Combinatorial Microarray Analysis Revealing Arabidopsis Genes Implicated in Cytokinin Responses through the His→Asp Phosphorelay Circuitry

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Abstract
In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep histidine kinase (AHK)→histidine-containing phosphotransmitter (AHP)→response regulator (ARR) phosphorelay signaling circuitry, which is initiated by the cytokinin receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His→Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we used a combinatorial microarray strategy by employing not only wild-type plants, but also certain transgenic lines in which the cytokinin-mediated His→Asp phosphorelay signaling circuitry has been genetically manipulated. These transgenic lines employed were ARR21-overexpressing and ARR22-overexpressing plants, each of which exhibits a characteristic phenotype with regard to the cytokinin-mediated His→Asp phosphorelay. The results of extensive microarray analyses with these plants allowed us systematically to identify a certain number of genes that were up-regulated at the level of transcription in response to cytokinin directly or indirectly. Among them, some representatives were examined further in wild-type plants to support the idea that certain genes encoding transcription factors are rapidly and specifically induced at the level of transcription by cytokinin in a manner similar to that of the type-A ARR genes, which are the hallmarks of the His→Asp phosphorelay signaling circuitry. Several interesting transcription factors were thus identified as being cytokinin responsive, including those belonging to the AP2/EREBP family, MYB family, GATA family or bHLH family. Including these, the presented list of cytokinin-up-regulated genes (214) will provide us with valuable bases for understanding the His→Asp phosphorelay in A. thaliana.