Abstract
Cytokinins are N(6)-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 (Arabidopsis thaliana). Microarray meta-analysis using 13 microarray experiments combined with empirically defined filtering criteria identified a set of 226 genes differentially regulated by cytokinin, a subset of which has 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 overrepresentation of type-B Arabidopsis response regulator binding elements, consistent with the role of type-B Arabidopsis response regulators 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 quantitative reverse transcription-polymerase chain reaction 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.
Highlights
Cytokinins are N6-substituted adenine derivatives that play diverse roles in plant growth and development
Type-B ARRs are activated by phosphorylation and mediate cytokinin-regulated gene expression, including the up-regulation of the type-A ARRs, which act as negative feedback elements in cytokinin signaling (Brandstatter and Kieber, 1998; D’Agostino et al, 2000; Sakai et al, 2000; Imamura et al, 2003; Tajima et al, 2004; To et al, 2004; Taniguchi et al, 2007; Argyros et al, 2008)
The method of application did have an effect on gene expression, as these experiments did tend to cluster together. This analysis revealed substantial differences in overall gene expression profiles based on the experimental designs and tissues used for these microarrays, which suggests that genes regulated by cytokinin across these diverse conditions will reflect robustly regulated genes
Summary
Cytokinins are N6-substituted adenine derivatives that play diverse roles in plant growth and development. Microarrays have been widely used in the genomewide analysis of hormone responses in Arabidopsis, including the response to altered cytokinin function (Rashotte et al, 2003; Kiba et al, 2004; Brenner et al, 2005; Lee et al, 2007; Taniguchi et al, 2007; Yokoyama et al, 2007; Argyros et al, 2008; Dello Ioio et al, 2008; Goda et al, 2008; Müller and Sheen, 2008; Argueso et al, 2010; Bishopp et al, 2011; Köllmer et al, 2011; Brenner and Schmülling, 2012) These data sets have identified many genes regulated by cytokinin, and several have been validated by other methods (Supplemental Table S1). A recent study presented a preliminary meta-analysis of cytokinintreated microarrays in Arabidopsis, though this study was limited to comparable growth conditions, treatment, and time points, and only a small subset of the genes identified were presented (Brenner et al, 2012)
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