Abscisic Acid In Arabidopsis Research Articles

TaABI5, a wheat homolog of Arabidopsis thaliana ABA insensitive 5, controls seed germination

Abscisic acid (ABA) response element (ABRE)-binding factors (ABFs) are basic region/leucine zipper motif (bZIP) transcription factors that regulate the expression of ABA-induced genes containing ABRE in their promoters. The amino acid sequence of the wheat bZIP protein, TaABI5, showed high homology to that of Arabidopsis ABA insensitive 5 (ABI5). TaABI5 was classified into the clade of ABI5s in Arabidopsis and rice, unlike TRAB1 of rice, Wabi5 of wheat, and HvABI5 of barley in the bZIP Group A family, by a phylogenetic analysis. TaABI5 was strongly expressed in seeds during the late ripening and maturing stages; however, its expression level markedly decreased after germination. An in situ hybridization analysis showed that TaABI5 mRNA accumulated in seed embryos, particularly the scutellum. In a transient assay using wheat aleurone cells, TaABI5 activated the promoter of Em containing ABRE, which is an embryogenesis abundant protein gene, indicating that TaABI5 acts as a transcription factor in wheat seeds. Furthermore, the seeds of transgenic Arabidopsis lines introduced with 35S:TaABI5 exhibited high sensitivity to ABA and the inhibition of germination. The seed dormancy of the transgenic Arabidopsis lines was stronger than that of Col. These results support TaABI5 playing an important role in mature seeds, particularly before seed germination, and acting as a functional ortholog to Arabidopsis ABI5.

Suppression of abscisic acid biosynthesis at the early infection stage of Verticillium longisporum in oilseed rape (Brassica napus).

Summary Verticillium longisporum infects oilseed rape (Brassica napus) and Arabidopsis thaliana. To investigate the early response of oilseed rape to the fungal infection, we determined transcriptomic changes in oilseed rape roots at 6 days post‐inoculation (dpi) by RNA‐Seq analysis, in which non‐infected roots served as a control. Strikingly, a subset of genes involved in abscisic acid (ABA) biosynthesis was found to be down‐regulated and the ABA level was accordingly attenuated in 6 dpi oilseed rape as compared with the control. Gene expression analysis revealed that this was mainly attributed to the suppression of BnNCED3‐mediated ABA biosynthesis, involving, for example, BnWRKY57. However, this down‐regulation of ABA biosynthesis could not be observed in infected Arabidopsis roots. Arabidopsis ABA‐ defective mutants nced3 and aao3 displayed pronounced tolerance to the fungal infection with delayed and impeded symptom development, even though fungal colonization was not affected in both mutants. These data suggest that ABA appears to be required for full susceptibility of Arabidopsis to the fungal infection. Furthermore, we found that in both 6 dpi oilseed rape and the Arabidopsis nced3 mutant, the salicylic acid (SA) signalling pathway was induced while the jasmonic acid (JA)/ethylene (ET) signalling pathway was concomitantly mitigated. Following these data, we conclude that in oilseed rape the V. longisporum infection triggers a host‐specific suppression of the NCED3‐mediated ABA biosynthesis, consequently increasing plant tolerance to the fungal infection. We believe that this might be part of the virulence strategy of V. longisporum to initiate/establish a long‐lasting compatible interaction with oilseed rape (coexistence), which appears to be different from the infection process in Arabidopsis.

Abscisic Acid Derivatives with Different Alkyl Chain Lengths Activate Distinct Abscisic Acid Receptor Subfamilies.

The plant hormone abscisic acid (ABA) regulates the development of various plant organs including seeds, roots, and fruits, and significantly contributes to abiotic stress responses, especially to drought. Since recent climate changes are adversely affecting crop cultivation, enhancement of plant stress tolerance by regulation of ABA signaling would be an important strategy. In the plant genome, ABA receptors are encoded by multiple genes constituting three subfamilies; however, functional differences among them remain unclear. To enhance desired effects of ABA, the biological functions of the receptor family warrant clarification. This study aimed to determine the functional differences among ABA receptors in plants. We screened small-molecule ligands binding to specific receptors, using a chemical array. In vitro evaluation of hit compounds using 11 Arabidopsis ABA receptors revealed that (+)-3'-alkyl ABAs served as agonists for different receptors depending on the length of their 3'-alkyl chains. Combinatorial in vitro and physiological effects of these compounds on the stomata, seeds, and seedlings indicated that, along with subfamily III, receptors of subfamily II are important to induce strong drought responses. Among (+)-3'-alkyl ABAs assessed herein, (+)-3'-butyl ABA induced a transcriptional response and stomatal closure but only slightly inhibited seed germination and growth, suggesting that it enhances drought tolerance. In silico docking simulation and site-directed mutagenesis revealed the amino acid residues contributing to the selective agonist activity of the (+)-3'-alkyl ABAs. These results provide novel insights into the structure and biological effects of 3'-derivatives of ABA and a basis for agrochemical development.

The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration.

Stomata are microscopic pores found on the surfaces of leaves that act to control CO2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, 'set' the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO2, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.

Abscisic Acid Regulates Auxin Distribution to Mediate Maize Lateral Root Development Under Salt Stress.

Roots are important plant organs. Lateral root (LR) initiation (LRI) and development play a central role in environmental adaptation. The mechanism of LR development has been well investigated in Arabidopsis. When we evaluated the distribution of auxin and abscisic acid (ABA) in maize, we found that the mechanism differed from that in Arabidopsis. The distribution of ABA and auxin within the primary roots (PRs) and LRs was independent of each other. Auxin localization was observed below the quiescent center of the root tips, while ABA localized at the top of the quiescent center. Furthermore, NaCl inhibited LRI by increasing ABA accumulation, which mainly regulates auxin distribution, while auxin biosynthesis was inhibited by ABA in Arabidopsis. The polar localization of ZmPIN1 in maize was disrupted by NaCl and exogenous ABA. An inhibitor of ABA biosynthesis, fluridone (FLU), and the ABA biosynthesis mutant vp14 rescued the phenotype under NaCl treatment. Together, all the evidence suggested that NaCl promoted ABA accumulation in LRs and that ABA altered the polar localization of ZmPIN1, disrupted the distribution of auxin and inhibited LRI and development.

The non-DNA binding bHLH transcription factor Paclobutrazol Resistances are involved in the regulation of ABA and salt responses in Arabidopsis

Abscisic acid (ABA) is the key hormone that regulating plant responses to abiotic stresses. Several basic helix-loop-helix (bHLH) transcription factors have been reported to regulate ABA signaling in Arabidopsis. Paclobutrazol Resistances (PREs) are non-DNA binding bHLH transcription factors involved in the regulation of plant response to several different plant hormones including gibberellin, brassinosteroid and auxin. Here, we show that PREs are involved in the regulation of ABA and salt responses in Arabidopsis. Quantitative RT-PCR results showed that the expression levels of PRE6 as well as several other PRE genes were reduced in response to ABA treatment, but increased to salt treatment. Seed germination assays indicated that ABA sensitivity is reduced in the pre6 mutants, but increased in transgenic plants overexpressing PRE6. On the other hand, the 35S:PRE6 transgenic plants showed enhanced tolerance to salt, whereas little, if any changes were observed in the pre6 mutants. Similar responses to ABA and salt treatments were observed in the pre2 mutants and the transgenic plants overexpressing PRE2, and a slight increased resistance to ABA in seed germination was observed in the pre2 pre6 double mutants. Taken together, our results suggest that at least some of the PRE genes are ABA responsive genes, and PREs may function redundantly to regulate ABA and salt responses in Arabidopsis.

Global metabolite profiling based on GC\u2013MS and LC\u2013MS/MS analyses in ABF3-overexpressing soybean with enhanced drought tolerance

Abscisic acid (ABA) is a phytohormone that plays an important role in the adaptive responses to abiotic stresses. We examined the metabolic changes in transgenic soybean that over-expressed Arabidopsis ABA responsive element-binding factor 3 (ABF3), which participates in drought tolerance. Transgenic and non-transgenic plants were exposed to a water deficit, and their metabolic differences were verified by untargeted GC‒MS and LC‒MS/MS analyses. A total of 64 and 476 primary and secondary metabolites from leaf extracts were identified based on GC‒MS and LC‒MS/MS platforms, respectively. Principal component analysis derived from both GC‒MS and LC‒MS/MS data showed a clearly greater separation in the metabolite profiles among three different degrees of drought stress. However, no discrimination of metabolites between transgenic and non-transgenic plants was apparent. Furthermore, except for some free amino acids, quantitative differences in relative levels of those metabolites were less than 50% between genotypes. These results suggest that, during periods of drought, overexpression of ABF3 in transgenic soybean might result in a negligible variance in primary and secondary metabolism when compared with its non-transgenic counterpart.

Expression Pattern and Function Analysis of AtPPRT1, a Novel Negative Regulator in ABA and Drought Stress Responses in Arabidopsis.

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.

The sHSP22 Heat Shock Protein Requires the ABI1 Protein Phosphatase to Modulate Polar Auxin Transport and Downstream Responses.

The phytohormones abscisic acid (ABA) and indole-3-acetic acid (IAA) response pathways interact synergistically or antagonistically to regulate plant development and environmental adaptation. Here, we show that ABI1, a key negative regulator of ABA signaling, is essential for auxin-modulated root development. We performed a microarray analysis using the loss-of-function mutant abi1-3 and Col-0 seedlings treated with IAA. For sHSP22, an endoplasmic reticulum (ER) small heat shock protein-encoding gene, the induction by IAA was dependent on ABI1shsp22 displayed enhanced sensitivity to ABA in primary root growth. In contrast, overexpression of full-length, but not truncated sHSP22 lacking signal peptide and ER-retention sequences, resulted in decreased ABA sensitivity. Overexpressed (OX) sHSP22 partially rescued the ABA hypersensitivity of abi1-3 In addition, sHSP22 is involved in auxin-regulated hypocotyl elongation at high temperature treatment. sHSP22 also affected accumulation of auxin efflux carrier PIN proteins due to potentiated intracellular trafficking. And sHSP22 OX lines initiated more lateral roots after auxin application. Our results suggest that sHSP22 regulates auxin response through modulating auxin polar transport, and ABI1-sHSP22 provides a novel module orchestrating ABA and auxin signaling crosstalk in Arabidopsis (Arabidopsis thaliana).

Interaction network of ABA receptors in grey poplar.

The plant hormone abscisic acid (ABA) is a key player in responses to abiotic stress. ABA regulates a plant's water status and mediates drought tolerance by controlling stomatal gas exchange, water conductance and differential gene expression. ABA is recognized and bound by the Regulatory Component of ABA Receptors (RCARs)/PYR1/PYL (Pyrabactin Resistance 1/PYR1-like). Ligand binding stabilizes the interaction of RCARs with type 2C protein phosphatases (PP2C), which are ABA co-receptors. While the core pathway of ABA signalling has been elucidated, the large number of different ABA receptors and co-receptors within a plant species generates a complexity of heteromeric receptor complexes that has not functionally been resolved in any plant species to date. In this study, we characterized ABA receptors and co-receptors of grey poplar (Populus x canescens [Ait.] Sm.) and their capacity to regulate ABA responses. We observed a high number of regulatory combinations of holo-receptor complexes, but also some preferential and selective RCAR-PP2C interactions. Poplar and Arabidopsis ABA receptor components revealed a strong structural and functional conservation. Heterologous receptor complexes of poplar and Arabidopsis components showed functionality invitro and regulated ABA-responsive gene expression in cells of both species. ABA-responsive promoters of Arabidopsis were also active in poplar, which was explored to generate poplar reporter lines expressing green fluorescent protein in response to ABA. The study presents a detailed analysis of receptor complexes of a tree species and shows high conservation of ABA receptor components between an annual and a perennial plant.

Abscisic acid negatively regulates post-penetration resistance of Arabidopsis to the biotrophic powdery mildew fungus.

Pytohormone abscisic acid (ABA) plays important roles in defense responses. Nonetheless, how ABA regulates plant resistance to biotrophic fungi remains largely unknown. Arabidopsis ABA-deficient mutants, aba2-1 and aba3-1, displayed enhanced resistance to the biotrophic powdery mildew fungus Golovinomyces cichoracearum. Moreover, exogenously administered ABA increased the susceptibility of Arabidopsis to G. cichoracearum. Arabidopsis ABA perception components mutants, abi1-1 and abi2-1, also displayed similar phenotypes to ABA-deficient mutants in resistance to G. cichoracearum. However, the resistance to G. cichoracearum is not changed in downstream ABA signaling transduction mutants, abi3-1, abi4-1, and abi5-1. Microscopic examination revealed that hyphal growth and conidiophore production of G. cichoracearum were compromised in the ABA deficient mutants, even though pre-penetration and penetration growth of the fungus were not affected. In addition, salicylic acid (SA) and MPK3 are found to be involved in ABA-regulated resistance to G. cichoracearum. Our work demonstrates that ABA negatively regulates post-penetration resistance of Arabidopsis to powdery mildew fungus G. cichoracearum, probably through antagonizing the function of SA.

Abscisic Acid Induces Resistance against Bamboo Mosaic Virus through Argonaute2 and 3.

Plant resistance to pathogens is tuned by defense-related hormones. Of these, abscisic acid (ABA) is well documented to moderate resistance against fungi and bacteria. However, ABA's contribution to resistance against viruses is pleiotropic. ABA affects callose deposition at plasmodesmata (therefore hindering the viral cell-to-cell movement), but here, we show that when callose synthase is down-regulated, ABA still induces resistance against infection with Bamboo mosaic virus (BaMV). By examining the potential connections between the ABA and RNA-silencing pathways in Arabidopsis (Arabidopsis thaliana), we showed that ABA regulates the expression of almost the whole ARGONAUTE (AGO) gene family, of which some are required for plant resistance against BaMV Our data show that BaMV infection and ABA treatment regulate the same set of AGOs, with positive effects on AGO1, AGO2, and AGO3, no effect on AGO7, and negative effects on AGO4 and AGO10 The BaMV-mediated regulation of AGO1, AGO2, and AGO3 is ABA dependent, because the accumulation of these AGOs in BaMV-infected ABA mutants did not reach the levels observed in infected wild-type plants. In addition, the AGO1-miR168a complex is dispensable for BaMV resistance, while AGO2 and AGO3 were important for ABA-mediated resistance. While most ago mutants showed increased susceptibility to BaMV infection (except ago10), ago1-27 showed reduced BaMV titers, which was attributed to the up-regulated levels of AGO2, AGO3, and AGO4 We have established that ABA regulates the expression of several members of the AGO family, and this regulation partially contributes to ABA-mediated resistance against BaMV These findings reveal another role for ABA in plants.

Analysis of plant hormone profiles in response to moderate dehydration stress.

Plant responses to dehydration stress are mediated by highly complex molecular systems involving hormone signaling and metabolism, particularly the major stress hormone abscisic acid (ABA) and ABA-dependent gene expression. To understand the roles of plant hormones and their interactions during dehydration, we analyzed the plant hormone profiles with respect to dehydration responses in Arabidopsis thaliana wild-type (WT) plants and ABA biosynthesis mutants (nced3-2). We developed a procedure for moderate dehydration stress, and then investigated temporal changes in the profiles of ABA, jasmonic acid isoleucine (JA-Ile), salicylic acid (SA), cytokinin (trans-zeatin, tZ), auxin (indole-acetic acid, IAA), and gibberellin (GA4 ), along with temporal changes in the expression of key genes involved in hormone biosynthesis. ABA levels increased in a bi-phasic pattern (at the early and late phases) in response to moderate dehydration stress. JA-Ile levels increased slightly in WT plants and strongly increased in nced3-2 mutant plants at 72h after the onset of dehydration. The expression profiles of dehydration-inducible genes displayed temporal responses in an ABA-dependent manner. The early phase of ABA accumulation correlated with the expression of touch-inducible genes and was independent of factors involved in the major ABA regulatory pathway, including the ABA-responsive element-binding (AREB/ABF) transcription factor. JA-Ile, SA, and tZ were negatively regulated during the late dehydration response phase. Transcriptome analysis revealed important roles for hormone-related genes in metabolism and signaling during dehydration-induced plant responses.

AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade.

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade essentially consists of three components: a MAPK kinase kinase (MAPKKK), a MAPK kinase, and a MAPK, connected to each other by the event of phosphorylation. Here, we report the characterization of a MAPKKK, ABA-INSENSITIVE PROTEIN KINASE1 (AIK1), which regulates abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana). T-DNA insertion mutants of AIK1 showed insensitivity to ABA in terms of both root growth and stomatal response. AIK1 functions in ABA responses via regulation of root cell division and elongation, as well as stomatal responses. The activity of AIK1 is induced by ABA in Arabidopsis and tobacco (Nicotiana benthamiana), and the Arabidopsis protein phosphatase type 2C, ABI1, a negative regulator of ABA signaling, restricts AIK1 activity by dephosphorylation. Bimolecular fluorescence complementation analysis showed that MPK3, MPK6, and AIK1 interact with MKK5. The single mutant seedlings of mpk6 and mkk5 have similar phenotypes to aik1, but mkk4 does not. AIK1 was localized in the cytoplasm and shown to activate MKK5 by protein phosphorylation, which was an ABA-activated process. Constitutively active MKK5 in aik1 mutant seedlings complements the ABA-insensitive root growth phenotype of aik1 The activity of MPK6 was increased by ABA in wild-type seedlings, but its activation by ABA was impaired in aik1 and aik1 mkk5 mutants. These findings clearly suggest that the AIK1-MKK5-MPK6 cascade functions in the ABA regulation of primary root growth and stomatal response.

Characterization of the calcineurin B-Like (CBL) gene family in maize and functional analysis of ZmCBL9 under abscisic acid and abiotic stress treatments

In plants, calcineurin B-like proteins (CBLs) play crucial roles in regulating calcium-signaling in response to various abiotic stresses by interacting with specific CBL-interacting protein kinases (CIPKs). However, the identities and functions of CBL gene family members in maize are largely unknown. Here, we identified from the maize genome 12 CBL genes. All 12 CBLs have conserved EF-hand domains, and half harbor myristoylation motifs. We further characterized the function of one CBL gene, ZmCBL9, which can be induced by salt, dehydration, glucose and abscisic acid (ABA) treatments. Overexpression of ZmCBL9 enhanced resistance or tolerance to ABA, glucose, salt and osmotic stress in Arabidopsis and complemented the hypersensitive phenotype of the Arabidopsis cbl9 mutant in response to ABA and abiotic stress. The ZmCBL9 gene negatively regulates the expression of genes in the ABA signaling, biosynthesis and catabolism pathways. Moreover, the ZmCBL9 protein is found to interact with eight maize CIPKs and these ZmCIPK genes were up-regulated by different stress treatments, including salt, dehydration, glucose, low potassium and ABA. These results suggest that ZmCBL9 may interact with various ZmCIPKs to regulate the abiotic stress and ABA response signaling in plants.

Contrasting transcriptional responses of PYR1/PYL/RCAR ABA receptors to ABA or dehydration stress between maize seedling leaves and roots.

BackgroundThe different actions of abscisic acid (ABA) in the aboveground and belowground parts of plants suggest the existence of a distinct perception mechanism between these organs. Although characterization of the soluble ABA receptors PYR1/PYL/RCAR as well as core signaling components has greatly advanced our understanding of ABA perception, signal transduction, and responses, the environment-dependent organ-specific sensitivity of plants to ABA is less well understood.ResultsBy performing real-time quantitative PCR assays, we comprehensively compared transcriptional differences of core ABA signaling components in response to ABA or osmotic/dehydration stress between maize (Zea mays L.) roots and leaves. Our results demonstrated up-regulation of the transcript levels of ZmPYLs homologous to dimeric-type Arabidopsis ABA receptors by ABA in maize primary roots, whereas those of ZmPYLs homologous to monomeric-type Arabidopsis ABA receptors were down-regulated. However, this trend was reversed in the leaves of plants treated with ABA via the root medium. Although the mRNA levels of ZmPYL1-3 increased significantly in roots subjected to polyethylene glycol (PEG)-induced osmotic stress, ZmPYL4-11 transcripts were either maintained at a stable level or increased only slightly. In detached leaves subjected to dehydration, the transcripts of ZmPYL1-3 together with ZmPYL5, ZmPYL6, ZmPYL10 and ZmPYL11 were decreased, whereas those of ZmPYL4, ZmPYL7 and ZmPYL8 were significantly increased. Our results also showed that all of the evaluated transcripts of PP2Cs and SnRK2 were quickly up-regulated in roots by ABA or osmotic stress; conversely they were either up-regulated or maintained at a constant level in leaves, depending on the isoforms within each family.ConclusionsThere is a distinct profile of PYR/PYL/RCAR ABA receptor gene expression between maize roots and leaves, suggesting that monomeric-type ABA receptors are mainly involved in the transmission of ABA signals in roots but that dimeric-type ABA receptors primarily carry out this function in leaves. Given that ZmPYL1 and ZmPYL4 exhibit similar transcript abundance under normal conditions, our findings may represent a novel mechanism for species-specific regulation of PYR/PYL/RCAR ABA receptor gene expression. A difference in the preference for core signaling components in the presence of exogenous ABA versus stress-induced endogenous ABA was observed in both leaves and roots. It appears that core ABA signaling components perform their osmotic/dehydration stress response functions in a stress intensity-, duration-, species-, organ-, and isoform-specific manner, leading to plasticity in response to adverse conditions and, thus, acclimation to life on land. These results deepen our understanding of the diverse biological effects of ABA between plant leaves and roots in response to abiotic stress at the stimulus-perception level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0764-x) contains supplementary material, which is available to authorized users.

AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana.

In plants, transcriptional regulation is the most important tool for modulating flavonoid biosynthesis. The AtMYB12 gene from Arabidopsis thaliana has been shown to regulate the expression of key enzyme genes involved in flavonoid biosynthesis, leading to the increased accumulation of flavonoids. In this study, the codon-optimized AtMYB12 gene was chemically synthesized. Subcellular localization analysis in onion epidermal cells indicated that AtMYB12 was localized to the nucleus. Its overexpression significantly increased accumulation of flavonoids and enhanced salt and drought tolerance in transgenic Arabidopsis plants. Real-time quantitative PCR (qRT-PCR) analysis showed that overexpression of AtMYB12 resulted in the up-regulation of genes involved in flavonoid biosynthesis, abscisic acid (ABA) biosynthesis, proline biosynthesis, stress responses and ROS scavenging under salt and drought stresses. Further analyses under salt and drought stresses showed significant increases of ABA, proline content, superoxide dismutase (SOD) and peroxidase (POD) activities, as well as significant reduction of H2O2 and malonaldehyde (MDA) content. The results demonstrate the explicit role of AtMYB12 in conferring salt and drought tolerance by increasing the levels of flavonoids and ABA in transgenic Arabidopsis. The AtMYB12 gene has the potential to be used to enhance tolerance to abiotic stresses in plants.

Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.

Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, group A protein phosphatases 2C (PP2Cs), and subclass III of SNF1-related protein kinase 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1-ABA-PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling.

Genetic interactions between DET1 and intermediate genes in Arabidopsis ABA signalling

Seed germination is regulated positively by light and negatively by the dormancy-promoting phytohormone abscisic acid (ABA). DE-ETIOLATED 1 (DET1) is a negative regulator of light signalling in Arabidopsis thaliana. In contrast, the bZIP transcription factor LONG HYPOCOTYL 5 (HY5) is a positive regulator of light signalling. HY5 also positively regulates ABA signalling by promoting the expression of ABA INSENSITIVE 5 (ABI5), a dormancy promoting transcription factor. Here we show that germination in det1 mutants is sensitive to ABA. Double mutant analysis indicates that det1 ABA sensitive germination requires HY5 and ABI5. DET1 forms a complex with DAMAGED DNA BINDING protein 1A/B (DDB1A/B). Another DDB1 complex containing DWA1 and 2 (DWD hypersensitive to ABA 1/2) has also been shown to negatively regulate ABA response. Double mutant analysis indicates that DWA1, DWA2, DDB1A, and DDB1B are also required for the det1 ABA sensitive germination phenotype. We also examined water loss in adult plants and found that the det1 rapid water loss phenotype is independent of HY5, ABI5, DWA1, DWA2, and DDB1B. These findings provide insight into interactions between ABA and light signalling in Arabidopsis.

Arabidopsis abscisic acid receptors play an important role in disease resistance

Stomata are natural pores of plants and constitute the entry points for water during transpiration. However, they also facilitate the ingress of potentially harmful bacterial pathogens. The phytohormone abscisic acid (ABA) plays a pivotal role in protecting plants against biotic stress, by regulating stomatal closure. In the present study, we investigated the mechanism whereby ABA influences plant defense responses to Pseudomonas syringae pv. tomato (Pst) DC3000, which is a virulent bacterial pathogen of Arabidopsis, at the pre-invasive stage. We found that overexpression of two ABA receptors, namely, RCAR4/PYL10-OX and RCAR5/PYL11-OX (hereafter referred to as RCARs), resulted in ABA-hypersensitive phenotypes being exhibited during the seed germination and seedling growth stages. Sensitivity to ABA enhanced the resistance of RCAR4-OX and RCAR5-OX plants to Pst DC3000, through promoting stomatal closure leading to the development of resistance to this bacterial pathogen. Protein phosphatase HAB1 is an important component that is responsible for ABA signaling and which interacts with ABA receptors. We found that hab1 mutants exhibited enhanced resistance to Pst DC3000; moreover, similar to RCAR4-OX and RCAR5-OX plants, this enhanced resistance was correlated with stomatal closure. Taken together, our findings demonstrate that alteration of RCAR4- or RCAR5-HAB1 mediated ABA signaling influences resistance to bacterial pathogens via stomatal regulation.