Transcription Factors BnaC09.FUL and BnaC06.WIP2 Antagonistically Regulate Flowering Time under Long-day Conditions in Brassica napus

Flowering time stands as a pivotal agronomic trait in rapeseed (Brassica napus L.). Timely flowering enables rapeseed to avoid weather-related losses, plant diseases, and insect pests. However, the molecular mechanisms governing flowering time remain largely unknown in B. napus. Here, a genome-wide association analysis of 165 diverse rapeseed accessions pinpointed BnaC09.FUL, a MADS-box transcription factor, as a promising candidate gene regulating flowering time in B. napus. Expression analysis revealed that the expression of BnaC09.FUL increased sharply in B. napus shoot apices close to bolting. Overexpression of BnaC09.FUL resulted in early flowering, while mutation of all six paralogs of BnaFUL led to delayed flowering in B. napus. Additionally, a zinc finger transcription factor, BnaC06.WIP2, was identified as an interaction protein of BnaC09.FUL, and overexpression of BnaC06.WIP2 delayed flowering in B. napus, with RNA-sequencing revealing its influence on a plethora of flowering-associated genes. Chromatin immunoprecipitation and transient dual-luciferase reporter assays demonstrated that BnaC06.WIP2 directly repressed the expression of BnaA05.SOC1, BnaC03.SOC1, BnaC04.SOC1, BnaC06.FT, BnaA06.LFY, BnaA08.CAL, BnaC03.CAL, and BnaC07.FUL and indirectly inhibited the expression of several other flowering-related genes. Genetic and molecular investigations highlighted an antagonistic relationship between BnaC09.FUL and BnaC06.WIP2 in regulating flowering in B. napus, primarily through direct regulation of BnaC03.SOC1, BnaA08.CAL, and BnaC03.CAL expression. Taken together, our findings provide a mechanism by which the BnaC09.FUL-BnaC06.WIP2 transcriptional regulatory module controls flowering time in B. napus

开花期是甘蓝型油菜（Brassica napus L.）的关键农艺性状。适时开花可使甘蓝型油菜规避气象灾害、植物病害及虫害，但目前学界对甘蓝型油菜中调控开花期的分子机制仍知之甚少。本研究通过对165份不同甘蓝型油菜种质资源开展全基因组关联分析（genome-wide association analysis），精准鉴定出MADS-box转录因子（MADS-box transcription factor）BnaC09.FUL作为调控甘蓝型油菜开花期的极具潜力的候选基因。表达分析结果显示，在临近抽薹的甘蓝型油菜茎尖中，BnaC09.FUL的表达量急剧升高。过表达BnaC09.FUL可使甘蓝型油菜提前开花，而敲除BnaFUL的全部6个旁系同源基因则会导致其开花延迟。此外，本研究鉴定出锌指转录因子（zinc finger transcription factor）BnaC06.WIP2是BnaC09.FUL的互作蛋白；过表达BnaC06.WIP2会延迟甘蓝型油菜的开花期，RNA测序（RNA-sequencing）结果显示其可调控大量开花相关基因的表达。染色质免疫共沉淀（chromatin immunoprecipitation）与瞬时双荧光素酶报告基因检测（transient dual-luciferase reporter assays）实验证实，BnaC06.WIP2可直接抑制BnaA05.SOC1、BnaC03.SOC1、BnaC04.SOC1、BnaC06.FT、BnaA06.LFY、BnaA08.CAL、BnaC03.CAL及BnaC07.FUL的表达，并间接抑制其他若干开花相关基因的转录。遗传与分子实验研究表明，BnaC09.FUL与BnaC06.WIP2在调控甘蓝型油菜开花期过程中存在拮抗关系，其核心调控通路为直接调控BnaC03.SOC1、BnaA08.CAL及BnaC03.CAL的表达。综上，本研究揭示了BnaC09.FUL-BnaC06.WIP2转录调控模块调控甘蓝型油菜开花期的分子机制。