Salinity Research

Background

Salinity is a major abiotic stress affecting crop plants worldwide. In Australia the problem is only going to get worse, with already 51% of Western Australian farms affected in some way by saline soils. Saline soils affect the growth of crop plants in two ways; the first, osmotic stress results in an immediate reduction in shoot growth; the second, ionic stress results from the build up of sodium (Na+) and chloride (Cl-) ions to toxic levels, which interfere with metabolic processes, such as enzyme activities and protein synthesis. Due to both of these stresses, crops grown on saline soils have significantly reduced yield.

Plants use three main mechanisms with which to tolerate salinity stress:
  • Osmotic tolerance – the ability to maintain growth whilst under osmotic stress by various, as yet unknown, mechanisms
  • Ion exclusion – reducing Na+ and Cl- accumulation in the shoot by manipulating root ion transport processes to minimise Na+ and Cl- delivery to the shoot
  • Ion tissue tolerance – tolerating the Na+ and Cl- that builds up in the leaf by compartmentalising them into organelles within a cell
 
The aim of the ACPFG Salt Focus Group is to understand and alter these salt tolerance mechanisms in Australian crop plants, and to create varieties that can survive and produce viable yields on saline soils. To do so, we need to identify genes and cellular processes involved in salinity tolerance in current crops and other tolerant lines.

Forward genetics team - Gene discovery

The forward genetics team is responsible for the identification of new and novel genes responsible for increasing a plant’s salinity tolerance. Screening of cultivars of wheat, barley, rice and near wild relatives has revealed major differences in these plants’ abilities to grow under salt stress. We have used this variation to identify quantitative trait loci (QTL), regions in the plant genome containing genes important for salt tolerance. Fine mapping of these regions allows us to narrow to the candidate gene responsible for the observed phenotypes, which we can then introduce into crop varieties through either breeding and/or genetic engineering. Similarly, comparing transcriptomic and metabolomic analyses between salt sensitive and salt tolerant lines also allow us to identify candidate genes. In a development that takes us beyond most laboratories in the world, we have developed techniques which allow us to efficiently separate different cell types that make up a plant’s tissue and extract mRNA from specific cell types for whole genome transcript profiling in an effort to identify cell type specific salt responsive genes.

Reverse genetics team - Gene characterisation

The reverse genetics team is responsible for the characterisation of genes identified as likely to increase salinity tolerance of plants. Currently, the group is focusing on genes that encode Na+ and Cl- transporters, such as the HKT, PPase, NSCCs and NHX gene families, and specific genes such as PpENA1. However, other genes likely to be involved in gene and protein regulation are also being studied, notably transcription factors. Candidate genes are knocked out or over-expressed in a variety of model and crop species (mainly wheat, barley, rice, and Arabidopsis) to investigate the effects on salinity tolerance. In the case of novel Na+ and Cl- transporters, these genes are additionally expressed in heterologous expression systems, such as yeast and Xenopus oocytes, for electrophysiological examination. Promoter::GUS and promoter::GFP fusions are also carried out to allow us to visualise the tissue and subcellular location of our genes of interest.

Control of gene expression team

A key step in the development of a plant with increased salinity tolerance is the ability to control when a gene is activated and in what tissue or even cell type. As many genes are involved in the transport of Na+ and Cl- into or out of cells, it can be seen that constitutive expression of a transporter throughout a plant would be detrimental, as while the gene’s expression in one tissue/cell type may be beneficial, in others it can be detrimental. Similarly, genes that are activated only when the plant experiences salt stress would also be more beneficial than a gene that is expressed all of the time. Better methods for the control of candidate genes for salinity tolerance are therefore required. Trials of such a system have been carried out in both Arabidopsis and rice using enhancer trap lines which express green fluorescent protein in a cell type-specific manner. By making use of this system, it is also possible to express genes important in salinity tolerance in specific cell types. The success of this approach has already been shown in Arabidopsis, where a Na+ transporter has been specifically over-expressed in root stelar cells, resulting in a significant reduction in shoot Na+ accumulation (Møller et al. 2009). Although it is not possible to use the enhancer trap system in wheat and barley, identification of gene promoters which have a cell type-specific pattern in cereals will also allow us to express our genes in specific cells and we have projects looking for both cell specific and stress inducible promoters in wheat and barley.

Delivery Team - Creating Salt Tolerant Plants

The delivery team is responsible for gathering together all the information gathered by the other teams and using it to produce salt tolerant plants. This team makes use of traditional breeding practices, as well as genetic engineering to introduce salinity tolerance traits into wheat and barley. Varieties of wheat and barley, that have been identified by the Forward Genetics team as being salinity tolerant, are bred with current Australian cultivars or elite pre-breeding lines to produce crop plants for field trials. In addition, transgenic wheat and barley plants are being created which use genes characterised by the Reverse genetics and Control of Gene Expression teams to improve the plant’s salinity tolerance. These plants are then tested in both glasshouse and field conditions.

Techniques 

  • QTL mapping
  • DNA and RNA extractions
  • Molecular markers
  • PCRs and restriction digests
  • Quantitative PCR
  • Flame photometry
  • ICP-AES
  • Confocal microscopy
  • LemnaTec high throughput, non-destructive phenotypic analysis
  • Fluorescently activated cell sorting
  • Microarrays
  • Transcript analysis
  • Cloning
  • Field work
  • Generation of transgenic plants
  • Electrophysiology

Forward Genetics

Reverse Genetics Gene Control Delivery
 Dr Bettina Berger Dr Andrew Jacobs Dr Andrew Jacobs Dr Yuri Shavrukov
 Dr Stuart Roy Dr Darren Plett   Dr Darren Plett
 Dr Yuri Shavrukov Dr Stuart Roy    
 Dr Megan Shelden      

For more information contact:

Dr Stuart Roy
Professor Mark Tester

Recent Publications 

  • Genc, Y., Oldach, K., Verbyla, A.P., Lott, G., Hassan, M., Tester, M., Wallwork, H. and McDonald, G.K. (2010) Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress, Theoretical and Applied Genetics, DOI 10.1007/s00122-010-1357-y
  • Tester, M. and Langridge, P. (2010) Breeding technologies to increase crop production in a changing world, Science 327: 818-822.
  • Genc, Y., Tester, M. and McDonald (2010) Calcium requirement of wheat in saline and non-saline conditions, Plant and Soil 327: 331-345.:793-804.
  • Jha D, Shirley N., Tester M. and Roy S.J, (2010) Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes linked to differences in the natural expression levels of transporters involved in sodium transport, Plant, Cell and Environment 33
  • Plett, D.C. and Møller, I.S. (2010) Na+ transport in glycophytic plants: what we know and would like to know, Plant, Cell and Environment 33: 612-626.
  • Shavrukov Y., Gupta N.K., Miyazaki J., Baho M.N., Chalmers K.J., Tester M., Langridge P., Collins N.C. (2010) HvNax3—a locus controlling shoot sodium exclusion derived from wild barley (Hordeum vulgare ssp. spontaneum). Functional and Integrative Genomics 10: 277-291.
  • Shavrukov Y., Langridge P., Tester M. (2009) Salinity tolerance and sodium exclusion in genus Triticum. Breeding Science 59: 671–678.
  • Møller, I.S., Gilliham, M., Jha, D., Mayo, G.G., Roy, S.J., Coates, J.C., Haseloff, J.and Tester, M. (2009) Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis. Plant Cell 21: 2163-2178
  • Plett, D., Berger, B. and Tester, M. (2009) Genetic determinants of salinity tolerance in crop plants. In Jenks, M. and Wood, A. Eds, Genes for plant abiotic stress. Wiley-Blackwell, pp83-111.
  • Widodo, Roessner, U., Patterson, J.H., Newbigin, E.J., Tester, M. and Bacic, A. (2009) Meatabolic responses to salt stress of the barley (Hordeum vulgate L.) cultivars, Sahara and Clipper, which differ in salinity tolerance. Journal of experimental Botany 60: 4089-4103.
  • Rajendran, K., Tester, M. & Roy, S.J. (2009) Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment 32: 237-249.
  • Munns R. & Tester M. (2008) Mechanisms of salinity tolerance. Annual Reviews of Plant Biology, 59, 651-681.
  • Roy, S.J. Gilliham, M., Berger, B., Essah, P.A., Cheffings, C., Miller, A.J., Widdowson, L., Davenport, R.J., Liu, L.-H., Skynner, M.J., Davies, J.M., Richardson, P., Leigh, R.A. & Tester, M. (2008) Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana. Plant, Cell & Environment 31: 861-871.
  • Tracy, F.E., Gilliham, M., Dodd, A.N., Webb, A.A.R. & Tester, M. (2008) Cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition.Plant, Cell & Environment 31: 1063-1073
  • Davenport R.J., Munoz-Mayor A., Jha D., Essah P.A., Rus A.N.A. & Tester M. (2007) The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis. Plant, Cell & Environment, 30, 497-507.
  • Byrt C.S., Platten J.D., Spielmeyer W., James R.A., Lagudah E.S., Dennis E.S., Tester M. & Munns R. (2007) HKT1;5-like cation transporters linked to Na+ exclusion loci in wheat, Nax2 and Kna1. Plant Physiol., 143, 1918-1928.
  • Johnson A.A.T., Hibberd J.M., Gay C., Essah P.A., Haseloff J., Tester M. & Guiderdoni E. (2005) Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system. The Plant Journal, 41, 779-789.

 

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