SILAC proteomics for quantitation of protein isoforms from alternative splicing in Arabidopsis seedlings

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Genetic variation is an important basis for biodiversity and phenotypic variation. Plant growth and productivity and how plants respond to external stimuli such as pathogens/pests or stress conditions depend 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 many aspects of plant development, viability, adaptability to external conditions, metabolism and physiology and the most recent estimate suggests that at least 60% of intron-containing genes in plants undergo AS. More and more examples are being described where AS regulates expression or functional protein diversity. As our overall knowledge of this important regulatory level increases, it becomes necessary to be able to investigate the impact and dynamic changes of AS at the protein level. There are particular challenges in identifying and quantifying peptides deriving from different protein isoforms generated by AS (e.g. the abundance of peptides that distinguish specific isoforms from peptides common to isoforms)Of various mass spectrometry (MS)-based quantitative proteomics methods, SILAC has a number of advantages for this specific application: many thousands of proteins are routinely detected, usually a significant fraction of proteins have multiple peptides, quantitation of proteins is relatively straightforward and good MS analysis software is available. In addition, SILAC,is able to quantify low abundance peptides and phosphopeptides, and analysis methods have been developed for analysis of protein isoforms in human cells. SILAC has had very limited use in plants (cell culture) due to inefficient labelling with stable-isotope-containing amino acids as plants are autotrophic. We have developed a method to obtain high levels of SILAC labelling (>90%) and, more significantly, in Arabidopsis seedlings (as opposed to cell cultures). This makes this frontline technology amenable to whole plant systems for the very first time and means that it can be applied to a wide range of areas of plant biology from development to responses to biotic and abiotic stresses, and facilitates the comparison of mutants at the protein level.In this proposal we will use this new modification of SILAC technology which allows it to be applied to plant seedlings to investigate alternative splicing at the protein level. We will analyse three sets of genetic lines where splicing factors or NMD factors are over-expressed or mutated and where we know from transcript analysis that the AS of many genes is significantly altered. These lines will maximise the opportunity for identifying isoform-specific peptides. There is currently very little data on protein isoform variants in plants and exploiting SILAC and using specific genetic lines will allow us to better understand the impact of AS at the protein level. Finally, we expect our plant SILAC system to be applied more widely to other, non-model plant species and to provide a new method to investigate biochemical questions of post-translational modification, protein turnover, microRNA effects on protein levels, and protein interaction networks.