Genome-wide analysis of short RNAs as modulators in dehydration stress tolerance using tolerant and genetic model systems

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Drought stress is a common adverse environmental condition that seriously affects crop productivity worldwide. The prediction is that the drought stress, in the form of unpredictable changes in rainfall or competition for fresh water with growing urban populations, will continue to be the major single abiotic factor likely to affect crop yields globally. Drought stress affects practically every aspect of plant growth and metabolism. Plant responses to water deficit depend upon factors such as duration and degree of stress, growth stage and time of stress exposure. Most environmental stresses result in water-deficit stress. Frozen soil can reduce water uptake and thus produce water stress; in the same way, salt accumulation in the soil decreases the water potential that makes soil water less available. In order to survive under water deficit conditions, plants have to maintain their water status to maintain ion homeostasis. The common responses to different stresses indicate similar functions of the gene products for plants under stress conditions involving water deficit. The existence of interacting signal perception and transduction pathways, which promote the plant stress response, is suggested by studies on gene expression during dehydration. Endogenous abscisic acid (ABA) levels increase as result of water deficit and it is thought to be involved in signal transduction. Up to now the protein coding regions have been analysed with respect to transgenic approaches to improve drought tolerance, but it has become clear that important regulatory determinants are missing. sRNAs have been recently recognised as important regulatory components of gene expression. There are two classes of sRNAs: microRNAs (miRNAs) and short interfering RNAs (siRNAs). miRNAs are endogenous regulatory sRNAs that derive from stem-loop regions of endogenous precursor transcripts. miRNAs anneal to the messages of protein-coding genes which result in the cleavage of the mRNAs. The genome wide analysis of sRNAs will allow us to identify novel sRNAs that are differentially accumulated in well watered and dried tissues. Therefore, sRNAs will be cloned and sequenced from well watered and dehydrated tissues of two different dehydration tolerant model species (C. plantagineum and M. truncatula) using high-throughput sequencing technology. The genome wide analysis of sRNAs will allow us to identify novel sRNAs that are differentially accumulated in well watered and dried tissues. The expression profile of known and novel sRNAs will be determined by microarray hybridisation and validated by Northern blots. Target genes will be predicted or experimentally determined for sRNAs that are differentially accumulated in well watered and dehydrated tissues. The biological relevance of the sRNA regulation of validated target genes will be analysed by over-expressing sRNAs or sRNA insensitive target genes. Several genomics tools are integrated in this approach, such as high-throughput sequencing and microarray hybridisation. This project is not feasible without these tools because the scale of sRNA regulation requires genome wide analysis. The advantage of the chosen methodology is that it will reveal novel sRNAs and the function of known and novel sRNAs in drought tolerance. Although it is more difficult to carry out this project using plant species without complete genome sequences, the advantages are that it allows (i) the identification of novel sRNAs that do not exist in Arabidopsis and rice and (ii) the analysis of sRNAs in two drought tolerant model species will reveal different networks regulating dehydration tolerance.