Program 1: Stress responses in adapted cereals

The fundamental approach in this Program will be to define existing mechanisms used by adapted cereal species to tolerate abiotic stresses and to attempt to transfer genes responsible for the tolerance into elite wheat and barley varieties.

Objectives:

The objectives of this program are to:

• Define stress perception mechanisms used in adapted wheats and barleys to ameliorate stress impact on productivity
• Examine stress response mechanisms imposed either individually or simultaneously at the genetic, transcriptional, proteome and metabolite levels
• Develop efficient processes to move from defined genetic loci conditioning stress tolerance to gene isolation
• Utilise the available adapted germplasm to define the basis for allelic diversity for abiotic stress tolerance
• Transfer alleles for stress tolerance to elite wheat and barley varieties.

Project 1.1. - Transcript Profiling

Plants have the ability to respond to environmental changes by altering the expression of complex gene networks through sensing environmental cues, signal transduction, and modification of biochemical pathways. These transcriptional changes can result in successful adaptations leading to tolerance in tolerant genotypes. To comprehensively understand stress adaptation processes, transcript profiling of both tolerant and sensitive responses under different abiotic stresses will be performed using microarray analysis. The outcomes of this project are to produce integrated transcript profiles of plant responses to multiple abiotic stresses to provide a comprehensive understanding of stress adaptation and clues for identification of genes, which are useful for improvement of abiotic stress tolerance.

• Initial work will focus on the development of the systems for expression profiling and the identification of germplasm for the differential screening.

Project 1.2 - Map-based cloning of abiotic stress tolerance genes

Natural variation at loci governing tolerance to various environmental stresses has been genetically mapped in wheat and barley. Although technically challenging, map-based cloning in the large-genome species barley and wheat has become much easier due to the availability of the rice genomic sequence and other tools. Potential target loci have been identified, and construction of segregating populations for fine-mapping genes of partial effect has been initiated.

The initial targets will be genes conferring tolerance to high soil boron, salinity and frost.

Program 2: Adaptation to extreme stress

The fundamental approach in this Program will be to define mechanisms used by grass species other than cereals to tolerate extreme environmental conditions and to transfer genes responsible for this tolerance into elite wheat and barley varieties.

Objectives:

The target species for gene discovery and functional genomics will be selected on the basis of a high level of adaptation to local conditions of abiotic stress, and from exotic grass species showing adaptation to extreme environments. More specific project objectives are:

• To isolate novel candidate genes and alleles conferring tolerance to abiotic stresses, such as drought, cold, salinity, sodicity, low soil fertility, mineral toxicities, etc. from wild relatives of wheat and barley, and from native and exotic species of grasses showing unique modes of adaptation to these abiotic stresses in extreme environments.
• To investigate functions of novel candidate genes and alleles through direct analysis in prokaryotic and eukaryotic expression systems.
• To identify protein characteristics and cellular metabolites correlated with stress tolerance through comparative molecular profiling and modelling of proteins and metabolites from adapted and non-adapted grasses.
• To develop new strategies for enhancing and extending stress tolerance well beyond that seen in existing germplasm in cereals wheat and barley, through introgression of chromosome segments from wild relatives of wheat and barley and through gain-of-function by transformation of novel candidate genes and alleles for the control of abiotic stress tolerance from native and exotic grasses.
• To define the function and adaptive features of key stress-related proteins at the molecular level, using X-ray crystallography, and use this information to design new alleles.
• To generate a proprietary database of derived gene sequences and a proprietary set of associated technologies for manipulation of stress tolerance in grains.

Project 2.1 - Grasses Adapted to Extreme Environments

This xenogenomics program aims to identify the genes controlling tolerance to salt stress in the blown grasses Lachnagrostis adamsonii (formerly Agrostis adamsonii ) and L. robusta , aluminium toxicity in weeping grass Microlaena stipoides , and cold and freezing stress in Antarctic hair grass Deschampsia antarctica . For each species collections of 4000-7000 ESTs have been derived from stressed plants. Genes with inferred roles in tolerance to extreme abiotic stresses have been identified and will be tested in transgenic material for their capacity to confer agronomically useful phenotypes in wheat and barley.

Project 2.2 - Allele Discovery

This project aims to use information developed from Program 1 to scan germplasm collections for novel alleles at target loci. At this stage the main emphasis has been on identifying germplasm, establishing screening procedures and obtaining seed of the key lines. Attention will be focused on improved screening of the databases and on building closer links with the breeding programs .

Program 3: Signalling Pathways and Stress Networks

This Program will draw on information generated from Programs 1 and 2, and will involve an evaluation of the genetic, cellular and biochemical networks that mediate abiotic stress perception and plant responses to the abiotic stresses.

Objectives

The overall objective of this program is to build a genetic and cellular picture of pathways involved in stress perception and signal transduction, and to identify key interconnecting points where signalling and response pathways intercept or interact. More specific objectives of this program are to:

• Identify genes responsive to abiotic stress at key stages of root, flower and grain development.
• Define metabolic responses to abiotic stress in key tissues and organs of stressed plants.
• Develop an overview of protein changes and interactions during abiotic stress responses.

Project 3.1 - The role of thioredoxins in the oxidative stress response of cereals

Thioredoxins are ubiquitous antioxidant proteins that contain a conserved dicysteine active site. The active site participates in dithiol/disulfide exchange reactions with a broad range of cellular substrates including peroxidases, kinases and transcription factors. This project aims to identify the stress-responsive biochemical pathways that are modulated by cytosolic thioredoxins in cereals.

Project 3.2 - Characterising monodehydroascorbate reductase (MDHAR) in Physcomitrella patens

Plant cells exposed to stress conditions often generate reactive oxygen species (ROS), which can be scavenged by oxidation of ascorbate. This process is catalysed by ascorbate peroxidase, but to maintain the scavenging potential of the ascorbate pool, ascorbate has to be re-reduced by MDHAR, using NAD(P)H as an electron donor. In addition, MDHAR has been shown to catalyse the reduction and thus detoxification of phenolic radicals. MDHAR activity is found in many cellular compartments and in different tissues, but only in plants and algae. The objective of the project is to characterise the different isoforms of MDHAR found in Physcomitrella , with respect to their physiological role under stress conditions and their biochemical properties.

The moss Physcomitrella has been established as a model organism, partly because it is capable of homologous recombination and thereby facilitatates studies using reverse genetics. The direct cloning of the different isoforms of MDHAR has been achieved.

Project 3.3 - Roles of plant callose synthases in stress tolerance

There are 12 callose synthase genes in Arabidopsis and previous work has indicated that these genes play a role in both biotic and abiotic stresses. The aim of this project is to characterise members of the family, to identify those genes involved in abiotic stress tolerance, and to define the mechanism of tolerance.

Project 3.4 - 14-3-3 The role of 14-3-3 proteins in barley stress responses

Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate the activities of a wide array of phosphorylated proteins via direct protein-protein interactions. They bind a range of transcription factors and other signalling proteins, and have roles in the regulation of plant development and stress responses. Elucidation of the role of each isoform of the 14-3-3 proteins will allow us to determine the pathways involved in stress response and/or the identification of individual gene products responsible for tolerance/resistance to cold and other abiotic stresses. Proteomics will become a major component of this project.

Project 3.5 - Protein kinases

Three cDNAs have been isolated via SSH (suppression subtractive hybridisation) from barley epidermis after challenging with Rhynchosporium secalis . They represent gene fragments with significant homology to receptor like kinases (RLKs). Preliminary work provided data on expression pattern and map location. A fourth RLK was identified in a yeast two-hybrid screen using a fungal elicitor as bait. Sequence analysis is currently underway with homologues from different susceptible and resistant barley cultivars. Further yeast two-hybrid screenings are in progress and will be backed up by co-immunoprecipitation experiments.

Project 3.6 - Proteomics in roots

At present, the mass spectrometry facility at the University of Melbourne is being optimised for the proteomic work. A MALDI-TOF machine, a tandem electrospray LC-MS/MS and a LC/LC-MS/MS are being tested, together with database searching capabilities. The aim is to investigate the proteomic make up of tissue samples from both freezing stress and boron tolerance projects

Experiments have commenced on the study of the differential response of different barley cultivars to boron toxicity, with the view to determining the mechanism responsible for boron tolerance/resistance. The proteomic profile will be monitored in both boron tolerant and boron intolerant barley cultivars in the presence and absence of boron in a hydroponic system. Differential proteomics of individual organelles will be studied to determine the effect of compartmentalization in the boron tolerance mechanism.

Project 3.7 - Regulatory elements

A number of different approaches will be used to identify novel gene regulatory elements in the form of promoters, enhancers within intron sequences and also factors that affect transcription of specific gene sequences or coordinate multiple steps in a pathway or network. Work has been focused on the development of yeast one-hybrid systems for the rapid identification of transcription factors. A new vector system has been constructed and tested using a known regulatory element from Arabidopsis. The system will now be tested for wheat and barley regulatory elements.

Program 4: Core Technologies

Overall, this program aims to provide technical expertise, efficient protocols and specialist knowledge for a range of applications that underpin the scientific research within the ACPFG.

These include:

• Bioinformatics and data handling from gene/protein discovery programs across all ACPFG Research Projects
• High throughput functional gene analysis, utilizing both transient expression of transgenes and stable transformation
• Heterologous expression systems for production of proteins, 3D structural analysis and antibody production for proteomics projects, studies of enzyme function and localization, and protein-protein interactions.

Program 5: Biotic Stress

Projects in this program fall outside of the ACPFG’s brief to work on abiotic stress tolerance, and are funded by additional grants.

Project 5.1 - Barley Diseases

Scald

The scald-causing pathogenic fungus, Rhynchosporium secalis is widespread in Europe, North America and Australia. Yield losses as high as 35-40% have been reported, however, losses of between 1-10% are more common and are a result of reduced grain weight. This project aims to isolate and understand the interplay of genes specifically involved in the regulation of resistance to R. secalis . Differentially expressed cDNAs have been identified in resistant and susceptible barley cultivars when challenged with the fungus by using suppression subtractive hybridization (SSH) and microarray analysis. So far about 30 clones show an interesting expression pattern and are candidates for further analysis to assign a specific function in the context of resistance to fungi and in comparison to other stresses. Our main focus is on the early response of the plant to the fungal attack, covering recognition of elicitors and subsequent signal transduction.

Powdery Mildew

The role of candidate genes will be compared between the necrotrophic R. secalis -barley system and the biotrophic powdery mildew ( Blumeria graminis ) and leaf rust ( Puccinia triticina ) systems. Stably transformed plants, mutants or transiently transformed tissue will be inoculated with spores of powdery mildew and/or leaf rust to assess their phenotype when challenged with biotrophic pathogens.

Leaf Rust

Suppression subtractive hybridisations have been used to enrich for differentially expressed genes in leaf rust resistant and susceptible bread wheat lines. cDNA libraries containing about 800 subtracted clones are currently stored as glycerol stock cultures for further investigations.