Kna1 Saltol Nax Torelant Salinity

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Chr 7H

bPb-1209-bPb-6821

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CM72 X Gairdner

Xue et al. (2009)

P40M38b

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Derkado X B83-12/21/5

Ellis et al. (2002)

identified as the sodium transporter OsHKT8 (Ren et al. 2005) . Combining superior alleles underlying the component traits could potentially result in higher levels of tolerance. Based on QTL-linked marker profile, Manneh et al. (2007) identified superior salt-tolerant genotypes of rice to improve selection efficiency while selecting for yield in stress environments. Wang et al. (2011) mapped 16 QTLs for salt tolerance of rice at the seed germination stage and four QTLs with major effect could be useful to improve stand establishment under saline areas. QTLs associated with tolerance at various developmental stages will be needed for more stable performance in salt affected areas.

Improving salt tolerance and productivity wheat is a major challenge in breeding program. In studies involving hexaploid, tetraploid, and diploid types, it was suggested that the D genome of wheat carries gene Kna1 that controls the relative concentration of K+ and Na+ in the shoots of plants grown in saline hydroponic culture (Shah et al. 1987. Gorham et al. 1987, 1997; Gorham 1994). This gene was mapped on to the chromosome 4DL (Dvorak et al. 1994) and then fine mapped as a single gene (Dubcovsky et al. 1996). Ma et al. +2007+ used a wheat RIL population derived from the cross Opata85 x W7984 (international mapping population) and identified 47 QTLs based on salt tolerance index, salt injury index, biomass, shoot length, root length, chlorophyll content, and proline content on all wheat chromosomes except 1B, 1D, 4B, 5D, and 7D. Ten of these QTLs were effective during germination stage and 37 QTLs were important at the seedling stage. In another study, genetic analysis of wheat Line 149 identified two major genes Nax1 and Nax2 for Na+ exclusion (Munns et al. 2003) . Nax1 was located on chromosome 2A by QTL analysis (Lindsay et al. 2004) and has been fine mapped as an Na+ transporter of the HKT gene family, HKT7 (Huang et al. 2006). Nax2 located on chromosome 5A was identified as HKT8 (Byrt et al. 2007). Nax1 removes Na+ from the xylem in roots and the lower parts of leaves, the leaf sheaths, while Nax2 removes Na+ from the xylem only in the roots (James et al. 2006). Nax2 has the same phenotype as Kna1, the QTL for Na+ exclusion and enhanced K+/Na+ selectivity in bread wheat, T. aestivum (Dvorak et al. 1994). Nax2 was shown to be homologous to Kna1 in T. aestivum, namely TaHKT8 (Byrt et al. 2007) . The HKT gene family encodes transporters in the plasma membrane that mediate the uptake of Na+ or K+ from the apoplast (Hauser and Horie 2010). They are important for cellular Na+ and K+ homeostasis, and their expression in the stele, particularly in the xylem parenchyma cells lining the xylem vessels, helps in retrieving Na+ from the transpiration stream and thus contributing to Na+ exclusion from leaves (Munns and Tester 2008; Hauser and Horie 2010). James et al. (2011) analyzed a population derived from crossing of a tetraploid durum wheat (Triticum turgidum ssp. durum) and hexaploid bread wheat (Triticum aestivum) by marker-assisted selection (MAS) for hexaploid plants containing either Nax1, Nax2, or both Nax1 and Nax2. Nax1 line decreased the leaf blade Na+ concentration by 50%, whereas Nax2 line decreased it by 30%, and both genes together decreased it by 60%. High Na+ sheath:blade ratio in Nax1 lines conferred extra advantage under waterlogged and saline conditions. The effect of Nax2 on lowering the Na+ concentration in bread wheat was surprising as this gene is already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat. A list of salinity-related QTLs for wheat and barley is given in Tables 4.2 and 4.3, respectively.

3.3.2 Marker-Assisted Selection

The application of QTL mapping provided the means to genetically dissect tolerance traits into discrete QTLs that can then be pyramided into high-yielding varieties using marker-assisted selection (MAS). Marker-assisted selection is the use of molecular markers linked to useful traits to select individuals with desirable genetic makeup during the variety development process. It provides a dramatic improvement in the efficiency with which breeders can select plants with desirable combination of genes. DNA markers should enhance the recovery rate of the isogenic recurrent genome after hybridization and facilitate the intro-gression of quantitative trait loci necessary to increase stress tolerance (Fig. 4.4). Molecular marker techniques were used successfully to transfer alleles of interest from wild relatives into commercial cultivars (Tanksley and McCouch 1997). Use of permanent mapping populations, such as recombinant inbred lines (RILs) or chromosomal segment substitution lines (CSSLs) enables testing stress-tolerant traits in replicated experiments across different environments, which can help in differentiating the QTLs based on their effectiveness at different stress levels. Once important QTL targets are identified, it can be used as single intro-gressions in a set of near-isogenic lines (NILs), which will help in identifying the complexity of different traits by limiting the variation between lines to focus only on the locus of interest.

BC1F1s X RP

MAS^

MAS^ Selfing MAS^ Selfing

[ Bulk BG2F2 populations | [ Bulk BC3F2 populations | ( BG4F2 populations ~]

Screening for salinity tolerance

BC4F1s

Screening for salinity tolerance

Fig. 4.4 A schematic representation of marker-assisted backcross breeding procedure for development of introgression Lines with enhanced salt tolerance. RP recurrent parent, DP donor parent, MAS Marker assisted selection

The recent advances in genomics have paved the way for clear and reliable methods for MAS in plants starting from QTL identification, NIL development, and fine-mapping to transferring the QTL into popular varieties using a precise marker-assisted backcrossing (MABC) strategy (Collard et al. 2005, 2008 Collard and Mackill 2008; ). MABC involves the manipulation of genomic regions involved in the expression of particular traits of interest through DNA markers, and combines the power of a conventional backcrossing program with the ability to differentiate parental chromosomal segments (Fig. 4.4).

3.4 Transgenic Approach

Progress in genomics is instrumental in discovery and characterization of large number of salt stress-related candidate genes offering unique opportunity for exploiting transgenic technology to enhance salinity tolerance in crop plants. Development of transgenic plants with improved tolerance to abiotic stresses has brought some hope for sustainable agriculture under harsh environmental conditions. Genetic engineering is an attractive option when genes of interest are present in cross barrier species, distant relatives, or non-plant sources (Bhatnagar-Mathur et al. 2008). It is also faster to introduce beneficial genes than the conventional or molecular breeding. Due to the complexity associated with salt tolerance mechanism, effort should be made to introduce and fine tune multiple genes with synergistic effect under suitable stress inducible promoters for controlling their expression at a specific time, in a specific organ, or under specific conditions of stress.

3.4.1 Discovery of Candidate Genes

When a plant is subjected to abiotic stresses, expressions of a number of genes are changed, resulting in altered levels of several proteins and metabolites inside the cell. Altered expression of these genes may be responsible for conferring protection or susceptibility to abiotic stresses. Candidate genes for salinity stress tolerance have been identified from prokaryotic extremophiles, lower eukaryotes, tolerant wild relatives, lan-draces, cultivars, and T-DNA mutant plants using high throughput techniques such as differential display polymerase chain reaction (DD-PCR), cDNA-amplified fragment length polymorphism (cDNA-AFLP), suppression subtractive hybridization (SSH), serial analysis of gene expression (SAGE), DNA microarray, and two-dimensional gel electrophoresis.

Differentially expressed genes under osmotic stress have been identified by DDRT-PCR in barley, sunflower, and pak-choi (Wei et al. 2001; Liu and Baird 2003; Qiu et al. 2009). Wei et al. (2001) identified a gene encoding the barley vacuolar ATPase subunit B (BSVAP) from salt-sensitive barley cultivar Maythorpe. In barley roots, this enzyme may be involved in the sequestration of Na+ and Ca2+ ions into the vacuole, since the proton gradient produced by the ATPase is used by Na+/H+ and Ca2+/H+ antiporters to drive the uptake of Na+ and of Ca2+ (Taiz 1992). Liu and Baird (2003) isolated 17 cDNA clones that are differentially expressed in sunflower under drought or salinity and 13 of these cDNAs were confirmed by quantitative RT-PCR to be expressed differentially in response to osmotic stress. Qiu et al. (2009) generated 101 cDNA fragments differentially expressed under salt stress in Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee), and found seven cDNA sequences highly homologous to some known expression genes or the genes related to the signaling pathways in plants under different abiotic stress.

Kumari et al. (2009) applied SSH technique in a salt-sensitive rice cultivar IR64 and a salt-tolerant landrace Pokkali and isolated 1,194 salinity-regulated cDNAs. Gene expression analysis of selected genes using macroarrays and Northern blots indicated that salinity tolerance of Pokkali may be due to constitutive overexpression of many salt responsive genes which are stress inducible in IR64. Few clones mapped near Saltol locus on chromosome 1. Using global gene expression analyses, Walia et al. (2005, 2007) observed gene expression changes in large number of genes under salinity stress in salt-sensitive genotypes compared with the tolerant lines. A total of 164 ESTs were upregulated in the tolerant line (FL478) under salinity stress during vegetative stage (Walia et al. 2005), whereas during panicle initiation stage, 292 genes were upregulated and 346 genes were downregulated in saltsensitive japonica Ml03. Interestingly, in the salt-tolerant j aponica only 54 were upregulated and 54 downregulated (Walia et al. 2007). Cotsaftis et al. (2011) carried out root-specific transcriptome studies in contrasting genotypes of rice (FL478, IR29, Pokkali and IR63731) under salinity stress and identified several gene families with known links to salinity tolerance.

Chen et al. (2003) used cDNA-AFLP to analyze differentially expressed genes in wheat under salinity stress and found a large number of gene fragments related to salt stress. One of the cDNA encoding glycogen synthase kinase-shaggy kinase (TaGSK1) was induced by NaCl stress as a part of the signal transduction component. This technique has also been used to isolate differentially expressed ESTs under salinity stress in soybean (Akoi et al. 2005) and Spartina alterniflora (Baisakh et al. 2008). An expressed sequence tag (EST) analysis in a grass halophyte Spartina alterniflora at early stages of salt stress (Baisakh et al. 2008) produced 1,227 quality ESTs of which 27% of ESTs represented genes for stress response. Transcript abundance analysis of eight known genes of various metabolic pathways and nine transcription factor genes showed temporal and tissue-dependent variation in expression under salinity stress.

A combination of genetic mapping and bulked transcriptome profiling was used by Pandit et al. (2010) resulting in identification of two genes, an integral transmembrane protein DUF6 and a chloride cotransporter, which colocalized within the QTL interval. Walia et al. (2006) performed expression analysis of genes in barley (Hordeum vulgare L.) during salinity stress at the seedling stage of barley using microarray. Genes, differentially regulated by salinity, were also associated with various abiotic stresses such as low temperature, heat stress, and drought stress. Expression level of genes related to jasmonic acid (JA) biosynthesis and jasmonic acid-responsive genes (JRGs) was found to be differentially regulated. Jasmonic acid may function as a "master switch" for stress-induced signaling pathway leading to changes in gene expression (Wasternack et al. 1998). It has been reported that JA and ABA affect gene expression in a synergistic manner through more or less independent signaling pathways (Ortel et al. 1999) , This indicates that ABA signaling pathway is also activated along with JA pathway in response to salinity stress in barley. Ozturk et al. (2002) analyzed drought and salinity-induced responses in barley (Hordeum vulgare L. cv. Tokak) transcriptome using cDNA microar-ray and reported alteration in 5% of the genes under salinity stress compared to 15% under drought stress. Upregulation under both drought and salt stress was restricted to ESTs for metallo-thionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress.

Yan et al. (2005) investigated the salt stress-responsive proteins in the root of rice (Oryza sativa L. cv. Nipponbare) in proteomics approach using 3-week-old seedlings treated with 150 mM NaCl for 24, 48 and 72 h. Two-dimensional gel electrophoresis showed 34 upregulated and 20 downregulated proteins. Mass spectrometry analysis could identify 12 spots representing 10 different proteins involved in regulation of carbohydrate, nitrogen, and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. Comparative proteomic analysis in two contrasting hybrid rice variety by Ruan et al. (2011) led to identification of new components involved in salt-stress signaling. One protein that was upreg-ulated during salt stress was homologous to cyclophilin 2 (OsCYP2). Chitteti and Peng (2007) reported the differential expression of phosphop-roteome under salinity stress in the root of rice. They identified 17 differentially upregulated and 11 differentially downregulated putative phosphoproteins. Witzel et al. (2009) conducted two-dimensional gel electrophoresis using a series of hydroponics-based salinity stress experiments in contrasting genetic mapping parents of barley cvs Steptoe and Morex. The proteome analysis of roots from both genotypes revealed cultivar-specific and salt stress-responsive protein expression. Twenty-six proteins could be identified by mass spectrometry. Among those, two proteins involved in the glutathione-based detoxification of reactive oxygen species (ROS) were more abundant in the tolerant genotype.

The identification of various abiotic stress-specific changes in gene expression has been achieved by comparing gene expression in non-induced and salinity stress-induced tissues or by comparing contrasting cultivars (Sahi et al. 2003; Baisakh et al. 2008, Karan et al. 2009; Kumari et al. 2009). In one such study, it was noted that less number of genes were induced under salt stress in Thellungiella halophila (a salt-tolerant relative of Arabidopsis) in comparison to Arabidopsis (Inan et al. 2004). This indicated that the stress tolerance of Thellungiella halophila may be due to constitutive overexpression of a few salt tolerance related genes which are stress inducible in Arabidopsis (Taji et al. 2004). Sengupta and Majumder (2009) analyzed changes in leaf protein expression under salt stress in the wild halophytic rice Porteresia coarctata and salt-sensitive Oryza sativa and identified 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis, and chaperone functions. These differentially regulated genes in different plant species may serve as candidates to improve salinity tolerance in crop plants using transgenic approach.

3.4.2 Transformation in Crop Plants

Most common genes used for genetic engineering of stress-tolerant plants include transcription factors, signal transduction genes, water channel proteins, ion transporters, detoxifying genes, molecular chaperones, dehydrins, and osmopro-tectants (Table 4.4).

Calcium acts as a secondary messenger in various signal transduction pathways in plants. Xu et al. (2011) isolated a calcium-binding multi-stress-responsive gene OsMSR2 from rice which

Table 4.4 Examples of transgenic intervention in crop plants to improve salt tolerance

Gene

Gene product

Gene source

Target plant

Effect of the transgene

Reference

HvCBF4

CBF transcription factor

Hordeum vulgare

Rice

Enhanced salinity, drought, and cold tolerance

Oh et al. (2007)

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