Identification of Cytokinin-Responsive Genes Using Microarray Meta-Analysis and RNA-Seq in Arabidopsis
Cytokinins are N6-substituted adenine derivatives that play diverse roles in plant growth and development. We sought to define a robust set of genes regulated by cytokinin, as well as to query the response of genes not represented on microarrays. To this end, we performed a meta- analysis of microarray data from a variety of cytokinin-treated samples and used RNA-seq to examine cytokinin-regulated gene expression in Arabidopsis thaliana. Microarray meta-analysis using thirteen microarray experiments combined with empirically-defined filtering criteria identified a set of 226 genes differentially regulated by cytokinin, a subset of which have previously been validated by other methods. RNA-seq validated about 73% of the up-regulated genes identified by this meta-analysis. In silico promoter analysis indicated an over- representation of type-B ARR binding elements, consistent with the role of type-B ARRs as primary mediators of cytokinin-responsive gene expression. RNA-seq analysis identified 73 cytokinin-regulated genes that were not represented on the ATH1 microarray. Representative genes were verified using qRT-PCR and NanoString analysis. Analysis of the genes identified reveals a substantial effect of cytokinin on genes encoding proteins involved in secondary metabolism, particularly those acting in flavonoid and phenylpropanoid biosynthesis, as well as in the regulation of redox state of the cell, particularly a set of glutaredoxin genes. Novel splicing events were found in members of some gene families that are known to play a role in cytokinin signaling or metabolism. The genes identified in this analysis represent a robust set of cytokinin- responsive genes that are useful in the analysis of cytokinin function in plants.