College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an 271018, China.
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China.
Plant Physiol. 2024 Nov 4;196(3):1802-1812. doi: 10.1093/plphys/kiae445.
Self-incompatibility (SI) is a mechanism in plants that prevents self-fertilization and promotes outcrossing. SI is also widely utilized in the breeding of Brassicaceae crops. Understanding the regulatory mechanisms of SI is essential but has been greatly restrained in most Brassicaceae crops due to inefficient transformation. In this study, we developed methods for examining signaling pathways and genes of pollen-stigma interactions in Brassicaceae crops lacking an efficient genetic transformation system. We pretreated excised stigmas of Brassica rapa (B. rapa L. ssp. Pekinensis) in vitro with chemicals to modify signaling pathways or with phosphorothioate antisense oligodeoxyribonucleotides (AS-ODNs) to modify the expression of the corresponding genes involved in pollen-stigma interactions. Using this method, we first determined the involvement of reactive oxygen species (ROS) in SI with the understanding that the NADPH oxidase inhibitor diphenyleneiodonium chloride, which inhibits ROS production, eliminated the SI of B. rapa. We further identified the key gene for ROS production in SI and used AS-ODNs targeting BrRBOHF (B. rapa RESPIRATORY-BURST OXIDASE HOMOLOGF), which encodes one of the NADPH oxidases, to effectively suppress its expression, reduce stigmatic ROS, and promote the growth of self-pollen in B. rapa stigmas. Moreover, pistils treated in planta with the ROS scavenger sodium salicylate disrupted SI and resulted in enlarged ovules with inbred embryos 12 d after pollination. This method will enable the functional study of signaling pathways and genes regulating SI and other pollen-stigma interactions in different Brassicaceae plants.
自交不亲和性（SI）是一种植物机制，可防止自花授粉并促进异交。SI 也广泛用于十字花科作物的育种。了解 SI 的调控机制至关重要，但由于转化效率低下，在大多数十字花科作物中受到了极大的限制。在这项研究中，我们开发了用于检查缺乏有效遗传转化系统的十字花科作物中花粉-柱头相互作用的信号通路和基因的方法。我们在体外预处理 Brassica rapa（B. rapa L. ssp. Pekinensis）的切除柱头，用化学物质修饰信号通路，或用硫代磷酸酯反义寡脱氧核苷酸（AS-ODN）修饰参与花粉-柱头相互作用的相应基因的表达。使用这种方法，我们首先确定了活性氧（ROS）在 SI 中的参与，因为 NADPH 氧化酶抑制剂二苯并碘（DPI）抑制 ROS 产生，消除了 B. rapa 的 SI。我们进一步确定了 SI 中 ROS 产生的关键基因，并使用靶向 BrRBOHF（B. rapa RESPIRATORY-BURST OXIDASE HOMOLOGF）的 AS-ODN，其编码 NADPH 氧化酶之一，有效地抑制其表达，减少柱头 ROS，并促进自花粉在 B. rapa 柱头中的生长。此外，体内用 ROS 清除剂水杨酸钠处理的雌蕊破坏了 SI，并导致授粉后 12 天胚珠增大，胚珠内有自交胚胎。该方法将使不同十字花科植物中调控 SI 和其他花粉-柱头相互作用的信号通路和基因的功能研究成为可能。
