Rice (Oryza sativa) Receptor for Activated C Kinase1B (OsRACK1B) Regulates Chlorophyll Catabolism and NADPH oxidase‐dependent H2O2 Signaling Pathways during Salinity Stress
Authors: Rahman Md Ahasanur , Ullah Hemayet 
Published: April 2020

The Receptor for Activated C Kinase1B (RACK1B) is a WD‐40 type scaffold protein that regulates diverse environmental stress signal transduction pathways. Arabidopsis RACK1A has been reported to interact with various proteins in salt stress and Light‐Harvesting Complex (LHC) pathways. However, the mechanism of how RACK1 contributes to the photosystem and chlorophyll metabolism in stress conditions remains elusive. Using T‐DNA‐mediated activation tagging transgenic rice (Oryza sativa L.) lines, we show that leaves from rice RACK1B gene (OsRACK1B) gain‐of‐function (OsRACK1B‐OX) plants exhibit the stay‐green phenotype by stabilizing chlorophyll under salinity stress. In contrast, leaves from down‐regulated OsRACK1B (OsRACK1B‐UX) plants display an accelerated yellowing phenotype accompanied by chlorophyll degradation. qRT‐PCR analysis revealed that genes encoding chlorophyll catabolic enzymes (CCEs) are differentially expressed in both OsRACK1B‐OX and OsRACK1B‐UX rice plants. In addition to CCEs, STAY‐GREEN (SGR) is a key component that forms the SGR‐CCE complex in senescing chloroplasts, which causes LHCII complex instability. Transcript and protein profiling revealed a significant upregulation of OsSGR in RACK1B‐UX plants than that in RACK1B‐OX plants during salt stress. The BiFC assay demonstrated that OsRACK1B interacts with OsSGR in planta in both cytoplasm and the nucleus. Our findings suggest that overexpression of OsRACK1B negatively regulates chlorophyll degradation, delays salinity‐induced senescence and OsRACK1B functions in this process, at least in part, by antagonizing the function of OsSGR through a direct interaction. Interestingly, histochemical staining revealed a global increase of hydrogen peroxide (H2O2) in plants overexpressing OsRACK1B compared to that of the wild‐type and the trend can be reversed by pre‐treatment leaves with diphenylidonium (DPI), a NADPH oxidase (NOX) inhibitor. In gel‐activity of NOX and Reactive Oxygen Species (ROS) scavenging enzymes revealed that OsRACK1B regulates NOX and ROS scavenging enzyme activity in a salt stress dependent manner indicating a complex interplay and a novel regulatory module involving OsRACK1, NOX dependent H2O2 signaling, and a balanced ROS scavenging enzyme activity. BiFC analysis demonstrated that RACK1 also interacts with N‐terminal region of NOX at the plasma membrane. We hypothesize that elevated level of H2O2 acts as a signaling molecule to counteract salt stress and chlorophyll catabolism in OsRACK1B transgenic leaves under salinity stress. Taken together, these results provide important insights into the molecular mechanisms of chlorophyll catabolism, salinity‐induced senescence which can be useful in circumventing the effect of salt on photosynthesis and in reducing yield penalty of important cereal crops, like rice, in global climate change conditions.Support or Funding InformationThis work was partially supported by National Science Foundation (NSF), USA grant to Howard University (Grant number MCB 0542312); Dept. of Biology and Graduate School of the Howard University.Proposed working model of how OsRACK1B delays chlorophyll breakdown through regulation of Stay‐Green (SGR) protein and Rboh/NADPH‐Oxidase‐dependent H2O2 signaling to cope with high salinity stress.OsRACK1B localizes in the nucleus and the cytoplasm in rice. During abiotic stresses such as high salinity, OsRACK1B directly or indirectly represses the transcripts of chlorophyll catabolic genes and activates defense‐related genes in the nucleus. After synthesized in the cytosol, OsRACK1B shuttles to the chloroplast where it binds to the Stay‐Green (SGR) protein and limits the availability of SGR to form SGR‐CCE‐LHCII multi‐protein complex, a prerequisite for chlorophyll breakdown. Simultaneously, OsRACK1B interacts with the N‐terminal region of Rboh/NADPH oxidase in the cytoplasm and regulates the NADPH oxidase‐dependent H2O2 production via the action of CuZn‐SOD resulting in a ROS dependent systemic wave inside the cell. At a certain level, H2O2 can act as a signaling molecule to generate a positive feedback loop for chloroplast homeostasis and may trigger retrograde signaling to regulate nuclear gene expression to protect the chloroplast and enhance stress tolerance of the plant. High salinity stress also induces stress phytohormone abscisic acid (ABA). OsRACK1B can also protect the photosystem complex through negative regulation of ABA signaling pathway. Solid and dotted lines indicate actual and hypothetical regulations of the signaling effect, respectively.Figure 1