Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 4T3, Canada.
Department of Biology, University of Western Ontario, 1151 Richmond Street, London, ON, N6A 3K7, Canada.
Plant Mol Biol. 2022 Dec;110(6):511-529. doi: 10.1007/s11103-022-01303-7. Epub 2022 Aug 17.
Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa.
我们的研究结果表明，SPL12 在调控紫花苜蓿（三叶草）的根瘤发育中起着至关重要的作用，并且 AGL6 是 SPL12 的靶标并被其下调。植物的根构型至关重要，因为它控制着养分循环、水利用效率以及对生物和非生物胁迫因素的响应。小 RNA，microRNA156（miR156）在植物中高度保守，通过沉默一组 SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE（SPL）转录因子来发挥作用。我们之前曾表明，过表达 miR156 的转基因紫花苜蓿（三叶草）植物表现出增加的结瘤、改善的固氮和增强的营养繁殖期间的根再生能力。在三叶草中，包括 SPL12 在内的十一个 SPL 的转录本被 miR156 靶向切割。在这项研究中，我们通过研究与 SPL12 转录本水平改变相关的转录组和表型变化，以及通过使用 SPL12 沉默和过表达三叶草植物来确定 SPL12 的调节靶标，来表征 SPL12 在根构型和结瘤中的作用。表型分析表明，沉默三叶草中的 SPL12 导致根再生、结瘤和固氮增加。此外，根据下一代测序介导的转录组分析和染色质免疫沉淀实验，确定编码 AGAMOUS-like MADS box 转录因子的 AGL6 是 SPL12 直接靶向的沉默靶标。总之，我们的研究结果表明，SPL12 和 AGL6 形成了一个遗传模块，调节三叶草的根发育和结瘤。
