ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in Arabidopsis

The phytohormone abscisic acid （ABA） plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been discovered, understanding of the ABA signaling pathway is still far from complete. In this work, we characterized the function of ROP11, a member of the plant-specific ROP small GTPases family, in the ABA signaling process. ROP11 is preferentially expressed in guard cells in all plant organs with stomata. Expression of a constitutively active ROP11 （CA-ROP11） suppresses ABA-mediated responses, whereas reduced expression of ROP11 or expression of its dominant-negative form （DN-ROP11） causes the opposite phenotypes. The affected ABA-mediated responses by ROP11 include seed germination, seedling growth, stomatal closure, induction of ABA-responsive genes, as well as plant response to drought stress. Furthermore, we showed that ROP11 and its closest-related family member, ROP10, act in parallel in mediating these responses. ABA treatment does not affect ROP11 transcription and protein abundance; however, it causes the accumulation of CA-ROP11 in the nucleus. These results demonstrated that ROP11 is a negative regulator of multiple ABA responses in Arabidopsis.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

A Dual-Function Transcription Factor, AtYY1, Is a Novel Negative Regulator of the Arabidopsis ABA Response Network

Abscisic acid （ABA） plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we iden- tified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 （YY1）, as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yyl mutant was more sensitive to ABA and NaCI than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYYI can bind the ABA REPRESSOR1 （ABR1） promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and saR-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 （ABI4） might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis.

The Asparagine-Rich Protein NRP Facilitates the Degradation of the PP6-type Phosphatase FyPP3 to Promote ABA Response in Arabidopsis

The phytohormone abscisic acid （ABA） plays critical roles in abiotic stress responses and plant develop- ment. In germinating seeds, the phytochrome-associated protein phosphatase, FyPP3, negatively regulates ABA signaling by dephosphorylating the transcription factor ABI5. However, whether and how FyPP3 is regulated at the posttranscriptional level remains unclear. Here, we report that an asparagine-rich protein, NRP, interacts with FyPP3 and tethers FyPP3 to SYP41/61-positive endosomes for subsequent degradation in the vacuole. Upon ABA treatment, the expression of NRP was induced and NRP-mediated FyPP3 turnover was accelerated. Consistently, ABA-induced FyPP3 turnover was abolished in an nrp null mutant. On the other hand, FyPP3 can dephosphorylate NRP in vitro, and overexpression of FyPP3 reduced the half-life of NRP in vivo. Genetic analyses showed that NRP has a positive role in ABA-mediated seed germination and gene expression, and that NRP is epistatic to FyPP3. Taken together, our results identify a new regulatory circuit in the ABA signaling network, which links the intracellular trafficking with ABA signaling.

Response of ABA content in rice leaves to water stress

The rice seeds of IR661 (indica); Yanjing 2 (japonica) and Shanyou 53 indica hybrid Fwere grown in 15 nursery trays (40×20×15cm~2 each), A water stress treatment was carried out at the leaf age 5 by excluding water for 120h. Leaf water potential (LWP) was determined with chamber pressure. Abscisic acid (ABA) content in rice leaves was measured with radioimmnoassay. The content of ABA in the leaves changed a little when LWP was larger than—0.02 MPa, but increased rapidly when LWP was lower than this value. It could be considered that the critical LWP for obvious accumulation of ABA in rice seedling leaves was about—0.02 MPa.

Long-distance ABA transport can mediate distal tissue responses by affecting local ABA concentrations

Environmental stresses that perturb plant Hwater relations influence abscisic acid(ABA) concentrations, but it is unclear whether long-distance ABA transport contributes to changes in local ABA levels. To determine the physiological relevance of ABA transport, we made reciprocal-and self-grafts of ABA-deficient flacca mutant and wild-type(WT) tomato plants, in which low phosphorus(P) conditions decreased ABA concentrations while salinity increased ABA concentrations. Whereas foliar ABA concentrations in the WT scions were rootstock independent under control conditions, salinity resulted in long-distance transport of ABA: flacca scions had approximately twice as much ABA when grafted on WT rootstocks compared to flacca rootstocks. Root ABA concentrations were scion dependent: both WT and flacca rootstocks had less ABA with the flacca mutant scion than with the WT scion under control conditions. In WT scions, whereas rootstock genotype had limited effects on stomatal conductance under control conditions, a flacca rootstock decreased leaf area of stressed plants, presumably due to attenuated root-to-shoot ABA transport. In flacca scions, a WT rootstock decreased stomatal conductance but increased leaf area of stressed plants, likely due to enhanced root-to-shoot ABA transport. Thus, long-distance ABA transport can affect responses in distal tissues by changing local ABA concentrations.

TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity

In plants, clade A type 2 C protein phosphatases(PP2 CAs) have emerged as major players in abscisic acid(ABA)-regulated stress responses by inhibiting protein kinase activity. However, how different internal and external environmental signals modulate the activity of PP2 CAs are not well known. The transmembrane kinase(TMK) protein4(TMK4), one member of a previously identified receptor kinase subfamily on the plasma membrane that plays vital roles in plant cell growth,directly interacts with PP2 CAs member(ABAInsensitive 2, ABI2). tmk4 mutant is hypersensitive to ABA in both ABA-inhibited seed germination and primary root growth, indicating that TMK4 is a negative regulator in ABA signaling pathway.Further analyses indicate that TMK4 phosphorylates ABI2 at three conserved Ser residues,thus enhancing the activity of ABI2. The phosphorylation-mimic ABI2~(S139DS140DS266D)can complement butnon-phosphorylated form ABI2~(S139AS140AS266) Acannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2 abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.

TaTIP41 and TaTAP46 positively regulate drought tolerance in wheat by inhibiting PP2A activity

Drought is a major environmental stress limiting global wheat(Triticum aestivum)production.Exploring drought tolerance genes is important for improving drought adaptation in this crop.Here,we cloned and characterized TaTIP41,a novel drought tolerance gene in wheat.TaTIP41 is a putative conserved component of target of rapamycin(TOR)signaling,and the Ta TIP41 homoeologs were expressed in response to drought stress and abscisic acid(ABA).The overexpression of Ta TIP41 enhanced drought tolerance and the ABA response,including ABA-induced stomatal closure,while its downregulation using RNA interference(RNAi)had the opposite effect.Furthermore,Ta TIP41 physically interacted with TaTAP46,another conserved component of TOR signaling.Like TaTIP41,TaTAP46 positively regulated drought tolerance.Furthermore,TaTIP41 and TaTAP46 interacted with type-2A protein phosphatase(PP2A)catalytic subunits,such as TaPP2A-2,and inhibited their enzymatic activities.Silencing TaPP2A-2 improved drought tolerance in wheat.Together,our findings provide new insights into the roles of TaTIP41 and TaTAP46 in the drought tolerance and ABA response in wheat,and their potential application in improving wheat environmental adaptability.

Functional analyses of an E3 ligase gene AIP2 from wheat in Arabidopsis revealed its roles in seed germination and pre‐harvest sprouting

Pre‐harvest sprouting（PHS） seriously affects wheat yield and quality of the grain. ABI3 is a key factor in the activation of seed development and repression of germination in Arabidopsis. An ABI3‐interacting protein（AIP2） could polyubiquitinate ABI3, impair seed dormancy and promote seed germination in Arabidopsis. In this study,two wheat AIP2 genes, TaAIP2A and TaAIP2B, were isolated.Subcellular localization assay and yeast two‐hybrid analysis revealed that TaAIP2A and TaAIP2B may function through interaction with wheat Viviporous‐1（TaVp1）. The transcripts TaAIP2A and TaAIP2B were more abundant in wheat PHS susceptible cultivars than that of resistant ones, and decreased gradually following seed development. Expression of TaAIP2A and TaAIP2B in Arabidopsis aip2‐1 mutant lines resulted in earlier flowering, promotion of seed germination,and reduced ABA sensitivity, respectively, somehow mimicking the phenotype of the wild type, with TaAIP2B having a stronger role in these aspects. Furthermore, the expression ofupstream genes ABI1 and ABI2 were upregulated, whereas that of downstream genes ABI3 and ABI5 were downregulated in both TaAIP2A and TaAIP2B complemented lines upon ABA treatment. These results suggested that wheat AIP2s could negatively regulate the ABA signaling pathway and play important roles in seed germination, and thus wheat PHS resistance finally.