Putative Abscisic Acid Receptor Research Articles

Functional characterization of ZjPYL8 from sour jujube: enhancing the sensitivity of stomata and roots to ABA in Arabidopsis thaliana

Abscisic acid (ABA) is a plant hormone that plays a crucial role in regulating plant growth, development, and adaptation to stress. The growth of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Chou, commonly known as Suanzao in Chinese, is significantly influenced by environmental factors, particularly drought and salt stresses. In this study, we isolated and characterized a putative ABA receptor, ZjPYL8, from Sour jujube. To investigate the effects of ZjPYL8 overexpression on ABA-responsive pathways, we introduced it into Arabidopsis thaliana (A. thaliana) and examined the resulting phenotypes. Our results demonstrated that overexpression of ZjPYL8 in A. thaliana led to a significant reduction in stomatal aperture and root length under ABA treatment, while the wild type (WT) was relatively insensitive to ABA. Moreover, ZjPYL8 transgenic plants exhibited shorter roots under salt treatment than the WT did. These findings suggest that the overexpression of ZjPYL8 in A. thaliana enhances the plant's resistance to stress and support the hypothesis that ZjPYL8 serves as a putative ABA receptor in Sour jujube, which may improve the plant's adaptability to drought and salt stresses. ZjPYL8 appears to mediate plant responses to ABA, similar to most ABA receptors in A. thaliana, such as stomatal closure and root length.

The Physiological and Molecular Mechanism of Abscisic Acid in Regulation of Fleshy Fruit Ripening.

The ripening of fleshy fruits is coupled with the degradation of both chlorophyll and cell walls, as well as changes in the metabolism of phenylpropanoids, flavonoids, starch/sucrose, and carotenoids. These processes are controlled by phytohormones and other factors, including abscisic acid (ABA), ethylene, auxin, polyamines, sugar, and reactive oxygen species. The ripening of climacteric fruits is controlled by ethylene and non-climacteric fruit ripening is regulated mainly by ABA. Also, ABA and ethylene may interact in both types of fruit ripening. ABA concentrations in fleshy fruits are regulated in response to developmental and environmental cues and are controlled by the relative rates of ABA biosynthesis and catabolism, the former mainly via 9-cis-epoxycarotenoid dioxygenases (NCEDs) and β-glucosidases and the latter via ABA 8'-hydroxylases (CYP707As) and β-glycosyltransferases. In strawberry fruit ripening, ABA is perceived via at least two receptors, Pyrabactin resistance (PYR)/PYR-like (PYL) and putative abscisic acid receptor (ABAR), which are linked separately to the conserved signaling pathway ABA-FaPYR1-FaABIl-FaSnRK2 and the novel signaling pathway ABA-FaABAR-FaRIPK1-FaABI4. Downstream signaling components include important transcription factors, such as AREB (ABA responsive element binding protein)/ABF (ABRE binding factors ABA responsive factor), ethylene response factor (ERF), and V-myb Myeloblastosis viral oncogene homolog (MYB), as well as ripening-related genes. Finally, a comprehensive model of ABA linked to ethylene, sugar, polyamines, auxin and reactive oxygen species in the regulation of strawberry fruit ripening is proposed. Next, new integrated mechanisms, including two ABA signaling pathways, ABA and ethylene signaling pathways, and ABA/ethylene to other phytohormones are interesting and important research topics in ripening, especially in non-climacteric fruits.

Arabidopsis exoribonuclease USB1 interacts with the PPR-domain protein SOAR1 to negatively regulate abscisic acid signaling.

Signaling by the phytohormone abscisic acid (ABA) involves pre-mRNA splicing, a key process of post-transcriptional regulation of gene expression. However, the regulatory mechanism of alternative pre-mRNA splicing in ABA signaling remains largely unknown. We previously identified a pentatricopeptide repeat protein SOAR1 (suppressor of the ABAR-overexpressor 1) as a crucial player downstream of ABAR (putative ABA receptor) in ABA signaling. In this study, we identified a SOAR1 interaction partner USB1, which is an exoribonuclease catalyzing U6 production for spliceosome assembly. We reveal that together USB1 and SOAR1 negatively regulate ABA signaling in early seedling development. USB1 and SOAR1 are both required for the splicing of transcripts of numerous genes, including those involved in ABA signaling pathways, suggesting that USB1 and SOAR1 collaborate to regulate ABA signaling by affecting spliceosome assembly. These findings provide important new insights into the mechanistic control of alternative pre-mRNA splicing in the regulation of ABA-mediated plant responses to environmental cues.

Overexpression of a sour jujube gene ZjPYR1, encoding a putative abscisic acid receptor, increases sensitivity of the stomata and roots to ABA in Arabidopsis thaliana

The abscisic acid (ABA) receptor binds to ABA in plants and can activate the ABA signaling pathway to initiate stress resistance. Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Chou) is an economic tree crop in North China, which is strongly adapted to drought and salt stress. Here, we cloned and overexpressed the ZjPYR1 gene in Arabidopsis thaliana. After ABA treatment, the accumulation of ZjPYR1 increased significantly, suggesting that ABA may stabilize ZjPYR1 in the plants. Compared with the wild-type, the heterologous transgenic lines showed smaller stomatal openings under ABA treatment and a shorter root length and lower germination rate under ABA and salt treatment. Based on these results, we speculate that the overexpression of ZjPYR1 in A. thaliana effectively enhanced the stress resistance of the plants, and furthermore, that ZjPYR1 is a putative ABA receptor in sour jujube that increases plant adaptability to drought and salt stress. We report that ZjPYR1, like most ABA receptors in A. thaliana, is involved in mediating plant responses to ABA, such as stomatal closure and root length.

Developmental Variation in Fruit Polyphenol Content and Related Gene Expression of a Red-Fruited versus a White-Fruited Fragaria vesca Genotype

Two cultivars of F. vesca, red-fruited Baron Solemacher (BS) and white-fruited Pineapple Crush (PC), were studied to compare and contrast the quantitative accumulation of major polyphenols and related biosynthetic pathway gene expression patterns during fruit development and ripening. Developing PC fruit showed higher levels of hydroxycinnamic acids in green stages and a greater accumulation of ellagitannins in ripe fruit in comparison to BS. In addition to anthocyanin, red BS fruit had greater levels of flavan-3-ols when ripe than PC. Expression patterns of key structural genes and transcription factors of the phenylpropanoid/flavonoid biosynthetic pathway, an abscisic acid (ABA) biosynthetic gene, and a putative ABA receptor gene that may regulate the pathway, were also analyzed during fruit development and ripening to determine which genes exhibited differences in expression and when such differences were first evident. Expression of all pathway genes differed between the red BS and white PC at one or more times during development, most notably at ripening when phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), flavanone-3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP:flavonoid-O-glucosyltransferase 1 (UFGT1) were significantly upregulated in the red BS fruit. The transcription factors MYB1 and MYB10 did not differ substantially between red and white fruit except at ripening, when both the putative repressor MYB1 and promoter MYB10 were upregulated in red BS but not white PC fruit. The expression of ABA-related gene 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) was higher in red BS fruit but only in the early green stages of development. Thus, a multigenic effect at several points in the phenylpropanoid/flavonoid biosynthetic pathway due to lack of MYB10 upregulation may have resulted in white PC fruit.

Ectopic expression of mutated type 2C protein phosphatase OsABI-LIKE2 decreases abscisic acid sensitivity in Arabidopsis and rice

Abscisic acid (ABA) is a phytohormone that is necessary for stress adaptation. Recent studies have reported that attenuated levels of ABA improved grain yield and seedling growth under low temperature in cereals. To improve plant growth under low temperature, we attempted to generate ABA-insensitive transgenic rice by expressing a clade A type 2C protein phosphatase (OsPP2C), OsABIL2, with or without the mutation equivalent to the Arabidopsis abi1-1 mutation. A yeast two-hybrid assay revealed that the interaction between OsABIL2 and a putative rice ABA receptor, OsPYL1, was ABA-dependent, and the interaction was lost with amino acid substitution from glycine to aspartic acid at the 183rd amino acid of the OsABIL2 protein, corresponding to abi1-1 mutation. The constitutive expression of OsABIL2 or OsABIL2G183D in Arabidopsis or rice decreased ABA sensitivity to differing degrees. Moreover, the transgenic rice expressing OsABIL2G183D exhibited improved seedling growth under low temperature, although the transgenic lines showed unfavorable traits, such as viviparous germination and elongated internodes. These results indicated that the introduction of abi1-1 type dominant mutation was also effective in OsABIL2 at decreasing ABA sensitivity in plants, and the attenuation of ABA sensitivity could be an alternative parameter to improve rice performance under low temperatures.

A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with the ABA receptor, FaABAR, to regulate fruit ripening in strawberry.

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA); however, its exact molecular mechanisms are yet not fully understood. In this study, a predicted leu-rich repeat (LRR) receptor-like kinase in strawberry, red-initial protein kinase 1 (FaRIPK1), was screened and, using a yeast two-hybrid assay, was shown to interact with a putative ABA receptor, FaABAR. This association was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation assays, and shown to occur in the nucleus. Expression analysis by real-time PCR showed that FaRIPK1 is expressed in roots, stems, leaves, flowers, and fruit, with a particularly high expression in white fruit at the onset of coloration. Down-regulation of FaRIPK1 expression in strawberry fruit, using Tobacco rattle virus-induced gene silencing, inhibited ripening, as evidenced by suppression of ripening-related physiological changes and reduced expression of several genes involved in softening, sugar content, pigmentation, and ABA biosynthesis and signaling. The yeast-expressed LRR and STK (serine/threonine protein kinase) domains of FaRIPK1 bound ABA and showed kinase activity, respectively. A fruit disc-incubation test revealed that FaRIPK1 expression was induced by ABA and ethylene. The synergistic action of FaRIPK1 with FaABAR in regulation of strawberry fruit ripening is discussed.

Molecular characterization of an AtPYL1-like protein, BrPYL1, as a putative ABA receptor in Brassica rapa

Abscisic acid (ABA)-induced physiological changes are conserved in many land plants and underlie their responses to environmental stress and pathogens. The PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors perceive the ABA signal and initiate signal transduction. Here, we show that the genome of Brassica rapa encodes 24 putative AtPYL-like proteins. The AtPYL-like proteins in Brassica rapa (BrPYLs) can also be classified into 3 subclasses. We found that nearly all BrPYLs displayed high expression in at least one tissue. Overexpression of BrPYL1 conferred ABA hypersensitivity to Arabidopsis. Further, ABA activated the expression of an ABA-responsive reporter in Arabidopsis protoplasts expressing BrPYL1. Overall, these results suggest that BrPYL1 is a putative functional ABA receptor in Brassica rapa.

FaPYR1 is involved in strawberry fruit ripening

Although the plant hormone abscisic acid (ABA) has been suggested to play a role in the ripening of non-climatic fruit, direct genetic/molecular evidence is lacking. In the present study, a strawberry gene homologous to the Arabidopsis ABA receptor gene PYR1, named FaPYR1, was isolated and characterized. The 627 bp cDNA includes an intact open reading frame that encodes a deduced protein of 208 amino acids, in which putative conserved domains were detected by homology analysis. Using tobacco rattle virus-induced gene silencing (VIGS), the FaPYR1 gene was silenced in strawberry fruit. Down-regulation of the FaPYR1 gene not only significantly delayed fruit ripening, but also markedly altered ABA content, ABA sensitivity, and a set of ABA-responsive gene transcripts, including ABI1 and SnRK2. Furthermore, the loss of red colouring in FaPYR1 RNAi (RNA interference) fruits could not be rescued by exogenously applied ABA, which could promote the ripening of wild-type fruits. Collectively, these results demonstrate that the putative ABA receptor FaPYR1 acts as a positive regulator in strawberry fruit ripening. It was also revealed that the application of the VIGS technique in strawberry fruit could be used as a novel tool for studying strawberry fruit development.

Abscisic Acid Plays an Important Role in the Regulation of Strawberry Fruit Ripening

The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells.

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

Changes in Kinetic Characteristics of the ABA Binding Protein(s) of the Grapevine Fruit during Different Stages of Fruit Development

By using the micro-volume radio-ligand binding essay, the changes in the kinetic characteristics of the abscisic acid (ABA)-binding protein(s) of the Kyhoh grapevine (Vitis vinifera × V. labrusca) fruit during the different stages of fruit development have been studied. The changes in the berry volume growth, concentration of sugar, organic acids, and ABA in fruit mesocarp have been surveyed, especially for studies of ABA-binding protein. The dissociation constant (Kd) and ABA binding maximum (Bmax) were determined by the Scatchard plots for ABA binding in microsomes of the fruit. They are Kd = 17.5, 50.0, 6.3, 13.3 nmol·L–1; Bmax = 98.6, 523.0, 41.6, 85.4 μmol·mg–1 protein, respectively, for the fruit developmental phase I, II, veraison, and phase III. The Scatchard plots showed a rectilinear function for all of the developmental phases including veraison, which suggests the sole ABA-binding site of high affinity for ABA in the fruit microsomes, but this site could either be only one kind of the same protein or consist of more kinds of different proteins for different developmental stages. The binding affinity of ABA-binding protein(s) for ABA was shown to be higher at veraison time than during other developmental phases; this binding affinity increased nearly by 10 times from phase II to veraison, while the concentration (Bmax) of the ABA-binding protein(s) decreased to the minimum at veraison. The very low concentration of ABA at veraison may be able to trigger the onset of fruit ripening due to the increase of the binding affinity of ABA-binding protein(s) for ABA at this time. The possible functions of the ABA-binding protein(s) for fruit development during the different developmental stages were discussed, and it is suggested that the protein(s) detected could be the putative ABA receptor(s) or transporter(s) for the action of this plant hormone in grapevine.