DataSheet_1_Overexpression of Medicago MtCDFd1_1 Causes Delayed Flowering in Medicago via Repression of MtFTa1 but Not MtCO-Like Genes.pdf

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

优化开花期对提升作物生产力至关重要，但目前学界对温带豆科植物开花调控机制的认知仍存在诸多空白。与冬性一年生拟南芥类似，苜蓿属植物（Medicago）在经历持续低温春化（vernalization, V）后，再经长日照（long-day, LD）光周期处理即可提早开花。在拟南芥中，光周期调控的开花过程由CONSTANS（CO）这一光周期开关触发，CO可直接激活编码可移动成花素且强效促花的FT基因（FLOWERING LOCUS T）。在拟南芥中，包括AtCDF1在内的多个循环DOF因子（CYCLING DOF FACTORs, CDFs）可通过功能冗余抑制CO的表达，进而下调FT的转录，直至在长日照条件下被蓝光诱导的F-BOX1/GIGANTEA（FKF1/GI）复合体降解。苜蓿属植物拥有FT的同源基因MtFTa1，其作为强效开花激活因子发挥功能。然而，MtFTa1的调控通路似乎并不依赖类CO基因。不过，豌豆的相关研究表明，CDFs仍可能参与温带豆科植物的开花时间调控。本研究聚焦苜蓿属MtCDF家族基因，重点解析MtCDFd1_1在开花时间调控与植株发育中的功能。在拟南芥中过量表达MtCDFd1_1可显著延迟开花，且该基因在苜蓿属植物中呈现昼夜周期性表达模式，于黎明时分达到表达峰值，这与AtCDF1等AtCDF家族基因的表达特征一致。但MtCDFd1_1缺乏预测的GI或FKF1结合结构域，提示其调控机制与AtCDF1可能存在差异。拟南芥中CDFs以功能冗余的方式发挥作用，温带豆科植物的CDFs大概率也存在功能冗余：在苜蓿Tnt1插入突变体材料中，单独敲除MtCDFd1_1或其他MtCDF家族基因均未观察到开花时间相关表型。不过，在苜蓿属植物中过量表达MtCDFd1_1，在经春化处理的长日照（vernalized long-day, VLD）诱导条件下会导致植株较野生型晚花，但在春化短日照（vernalized short days, VSDs）条件下无此效应，使转基因植株变为对日长不敏感型。转基因植株中类CO基因的表达未受影响，但受长日照诱导的MtFTa1、MtFTb1、MtFTb2及MtSOC1a的表达量均显著下调。同时携带Mtfta1突变与35S:MtCDFd1_1过表达载体的植株，其开花时间与仅携带Mtfta1突变的植株无显著差异。上述结果表明，35S:MtCDFd1_1在VLD条件下通过抑制MtFTa1的表达来调控开花时间。综上，本研究表明，苜蓿属的MtCDF家族基因可通过功能冗余的方式抑制类FT基因（尤其是MtFTa1）的表达，从而参与光周期调控开花的过程，且该调控通路不依赖CO，这与拟南芥的调控模型存在显著差异。