Monomerization of abscisic acid receptors through CARKs‐mediated phosphorylation

Abstract

Summary Cytosolic ABA Receptor Kinases (CARKs) play a pivotal role in abscisic acid (ABA)‐dependent pathway in response to dehydration, but their regulatory mechanism in ABA signaling remains unexplored. In this study, we showed that CARK4/5 of CARK family physically interacted with ABA receptors (RCARs/PYR1/PYLs), including RCAR3, RCAR11–RCAR14, while CARK2/7/11 only interacted with RCAR11–RCAR14, but not RCAR3. It indicates that the members in CARK family function redundantly and differentially in ABA signaling. RCAR12 can form heterodimer with RCAR3 in vitro and in vivo. Moreover, the members of CARK family can form homodimer or heterodimer in a kinase activity dependent manner. ITC (isothermal titration calorimetry) analysis demonstrated that the phosphorylation of RCAR12 by CARK1 enhanced the ABA binding affinity. The phosphor‐mimic RCAR12T105D significantly displayed ABA‐induced inhibition of the phosphatase ABI1 (ABA insensitive 1) activity, leading to upregulation of ABA‐responsive genes RD29A and RD29B in cark157:RCAR12T105D transgenic plants, which exhibited ABA hypersensitive phenotype. The transcription factor ABI5 (ABA insensitive 5) activates the transcriptions of CARK1 and CARK3 by binding to ABA‐response elements (ABREs) of their promoters. Collectively, our data imply that the dimeric CARKs phosphorylate homodimer or heterodimer ABA receptors, leading to monomerization for triggering ABA responses in Arabidopsis.

CARK3-mediated ADF4 regulates hypocotyl elongation and soil drought stress in Arabidopsis

“…CARK3 has been shown to phosphorylate ABA receptors to mediate ABA signaling pathway ( Li et al., 2022 ). ADF4 has revealed to be phosphorylated by CKL2 ( Arabidopsis thaliana casein kinase 1-like protein 2) and CPK3 (Arabidopsis calcium-dependent protein kinase 3), showing multiple potential Ser/Thr phosphorylation sites ( Guo et al., 2016 ; Lu et al., 2020 ).…”

“…To test the interaction of CARK3 with ADF4 in vitro , GST-CARK3, GST and His-ADF4 were purified as previously described ( Li et al., 2022 ; Yao et al., 2022 ). 5 μg GST-CARK3 or GST bound glutathione-agarose beads were incubated with His-ADF4 in binding buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 0.1% Tween 20) at room temperature for 30 min and washed 10 times with binding buffer.…”

“…Stomatal aperture was carried out as previously described ( Li et al., 2022 ). 3-week old leaves were peeled and then incubated in opening buffer (50 mM KCl, 10 μM CaCl 2 , 10 mM MES pH 6.15) for 3 h to induce stomatal opening.…”

“…Our previous studies have shown that CAK3 kinase protein could form dimer to phosphorylate ABA (abscisic acid) receptors, and subsequently trigger ABA signaling pathway ( Li et al., 2022 ). Here, we observed that CARK3 T-DNA mutants exhibited long hypocotyl compared with the wild-type plants.…”

FtbZIP85 Is Involved in the Accumulation of Proanthocyanidin by Regulating the Transcription of FtDFR in Tartary Buckwheat

“…The coding region of FtbZIP85 was cloned into pGBKT7 (Clontech, Mountain View, CA, USA) as the prey. The bait and prey constructs were co-transformed into the yeast strain AH109, using the previously described method [ 31 ]. The yeast strains were grown on a selection medium lacking SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade.…”