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Uridine diphosphate‐glucosyltransferases (UGTs) maintain abscisic acid (ABA) homeostasis in Arabidopsis thaliana by converting ABA to abscisic acid‐glucose ester (ABA‐GE). UGT71C5 plays an important role in the generation of ABA‐GE. Abscisic acid receptors are crucial upstream components of the ABA signaling pathway, but how UGTs and ABA receptors function together to modulate ABA levels is unknown. Here, we demonstrated that the ABA receptors RCAR12/13 and UGT71C5 maintain ABA homeostasis in Arabidopsis following rehydration under drought stress. Biochemical analyses show that UGT71C5 directly interacted with RCAR8/12/13 in yeast cells, and the interactions between UGT71C5 and RCAR12/13 were enhanced by ABA treatment. Enzyme activity analysis showed that ABA‐GE contents were significantly elevated in the presence of RCAR12 or RCAR13, suggesting that these ABA receptors enhance the activity of UGT71C5. Determination of the content of ABA and ABA‐GE in Arabidopsis following rehydration under drought stress revealed that ABA‐GE contents were significantly higher in Arabidopsis plants overexpressing RCAR12 and RCAR13 than in non‐transformed plants and plants overexpressing RCAR11 following rehydration under drought stress. These observations suggest that RCAR12 and RCAR13 enhance the activity of UGT71C5 to glycosylate excess ABA into ABA‐GE following rehydration under drought stress, representing a rapid mechanism for regulating plant growth and development. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) plays a vital role in responses to abiotic stresses that allow plants to cope with environmental challenges. In this study, we analyzed ABA receptors of subfamily III as the potential targets of Cytosolic ABA Receptor Kinase 1 (CARK1). We previously found that CARK1 phosphorylated the subfamily III member RCAR11 at a distinct threonine residue (T78). Our study now shows the physical interaction of CARK1 with the receptors RCAR12/13/14 in vitro and in vivo. The catalytically inactive form CARK1-N204A did not interact with the receptors. Phosphorylation of these ABA receptors in vitro occurred at a serine/threonine amino acid residue corresponding to T78 in RCAR11, which is located in the loop of β3 within a conserved site. Further analysis revealed that the phosphorylation of RCAR11T78 could increase the sensitivity of the pyr1pyl1pyl2pyl4 quadruple mutant (1124) to ABA, including the inhibition of root elongation and increasing drought tolerance. The analysis of CARK1 :1124 complementation and the expression of ABA-related genes indicated that CARK1 could rescue the insensitivity of 1124 to ABA. Our results indicate that CARK1 tends to phosphorylate subfamily III ABA receptors, and the phosphosites RCAR11T78, RCAR12T105, RCAR13T101, and RCAR14S81 are the major sites involved in the activation of the ABA response pathway. [ABSTRACT FROM AUTHOR]

Key message: The RING-type E3 ligase AtARRE participates in the plant ABA responding as a negative regulator.Abstract: Ubiquitination protease system (UPS) is significant in post-transcriptional regulation. In UPS, E3 ligase recognizes the substrate protein and mediates the polyubiquitin chain onto the substrate. Here, we identified a new gene, named Arabidopsis thaliana ABA-related RING-type E3 ligase (AtARRE), which induced by ABA and NaCl. AtARRE encodes a functional RING-type E3 ligase protein localized in nucleus and plasma membrane of Arabidopsis. Physiological analysis demonstrated that mutation of AtARRE (T-DNA insert mutants atarre-1 and atarre-2) caused plants hypersensitivity to ABA, including enhanced stomatal closure, reduced root elongation and seed germination. However, overexpression of AtARRE transgenic lines caused plants hyposensitive to ABA compared with WT and mutant atarre plants. Under the treatment of ABA, the transcript abundances of ABA-responsive genes RD29A, RD29B, RD22 and ABI5 in atarre mutant plants were markedly higher than those of WT and AtARRE overexpression lines. Hence, these results indicate that AtARRE acts as a negative regulator of ABA-mediated stress responses in Arabidopsis. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability. [ABSTRACT FROM AUTHOR]

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)-insensitive RING protein 4 ( AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4-RING finger domain in its C-terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T-DNA insertion mutant atairp4 effectively recovered the ABA-associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild-type and atairp4 mutant plants. In addition, the expression levels of ABA- and drought-induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild-type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA-mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is a vital phytohormone that accumulates in response to various biotic and abiotic stresses, as well as plant growth. In Arabidopsis thaliana, there are 14 members of the ABA receptor family, which are key positive regulators involved in ABA signaling. Besides reduced drought stress tolerance, the quadruple and sextuple mutants (pyr1pyl1pyl2pyl4 (1124) and pyr1pyl1pyl2pyl4pyl5pyl8 (112458) show abnormal growth phenotypes, such as decreases in yield and height, under non-stress conditions. However, it remains unknown whether ABA receptors mediate ABA signaling to regulate plant growth and development. Here, we showed the primary metabolite profiles of 1124, 112458 and wild-type (WT) plants grown under normal conditions. The metabolic changes were significantly different between ABA receptor mutants and WT. Guanosine, for the biosynthesis of cyclic guanosine 3′,5′-monophosphate (cGMP), is an important second messenger that acts to regulate the level of ABA. In addition, other amino acids were increased in the 112458 mutant, including proline. These results, together with phenotype analysis, indicated that ABA receptors are involved in ABA signaling to modulate metabolism and plant growth under normal conditions. [ABSTRACT FROM AUTHOR]

Drought is a major limiting factor for plant growth and crop productivity. Many Calcineurin B-like interacting protein kinases (CIPKs) play crucial roles in plant adaptation to environmental stresses. It is particularly essential to find the phosphorylation targets of CIPKs and to study the underlying molecular mechanisms. In this study, we demonstrate that CIPK11 acts as a novel component to modulate drought stress in plants. The overexpression of CIPK11 (CIPK11OE) in Arabidopsis resulted in the decreased tolerance of plant to drought stress. When compared to wild type plants, CIPK11OE plants exhibited higher leaf water loss and higher content of reactive oxygen species (ROS) after drought treatment. Additionally, a yeast two hybrid screening assay by using CIPK11 as a bait captures Di19-3, a Cys2/His2-type zinc-finger transcription factor that is involved in drought stress, as a new interactor of CIPK11. Biochemical analysis revealed that CIPK11 interacted with Di19-3 in vivo and it was capable of phosphorylating Di19-3 in vitro. Genetic studies revealed that the function of CIPK11 in regulating drought stress was dependent on Di19-3. The transcripts of stress responsive genes, such as RAB18, RD29A, RD29B, and DREB2A were down-regulated in the CIPK11OE plants. Whereas overexpression of CIPK11 in di19-3 mutant background, expression levels of those marker genes were not significantly altered. Taken together, our results demonstrate that CIPK11 partly mediates the drought stress response by regulating the transcription factor Di19-3. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) plays a fundamental role in plant growth and development, as well as in the responses to abiotic stresses. Previous studies have revealed that many components in ABA and drought stress signaling pathways are ubiquitinated by E3 ligases. In this study, AtPPRT1, a putative C3HC4 zinc-finger ubiquitin E3 ligase, was explored for its role in abiotic stress response in Arabidopsis thaliana. The expression of AtPPRT1 was induced by ABA. In addition, the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter was more active in the root hair zone and root tips of primary and major lateral roots of young seedlings in the presence of ABA. The assays for seed germination, stomatal aperture, root length, and water deficit demonstrated that the AtPPRT1-overexpressing Arabidopsis was insensitive to ABA and sensitive to drought stress compared with wild-type (WT) plants. The analysis by quantitative real-time PCR (qRT-PCR) revealed that the expression of three stress-inducible genes (AtRAB18, AtERD10, and AtKIN1) were upregulated in the atpprt1 mutant and downregulated in AtPPRT1-overexpressing plants, while two ABA hydrolysis genes (AtCYP707A1 and AtCYP707A3) were downregulated in the atpprt1 mutant and upregulated in AtPPRT1-overexpressing plants in the presence of ABA. AtPPRT1 was localized in the mitochondria. Our findings indicate that AtPPRT1 plays a negative role in ABA and drought stress responses. [ABSTRACT FROM AUTHOR]

Drought stress hinders plant growth and development, and abscisic acid (ABA) stimulates plants to respond to drought. Here, to increase plant tolerance to drought, we designed three synthetic promoters (Ap, Dp, ANDp) to determine transcription activity and drought stress resistance in plants resulting from combinations of (1) synthetic promoters and (2) the functional genes CARK1 (cytosolic ABA receptor kinase 1) and RCAR11 (regulatory components of ABA receptor 11). Transient expression of eGFP and the dual-luciferase assay demonstrated that the basal transcriptional activities of Ap and ANDp were present at low levels under normal conditions, while the synthetic promoters were apparently induced upon either treatment of exogenous ABA or co-transformation with effector DREB2A (dehydration-responsive element binding protein 2A). Analysis of the transgenic plants (Ap:CARK1, Dp:CARK1, ANDp:CARK1, and Dp:RCAR11-Ap:CARK1) showed that the synthetic promoters Ap, Dp, and ANDp increased the expression of exogenous genes in transgenic plants upon treatment of ABA or d-mannitol. ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 transgenic plants were sensitive to ABA and d-mannitol during cotyledon greening and root growth. A drought tolerance assay revealed that ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 exhibited a higher survival rate than others upon drought stress. These results indicate that the combinations ANDp:CARK1 and Dp:RCAR11-Ap:CARK1 can be used to generate drought stress resistance in plants. [ABSTRACT FROM AUTHOR]

Arabidopsis abscisic acid ABA-Insensitive RING Proteins (AtAIRP1–4) are RING E3s that play significant roles in ABA-signaling pathways. However, it is still unclear whether they have other functions. Here, AtAIRP4 was determined to play a role in response to gibberellin A3 (GA 3) in Arabidopsis thaliana. After proAtAIRP4::GUS transgenic lines were treated with GA 3 , the GUS activity decreased in hypocotyls. Increased hypocotyl elongation in response to GA 3 seen in WT was not observed in the AtAIRP4 -overexpression lines, whereas AtAIRP4 -overexpression lines were hypersensitive to Paclobutrazol (PAC, an inhibitor of GA biosynthesis) during the seed germination stage. Additionally, AtAIRP4 -overexpressing lines showed the lowest level of primary root elongation in the presence of GA 3. The levels of endogenous GA 3 in 35S::AtAIRP4 lines were lower than those in wild-type. In addition, among the plants, the mRNA levels of the GA synthetic gene GIBBERELLIN 20-OXIDASE1 (GA20ox1) was the lowest in overexpressing line. However, the expression of the response gene DELLA RGA-LIKE3 (RGL3) was the highest in overexpressing lines after treatment with GA 3. Thus, AtAIRP4 plays a negative role in GA-mediated hypocotyl elongation and root growth, and it inhibits the synthesis of endogenous biologically active GA 3 to some extent. [ABSTRACT FROM AUTHOR]