The focus for this research is to develop and apply statistical methods to analyze and interpret high-throughput biological data. We developed a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. This computational approach is not only applicable to DNA microarray analysis but is also applicable to proteomics data or any other high-throughput analysis methodology. The suppression of APY1 and APY2 in mutants expressing an inducible RNAi system resulted in plants with a dwarf phenotype and disrupted auxin distribution, and we used these mutants to discover what genes changed expression during growth suppression. We evaluated the gene expression changes of apyrase-suppressed RNAi mutants that had been grown in the light and in the darkness, using the NimbleGen Arabidopsis thaliana 4-Plex microarray, respectively. We compared the two sets of large-scale expression data and identified genes whose expression significantly changed after apyrase suppression in light and darkness, respectively. Our results allowed us to highlight some of the genes likely to play major roles in mediating the growth changes that happen when plants drastically reduce their production of APY1 and APY2, some more associated with growth promotion and others, such as stress-induced genes, more associated with growth inhibition. There is a strong rationale for ranking all these genes as prime candidates for mediating the inhibitory growth effects of suppressing apyrase expression, thus the NimbleGen data will serve as a catalyst and valuable guide to the subsequent physiological and molecular experiments that will be needed to clarify the network of gene expression changes that accompany growth inhibition. / text

Adenosine triphosphate (ATP) is well recognized for its role as the primary cellular energy currency. However, studies dating back to 1929 have reshaped our understanding of ATP as not only an energy source, but also as a signaling agent. Among the most important of these discoveries are animal purinergic receptors (P2X and P2Y receptors) that perceive extracellular ATP (eATP), primarily in the nervous system. Though eATP is an established receptor agonist in animals and applied poorly hydrolyzable ATP analogs have numerous effects on growth in plants, eATP is not widely accepted as a signal in plants where no purinoceptor has been identified. Here, enriched outside-out plasma membrane vesicles were isolated and proteins labeled with a radioactive ATP analog (8N₃ATP[α²³P]) to identify a putative purinoceptor-like receptor. We used etiolated seedlings to capture proteins from plant tissue that was actively growing and used sodium carbonate washes to separate peripheral and integral membrane proteins. With this method, we have generated lists of plasma membrane ATP binding proteins, and therefore possible eATP receptors. Ectoapyrases are phosphohydrolases thought to regulate eATP in both animals and plants. Here, we also investigated the expression and role of the candidate ectoapyrases AtAPY1 and AtAPY2 in guard cells and stomatal responses. AtAPY1 and AtAPY2 transcript and protein expression was confirmed in guard cells. Early genetic studies using an apy2 knock out with induced RNAi-silencing of APY1 suggest a role for these apyrases in stomatal regulation. In response to treatment with five hours light, the apyrase-suppressed line features wider stomatal aperture when compared to WS wild-type. / text