Drought Tolerance In Transgenic Arabidopsis Research Articles

Expression of Foxtail Millet bZIP Transcription Factor SibZIP67 Enhances Drought Tolerance in Arabidopsis.

Foxtail millet is a drought-tolerant cereal and forage crop. The basic leucine zipper (bZIP) gene family plays important roles in regulating plant development and responding to stresses. However, the roles of bZIP genes in foxtail millet remain largely uninvestigated. In this study, 92 members of the bZIP transcription factors were identified in foxtail millet and clustered into ten clades. The expression levels of four SibZIP genes (SibZIP11, SibZIP12, SibZIP41, and SibZIP67) were significantly induced after PEG treatment, and SibZIP67 was chosen for further analysis. The studies showed that ectopic overexpression of SibZIP67 in Arabidopsis enhanced the plant drought tolerance. Detached leaves of SibZIP67 overexpressing plants had lower leaf water loss rates than those of wild-type plants. SibZIP67 overexpressing plants improved survival rates under drought conditions compared to wild-type plants. Additionally, overexpressing SibZIP67 in plants displayed reduced malondialdehyde (MDA) levels and enhanced activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) under drought stress. Furthermore, the drought-related genes, such as AtRD29A, AtRD22, AtNCED3, AtABF3, AtABI1, and AtABI5, were found to be regulated in SibZIP67 transgenic plants than in wild-type Arabidopsis under drought conditions. These data suggested that SibZIP67 conferred drought tolerance in transgenic Arabidopsis by regulating antioxidant enzyme activities and the expression of stress-related genes. The study reveals that SibZIP67 plays a beneficial role in drought response in plants, offering a valuable genetic resource for agricultural improvement in arid environments.

TaMYB44-5A reduces drought tolerance by repressing transcription of TaRD22-3A in the abscisic acid signaling pathway.

TaMYB44-5A identified as a transcription factor negatively regulates drought tolerance in transgenic Arabidopsis. Drought can severely reduce yields throughout the wheat-growing season. Many studies have shown that R2R3-MYB transcription factors are involved in drought stress responses. In this study, the R2R3-MYB transcription factor MYB44-5A was identified in wheat (Triticum aestivum L.) and functionally analyzed. Three homologs of TaMYB44 were isolated, all of which localized to the nucleus. Overexpression of TaMYB44-5A reduced drought tolerance in Arabidopsis thaliana. Further analysis showed that TaMYB44-5A reduced the sensitivity of transgenic Arabidopsis to ABA. Genetic and transcriptional regulation analyses demonstrated that the expression levels of drought- and ABA-responsive genes were downregulated by TaMYB44-5A, and TaMYB44-5A directly bound to the MYB-binding site on the promoter to repress the transcription level of TaRD22-3A. Our results provide insights into a novel molecular pathway in which the R2R3-MYB transcription factor negatively regulates ABA signaling in response to drought stress.

Characterization of DREB family genes in Lotus japonicus and LjDREB2B overexpression increased drought tolerance in transgenic Arabidopsis

BackgroundDrought stress affects plant growth and development. DREB proteins play important roles in modulating plant growth, development, and stress responses, particularly under drought stress. To study the function of DREB transcription factors (TFs), we screened key DREB-regulating TFs for drought in Lotus japonicus.ResultsForty-two DREB TFs were identified, and phylogenetic analysis of proteins from L. japonicus classified them into five subfamilies (A1, A2, A4, A5, A6). The gene motif composition of the proteins is conserved within the same subfamily. Based on the cis-acting regulatory element analysis, we identified many growth-, hormone-, and stress-responsive elements within the promoter regions of DREB. We further analyzed the expression pattern of four genes in the A2 subfamily in response to drought stress. We found that the expression of most of the LjDREB A2 subfamily genes, especially LjDREB2B, was induced by drought stress. We further generated LjDREB2B overexpression transgenic Arabidopsis plants. Under drought stress, the growth of wild-type (WT) and overexpressing LjDREB2B (OE) Arabidopsis lines was inhibited; however, OE plants showed better growth. The malondialdehyde content of LjDREB2B overexpressing lines was lower than that of the WT plants, whereas the proline content and antioxidant enzyme activities in the OE lines were significantly higher than those in the WT plants. Furthermore, after drought stress, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than those in the WT plants.ConclusionsOur results facilitate further functional analysis of L. japonicus DREB. LjDREB2B overexpression improves drought tolerance in transgenic Arabidopsis. These results indicate that DREB holds great potential for the genetic improvement of drought tolerance in L. japonicus.

Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress

BackgroundThe early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed.ResultsIn this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription.ConclusionsThis study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Grape SnRK2.7 Positively Regulates Drought Tolerance in Transgenic Arabidopsis.

In this study, we obtained and cloned VvSnRK2.7 by screening transcriptomic data to investigate the function of the grape sucrose non-fermenting kinase 2 (SnRK2) gene under stress conditions. A yeast two-hybrid (Y2H) assay was used to further screen for interaction proteins of VvSnRK2.7. Ultimately, VvSnRK2.7 was heterologously expressed in Arabidopsis thaliana, and the relative conductivity, MDA content, antioxidant enzyme activity, and sugar content of the transgenic plants were determined under drought treatment. In addition, the expression levels of VvSnRK2.7 in Arabidopsis were analyzed. The results showed that the VvSnRK2.7-EGFP fusion protein was mainly located in the cell membrane and nucleus of tobacco leaves. In addition, the VvSnRK2.7 protein had an interactive relationship with the VvbZIP protein during the Y2H assay. The expression levels of VvSnRK2.7 and the antioxidant enzyme activities and sugar contents of the transgenic lines were higher than those of the wild type under drought treatment. Moreover, the relative conductivity and MDA content were lower than those of the wild type. The results indicate that VvSnRK2.7 may activate the enzyme activity of the antioxidant enzyme system, maintain normal cellular physiological metabolism, stabilize the berry sugar metabolism pathway under drought stress, and promote sugar accumulation to improve plant resistance.

Feedback regulation of the isoprenoid pathway by SsdTPS overexpression has the potential to enhance plant tolerance to drought stress.

In order to maintain the dynamic physiological balance, plants are compelled to adjust their energy metabolism and signal transduction to cope with the abiotic stresses caused by complex and changeable environments. The diterpenoid natural compound and secondary metabolites, sclareol, derived from Salvia sclarea, has gained significant attention owing to its economic value as a spice material and diverse physiological activities. Here, we focused on the roles and regulatory mechanisms of the sclareol diterpene synthase gene SsdTPS in the resistance of S. sclarea to abiotic stresses. Our results suggested that abiotic stresses could induce the response and upregulation of SsdTPS expression and isoprenoid pathway in S. sclarea. Ectopic expression of SsdTPS conferred drought tolerance in transgenic Arabidopsis, compared with wild-type. Overexpression of SsdTPS enhanced the transcription of ABA signal transduction synthetic regulators and induced the positive feedback upregulating key regulatory genes in the MEP pathway, thereby promoting the increase of ABA content and improving drought tolerance in transgenic plants. In addition, SsdTPS-overexpressed transgenic Arabidopsis improved the responses of stomatal regulatory genes and ROS scavenging enzyme activities and gene expression to drought stress. This promoted the stomatal closure and ROS reduction, thus enhancing water retention capacity and reducing oxidative stress damage. These findings unveil the potentially positive role of SsdTPS in orchestrating multiple regulatory mechanisms and maintaining homeostasis for improved abiotic stress resistance in S. sclarea, providing a novel insight into strategies for promoting drought resistance and cultivating highly tolerant plants.

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.

Genome-wide analysis of the U-box E3 ligases gene family in potato (Solanum tuberosum L.) and overexpress StPUB25 enhance drought tolerance in transgenic Arabidopsis.

Plant U-box (PUB) E3 ubiquitin ligases have vital effects on various biological processes. Therefore, a comprehensive and systematic identification of the members of the U-box gene family in potato will help to understand the evolution and function of U-box E3 ubiquitin ligases in plants. This work identified altogether 74 PUBs in the potato (StPUBs) and examined their gene structures, chromosomal distributions, and conserved motifs. There were seventy-four StPUB genes on ten chromosomes with diverse densities. As revealed by phylogenetic analysis on PUBs within potato, Arabidopsis, tomato (Solanum lycopersicum), cabbage (Brassica oleracea), rice (Oryza sativa), and corn (Zea mays), were clustered into eight subclasses (C1-C8). According to synteny analysis, there were 40 orthologous StPUB genes to Arabidopsis, 58 to tomato, 28 to cabbage, 7 to rice, and 8 to corn. In addition, RNA-seq data downloaded from PGSC were utilized to reveal StPUBs' abiotic stress responses and tissue-specific expression in the doubled-monoploid potato (DM). Inaddition, we performed RNA-seq on the 'Atlantic' (drought-sensitive cultivar, DS) and the 'Qingshu NO.9' (drought-tolerant cultivar, DT) in early flowering, full-blooming, along with flower-falling stages to detect genes that might be involved in response to drought stress. Finally, quantitative real-time PCR (qPCR) was carried out to analyze three candidate genes for their expression levels within 100mM NaCl- and 10% PEG 6000 (w/v)-treated potato plantlets for a 24-h period. Furthermore, we analyzed the drought tolerance of StPUB25 transgenic plants and found that overexpression of StPUB25 significantly increased peroxidase (POD) activity, reduced ROS (reactive oxygen species) and MDA (malondialdehyde) accumulation compared with wild-type (WT) plants, and enhancing drought tolerance of the transgenic plants. In this study, three candidate genes related to drought tolerance in potato were excavated, and the function of StPUB25 under drought stress was verified. These results should provide valuable information to understand the potato StPUB gene family and investigate the molecular mechanisms of StPUBs regulating potato drought tolerance.

Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress.

Drought stress is an important factor that reduces plant biomass production and quality. As one of the most important economic forage grasses, orchardgrass (Dactylis glomerata) has high drought tolerance. Auxin/indole-3-acetic acid (Aux/IAA) is one of the early responsive gene families of auxin and plays a key role in the response to drought stress. However, the characteristics of the Aux/IAA gene family in orchardgrass and their potential function in responding to drought stress remain unclear. Here, 30 Aux/IAA members were identified in orchardgrass. Segmental duplication may be an important driving force in the evolution of the Aux/IAA gene family in orchardgrass. Some Aux/IAA genes were induced by IAA, drought, salt, and temperature stresses, implying that these genes may play important roles in responding to abiotic stresses. Heterologous expression in yeast revealed that DgIAA21 can reduce drought tolerance. Similarly, the overexpression of DgIAA21 also reduced drought tolerance in transgenic Arabidopsis, which was supported by lower total chlorophyll content and relative water content as well as higher relative electrolyte leakage and malondialdehyde content (MDA) than Col-0 plants under drought conditions. The results of this study provided valuable insight into the function of DgIAAs in response to drought stress, which can be further used to improve forage grass breeding programs.

Genome-wide identification of the mango pathogenesis-related 1 (PR1) gene family and functional analysis of MiPR1A genes in transgenic Arabidopsis

Pathogen-related protein 1 (PR1) is an essential component of plant defense mechanisms against a variety of biotic and abiotic stresses. However, knowledge of PR1 in mango (Mangifera indica L), one of the most important horticultural crops, is limited. In the present study, a total of 10 PR1 family members were first discovered by bioinformatics analysis of the mango genome. Chromosomal mapping revealed that the MiPR1 genes were unevenly distributed on the five chromosomes of mango. The conserved domain and genetic structure analysis showed that all ten MiPR1 genes have a conserved CAP domain and intron-free. Cis element analysis indicated that several stress response elements were found in these promoters. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis showed that the MiPR1 genes have differences in expression patterns among the organs and under low temperature, polyethylene glycol (PEG), sodium chloride (NaCl) and anthracnose treatments. Furthermore, MiPR1A was selected for further functional analysis. Overexpression of MiPR1A increased salt and drought tolerance in transgenic Arabidopsis, superoxide dismutase (SOD) activity and proline content were significantly upregulated and malondialdehyde (MDA) content was significantly downregulated in transgenic Arabidopsis compared with control lines. In addition, MiPR1A enhanced resistance to Colletotrichum gloeosporioides. Taken together, these findings provide insight into the potential functional roles of the MiPR1A gene in biotic and abiotic stress tolerance in mango.

Overexpression of a WRKY transcription factor McWRKY57-like from Mentha canadensis L. enhances drought tolerance in transgenic Arabidopsis

BackgroundDrought has become a major environmental problem affecting crop production. Members of the WRKY family play important roles in plant development and stress responses. However, their roles in mint have been barely explored.ResultsIn this study, we isolated a drought-inducible gene McWRKY57-like from mint and investigated its function. The gene encodes a group IIc WRKY transcription factor, McWRKY57-like, which is a nuclear protein with a highly conserved WRKY domain and a C2H2 zinc-finger structure, and has transcription factor activity. Its expression levels were examined in different tissues of mint and under the treatment of mannitol, NaCl, abscisic acid, and methyl jasmonate. We found that McWRKY57-like overexpression in Arabidopsis significantly increased drought tolerance. Further studies showed that under drought stress, McWRKY57-like-overexpressing plants had higher chlorophyll, soluble sugar, soluble protein, and proline contents but lower water loss rate and malondialdehyde content than wild-type plants. Moreover, the activities of antioxidant enzymes catalase, superoxide dismutase, and peroxidase were enhanced in McWRKY57-like transgenic plants. Furthermore, qRT-PCR analysis revealed that the drought-related genes AtRD29A, AtRD29B, AtRD20, AtRAB18, AtCOR15A, AtCOR15B, AtKIN2, and AtDREB1A were upregulated in McWRKY57-like transgenic plants than in wild-type Arabidopsis under simulated drought conditions.ConclusionThese data demonstrated that McWRKY57-like conferred drought tolerance in transgenic Arabidopsis by regulating plant growth, osmolyte accumulation and antioxidant enzyme activities, and the expression of stress-related genes. The study indicates that McWRKY57-like plays a positive role in drought response in plants.

AgMYB5, an MYB transcription factor from celery, enhanced β-carotene synthesis and promoted drought tolerance in transgenic Arabidopsis

BackgroundWater shortage caused by global warming seriously affects the yield and quality of vegetable crops. β-carotene, the lipid-soluble natural product with important pharmacological value, is abundant in celery. Transcription factor MYB family extensively disperses in plants and plays regulatory roles in carotenoid metabolism and water scarcity response.ResultsHere, the AgMYB5 gene encoding 196 amino acids was amplified from celery cv. ‘Jinnanshiqin’. In celery, the expression of AgMYB5 exhibited transactivation activity, tissue specificity, and drought-condition responsiveness. Further analysis proved that ectopic expression of AgMYB5 increased β-carotene content and promoted drought tolerance in transgenic Arabidopsis thaliana. Moreover, AgMYB5 expression promoted β-carotene biosynthesis by triggering the expression of AtCRTISO and AtLCYB, which in turn increased antioxidant enzyme activities, and led to the decreased contents of H2O2 and MDA, and the inhibition of O2− generation. Meanwhile, β-carotene accumulation promoted endogenous ABA biosynthesis of transgenic Arabidopsis, which resulted in ABA-induced stomatal closing and delayed water loss. In addition, ectopic expression of AgMYB5 increased expression levels of AtERD1, AtP5CS1, AtRD22, and AtRD29.ConclusionsThe findings indicated that AgMYB5 up-regulated β-carotene biosynthesis and drought tolerance of Arabidopsis.

An apple NITRATE REDUCTASE 2 gene positively regulates nitrogen utilization and abiotic stress tolerance in Arabidopsis and apple callus

Nitrogen (N) is an essential element that plays an important role in crop biomass accumulation and quality formation. Increased crop yield is relied on excessive application of fertilizers, which usually leads to environmental pollution and unsustainable development. Thus, identification and characterization of genes involved in promoting nitrogen use efficiency is of high priority in crop breeding. The activity of nitrate reductase (NR) plays a critical role in nitrogen metabolism. In model plant Arabidopsis, NITRATE REDUCTASE 2 (NIA2), one of the two NRs, is responsible for about 90% of the NR activity. In this study, MdNIA2 gene in apple (Malus domestica) genome was screened out and identified by using AtNIA2 as bait. Phylogenetic analysis revealed that MdNIA2 had the closest evolutionary relationship with MbNIA from Malus baccata. Ectopic expression of MdNIA2 in Arabidopsis elevated the nitrogen use efficiency and increased root hair elongation and formation, resulting in promoted plant growth. Furthermore, the overexpression of MdNIA2 improved salt and drought tolerance in transgenic Arabidopsis and improved the salt tolerance of transgenic apple callus, and MdNIA2-reagualted NO metabolism might contribute to the abiotic stress tolerance. Overall, our data indicate the critical role of MdNIA2 in regulating nitrogen utilization efficiency and abiotic stress responses.

Genome-wide characterization of Alfin-like (AL) genes in apple and functional identification of MdAL4 in response to drought stress.

Eleven Alfin-like (AL) genes were obtained from apple and MdAL4 was selected for improving drought stress tolerance of transgenic apple callus and Arabidopsis. Drought is an important environmental factor affecting plant growth all over the world. Alfin-like (AL) have well-documented functions in abiotic stress response, but their drought stress tolerance in apple (Malus domestica) are poorly understood. According to the transcriptome data, 11 MdAL genes containing conserved Alfin and PHD-finger domain were identified in apple and divided into three subgroups with a total of 35 members from different species. Subsequently, gene structures, conserved amino acid sequences, promoter cis-acting elements, and gene evolution events were analyzed. Based on differential expression of MdALs in response to abiotic stresses, MdAL4, which was highly expressed under drought, was further cloned and investigated. MdAL4 encoding nuclear-localized protein conferred enhanced drought tolerance in overexpressing transgenic calli of apple 'Orin'. Moreover, the ectopic expression of MdAL4 improved the drought tolerance of transgenic Arabidopsis, as judged from remarkably decreased malonaldehyde (MDA) content and electrolyte leakage in MdAL4 overexpressing plants relative to WT. Furthermore, MdAL4 possibly could bind to promoter regions of ROS-scavenging and stress-related genes to improve drought tolerance. Additionally, we found in silico evidence that three proteins containing the WD40 domain that interact with MdAL4. Based on these results, MdAL4 was identified as a positive regulator for improving drought stress of apple.

A Novel bHLH Transcription Factor PtrbHLH66 from Trifoliate Orange Positively Regulates Plant Drought Tolerance by Mediating Root Growth and ROS Scavenging

Drought limits citrus yield and fruit quality worldwide. The basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in plant response to drought stress. However, few bHLH TFs related to drought response have been functionally characterized in citrus. In this study, a bHLH family gene, named PtrbHLH66, was cloned from trifoliate orange. PtrbHLH66 contained a highly conserved bHLH domain and was clustered closely with bHLH66 homologs from other plant species. PtrbHLH66 was localized to the nucleus and had transcriptional activation activity. The expression of PtrbHLH66 was significantly induced by polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments. Ectopic expression of PtrbHLH66 promoted the seed germination and root growth, increased the proline and ABA contents and the activities of antioxidant enzymes, but reduced the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) under drought stress, resulting in enhanced drought tolerance of transgenic Arabidopsis. In contrast, silencing the PtrbHLH66 homolog in lemon plants showed the opposite effects. Furthermore, under drought stress, the transcript levels of 15 genes involved in ABA biosynthesis, proline biosynthesis, ROS scavenging and drought response were obviously upregulated in PtrbHLH66 ectopic-expressing Arabidopsis but downregulated in PtrbHLH66 homolog silencing lemon. Thus, our results suggested that PtrbHLH66 acted as a positive regulator of plant drought resistance by regulating root growth and ROS scavenging.

The sweet potato B-box transcription factor gene IbBBX28 negatively regulates drought tolerance in transgenic Arabidopsis.

B-box (BBX) which are a class of zinc finger transcription factors, play an important role in regulating of photoperiod, photomorphogenesis, and biotic and abiotic stresses in plants. However, there are few studies on the involvement of BBX transcription factors in response to abiotic stresses in sweet potato. In this paper, we cloned the DNA and promoter sequences of IbBBX28. There was one B-box conserved domain in IbBBX28, and the expression of IbBBX28 was induced under drought stress. Under drought stress, compared to wild type Arabidopsis, the protective enzyme activities (SOD, POD, and CAT) were all decreased in IbBBX28-overexpression Arabidopsis but increased in the mutant line bbx28, while the MDA content was increased in the IbBBX28-overexpression Arabidopsis and decreased in the bbx28. Moreover, the expression levels of the resistance-related genes showed the same trend as the protective enzyme activities. These results showed that IbBBX28 negatively regulates drought tolerance in transgenic Arabidopsis. Additionally, the yeast two-hybrid and BiFC assays verified that IbBBX28 interacted with IbHOX11 and IbZMAT2. The above results provide important clues for further studies on the role of IbBBX28 in regulating the stress response in sweet potato.

The wheat WRKY transcription factor TaWRKY1-2D confers drought resistance in transgenic Arabidopsis and wheat (Triticum aestivum L.)

The WRKY transcription factor family has been associated with a variety of plant biological processes, such as biotic and abiotic stress responses. In this study, 13 wheat TaWRKY DEGs in transcriptome data before and after drought stress, namely TaWRKY1 to TaWRKY8, including various copies, were identified and classified as Group I, II, or III. TaWRKY1-2D overexpression enhanced drought tolerance in transgenic Arabidopsis. Moreover, the AtRD29A, AtP5CS1, AtPOD1, AtCAT1, and AtSOD (Cu/Zn) genes, which are related to the stress response and antioxidant system, were significantly upregulated in TaWRKY1-2D transgenic Arabidopsis under drought stress. TaWRKY1-2 silencing in wheat increases the MDA content, reduces the contents of proline and chlorophyll and the activities of antioxidant enzymes, and inhibits the expression levels of antioxidant (TaPOD, TaCAT, and TaSOD (Fe))- and stress-related genes (TaP5CS) under drought stress. Yeast two-hybrid screening revealed TaDHN3 as an interaction partner of TaWRKY1-2D; their interaction was further confirmed using yeast two-hybrid and bimolecular fluorescence complementation. Furthermore, TaWRKY1-2D may play essential roles in wheat drought tolerance through posttranslational regulation of TaDHN3. Overall, these findings contribute to our knowledge of the WRKY family in wheat and identify TaWRKY1-2D as a promising candidate gene for improving wheat breeding to generate drought-tolerant wheat.

FtNAC31, a Tartary buckwheat NAC transcription factor, enhances salt and drought tolerance in transgenic Arabidopsis

Tartary buckwheat [Fagopyrum tataricum (L.) Gaertn.] is a pseudocereal with strongly abiotic resistance. NACs, one of the largest plant-specific transcription factors (TFs), are involved in various stress responses. However, the characteristics and regulatory mechanisms of NAC TFs remain unclarified clearly in Tartary buckwheat (TB). In this study, it validated that salt, drought, and abscisic acid (ABA) stress significantly up-regulated the expression of NAC TF gene FtNAC31. Its coding protein has a C-terminal transactivated domain and localized in the nucleus, suggesting that FtNAC31 might play a transcriptional activation role in TB. Notably, overexpression of FtNAC31 lowered the seed germination rate upon ABA treatment and enhanced the tolerance to salt and drought stress in transgenetic Arabidopsis. Furthermore, under various stresses, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in FtNAC31 overexpressed lines exhibited a sharp increase trend. Meanwhile, the expression levels of several stress-associated genes including RD29A, RD29B, RD22, DREB2B, NCED3, and POD1, were dramatically upregulated in lines overexpressing FtNAC31. Altogether, overproduction of FtNAC31 could enhance the resistance to salt and drought stresses in transgenic Arabidopsis, which most likely functioned in an ABA-dependent way.

OsHsfB4b Confers Enhanced Drought Tolerance in Transgenic Arabidopsis and Rice.

Heat shock factors (Hsfs) play pivotal roles in plant stress responses and confer stress tolerance. However, the functions of several Hsfs in rice (Oryza sativa L.) are not yet known. In this study, genome-wide analysis of the Hsf gene family in rice was performed. A total of 25 OsHsf genes were identified, which could be clearly clustered into three major groups, A, B, and C, based on the characteristics of the sequences. Bioinformatics analysis showed that tandem duplication and fragment replication were two important driving forces in the process of evolution and expansion of the OsHsf family genes. Both OsHsfB4b and OsHsfB4d showed strong responses to the stress treatment. The results of subcellular localization showed that the OsHsfB4b protein was in the nucleus whereas the OsHsfB4d protein was located in both the nucleus and cytoplasm. Over-expression of the OsHsfB4b gene in Arabidopsis and rice can increase the resistance to drought stress. This study provides a basis for understanding the function and evolutionary history of the OsHsf gene family, enriching our knowledge of understanding the biological functions of OsHsfB4b and OsHsfB4d genes involved in the stress response in rice, and also reveals the potential value of OsHsfB4b in rice environmental adaptation improvement.

Genome-wide identification and functional analyses of heat shock transcription factors involved in heat and drought stresses in ryegrass

Italian ryegrass ( Lolium multiflorum ) is one of the most important and widely cultivated cool-season forage and turfgrass around the world. Heat and drought are two key factors limiting its growth and distribution. Heat shock transcription factors ( Hsfs ) are important regulators involved in various abiotic stresses; however, how the Hsf family responds to heat and drought stresses and the roles of LmHsfs in heat and drought tolerance remain to be determined in Italian ryegrass . In the present study, 16 putative Hsf genes were identified by whole-genome bioinformatics analysis, including eight HsfAs , five HsfBs , and three HsfCs . The subcellular localization, physiological and biochemical characteristics, conserved domains, gene structure, and phylogenetic relationship of the Hsf family were systematically analyzed in silico. Gene expression profiling analyses revealed that all Hsfs were responsive to heat and drought stresses. Most Hsfs , including LmHsfA2 , LmHsfA3 , LmHsfA4 , LmHsfA5 , LmHsfA6 , LmHsfA7 , LmHsfA8 , LmHsfB1 , and LmHsfB2 , were induced to the highest expression levels at 1 h of heat stress treatment, while the majority of LmHsfs were up-regulated with a peak at 10 d of drought stress treatment. Subsequently, the biological function of LmHsfA5 which was induced by both heat and drought stresses was analyzed. Heterologous overexpression of LmHsfA5 improved heat and drought tolerance in transgenic Arabidopsis . The qRT-PCR analysis showed that the expression of APX2 and HSP18.2 were significantly induced in LmHsfA5- overexpressing lines under both normal and heat stress conditions. What’s more, yeast one-hybrid, dual-luciferase assay, and electrophoretic mobility shift assay exhibited that LmHsfA5 could bind to the promoters of LmHSP18.2 and LmAPX2 and induce their expression, indicating that LmHsfA5 may positively regulate heat and drought tolerance via directly activating the expression of LmHSP18.2 and LmAPX2 in ryegrass. This study provides a basis for further research on LmHsfs functions in abiotic stress. • 16 LpHsfs were identified by whole-genome bioinformatics analysis. • All LmHsfs were responsive to heat and drought stresses. • Heterologous expression of LmHsfA5 improved heat and drought tolerance in Arabidopsis. • LmHsfA5 directly activates the expression of LmHSP18.2 and LmAPX2 .