Breeding approaches for improving abiotic stress tolerance in crop species are evolving at a rapid pace. With the development of molecular technologies, transgenic approaches have become a prominent part of many research initiatives. Genetic transformation currently assists in the study of cellular mechanisms underlying salt, waterlogging and inundation tolerance, and there have been many potentially beneficial genes identified for genetic transformation (Dennis et al., 2000; Yamaguchi and Blumwald, 2005; Munns, 2005; Agarwal and Grover, 2006; Munns and Tester, 2008; Flowers and Colmer, 2008). Table 6.2 details a range of candidate genes for salinity, waterlogging and inundation and their expected function in crop plants. However, in spite of the increasing volume of knowledge on candidate genes and their function in plants, the future role of transgenically
Table 6.2. Selected genes associated with salinity, waterlogging and inundation. Candidate
Salinity NHX Vacuolar Na+/H+ The NHX antiporters (NHX1:5) transport Na+ across antiporter the tonoplast and into vacuoles and are driven by an electrochemical gradient of protons. The overexpression of AtNHX1 in transgenic Arabidopsis has been shown to improve salt tolerance; plant growth and development were unaffected at up to 200 mM NaCl. Field trials have also shown benefits of the transgenic expression of NHX in wheat through improvements in yield under salt stress (Munns, 2005; Flowers and Colmer, 2008; Munns and Tester, 2008).
SOS Plasma membrane The SOS1 transporter is important for the extrusion Na+/H+ antiporter of Na+ from plant cells. It is responsible for the exchange of Na+ and H+ ions across the plasma membrane (Na+ outwards). The activation of this transporter is regulated by the SOS2 and SOS3 genes, allowing the cell to respond to different cellular conditions. The genes have the potential to enable the efflux of Na+ from roots, however, the exact role of SOS1 in salinity tolerance remains uncertain (Munns, 2005; Flowers and Colmer, 2008; Munns and Tester, 2008). HKT1 was originally isolated from wheat roots by expression cloning, and at low external Na+ concentrations plays a role in K+ uptake from soil and nutrient transfer of K+ into leaves. However, the activity of HKT1 is also known to facilitate Na+ influx into tissues in high Na+ environments. Functional analysis in Arabidopsis suggests that the gene may also be involved in Na+ recirculation from shoots to roots and maintenance of shoot K+ homoeostasis (Munns, 2005; Flowers and Colmer, 2008; Munns and Tester, 2008). LCT1 Low-affinity cation Studies with yeast cells indicate that the LCT1
transporter transporter is located on the plasma membrane and is responsible for the transport of Na+, K+, Ca2+ and Cd2+. While the exact physiological role of LCT1 has not yet been established, it is known that LCT1 is an important contributor to Na+ influx in wheat at high external Na+ concentrations and preliminary investigations indicate that modification of the selectivity of LCT1 has the potential for improving salt tolerance in plants (Flowers and Colmer, 2008).
AVP1 Vacuolar AVP1 has the ability to increase the vacuole
H+-pyrophosphatase transmembrane proton gradient, increasing the (PPiase) capacity for sequestration of cations in the vacuole, and thus reducing the toxic effects of Na+ in the cytosol. PPiase proton pumps appear to be important for enhancing salt tolerance as they generate the primary driving force for Na+ transport via proteins such as SOS1 and NHX1 (Munns, 2005; Munns and Tester, 2008).
HKT High-affinity K+ transporter
Table 6.2. Continued
Stress gene Protein family
Role in plant function
Waterlogging PDC and inundation
Pyruvate decarboxylase (PDC) (alcohol fermentation)
Alcohol dehydrogenase (ADH) (alcohol fermentation)
Sucrose synthase (SuSy) (carbohydrate metabolism)
PDC (2-oxo-acid carboxylase) is the first enzyme channelling carbohydrates towards alcoholic fermentation and is considered to be the rate-limiting step in this pathway. A number of different plant PDC genes have been cloned and sequenced. Maize and rice are the most extensively analysed plant systems for the characterization of PDC enzymes and their corresponding genes. It has been hypothesized that change in the subunit composition confers upon rice seedlings the capacity to carry out active ethanol fermentation during prolonged treatment with anoxia (Dennis et al., 2000; Agarwal and Grover, 2006).
ADH leads to the conversion of acetaldehyde to ethanol in the final step of the alcoholic fermentation pathway. The increased expression of ADH genes in response to O2 deprivation has been identified and studied in many crop species, including barley, rice, maize, cotton and tomato (Dennis et al., 2000; Agarwal and Grover, 2006).
Increased SuSy activity after the onset of hypoxia has been documented in many crop species including wheat, maize, rice and potato. SuSy exists in the cytoplasm of many non-photosynthetic tissues, where it increases sucrose cleavage, providing carbohydrates for alcoholic fermentation and the synthesis of storage and structural polymers (Dennis et al., 2000; Agarwal and Grover, 2006).
Haemoglobins are known for their ability to act as O2 carriers to facilitate O2 delivery. At low O2 tensions they may also act as O2 sensors to regulate gene expression. Transgenic studies in lucerne and maize indicate a beneficial role of haemoglobins in nitric oxide regulation and root growth under low O2 stress (Dennis et al., 2000; Agarwal and Grover, 2006)._
engineered salinity, waterlogging or inundation tolerance in crop species is uncertain. The complexity of plant response and the environment suggests that single gene modifications may not contribute a significant improvement in salt, waterlogging or inundation tolerance. Nevertheless, the process of genetic modification may contribute some advantage if the gene is involved in signalling and regulatory pathways (Seki et al., 2003), has a pleiotropic effect, or encodes a protein conferring stress tolerance (Wang et al., 2004)
or enzymes leading to the synthesis of functional and structural metabolites (Apse and Blumwald, 2002). In fact, claims of improved tolerance through genetic modification have been made, but are difficult to substantiate due to experimental designs and data sets that do not represent the target environment (Flowers, 2004). Meanwhile, there are other promising breeding approaches that may be utilized such as targeting physiological traits within conventional breeding programmes (Fig. 6.4).
Blum (1989) and Yeo et al. (1990) suggested that targeting physiological parameters would simplify the genetics and breeding procedures for tolerance to abiotic stress. The approach has become an important component of international wheat breeding initiatives (Reynolds and Pfeiffer, 2000; Reynolds and Trethowan, 2007) and has also been used as a basis for rice selections (Dedolph and Hettel, 1997). Key components of physiological trait breeding include: (i) design of a model encompassing physiological traits contributing to tolerance in a crop species; (ii) identification of variation for the traits within the physiological model; and (iii) evaluation of the potential genetic gains contributed by each of the components of the model, so that traits may be combined in such a way as to maximize additive genetic gains. Flowers and Yeo (1995) advocate the use of a physiological trait-based breeding approach and report on the feasibility of increasing the resistance of salt-sensitive species; this approach has been successful in rice (Gregorio et al., 2002). In the past there have been several challenges inhibiting the successful implementation of this approach, including the need for time-consuming or destructive physiological screens. However, technology is now better able to overcome some of these limitations through the development and increased efficiency of MAS (Bonnett et al., 2005; Kuchel et al., 2005) and high-throughput phenotypic analysis (Babar et al., 2006; Ruuska et al., 2006; Olivares-Villegas et al., 2007).
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