Plant Alternative Splicing and Abiotic Stress (PASAS)

Genetic variation is an important basis for biodiversity and phenotypic variation. How plants respond to external stimuli such as attack by pathogens/pests or stress conditions depends on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels. Alternative splicing affects all aspects of plant development, viability and adaptability to external conditions including abiotic stress. Nevertheless, AS in plants has been largely over-looked as a major contributor to global gene expression control despite its influence on transcript levels and on generation of differential protein functions. Current estimates suggest that a significant proportion of plant genes (35%) undergo alternative splicing. We have shown that this is clearly underestimated such that substantially more genes undergo AS and many genes have more AS events than have been described to date. In addition, alternative splicing patterns of many genes are regulated by different stress conditions and by altered levels of proteins which are involved in splicing. It is, therefore, essential to have a concerted effort to discover and identify AS events and to develop methods to accurately measure changes in AS at global and gene-specific levels. We will approach this by applying genomics and bioinformatics tools: 454 sequencing, whole genome tiling arrays, an RT-PCR system to quantify AS transcript levels and algorithms for comparing ESTs, to plants experiencing different abiotic stresses and with altered expression of splicing factors. We will identify new, un-annotated AS events, assess the technologies for their potential to provide quantitative analysis of AS and use these tools to study changes in AS under abiotic stress. We will also use these technologies to extend our examinations of the roles of specific trans-acting alternative splicing factors in regulating AS. The proposal is directly relevant to the themes of the ERA-PG call and brings together four research groups already involved in plant alternative splicing research and abiotic stress. The measurable outcomes will be novel, previously undescribed AS events deposited in appropriate databases; techniques for studying changes in AS; a database of AS events with their frequency and consequences; and information on the relationship between transcript levels and AS for many Arabidopsis genes. An overall outcome of this research will be a significantly increased awareness of the importance of AS and the need to consider AS and its consequences in post-transcriptional control of plant gene expression. Knowledge of the complexity and subtlety of all aspects of gene regulation is important in understanding how plants grow and survive and in the prediction of responses to changing environments and such awareness is the cornerstone of future plant breeding policies.

Alternative splicing (AS) affects all aspects of plant growth, development and responses to abiotic and biotic stress. For example, AS in plants occurs in flowering time control, regulation of the circadian clock, disease resistance, various metabolic enzymes as well as in both transcription factors and splicing factors. AS can affect mRNA stability and protein expression, activity, localization and ability to interact with other proteins. In addition, AS is dynamic and patterns of many genes are regulated by different stress conditions and by altered levels of proteins which are involved in splicing. Although current estimates of the number of plant genes which undergo alternative splicing (35%) represents a substantial fraction of plant genes, we have shown that this figure is an underestimate and have detected numerous previously un-annotated alternative splicing events. Key goals in plant alternative splicing are therefore to identify as many AS events as possible (discovery) and develop methods to accurately measure changes in alternative splicing under different conditions at global and gene-specific levels (quantitation). The main partners in the project will take a co-ordinated approach in the application of genomics and bioinformatic approaches to AS discovery and quantitation. These include 454 (the most appropriate new generation sequencing platform for AS discovery) (Barta, Brown), whole genome tiling arrays (Fluhr), an RT-PCR system to quantify AS transcript levels (Brown) and algorithms for comparing ESTs, handling 454 sequences and extracting AS data from tiling arrays (Fluhr, Barta). The same plant material will be used in the different platforms to allow direct comparison. We will identify novel AS events, assess the technologies for their ability to quantify AS and use these tools to study changes in AS in plants grown under abiotic stress and in mutants of specific trans-acting alternative splicing factors.