Calcium-dependent Protein Kinase 5 (CPK5) positively modulates drought tolerance through phosphorylating ABA-Responsive Element Binding Factors in oilseed rape (Brassica napus L.).

Drought is an environmental stress that causes severe crop loss. Drought stress can induce abscisic acid (ABA) accumulation and cytoplasmic calcium oscillation. Calcium-dependent protein kinases (CPKs) constitute a group of Ser/Thr protein kinases decoding calcium signals. However, the function and molecular mechanisms of most CPKs in oilseed rape (Brassica napus) remain unknown. Here, we report the functional characterization of BnaCPK5 in drought stress tolerance. BnaCPK5 belongs to Group I of the CPK family and was localized at the plasma membrane and nuclei. Overexpression of BnaCPK5 enhanced drought stress tolerance compared with the control. A screening of interacting proteins identified that BnaCPK5 interacted strongly with two ABA-Responsive Element Binding Factors (ABF/AREBs), BnaABF3 and BnaABF4. BnaCPK5 was shown to phosphorylate both BnaABF3 and BnaABF4 in a kinase assay. Further, it was found that the phosphorylation of BnaABF3 and BnaABF4 by BnaCPK5 increased their transcriptional activities against the famous drought stress marker gene, Responsive to Dehydration (RD) 29B and protein stability. Taken together, these data demonstrate that BnaCPK5 acts as a positive regulator of drought tolerance by, at least in part, phosphorylating two core ABA-signaling components to modulate Late-Embryogenesis Abundant (LEA)-like RD29B expression.

Rapeseed calcium-dependent protein kinase CPK6L modulates reactive oxygen species and cell death through interacting and phosphorylating RBOHD.

Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD.