Richard A. James

Screening methods for salinity tolerance: a case study with tetraploid wheat

Fast and effective glasshouse screening techniques that could identify genetic variation in salinity tolerance were tested. The objective was to produce screening techniques for selecting salt-tolerant progeny in breeding programs in which genes for salinity tolerance have been introduced by either conventional breeding or genetic engineering. A set of previously unexplored tetraploid wheat genotypes, from five subspecies of Triticum turgidum, were used in a case study for developing and validating glasshouse screening techniques for selecting for physiologically based traits that confer salinity tolerance. Salinity tolerance was defined as genotypic differences in biomass production in saline versus non-saline conditions over prolonged periods, of 3–4 weeks. Short-term experiments (1 week) measuring either biomass or leaf elongation rates revealed large decreases in growth rate due to the osmotic effect of the salt, but little genotypic differences, although there were genotypic differences in long-term experiments. Specific traits were assessed. Na+ exclusion correlated well with salinity tolerance in the durum subspecies, and K+/Na+ discrimination correlated to a lesser degree. Both traits were environmentally robust, being independent of root temperature and factors that might influence transpiration rates such as light level. In the other four T. turgidum subspecies there was no correlation between salinity tolerance and Na+ accumulation or K+/Na+ discrimination, so other traits were examined. The trait of tolerance of high internal Na+ was assessed indirectly, by measuring chlorophyll retention. Five landraces were selected as maintaining green healthy leaves despite high levels of Na+ accumulation. Factors affecting field performance of genotypes selected by trait-based techniques are discussed.

Avenues for increasing salt tolerance of crops, and the role of physiologically based selection traits

Increased salt tolerance is needed for crops grown in areas at risk of salinisation. This requires new genetic sources of salt tolerance, and more efficient techniques for identifying salt-tolerant germplasm, so that new genes for tolerance can be introduced into crop cultivars. Screening a large number of genotypes for salt tolerance is not easy. Salt tolerance is achieved through the control of salt movement into and through the plant, and salt-specific effects on growth are seen only after long periods of time. Early effects on growth and metabolism are likely due to osmotic effects of the salt, that is to the salt in the soil solution. To avoid the necessity of growing plants for long periods of time to measure biomass or yield, practical selection techniques can be based on physiological traits. We illustrate this with current work on durum wheat, on selection for the trait of sodium exclusion. We have explored a wide range of genetic diversity, identified a new source of sodium exclusion, confirmed that the trait has a high heritability, checked for possible penalties associated with the trait, and are currently developing molecular markers. This illustrates the potential for marker-assisted selection based on sound physiological principles in producing salt-tolerant crop cultivars.

Wheat grain yield on saline soils is improved by an ancestral Na+ transporter gene

The ability of wheat to maintain a low sodium concentration ([Na+]) in leaves correlates with improved growth under saline conditions. This trait, termed Na+ exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na+ exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na+] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na+-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na+ from the xylem and reduce transport of Na+ to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na+] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus.

Control of Sodium Transport in Durum Wheat

In many species, salt sensitivity is associated with the accumulation of sodium (Na+) in photosynthetic tissues. Na+ uptake to leaves involves a series of transport steps and so far very few candidate genes have been implicated in the control of these processes. In this study, Na+ transport was compared in two varieties of durum wheat (Triticum turgidum) L. subsp. durum known to differ in salt tolerance and Na+ accumulation; the relatively salt tolerant landrace line 149 and the salt sensitive cultivar Tamaroi. Genetic studies indicated that these genotypes differed at two major loci controlling leaf blade Na+ accumulation (R. Munns, G.J. Rebetzke, S. Husain, R.A. James, R.A. Hare [2003] Aust J Agric Res 54: 627–635). The physiological traits determined by these genetic differences were investigated using measurements of unidirectional 22Na+ transport and net Na+ accumulation. The major differences in Na+ transport between the genotypes were (1) the rate of transfer from the root to the shoot (xylem loading), which was much lower in the salt tolerant genotype, and (2) the capacity of the leaf sheath to extract and sequester Na+ as it entered the leaf. The genotypes did not differ significantly in unidirectional root uptake of Na+ and there was no evidence for recirculation of Na+ from shoots to roots. It is likely that xylem loading and leaf sheath sequestration are separate genetic traits that interact to control leaf blade Na+.

Genetic variation for improving the salt tolerance of durum wheat

Durum wheat (AB genomes) is more salt-sensitive than bread wheat (ABD genomes), a feature that restricts its expansion into areas with sodic or saline soils. Salt tolerance in bread wheat is linked with a locus on the D genome that results in low Na+ uptake and enhanced K+/Na+ discrimination. In order to introduce salt tolerance into current durum wheats from sources other than the D genome, a search for genetic variation in salt tolerance was made across a wide range of tetraploids representing 5 Triticum turgidum sub-species (durum, carthlicum, turgidum, turanicum, polonicum). Selections were screened for low Na+ uptake and enhanced K+/Na+ discrimination. This was assessed in seedlings grown in 150 mМ NaCl with supplemental Ca2+, by measuring the Na+ and K+ accumulated in the blade of a given leaf over 10 days. Large and repeatable genetic variation was found. Low Na+ accumulation and high K+/Na+ discrimination of similar magnitude to that of bread wheat was found in the sub-species durum. These selections have the potential for improving salt tolerance in durum wheat breeding programs.

Physiological Characterization of Two Genes for Na+ Exclusion in Durum Wheat, Nax1 and Nax2

Durum wheat (Triticum turgidum L. subsp. durum Desf.) Line 149 contains two novel major genes for excluding Na+ from leaf blades, named Nax1 and Nax2. The genes were separated into families containing a single gene and near-isogenic homozygous lines were selected. Lines containing either Nax1 or Nax2 had lower rates of Na+ transport from roots to shoots than their near-isogenic pairs due to lower rates of net loading of the xylem, not to lower rates of net uptake from the soil or higher rates of retranslocation in the phloem. Nax1 and Nax2 lines also had higher rates of K+ transport from root to shoot, resulting in an enhanced discrimination of K+ over Na+. Lines containing Nax1 differed from those containing Nax2 by unloading Na+ from the xylem as it entered the shoot so that Na+ was retained in the base of the leaf, leading to a high sheath to blade ratio of Na+ concentration. Gradients in tissue concentrations of Na+ along the leaf suggested that Na+ was continually removed from the xylem. The Nax2 line did not retain Na+ in the base of the leaf, suggesting that it functioned only in the root. The Nax2 gene therefore has a similar function to Kna1 in bread wheat (Triticum aestivum).

Factors affecting CO2 assimilation, leaf injury and growth in salt-stressed durum wheat

To examine the factors that affect tolerance to high internal salt concentrations, two tetraploid wheat genotypes that differ in the degree of salt-induced leaf injury (Wollaroi and Line 455) were grown in 150 mM NaCl for 4 weeks. Shoot biomass of both genotypes was substantially reduced by salinity, but genotypic differences appeared only after 3 weeks, when durum cultivar Wollaroi showed greater leaf injury and a greater reduction in biomass than Line 455. Ion accumulation, water relations, chlorophyll fluorescence and gas exchange were followed on one leaf (leaf 3) throughout its life. Salinity caused a large decrease in stomatal conductance (gs) of both genotypes. This was not due to poor water relations, as leaf turgor of both genotypes was higher in the salt treatment than in controls, so chemical signals were likely to have caused the decrease in gs. Reductions in assimilation rate were initially due to gs and, with time, were due to a combination of stomatal and non-stomatal limitations. The non-stomatal limitations were associated with a build up of Na+ above 250 mM. The efficiency of PSII photochemistry in Line 455 was unaffected throughout. However, in Wollaroi, the potential and actual quantum yield of PSII photochemistry began to decline as the leaf aged and the thermal energy dissipation of excess light energy (NPQ) increased. This coincided with high Na+ and Cl- concentrations in the leaf and with chlorophyll degradation, indicating that these later reductions in CO2 assimilation in Wollaroi were a consequence of a direct toxic ion effect. The earlier reduction in CO2 assimilation and greater leaf injury explain why growth of Wollaroi was less than Line 455. The most sensitive indicator of salinity stress was gs, followed by CO2 assimilation, with fluorescence parameters other than NPQ being no more sensitive than chlorophyll itself.

New phenotyping methods for screening wheat and barley for beneficial responses to water deficit

This review considers stomatal conductance as an indicator of genotypic differences in the growth response to water stress. The benefits of using stomatal conductance are compared with photosynthetic rate and other indicators of genetic variation in water stress tolerance, along with the use of modern phenomics technologies. Various treatments for screening for genetic diversity in response to water deficit in controlled environments are considered. There is no perfect medium: there are pitfalls in using soil in pots, and in using hydroponics with ionic and non-ionic osmotica. Use of mixed salts or NaCl is recommended over non-ionic osmotica. Developments in infrared thermography provide new and feasible screening methods for detecting genetic variation in the stomatal response to water deficit in controlled environments and in the field.

A Sodium Transporter (HKT7) Is a Candidate for Nax1, a Gene for Salt Tolerance in Durum Wheat

Durum wheat (Triticum turgidum subsp. durum) is more salt sensitive than bread wheat (Triticum aestivum). A novel source of Na+ exclusion conferring salt tolerance to durum wheat is present in the durum wheat Line 149 derived from Triticum monococcum C68-101, and a quantitative trait locus contributing to low Na+ concentration in leaf blades, Nax1, mapped to chromosome 2AL. In this study, we used the rice (Oryza sativa) genome sequence and data from the wheat expressed sequence tag deletion bin mapping project to identify markers and construct a high-resolution map of the Nax1 region. Genes on wheat chromosome 2AL and rice chromosome 4L had good overall colinearity, but there was an inversion of a chromosomal segment that includes the Nax1 locus. Two putative sodium transporter genes (TmHKT7) related to OsHKT7 were mapped to chromosome 2AL. One TmHKT7 member (TmHKT7-A1) was polymorphic between the salt-tolerant and -sensitive lines, and cosegregated with Nax1 in the high-resolution mapping family. The other TmHKT7 member (TmHKT7-A2) was located within the same bacterial artificial chromosome contig of approximately 145 kb as TmHKT7-A1. TmHKT7-A1 and -A2 showed 83% amino acid identity. TmHKT7-A2, but not TmHKT7-A1, was expressed in roots and leaf sheaths of the salt-tolerant durum wheat Line 149. The expression pattern of TmHKT7-A2 was consistent with the physiological role of Nax1 in reducing Na+ concentration in leaf blades by retaining Na+ in the sheaths. TmHKT7-A2 could control Na+ unloading from xylem in roots and sheaths.

Major genes for Na+ exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na+ accumulation in bread wheat leaves under saline and waterlogged conditions

Two major genes for Na(+) exclusion in durum wheat, Nax1 and Nax2, that were previously identified as the Na(+) transporters TmHKT1;4-A2 and TmHKT1;5-A, were transferred into bread wheat in order to increase its capacity to restrict the accumulation of Na(+) in leaves. The genes were crossed from tetraploid durum wheat (Triticum turgidum ssp. durum) into hexaploid bread wheat (Triticum aestivum) by interspecific crossing and marker-assisted selection for hexaploid plants containing one or both genes. Nax1 decreased the leaf blade Na(+) concentration by 50%, Nax2 decreased it by 30%, and both genes together decreased it by 60%. The signature phenotype of Nax1, the retention of Na(+) in leaf sheaths resulting in a high Na(+) sheath:blade ratio, was found in the Nax1 lines. This conferred an extra advantage under a combination of waterlogged and saline conditions. The effect of Nax2 on lowering the Na(+) concentration in bread wheat was surprising as this gene is very similar to the TaHKT1;5-D Na(+) transporter 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.