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Pleiotropic effects of overexpressing a legume-specific Cycling DOF Factor MsCDFc1 on flowering and forage quality in alfalfa

Xu Jiang1, Huiting Cui2, Lili Zhang1, Zhen Wang3, Xue Wang1, Mingna Li1, Tiejun Zhang1, Ruicai Long1, Qingchuan Yang1, Junmei Kang1#

1 Chinese Academy of Agricultural Sciences Institute of Animal Science, Beijing 100193, China.

2 Henan Institute of Science and Technology, School of Agriculture, Xinxiang, Henan 453003, China.

3 University of Nebraska-Lincoln, Department of Agronomy and Horticulture, Nebraska Lincoln, NE 68583, USA.

 Highlight

1. Fifteen CDFs were identified in alfalfa, with duplication events driving CDF gene family expansion.

2. MsCDFc1 delays flowering in alfalfa under LD conditions through MsE1 and MsFTbut not through MsCOLs.

3. MsCDFc1-overexpressed late-flowering alfalfa has improved quality.

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摘要  

背景:紫花苜蓿(Medicago sativa L.)作为光周期敏感型长日照开花豆科饲草作物资源,因高产、优良的饲用特性及显著经济效益而被广泛栽培。然而,过早开花显著制约其生物量积累与饲用品质。循环DOF因子(Cycling DOF Factors, CDFs)作为调控高等植物光周期开花等核心生理过程的关键转录因子,但在调控紫花苜蓿开花的功能仍不明确,需要进一步研究。

方法和结果:本研究通过全基因组分析,在紫花苜蓿基因组中系统鉴定了15个MsCDF基因,其数量约为已报道高等植物CDF基因家族成员的三倍,基因复制事件被证实为MsCDF家族扩张的主要驱动力。进化分析揭示,C亚家族MsCDFs为豆科植物所特有,暗示其在豆科植物中可能具有独特的功能分化。表达模式分析表明,MsCDFc1基因表达呈现显著昼夜节律性,且在长日照条件下特定时段的转录水平显著高于短日照条件。通过异源过表达及同源转化体系验证,MsCDFc1的过表达可显著延迟模式植物拟南芥及紫花苜蓿的开花时间,显著降低了紫花苜蓿中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量,改善饲草消化率,提升了牧草粗蛋白含量与相对饲用价值(RFV)。遗传机制解析发现,MsCDFc1通过降低豆科植物特异性转录因子MsE1和成花素基因MsFTa1表达,而不是CO-Like基因,延迟紫花苜蓿开花。MsCDFc1的不与MsFTa1MsFTb1启动子结合。酵母双杂交表明,其C端推测的FKF1结合结构域的N417发生突变,导致不与MsFKF1相互作用。研究结果表明,MsCDFc1作为豆科植物特有的关键调控因子,在整合紫花苜蓿光周期信号、延迟开花、提升饲用品质方面具有重要生物学功能。本研究首次系统地对紫花苜蓿 CDF 基因家族成员进行鉴定和分析,并利用遗传学方法阐明了MsCDFc1基因在长日照下通过调控MsFTa1MsE1基因,而非MsCO–Like基因,延迟紫花苜蓿开花时间、改善牧草品质,揭示了豆科植物中 CDF 介导的开花调控新机制。



Abstract  

Alfalfa (Medicago sativa L.), a photoperiod sensitive long-day (LD) flowering legume forage crop, is widely cultivated for its high-yield, -quality, and -related economic benefits. However, early flowering affects the biomass yield and quality of alfalfa. Cycling DOF Factors (CDFs) play critical roles in multiple fundamental processes in higher plants, including photoperiodic flowering time regulation. Here, we identified 15 CDFs in the alfalfa genome, which is approximately three times than the number of higher plants. Duplication events are the primary driving force behind the expansion of the CDF gene family in alfalfa. Evolutionary analysis revealed that MsCDFs in the C subclade is exclusively present in leguminous plants, suggesting their diverse functions within the legume family. Among them, MsCDFc1 mRNA exhibited a rhythmic expression pattern and its mRNA levels predominantly expressed than other members. MsCDFc1 protein localized to the nucleus and exhibited no transactivation in vitro. We demonstrated that under LD conditions, MsCDFc1 has a conserved function of flowering time regulation, as overexpressed plants (Arabidopsis and alfalfa) showed delay (P<0.05) in flowering time. Therefore, the quality of the late-flowering alfalfa was improved by reduced (P<0.05) levels of neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin contents at initial flowering stage. Further investigations showed that the late flowering in the over- expressed plant was correlated with the reduced (P<0.05) transcript levels of the MsFTa1 and MsE1 gene but in a MsCO-Like independent manner. Furthermore, MsCDFc1 does not interact with MsFKF1 or bind to the MsFTa1 and MsFTb1 promoters, suggesting functional divergence from the Arabidopsis model.

Keywords:  CDF       alfalfa              gene family              late flowering              forage quality  
Online: 04 September 2025  
Fund: 

This work was supported by the National Natural Science Foundation of China (32071868).

Cite this article: 

Xu Jiang, Huiting Cui, Lili Zhang, Zhen Wang, Xue Wang, Mingna Li, Tiejun Zhang, Ruicai Long, Qingchuan Yang, Junmei Kang. 2025. Pleiotropic effects of overexpressing a legume-specific Cycling DOF Factor MsCDFc1 on flowering and forage quality in alfalfa. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.09.007

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