Small RNAs (sRNAs) are essential for regulating plant growth and development, and they possess the notable ability to travel long distances within organisms to regulate target gene expression. Our study examined the dcl2 mutant, a key enzyme in sRNA biogenesis, to determine the role of the DCL2 protein in sRNA synthesis and to identify mobile sRNAs under DCL2 regulation. Through grafting experiments between dcl2 mutants and wild-type soybean plants, coupled with sRNA sequencing, we identified 14,105 sRNAs significantly affected by DCL2 and discovered 375 mobile sRNAs under its regulation. Degradome analysis provided deeper insights into the regulatory effects of these mobile sRNAs on their target genes, enabling us to understand their potential influences on plant development and stress responses. Leveraging the systemic movement of sRNAs from roots to shoots, we propose a novel strategy for manipulating gene expression in aboveground tissues. Overall, our research findings not only deepen our understanding of the complex regulatory networks involving mobile sRNAs regulated by DCL2, but also provide a new strategy for gene regulation, which could have a positive impact on agricultural biotechnology.

Alfalfa (Medicago sativa L.) is one of the most extensively grown leguminous forage worldwide. Environmental saline-alkali stress significantly influences the growth, development, and yield of alfalfa, posing a threat to its agricultural production. However, little is known about the potential mechanisms by which alfalfa responds to saline-alkali stress. Here, we investigated these mechanisms by cloning a saline-alkali-induced flavonol synthase gene (MsFLS13) from alfalfa, which was previously reported to be significantly upregulated under saline-alkali stress, and examining its function in the saline-alkali response. Overexpression of MsFLS13 in alfalfa promoted plant tolerance to saline-alkali stress by enhancing flavonol accumulation, antioxidant capacity, osmotic balance, and photosynthetic efficiency. Conversely, MsFLS13 inhibition using RNA interference reduced flavonol synthase activity and inhibited hairy root growth under saline-alkali stress. Yeast one-hybrid and dual-luciferase reporter assays indicated that the R2R3-MYB MsMYB12 transcription factor activates MsFLS13 expression by binding to the MBS motif in the MsFLS13 promoter. Further analysis revealed that abscisic acid mediates the saline-alkali stress response partially by inducing MsMYB12 and MsFLS13 expression, which consequently increases flavonol levels and maintains antioxidant homeostasis in alfalfa. Collectively, our findings highlight the crucial role of MsFLS13 in alfalfa in response to saline-alkali stress and provide a novel genetic resource for creating saline-alkali-resistant alfalfa through genetic engineering.