Identification of Cytokinin-Responsive Genes Using Microarray Meta-Analysis and RNA-Seq in Arabidopsis      

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 cytokininregulated 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 cytokininresponsive genes that are useful in the analysis of cytokinin function in plants.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

“…Previous results showing that a local BAP treatment of white clover roots led to cortical cell divisions, CHS induction, and flavonoid accumulation in the dividing cells also support this model (Mathesius et al, 2000). In addition, in Arabidopsis, expression of some flavonoid genes was also found to be under the control of cytokinins (Bhargava et al, 2013). However, it is likely that BAP treatment does not correctly mimic the induction of specific endogenous cytokinins during nodule initiation (van Zeijl et al, 2015), since BAP also induced changes in flavonoid gene expression in the cre1 mutant.…”

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway

“…5 A-C). At this time there is no evidence to connect TCP20 with cytokinin, as TCP20 mRNA shows no response to cytokinin (70). There are several lines of evidence that TCP20 does regulate NRT1.1.…”

The Yin-Yang of Hormones: Cytokinin and Auxin Interactions in Plant Development

“…In most plant species, cytokinin oxidase is encoded by a multigene family whose members show distinct patterns of expression, intracellular locations, and enzymatic properties Kowalska et al, 2010). Several genes encoding cytokinin oxidases are induced rapidly upon cytokinin and auxin treatment in rice (Oryza sativa) and Arabidopsis (Tsai et al, 2012;Bhargava et al, 2013;Gao et al, 2014).…”

“…This is achieved through interactions at the level of signaling and biosynthesis, though it is clear that there remains much to learn. For example, no studies have described all aspects of the interaction between these hormones (i.e., biosynthesis, transport, signaling, and degradation); indeed, additional points of regulation are known, such as the induction of several LOG and CKX genes by auxin, that have not as yet been linked to a particular developmental process (Figure 1) (Bhargava et al, 2013). Mathematical modeling has helped reveal the circuitry of these interactions and will likely continue to contribute to our understanding of how auxin and cytokinin act to bring about integrated responses.…”