Binding of 14-3-3κ to ADF4 is involved in the regulation of hypocotyl growth and response to osmotic stress in Arabidopsis.

14-3-3 proteins, a family of conserved molecules in eukaryotes, target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. ADF4, as one of Actin-Depolymerizing Factor (ADF) family of proteins, is involved in plant development, and response to biotic and abiotic stresses. Here, we show that 14-3-3κ specially interacted with ADF4 in vitro and in vivo. The 14-3-3κ×adf4 double mutant displayed less F-actin bundle and shorter hypocotyl compared with adf4 mutant, indicating that 14-3-3κ acts upstream of ADF4 to mediate the hypocotyl growth in the dark-grown seedlings. Under the osmotic stress, 14-3-3κ mutants displayed less survival rate than wild-type plants. The adf4 mutants exhibited markedly enhanced survival rate under osmotic treatment, while ADF4-overexpressing plants displayed the opposite results, indicating that ADF4 plays a negative role in response to osmotic stress in Arabidopsis. The interaction between ADF4 and 14-3-3κ inhibited the association of ADF4 with actin filament. Moreover, the in vitro phosphorylation assay demonstrates that the phosphorylation of ADF4 by CASEIN KINASE1-LIKE PROTEIN2 (CKL2) was enhanced by binding 14-3-3κ. Collectively, our data infer a fundamental role for the interaction between 14-3-3κ and ADF4 in regulating hypocotyl growth and osmotic tolerance of plants.

Monomerization of abscisic acid receptors through CARKs-mediated phosphorylation.

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.

Abscisic acid receptors are involves in the Jasmonate signaling in Arabidopsis.

The phytohormones jasmonates (JAs) act as important molecules of elicitors for the chlorophyll degradation and anthocyanin biosynthesis. JAs do usually not act independently but integrate in complex networks linking to other hormonal signaling transduction. Here, the crosstalk was detected between the JAs (jasmonic acid) and abscisic acid (ABA) signaling pathways in the mediation of chlorophyll degradation and anthocyanin biosynthesis. In this study, we found that the ABA receptor mutants, pyr1pyl1pyl2pyl4 (1124) and pyr1pyl1ply2pyl4pyl5pyl8 (112458) showed less level of chlorophyll and anthocyanin than the wild-type plants, while gain-of-function of RCAR13 transgenic lines inhibited chlorophyll degradation and enhanced anthocyanin accumulation after MeJA treatment. The amidohydrolases, including ILL6 and IAR3 and cytochrome P450 (CYP94B3), encoding JA-Ile catabolism were markedly depressed by ABA receptors. While transcripts of the enzymes for activation of anthocyanin biosynthesis pathway were analyzed, the results indicating that JA biosynthetic genes, including allene oxide synthase (AOS), LOX3 and LOX4 were enhanced by the link of JAs and ABA receptors. Moreover, the ABA receptors are also involved in JAs signal transduction through the regulation of COI, JAZ and MYC2 transcripts. These findings elucidate a connection between a core component of the ABA signaling pathway and JA responses.

Characterization of abscisic acid (ABA) receptors and analysis of genes that regulate rutin biosynthesis in response to ABA in Fagopyrum tataricum.

Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) is a nutritional crop, which has high rutin, and is good for health. Until now, plant genetic engineering is insufficient for Tartary buckwheat. Abscisic acid (ABA), as one of phytohormones, is involved in the regulation of plant growth and development, and responses to diverse environmental challenges. Although ABA receptors have been well characterized in Arabidopsis, it is little understood in Tartary buckwheat. In this study, we identified 12 ABA receptors, designated as FtRCAR1 through FtRCAR12 in Tartary buckwheat. FtRCARs are divided into three subfamily. Based on the similarity, we could predict that FtRCARs comprise of the monomeric (FtRCAR1, 3, 4, 5, 9, 10, 11 and 12) and the dimeric (FtRCAR2, 7 and 8) state in solution. The analysis of the transcript pattern indicated that most of FtRCARs were significantly variable among the root, stem, leaf, flower and seed, while FtRCAR4 transcript was undetectable under in all tissues. The transcript levels of FtRCARs under ABA treatment indicated that most FtRCARs transcripts were depressed, indicating a possible feedback regulation of ABA signaling. The analysis of rutin biosynthesis related-genes indicated that ABA up-graduated CHS, CHI, F3'H, F3H and FLS transcript levels, while transcripts of 4CL and PAL were down-regulated. In addition, the transcription factors that mediated the rutin biosynthesis related-genes were also regulated by exogenous ABA. Thus, the identification and the characterization of FtRCARs would enable us to further understand the role of ABA signal in Tartary buckwheat.