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Honours supervisors and projects 2008
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Environmental regulation of gene expression in Arabidopsis and resurrection plants
Telephone: 9905 3851
Email: alan.neale@sci.monash.edu.au
(Dr A.D. Neale in collaboration with Prof. John D Hamill and Dr Cecilia Blomstedt)
Anhydrobiotic organisms can survive in very dry deserts or freezing polar areas by entering a state of total metabolic inactivity (“suspended animation”) which is often induced by desiccation. Anhydrobiotic organisms are found in a variety of biological taxa, including both unicellular and multicellular organisms in both the animal and plant kingdom (Clegg, 2001). Projects in this area will focus on characterising the molecular genetic response to severe drought stress of a remarkable group of plants termed resurrection grasses. These plants can survive complete desiccation and also are tolerant of high levels of salt stress. Drought-stress, often accompanied by high salinity, is increasingly being recognised as a critical factor for sustaining agricultural production in many regions of the world which may experience significant climate change over the forthcoming decades. Knowledge relating to the various mechanisms that enable plants to cope with such stresses is crucial from both a fundamental and an applied perspective.
Many of the early genetic responses to mild water loss are analogous in both resurrection plants and drought-sensitive plants such as Arabidopsis (Neale et al., 2000). It is noteworthy however that Arabidopsis can only survive significant water loss for a few hours and is unable to recover from extended periods of desiccation (Yamaguchi- Shinozaki et al., 1992). In contrast, the drought- and salt-tolerant resurrection grass Sporobolus stapfianus can fully and rapidly recover from ≥ 95% water-loss, even after an extended period of time in the desiccated state (Gaff & Loveys, 1993). This is because individual cells of this resurrection plant exhibit protoplasmic drought-tolerance which allows the plant to systematically reduce growth and retain viable leaf and root tissue during periods of drought, and then reinstitute growth within hours of water becoming available (Ghasempour et al., 2001).
Determining which genes have an adaptive role in establishing tolerance to cellular dehydration is of critical importance (Oliver et al., 2004). In angiosperms such as S. stapfianus, desiccation tolerance is believed to have evolved from the ectopic expression in vegetative tissues of genetic processes associated with the seed developmental program, as species spread into arid areas (Oliver et al., 2000). This suggests that desiccation tolerance in these resurrection plants may be conferred by a unique pattern of regulatory gene expression rather than the presence of unique structural genes. Knowledge of regulatory genes likely to control desiccation-tolerant gene expression patterns in resurrection plants in general is very limited. Nevertheless, use of these extremophiles is required to identify genes specific to the desiccation tolerant state. Ultimately an understanding of the function of these genes and how their expression is regulated will throw light on the mysteries of desiccation tolerance. With an understanding of the molecular mechanisms which allow this monocotyledonous resurrection plant to successfully cope with water loss we will be in a strong position to adapt components of this genetic program to agriculture to minimise crop injury and loss of productivity caused by water deficit.
In this project we take advantage of the finding that S. stapfianus leaf protoplasmic drought-tolerance is induced at a water deficit of 60% relative water content (RWC) but only when the leaf tissue is drying on the intact plant (Gaff & Loveys, 1993). Leaf tissue dried at the same rate to 60% RWC following detachment is desiccation sensitive. This provides a unique means of distinguishing, amongst a large number of drought responsive genes, those genes which may be critical to protoplasmic drought-tolerance. A number of genes which are induced in these grass plants in response to severe drought stress have been recovered and characterising these genes will allow us to deduce how their encoded protein products may contribute to drought and salt tolerance. The yeast one hybrid system has also been used to identify genes encoding transcription factor proteins which control expression in severely drought-stressed tissues.
Work in this area involves a molecular analysis of selected gene and associated regulatory DNA sequences and recovery and characterisation of full length genes encoding DNA binding proteins which play a role in the regulation of gene expression in drought and salt stressed tissues. Transgenic experiments conducted in Arabidopsis, rice and other plant systems will investigate the effects of over expressing specific gene sequences that we have isolated from the African resurrection grass Sporobolus stapfianus in tissues of drought/salt tolerant species to assess the extent to which stress tolerance is increased.
(Dr Alan D. Neale in collaboration with Dr Ros Gleadow, Prof. John D Hamill & Dr Cecilia Blomstedt).
Forage sorghum is a fast growing crop with a high water use efficiency and is grown throughout the world as a pasture plant, especially in the dry tropics. In Australia, the total forage sorghum area is approx 400,000 ha. With increasingly warm and dry conditions predicted for the next century, it is likely that it will become even more widely planted.
The problem is that sorghum produces prussic acid (hydrogen cyanide), particularly when plants experience non-optimal environmental conditions. The prussic acid is stored in a stable, non-toxic form as a cyanogenic glycoside called dhurrin (see Fig. 1). Dhurrin breaks down to release toxic cyanide when plant tissue is damaged, for example when the leaves are chewed, in a process known as cyanogenesis (Fig. 1). Cyanogenesis is primarily a herbivore defence mechanism (Gleadow and Woodrow 2002) although cyanogenic glycosides may also play a role in storing reduced nitrogen (Selmar et al. 1988). Cattle and sheep are able to tolerate a certain amount of prussic acid (Westley 1988). However, under adverse environmental conditions, such as drought, the concentrations become elevated, resulting in respiratory poisoning and possible death of stock (Mackay 2005). It is estimated that about 10% of the crop is never grazed because of fears of prussic acid poisoning. Some deaths do occur but the biggest impact is not utilising crops, especially as this is usually when crops are moisture stressed and there is little alternative feed.
Similar problems occur for people in a number of third world countries where the major staple Cassava may contain lethal levels of the cyanogenic glucosides especially in times of drought. People eating improperly processed cassava root suffer from debilitating afflictions such as paralysis, impaired vision, goiter, and cretinism.
By using expertise in molecular genetics, biochemistry, ecophysiology and agriculture, this project takes an integrated approach to the regulation of cyanogenesis in the important agricultural plant Sorghum. The project aims to address the substantial problem of sorghum toxicity to livestock and also answer some fundamental questions about the allocation of resources to defence-related secondary metabolism in plants The genes responsible for dhurrin synthesis have been identified and characterised. It should therefore be possible to assess whether dhurrin concentration is actively or passively regulated by examining the alterations in mRNA transcription under adverse environmental conditions. Moreover, identification of genes responsible for dhurrin production, catabolism, storage and detoxification, in conjunction with recent developments in our understanding of plant drought responses (e.g. Neale et al. 2000) present a number of different ways of developing a constitutively low-cyanogenic Sorghum line that does not accumulate dhurrin as a response to water-deficit. We also hope to identify mutants with enhanced cyanogenic capacity for use in soil biofumigation. The use of agricultural chemicals is expensive and, in some cases, environmentally unfriendly. Any reduction in the use of fumigants to kill nematodes or other soil-borne pests would be of benefit economically and help to develop environmentally sustainable practices. Some farmers already exploit the cyanogenic capacity of sorghum as a natural soil fumigant by ploughing uneaten plants into the ground (Only the above-ground plant parts contain significant amounts of dhurrin). If we identify plants with extremely high prussic acid concentrations then this could give rise to an independent product —an ‘organic’ soil fumigant.
THE AIM of this project is to:
(2) Mutagenesis: The TILLING method utilises a chemical mutagen to introduce
random single nucleotide alterations in the DNA of an organism (Henikoff et al. 2004; Till et al., 2004; McManus et al. under review). Mutagenised plants will be screened for induced mutations in particular genes of interest. DNA is isolated from leaf tissue of several plants and utilized as templates for PCR using a proof-reading enzyme and fluorescent labelled primers. To detect mutations within the amplified regions, the PCR products are heated and cooled to allow duplexes to form. Digestion with the enzyme CEL1 targets mismatches in these duplexes, resulting in fragmentation of the amplified region. Electrophoresis will reveal those plants that contain CEL 1 fragmented products using a LI-COR slab gel analyser. The selected lines that contain the mutations of interest can then be used in conventional breeding programs. In addition to screening mutants for the desired genes, in collaboration with overseas groups who have experience in this area, we will also examine any downstream effects on the metabolome and transcriptome
Identification of novel genetically altered sorghum will allow the development of high yielding, safe and nutritious forage sorghum varieties as well as lines with enhanced capacity to synthesise dhurrin for use as natural soil biofumigants. These varieties will not contain recombinant DNA and will be of particular interest to the increasingly important organic farming component of the agricultural sector. The findings from this project may also ultimately be useful for controlling cyanogenesis in cassava.
