Modulates Abscisic Acid Signaling Research Articles

MdMYB44-like positively regulates salt and drought tolerance via the MdPYL8-MdPP2CA module in apple.

Abscisic acid (ABA) is involved in salt and drought stress responses, but the underlying molecular mechanism remains unclear. Here, we demonstrated that the overexpression of MdMYB44-like, an R2R3-MYB transcription factor, significantly increases the salt and drought tolerance of transgenic apples and Arabidopsis. MdMYB44-like inhibits the transcription of MdPP2CA, which encodes a type 2C protein phosphatase that acts as a negative regulator in the ABA response, thereby enhancing ABA signaling-mediated salt and drought tolerance. Furthermore, we found that MdMYB44-like and MdPYL8, an ABA receptor, form a protein complex that further enhances the transcriptional inhibition of the MdPP2CA promoter by MdMYB44-like. Significantly, we discovered that MdPP2CA can interfere with the physical association between MdMYB44-like and MdPYL8 in the presence of ABA, partially blocking the inhibitory effect of the MdMYB44-like-MdPYL8 complex on the MdPP2CA promoter. Thus, MdMYB44-like, MdPYL8, and MdPP2CA form a regulatory loop that tightly modulates ABA signaling homeostasis under salt and drought stress. Our data reveal that MdMYB44-like precisely modulates ABA-mediated salt and drought tolerance in apples through the MdPYL8-MdPP2CA module.

The pepper phosphatidyl ethanolamine-binding proteins CaMFT02 and CaMFT03 have distinct roles in responses to drought and salt stresses

Plants have established defense systems to overcome diverse environmental threats. Abscisic acid (ABA) is one of the representative phytohormones that involves in stress response. Here, we isolated and verified the function of pepper CaMFT (Capsicum annuum Mother of FT and TFL1) genes, which negatively modulate ABA-mediated drought and salt tolerance. MFT is part of the phosphatidyl ethanolamine-binding protein (PEBP) family, which also contains the flowering locus T (FT) and terminal flower 1 (TFL1) subfamilies. While FT and TFL1 have been identified as regulators of flowering time in Arabidopsis, the biological function of MFT is largely unknown. To elucidate how CaMFT02 acts in ABA signaling, along with drought and salt stress responses, we conducted loss-of-function and gain-of-function assays with pepper and Arabidopsis, respectively. CaMFT02-silenced pepper exhibited increased ABA sensitivity and drought tolerance, whereas CaMFT02-overexpressing Arabidopsis showed ABA hyposensitivity and drought hypersensitivity. Silencing of CaMFT03, a CaMFT02 homolog, improved salt stress tolerance in pepper, and this negative regulatory role of CaMFT03 in salt stress tolerance was also observed in CaMFT03-overexpressing Arabidopsis. Taken together, our findings indicate that CaMFT02 negatively regulates drought stress tolerance via ABA signaling in pepper by modulating ABA signaling and suggest that CaMFT03 plays a similar negative regulatory role in salt stress tolerance.

A novel ABA-induced transcript factor from Millettia pinnata, MpAITR1, enhances salt and drought tolerance through ABA signaling in transgenic Arabidopsis

Abiotic stress, such as salt and drought stress, seriously limits plant growth and crop yield. Abscisic acid (ABA) is essential in regulating plant responses to abiotic stress via signal perception, transduction, and transcriptional regulation. Pongamia (Millettia pinnata) is a kind of semi-mangrove plant with strong stress tolerance and can grow in fresh and sea water. However, the molecular mechanism of the ABA signaling pathway mediating the environmental tolerance of Pongamia is still scarce so far. AITR (ABA-Induced Transcription Repressor) was a recently identified small conserved family of transcription factor in angiosperms, which played controversial roles in response to abiotic stresses in different species. Here, we identified an ABA-induced gene, MpAITR1, which encoded a nucleus localization transcriptional factor in Pongamia. MpAITR1 was highly induced by ABA and salt treatments in roots and leaves. Heterologous expression of MpAITR1 in Arabidopsis increased sensitivity to ABA, moreover, enhanced tolerance to salt and drought stress. The expression levels of some ABA-responsive and stress-responsive genes were altered in transgenic plants compared to wild-type plants under the ABA, salt, and drought stress, which was consistent with the stress-tolerant phenotype of transgenic plants. These results reveal that MpAITR1 positively modulates ABA signaling pathways and enhances the tolerance to salt and drought stress by regulating downstream target genes. Taken together, MpAITR1 from the semi-mangrove plant Pongamia serves as a potential candidate for stress-tolerant crop breeding.

Transcription factor RcNAC091 enhances rose drought tolerance through the abscisic acid-dependent pathway.

NAC (NAM, ATAF1,2, and CUC2) transcription factors (TFs) play critical roles in controlling plant growth, development, and abiotic stress responses. However, few studies have examined NAC proteins related to drought stress tolerance in rose (Rosa chinensis). Here, we identified a drought- and abscisic acid (ABA)-induced NAC TF, RcNAC091, that localizes to the nucleus and has transcriptional activation activity. Virus-induced silencing of RcNAC091 resulted in decreased drought stress tolerance, and RcNAC091 overexpression had the opposite effect. Specifically, ABA mediated RcNAC091-regulated drought tolerance. A transcriptomic comparison showed altered expression of genes involved in ABA signaling and oxidase metabolism in RcNAC091-silenced plants. We further confirmed that RcNAC091 directly targets the promoter of RcWRKY71 in vivo and in vitro. Moreover, RcWRKY71-slienced rose plants were not sensitive to both ABA and drought stress, whereas RcWRKY71-overexpressing plants were hypersensitive to ABA, which resulted in drought-tolerant phenotypes. The expression of ABA biosynthesis- and signaling-related genes was impaired in RcWRKY71-slienced plants, suggesting that RcWRKY71 might facilitate the ABA-dependent pathway. Therefore, our results show that RcWRKY71 is transcriptionally activated by RcNAC091, which positively modulates ABA signaling and drought responses. The results of this study provide insights into the roles of TFs as functional links between RcNAC091 and RcWRKY71 in priming resistance; our findings also have implications for the approaches to enhance the drought resistance of roses.

Allelic variation in transcription factor PtoWRKY68 contributes to drought tolerance in Populus.

Drought stress limits woody species productivity and influences tree distribution. However, dissecting the molecular mechanisms that underpin drought responses in forest trees can be challenging due to trait complexity. Here, using a panel of 300 Chinese white poplar (Populus tomentosa) accessions collected from different geographical climatic regions in China, we performed a genome-wide association study (GWAS) on seven drought-related traits and identified PtoWRKY68 as a candidate gene involved in the response to drought stress. A 12-bp insertion and/or deletion and three nonsynonymous variants in the PtoWRKY68 coding sequence categorized natural populations of P. tomentosa into two haplotype groups, PtoWRKY68hap1 and PtoWRKY68hap2. The allelic variation in these two PtoWRKY68 haplotypes conferred differential transcriptional regulatory activities and binding to the promoters of downstream abscisic acid (ABA) efflux and signaling genes. Overexpression of PtoWRKY68hap1 and PtoWRKY68hap2 in Arabidopsis (Arabidopsis thaliana) ameliorated the drought tolerance of two transgenic lines and increased ABA content by 42.7% and 14.3% compared to wild-type plants, respectively. Notably, PtoWRKY68hap1 (associated with drought tolerance) is ubiquitous in accessions in water-deficient environments, whereas the drought-sensitive allele PtoWRKY68hap2 is widely distributed in well-watered regions, consistent with the trends in local precipitation, suggesting that these alleles correspond to geographical adaptation in Populus. Moreover, quantitative trait loci analysis and an electrophoretic mobility shift assay showed that SHORT VEGETATIVE PHASE (PtoSVP.3) positively regulates the expression of PtoWRKY68 under drought stress. We propose a drought tolerance regulatory module in which PtoWRKY68 modulates ABA signaling and accumulation, providing insight into the genetic basis of drought tolerance in trees. Our findings will facilitate molecular breeding to improve the drought tolerance of forest trees.

Insights into cryptochrome modulation of ABA signaling to mediate dormancy regulation in Marchantia polymorpha.

The acquisition of dormancy capabilities has enabled plants to survive in adverse terrestrial environmental conditions. Dormancy accumulation and release is coupled with light signaling, which is well studied in Arabidopsis, but it is unclear in the distant nonvascular relative. We study the characteristics and function on dormancy regulation of a blue light receptor cryptochrome in Marchantia polymorpha (MpCRY). Here, we identified MpCRY via bioinformatics and mutant complement analysis. The biochemical characteristics were assessed by multiple protein-binding assays. The function of MpCRY in gemma dormancy was clarified by overexpression and mutation of MpCRY, and its mechanism was analyzed via RNA sequencing and quantitative PCR analyses associated with hormone treatment. We found that the unique MpCRY protein in M. polymorpha undergoes both blue light-promoted interaction with itself (self-interaction) and blue light-dependent phosphorylation. MpCRY has the specific characteristics of blue light-induced nuclear localization and degradation. We further demonstrated that MpCRY transcriptionally represses abscisic acid (ABA) signaling-related gene expression to suppress gemma dormancy, which is dependent on blue light signaling. Our findings indicate that MpCRY possesses specific biochemical and molecular characteristics, and modulates ABA signaling under blue light conditions to regulate gemma dormancy in M. polymorpha.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

CycC1;1 negatively modulates ABA signaling by interacting with and inhibiting ABI5 during seed germination.

Regulation of seed germination is important for plant survival and propagation. ABSCISIC ACID (ABA) INSENSITIVE5 (ABI5), the central transcription factor in the ABA signaling pathway, plays a fundamental role in the regulation of ABA-responsive gene expression during seed germination; however, how ABI5 transcriptional activation activity is regulated remains to be elucidated. Here, we report that C-type Cyclin1;1 (CycC1;1) is an ABI5-interacting partner affecting the ABA response and seed germination in Arabidopsis (Arabidopsis thaliana). The CycC1;1 loss-of-function mutant is hypersensitive to ABA, and this phenotype was rescued by mutation of ABI5. Moreover, CycC1;1 suppresses ABI5 transcriptional activation activity for ABI5-targeted genes including ABI5 itself by occupying their promoters and disrupting RNA polymerase II recruitment; thus the cycc1;1 mutant shows increased expression of ABI5 and genes downstream of ABI5. Furthermore, ABA reduces the interaction between CycC1;1 and ABI5, while phospho-mimic but not phospho-dead mutation of serine-42 in ABI5 abolishes CycC1;1 interaction with ABI5 and relieves CycC1;1 inhibition of ABI5-mediated transcriptional activation of downstream target genes. Together, our study illustrates that CycC1;1 negatively modulates the ABA response by interacting with and inhibiting ABI5, while ABA relieves the CycC1;1 interaction with and inhibition of ABI5 to activate ABI5 activity for the ABA response, thereby inhibiting seed germination.

NUA and ESD4 negatively regulate ABA signaling during seed germination

The phytohormone abscisic acid (ABA) plays important roles in plant growth, development and adaptative responses to abiotic stresses. SNF1-related protein kinase 2s (SnRK2) are key components that activate the ABA core signaling pathway. NUCLEAR PORE ANCHOR (NUA) is a component of the nuclear pore complex (NPC) that involves in deSUMOylation through physically interacting with the EARLY IN SHORT DAYS 4 (ESD4) SUMO protease. However, it is not clear how NUA functions with SnRK2 and ESD4 to regulate ABA signaling. In our study, we found that nua loss-of-function mutants exhibited pleiotropic ABA-hypersensitive phenotype. We also found that ABA-responsive genes remarkably up-regulated in nua by exogenous ABA. The nua snrk2.2 snrk2.3 triple mutant and nua abi5 double mutant partially rescued the ABA-hypersensitive phenotype of nua, thereby suggesting that NUA is epistatic to SnRK2s. Additionally, we observed that esd4-3 mutant was also ABA-hypersensitive. NUA and ESD4 were further demonstrated to physically interact with SnRK2s and negatively regulate ABA signaling by reducing SnRK2s stability. Taken together, our findings uncover a new regulatory mechanism that can modulate ABA signaling.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 Modulates Abscisic Acid Signaling by Inducing PYR1 Monomerization and Association With ABI1 in Arabidopsis.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 (BAK1) is a versatile kinase involved in many different plant developmental responses. Previously, we showed that BAK1 interacts with open stomata 1 (OST1), a cytoplasmic kinase, to promote abscisic acid (ABA)-induced stomatal closure. ABA is a plant hormone that primarily regulates stress responses and is recognized by the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTORS (RCAR), which activates ABA signaling. Here, we demonstrated that BAK1 interacts with PYR1 and phosphorylates PYR1 in response to ABA in plants. We identified T137 and S142 of PYR1 as the phosphosites targeted by BAK1. Using phosphomimetic (PYR1DD) and phospho-dead (PYR1AA) PYR1 compared with wild-type PYR1, we showed that transgenic plants overexpressing a phosphomimetic PYR1 exhibited hypersensitivity to the inhibition of ABA-induced root growth and seed germination and increased ABA-induced stomatal closure and ABA-inducible gene expression. As underlying reasons for these phenomena, we further demonstrated that phosphorylated PYR1 existed in a monomeric form, in which ABA binding was increased, and the degree of complex formation with ABI1 was also increased. These results suggest that BAK1 positively modulates ABA signaling through interaction with PYR1, in addition to OST1.

The deubiquitinases UBP12 and UBP13 integrate with the E3 ubiquitin ligase XBAT35.2 to modulate VPS23A stability in ABA signaling.

Ubiquitination-mediated protein degradation in both the 26S proteasome and vacuole is an important process in abscisic acid (ABA) signaling. However, the role of deubiquitination in this process remains elusive. Here, we demonstrate that two deubiquitinating enzymes (DUBs), ubiquitin-specific protease 12 (UBP12) and UBP13, modulate ABA signaling and drought tolerance by deubiquitinating and stabilizing the endosomal sorting complex required for transport-I (ESCRT-I) component vacuolar protein sorting 23A (VPS23A) and thereby affect the stability of ABA receptors in Arabidopsis thaliana. Genetic analysis showed that VPS23A overexpression could rescue the ABA hypersensitive and drought tolerance phenotypes of ubp12-2w or ubp13-1. In addition to the direct regulation of VPS23A, we found that UBP12 and UBP13 also stabilized the E3 ligase XB3 ortholog 5 in A. thaliana (XBAT35.2) in response to ABA treatment. Hence, we demonstrated that UBP12 and UBP13 are previously unidentified rheostatic regulators of ABA signaling and revealed a mechanism by which deubiquitination precisely monitors the XBAT35/VPS23A ubiquitination module in the ABA response.

COP1 positively regulates ABA signaling during Arabidopsis seedling growth in darkness by mediating ABA-induced ABI5 accumulation.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a well-characterized E3 ubiquitin ligase, is a central repressor of seedling photomorphogenic development in darkness. However, whether COP1 is involved in modulating abscisic acid (ABA) signaling in darkness remains largely obscure. Here, we report that COP1 is a positive regulator of ABA signaling during Arabidopsis seedling growth in the dark. COP1 mediates ABA-induced accumulation of ABI5, a transcription factor playing a key role in ABA signaling, through transcriptional and post-translational regulatory mechanisms. We further show that COP1 physically interacts with ABA-hypersensitive DCAF1 (ABD1), a substrate receptor of the CUL4-DDB1 E3 ligase targeting ABI5 for degradation. Accordingly, COP1 directly ubiquitinates ABD1 in vitro, and negatively regulates ABD1 protein abundance in vivo in the dark but not in the light. Therefore, COP1 promotes ABI5 protein stability post-translationally in darkness by destabilizing ABD1 in response to ABA. Interestingly, we reveal that ABA induces the nuclear accumulation of COP1 in darkness, thus enhancing its activity in propagating the ABA signal. Together, our study uncovers that COP1 modulates ABA signaling during seedling growth in darkness by mediating ABA-induced ABI5 accumulation, demonstrating that plants adjust their ABA signaling mechanisms according to their light environment.

The Arabidopsis circadian clock protein PRR5 interacts with and stimulates ABI5 to modulate abscisic acid signaling during seed germination.

Seed germination and postgerminative growth require the precise coordination of multiple intrinsic and environmental signals. The phytohormone abscisic acid (ABA) suppresses these processes in Arabidopsis thaliana and the circadian clock contributes to the regulation of ABA signaling. However, the molecular mechanism underlying circadian clock-mediated ABA signaling remains largely unknown. Here, we found that the core circadian clock proteins PSEUDO-RESPONSE REGULATOR5 (PRR5) and PRR7 physically associate with ABSCISIC ACID-INSENSITIVE5 (ABI5), a crucial transcription factor of ABA signaling. PRR5 and PRR7 positively modulate ABA signaling redundantly during seed germination. Disrupting PRR5 and PRR7 simultaneously rendered germinating seeds hyposensitive to ABA, whereas the overexpression of PRR5 enhanced ABA signaling to inhibit seed germination. Consistent with this, the expression of several ABA-responsive genes is upregulated by PRR proteins. Genetic analysis demonstrated that PRR5 promotes ABA signaling mainly dependently on ABI5. Further mechanistic investigation revealed that PRR5 stimulates the transcriptional function of ABI5 without affecting its stability. Collectively, our results indicate that these PRR proteins function synergistically with ABI5 to activate ABA responses during seed germination, thus providing a mechanistic understanding of how ABA signaling and the circadian clock are directly integrated through a transcriptional complex involving ABI5 and central circadian clock components.

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

In plants, clade A type 2C protein phosphatases (PP2CAs) 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 PP2CAs are not well known. The transmembrane kinase (TMK) protein 4 (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 PP2CAs member (ABA-Insensitive 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 ABI2S139DS140DS266D can complement but non-phosphorylated form ABI2S139AS140AS266A cannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.

Transcriptional Regulation of Protein Phosphatase 2C Genes to Modulate Abscisic Acid Signaling.

The plant hormone abscisic acid (ABA) triggers cellular tolerance responses to osmotic stress caused by drought and salinity. ABA controls the turgor pressure of guard cells in the plant epidermis, leading to stomatal closure to minimize water loss. However, stomatal apertures open to uptake CO2 for photosynthesis even under stress conditions. ABA modulates its signaling pathway via negative feedback regulation to maintain plant homeostasis. In the nuclei of guard cells, the clade A type 2C protein phosphatases (PP2Cs) counteract SnRK2 kinases by physical interaction, and thereby inhibit activation of the transcription factors that mediate ABA-responsive gene expression. Under osmotic stress conditions, PP2Cs bind to soluble ABA receptors to capture ABA and release active SnRK2s. Thus, PP2Cs function as a switch at the center of the ABA signaling network. ABA induces the expression of genes encoding repressors or activators of PP2C gene transcription. These regulators mediate the conversion of PP2C chromatins from a repressive to an active state for gene transcription. The stress-induced chromatin remodeling states of ABA-responsive genes could be memorized and transmitted to plant progeny; i.e., transgenerational epigenetic inheritance. This review focuses on the mechanism by which PP2C gene transcription modulates ABA signaling.

The UBC27–AIRP3 ubiquitination complex modulates ABA signaling by promoting the degradation of ABI1 in Arabidopsis

Abscisic acid (ABA) is the key phytohormone in plant drought tolerance and stress adaptation. The clade A protein phosphatase 2Cs (PP2Cs) like ABI1 (ABA-INSENSITIVE 1) work as coreceptors of ABA and regulate multiple ABA responses. Ubiquitination of ABI1 has been proven to play important regulatory roles in ABA signaling. However, the specific ubiquitin conjugating enzyme (E2) involved is unknown. Here, we report that UBC27 is an active E2 that positively regulates ABA signaling and drought tolerance. UBC27 forms the E2-E3 pair with the drought regulator RING E3 ligase AIRP3. Both UBC27 and AIRP3 interact with ABI1 and affect the ubiquitination and degradation of ABI1. ABA activates the expression of UBC27, inhibits the proteasome degradation of UBC27, and enhances the interaction between UBC27 and ABI1 to increase its activity. These findings uncover a regulatory mechanism in ABA signaling and drought response and provide a further understanding of the plant ubiquitination system and ABA signaling pathway.

Molecular Mechanism Underlying the Synergetic Effect of Jasmonate on Abscisic Acid Signaling during Seed Germination in Arabidopsis

Abscisic acid (ABA) is known to suppress seed germination and post-germinative growth of Arabidopsis (Arabidopsis thaliana), and jasmonate (JA) enhances ABA function. However, the molecular mechanism underlying the crosstalk between the ABA and JA signaling pathways remains largely elusive. Here, we show that exogenous coronatine, a JA analog structurally similar to the active conjugate jasmonate-isoleucine, significantly enhances the delayed seed germination response to ABA. Disruption of the JA receptor CORONATINE INSENSITIVE1 or accumulation of the JA signaling repressor JASMONATE ZIM-DOMAIN (JAZ) reduced ABA signaling, while jaz mutants enhanced ABA responses. Mechanistic investigations revealed that several JAZ repressors of JA signaling physically interact with ABSCISIC ACID INSENSITIVE3 (ABI3), a critical transcription factor that positively modulates ABA signaling, and that JAZ proteins repress the transcription of ABI3 and ABI5. Further genetic analyses showed that JA activates ABA signaling and requires functional ABI3 and ABI5. Overexpression of ABI3 and ABI5 simultaneously suppressed the ABA-insensitive phenotypes of the coi1-2 mutant and JAZ-accumulating (JAZ-ΔJas) plants. Together, our results reveal a previously uncharacterized signaling module in which JAZ repressors of the JA pathway regulate the ABA-responsive ABI3 and ABI5 transcription factors to integrate JA and ABA signals during seed germination and post-germinative growth.

ESCRT-I Component VPS23A Is Targeted by E3 Ubiquitin Ligase XBAT35 for Proteasome-Mediated Degradation in Modulating ABA Signaling

A myriad of abiotic stress responses in plants are controlled by abscisic acid (ABA) signaling. ABA receptors can be degraded by both the 26S proteasome pathway and vacuolar degradation pathway after processing via the endosomal sorting complex required for transport (ESCRT) proteins. Despite being essential for ABA signaling, the upstream regulators of ESCRTs remain unknown. Here, we report that the ESCRT-I component VPS23A is an unstable protein that is degraded via the ubiquitin-proteasome system (UPS). The UEV domain of VPS23A physically interacts with the two PSAP motifs of XBAT35, an E3 ubiquitin ligase, and this interaction results in the deposition of K48 polyubiquitin chains on VPS23A, marking it for degradation by 26S proteasomes. We showed that XBAT35 in plants is a positive regulator of ABA responses that acts via the VPS23A/PYL4 complex, specifically by accelerating VPS23A turnover and thereby increasing accumulation of the ABA receptor PYL4. This work deciphers how an ESCRT component is regulated in plants and deepens our understanding of plant stress responses by illustrating a mechanism whereby crosstalk between the UPS and endosome-vacuole-mediated degradation pathways controls ABA signaling.

CDK8 is associated with RAP2.6 and SnRK2.6 and positively modulates abscisic acid signaling and drought response in Arabidopsis.

CDK8 is a key subunit of Mediator complex, a large multiprotein complex that is a fundamental part of the conserved eukaryotic transcriptional machinery. However, the biological functions of CDK8 in plant abiotic stress responses remain largely unexplored. Here, we demonstrated CDK8 as a critical regulator in the abscisic acid (ABA) signaling and drought response pathways in Arabidopsis. Compared to wild-type, cdk8 mutants showed reduced sensitivity to ABA, impaired stomatal apertures and hypersensitivity to drought stress. Transcriptomic and chromatin immunoprecipitation analysis revealed that CDK8 positively regulates the transcription of several ABA-responsive genes, probably through promoting the recruitment of RNA polymerase II to their promoters. We discovered that both CDK8 and SnRK2.6 interact physically with an ERF/AP2 transcription factor RAP2.6, which can directly bind to the promoters of RD29A and COLD-REGULATED 15A (COR15A) with GCC or DRE elements, thereby promoting their expression. Importantly, we also showed that CDK8 is essential for the ABA-induced expression of RAP2.6 and RAP2.6-mediated upregulation of ABA-responsive genes, indicating that CDK8 could link the SnRK2.6-mediated ABA signaling to RNA polymerase II to promote immediate transcriptional response to ABA and drought signals. Overall, our data provide new insights into the roles of CDK8 in modulating ABA signaling and drought responses.

Structural determinants for pyrabactin recognition in ABA receptors in Oryza sativa.

We determined the structure of OsPYL/RCAR3:OsPP2C50 complex with pyrabactin. Our results suggest that aless-conserved phenylalanine of OsPYL/RCAR subfamily I is one of considerations of ABA agonist development for Oryza sativa. Pyrabactin is a synthetic chemical mimicking abscisic acid (ABA), a naturally occurring phytohormone orchestrating abiotic stress responses. ABA and pyrabactin share the same pocket in the ABA receptors but pyrabactin modulates ABA signaling differently, exhibiting both agonistic and antagonistic effects. To explore structural determinants of differential functionality of pyrabactin, we determined the crystal structure of OsPYL/RCAR3:pyrabactin:OsPP2C50, the first rice ABA receptor:co-receptor complex structure with a synthetic ABA mimicry. The water-mediated interaction between the wedging Trp-259 of OsPP2C50 and pyrabactin is lost, undermining the structural integrity of the ABA receptor:co-receptor. The loss of the interaction of the wedging tryptophan of OsPP2C with pyrabactin appears to contribute to the weaker functionality of pyrabactin. Pyrabactin in the OsPYL/RCAR3:OsPP2C50 complex adopts a conformation different from that in ABA receptors from Arabidopsis. Phe125, specific to the subfamily I of OsPYL/RCARs in the ABA binding pocket, appears to be the culprit for the differential conformation of pyrabactin. Although the gate closure essential for the integrity of ABA receptor:co-receptor is preserved in the presence of pyrabactin, Phe125 apparently restricts accessibility of pyrabactin, leading to decreased affinity for OsPYL/RCAR3 evidenced by phosphatase assay. However, Phe125 does not affect conformation and accessibility of ABA. Yeast two-hybrid, germination and gene transcription analyses in rice also support that pyrabactin imposes a weak effect on the control of ABA signaling. Taken together, our results suggest that phenylalanine substitution of OsPYL/RCARs subfamily I may be one of considerations for ABA synthetic agonist development.