MdWRKY9 overexpression confers intensive dwarfing in the M26 rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression

Corresponding author: Jin Kong

• Dwarfing rootstocks (砧木) enable high-density planting and are therefore highly desirable in modern apple (Malus domestica) production. M26 is a semi-dwarfing (半矮化) rootstock that is used worldwide, but identifying intensive dwarfing rootstock is a major goal of apple breeding programs.

• Herein, we show that MdWRKY9 mediates dwarfing by directly inhibiting the transcription of the brassinosteroid (BR) rate-limiting synthetase MdDWF4 and reducing BR production.

• We found that the transcriptional factor MdWRKY9 is highly expressed in all tested dwarfing rootstocks. Transgenic lines of M26 rootstock overexpressing MdWRKY9 exhibit further dwarfing, which resulted from the reduced BR levels and was reversed via exogenous brassinolide treatment. Both an in vivo (体内) chromatin immunoprecipitation (ChIP) analysis and an in vitro (体外) electrophoretic mobility shift assay (EMSA; 电泳迁移分析法) indicated that MdWRKY9 binds to the promoter of MdDWF4. Furthermore, MdWRKY9 repressed MdDWF4 expression in stable transgenic apple plants as determined by quantitative PCR. In addition, RNA-interfered expression of MdWRKY9 in transiently transformed apple calli (愈伤组织) led to a significant increase of MdDWF4, suggesting MdWRKY9 plays a critical role in regulating the expression of MdDWF4.

• We report a novel dwarfing mechanism in perennial woody plants that involves WRKY-controlled BR production, and present a new dwarfing M26 rootstock for potential applications in apple production.

矮化的砧木可以进行高密度的种植，因此在现代苹果产业中很有应用潜力。M26是一个半矮化砧木，在全世界范围内广泛使用，但鉴定更加矮化的砧木是苹果育种项目中一个主要目标。

本文研究显示MdWRKY9通过直接抑制油菜素甾醇BR限速合成酶MdDWF4的转录和减少BR的产量介导植株矮化。

作者发现转录因子MdWRKY9在所有测试的矮化砧木中均高表达。M26砧木高表达MdWRKY9转基因植株均会进一步矮化，而这是因为BR水平的降低，并且能够通过外源油菜素内酯处理恢复矮化。体内染色质免疫沉淀ChlP分析和体外电泳迁移分析法EMSA显示MdWRKY9会结合到MdDWF4基因的启动子。此外，定量PCR显示在稳定的转基因苹果中，MdWRKY9会抑制MdDWF4基因的表达。另外，在转基因苹果愈伤组织中实施RNA干扰MdWRKY9基因的表达显著提高了MdDWF4基因的表达，显示MdWRKY9在调控MdDWF4基因上起到重要作用。

作者报道了一个由WRKY基因控制的BR量介导的多年生木本植物的矮化机制，并且为苹果产业提供了一个潜在的新的M26矮化砧木。

通讯：孔瑾 (http://yyxy.cau.edu.cn/yyxy/yuanyi/teacher/183)

个人简介：1991/07 - 1995/06，东北林业大学，生物学学士；1995/07 - 2000/06，东北林业大学，植物学博士（硕博连读）；2000/06 - 2003/04，中国科学院遗传与发育生物学研究所，博士后；2003/04 - 2005/05，英国布里斯托大学（University of Bristol），博士后；2005-2015年，中国农业大学，果树系，副教授；2016-至今，中国农业大学，果树系，教授，博士生导师。

doi: 10.1111/nph.14891