Identification of BABY BOOM downstream targets

A glucocorticoid-regulated BBM protein (35S:BBM-GR) was used in combination with microarray analysis to identify genes directly activated by BBM. We employed the system described by (Lloyd et al., 1994) in which dexamethasone (DEX) and cycloheximide (CHX) are applied together to respectively, induce nuclear localization of the BBM-GR protein and prevent translation of the primary targets mRNAs. In this way it is possible to identify direct targets of a transcriptional activator by comparing gene expression profiles between DEX+CHX-treated transgenic and wild-type tissues. The ability of the 35S:BBM GR construct to induce somatic embryogenesis in Arabidopsis seedlings was determined by phenotypic observation of 35S:BBM GR seeds germinated and grown in the presence of 10 µM dexamethasone (DEX). As in 35S:BBM seedlings, we observed somatic embryo formation on the cotyledons, first leaves and shoot meristem of DEX-treated 35S:BBM GR seedlings. We identified a set of 20 genes (including BBM itself) and our analysis indicates that BBM directly activates a signaling pathway comprising transcription factors and other signaling molecules, but which does not initially include genes known to induce somatic embryogenesis, such as LEC1, LEC2 or WUS. The functions of the BBM target genes are unknown, however a number of them have recently been identified in microarray screens for meristem-expressed genes. The identification of BBM-interacting partners and downstream targets provides new tools for unraveling pathways related to plant cell growth and organogenesis. Keywords: transcriptional activation To identify candidate BBM targets, we treated 4 day old 35S:BBM-GR seedlings for 8 hours with 10 µM DEX in the presence of 10 µM CHX and compared the mRNA population from these seedlings using Arabidopsis Operon microarrays (http://www.ag.arizona.edu/microarray/) to that of 4 day old wild-type seedlings treated in the same way. The seed batches used in this analysis showed 100% EFS when grown continuously on media with 10 µM DEX. Four day old seedlings were chosen as the experimental material because they are highly responsive to BBM GR activation and provide sufficient RNA for the microarray experiments. The biological reproducibility of the data was monitored using two independent single locus 35S:BBM GR lines. The technical reproducibility of the data was monitored using two independently DEX + CHX-treated samples from each transgenic line. Dye effects were monitored in a dye-swap experiment using one of the four RNA samples from the two biological replicates

本研究采用糖皮质激素调控的BBM蛋白（35S:BBM-GR）结合基因芯片分析（microarray analysis），以鉴定BBM直接激活的靶基因。我们采用Lloyd等人（1994）报道的实验体系，即同时施加地塞米松（dexamethasone, DEX）与环己酰亚胺（cycloheximide, CHX），分别诱导BBM-GR蛋白的核定位，并阻断初级靶标mRNA的翻译。通过比较DEX+CHX处理的转基因与野生型组织的基因表达谱，即可鉴定转录激活因子BBM的直接靶基因。我们通过对在10 μM地塞米松（DEX）存在下发芽并生长的35S:BBM-GR种子进行表型观察，验证了35S:BBM-GR载体诱导拟南芥幼苗体细胞胚胎发生（somatic embryogenesis）的能力。与35S:BBM幼苗一致，经DEX处理的35S:BBM-GR幼苗的子叶、初生叶及茎尖分生组织上均出现了体细胞胚结构。本研究共鉴定得到20个基因（包含BBM自身），分析结果显示BBM可直接激活一条包含转录因子与其他信号分子的信号通路，但该通路初始阶段并不包含已知的体细胞胚胎发生诱导基因，如LEC1、LEC2或WUS。目前BBM靶基因的功能尚不明确，但其中多个基因已在近期的分生组织表达基因芯片筛选中被鉴定出来。对BBM互作蛋白与下游靶标的鉴定，为解析植物细胞生长与器官发生相关通路提供了新的研究工具。关键词：转录激活 为鉴定候选BBM靶基因，我们将4日龄的35S:BBM-GR幼苗在10 μM CHX存在下用10 μM DEX处理8小时，并采用拟南芥Operon基因芯片（http://www.ag.arizona.edu/microarray/），比较该处理组幼苗与经相同方式处理的4日龄野生型幼苗的mRNA群体。本研究中使用的种子批次在持续置于含10 μM DEX的培养基上生长时，胚胎形成效率（EFS）达100%。选择4日龄幼苗作为实验材料，是因其对BBM-GR激活响应性极强，且可提供足够的RNA用于基因芯片实验。数据的生物学重复性通过两个独立的单座位35S:BBM-GR株系进行验证；技术重复性则通过每个转基因株系的两个独立DEX+CHX处理样本进行监测。染料效应通过染料互换实验进行监测，该实验采用了来自两个生物学重复的四份RNA样本中的一份。