Dehydration-responsive Element-binding Protein Research Articles

Genome-wide identification and evolutionary analysis of the AP2/EREBP, COX and LTP genes in Zea mays L. under drought stress

AP2 (APETALA2)/EREBP (ethylene-responsive element-binding protein), cytochrome c oxidase (COX) and nonspecific lipid transfer proteins (LTP) play important roles in the response to drought stress. This is the first study to identify the COX gene in Zea mays L. via genome-wide analysis. The qRT‒PCR results indicated that AP2/EREBP, COX and LTP were downregulated, with fold changes of 0.84, 0.53 and 0.31, respectively, after 12 h of drought stress. Genome-wide analysis identified 78 AP2/EREBP, 6 COX and 10 LTP genes in Z. mays L. Domain analysis confirmed the presence of the AP2 domain, Cyt_c_Oxidase_Vb domain and nsLTP1 in the AP2/EREBP, COX and LTP proteins, respectively. The AP2/EREBP protein family (AP2) includes five different domain types: the AP2/ERF domain, the EREBP-like factor (EREBP), the ethylene responsive factor (ERF), the dehydration responsive element binding protein (DREB) and the SHN SHINE. Synteny analysis of the AP2/EREBP, COX and LTP genes revealed collinearity orthologous relationships in O. sativa, H. vulgare and A. thaliana. AP2/EREBP genes were found on the 10 chromosomes of Z. mays L. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10. In the present study, the Ka/Ks ratios of the AP2/EREBP paralogous pairs indicated that the AP2/EREBP genes were influenced primarily by purifying selection, which indicated that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4 paralogous pairs indicate that the COX-3/COX-4 genes were influenced primarily by Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs indicate that these genes were influenced primarily by purifying selection, while the Ka/Ks ratios of the LTP-2/LTP-6 paralogous pairs indicate that these genes were influenced primarily by Darwinian selection. The duplication time of the AP2/EREBP paralogous gene pairs in Z. mays L. ranged from approximately 9.364 to 100.935 Mya. The duplication time of the COX-3/COX-4 paralogous gene pair was approximately 5.217 Mya. The duplication time of the LTP paralogous gene pairs ranged from approximately 19.064 to 96.477 Mya. The major focus of research is to identify the genes that are responsible for drought stress tolerance to improve maize for drought stress tolerance. The results of the present study will improve the understanding of the functions of the AP2/EREBP, COX and LTP genes in response to drought stress.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Screening cotton genotypes for their drought tolerance ability based on the expression level of dehydration-responsive element-binding protein and proline biosynthesis-related genes and morpho-physio-biochemical responses.

Drought stress adversely affects growth, development, productivity, and fiber quality of cotton (Gossypium hirsutum L). Breeding strategies to enhance drought tolerance require an improved knowledge of plant drought responses necessitating proper identification of drought-tolerant genotypes of crops, including cotton. The objective of this study was to classify the selected cotton genotypes for their drought tolerance ability based on morpho-physio-biochemical traits using Hierarchical Ward's cluster analysis. Five genotypes of cotton (Takfa 3, Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5) were selected as plant materials, and were grown under well-watered (WW; 98 ± 2% field capacity) and water-deficit (WD; 50 ± 2% field capacity) conditions for 16days during the flower initiation stage. Data on morpho-physio-biochemical parameters and gene expression levels for these parameters were collected, and subsequently genotypes were classified either as a drought tolerant or drought susceptible one. Upregulation of GhPRP (proline-rich protein), GhP5CS (Δ1-pyrroline-5-carboxylate synthetase), and GhP5CR (Δ1-pyrroline-5-carboxylate reductase) in relation to free proline enrichment was observed in Takfa 3 genotype under WD condition. An accumulation of free proline, total soluble sugar, and potassium in plants under WD conditions was detected, which played a key role as major osmolytes controlling cellular osmotic potential. Magnesium and calcium concentrations were also enriched in leaves under WD conditions, functioning as essential elements and regulating photosynthetic abilities. Leaf greenness, net photosynthetic rate, stomatal conductance, and transpiration rate were also declined under WD conditions, leading to growth retardation, especially aboveground traits of Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5 genotypes. An increase in leaf temperature (1.1 - 4.0°C) and crop water stress index (CWSI > 0.75) in relation to stomatal closure and reduced transpiration rate was recorded in cotton genotypes under WD conditions compared with WW conditions. Based on the increase of free proline, soluble sugar, leaf temperature, and CWSI, as well as the decrease of aboveground growth traits and physiological attributes, five genotypes were categorized into two cluster groups: drought tolerant (Takfa 3) and drought susceptible (Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5). The identified drought-tolerant cotton genotype, namely, Takfa 3, may be grown in areas experiencing drought conditions. It is recommended to further validate the yield traits of Takfa 3 under rainfed field conditions in drought-prone environments.

Picea wilsonii NAC31 and DREB2A Cooperatively Activate ERD1 to Modulate Drought Resistance in Transgenic Arabidopsis.

The NAC family of transcription factors (TFs) regulate plant development and abiotic stress. However, the specific function and response mechanism of NAC TFs that increase drought resistance in Picea wilsonii remain largely unknown. In this study, we functionally characterized a member of the PwNAC family known as PwNAC31. PwNAC31 is a nuclear-localized protein with transcriptional activation activity and contains an NAC domain that shows extensive homology with ANAC072 in Arabidopsis. The expression level of PwNAC31 is significantly upregulated under drought and ABA treatments. The heterologous expression of PwNAC31 in atnac072 Arabidopsis mutants enhances the seed vigor and germination rates and restores the hypersensitive phenotype of atnac072 under drought stress, accompanied by the up-regulated expression of drought-responsive genes such as DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A) and ERD1 (EARLY RESPONSIVE TO DEHYDRATION STRESS 1). Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that PwNAC31 interacts with DREB2A and ABF3 (ABSCISIC ACID-RESPONSIVE ELEMENT-BINDING FACTOR 3). Yeast one-hybrid and dual-luciferase assays showed that PwNAC31, together with its interaction protein DREB2A, directly regulated the expression of ERD1 by binding to the DRE element of the ERD1 promoter. Collectively, our study provides evidence that PwNAC31 activates ERD1 by interacting with DREB2A to enhance drought tolerance in transgenic Arabidopsis.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Phytosulfokine promotes fruit ripening and quality via phosphorylation of transcription factor DREB2F in tomato.

Phytosulfokine (PSK), a plant peptide hormone with a wide range of biological functions, is recognized by its receptor PHYTOSULFOKINE RECEPTOR 1 (PSKR1). Previous studies have reported that PSK plays important roles in plant growth, development, and stress responses. However, the involvement of PSK in fruit development and quality formation remains largely unknown. Here, using tomato (Solanum lycopersicum) as a research model, we show that exogenous application of PSK promotes the initiation of fruit ripening and quality formation, while these processes are delayed in pskr1 mutant fruits. Transcriptomic profiling revealed that molecular events and metabolic pathways associated with fruit ripening and quality formation are affected in pskr1 mutant lines and transcription factors are involved in PSKR1-mediated ripening. Yeast screening further identified that DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2F (DREB2F) interacts with PSKR1. Silencing of DREB2F delayed the initiation of fruit ripening and inhibited the promoting effect of PSK on fruit ripening. Moreover, the interaction between PSKR1 and DREB2F led to phosphorylation of DREB2F. PSK improved the efficiency of DREB2F phosphorylation by PSKR1 at the tyrosine-30 site, and the phosphorylation of this site increased the transcription level of potential target genes related to the ripening process and functioned in promoting fruit ripening and quality formation. These findings shed light on the involvement of PSK and its downstream signaling molecule DREB2F in controlling climacteric fruit ripening, offering insights into the regulatory mechanisms governing ripening processes in fleshy fruits.

The silicon regulates microbiome diversity and plant defenses during cold stress in Glycine max L.

With climate change, frequent exposure of bioenergy and food crops, specifically soybean (Glycine max L.), to low-temperature episodes is a major obstacle in maintaining sustainable plant growth at early growth stages. Silicon (Si) is a quasi-essential nutrient that can help to improve stress tolerance; however, how Si and a combination of cold stress episodes influence plant growth, plant physiology, and microbiome diversity has yet to be fully discovered. The soybean plants were exposed to cold stress (8-10°C) with or without applying Si, and the different plant organs (shoot and root) and rhizospheric soil were subjected to microbiome analysis. The plant growth, physiology, and gene expression analysis of plant defenses during stress and Si were investigated. We showed that cold stress significantly retarded soybean plants' growth and biomass, whereas, Si-treated plants showed ameliorated negative impacts on plant growth at early seedling stages. The beneficial effects of Si were also evident from significantly reduced antioxidant activities - suggesting lower cold-induced oxidative stress. Interestingly, Si also downregulated critical genes of the abscisic acid pathway and osmotic regulation (9-cis-epoxy carotenoid dioxygenase and dehydration-responsive element binding protein) during cold stress. Si positively influenced alpha and beta diversities of bacterial and fungal microbiomes with or without cold stress. Results showed significant variation in microbiome composition in the rhizosphere (root and soil) and phyllosphere (shoot) in Si-treated plants with or without cold stress exposures. Among microbiome phyla, Proteobacteria, Bacteroidota, and Ascomycota were significantly more abundant in Si treatments in cold stress than in control conditions. For the core microbiome, we identified 179 taxa, including 88 unique bacterial genera in which Edaphobacter, Haliangium, and Streptomyces were highly abundant. Enhanced extracellular enzyme activities in the cold and Si+cold treatments, specifically phosphatase and glucosidases, also reflected the microbiome abundance. In conclusion, this work elucidates cold-mediated changes in microbiome diversity and plant growth, including the positive impact Si can have on cold tolerance at early soybean growth stages - a step toward understanding crop productivity and stress tolerance.

NFXL1 functions as a transcriptional activator required for thermotolerance at reproductive stage in Arabidopsis.

Plants are highly susceptible to abiotic stresses, particularly heat stress during the reproductive stage. However, the specific molecular mechanisms underlying this sensitivity remain largely unknown. In the current study, we demonstrate that the Nuclear Transcription Factor, X-box Binding Protein 1-Like 1 (NFXL1), directly regulates the expression of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A (DREB2A), which is crucial for reproductive thermotolerance in Arabidopsis. NFXL1 is upregulated by heat stress, and its mutation leads to a reduction in silique length (seed number) under heat stress conditions. RNA-Seq analysis reveals that NFXL1 has a global impact on the expression of heat stress responsive genes, including DREB2A, Heat Shock Factor A3 (HSFA3) and Heat Shock Protein 17.6 (HSP17.6) in flower buds. Interestingly, NFXL1 is enriched in the promoter region of DREB2A, but not of either HSFA3 or HSP17.6. Further experiments using electrophoretic mobility shift assay have confirmed that NFXL1 directly binds to the DNA fragment derived from the DREB2A promoter. Moreover, effector-reporter assays have shown that NFXL1 activates the DREB2A promoter. The DREB2A mutants are also heat stress sensitive at the reproductive stage, and DEREB2A is epistatic to NFXL1 in regulating thermotolerance in flower buds. It is known that HSFA3, a direct target of DREB2A, regulates the expression of heat shock proteins genes under heat stress conditions. Thus, our findings establish NFXL1 as a critical upstream regulator of DREB2A in the transcriptional cassette responsible for heat stress responses required for reproductive thermotolerance in Arabidopsis.

Molecular identification of twenty NtDREB homologs and overexpression of NtDREB_A2.1 improved plant growth in response to cold-stress and P-nutrition limitation

Being as a useful model plant for biological study and significant industrial-crop that is sensitive to low temperature, tobacco is frequently adopted to evaluate functions of interested genes from other species by heterologous expression. Dehydration-responsive element binding (DREB) proteins are a type of transcription factors involved in plant responses to abiotic/biotic stimuli. However, genetic information and molecular-physiological roles of tobacco DREBs are little described. Here, we identified 20 NtDREBs containing a typical AP2-domain and phylogenetically classified them into five subgroups (A2-A6). Inspecting promoter sequences of NtDREBs revealed a different enrichment of certain abiotic stress-related cis-elements including ‘MeJA- and ABA-responsive and A/T-rich binding’ components. Analysis of publicly available RNA-seq data showed that many NtDREBs were transcriptionally regulated by cold-treatment. Furthermore, we assessed physiological roles in tobacco of NtDREB_A2.1, because of its rapid and/or strong expression responsive to low temperature and plant P- and N-nutritional status. Overexpression of NtDREB2.1 in tobacco cultivated under 15 °C improved the growth with increased plant-height and leaf-area compared to WT. Interestingly, NtDREB_2A.1 overexpression negatively influenced plant growth under conditions of low/high N-supply and 15 °C, whereas NtDREB_2A.1-transgenic lines could effectively utilize external low- and/or high P for better growth with increased N- and P-accumulation under 15–25 °C. Our work added not only new members to the DREB-subfamily but a valuable molecular-target for engineering of crops, aiming at improvement of plant cold-tolerance and utilization of soil phosphorus occurring at low available levels for plants in most soils.

Life-Cycle Multiomics ofRice Shoots Reveals Growth Stage-Specific Effects ofDrought Stress andTime-Lag Drought Responses.

Field-grown rice plants are exposed to various stresses at different stages of their life cycle, but little is known about the effects of stage-specific stresses on phenomes and transcriptomes. In this study, we performed integrated time-course multiomics on rice at 3-d intervals from seedling to heading stage under six drought conditions in a well-controlled growth chamber. Drought stress at seedling and reproductive stages reduced yield performance by reducing seed number and setting rate, respectively. High temporal resolution analysis revealed that drought response occurred in two steps: a rapid response via the abscisic acid (ABA) signaling pathway and a slightly delayed DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN (DREB) pathway, allowing plants to respond flexibly to deteriorating soil water conditions. Our long-term time-course multiomics showed that temporary drought stress delayed flowering due to prolonged expression of the flowering repressor gene GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) and delayed expression of the florigen genes HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Our life-cycle multiomics dataset on rice shoots under drought conditions provides a valuable resource for further functional genomic studies to improve crop resilience to drought stress.

The Knockdown of AUXIN RESPONSE FACTOR 2 Confers Enhanced Tolerance to Salt and Drought Stresses in Tomato (Solanum lycopersicum L.).

Auxin response factors (ARFs) act as key elements of the auxin-signaling pathway and play important roles in the process of a plant's growth, development, and response to environmental conditions. We studied the implication of the SlARF2 gene in the tomato response to salt (150 mM of NaCl) and drought (15% PEG 20000) stresses. The functional characterization of SlARF2 knockdown tomato mutants revealed that the downregulation of this gene enhanced primary root length and root branching and reduced plant wilting. At the physiological level, the arf2 mutant line displayed higher chlorophyll, soluble sugars, proline, and relative water contents as well as lower stomatal conductance and a decreased malondialdehyde content. Moreover, SlARF2 knockdown tomato mutants demonstrated higher activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) under salt and drought stresses than the wild type. Indeed, the stress tolerance of the arf2 mutant was also reflected by the upregulation of stress-related genes involved in ROS scavenging and plant defense, including SOD, CAT, dehydration-responsive element-binding protein, and early responsive to dehydration, which can ultimately result in a better resistance to salt and drought stresses. Furthermore, the transcriptional levels of the Δ1-pyrroline-5-carboxylate synthase (P5CS) gene were upregulated in the arf2 mutant after stress, in correlation with the higher levels of proline. Taken together, our findings reveal that SlARF2 is implicated in salt and drought tolerance in tomato and provides some considerable elements for improving the abiotic stress tolerance and increasing the crop yields of tomato.

Tea-based biochar-mediated changes in cation diffusion homeostasis in rice grown in heavy metal (loid) contaminated mining soil

Foreseeable future scenarios highlight the urgency of applying eco-safe avoidance methods or tolerance to heavy metal(loid) (HM) stress in agricultural production areas of contamination. The analyses show that the Ni, Mn, As, and Cr concentrations detected in the soils of the paddy fields in the Black Sea region vary between 123.60 and 263.30; 687–1271; 8.90–14.50; 162.00–340.00 mg kg−1 proving high accumulation of Ni, Mn, As, Cr in rice. Overconsumption of rice farmed extensively on these soils might also lead to human HM-related health problems. Therefore, in the current study, the approach of using tea-based biochar (BC) proven to have one of the most significant potentials as a soil amendment to reduce HM transmission to in-vitro-grown rice plants was investigated in the soil medium naturally contaminated with HMs. The tea-BC was produced from readily available local black tea waste of a conventional fermentation process and applied in the in-vitro experiments. Among the tested doses examined, 1% tea-BC showed a more positive effect on rice plant growth and development characterized by a better relative growth rate (59.7 and 84 mg g−1 d−1 for root and shoot tissues), photosynthetic pigment intactness (62.48 μg mL−1), cellular membrane integrity (93%), and relative water (96%) than the other rates (0% BC, 3%BC, 5%BC). The mRNA expression data highlights the probability of a cation diffusion facilitator (CDF) (OsMTP11) in concert with catalase isozyme (CATa) and dehydration-responsive element binding protein (DREB1a) linking the HM detoxification, oxidative defense, and dehydration pathways with the help of tea-BC. At the optimum concentration (1%BC), this approach might reduce HM accumulation levels of crops planted in HM-contaminated farmlands.

Overexpression of an autophagy-related gene DiATG3 from Davidia involucrata improves plant thermotolerance by enhancing the accumulation of polyamines and regulating genes in calcium and MAPK signaling pathways

Autophagy, a conserved eukaryotic mechanism for degrading intracellular constituents, plays prominent roles in plant abiotic stress tolerance. This study aimed to isolate an autophagy-related gene DiATG3 from a cool-adapted endangered plant species, dove tree (Davidia involucrata Baill.), and to investigate the physiological functions and regulatory mechanisms of DiATG3 for improving heat tolerance. DiATG3 was found to mainly localize to cytoplasm and possess the N-terminal domain, the catalytic domain with the HPC motif, and the C-terminal domain with the FLKF motif. Elevated transcript level of DiATG3 was detected in D. involucrata treated with heat stress. The constitutive overexpression of DiATG3 in Arabidopsis led to elevated levels of autophagy and conferred tolerance under heat stress. Furthermore, this thermotolerant phenotype was associated with improved photosynthetic capacity, higher antioxidant enzyme activities, and increased accumulation of polyamines. Transcriptome analysis suggest that activated calcium signaling and mitogen-activated protein kinase (MAPK) cascade may act as a central hub necessary for DiATG3-mediated heat tolerance. In addition, DiATG3 overexpression resulted in up-regulated expression of stress-related genes including heat shock factor A3 (AtHSFA3), dehydration-responsive element-binding protein 2C (AtDREB2C), AtWRKY26, and AtWRKY33 after heat treatment. Our study could yield insights into the engagement of plant autophagy during abiotic stress responses and contribute to breeding and engineering thermotolerant endangered plant species.

MaDREB1F confers cold and drought stress resistance through common regulation of hormone synthesis and protectant metabolite contents in banana.

Adverse environmental factors severely affect crop productivity. Improving crop resistance to multiple stressors is an important breeding goal. Although CBFs/DREB1s extensively participate in plant resistance to abiotic stress, the common mechanism underlying CBFs/DREB1s that mediate resistance to multiple stressors remains unclear. Here, we show the common mechanism for MaDREB1F conferring cold and drought stress resistance in banana. MaDREB1F encodes a dehydration-responsive element binding protein (DREB) transcription factor with nuclear localization and transcriptional activity. MaDREB1F expression is significantly induced after cold, osmotic, and salt treatments. MaDREB1F overexpression increases banana resistance to cold and drought stress by common modulation of the protectant metabolite levels of soluble sugar and proline, activating the antioxidant system, and promoting jasmonate and ethylene syntheses. Transcriptomic analysis shows that MaDREB1F activates or alleviates the repression of jasmonate and ethylene biosynthetic genes under cold and drought conditions. Moreover, MaDREB1F directly activates the promoter activities of MaAOC4 and MaACO20 for jasmonate and ethylene syntheses, respectively, under cold and drought conditions. MaDREB1F also targets the MaERF11 promoter to activate MaACO20 expression for ethylene synthesis under drought stress. Together, our findings offer new insight into the common mechanism underlying CBF/DREB1-mediated cold and drought stress resistance, which has substantial implications for engineering cold- and drought-tolerant crops.

Enhanced osmotic adjustment, antioxidant defense, and photosynthesis efficiency under drought and heat stress of transgenic cowpea overexpressing an engineered DREB transcription factor

Cowpea is sensitive to drought and heat stress, particularly at the reproductive stages of development. Both stresses limit growth and yield, and their effect is more devastating when occurring concurrently. Dehydration-responsive element-binding protein 2A (DREB2A) is an important signaling hub integrating information about two different abiotic stresses, drought and heat. We identified VuDREB2A as a canonical DREB ortholog in cowpea, activating downstream stress-responsive genes by binding to DREs in their promoter. Post-translational modification of a negative regulatory domain (NRD) within the VuDREB2A protein prevents its degradation. Targeted deletion of the NRD produces a stable and constitutively active form VuDREB2A-CA. However, there is very little evidence of its practical utility under field conditions. This study overexpressed the VuDREB2A-CA in a popular cowpea variety and conducted drought- and heat-tolerance experiments across various stress regimes. Transgenic cowpea exhibited significant tolerance with consistently higher yield when exposed to over 30-d drought stress and 3-d exposure to high temperature (28 °C˗52 °C) without any pleiotropic alterations. The transgenic lines showed higher photosynthetic efficiency, osmotic adjustment, antioxidant defense, thermotolerance, and significantly higher survival and increased biomass than the wild type. Late embryogenesis abundant 5, heat shock protein 70, dehydrin, mitogen-activated protein kinase 2/4, isoflavonoid reductase, and myoinositol phosphate synthase were upregulated in transgenic lines under drought and heat stress. Through transcriptome analysis of the transgenic lines, we found significant up-regulation of various stress-responsive cowpea genes, having DRE in their promoter. Our results suggest that overexpression of VuDREB2A could improve cowpea production under drought and high temperatures.

The cold-stress responsive gene DREB1A involved in low-temperature tolerance in Xinjiang wild walnut.

BackgroundLow-temperatures have the potential to be a serious problem for plants and can negatively affect the normal growth and development of walnuts. DREB1/CBF (Dehydration Responsive Element Binding Protein 1/C-repeat Binding Factor), one of the most direct transcription factors in response to low-temperature stress, may improve the resistance of plants to low-temperatures by regulating their functional genes. However, few studies have been conducted in walnut. The Xinjiang wild walnut is a rare wild plant found in China, with a large number of excellent trait genes, and is hardier than cultivated walnuts in Xinjiang.MethodsIn this work, we identified all of the DREB1 members from the walnut genome and analyzed their expression levels in different tissues and during low-temperature stress on the Xinjiang wild walnut. The JfDREB1A gene of the Xinjiang wild walnut was cloned and transformed into Arabidopsis thaliana for functional verification.ResultsThere were five DREB1 transcription factors in the walnut genome. Among them, the relative expression level of the DREB1A gene was significantly higher than other members in the different tissues (root, stem, leaf) and was immediately un-regulated under low-temperature stress. The overexpression of the JfDREB1A gene increased the survival rates of transgenic Arabidopsis lines, mainly through maintaining the stability of cell membrane, decreasing the electrical conductivity and increasing the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the expression levels of cold-inducible genes like AtKIN1, AtERD10, AtRD29A, AtCOR15A and AtCOR47, were significantly increased. These results showed that the JfDREB1A gene may play an important role in the response to cold stress of the Xinjiang wild walnut. This study contributes to our understanding of the molecular mechanism of the Xinjiang wild walnut’s response to low-temperature stress and will be beneficial for developing walnut cultivars with improved cold resistance.

Sugarcane ScDREB2B-1 Confers Drought Stress Tolerance in Transgenic Nicotiana benthamiana by Regulating the ABA Signal, ROS Level and Stress-Related Gene Expression

The dehydration-responsive element-binding protein (DREB) is a subgroup member of the AP2/ERF family and actively participates in the response of plants to abiotic stress. Although DREB genes have been studied in a variety of plant species, there are few reports of DREB genes in sugarcane (Saccharum spp.). In this study, a novel full-length cDNA sequence of the ScDREB2B-1 gene was cloned from the Saccharum hybrid ROC22, whose encoding protein contained only one AP2-conserved domain and was clustered into the DREB (A-2) subgroup. The diverse promoter elements in the ScDREB2B-1 gene and the accumulated transcripts of its homologous gene (SsAP2/ERF-107) in S. spontaneum under drought stress suggest that the ScDREB2B-1 gene may play a role in drought response. In addition, reverse transcription quantitative PCR analysis showed that the expression level of the ScDREB2B-1 gene was upregulated in the root and leaf of ROC22 under polyethylene glycol, sodium chloride and abscisic acid (ABA) treatments. The yeast two-hybrid experiment demonstrated that ScDREB2B-1 had transcriptional self-activation activity. Compared with wild-type plants, the overexpression of the ScDREB2B-1 gene improved the drought tolerance of the transgenic Nicotiana benthamiana by activating the ABA pathway to enhance the expression of the ABA-responsive gene (NbNCED) and ABA content, regulate the intracellular reactive oxygen species (ROS) level (enhance the transcripts of ROS synthase-related gene NbRbohB and the activities of catalase, peroxidase and superoxide dismutase) and increase the relative water content, proline content and expression level of osmotic stress-related genes (NbERD and NbLEA). Collectively, our data indicate that ScDREB2B-1 is a stress-inducible and ABA-responsive transcription factor gene that responds to drought stress by regulating ABA signaling, ROS levels and stress-related gene expression. This study contributes to a better understanding of the biological function of ScDREB2B-1, which could serve as a foundation for future resistance breeding in sugarcane.

Genome-wide analysis of the DREB family genes and functional identification of the involvement of BrDREB2B in abiotic stress in wucai (Brassica campestris L.)

Dehydration responsive element binding protein (DREB) is a significant transcription factor class known to be implicated in abiotic stresses. In this study, we systematically conducted a genome-wide identification and expression analysis of the DREB gene family, including gene structures, evolutionary relationships, chromosome distribution, conserved domains, and expression patterns. A total of 65 DREB family gene members were identified in Chinese cabbage (Brassica rapa L.) and were classified into five subgroups based on phylogenetic analysis. Through analysis of the conserved domains of BrDREB family genes, only one exon existed in the gene structure. Through the analysis of cis-acting elements, these genes were mainly involved in hormone regulation and adversity stress. In order to identify the function of BrDREB2B, overexpressed transgenic Arabidopsis was constructed. After different stress treatments, the germination rate, root growth, survival rate, and various plant physiological indicators were measured. The results showed that transgenic Arabidopsis thaliana plants overexpressing BrDREB2B exhibited enhanced tolerance to salt, heat and drought stresses. Taken together, our results are the first to report the BrDREB2B gene response to drought and heat stresses in Chinese cabbage and provide a basis for further studies to determine the function of BrDREBs in response to abiotic stresses.

CRISPR/Cas9-mediated mutation in auxin efflux carrier OsPIN9 confers chilling tolerance by modulating reactive oxygen species homeostasis in rice.

Phytohormone auxin plays a vital role in plant development and responses to environmental stresses. The spatial and temporal distribution of auxin mainly relies on the polar distribution of the PIN-FORMED (PIN) auxin efflux carriers. In this study, we dissected the functions of OsPIN9, a monocot-specific auxin efflux carrier gene, in modulating chilling tolerance in rice. The results showed that OsPIN9 expression was dramatically and rapidly suppressed by chilling stress (4°C) in rice seedlings. The homozygous ospin9 mutants were generated by CRISPR/Cas9 technology and employed for further research. ospin9 mutant roots and shoots were less sensitive to 1-naphthaleneacetic acid (NAA) and N-1-naphthylphthalamic acid (NPA), indicating the disturbance of auxin homeostasis in the ospin9 mutants. The chilling tolerance assay showed that ospin9 mutants were more tolerant to chilling stress than wild-type (WT) plants, as evidenced by increased survival rate, decreased membrane permeability, and reduced lipid peroxidation. However, the expression of well-known C-REPEAT BINDING FACTOR (CBF)/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1 (DREB)-dependent transcriptional regulatory pathway and Ca2+ signaling genes was significantly induced only under normal conditions, implying that defense responses in ospin9 mutants have probably been triggered in advance under normal conditions. Histochemical staining of reactive oxygen species (ROS) by 3′3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) showed that ospin9 mutants accumulated more ROS than WT at the early stage of chilling stress, while less ROS was observed at the later stage of chilling treatment in ospin9 mutants. Consistently, antioxidant enzyme activity, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), improved significantly during the early chilling treatments, while was kept similar to WT at the later stage of chilling treatment, implying that the enhanced chilling tolerance of ospin9 mutants is mainly attributed to the earlier induction of ROS and the improved ROS scavenging ability at the subsequent stages of chilling treatment. In summary, our results strongly suggest that the OsPIN9 gene regulates chilling tolerance by modulating ROS homeostasis in rice.

NtDREB-1BL1 Enhances Carotenoid Biosynthesis by Regulating Phytoene Synthase in Nicotiana tabacum.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in Nicotiana tabacum K326, NtDREB-1BL1, which regulates carotenoids biosynthesis by binding to the NtPSY promoter. The NtDREB-1BL1 transcript was widely distributed in leaves by Real-time PCR. Confocal image revealed that NtDREB-1BL1 was localized in the nucleus. The chromatin immunoprecipitation (ChIP) with the qPCR technique indicated that NtDREB-1BL1 could anchor the promoter region of NtPSY. Overexpression (NtDREB-1BL1 OE) and RNA interference (NtDREB-1BL1 RNAi) of NtDREB-1BL1 were performed to evaluate its biological function in N. tabacum. Both carotenoid and chlorophyll contents increased in transgenic plants of NtDREB-1BL1 OE compared with wild-type (WT) plants, with the augment of the genes involved in carotenoid biosynthesis. In contrast, the contents of carotenoid and chlorophyll significantly decreased in transgenic plants of NtDREB-1BL1 RNAi compared to WT, along with the decline in the expression of genes related to carotenoid biosynthesis. Moreover, transgenic plants of NtDREB-1BL1 OE exhibited enhanced tolerance under drought stress, with the weakened tolerance of drought stress in transgenic plants of NtDREB-1BL1 RNAi. In conclusion, our results illustrated the new role of transcription factor NtDREB-1BL1 in improving carotenoid biosynthesis through regulating NtPSY expression.