Understanding the molecular basis of drought tolerance in soybean

Transcription

1 Understanding the molecular basis of drought tolerance in soybean John McLean a, A.T. James a, G.P. Xue a, D. Maclean b. a CSIRO Tropical Agriculture, Indooroopilly b Department of Biochemistry, The University of Queensland Abstract Soybean is relatively sensitive to drought stress and this often limits yield in droughtprone environments. There is genotypic variation in Glycine spp. for drought tolerance. However, it is costly to screen breeding material for this trait. In order to make it easier and quicker to recover drought tolerant lines in the breeding program, it would be useful to know which genes are involved in the drought response of soybean and how the expression of these genes differs between tolerant and sensitive genotypes. A study is currently being undertaken to examine the differential expression of genes induced by drought in the moderately drought tolerant line Glycine max G2120 versus the drought sensitive variety Glycine max Valder. A wild relative of soybean with strong drought tolerance (Glycine latifolia) will also be compared with the cultivated soybean for differential expression of drought induced genes. A powerful technique called suppression subtractive hybridization enables the generation of a cdna library that contains genes that are expressed in one genotype but expressed at lower levels (or not at all) in another. To determine the critical stress stages of soybean for isolating drought tolerant, genotype-specific genes it is important to examine expression patterns of drought-inducible genes during various stages of drought stress. A preliminary study therefore is looking at the difference between mrna levels of tolerant and sensitive genotypes for known genes that are induced by drought. Once gene which are expressed specifically or at higher levels in drought tolerant genotypes are identified, these genes may be used to develop gene-specific markers for screening for the drought tolerance trait. Soybean and Drought There is an increasing demand for soybean in Australia, particularly for use in animal feed. However, at present, soybean production is restricted to irrigated areas or as a nonirrigated crop in higher-rainfall areas (Brinsmead et al. 1991). Non-irrigated crops, especially in inland cropping regions, are always at risk due to the irregularity of rainfall in Australia. To broaden the growing area of the crop it would be beneficial to improve the drought tolerance of soybean. For this reason it would be useful to identify genes involved in drought tolerance and to determine which genes enable one cultivar to be more drought tolerant than another. Yields of soybean are commonly limited by water stress. A study by (James et al. 1994) showed the following important aspects of soybean response to drought. Soybean has a moderate to high critical relative water content (RWC), moderate to high epidermal

2 conductance (Ge) and moderate to very high osmotic adjustment. Osmotic adjustment allows the plant to extract more water from the soil and maintain turgor, prolonging the period that the stomates remain open. As a result, soil water is rapidly depleted eventually resulting in stomatal closure. The high Ge of soybean means that even after the stomates have closed the leaf will continue to lose significant amounts of water and quickly die due to the high critical RWC of soybean. Many of the wild relatives of soybean, however, show much greater drought tolerance than cultivated soybean (James 2000). A better understanding of the genetic basis for this drought tolerance could provide useful information for improving the drought tolerance of cultivated varieties. However, molecular mechanisms underlying the drought tolerance trait are still largely unknown. Morphological and physiological changes of plants in response to drought require alterations in expression of a large number of genes. Table 1: Genes that will be examined for expression patterns in drought tolerant and sensitive genotypes during drought stress. Accession Number AW AW U08108 AF AF AI AI M64987 Gene Function EST homologue of pyrroline-5-carboxylate synthetase gene (P5CS) from mothbean. Involved in the biosynthesis of proline, a compatible solute (Hu et al. 1992). EST homologue of manganese superoxide dismutase gene (SOD) from wheat that protects cells from superoxide radicals (Wu et al. 1999). Characterised soybean gene for late embryogenesis abundant protein (LEA) belonging to the D95 family of LEAs. Potential desiccation protective effects (Maitra and Cushman 1994) Characterised dehydrin gene from soybean with possible cellular protection properties. Characterised 9-cis-epoxycarotenoid dioxygenase gene from Phaseolus vulgaris. Catalyzes a key regulatory step in ABA biosynthesis (Qin and Zeevaart 1999). EST homologue of a HD-Zip protein. Potential role as a transcription factor involved in expression of ABA-responsive genes. EST homologue of ATMYB2 gene from Arabidopsis thaliana that codes for a transcription factor that controls the ABA-dependent expression of some drought-inducible genes (Abe et al. 1997). Characterised soybean calcium dependent protein kinase gene (CDPK). Involved in signal transduction under water stress (Urao et al. 1994).

3 AI U39858 V00450 EST homologue of a phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K). Involved in signal transduction under water stress (Mikami et al. 1998). Characterised rbcs gene from soybean. Negatively regulated by ABA and water stress (Bartholomew et al. 2000). Characterised actin gene from soybean. Structural gene that should be expressed ubiquitously. Drought-inducible genes Many drought inducible genes have been identified in a variety of plant species. By searching databases such as GenBank we can find expressed sequence tags (ESTs) from soybean that are homologous to drought-inducible genes from another plant species. By making RNA probes for these homologues we can determine the effect of drought on the expression of these genes. The genes that are to be examined in this study are outlined in Table 1. These genes can be roughly grouped according to their functions into four classes - compatible solutes, scavenging reactive intermediates, cellular protectors and regulatory genes. Compatible solutes. Osmotic stress can occur when depleting soil water results in an increased solute concentration in the soil and a corresponding reduced water potential. As a result plants under this stress will lose turgor unless they can osmotically adjust. During osmotic adjustment plants actively accumulate compatible solutes such as sugars, proline and glycine betaine (Blum 1996;Hu et al. 1992) to maintain an osmotic gradient without affecting metabolic activity. Scavenging reactive intermediates. Oxidative stress occurs as a result of the inhibition of photosynthesis brought about by limited water availability (Holmberg and Bülow Leif 1998). When photosynthesis is impaired but chloroplasts are exposed to excitation energy oxygen is photoreduced (Holmberg and Bülow Leif 1998). The reactive oxygen intermediates that are produced cause tissue damage in plants. Schake et al. (1996) have shown that the expression of certain antioxidative enzymes can provide protection. Cellular protectors. This refers to the group of genes that code for certain groups of late embryogenesis abundant (LEA) proteins that are induced by drought (including one subgroup of LEAs called dehydrins). The exact function of these proteins is still not clear. Kermode (1997) suggested that some of the LEA proteins act as agents that solvate cellular components like membranes and proteins (possibly via their ability to form amorphous coils), thus protecting them from damage or disruption in the absence of water. The proteins form random coils (which are thought to have a water-binding capacity) that may enable the shape of some LEA proteins to conform to that of other

4 structures and provide a cohesive layer with greater stability than would be formed with sugars (Kermode 1997). Other LEA proteins may be involved in sequestering ions that accumulate in desiccating tissues (Maitra and Cushman 1994). Regulatory genes. This is a loose grouping of genes that code for transcription factors, protein kinases involved in signal transduction, and enzymes involved in abscisic acid (ABA) biosynthesis. Numerous studies have shown that ABA is produced under water deficit and regulates gene expression during drought response (Chandler and Robertson 1994). The gene for 9-cis epoxycarotenoid dioxygenase (which catalyzes a key step in ABA biosynthesis) is up-regulated during water stress and the increase in transcript levels correlate with increased ABA expression (Iuchi et al. 2000). It is now hypothesised that at least four independent signal pathways function in the activation of stress-inducible genes under dehydration conditions - two are ABAdependent and two are ABA-independent (Shinozaki et al. 1998). Different transcription factors and protein kinases are required in all of the signal transduction pathways for expression of the drought-inducible genes. Some of these have been characterised (Mikami et al. 1998; Shinozaki et al. 1998; Soderman et al. 1996; Soderman et al. 1999; Urao et al. 1994) but further molecular analysis could provide better understanding of how they interact and possibly reveal new regulatory genes. Determining Critical Stress Stages For Analysis of Gene Expression To isolate differentially expressed genes a plant must be subjected to the appropriate environmental conditions so that the genes of interest will be expressed. Different drought-inducible genes may be expressed at different stages of stress. Therefore it is important to identify what stage of stress RNA should be isolated in order to find a particular type of drought-inducible gene. Relative water content (RWC) is an easily measured and accurate indication of a plant s level of water stress. By extracting RNA from plants within given ranges of RWC and probing for known drought-inducible genes we can gauge when similar or related genes may also be expressed. The proposed ranges of RWC that will be probed are % (unstressed), 70-80% (early stress), 40-50% (mid-stress), 30-40% (late stress), 20-30% (very late stress). Development of Gene-Specific Markers As drought tolerance is a quantitative trait, the relative capacity of tolerance is likely, in most cases, to be correlated with expression levels of genes underlining the tolerant trait rather than just presence or absence of specific genes. Labhilili et al. (1995) reported that expression levels of some dehydration-inducible mrnas differ between drought-tolerant and sensitive cultivars under drought stress. This would form the basis for developing gene-specific markers to quantify the tolerant trait. In fact, gene-specific markers for quantifying the freezing-tolerance trait (also a quantitative trait) in wheat have been reported (Sarhan et al. 1997). These researchers have found that the accumulation of the

5 WCS120 proteins in more than 20 genotypes of wheat during cold-acclimatisation is positively correlated with the capacity of each genotype's freezing tolerance. This genespecific marker is being used for accessing segregation of freezing tolerance in different crosses. These data would support the feasibility of developing gene-specific markers. Gene-specific markers based on genetic variation at the molecular level between tolerant and sensitive genotypes will be useful for marker-assisted breeding of soybean in the future. These new generations of gene-specific markers may allow us to quantify the tolerance trait based on correlation of expression levels of certain genes with individual morphological and physiological traits. Development of gene-specific markers requires isolation of genes that are expressed only or at higher levels in drought tolerant genotypes, but are not expressed or expressed at lower levels in sensitive genotypes. There are a number of strategies for isolation of drought up-regulated and tolerant genotype-specific genes (cdnas). A newly developed and highly effective procedure called "suppression subtractive hybridisation" (SSH) can be used to achieve this aim (Diatchenko et al. 1996). The SSH method combines normalisation and subtraction in a single procedure for the generation of subtracted cdna libraries. The normalisation step equalises the abundance of cdnas within a target population (e.g. cdnas from a drought tolerant genotype). The subtraction step excludes the common cdna sequences between the target (the tolerant genotype) and driver (e.g. the cdnas from a drought sensitive genotype) populations with high enrichment of genotype-specific cdnas in one round of substrative hybridisation. With this method, we hope to isolate a number of drought-inducible genotype-specific genes to be used for developing gene-specific markers for screening the drought tolerance trait for soybean breeding program. Acknowledgements Funding for this research is provided in part by the Grains Research and Development Corporation. References Abe, H., Yamaguchi, S. K., Urao, T., Iwasaki, T., Hosokawa, D., and Shinozaki, K. (1997). Role of arabidopsis MYC and MYB homologs in drought-and abscisic acidregulated gene expression. Plant cell 9, Bartholomew, D. M., Bartley, G. E., and Scolnik, P. A. (2000). Abscisic acid control of rbcs and cab transcription in tomato leaves. Plant Physiology 96, Blum, A. (1996). Crop responses to drought and interpretation of adaption. Plant Growth Regulation 20, Brinsmead, R. B., Brouwer, J. B., Hawthorne, W. A., Homes, J. H. G., Knights, E. J., and Walton, G. H. Grain Legumes - Breeding for adaptability, productivity, quality and marketability of grain legumes in Australia Grains Council of Australia.

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