Identification of genes cooperatively regulated by cytokinin and HD-ZIP III transcription factors

In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.

在拟南芥（Arabidopsis thaliana）中，细胞分裂素响应型B型ARR转录因子与REVOLUTA（REV）等HD-ZIP III类转录因子协同作为茎再生的核心调控因子。为鉴定ARR-HD-ZIP III转录复合物的下游靶基因，我们采用了REV的诱导表达株系35S::FLAG-GR-rREV，该株系通过35S启动子表达经FLAG标签标记的、miR165/6不可靶向的REV突变体（rREV）与GR的融合蛋白。地塞米松（DEX）处理可促使FLAG-GR-rREV融合蛋白从细胞质易位至细胞核，从而激活REV的功能。我们将35S::FLAG-GR-rREV幼苗分别用6-苄氨基腺嘌呤（6-BA，一种细胞分裂素）、地塞米松（DEX）以及6-BA与DEX的混合液处理2小时，提取总RNA后进行安捷伦（Agilent）拟南芥基因表达芯片分析，筛选出表达差异倍数>1.5倍、P值<0.05的差异表达基因。我们选取10日龄的35S::FLAG-GR-rREV植株，分别用6-苄氨基腺嘌呤（6-BA）、地塞米松（DEX）以及6-BA与DEX的混合液处理2小时；DEX处理可通过使FLAG-GR-rREV融合蛋白从细胞质易位至细胞核，实现REV的激活。使用RNeasy Mini Kit提取总RNA，并将其与安捷伦拟南芥基因表达芯片进行杂交；差异表达基因的判定标准为表达差异倍数达1.5倍以上且P值<0.05，实验设置了生物学重复。