Endocytosis of superoxide vesicle by receptor protein kinase in age-dependent cell death in Arabidopsis

Abstract

Leaf senescence, which constitutes the final stage of leaf development, involves programmed cell death (PCD) and is intricately regulated by various internal and environmental signals that are incorporated with age-related information. Abscisic acid (ABA) plays diverse and important physiological roles in plants, and is involved in various developmental events and stress responses. ABA has long been regarded as a positive regulator of leaf senescence. However, the cellular mediators of ABA-induced senescence have not been identified. We sought to understand the ABA-induced senescence signaling process in Arabidopsis by examining the function of an ABA- and age-induced gene, RPK1, which encodes a membrane-bound, leucine-rich repeat-containing receptor kinase. Loss-of-function mutants in RPK1 were significantly delayed in age-dependent senescence. Furthermore, rpk1 mutants exhibited reduced sensitivity to ABA-induced senescence but little to jasmonic acid- or ethylene-induced senescence. RPK1 thus mediates ABA-induced leaf senescence as well as age-induced leaf senescence. Conditional overexpression of RPK1 at mature stage clearly accelerated senescence and cell death, whereas induction of RPK1 at an early developmental stage retarded growth without triggering senescence symptoms. Therefore, RPK1 plays different roles at different stages of development. Consistently, exogenously applied ABA affected leaf senescence in old leaves but not in young leaves. The results, together, showed that membrane-bound RPK1 functions in ABA-dependent leaf senescence. Furthermore, the effect of ABA and ABA-inducible RPK1 on leaf senescence is dependent on the age of the plant, which in part explains the mechanism of functional diversification of ABA action. Superoxide is one of the reactive oxygen species and has been implicated as an important signaling molecule in various cellular responses. However, little is known about how this reactive, unstable, membrane non- permeable anion is incorporated into specific cellular signaling systems. Here, we demonstrate that the Arabidopsis receptor kinase RPK1 mediates production of superoxide-containing vesicles at the plasma membrane for diverse cellular responses including age-dependent programmed cell death. The superoxide-containing vesicles undergo endocytosis and are associated with microtubules, leading to induction of a senescence-induced kinase gene, SIRK. Unexpectedly, the major superoxide source in the leaf plasma membrane, NADPH oxidase AtrbohD, is not involved in RPK1-mediated superoxide production but, instead, inhibits RPK1-mediated SIRK induction and cell death. These results indicate that various sources of membrane superoxide are utilized to signal distinct, even antagonistic, outcomes. Together with the report on the presence of hydrogen peroxide-containing vesicles, our results imply that ROS-containing vesicles may be a general cellular mechanism by which cells deliver ROS to specific targets without causing non- specific damage to other cellular components.