Project description:Plant basic helix-loop-helix (bHLH) transcription factors are involved in physiological and developmental processes, and also play essential roles in abiotic stresses. However, their exact roles in abiotic stress are still need to be elucidated, and most of bHLHs have not been functionally characterized. In the present study, we characterized the functional role of AtbHLH112 in response to abiotic stresses. AtbHLH112 is a nuclear-localized protein, and its nuclear-localization is induced by salt, drought and ABA. Besides binding to E-box motif, AtbHLH112 is found to bind to a novel motif with the sequence M-bM-^@M-^\GG[GT]CC[GT][GA][TA]CM-bM-^@M-^] (GCG-box), and the binding affinity is induced by salt and ABA. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by upregulating the expression of P5CS genes and decreasing the expression of P5CDH and PRODH genes to increase proline levels, and via enhancing the expression of POD and SOD genes to improve ROS scavenging ability. All data together suggested that AtbHLH112 regulates the expression of genes through binding to GCG-box and E-box to mediate the physiological stress responses, including proline biosynthesis and ROS scavenging pathways to enhance stress tolerance. Differentially expression genes of AtbHLH112-overexpression plants, mutant (SALK_033618C) plants and wild type of Columbia Arabidopsis thaliana were measured under salt stressed and normal condition for 3 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
Project description:Plant basic helix-loop-helix (bHLH) proteins play essential roles in physiological and developmental processes and are also involved in abiotic stresses. However, their exact roles in abiotic stress are still not fully understood, and most of them have not been functionally characterised. In the present study, we characterised the functional role of AtbHLH112 in response to abiotic stress. A WRKY gene, AtWRKY66, can regulate the expression of the AtbHLH112 via binding to W-box motifs present in its promoter. AtbHLH112 is a nuclear-localised protein, and its nuclear localisation is increased upon exposure to NaCl, mannitol and ABA. In addition to binding to the G-box motif, AtbHLH112 is found to bind to a novel motif M-bM-^@M-^\GGGCCGGTCM-bM-^@M-^] (named the GCG-box) to regulate gene expression. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with tolerance to salt and drought. AtbHLH112 can confer stress tolerance via enhanced expression of POD and SOD genes to improve ROS scavenging ability and via upregulated expression of P5CS genes and decreased expression of P5CDH and PRODH genes to improve proline levels. Our data suggested that AtbHLH112 regulates the expression of genes via binding to the G-box and the GCG-box to improve stress-related pathways, such as ROS scavenging and proline biosynthesis. Differentially expression genes of AtbHLH112-overexpression plants and mutant (SALK_033618C) plants of Arabidopsis thaliana were measured under salt stressed and normal condition for 3 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
Project description:Plant basic helix-loop-helix (bHLH) transcription factors are involved in physiological and developmental processes, and also play essential roles in abiotic stresses. However, their exact roles in abiotic stress are still need to be elucidated, and most of bHLHs have not been functionally characterized. In the present study, we characterized the functional role of AtbHLH112 in response to abiotic stresses. AtbHLH112 is a nuclear-localized protein, and its nuclear-localization is induced by salt, drought and ABA. Besides binding to E-box motif, AtbHLH112 is found to bind to a novel motif with the sequence “GG[GT]CC[GT][GA][TA]C” (GCG-box), and the binding affinity is induced by salt and ABA. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by upregulating the expression of P5CS genes and decreasing the expression of P5CDH and PRODH genes to increase proline levels, and via enhancing the expression of POD and SOD genes to improve ROS scavenging ability. All data together suggested that AtbHLH112 regulates the expression of genes through binding to GCG-box and E-box to mediate the physiological stress responses, including proline biosynthesis and ROS scavenging pathways to enhance stress tolerance.
Project description:Plant basic helix-loop-helix (bHLH) proteins play essential roles in physiological and developmental processes and are also involved in abiotic stresses. However, their exact roles in abiotic stress are still not fully understood, and most of them have not been functionally characterised. In the present study, we characterised the functional role of AtbHLH112 in response to abiotic stress. A WRKY gene, AtWRKY66, can regulate the expression of the AtbHLH112 via binding to W-box motifs present in its promoter. AtbHLH112 is a nuclear-localised protein, and its nuclear localisation is increased upon exposure to NaCl, mannitol and ABA. In addition to binding to the G-box motif, AtbHLH112 is found to bind to a novel motif “GGGCCGGTC” (named the GCG-box) to regulate gene expression. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with tolerance to salt and drought. AtbHLH112 can confer stress tolerance via enhanced expression of POD and SOD genes to improve ROS scavenging ability and via upregulated expression of P5CS genes and decreased expression of P5CDH and PRODH genes to improve proline levels. Our data suggested that AtbHLH112 regulates the expression of genes via binding to the G-box and the GCG-box to improve stress-related pathways, such as ROS scavenging and proline biosynthesis.
Project description:Arabidopsis AtbHLH112 protein binds to the G-box and a novel motif GCG-box to regulate gene expression in response to abiotic stress
Project description:Campeiostachys nutans, a dominant perennial grass in the Qinghai-Tibet Plateau, exhibits high tolerance to salt stress. The Salt Overly Sensitive (SOS) pathway is key to plant salt stress tolerance. However, the pivotal role of the SOS pathway in response to salt stress in C. nutans remains unknown. Here, we identified CnbHLH130 as a novel transcriptional activator of CnCBL10, directly binds to the G-box motif in the promoter. CnbHLH130 responds to salt stress and positively regulate salt tolerance in rice and C. nutans. Interestingly, we found CnCBL10 and CnCIPK4 interact with CnbHLH130 by a Y2H screening assay. The interactions were confirmed by split-luciferase complementation (split-LUC), Pull-down, Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. Moreover, CnbHLH130 enhanced the interaction between CnCBL10 and CnCIPK4, which further phosphorylate and activate Na+/H+ antiporter CnSOS1 to exclude excess cytosolic Na+ from cells in the shoots. Genetic evidence showed that CnCBL10, CnCIPK4 and CnbHLH130 coordinately regulates salt tolerance in plants. In summary, this study demonstrated that CnbHLH130 acts as a novel core component and transcriptional activator regulating CnCBL10-CnCIPK4 mediated SOS pathway, thus conferring to the salt tolerance in C. nutans. This work advanced our understandings of how an alpine plant greatly survived in the Qinghai-Tibet Plateau by concise regulation of the SOS pathway in response to salt stress.
Project description:Perturbation of the cellular redox state by stress conditions is sensed by redox-sensitive proteins so that the cell can physiologically respond to stressors; however, the mechanisms linking sensing to response remain poorly understood in plants. Here we report that the transcription factor bZIP68 underwent in vivo oxidation in Arabidopsis cells under oxidative stress which is dependent on its redox-sensitive Cys320 residue. bZIP68 is primarily localized to the nucleus under normal conditions in Arabidopsis seedlings and oxidative stress reduces its accumulation in the nucleus and increases its cytosolic localization. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that bZIP68 primarily binds to promoter regions containing the core G-box (CACGTG) or G-box-like motif of the genes involved in abiotic and biotic stress responses, photosynthesis, biosynthetic processes, and transcriptional regulation. The bzip68 mutant displayed slower growth but enhanced tolerance to oxidative stress. The results from the ChIP-seq and phenotypic and transcriptome comparison between the bzip68 mutant and wildtype indicate that bZIP68 normally suppresses expression of stress tolerance genes and promotes expression of growth-related genes, whereas its inactivation enhances stress tolerance but suppresses growth. bZIP68 might balance stress tolerance with growth through the extent of its oxidative inactivation according to the environment.
Project description:The ubiquitination pathway regulates growth, development, and stress responses in plants, and the U-box protein family of ubiquitin ligases has important roles in this pathway. Here, 64 putative U-box proteins were identified in the Medicago truncatula genome. In addition to the conserved U-box motif, other functional domains, such as the ARM, kinase, KAP, and WD40 domains, were also detected. Phylogenetic analysis of the M. truncatula U-box proteins grouped them into six subfamilies, and chromosomal mapping and synteny analyses indicated that tandem and segmental duplications may have contributed to the expansion and evolution of the U-box gene family in this species. Using RNA-seq data from M. truncatula seedlings subjected to three different abiotic stresses, we identified 33 stress-inducible plant U-box genes (MtPUBs). Specifically, 25 salinity-, 15 drought-, and 16 cold-regulated MtPUBs were detected. Among them, MtPUB10, MtPUB17, MtPUB18, MtPUB35, MtPUB42, and MtPUB44 responded to all three stress conditions. Expression profiling by real-time PCR was consistent with the RNA-seq data, and stress-related elements were identified in the promoter regions. The present findings strongly indicate that U-box proteins play critical roles in abiotic stress response in M. truncatula.
Project description:Plant homeodomain (PHD) finger proteins affect growth and development by regulating transcription and reading epigenetic modifications of histones, but their functions in abiotic stress responses remain largely unclear. Here we characterize seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in the response to abiotic stresses. ALs localize to the nucleus and repress transcription. Except AL6, all the ALs bind to G-box element. Changes of the amino acids at positions 34 and 35 in AL6 cause the loss of G-box binding ability. Expression of the ALs responded differently to osmotic stress, salt, cold and abscisic acid treatments. AL5 was induced by multiple stresses, and AL5-overexpressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Also, al5 mutants showed reduced stress tolerance. ChIP-Seq assay helps find the direct targets of AL5. Polyclonal antibody of AL5 protein was used to perform ChIP experiment. Two samples were analyzed, AL5 OE sample and its knock out mutant. Data was analyzed as OE sample Vs mutant sample and help find targets of AL5 protein.
Project description:High temperature and drought are the primary yield-limiting environmental constraints for staple food crops. Heat shock transcription factors (HSF) terminally regulate the plant abiotic stress responses to maintain growth and development under extreme environmental conditions. HSF genes of Subclass A2 predominantly express under heat stress (HS) and activate the transcriptional cascade of defense-related genes. In this study, a highly heat-inducible HSF, HvHSFA2e was constitutively expressed in barley (Hordeum vulgareL.) to investigate its role in abiotic stress response and plant development. Transgenic barley plants displayed enhanced heat and drought tolerance in terms of increased chlorophyll content, improved membrane stability, reduced lipid peroxidation, and less accumulation of ROS in comparison to wild-type (WT) plants. Transcriptome analysis revealed that HvHSFA2e positively regulates the expression of abiotic stress-related genes encoding HSFs, HSPs, and enzymatic antioxidants, contributing to improved stress tolerance in transgenic plants. The major genes of ABA biosynthesis pathway, flavonoid, and terpene metabolism were also upregulated in transgenics. Our findings show that HvHSFA2e mediated upregulation of heat-responsive genes, modulation in ABA and flavonoid biosynthesis pathways enhance drought and heat stress tolerance.