Department of Biology, Howard University, Washington, DC 20059, USA.
Department of Biological Sciences, Hampton University, Hampton, VA 23668, USA.
Int J Mol Sci. 2022 Jul 30;23(15):8455. doi: 10.3390/ijms23158455.
The scaffold protein receptor for Activated C Kinase1 (RACK1) regulates multiple aspects of plants, including seed germination, growth, environmental stress responses, and flowering. Recent studies have revealed that RACK1 is associated with NADPH-dependent reactive oxygen species (ROS) signaling in plants. ROS, as a double-edged sword, can modulate several developmental pathways in plants. Thus, the resulting physiological consequences of perturbing the RACK1 expression-induced ROS balance remain to be explored. Herein, we combined molecular, pharmacological, and ultrastructure analysis approaches to investigate the hypothesized connection using T-DNA-mediated activation-tagged RACK1B overexpressed (OX) transgenic rice plants. In this study, we find that OsRACK1B-OX plants display reduced pollen viability, defective anther dehiscence, and abnormal spikelet morphology, leading to partial spikelet sterility. Microscopic observation of the mature pollen grains from the OX plants revealed abnormalities in the exine and intine structures and decreased starch granules in the pollen, resulting in a reduced number of grains per locule from the OX rice plants as compared to that of the wild-type (WT). Histochemical staining revealed a global increase in hydrogen peroxide (HO) in the leaves and roots of the transgenic lines overexpressing OsRACK1B compared to that of the WT. However, the elevated HO in tissues from the OX plants can be reversed by pre-treatment with diphenylidonium (DPI), an NADPH oxidase inhibitor, indicating that the source of HO could be, in part, NADPH oxidase. Expression analysis showed a differential expression of the NADPH/respiratory burst oxidase homolog D (RbohD) and antioxidant enzyme-related genes, suggesting a homeostatic mechanism of HO production and antioxidant enzyme activity. BiFC analysis demonstrated that OsRACK1B interacts with the N-terminal region of RbohD in vivo. Taken together, these data indicate that elevated OsRACK1B accumulates a threshold level of ROS, in this case HO, which negatively regulates pollen development and fertility. In conclusion, we hypothesized that an optimal expression of RACK1 is critical for fertility in rice plants.
支架蛋白受体激活的 C 激酶 1（RACK1）调节植物的多个方面，包括种子萌发、生长、环境胁迫响应和开花。最近的研究表明，RACK1 与植物中 NADPH 依赖性活性氧物种（ROS）信号有关。ROS 作为一把双刃剑，可以调节植物中的几个发育途径。因此，扰乱 RACK1 表达诱导的 ROS 平衡所产生的生理后果仍有待探索。在此，我们结合分子、药理学和超微结构分析方法，使用 T-DNA 介导的激活标记 RACK1B 过表达（OX）转基因水稻植物来研究假设的联系。在这项研究中，我们发现 OsRACK1B-OX 植物表现出花粉活力降低、花药开裂缺陷和小穗形态异常，导致部分小穗不育。对 OX 植物成熟花粉粒的显微镜观察显示，外壁和内壁结构异常，花粉中的淀粉粒减少，导致 OX 水稻植物每个小穗室的粒数减少。组织化学染色显示，与野生型（WT）相比，过表达 OsRACK1B 的转基因系的叶片和根部的过氧化氢（HO）总量增加。然而，用 NADPH 氧化酶抑制剂二苯基碘鎓（DPI）预处理可以逆转 OX 植物组织中升高的 HO，表明 HO 的来源部分可能是 NADPH 氧化酶。表达分析显示 NADPH/呼吸爆发氧化酶同源物 D（RbohD）和抗氧化酶相关基因的差异表达，表明 HO 产生和抗氧化酶活性的一种动态平衡机制。BiFC 分析表明 OsRACK1B 在体内与 RbohD 的 N 端区域相互作用。综上所述，这些数据表明，升高的 OsRACK1B 积累了 ROS 的阈值水平，在这种情况下是 HO，其负调节花粉发育和育性。总之，我们假设 RACK1 的最佳表达对水稻植物的育性至关重要。
