolerance mechanism has a multi-gene trait, then wide mapping of some specific traits in different populations of the same plant species may identify the common loci (Langridge et al., 2006). Large segregating populations are needed for positional cloning through high-resolution linkage maps, and combining tasks such as identification of loci and positional cloning may be the alternative easier way in the future for plant breeding to induce abiotic stress tolerance (Roy et al., 2011).

1.5.3 Expression sequence tags

Although the genetic sequencing was found to be a good source of genome information, the large size of the genome of some plant species or the non-coding part of the genome sequence was still a limitation related to the genetic information. Plants with larger genomes are unpopular or are difficult to sequence because they contain copies of large repeats of genes, which do not provide information of coding genes; the purpose of sequencing is to get the information for discovery and characterization of the genes for proteomics. This has led to the progression to mass mode to anticipate the actual regions of protein coding constituents in a genome. Therefore, an expression sequence tags (EST)-based study was developed as the genomics approach to overcome the problems associated with the large genome sequence or noncoding part of the genetic sequence (Rudd, 2003).

The early 1980s saw the initial use of cDNA as a method to explore gene discovery (Putney et al., 1983), which in 1990 was further extended by supporting the implication of a high-throughput approach for transcript profiling as characterization of the coding region of human genome would involve messengers from the expressed genes (Brenner, 1990). Continuing this study, Adams et al. (1991) described the term EST, correlated with gene discovery and the human genome project. Since these reports, more than 10 million ESTs have been sequenced from more than 500 distinctly annotated species (fungi, plants, animals) (Rudd, 2003).

Langridge et al. (2006) considered ESTs and cDNA libraries as “Electronic Northern” and John and Spangenberg, (2005) considered them as “in silico Northern analysis,” producing a large number of sequences appraising gene expression and being a useful method for prevalence of transcript abundance at the primary level. Analysis of EST data shows that the homologous gene shows variation among the genes and also that they are tissue specific (Mochida et al., 2004). There are a large number of data produced by the ESTs and there is a report of 449,101 ESTs for drought, 312,353 ESTs for salt, 103,898 ESTs for low temperature, 252,595 ESTs for high temperature, 19,384 ESTs for nutrient deficiency, and 135,578 ESTs for light stress on the National Center for Biotechnology Information browser (see http://www.ncbi.nlm.nih.gov/).

EST is a simple and easy way to obtain the sequencing of complicated and targeted plant species under abiotic stress conditions. It provides the mRNA with abundant expression profile of specific sequences from cDNA under specific conditions. To observe the expression of clones differentially expressing certain genes, libraries are constructed with cDNA that contain 10,000 clones acquired from targeted plant species under specific abiotic stress compared with the control (Pariset et al., 2009). Ubiquitously expressing housekeeping genes within the cells may also be considered for validation studies. It depends upon sampling of the plant tissues or organs taken from roots, leaves, etc., under salt, drought, frost, or other abiotic stresses.

Complete analysis of the core gene assembly is a complicated issue and difficult to resolve. Sampling of all transcribing genes is only possible if mRNA is gathered from all available types of cells at each and every growing stage of the plant under as many combinations of biological and environmental trials as possible. This is very complex and can only be possible through extensive experimental planning and comprehensive collection of cell types under a wider variety of environmental threats. These assemblies of cDNA and ESTs are expected to comprise the differentially expressed genes, which are stress induced and expected to play key a role in plants’ tolerance mechanisms.

Demonstration of the host gene within a library and the quality of sequence are the limiting factors associated with EST technology and this is difficult to overcome due to the difficulty in observing any transcript from a tissue under some specific stress, and the sequencing may contain a background of incorrect (partially or incomplete) sequences. Initially, the EST sequencing was preferred for the 5′ end due to the coding or conserved regions, but with the advancements in sequencing technology, EST sequencing at both ends is becoming prevalent due to high-throughput sequencing of plants’ ESTs. It may also provide more distinctive sequences and hence be the possible solution to sequencing problems within ESTs. Hence, this is the link to the genome if the complete sequence of the genome is lacking.

1.5.4 Microarray

Limitations for improvements or development of salt-tolerant crops are the partial understanding of physiological criterion that reveals the genetic prospective and/or hereditary restrictions in combination with conventional breeding under abiotic stress-growing medium. But the turning point in understanding the key traits responsible for reducing productivity under abiotic stress is the meaningful approach to assimilate the plant’s responses at the physiological as well as molecular level. Combining this approach with traditional breeding will boost the improvements in productivity (Araus et al., 2002). This will also help to produce crops suitable for our environment and for sustainable agriculture. “Omics” (metabolomics, transcriptomics, proteomics, and genomics) is involved throughout stress responses in plants (Saeed et al., 2012). A number of abiotic stress responsive genes with known structure and functions have been identified after the advent of extensive projects on genomics and helped to unravel the complexity of many associated genes (Hamel et al., 2006). In addition to identifying genes, high-throughput studies are aimed at the regulation of genes at the transcriptional level (Yanik et al., 2013). Proteome analyses reveal several proteins as showing peculiarity in their expression patterns under drought conditions (Zadraznik et al., 2013). Careful interpretation of these analyses has brought to light various mechanisms that operate during drought and other environmental challenges at both the molecular and cellular level. Recent advances in genomics research have progressively contributed to enhance the genetic improvements of the agriculturally important crops and a number of other promising plant species.

Microarray technology has been used over the past 20 years to identify the expression of high-throughput genes or to measure the expression of thousands of genes in a single experiment. These studies may be of cDNA inserts, fragments of short 20-25mers, or long oligonucleotides of 60-80mers (Lockhart and Winzeler, 2000). The model plant Arabidopsis played a key role in the identification of many stress-induced genes. But now this technology is being used to identify other abiotic stress-related genes in a number of plant species such as cotton (Arpat et al., 2004; Udall et al., 2006; Zhang et al., 2008; Payton et al., 2011; Barozai and Husnain, 2012), model plant Arabidopsis (Bray, 2002), rice (Gorantla et al., 2007; Hadiarto and Tran, 2011), beans (Micheletto et al., 2007; Sanchez et al., 2011), alfalfa (Kersey, 2004), and many others (Oh et al., 2010).

A variety of microarray platforms is available, i.e., oligonucleotide and cDNA. Oligonucleotide microarray consists of an abundance of mRNA transcripts in some specific tissue or cell for differential expression of several genes at one time. The “array or grid” of linearized complementary DNA from genes under investigation is plotted onto the glass slides. A single slide may have an array of thousands of spots. Private companies (Affymetrix, Agilent, and NimbleGen) are also available to design and spot the arrays. The in-house printing may be cheaper than from the private companies. Spotted slides are hybridized with the mRNA extracted from the sample to be investigated. After hybridization, the expression of the differentially expressed genes will be investigated by correlating the spots of complementary DNA and abundance of mRNA transcript from the labeled sample (Lipshutz et al., 1999). Complex and extensive computational and statistical analysis is compulsory for the exploration of data from such experiments.

Several batches of cDNA- or EST-based microarray data have developed in cotton for fiber, plant growth and development, abiotic stresses, and pathogen-related genes (Zhang et al., 2008; Rodriguez-Uribe et al., 2011). Cotton fiber genes have been studied at different developmental stages such as 10 and 24 days post-anthesis and it was found that gene expression was altered progressively from initiation to elongation. Microarray results showed the developmental differences at primary to secondary cell wall metabolism. The number of genes up- or down-regulated confirmed that this process is associated with the plant developmental stage (Arpat et al., 2004). A number of ESTs have been collected to generate cDNA microarray from different plant species subjected to a number of abiotic stresses such as freezing, salt, water deficit, and metal toxicity. Microarray was developed by spotting unigene, and a transcript profile was done to classify the genes expressing under subjected abiotic stresses. Functional categorization of a number of candidate genes expressing have been performed and the method was found to be a useful tool for sequence annotation and transcriptome analysis (John and Spangenberg, 2005).

Identification and characterization of the genes involved in different metabolic processes have facilitated understanding of the basic molecular mechanism involved and thus breeders can improve the genetic capability of the crops (Parida and Das, 2005; Yokotani et al., 2013). Certain regulatory pathways are common and shared across the species, such as a microarray of certain species of wheat showing expression of enzymes and osmolytes under water deficit, which suggests that these mechanisms are genotype, duration, severity, and type of stress exposure dependent (Mohammadi et al., 2007). Insufficient identification of transcripts sometimes means that there is scarce selection of a cDNA array based on EST selection. For this solution, large-scale EST selection and sequencing from different tissues at different developmental growth stages are required (Cushman and Bohnert, 2000). There is evidence that many of the genes identified through microarray have confirmed the tolerance of abiotic stress in transgenic plants (Bar et al., 2013). For those species where representative array is not available for analysis or comparison, cross-species hybridization and analysis are now available on the publicly available repositories. Model species such as Arabidopsis, rice, etc., play an important role in this limitation (Pariset et al., 2009), as whole genome of Arabidopsis was complete in 2000, so all the predicted genes could be monitored for expression profiles in a single microarray experiment at one time (Hirayama and Shinozaki, 2010). In a broader sense, the abiotic stress-induced genes identified through microarray can be classified as the first group of proteins (heat shock proteins and late embryogenesis abundant proteins) transcribed directly under abiotic stress. The second group consists of the regulatory proteins related to signal transduction pathways (MAPK, enzymes, and transcription factors). These functional groups of genes competently stimulate the cellular machinery of plants to respond to abiotic stress.

There are public repositories, like GEO (Gene Expression Omnibus) and Array Express, where microarray data can be deposited. These databases are easily available and accessible to the public, which is helpful for further hypothesis and research findings that may lead to major conclusions (Mah et al., 2004; Sanchez et al., 2011; Tseng et al., 2012). Table 1.2 summarizes the different plant species under salt and/or in combination with multiple abiotic stresses as reported on the GEO Data Set record (http://www.ncbi.nlm.nih.gov/gds). There are certain standards for data submission like experimental design, data input, normalization, and data annotation, so that scientists from different backgrounds can access the data in the same pattern and genes have been identified related to responsiveness to abiotic stresses alone or in combination with different plant species. The significance of microarray technology can be further strengthened due to the publication and availability of 88,696 free full text articles over the last 5 years (http://www.ncbi.nlm.nih.gov/gquery/?term=microarray) compared with only 4800 articles in 2000–2003 (Mah et al., 2004).

The use of microarrays in future functional genomics tools will probably lead to gene regulation at the transcriptional level for different tissues in cotton and other agriculturally important crops. However, these microarray-based methods are expected to be innovative when they become an essential part of the research/experimental activities of a routine molecular biology laboratory.

1.5.5 Proteomics and metabolomics

Plants will always be open to environmental threats as they are exposed to various abiotic stresses. Adaptation to these stresses is a complex process at the cellular and physiological level, and the acquired modifications in gene expression monitor the alterations in protein profiles (Parker et al., 2006). Proteomics and metabolomics are comparatively new research areas to emerge from the post-genomic era and there are only a few published reports on these applications to abiotic stress tolerance (Bahrman et al., 2004). Hence, these innovative “omics” perspectives have provoked curiosity among genomics scientists and opened up new horizons in plant stress biology (Khan et al., 2007). While transcriptomic studies are in progress to unravel gene transcription and regulation, gene functions and expression profiles can certainly be accomplished by proteomic study, as the primary protein sequences undergo proteolysis after post-translational modification. Accordingly, protein-level quantification of expressed genes is essentially done to measure the plant’s response to abiotic stress.

Proteomics techniques such as GC-MS and 2D gel electrophoresis are well known for protein profiling. A number of key proteins could be identified and quantified in a single gel for a specific subject area. For example, Salekdeh et al. (2002) identified 3000 proteins from rice drought and salt-stressed plants from a single gel: over 1000 were quantified among those and 42 were found worthy of further study related to their abundance or position in response to the subjected stress. Defined tissues of wheat resolved significant proteins through proteomic studies. Those proteins identified by proteomic methods have proven their functional involvement in abiotic stress tolerance mechanisms (Skylas et al., 2005; Woo et al., 2003).

Abiotic stresses impact the plant’s physiological processes such as photosynthesis—the effects on the Calvin cycle and results in reduction of plant growth and development. To adapt or to cope with these conditions, protein profiles regulating the mechanism of glycolysis and amino acid biosynthesis are up-regulated for ATP synthesis and osmotic adjustments. Molecular chaperones and C₄ photosynthesis encoding proteins are up-regulated as defense mechanisms against salt in maize, and such genes may be the valuable source for genetic transformation to induce salt tolerance to other agronomically important crops. Soybean and potatoes are relatively salt sensitive and up- or down-regulation of certain protein profiles suggests that these crops are sensitive to photosynthesis and protein biosynthesis-related proteins, but tolerant to mechanisms like water potential, osmotic adjustments, and antioxidants (Sobhanian et al., 2011).

Plasma membrane plays a key role in regulating several cellular processes; therefore, it is considered to be a prime location of damage after abiotic stress such as cold. Freezing or cold is also a serious threat to crops especially to the cold-sensitive/susceptible species that do not adapt to severe cold temperatures due to limited functions to modify the cellular machinery. Tolerant species may have the adaptation mechanism as they may alter the plasma membrane, and hence many key proteins may be involved in the pathway. So, proteomic skills are useful to identify the regulatory and functional protein in the freezing mechanism, and identification of those candidate genes may help to alter the plasma membrane functions under low temperature stress. Hence, this research application is of key importance for plant molecular breeding strategies to induce the low temperature tolerance mechanism in crop plants (Takahashi et al., 2013). To induce abiotic stress tolerance to crops, different plant species should be identified for different types of stresses, and the approaches for tolerance levels should be listed. This would be accomplished with rigorous research on each plant species’ response to specific stresses. The evaluation of susceptible and tolerant cultivars/species should be done for proteome profiling in order to reveal the distinctive mechanisms involved in this challenge.

Plant growth and development is affected by exposure to salts and plants may experience various symbiotic restraints; these include water deficit, ionic toxicity, and oxidation stress, which lead to metabolic and nutrient discrepancy that culminates in a complex physiological disorder. Metabolomics is another area in the genomics approach that deals with the metabolic changes in plant responses to abiotic stresses, which suggests that thorough profiling of metabolites may offer enormous understanding for stress tolerance pathways. This is a relatively innovative area and there are not too many reports on its application. However, one of the available reports found 88 main metabolites from the extract of rice leaves, most of them covered pathways of sugar and amino acid metabolism (Sato et al., 2004). This was further confirmed by Sanchez et al. (2010), in that polyols, sugars, and specific amino acids are accumulated and usually termed as compatible solutes or osmolytes for osmotic adjustments. Further, these metabolites are also involved to regulate physiological responses such as ROS, cell membrane stability, protein protection from degradation, and cell signaling. Accumulation or decreased metabolites is also specific for distinct species, so it may help to identify the stress-susceptible or -tolerant species (Szabados and Savouré, 2010).

Carbohydrates and secondary metabolites play key roles during the post-harvest of fruit crops. Understanding the molecular pathways involved in the shelf-life of fruit can help to improve fruit quality (softening and ripening). Sorbitol concentration was reduced due to down-regulation of sorbitol dehydrogenase, and sugar was increased due to up-regulation of sucrose synthase in the transgenic apples harboring the aldose-6-phosphate reductase (A6PR) gene with antisense orientation, which led to improvement in photosynthesis and CO₂ that produced good vegetative growth (Zhou et al., 2006). Sucrose contents were reduced with increased polymerization of pro-anthocyanidins with improved volatile compounds, such as carotenoid and shikimate-derived volatiles in grapevines-overexpressed Adh alcohol dehydrogenase, known to be regulated progressively under environmental threats. Strawberry, due to its small genome and faster growth, is a preferred model among fruit crops for functional genomics. A small subunit of ADP-glucose pyrophosphorylase (AGPase) was regulated under the promoter ascorbate peroxidase (APX), which overexpressed the pyrophosphate mechanism. Assessing the metabolic pathway reveals that fructose-6-phosphate 1-phosphotransferase (PFP) is a cytosolic enzyme that catalyzes the glycolysis, which leads to the accumulation of sugars and degrades the starch contents in transgenic plants (Basson et al., 2011).

The flavanoid compound family contains one of the water soluble pigments—anthocyanin—which are responsible for the color trait in fruits and flowers. These flavonoids are regulated and expressed through MYB well-known transcription factors. Strawberry transgenic plants harboring FaMYB10 showed enhanced levels of anthocyanins in different plant parts, but the silencing of anthocyanidin synthase (MdANS) in red-leaved apple produced necrotic leaves, and flavonoids/anthocyanins-altered metabolic pathway was observed (Szankowski et al., 2009b; Lin-Wang et al., 2010). Transgenic plants have been used to analyze the flavonoids and other metabolic profiling, up- or down-regulated genes, and associated metabolic pathways, and this study might be helpful to unravel the unknown genes and metabolites involved during the plants’ stress challenges. Hence, the genome sequences of many plant species are available and some of them are important as they contain genes involved for the production of particular components completely or partially known or unknown. Therefore this may likely to expose regulatory or functional mechanisms relevant to functions of metabolites in whole or plant parts.

1.5.6 Transgenic plants for improved salinity tolerance

Abiotic stress causes significant plant yield losses and hence lowers crop production (Orsini et al., 2010). In addition to desiccation, ionic toxicity and ROS stress plants also face metabolic and nutrient mismanagement and all these lead to different physiological responses (Ashraf, 2004; Sanchez et al., 2011). Therefore, production of crops tolerant to stress is the ultimate and important solution for sustainable agriculture to meet the demands of the growing population (Aquino et al., 2011). Molecular biology, genomics, or biotechnology applications in combination with plant breeding are being successively used to produce transgenic crops/plants to overcome this crop productivity threat. Genomics approaches have provided insights into the functional regulation of the key genes participating in the stress tolerance mechanism of plants. New attempts have been reported by using genomics approaches such as marker-free selection of plants, tissue-specific promoters, and induction of disease resistance (fungal, bacterial, and viral) (Gambino and Gribaudo, 2012). Table 1.3 summarizes a number of genes identified individually or in combination with abiotic and biotic stress tolerance in different plants.

Plants are adapted to saline environmental conditions in a number of ways and among these is the compartmentalization of Na⁺ into the vacuole, which helps to maintain the osmotic balance. Transgenic legume forage (Medicago sativa) was developed with AVP1, a vacuolar H⁺-pyrophosphatase (H⁺-PPase) gene from Arabidopsis thaliana, to adapt to saline and arid soils (Bao et al., 2009). Transgenic plants were developed which expressed the AtNHX1 gene (a vacuolar Na⁺/H⁺ antiporter). This gene has been reported to reduce the cytosolic Na⁺ by sequestering Na⁺ in the vacuole (Asif et al., 2011; Jha et al., 2011). The transgenic plants were produced with better germination rates and showed improved fresh and dry biomass in severe saline conditions (Xue et al., 2004). Another plasma membrane Na⁺/H⁺ antiporter, i.e., the SOS1 gene from Arabidopsis thaliana, was tested into the model plant tobacco and showed improved germination, vegetative growth, and chlorophyll contents in transgenic plants as compared to the non-transgenic (Pons et al., 2011; Yue et al., 2012). By reducing the expression of the sodium/proton antiporter SOS1 through genetic engineering affects the numerous pathways, indicating a role for SOS1 that exceeds its known function as an antiporter (Oh et al., 2009, 2010). Tissue-specific expressions for the SOS genes may contribute a significant role to Na⁺ regulation (Kumar et al., 2009). The higher amount of Na⁺ and Cl⁻ separately under NaCl treatment restricts the plants’ growth and productivity through different but simultaneous mechanisms. It has been documented that a higher level of Na⁺ hampers K⁺ and Ca²⁺ nutrients whereas a high concentration of Cl⁻ degrades the chlorophyll and decreases the photosynthesis (Tavakkoli et al., 2010).

Cyclophilin (Cyp) genes belong to the ubiquitous proteins family and are capable of peptidyl-prolyl isomerase activity, which helps to catalyze the cis/trans isomerize, the peptide bond at proline residues and protein folding. These Cyps have been reported to regulate gene functions at cell division, signaling, transcriptional regulation, and pre-mRNA splicing under different environmental stresses including temperature, salt, and drought (Zhu et al., 2011a). There is the possibility that some of the genes perform vital roles to acclimatize and tolerate the stressful environment, but this is not likely to conclude the stress transcriptome of a genus on the basis of only one species (Sanchez et al., 2011). Proline and trehalose accumulation has been reported in response to plant adaptation, and related genes expression has been found increased and this could be applied to help the plants cope under abiotic stresses (Nounjan et al., 2012). The MtSAP1 gene isolated from Medicago truncatula has been found overexpressing in tobacco transgenic seedlings and showed tolerance to extreme temperature, osmotic, and salt stresses (Charrier et al., 2013).

Transcription factors are considered as the potent application for the plants to adapt under different abiotic stresses (Martin et al., 2012; Mizoi and Yamaguchi-Shinozaki, 2013; Naika et al., 2013). Transcription factors genes of the basic leucine zipper (bZIP) and zinc finger protein regulate the pathways responsible for plant growth under abiotic stress and metal toxicity. ZmbZIP72, a transcription factor from maize bZIP, has showed improved tolerance in Arabidopsis under different types of abiotic stresses (Ying et al., 2012). ABI5 is a transcription factor of the bZIP family, which binds with the ABA response element (ABRE) present in AtEm6 gene, hence regulating its expression. AtEm6 is reported to increase salt tolerance by manipulating calcium-dependent protein kinases (Tang and Page, 2013). The NAC is found to be one of the largest transcription factor families and its proteins are characterized by a highly conserved DNA-binding domain. These are abundant only in plants and perform key roles during different plant developmental stages. TaNAC2, an NAC transcription factor, was isolated from wheat and then overexpression was observed in the model plant Arabidopsis under different abiotic stresses including salt, drought, ABA, etc. (Mao et al., 2012). The DREB-1A transcription factor gene has enriched the drought tolerance in transgenic wheat (Shen et al., 2003). Another illustration is the overexpression of ornithine aminotransferase for salt- and water-deficit tolerance in Arabidopsis, which further has been transformed into wheat to induce the tolerance mechanism (Roosens et al., 1998). Another transcription factor gene from the ethylene responsive factor W6 gene from wheat showed overexpression in tobacco and improved the tolerance mechanism under saline stress condition.

The changes in biochemical and physiological parameters observed in transgenic plants compared with non-transgenic plants found the up-regulation of transgene in transgenic plants (Yan et al., 2008). The Stress responsive Transcription Data Base (STIFDB) is a collection of identified stress-related signals, responsive genes, and transcription factors. It catalogues all the available information on putative binding sites of the stress responsive transcription factors in the model species Arabidopsis (Shameer et al., 2009). The updated version of this database is STIFDB2, which contains supplementary information related to other agriculturally important crop species, i.e., maize, sorghum and soybean, novel stress-related signals, newly identified transcription factors and their regulatory sites, and addition of the stress responsive genes from microarray-based experiments (Naika et al., 2013). All this information will be helpful for plant and computational biologists to further understand plants’ stress-related mechanisms.

Induction for production of certain enzymes like glutamate synthase, proline, or regulation of organic solute such as proline and glycinebetain and antioxidant enzymes under salt and other abiotic stresses is regulated by signal transduction pathway (Misra and Saxena, 2009; Yang et al., 2013). Salicylic acid is reported to be part of the signaling pathway and considered to take part in improving the functions related to photosynthesis, which ultimately would help to improve the contrary effects of salts and thus help to improve the plant defense mechanism by improving the physiological, metabolic, and biochemical pathway (Idrees et al., 2011). The combined effect of cassava Cu/Zn superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) improved cytosolic expression and maintained the ROS scavengers by showing improved shelf-life of cassava roots to combat abiotic stress. SOD, CAT, proline accumulation, and water-related attributes were improved and lowered the malendialdehyde in transgenic cassava under cold stress (Xu et al., 2013).

Ethylene production has a key function at post-harvest and affects the shelf-life of fruits. This induces several genes altogether, which transcribes and regulates the functional genes affecting the storage of fruits. ACS (ACC synthase; ACC-1-aminocyclopropane-1-carboxylic acid) and ACO (ACC oxidase) are the basic enzymes known for ethylene biosynthesis. Transgenic apple plants silenced with either of these enzymes produced less ethylene due to suppression of volatile ester synthesis, and fruit maturity features were observed (Dandekar et al., 2004; Johnston et al., 2009). ACO suppression was done by RNAi in kiwi and papaya fruits and volatile production was reduced, which led to reduced ethylene production and improved shelf-life of the fruits thereby extending and fruit storage (López-Gómez et al., 2009; Atkinson et al., 2011)

Plant growth and development under abiotic stresses is also regulated by the hormonal activation and in this regard brassinosteroid (BR) is reported to interact with ABA; various BR receptive genes are also responsive to ABA (Cui et al., 2012). These BRs are steroidal compounds, ubiquitous, distributed in free form, and conjugated to starch and lipids and thus help the plants to adapt under salt and other abiotic stresses (Hayat et al., 2012). ABA is considered a stress regulating plant hormone and plays a significant role in changing gene expression profile and cellular responses, as this is the most studied plant hormone under abiotic stress. NCEDs (9-cis-epoxycarotenoid di-oxygenases) and P450 CYP707As are the two enzymes involved in ABA biosynthetic and catabolic pathways, respectively, and their genes are activated after abiotic stresses. When the plants are growing under normal conditions, ABA exists within vacuoles and apoplasts as ABA glucosyl ester, which is an inactive form released by the action of β-glucosidase when the plants are dehydrated (Hirayama and Shinozaki, 2010).

There are other reports suggesting that some of the enzyme-related genes like betaine aldehyde dehydrogenase (BADH), pyrroline-5-carboxylate synthetase (P5CS), mannitol-1-phosphodehydrogenase (mtlD), 6-sorbitol dehydrogenase phosphatase (gutD), and late embryogenesis LEA, HVA1, and ME-leaN4 have been overexpressing among different crops under different stress condition (Swire-Clark and Marcotte, 1999; Prabhavathi et al., 2002; Sawahel and Hassan, 2004; Oraby et al., 2005; Park et al., 2005; Yan et al., 2008).

Even though inducing abiotic stress tolerance in plants through genetic engineering is quite successful in many agriculturally important crops, this approach will simply be authenticated after validation of the results of successive generations produced in field trials. When plants are grown as part of the plant community under field conditions and are subjected to multiple stresses, then various essential abiotic stress reactions induced in plants relate to their performance as a crop. Thus, the initial outcomes of the genes from model species are still to be studied to reach factual consideration.