Abscisic Acid Receptor Research Articles

Tuning to the Signal of Stress: Subcellular Regulation of Abscisic Acid Receptor Abundance by E3 Ubiquitin Ligases.

Developmental and environmental changes are communicated systemically throughout the plant by various signals, ranging from electric currents to plant hormones. As the responses elicited by the systemic signals depend on the tissue and subcellular context, the signal needs to be adjusted at every

Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes.

Few proteins have been characterized as abscisic acid transporters. Several of them are NRT1/PRT Family (NPF) transporters which have been characterized in yeast using reporter systems. Because several members of the NPF4 subfamily members were identified in yeast as ABA transporters, here, we screened for ABA transport activity the seven members of the NPF4 subfamily in Xenopus oocytes using cRNA injection and 3H-ABA accumulation. The ABA transport capacities of NPF4.2, NPF4.5, NPF4.6, and NPF4.7 were confirmed. The transport properties of NPF4.5 and NPF4.6 were studied in more detail. Both ABA transporter activities are pH-dependent and slightly pH-dependent apparent Km around 500 μM. There is no competitive inhibition of the ABA-analogs pyrabactin and quinabactin on ABA accumulation demonstrating a different selectivity compared to the ABA receptors. Functional expression of these ABA transporters in Xenopus oocyte is an opportunity to start structure–function studies and also to identify partner proteins of these hormone transporters.

Functional characteristics of <italic>TaPYR1</italic>, an abscisic acid receptor family gene in mediating wheat tolerance to drought stress

ABA receptors are involved in the mediation of ABA signaling transduction through interaction with abscisic acid (ABA) molecules induced by osmotic stresses and play critical roles in regulating the drought stress tolerance in plants. In this study, TaPYR1 , an ABA family gene in wheat that was shown to be differentially expressed in our previous transcriptome analysis was used to analyze its molecular property, expression patterns under drought stress condition, and functions in mediating plant adaptation to drought stress. TaPYR1 shares high similarities to its plant counterparts at amino acid level. TaPYR1 protein contains the conserved domains specified by the plant PYR proteins and was targeted onto the plasma membrane after endoplasmic reticulum (ER) assortment. The expression of TaPYR1 was induced in both roots and leaves under drought, with the highest expression levels at 48 h of drought treatment. Transgene analysis on TaPYR1 was performed to assess the gene function in mediating plant drought tolerance. Compared with wild type (WT), the tobacco lines overexpressing TaPYR1 enhanced growth vigor and increased fresh and dry weight under drought stress. In addition, the transgenic lines with TaPYR1 overexpression also increased photosynthetic function, enhanced activities of cellular antioxidant enzymes, and elevated the contents of osmolytes (i.e., proline and soluble sugar) under drought condition. Our investigation suggests that TaPYR1 transcriptively responds to drought stress signaling and plays an important role in regulating plant drought adaptation by improving the associated physiological processes.

Overexpression of the persimmon abscisic acid β-glucosidase gene (DkBG1) alters fruit ripening in transgenic tomato.

β-Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) β-glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA-BG in fruits is still unclear. In this study, through RNA-seq analysis of persimmon fruit, 10 full-length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA-glucose ester (ABA-GE) to release free ABA. Compared with wild-type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1-OE) accelerated fruit ripening onset by 3-4days by increasing ABA levels at the pre-breaker stage and induced early ethylene release compared with wild-type fruits. DkBG1-OE altered the expression of ripening regulator NON-RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA-GE.

PHYTOCHROME-INTERACTING FACTORS Interact with the ABA Receptors PYL8 and PYL9 to Orchestrate ABA Signaling in Darkness

PHYTOCHROME-INTERACTING FACTORS (PIFs) are a group of basic helix-loop-helix transcription factors that can physically interact with photoreceptors, including phytochromes and cryptochromes. It was previously demonstrated that PIFs accumulated in darkness and repressed seedling photomorphogenesis, and that PIFs linked different photosensory and hormonal pathways to control plant growth and development. In this study, we show that PIFs positively regulate the ABA signaling pathway during the seedling stage specifically in darkness. We found that PIFs positively regulate ABI5 transcript and protein levels in darkness in response to exogenous ABA treatment by binding directly to the G-box motifs in the ABI5 promoter. Consistently, PIFs and the G-box motifs in the ABI5 promoter determine ABI5 expression in darkness, and overexpression of ABI5 could rescue the ABA-insensitive phenotypes of pifq mutants in the dark. Moreover, we discovered that PIFs can physically interact with the ABA receptors PYL8 and PYL9, and that this interaction is not regulated by ABA. Further analyses showed that PYL8 and PYL9 promote PIF4 protein accumulation in the dark and enhance PIF4 binding to the ABI5 promoter, but negatively regulate PIF4-mediated ABI5 activation. Taken together, our data demonstrate that PIFs interact with ABA receptors to orchestrate ABA signaling in darkness by controlling ABI5 expression, providing new insights into the pivotal roles of PIFs as signal integrators in regulating plant growth and development.

Grapevine ABA receptor VvPYL1 regulates root hair development in Transgenic Arabidopsis

Root architecture is very important for plant growth. In this study, we characterized the process of root formation in grapevine (Vitis vinifera L.). Continuous observation of root morphology during development revealed that the establishment of root system could be divided into five stages: initial cultivation (stage I), preliminary development (stage II), even change (stage III), root system formation (stage IV), and root architecture stability (stage V). The level of abscisic acid (ABA) increased from stages II to IV and was stable at stage V. Quantitative expression analysis of 11 genes encoding ABA-related rate-limiting enzymes in different tissues showed that the expression of VvPYL1 was the highest in roots. Spatiotemporal expression analysis showed that VvPYL1 was highly expressed during stages II and III. Furthermore, VvPYL1 was highly expressed in lateral roots of grapevine seedlings in tissue culture. Overexpression of VvPYL1 in Arabidopsis thaliana resulted in longer root hairs compared with wild-type plants. Moreover, the root hair length of transgenic lines was hypersensitive to exogenously applied ABA. Additionally, VvPYL1 overexpressing plants showed greater drought tolerance and longer root hairs than wild-type plants under osmotic stress. These results suggest that VvPYL1 may play a key role in root development and drought resistance.

Characterization of Arabidopsis thaliana Root-Related Mutants Reveals ABA Regulation of Plant Development and Drought Resistance

The plant hormone abscisic acid (ABA) regulates many processes of plant growth and development. ABA receptors have been identified in studies of the ABA response of stomatal movement, but the underlying mechanisms of ABA-regulated root growth and development are unclear. To address these questions, we screened for Arabidopsis thaliana mutants based on the sensitivity of root growth to exogenous ABA, using ethyl methanesulfonate-mutagenized (EMS) and T-DNA insertion mutant libraries. We identified 11 mutants, termed roa1–roa11, with Root growth Overly sensitive to ABA (ROA) phenotypes, and cloned two of the ROA genes, one by thermal asymmetric interlaced PCR technology (ROA3) and the other by map-based cloning (ROA9). The roa mutants were also found to have defects in other major ABA responses, including ABA-mediated seed germination and drought resistance. The roa mutants provide crucial genetic material for further studies of ABA signaling and regulatory mechanisms in root growth and development.

Role of an Amur grape CBL-interacting protein kinase VaCIPK02 in drought tolerance by modulating ABA signaling and ROS production

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) play critical roles in mediating plant responses to various biotic and abiotic stresses. However, the underlying molecular mechanisms by which these complex functions and the potential cross talk between the diverse downstream signaling networks are regulated still remain elusive. Here we characterized a VaCIPK02 from Vitis amurensis, and show it functions in salt and drought stresses. We have identified that, at least, the domain at C terminus of VaCIPK02 involved in transcriptional activation. VaCIPK02 could strongly interact with four Ca2+ sensor proteins, VaCBL1, 4, 5 and 8, respectively. A Y2H-based screen to isolate genes encoding VaCIPK02-interacting proteins identified 12 additional partners, including an ABA receptor PYL9, which interacts with VaCIPK02 in vivo. Constitutive expression of VaCIPK02 in Arabidopsis showed extremely sensitive to salt stress, but displayed increased tolerance to drought stress accompanied by ABA-hypersensitive stomata closure, improved water-conserving capacity, and increased cellular ABA levels. Moreover, overexpression of VaCIPK02 caused reduced cellular levels of ABA-induced reactive oxygen species (ROS) by regulating the expression levels of many ABA-responsive genes to scavenge and modulate ROS levels. Our results provide evidence for the involvement of VaCIPK02 along with its diverse interacting proteins in regulating salt and drought stress signal pathway.

Exogenous Abscisic Acid Regulates Distribution of 13C and 15N and Anthocyanin Synthesis in 'Red Fuji' Apple Fruit Under High Nitrogen Supply.

In order to improve the problem of poor coloring caused by high fruit nitrogen in apple production, we studied the effects of different concentrations of abscisic acid (ABA: 0, 50, 100, and 150 mg/L) and fluridone (ABA biosynthesis inhibitor) on the fruit of ‘Red Fuji’ apple (Malus Domestica Borkh.) in the late stage of apple development (135 days after blooming) in 2017 and 2018. The effects of these treatments on the distribution of 13C and 15N and anthocyanin synthesis in fruit were studied. The results showed that the expression levels of ABA synthesis and receptor genes in the peel and flesh were upregulated by exogenous ABA treatment. An appropriate concentration of ABA significantly increased the expression of anthocyanin synthesis genes and transcription factors and increased the content of anthocyanin in the peel. The results of 13C and 15N double isotope labeling showed that exogenous ABA coordinated the carbon–nitrogen nutrient of apple fruit in the late stage of the development, reduced the accumulation of fruit nitrogen, increased the accumulation of fruit carbon and sugar, provided a substrate for anthocyanin synthesis, or promoted anthocyanin synthesis through the sugar signal regulation mechanism. Comprehensive analysis showed that the application of 100 mg/L ABA effectively improved the problem of poor coloring caused by high fruit nitrogen in the late stage of apple development and is beneficial to the accumulation of carbon in fruit and the formation of color.

Degradation of Abscisic Acid Receptors Through the Endosomal Pathway.

Turnover of membrane proteins or soluble proteins associated to plasma membrane involves clathrin-mediated endocytosis (CME), endosomal trafficking, and vacuolar degradation. Thus, endocytic and endosomal trafficking regulate numerous physiological processes, including mineral transport, hormone signaling, and pathogen response. Abscisic acid (ABA) signaling is triggered upon ABA perception by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), which are soluble proteins that can associate to membrane by interaction with members of the C2-domain ABA-related (CAR) protein family and the RING finger of seed longevity (RSL1) E3 ubiquitin ligase. Half-life of PYR/PYL/RCAR ABA receptors is regulated by ubiquitination and degradation in different subcellular compartments. In particular, pharmacological, genetic, and cell biology approaches have been used to study the different steps that encompass from CME to receptor degradation in the vacuole. In this chapter, we will focus on (1) coimmunoprecipitation (co-IP) assays of clathrin heavy chain (CHC) subunits together with HA-tagged PYL4 ABA receptor and (2) analysis of PYL4 delivery to the vacuole using the TMD23-Ub marker.

Functional enhancement of abscisic acid receptor confers water-saving and drought tolerance in wheat

Functional enhancement of abscisic acid receptor confers water-saving and drought tolerance in wheat

Design of potent ABA receptor antagonists based on a conformational restriction approach.

The physiological functions of the plant hormone abscisic acid (ABA) are triggered by interactions between PYR/PYL/RCAR receptors (PYLs) and group-A protein phosphatases 2C (PP2Cs). PYL agonists/antagonists capable of inducing/disrupting these interactions would be valuable in investigating the regulatory mechanisms of ABA signaling. Previously, we developed (+)-PAO4, a high-affinity PYL antagonist, by conformationally restricting the S-hexyl chain of our first reported PYL antagonist, 3'-hexylsulfanyl-ABA. Although (+)-PAO4 shows a greater binding affinity for Arabidopsis PYL5 compared with 3'-hexylsulfanyl-ABA, it is not able to completely block the ABA responses both in vitro and in vivo. Therefore, we designed novel conformationally restricted PYL antagonists in which the O-butyl chain of (+)-PAO4 was replaced with a pentyl (PAC4), a pentyne (PAT3) or a pentadiyne (PATT1) chain. (+)-PAT3 and (+)-PATT1 suppressed the ABA-induced inhibition of Arabidopsis seed germination more strongly than (+)-PAO4, but contrary to expectations, the affinity of each compound for PYL5 was almost the same as that of (+)-PAO4. Subsequent biochemical analyses revealed that unlike (+)-PAO4, (+)-PAT3 and (+)-PATT1 completely abolished ABA-induced PYL-PP2C interactions without partial agonistic activities. The superior PYL antagonist functions of (+)-PAT3 and (+)-PATT1 over (+)-PAO4 may explain their potent antagonistic activities against exogenous ABA in vivo. Furthermore, (+)-PAT3 and (+)-PATT1 also suppressed ABA responses in rice, indicating that both compounds are useful chemical tools for ABA-signaling studies, not only in dicots but also in monocots.

Enhancement of endogenous SA accumulation improves poor-nutrition stress tolerance in transgenic tobacco plants overexpressing a SA-binding protein gene

Salicylic acid (SA) plays an important role in the response of plants to abiotic stresses. Starvation stress affects plant cell metabolic activities, which further limits the normal growth and development of plants. It was reported that SA might play a regulatory role in the process of plant against starvation stress, but the mechanism involved in this process is still unclear. Thus, in this study, the transgenic plants overexpressing a SA binding protein 2 (SABP2) gene were exposed to starvation stress and the transgenic lines showed starvation-tolerant phenotype. Compared with wild-type (WT) plants, transgenic plants showed better growth status under poor-nutrition stress. Transgenic plants also showed more vigorous roots than WT plants. Physiological tests indicated that the transgenic plants showed higher relative water content (RWC), chlorophyll content, photosynthetic capacity, endogenous SA content, and lower ROS level compared to WT plants. Transcriptome analysis of tobacco plants identified 3, 748 differentially expressed genes (DEGs) between transgenic and WT plants under starvation stress. These DEGs are mainly involved in glycolysis/gluconeogenesis pathway group, MAPK signaling pathway group and plant hormone signal transduction pathway group. As determined by qPCR, up-regulated expression of fifteen genes such as abscisic acid receptor PYR1-like gene (NtPYR1-like), bidirectional sugar transporter N3-like gene (NtSWEETN3-like) and superoxide dismutase [Fe] chloroplastic-like gene (NtFeSOD-like), etc., was observed in transgenic plants under poor-nutrition stress which was in accordance with RNA-sequencing results. The modified pathways involved in plant hormone signaling are thought to be at least one of the main causes of the increased starvation tolerance of transgenic tobacco plants with altered SA homeostasis.

Structure-Guided Design of ABA Receptor Agonists for the Control of Plant Water Use

Key words ABA receptor agonist - virtual screening - plant biology - structure-guided design

Root Growth Adaptation is Mediated by PYLs ABA Receptor-PP2A Protein Phosphatase Complex.

Plant root architecture dynamically adapts to various environmental conditions, such as salt‐containing soil. The phytohormone abscisic acid (ABA) is involved among others also in these developmental adaptations, but the underlying molecular mechanism remains elusive. Here, a novel branch of the ABA signaling pathway in Arabidopsis involving PYR/PYL/RCAR (abbreviated as PYLs) receptor‐protein phosphatase 2A (PP2A) complex that acts in parallel to the canonical PYLs‐protein phosphatase 2C (PP2C) mechanism is identified. The PYLs‐PP2A signaling modulates root gravitropism and lateral root formation through regulating phytohormone auxin transport. In optimal conditions, PYLs ABA receptor interacts with the catalytic subunits of PP2A, increasing their phosphatase activity and thus counteracting PINOID (PID) kinase‐mediated phosphorylation of PIN‐FORMED (PIN) auxin transporters. By contrast, in salt and osmotic stress conditions, ABA binds to PYLs, inhibiting the PP2A activity, which leads to increased PIN phosphorylation and consequently modulated directional auxin transport leading to adapted root architecture. This work reveals an adaptive mechanism that may flexibly adjust plant root growth to withstand saline and osmotic stresses. It occurs via the cross‐talk between the stress hormone ABA and the versatile developmental regulator auxin.

The Shaker Type Potassium Channel, GORK, Regulates Abscisic Acid Signaling in Arabidopsis.

Evolution of adaptive mechanisms to abiotic stress is essential for plant growth and development. Plants adapt to stress conditions by activating the abscisic acid (ABA) signaling pathway. It has been suggested that the ABA receptor, clade A protein phosphatase, SnRK2 type kinase, and SLAC1 anion channel are important components of the ABA signaling pathway. In this study, we report that the shaker type potassium (K+) channel, GORK, modulates plant responses to ABA and abiotic stresses. Our results indicate that the full length of PP2CA is needed to interact with the GORK C-terminal region. We identified a loss of function allele in gork that displayed ABA-hyposensitive phenotype. gork and pp2ca mutants showed opposite responses to ABA in seed germination and seedling growth. Additionally, gork mutant was tolerant to the NaCl and mannitol treatments, whereas pp2ca mutant was sensitive to the NaCl and mannitol treatments. Thus, our results indicate that GORK enhances the sensitivity to ABA and negatively regulates the mechanisms involved in high salinity and osmotic stresses via PP2CA-mediated signals.

Ectopic expression of Medicago truncatula homeodomain finger protein, MtPHD6, enhances drought tolerance in Arabidopsis

BackgroundThe plant homeodomain (PHD) finger is a Cys4HisCys3-type zinc finger which promotes protein-protein interactions and binds to the cis-acting elements in the promoter regions of target genes. In Medicago truncatula, five PHD homologues with full-length sequence were identified. However, the detailed function of PHD genes was not fully addressed.ResultsIn this study, we characterized the function of MtPHD6 during plant responses to drought stress. MtPHD6 was highly induced by drought stress. Ectopic expression of MtPHD6 in Arabidopsis enhanced tolerance to osmotic and drought stresses. MtPHD6 transgenic plants exhibited decreased water loss rate, MDA and ROS contents, and increased leaf water content and antioxidant enzyme activities under drought condition. Global transcriptomic analysis revealed that MtPHD6 reprogramed transcriptional networks in transgenic plants. Expression levels of ABA receptor PYR/PYLs, ZINC FINGER, AP2/EREBP and WRKY transcription factors were mainly up-regulated after transformation of MtPHD6. Interaction network analysis showed that ZINC FINGER, AP2/EREBP and WRKY interacted with each other and downstream stress induced proteins.ConclusionsWe proposed that ZINC FINGER, AP2/EREBP and WRKY transcription factors were activated through ABA dependent and independent pathways to increase drought tolerance of MtPHD6 transgenic plants.

Overexpression of ABA Receptor PYL10 Gene Confers Drought and Cold Tolerance to Indica Rice.

Abscisic acid (ABA) plays versatile functions in regulating plant development and tolerance to various biotic and abiotic stresses. Towards elucidating the functions of one of the ABA receptors (ABARs) in rice, OsPYL10 was cloned from drought tolerant rice cv. Nagina 22 and was overexpressed under stress inducible RD29A promoter in a mega rice variety MTU1010 by using Agrobacterium mediated genetic transformation. Four single copy transgenic lines selected based on Southern blot analysis were used for physiological and molecular analysis. PYL10 receptor appears to regulate its ligand ABA accumulation as PYL10 overexpressing transgenics accumulated 2–3.3-fold higher levels of ABA than that of WT in flag leaf at anthesis under non-stress conditions. The enhanced accumulation of ABA was associated with enhanced expression of genes for ABA biosynthesis viz., ZEP1, NCED1, NCED2, NCED3, and NCED4 in transgenics than in WT plants. At seedling stage, PYL10 transgenics showed significantly higher survival rate under cold stress as compared with WT plants. qRT-PCR analysis showed that expression levels of cold responsive genes viz., DREB1F, MYB3R2, TPP1, COR410, DEHYDRIN, and LEA3 were significantly higher in PYL10 overexpressing transgenic lines as compared to WT plants under cold stress. PYL10 transgenic and WT plants grown in the same pot were subjected to -80 kPa drought stress and recovery treatments at vegetative and reproductive stages. At vegetative stage drought stress, three overexpressing lines showed significantly higher grain yield (40–58%) and at reproductive stage drought stress one of these overexpression lines showed two-fold higher grain yield than that of WT plants. Excised leaf water loss analysis showed that PYL10 transgenic lost about 20% less water than WT plants. At reproductive stage, OsPYL10 transgenic maintained higher RWC, membrane stability index, chlorophyll content, and accumulated lower amount of MDA and H2O2 as compared with WT plants. qRT-PCR analysis showed that expression levels of RAB16, Dehydrin, LEA3, and ABA45 were higher in PYL10 transgenics as compared with WT plants under drought stress. Thus, overall results showed that OsPYL10 overexpression has potential to improve both drought and cold stress tolerance of indica rice.

Genome-wide phylogenetic and structural analysis reveals the molecular evolution of the ABA receptor gene family.

The plant hormone abscisic acid (ABA) plays a crucial role during the plant life cycle as well as in adaptive responses to environmental stresses. The core regulatory components of ABA signaling in plants are the pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor family (PYLs), which comprise the largest plant hormone receptor family known. They act as negative regulators of members of the protein phosphatase type 2C family. Due to the biological importance of PYLs, many researchers have focused on their genetic redundancy and consequent functional divergence. However, little is understood of their evolution and its impact on the generation of regulatory diversity. In this study, we identify positive selection and functional divergence in PYLs through phylogenetic reconstruction, gene structure and expression pattern analysis, positive selection analysis, functional divergence analysis, and structure comparison. We found the correlation of desensitization of PYLs under specific modifications in the molecular recognition domain with functional diversification. Hence, an interesting antagonistic co-evolutionary mechanism is proposed for the functional diversification of ABA receptor family proteins. We believe a compensatory evolutionary pathway may have occurred.

Toward Development of Fluorescence-Quenching-Based Biosensors for Drought Stress in Plants.

Abscisic acid (ABA) is a drought stress signaling molecule, and simple methods for detecting its levels could benefit agriculture. Here, we present proof-of-concept detection for ABA in aqueous solutions by the use of a mixture of Cyanine 5.5 (Cy5.5) fluorophore- and BHQ3 quencher-conjugated endogenous ABA receptor pyrabactin resistance 1 like proteins (PYL3). These dye-conjugated PYL3 protein form dimers in solutions without ABA and monomerize upon ABA binding. When they are in dimers, fluorescence of Cy5.5 is either nearly completely quenched by the BHQ3 or 20% quenched by another Cy5.5. Consequently, mixtures of equal amounts of the two protein conjugates were used to detect ABA in aqueous solution. As the ABA concentration increased from <1 μM to 1 mM, the intensity of fluorescence detected at around 680 nm from the mixture was more than doubled as a result of ABA-induced monomerization, which leads to halt of quenching and recovery of fluorescence of Cy5.5 in monomers. Kinetic modeling was used to simulate the fluorescence response from the mixture and the results generally agree with the experimentally observed trend. This work demonstrates that fluorescence measurements of a single dissociation reaction in one spectral region are adequate to assess the ABA concentration of a solution.