Abscisic acid-induced degradation of Arabidopsis guanine nucleotide exchange factor requires calcium-dependent protein kinases

Abstract

Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1, thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple-mutant plants in the calcium-dependent protein kinases (CPKs) disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, partially impairs ABA inhibition of cotyledon emergence. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 overexpression phenotype in root hair development in Our findings establish the physiological and molecular functions and relevance of CPKs in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in ABA signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.

Properties and functions of calcium‐dependent protein kinases and their relatives inArabidopsis thaliana

“…Importantly, the altered root hair development triggered by overexpressing RopGEF1 was enhanced in a cpk3,4,6,11 background and reduced by expressing back CPK4 WT but not inactive kinase (Li et al ., 2018c). These data clearly place multiple CPKs upstream of RopGEF1 to trigger its phospho‐dependent degradation, although the involvement of CPK6 is still uncertain given its inability to phosphorylate RopGEF1 in vitro (Li et al ., 2018c). Thus, several CPKs promote root hair growth by inhibiting ROP11 either through the stimulation of RhoGDI or the degradation of RopGEF1.…”

“…ABA‐induced degradation of RopGEF1 can thus facilitate ABA signaling. Consistent with AtCPK3/4/6/11 promoting RopGEF1 degradation (see part III), the cpk3,4,6,11 mutant was insensitive to ABA, which was exacerbated by overexpressing RopGEF1 in the quadruple mutant (Li et al ., 2018c). Nonetheless, it is not clear whether AtCPK3 and AtCPK6 are required in this process because AtCPK3 was not tested in a kinase assay and AtCPK6 was unable to phosphorylate RopGEF1 in vitro .…”

“…The phosphomimic variant, which exhibited increased affinity for ROP, could rescue the defective root hair phenotype of the gdi mutant unlike the phosphodead variant, suggesting a positive role of AtCPK3 by stimulating RhoGDI. Moreover, the cpk11 single mutant exhibited a weak root hair phenotype (Vijayakumar et al ., ), which was strengthened in the cpk3,4,6,11 quadruple mutant, suggesting redundant functions for those kinases (Li et al ., 2018c). On the other hand, AtCPK4/7/10/11 phosphorylated RopGEF1 in vitro , which triggered its degradation in vivo (Li et al ., 2018c).…”

“…Moreover, the cpk11 single mutant exhibited a weak root hair phenotype (Vijayakumar et al ., ), which was strengthened in the cpk3,4,6,11 quadruple mutant, suggesting redundant functions for those kinases (Li et al ., 2018c). On the other hand, AtCPK4/7/10/11 phosphorylated RopGEF1 in vitro , which triggered its degradation in vivo (Li et al ., 2018c). While AtCPK4 targeted S48/S51/S52 in vitro , only S51 phosphorylation was lost in cpk3,4,6,11 , indicating that S51 is the main CPK target in vivo .…”

“…While AtCPK4 targeted S48/S51/S52 in vitro , only S51 phosphorylation was lost in cpk3,4,6,11 , indicating that S51 is the main CPK target in vivo . Importantly, the altered root hair development triggered by overexpressing RopGEF1 was enhanced in a cpk3,4,6,11 background and reduced by expressing back CPK4 WT but not inactive kinase (Li et al ., 2018c). These data clearly place multiple CPKs upstream of RopGEF1 to trigger its phospho‐dependent degradation, although the involvement of CPK6 is still uncertain given its inability to phosphorylate RopGEF1 in vitro (Li et al ., 2018c).…”