Global expression profiling applied to the analysis of Arabidopsis stamen development

To obtain detailed information about gene expression during stamen development in Arabidopsis thaliana, we compared, by microarray analysis, the gene expression profile of wild-type inflorescences to those of the floral mutants apetala3, sporocyteless/nozzle, and male sterile 1, in which different aspects of stamen formation are disrupted. These experiments led to the identification of groups of genes with predicted expression at early, intermediate and late stages of stamen development. Additional experiments aimed at characterizing gene expression specifically during microspore formation. To this end, we compared the gene expression profiles of wild-type flowers of distinct developmental stages to those of the male sterile 1 mutant. Computational analysis of the datasets derived from this experiment led to the identification of genes that are likely involved in the control of key developmental processes during microsporogenesis. Keywords: mutant comparison, developmental series To identify genes expressed during distinct stages of stamen development at a genome-wide scale, we compared, by microarray analysis, the gene expression profiles of flowers of ap3, spl/nzz, and ms1 mutants to that of wild-type flowers. For the identification of transcripts expressed at early stages of stamen development, we dissected older flowers from inflorescences of ap3 mutant plants and the wild type and then collected the inflorescence meristem and floral buds up to early stage 10 for analysis. We refer to these tissue samples as ap3 early stages, or ap3 es. The second mutant included in our analysis, spl/nzz, shows defects at the earliest steps of sporogenesis. Thus, we expected that genes expressed in sporogenous tissues would be down-regulated in mutant flowers compared to the wild type. Lastly, we analyzed the ms1 mutant to identify genes expressed during pollen development and maturation. For both ms1and spl/nzz, as well as for the wild type, tissue samples containing the inflorescence meristem and floral buds up to stage 13 were collected for analysis. To gain more detailed insights into the transcriptional program underlying microspore development in Arabidopsis, we made further use of the ms1 mutant, which lacks viable pollen. We collected flowers from ms1 and wild-type plants and pooled them according to their developmental stages into 7 tissue samples, where sample 1 contained mature (stage 13) flowers and subsequent samples contained successively younger flowers, with sample 7 containing the youngest floral buds and the inflorescence meristem. Gene expression profiling experiments were done using oligonucleotide microarrays, as described previously (Wellmer et al. (2006) PLoS Genetics 2, e117. In all cases, at least three independent biological samples were used in separate hybridizations, and the dyes used for labeling of the co-hybridized samples were switched in the replicate experiments.

为获取拟南芥（Arabidopsis thaliana）雄蕊发育过程中基因表达的详细信息，本研究通过微阵列分析（microarray analysis）比较了野生型花序与花突变体apetala3（简称ap3）、sporocyteless/nozzle（简称spl/nzz）以及雄性不育1（male sterile 1，简称ms1）的基因表达谱，上述突变体的雄蕊形成过程在不同环节受到破坏。本实验最终鉴定出在雄蕊发育早期、中期和晚期具有预测表达模式的基因簇。

为进一步表征小孢子形成阶段特异性的基因表达情况，我们将不同发育阶段的野生型花与ms1突变体的基因表达谱进行对比。对该实验产生的数据集开展计算分析后，我们鉴定出可能参与调控小孢子发生过程关键发育事件的基因。关键词：突变体对比、发育序列

为在全基因组范围鉴定雄蕊发育不同阶段的表达基因，我们通过微阵列分析比较了ap3、spl/nzz及ms1突变体花与野生型花的基因表达谱。为鉴定雄蕊发育早期阶段的表达转录本，我们从ap3突变体与野生型植株的花序中移除成熟老花，收集花序分生组织及发育至早期10期的花芽作为分析样本，将此类组织样本命名为ap3早期样本（ap3 es）。本研究纳入的第二个突变体spl/nzz在孢子发生的最早步骤即出现发育缺陷，因此我们推测，与野生型相比，孢子发生组织特异性表达的基因在该突变体花中呈现下调表达。最后，我们通过分析ms1突变体来鉴定花粉发育与成熟阶段的表达基因。针对ms1、spl/nzz及野生型，我们均收集包含花序分生组织及发育至13期的花芽的组织样本用于后续分析。

为更深入解析拟南芥小孢子发育背后的转录调控程序，我们进一步利用了无存活花粉的ms1突变体。我们从ms1突变体与野生型植株中采集花组织，并按发育阶段分为7个样本：样本1包含成熟（13期）花，后续样本依次对应更幼嫩的花，样本7则包含最幼嫩的花芽与花序分生组织。本研究采用寡核苷酸微阵列（oligonucleotide microarray）进行基因表达谱分析，具体方法参照此前发表的研究（Wellmer等(2006) PLoS Genetics 2, e117）。所有实验均至少使用3份独立生物学重复样本开展独立杂交实验，且重复实验中互换了共杂交样本的标记荧光染料。