An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis.
ABSTRACT: Plant hormone abscisic acid (ABA) plays a crucial role in modulating plant responses to environmental stresses. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families that regulate multiple aspects of plant growth and development, as well as of plant metabolism in Arabidopsis. Several bHLH transcription factors have been shown to be involved in the regulation of ABA signaling. We report here the characterization of bHLH129, a bHLH transcription factor in Arabidopsis. We found that the expression level of bHLH129 was reduced in response to exogenously applied ABA, and elevated in the ABA biosynthesis mutant aba1-5. Florescence observation of transgenic plants expressing bHLH129-GFP showed that bHLH129 was localized in the nucleus, and transient expression of bHLH129 in protoplasts inhibited reporter gene expression. When expressed in Arabidopsis under the control of the 35S promoter, bHLH129 promoted root elongation, and the transgenic plants were less sensitivity to ABA in root elongation assays. Quantitative RT-PCR results showed that ABA response of several genes involved in ABA signaling, including ABI1, SnRK2.2, SnRK2.3 and SnRK2.6 were altered in the transgenic plants overexpressing bHLH129. Taken together, our study suggests that bHLH129 is a transcription repressor that negatively regulates ABA response in Arabidopsis.
Project description:Drought is a major threat to plant growth and crop productivity. The phytohormone abscisic acid (ABA) plays a critical role in plant response to drought stress. Although ABA signaling-mediated drought tolerance has been widely investigated in Arabidopsis thaliana, the feedback mechanism and components negatively regulating this pathway are less well understood. Here we identified a member of Arabidopsis HD-ZIP transcription factors HAT1 which can interacts with and be phosphorylated by SnRK2s. hat1hat3, loss-of-function mutant of HAT1 and its homolog HAT3, was hypersensitive to ABA in primary root inhibition, ABA-responsive genes expression, and displayed enhanced drought tolerance, whereas HAT1 overexpressing lines were hyposensitive to ABA and less tolerant to drought stress, suggesting that HAT1 functions as a negative regulator in ABA signaling-mediated drought response. Furthermore, expression levels of ABA biosynthesis genes ABA3 and NCED3 were repressed by HAT1 directly binding to their promoters, resulting in the ABA level was increased in hat1hat3 and reduced in HAT1OX lines. Further evidence showed that both protein stability and binding activity of HAT1 was repressed by SnRK2.3 phosphorylation. Overexpressing SnRK2.3 in HAT1OX transgenic plant made a reduced HAT1 protein level and suppressed the HAT1OX phenotypes in ABA and drought response. Our results thus establish a new negative regulation mechanism of HAT1 which helps plants fine-tune their drought responses.
Project description:The phytohormone abscisic acid (ABA) is an essential part of the plant response to abiotic stressors such as drought. Upon the perception of ABA, pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins interact with co-receptor protein phosphatase type 2Cs to permit activation Snf1-related protein kinase2 (SnRK2) kinases, which switch on ABA signaling by phosphorylating various target proteins. Thus, SnRK2 kinases are central regulators of ABA signaling. However, the mechanisms that regulate SnRK2 degradation remain elusive. Here, we show that SnRK2.3 is degradated by 26S proteasome system and ABA promotes its degradation. We found that SnRK2.3 interacts with AtPP2-B11 directly. AtPP2-B11 is an F-box protein that is part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex that negatively regulates plant responses to ABA by specifically promoting the degradation of SnRK2.3. AtPP2-B11 was induced by ABA, and the knockdown of AtPP2-B11 expression markedly increased the ABA sensitivity of plants during seed germination and postgerminative development. Overexpression of AtPP2-B11 does not affect ABA sensitivity, but inhibits the ABA hypersensitive phenotypes of SnRK2.3 overexpression lines. These results reveal a novel mechanism through which AtPP2-B11 specifically degrades SnRK2.3 to attenuate ABA signaling and the abiotic stress response in Arabidopsis.
Project description:BACKGROUND:To maintain sweetpotato (Ipomoea batatas (L.) Lam) growth and yield, sucrose must be transported from the leaves to the roots. Sucrose transporters or carriers (SUTs or SUCs) transport sucrose and are involved in plant growth and response to abiotic stress. However, the mechanisms of SUTs in sweetpotato abiotic stress resistance remains to be determined. RESULTS:In the present study, we cloned a novel IbSUT4 gene; the protein encoded by this gene is localized in the tonoplast and plasma membrane. The plant growth was promoted in the IbSUT4 transgenic Arabidopsis thaliana lines, with increased expression of AtFT, a regulator of flowering time in plants. Over-expression of IbSUT4 in Arabidopsis thaliana resulted in higher sucrose content in the roots and lower sucrose content in the leaves, as compared to the wild-type (WT) plants, leading to improved stress tolerance during seedling growth. Moreover, we systematically analyzed the mechanisms of IbSUT4 in response to abiotic stress. The results suggest that the ABRE-motif was localized in the IbSUT4 promoter region, and the expression of the ABA signaling pathway genes (i.e., ABF2, ABF4, SnRK2.2, SnRK2.3, and PYL8/RCAR3) were induced, and the expression of ABI1 was inhibited. CONCLUSIONS:Our dates provide evidence that IbSUT4 is not only involved in plant growth but also is an important positive regulator in plant stress tolerance through the ABF-dependent ABA signaling pathway.
Project description:Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H2O2 level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.
Project description:Containing both AP2 domain and B3 domain, RAV (Related to ABI3/VP1) transcription factors are involved in diverse functions in higher plants. A total of eight TsRAV genes were isolated from the genome of Thellungiella salsuginea and could be divided into two groups (A- and B-group) based on their sequence similarity. The mRNA abundance of all Thellungiella salsuginea TsRAVs followed a gradual decline during seed germination. In Thellungiella salsuginea seedling, transcripts of TsRAVs in the group A (A-TsRAVs) were gradually and moderately reduced by salt treatment but rapidly and severely repressed by ABA treatment. In comparison, with a barely detectable constitutive expression, the transcriptional level of TsRAVs in the group B (B-TsRAVs) exhibited a moderate induction in cotyledons when confronted with ABA. We then produced the "gain-of-function" transgenic Arabidopsis plants for each TsRAV gene and found that only 35S:A-TsRAVs showed weak growth retardation including reduced root elongation, suggesting their roles in negatively controlling plant growth. Under normal conditions, the germination process of all TsRAVs overexpressing transgenic seeds was inhibited with a stronger effect observed in 35S:A-TsRAVs seeds than in 35S:B-TsRAVs seeds. With the presence of NaCl, seed germination and seedling root elongation of all plants including wild type and 35S:TsRAVs plants were retarded and a more severe inhibition occurred to the 35S:A-TsRAV transgenic plants. ABA treatment only negatively affected the germination rates of 35S:A-TsRAV transgenic seeds but not those of 35S:B-TsRAV transgenic seeds. All 35S:TsRAVs transgenic plants showed a similar degree of reduction in root growth compared with untreated seedlings in the presence of ABA. Furthermore, the cotyledon greening/expansion was more severely inhibited 35S:A-TsRAVs than in 35S:B-TsRAVs seedlings. Upon water deficiency, with a wider opening of stomata, 35S:A-TsRAVs plants experienced a faster transpirational water loss than wild type and 35S:B-TsRAVs lines. Taken together, our results suggest that two groups of TsRAVs perform distinct regulating roles during plant growth and abiotic defense including drought and salt, and A-TsRAVs are more likely than B-TsRAVs to act as negative regulators in the above-mentioned biological processes.
Project description:Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABAregulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.
Project description:The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3.
Project description:Trehalose plays important roles in plant growth and stress responses and is synthesized from trehalose-6-phosphate by trehalose-6-phosphate phosphatase (TPP). Here, we show that trehalose and abscisic acid (ABA) have synergistic effects on root growth and stomatal closure. The Arabidopsis thaliana genome contains ten genes encoding TPPs and the expression level of one, TPPE, and trehalose contents increased in response to ABA. In the presence of ABA, the ABA-responsive transcription factor ABA RESPONSE ELEMENT BINDING FACTOR2 (ABF2) directly binds to the TPPE promoter to activate its expression. Genetic analysis revealed that TPPE acts downstream of ABF2, which is supported by the findings that TPPE expression and trehalose content are reduced in the abf2 mutant and that a mutation in TPPE abolished the ABA-sensitive root elongation phenotype of 35S:ABF2 plants. Reactive oxygen species (ROS) accumulation in response to ABA failed to occur in tppe mutant plants, suggesting that TPPE is involved in ABA-controlled root elongation and stomatal movement by inducing ROS accumulation. This study uncovers a new branch of the ABA signaling pathway and provides a molecular basis for the role of trehalose in plant responses to abiotic stress.
Project description:Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 double mutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.
Project description:<label>BACKGROUND</label>Many transcription factors (TFs), such as those in the basic helix-loop-helix (bHLH) family, are important for regulating plant growth and plant responses to abiotic stress. The expression of OsbHLH035 is induced by drought and salinity. However, its functional role in rice growth, development, and the salt response is still unknown.<label>RESULTS</label>The bHLH TF OsbHLH035 is a salt-induced gene that is primarily expressed in germinating seeds and seedlings. Stable expression of GFP-fused OsbHLH035 in rice transgenic plants revealed that this protein is predominantly localized to the nucleus. Osbhlh035 mutants show delayed seed germination, particularly under salt-stress conditions. In parallel, abscisic acid (ABA) contents are over-accumulated, and the expression of the ABA biosynthetic genes OsABA2 and OsAAO3 is upregulated; furthermore, compared with that in wild-type (WT) seedlings, the salt-induced expression of OsABA8ox1, an ABA catabolic gene, in germinating Osbhlh035 mutant seeds is downregulated. Moreover, Osbhlh035 mutant seedlings are unable to recover from salt-stress treatment. Consistently, sodium is over-accumulated in aerial tissues but slightly reduced in terrestrial tissues from Osbhlh035 seedlings after salt treatment. Additionally, the expression of the sodium transporters OsHKT1;3 and 1;5 is reduced in Osbhlh035 aerial and terrestrial tissues, respectively. Furthermore, genetic complementation can restore both the delayed seed germination and the impaired recovery of salt-treated Osbhlh035 seedlings to normal growth.<label>CONCLUSION</label>OsbHLH035 mediates seed germination and seedling recovery after salt stress relief through the ABA-dependent and ABA-independent activation of OsHKT pathways, respectively.