Identification of genes regulated by the MADS transcription factor (TF), SEPALLATA3, in the context of the double sep1sep2 and triple sep1sep2sep3 mutant background, by RNA-Seq analysis

These analyses allowed the identification of more than 2000 genes regulated by the MADS TF, SEPALLATA3 (SEP3). Although the involvement of SEP3 in all flower organ development is well established, the single sep3 mutant shows no flower phenotype, due to redundancy with other SEPALLATA genes. The triple mutant, sep1sep2sep3, shows no floral organ development except sepals, while the sep1sep2 mutant showed characteristic WT flower with, from the outer to the inner whorl of the flower, 4 sepals, 4 petals, 6 stamens and one gynoecium. We performed RNA-seq analysis on sep1sep2, sep1sep2sep3 and on sep1sep2sep3 lines complemented with SEP3 to highlight genes involved in floral organ development and controlled by SEP3. For this purpose we compared gene expression between the triple and the double mutant, and between triple mutant lines complemented with SEP3 and the triple mutant. Genes were considered regulated by SEP3 when the log FC was between 1 and -1 and the FDR <0.05. Using these criteria, we obtained a list of >2000 genes regulated by SEP3. RNA seq analysis were performed on inflorescence meristem and flower buds up to stage 10-11 harvested from Arabidopsis plants grown in parralel in long days conditions. Experiments were performed on 2 replicates from sep1sep2 mutant, 2 replicates from sep1sep2sep3 triples mutant, and 2 independantes complemented lines expressing SEP3 in the sep1sep2sep3 background (considered as 2 replicats). These lines are described in Hugouvieux et al, Nucleic Acid Research, 2018:1;46(10):4966-4977. doi: 10.1093/nar/gky205. Libraries and sequencing were performed by Genewiz (Illumina High SEq, 2X150 bp configuration).

本研究通过相关分析鉴定出2000余个受MADS转录因子（MADS TF）SEPALLATA3（SEP3）调控的基因。尽管SEP3在所有花器官发育中的作用已得到广泛证实，但由于与其他SEPALLATA家族基因存在功能冗余，单突变体sep3并未表现出花发育异常表型。三重突变体sep1sep2sep3仅能形成萼片，无法发育其他花器官；而双突变体sep1sep2则表现出典型的野生型（Wild Type, WT）花结构：从外至内的花轮依次为4枚萼片、4枚花瓣、6枚雄蕊以及1枚雌蕊。为筛选参与花器官发育且受SEP3调控的基因，我们对双突变体sep1sep2、三重突变体sep1sep2sep3，以及在sep1sep2sep3背景中互补表达SEP3的株系进行了RNA测序（RNA-seq）分析。为此，我们分别比较了三重突变体与双突变体的基因表达差异，以及互补表达SEP3的三重突变体株系与三重突变体的基因表达差异。当基因的对数倍变化值（log Fold Change, log FC）介于-1至1之间，且错误发现率（False Discovery Rate, FDR）小于0.05时，即认定该基因受SEP3调控。基于上述筛选标准，我们最终得到了包含2000余个受SEP3调控基因的列表。我们从在长日照条件下平行培养的拟南芥（Arabidopsis）植株中采集了花序分生组织以及发育至10-11期的花芽，用于RNA-seq分析。本实验设置了如下生物学重复：双突变体sep1sep2、三重突变体sep1sep2sep3各2次生物学重复；以及在sep1sep2sep3背景中表达SEP3的2株独立互补株系（视为2次生物学重复）。上述株系的详细信息参见Hugouvieux等人发表于《核酸研究》（Nucleic Acid Research）2018年的研究：1;46(10):4966-4977，DOI: 10.1093/nar/gky205。测序文库构建及测序工作由Genewiz公司完成，采用Illumina High SEq平台，2×150 bp双端测序模式。