Procedimientos metodológicos para el desarrollo de las pruebas

Tissue tolerance is a term used to describe the plant's ability to grow under salt conditions (Munns and Tester, 2008). The tissue tolerance mechanism is about ensuring the survival of old leaves and improving growth (Munns and Tester, 2008). The compartmentalisation

of Na+ _{and Cl}- _{ions at the cellular and intracellular level is requisite in tissue tolerance phase}

in order to prevent the toxicity effects of higher concentrations of these ions to the mesophyll cells in the leaf (Munns and Tester, 2008). Also significant is an increase in accumulation of compatible solutes within the cytoplasm under salt stress. The compatible solutes are examples of small molecules, water soluble, derivatives of sugars, phenolic amino acids, and other complex sugars (Ashraf and Foolad, 2007). They play an important role in osmo- tolerance, protecting enzymes from denaturation, thereby ensuring membrane and macromolecules stability mediating the osmotic adjustment (Ashraf and Foolad, 2007). Their main functions are not limited to balancing and osmotic adjustment as they are involved in regulation of hydrophilicity, and replacement of water molecules at the surface of proteins and membranes, by creating and serving as low molecular weight chaperones (Hasegawa et al., 2000). Studies have also reported their involvement in a scavenging function against the ROS in the stabilisation of cellular structures (Hasegawa et al., 2000; Zhu et al., 2015). Their unique characteristics have attributed to their neutral nature, therefore high accumulation of compatible solutes do not affect cellular processes (Sakamoto and Murata, 2002). Examples of the known compatible solutes include; proline and glycine betaine (GB) levels of both have been shown to increase significantly under salt and drought stresses (Munns, 2002; Sakamoto and Murata, 2002). They constituted a significant amount of the metabolites reported to be increased in studies involving durum wheat under salt stress (Sairam and Tyagi, 2004; Ashraf and Foolad, 2007). Halophytes have also been reported to have an improved osmotic pressure, which was attributed to the increasing accumulation of compatible solutes such as proline and GB (Flowers et al., 1977). In the glycophytes, studies have shown the concentrations of the compatible solutes are lower compared to halophytes. Durum wheat has been shown to have improved osmotic adjustment through a high proline content (39%), on the other hand, GB has shown to

contribute up to 16% of the osmotic balance in both cytoplasmic compartments of both older and younger leaves tissues (Carillo et al., 2008).

1.3.8 Molecular Basis of Regulatory Networks in Plant Salt Stress

Signalling Pathways

The plant's genetic response to salt stress is mechanistically complicated and has been shown to involved strict regulation of significant players, e.g., proteins and RNAs. The mechanism has evolved to act at different stages of regulation that could be described by central dogma, where transcription leads to mRNA synthesis followed by post-translational modification, translation, and post-translational modification where a protein is coded and decoded (Gupta and Huang, 2014). Knowledge of the regulation of gene expression at the mRNA level has provided insight on how plants behave in response to extreme changes due to environmental cues (Gupta and Huang, 2014). Proper profiling of transcriptional processes has been widely used to screen out the candidate genes involved in abiotic stress responses. Information on salt-responsive genes, transcription factors, up-regulation or down-regulation have been documented using microarray data and transcriptomic profiling (Johnson et al., 2006). Furthermore, advances in molecular biology techniques and approaches such as functional genomics have contributed significantly towards identification, cloning, and characterisation of these genes (Johnson et al., 2006). The role of transcription factors in gene expression under salt conditions have been reported and the interaction has been considered to be the most crucial in up- and down-regulation of key genes, which could determine the plant's adaptability to a salt environment (Johnson et al., 2006). For example, zinc finger genes; bZIP, WRKY, AP2, NAC, C2H2, and MYB and DREB family proteins have been considered to be the most stress-responsive members

to have the capacity to alter gene expression by Cis-acting specific binding in the promoter region of many genes (Johnson et al., 2006).

Moreover, a quite significant number of drought/salt stress inducible-genes have been discovered through microarray analysis of Arabidopsis transcriptome and other grasses, such as rice (Oryzasativa) (Takasaki et al., 2010). They have been reported to play vital roles in plants stress tolerance and have the potential to coordinate genes expression through signal transduction in many plant cells (Xiong et al., 2003; Takasaki et al., 2010). Efforts have been undertaken to unmasked the molecular mechanisms involved in the regulation and control of gene expression in response to abiotic stresses (Takasaki et al., 2010). The initial studies have been reported and aimed to establish the key role of cis- and acting elements and how they affect modulation of the stress response by using model dicot plant Arabidopsis thaliana; and some studies involving rice (Oryzasativa) (Egawa et al., 2006; Agarwal et al., 2007).

The transcription factors have shown to bind to the cis-acting elements, these are elements involved in transcription of stress-gene in the promoter region of a target genes thereby affecting its expression, and the complete process of transcriptional regulations is known as the regulon. Several such regulons are actively involved in response to abiotic stress have been identified using the model plant Arabidopsis thaliana (Nakashima et al., 2009).

Figure 1.6: Schematic diagram of the plant regulatory networks induced by drought/salinity stress, which involves the activation of both pathways of Abscisic acid- dependent (ABA-dependent) and Abscisic acid independent pathways. This involves the binding of transcription factors (TFs), such as MYB2, MYC2, NAC, AREB/ABF, NAC HD-ZIP and DREB2 to specific cis-acting elements (MYBR, MYCR, ABRE/ABF and DRE/CRT) which leads to the expression of drought/salinity stress-responsive gene such as: RD22, Gly, RD29B, and RD20A respectively (Nakashima et al., 2009).

The transcriptional regulatory networks established by using a model of signalling cascade pathways have shown to comprised two critical operational paths, that have shown to become activated in response to either abiotic/biotic stresses. These have been referred to as ABA-dependent and ABA-independent signalling pathways respectively. This pathway regulates signal transduction and eventual expression of elements responsive to genes including RD29B and RD20A genes. MYB2 and MYC2, are also involved through ABA- dependent stress-inducible genes that have shown to influence the appearance of the RD22 genes. Additionally, MYC2 has also been shown to be involved in the metabolic pathway related to JA-inducible genes that are triggered following most biotic stress's especially those that are pathogen related. Other important transcription factors that have shown to play vital roles include, RD26 NACs, which operate through dual pathways; ABA- dependent and JA-inducible gene expression, in response to stresses (Nakashima et al.,

Drought, High salinity

Signal

ABA-dependent pathway ABA-independent pathway

MYB2, MYC2 (MYB, MYC) NAC (RD26) AREB/ABF(bZIP) ABF ABRE NAC

HD-ZIP (AP2/ERF)DREB2

DRE/CRT

RD22 Gly RD29B, RD20A RD29B

MYCR MYBR

2009). The cross-talking between abiotic and biotic stress responses involves activities of MYC2 and NAC transcription factors. In the ABA-independent pathway, DREB2s are the main transcription factors; and are activated in response to dehydration and high salt stresses. Furthermore, dehydration response elements (DREs) have been shown to become operational via cis-acting elements operating through the same pathway to modulate and regulate genes for drought and salt stress. (Nakashima et al., 2009).