Alfalfa (Medicago sativa L.) is a globally pivotal legume forage. Selenium (Se), an essential trace element for humans and animals, can significantly enhance the growth and development of alfalfa. Chlorophyll is the central pigment of plant photosynthesis. Previous research on chlorophyll synthesis in alfalfa has mainly focused on transcriptional regulation, environmental factors (light, nutrient availability), and phytohormone signaling, while fewer studies have been conducted at the post-transcriptional level. Through whole transcriptome sequencing analysis, microRNAs (miRNAs) were identified as positively responsive to Se. This study focused on the regulation of chlorophyll synthesis by the miR171-SCL6 module in alfalfa. β-glucuronidase staining and dual-luciferase assays revealed that MsmiR171 negatively regulated the transcript levels of the SCARECROW-LIKE 6 transcription factor MsSCL6. Subcellular localization analysis revealed that MsSCL6 was mainly in the cell nucleus. Functional analyses demonstrated that MsmiR171 promoted chlorophyll synthesis and photosynthesis in alfalfa, while MsSCL6 negatively regulated chlorophyll synthesis. Notably, Se treatment upregulated MsmiR171 expression, downregulated MsSCL6 expression, and enhanced chlorophyll accumulation. qRT-PCR analysis revealed differential expression of MsPOR in MsmiR171 and MsSCL6 overexpression or silencing plants. Combined yeast one-hybrid and dual-luciferase assays demonstrated that MsSCL6 transcriptionally represses MsPOR through direct promoter binding, suppressing chlorophyll accumulation. In summary, this study for the first time revealed the mechanism of the MsmiR171-MsSCL6-MsPOR module mediating Se-regulated chlorophyll biosynthesis in alfalfa. These findings provide a theoretical foundation and technical guidance for alfalfa breeding and the production of Se-enriched forage.

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