Receptor for Activated C Kinase1B (OsRACK1B) Impairs Fertility in Rice through NADPH-Dependent H₂O₂ Signaling Pathway

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 (H₂O₂) in the leaves and roots of the transgenic lines overexpressing OsRACK1B compared to that of the WT. However, the elevated H₂O₂ in tissues from the OX plants can be reversed by pre-treatment with diphenylidonium (DPI), an NADPH oxidase inhibitor, indicating that the source of H₂O₂ 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 H₂O₂ 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 H₂O₂, which negatively regulates pollen development and fertility. In conclusion, we hypothesized that an optimal expression of RACK1 is critical for fertility in rice plants.