miR171-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 regulate somatic embryogenesis in citrus

背景回顾：Somatic embryogenesis (SE) is a key regeneration pathway in various biotechnology approaches to crop improvement, especially for economically important perennial woody crops like citrus. 

提出问题：However, maintenance of SE capability has long been a challenge and becomes a bottleneck in biotechnology-facilitated plant improvement. 

结果1-miR171-CsSCL2/3-SE：In the embryogenic callus (EC) of citrus, we identified two csi-miR171c targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 (CsSCL2/3), which exert positive feedback regulation on csi-miR171c expression. Suppression of CsSCL2 expression by RNA interference (RNAi) enhanced SE in citrus callus. 

结果2-CcSCL2/3-CsClot-ROS-SE：A thioredoxin superfamily protein CsClot was identified as an interactive protein of CsSCL2/3. Overexpression of CsClot disturbed reactive oxygen species (ROS) homeostasis in EC and enhanced SE. 

结果3-CsSCL2/3-再生因子-SE：ChIP-Seq and RNA-Seq identified 660 genes directly suppressed by CsSCL2 that were enriched in biological processes including development related processes, auxin signaling pathway and cell wall organization. CsSCL2/3 bound to the promoters of regeneration-related genes, such as WUSCHEL-RELATED HOMEOBOX 2 (CsWOX2), CsWOX13 and Lateral Organ Boundaries Domain 40 (LBD40), and repressed their expression. 

结论：Overall, CsSCL2/3 modulate ROS homeostasis through the interactive protein CsClot and directly suppress the expression of regeneration-related genes, thus regulating SE in citrus. We uncovered a regulatory pathway of miR171c-targeted CsSCL2/3 in SE, which shed light on the mechanism of SE and regeneration capability maintenance in citrus.

体细胞胚胎发生是一种再生途径，被广泛用于作物改良，尤其是经济重要性的多年生木本作物，比如柑橘。但是，对于体胚发生能力的维持是一个挑战，已经成为了这一技术应用于作物改良的瓶颈。在柑橘的胚性愈伤组织中，作者鉴定到了两个由csi-miR171c靶向的SCR-like基因CsSCL2/3，这两个基因对于csi-miR171c表达存在正反馈调节。通过RNAi技术抑制柑橘愈伤中CsSCL2的表达，能够增强其体胚发生能力。另外，作者还鉴定到了CsSCL2/3的一个互作蛋白，即硫氧还蛋白CsClot。通过过表达CsClot能够扰乱胚性愈伤中的ROS稳态，增强体胚发生能力。ChIP-Seq和RNA-Seq鉴定到了660直接受到CsSCL2抑制的基因，这些基因富集再生长素信号转导和细胞壁组织相关的发育过程中。CsSCL2/3能够结合CsWOX2、CsWOX13及LBD40等再生相关基因的启动子上，抑制这些基因的表达。综上，CsSCL2/3通过与CsClot蛋白的互作介导ROS稳态的调控，同时直接抑制再生相关的基因，继而调控柑橘的体胚发生。本文的研究结果揭示了一个由miR171c靶向的CsSCL2/3作用于柑橘体胚发生的调控通路，这一结果对于未来柑橘再生能力的维持及体胚发生机理的研究提供了新的视野。

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