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Drought Reverse Genetics
Because tolerance to drought has many confounding complexities both at the genetic and environmental levels, dissecting the molecular and physiological mechanisms controlling this trait are very challenging and time-consuming.
One means of simplifying these analyses is to investigate classes of genes of known molecular function and to determine individually their effect on various drought-related phenotypes. This approach termed “reverse genetics” incorporates recent advances in recombinant DNA technologies and transgenesis. Central to this strategy are tools to isolate gene sequences and their regulatory elements and report their activity in vitro or in planta.
For example, both yeast-1-hybrid (Y1H) and yeast-2-hybrid (Y2H) libraries constructed of wheat and barley cDNAs have been used to identify various transcription factor (TF) complexes that bind drought-responsive gene switches (Lopato et al., 2006). Overexpression of individual TFs in planta using constitutive promoters (Ubiquitin and CaMV 35S promoters) has demonstrated that these TFs can ectopically trans-activate portions of the drought-responsive transcriptional network.
Unfortunately, constitutive overexpression of these TFs often results in unwanted pleiotropic side effects such as delayed phenological development (Morran et al., 2010). To minimize such effects, TF expression in planta has been tailored to overexpress upon drought stress (stress inducible Rab17 promoter).
Further research aims to temporally and spatially fine-tune transgene expression and generate a suite of well-characterized promoters for this purpose. Collections of stress inducible and tissue-specific promoters are currently being tested in wheat and barley using transcriptional reporter genes gfp and gus (Li et al., 2008; Rai et al., 2009; Kovalchuk et al., 2009; Kovalchuk et al., 2010). Mapping of stress specific cis-elements in some of these promoters and the isolation of their cognate transcription factors using Y1H system will help to understand mechanisms and specificity of promoter activation under different stresses, reason of high levels of basal activity of some promoters and differences in levels of expression of the same genes in drought tolerant and drought sensitive cultivars. Gained knowledge about promoter structure and function will permit to change their characteristics by introducing changes in promoter size and in number of active cis-elements (generation of artificial promoters) with the aim to adopt them for particular biotechnological purposes and decrease risk of their activation in transgenic plants by other stresses or stimuli.
Another avenue through which to fine-tune the drought responsive gene transcription is at the post-translational level whereby targeted alteration of TF-cis element binding domains or TF-TF interaction domains may help generate novel combinations that eliminate unwanted pleiotropic side effects, decrease dependence of protein-DNA interactions from modifying enzymes, elucidate mechanisms of passive and active repression, and understand role of different subunits in multisubunit transcription complexes.
Much insight into stress response gene networks and biochemical pathways has been gained through functional genomics studies of model species including Arabidopsis and rice. Gain- and loss-of-function mutant populations from these model species have proven essential for the identification of core regulatory components of drought response networks and metabolic pathways.
Abscisic acid (ABA) biosynthesis for example, is one such metabolite pathway that is upregulated upon water deficit. The key rate-limiting enzymatic steps of this pathway have been determined and manipulated using reverse genetic methodologies to gain a greater understanding of ABA’s role during drought stress (Shinozaki et al., 2007).
Our research focuses on several metabolite pathways identified in model species as critical for a plants co-ordinated response to drought stress. These drought-related metabolite pathways have also been identified within transcriptomic, proteomic, metabolomic data collated from our forward genetics program. We aim to modulate these metabolite pathways by gene-stacking rate-limiting enzymes and to use pathway mutants to dissect the relative importance of each metabolite with respect to the targeted drought tolerance component trait. The drought component trait will be assessed using high-resolution plant phenotyping (APPF).
International collaboration through Pioneer Hybrid International/DuPont http://www.acpfg.com.au/uploads/documents/news/PION%2011-10%20JointACPFGDuPontReleasevFinal.pdf
Pioneer Hybrid International/DuPont (USA)
Shinozaki K and Yamaguchi-Shinozaki K. Gene networks involved in drought stress response and tolerance J. Exp. Bot. 2007 58:221-7.
Lopato, S., Bazanova, N., Morran, S., Milligan, A. S., Shirley, N. and Langridge, P. (2006) Isolation of plant transcription factors using a modified yeast one-hybrid system. Plant Methods 2:3-17.
Morran S, Eini O, Pyvovarenko T, Parent B, Singh R, Ismagul A, Eliby S, Shirley N, Langridge P, Lopato S. Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors Plant Biotechnol J. 2010 doi: 10.1111/j.1467-7652.2010.00547.x