Tolerance In Transgenic Arabidopsis Research Articles

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.

MpNAC1, a transcription factor from the mangrove associate Millettia pinnata, confers salt and drought stress tolerance in transgenic Arabidopsis and rice

Pongamia (Millettia pinnata Syn. Pongamia pinnata), a mangrove associate plant, exhibits good stress tolerance, making it a treasure of genetic resources for crop improvement. NAC proteins are plant-specific transcription factors, which have been elucidated to participate in the regulation and tolerance of abiotic stresses (such as salt and drought). Here, we identified a salt-induced gene from Pongamia, MpNAC1, which encodes an NAC factor sharing five highly conserved domains with other NACs and exhibits close homology to AtNAC19/AtNAC55/AtNAC72 in Arabidopsis. MpNAC1 showed nuclear localization and transcriptional activator activity. MpNAC1-overexpressing Arabidopsis exhibited significantly stronger salt and drought tolerance compared with wild-type plants. The expression levels of stress-responsive genes were activated in transgenic Arabidopsis. Furthermore, the heterologous expression of MpNAC1 also enhanced the salt and drought tolerance of transgenic rice. The major agronomic traits, such as plant height and tiller number, panicle length, grain size, and yield, were similar between the transgenic lines and wild type under normal field growth conditions. RNA-Seq analysis revealed that MpNAC1 significantly up-regulated stress-responsive genes and activated the biosynthesis of secondary metabolites such as flavonoids, resulting in increased stress tolerance. Taken together, the MpNAC1 increased salt and drought stress tolerance in transgenic plants and did not retard the plant growth and development under normal growth conditions, suggesting the potential of MpNAC1 in breeding stress-resilient crops.

Overexpression of the Panax ginseng CyP gene enhances abiotic and biotic stress tolerance in transgenic Arabidopsis

To explore the response of the cyclophilin (CyP) gene to abiotic and biotic stress, transgenic Arabidopsis was exposed to salt stress and inoculated with Phytophthora cactorum. The CyP gene enhanced salt tolerance by increasing proline content and slowing chlorophyll decline compared to wild-type plants. Under P. cactorum infection, the CyP protein interacted with spores and restricted spore germination to enhance resistance to P. cactorum. Thus, the CyP protein positively regulates gene expression in the abiotic and biotic stress tolerance networks. The CyP gene may be valuable for ginseng breeding programs aiming to develop new cultivars with enhanced stress tolerance.

VvERF117 positively regulates grape cold tolerance through direct regulation of the antioxidative gene BAS1

Cold stress significantly threatens grape quality, yield, and geographical distribution. Although ethylene-responsive factors (ERFs) are recognized for their pivotal roles in cold stress, the regulatory mechanisms of many ERFs contributing to tolerance remain unclear. In this study, we identified the cold-responsive gene VvERF117 and elucidated its positive regulatory function in cold tolerance. VvERF117 exhibits transcriptional activity and localizes to the nucleus. VvERF117 overexpression improved cold tolerance in transgenic Arabidopsis, grape calli, and grape leaves, whereas VvERF117 silencing increased cold sensitivity in grape calli and leaves. Furthermore, VvERF117 overexpression remarkably upregulated the expression of several stress-related genes. Importantly, BAS1, encoding a 2-Cys peroxidase (POD), was confirmed as a direct target gene of VvERF117. Meanwhile, compared to the wild-type, POD activity and H2O2 content were remarkably increased and decreased in VvERF117-overexpressing grape calli and leaves, respectively. Conversely, VvERF117 silencing displayed the opposite trend in grape calli and leaves under cold stress. These findings indicate that VvERF117 plays a positive role in cold resistance by, at least in part, enhancing antioxidant capacity through regulating the POD-encoding gene VvBAS1, leading to effective mitigation of reactive oxygen species.

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.

A Comprehensive Analysis of the Peanut SQUAMOSA Promoter Binding Protein-like Gene Family and How AhSPL5 Enhances Salt Tolerance in Transgenic Arabidopsis.

SPL (SQUAMOSA promoter binding protein-like), as one family of plant transcription factors, plays an important function in plant growth and development and in response to environmental stresses. Despite SPL gene families having been identified in various plant species, the understanding of this gene family in peanuts remains insufficient. In this study, thirty-eight genes (AhSPL1-AhSPL38) were identified and classified into seven groups based on a phylogenetic analysis. In addition, a thorough analysis indicated that the AhSPL genes experienced segmental duplications. The analysis of the gene structure and protein motif patterns revealed similarities in the structure of exons and introns, as well as the organization of the motifs within the same group, thereby providing additional support to the conclusions drawn from the phylogenetic analysis. The analysis of the regulatory elements and RNA-seq data suggested that the AhSPL genes might be widely involved in peanut growth and development, as well as in response to environmental stresses. Furthermore, the expression of some AhSPL genes, including AhSPL5, AhSPL16, AhSPL25, and AhSPL36, were induced by drought and salt stresses. Notably, the expression of the AhSPL genes might potentially be regulated by regulatory factors with distinct functionalities, such as transcription factors ERF, WRKY, MYB, and Dof, and microRNAs, like ahy-miR156. Notably, the overexpression of AhSPL5 can enhance salt tolerance in transgenic Arabidopsis by enhancing its ROS-scavenging capability and positively regulating the expression of stress-responsive genes. These results provide insight into the evolutionary origin of plant SPL genes and how they enhance plant tolerance to salt stress.

Positive Regulatory Roles of Manihot esculenta HAK5 under K+ Deficiency or High Salt Stress.

HAK/KUP/KT family members have been identified as playing key roles in K+ uptake and salt tolerance in numerous higher plants. However, their functions in cassava (Manihot esculenta Cantz) remain unknown. In this study, a gene encoding for a high-affinity potassium transporter (MeHAK5) was isolated from cassava and its function was investigated. Subcellular localization analysis showed that MeHAK5 is a plasma membrane-localized transporter. RT-PCR and RT-qPCR indicated that MeHAK5 is predominantly expressed in cassava roots, where it is upregulated by low potassium or high salt; in particular, its highest expression levels separately increased by 2.2 and 2.9 times after 50 µM KCl and 150 mM NaCl treatments. When heterologously expressed in yeast, MeHAK5 mediated K+ uptake within the cells of the yeast strain CY162 and rescued the salt-sensitive phenotype of AXT3K yeast. MeHAK5 overexpression in transgenic Arabidopsis plants exhibited improved growth and increased shoot K+ content under low potassium conditions. Under salt stress, MeHAK5 transgenic Arabidopsis plants accumulated more K+ in the shoots and roots and had reduced Na+ content in the shoots. As a result, MeHAK5 transgenic Arabidopsis demonstrated a more salt-tolerant phenotype. These results suggest that MeHAK5 functions as a high-affinity K+ transporter under K+ starvation conditions, improving K+/Na+ homeostasis and thereby functioning as a positive regulator of salt stress tolerance in transgenic Arabidopsis. Therefore, MeHAK5 may be a suitable candidate gene for improving K+ utilization efficiency and salt tolerance.

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.

Overexpression of PvWAK3 from seashore paspalum increases salt tolerance in transgenic Arabidopsis via maintenance of ion and ROS homeostasis

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species with extreme salt tolerance, but investigations on its salt tolerance mechanism are limited. A salt induced PvWAK3 from halophyte seashore paspalum was identified in this study. Overexpression of PvWAK3 in Arabidopsis led to increased salt tolerance. Transgenic plants had higher levels of seed germination rate, root length, number of lateral roots, shoot weight, survival rate, Fv/Fm, ETR, and NPQ compared with the wild type (WT) under salt stress. Na+ content was increased and K+ content was decreased after salinity treatment, with lower levels of Na+ and Na+/K+ ratio but higher level of K+ in transgenic plants than in WT under salt stress. The improved maintenance of Na+ and K+ homeostasis was associated with the higher transcript levels of K + -Uptake Permease 4 (KUP4), Potassium Transport 2/3 (AKT2), Salt Overly Sensitive 1 (SOS1) and High-Affinity K + Transporter 5 (HAK5) in transgenic plants compared with WT. Superoxide dismutase (SOD), catalase (CAT) and ascorbate-peroxidase (APX) activities, proline concentration, and P5CS1 transcript were increased after salinity treatment, with higher levels in transgenic lines compared with WT, which led to reduced accumulation of O2·− and H2O2 under salt stress. It is suggested that PvWAK3 regulates salt tolerance positively, which is associated with promoted Na+ and K+ homeostasis, activated antioxidant enzymes, and proline biosynthesis under salt stress.

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.

BcaSOD1 enhances cadmium tolerance in transgenic Arabidopsis by regulating the expression of genes related to heavy metal detoxification and arginine synthesis

Cadmium (Cd), which is a nonessential heavy metal element for organisms, can have a severe impact on the growth and development of organisms that absorb excessive Cd. Studies have shown that Brassica carinata, a semiwild oil crop, has strong tolerance to various abiotic stresses, and RNA-seq has revealed that the B. carinata superoxide dismutase gene (BcaSOD1) likely responds to Cd stress. To elucidate the BcaSOD1 function involved in tolerance of Cd stress, we cloned the coding sequences of BcaSOD1 from a purple B. carinata accession and successfully transferred it into Arabidopsis thaliana. The subcellular localization results demonstrated that BcaSOD1 was primarily located in the plasma membrane, mitochondria and nucleus. Overexpression of BcaSOD1 in transgenic Arabidopsis (OE) effectively decreased the toxicity caused by Cd stress. Compared to the WT (wild type lines), the OE lines exhibited significantly increased activities of antioxidant enzymes (APX, CAT, POD, and SOD) after exposure to 2.5 mM CdCl2. The Cd content of underground (root) in the OE line was dominantly higher than that in the WT; however, the Cd content of aboveground (shoot) was comparable between the OE and WT types. Moreover, the qRT‒PCR results showed that several heavy metal detoxification-related genes (AtIREG2, AtMTP3, AtHMA3, and AtNAS4) were significantly upregulated in the roots of OE lines under Cd treatment, suggesting that these genes are likely involved in Cd absorption in the roots of OE lines. In addition, both comparable transcriptome and qRT-PCR analyses revealed that exogenous BcaSOD1 noticeably facilitates detoxification by stimulating the expression of two arginine (Arg) biosynthesis genes (AtGDH1 and AtGDH2) while inhibiting the expression of AtARGAH1, a negative regulator in biosynthesis of Arg. The Arg content was subsequently confirmed to be significantly enhanced in OE lines under Cd treatment, indicating that BcaSOD1 likely strengthened Cd tolerance by regulating the expression of Arg-related genes. This study demonstrates that BcaSOD1 can enhance Cd tolerance and reveals the molecular mechanism of this gene, providing valuable insights into the molecular mechanism of Cd tolerance in plants.

Unveiling differential expression profiles of the wheat DOG1 gene family and functional analysis of the association between TaDOG1-1 and heat stress tolerance in transgenic Arabidopsis

High temperatures can significantly impact wheat growth and grain yields during the grain-filling stage. In this study, we identified genes that respond to high-temperature stress during the grain-filling stage. We also identified and characterized 24 novel genes of the DOG1 gene family in hexaploid wheat. Motif analysis and conserved domain search revealed substantial similarities among TaDOG1 family members. Phylogenetic analysis demonstrated the evolutionary conservation of the TaDOG1 family across various plant species. Tissue-specific expression profiling indicated consistent patterns, with TaDOG1 genes predominantly expressed in stem tissues. Only TaDOG1-1 exhibited enhanced expression, particularly during hard dough and ripening stages. TaDOG1-1 and TaDOG1-7 exhibited increased expression under heat stress during the grain-filling stage, indicating their heat-responsive nature. Cis-element analysis revealed potential regulatory motifs, suggesting the involvement of TaDOG1-1 and TaDOG1-7 in stress tolerance mechanisms. Yeast two-hybrid screening revealed interacting proteins, including stress-responsive and grain development-associated proteins. To understand the biological function, we overexpressed TaDOG1-1 in Arabidopsis plants and observed enhanced thermotolerance under basal heat stress. Under heat stress, the transgenic plants exhibited increased biomass and elevated expression levels of heat-responsive genes. Furthermore, TaDOG1-1–overexpressing plants showed improved survival rates under soil heat stress, along with a greater accumulation of antioxidant enzymes in leaves. In this study, the identification and functions of the DOG1 gene family provide valuable insights for developing genetic engineering strategies aimed at improving wheat yield under high-temperature stress.

Populus euphratica CPK21 interacts with heavy metal stress-associated proteins to mediate Cd tolerance in Arabidopsis

High cadmium (Cd, 50 µM) caused transcriptional upregulation of Ca2+-dependent protein kinase 21 (CPK21) in roots and leaves of Populus euphratica. The promoter region (1803 bp) of PeCPK21 was engineered to fuse PeCPK21-pro::GUS and transformed in Arabidopsis. The activity of GUS significantly increased in roots, cotyledons, and hypocotyls under 100 μM CdCl2 treatment. The 1599 bp coding sequence of PeCPK21 was transferred into Arabidopsis, and overexpression of PeCPK21 increased Cd tolerance as evidenced by less reduction in root length and plant growth under CdCl2 stress conditions (100 µM, 7 d). PeCPK21-interacting proteins were enriched by HaloTag pull-down and corresponding expression profiles after Cd exposure indicate that PeCPK21 interacts with heavy metal stress-associated proteins (HMAPs) to mediate Cd tolerance in transgenic Arabidopsis. PeCPK21 interacted with the Cd-regulated cation/heavy metal transport proteins annexin, OPT3, COPT5, and PDF2.2 to limit Cd uptake and accumulation in transgenic plants. PeCPK21 may also interact with the V-type proton ATPase subunits, VHA-B1, VHA-C, and AVA-P2, which were transcriptionally upregulated by Cd to accelerate vacuolar Cd compartmentalization. Cd-triggered H2O2 production was limited in plants overexpressing PeCPK21, which was due to the activated antioxidant enzymes. The interaction of PeCPK21 with the antioxidant enzymes TAPX, APX1, APX2, TRXM4, GPX3, CDSP32, and PRXQ, which showed transcriptional upregulation by Cd, could enhance antioxidant defense in transgenic Arabidopsis. It is also possible that PeCPK21 interacts with plasma membrane intrinsic proteins, PIP1–1, PIP2A, and PIP2–7, which had an Cd-enhanced transcript, to improve water status in transgenic Arabidopsis. In summary, we hypothesize that the calcium sensor PeCPK21 can attenuate Cd toxicity by interacting with HMAPs to effectively limit Cd accumulation, increase ROS scavenging, and improve water status in transgenic Arabidopsis.

Identification of Whirly transcription factors in Triticeae species and functional analysis of TaWHY1-7D in response to osmotic stress.

Osmotic stress poses a threat to the production and quality of crops. Whirly transcription factors have been investigated to enhance stress tolerance. In this study, a total of 18 Whirly genes were identified from six Triticeae species, which were classified into Whirly1 and Whirly2. The exon-intron structure, conserved motif, chromosomal location, collinearity, and regulatory network of Whirly genes were also analyzed. Real-time PCR results indicated that TaWHY1 genes exhibited higher expression levels in leaf sheaths and leaves during the seedling stage, while TaWHY2 genes were predominantly expressed in roots. Under PEG stress, the expression levels of TaWHY1-7A, TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D were increased, TaWHY1-7D was reduced, and TaWHY1-4A had no significant change. All TaWHY genes were significantly up-regulated in response to NaCl stress treatment. In addition, TaWHY1-7A and TaWHY1-7D mainly enhanced the tolerance to oxidative stress in yeast cells. TaWHY2s mainly improved NaCl stress tolerance and were sensitive to oxidative stress in yeast cells. All TaWHYs slightly improved the yeast tolerance to d-sorbitol stress. The heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis. In conclusion, these results provide the foundation for further functional study of Whirly genes aimed at improving osmotic stress tolerance in wheat.

PUB63, A Novel Early Heat Responsive Ubiquitin E3 Ligase from Rice Implicates Heat Stress Tolerance

Heat stress has emerged as a major environmental challenge triggering reduced crop yields, compromised growth, and negatively affecting the development of crops. The cellular response to heat stress involves transcriptional and translational reprogramming and regulation of protein homeostasis which is crucial for the heat stress response and leads to the differential expression of many genes associated with protein turnover mechanisms. Plant U-box (PUB) proteins are a class of the Ubiquitin-E3 ligase involved in the Ubiquitin-proteasome pathway. In the present study, we investigated the role of a PUB protein from rice, OsPUB63, involved in heat stress responses in rice. We also identified all PUB coding genes from O. sativa indica and compared with O. sativa japonica. A quantitative real time PCR demonstrated, OsPUB63 as an early heat inducible gene in rice and the subcellular localization study confirmed its presence in the nucleus and cytosol. OsPUB63 was demonstrated as a TPR repeat containing protein with molecular weight of 39.3 KDa. Using a combination of genetic, biochemical, and physiological assays, our work establishes OsPUB63 as crucial in regulating the heat stress response in rice. Specifically, we show that OsPUB63 enhances the heat stress tolerance in transgenic Arabidopsis by promoting the accumulation of proline and reduced MDA content. Our work has identified PUB63 as the first PUB gene family E3 ligase from rice which is implicated in heat stress response and provide a crucial target for improving crop yields under high-temperature conditions in rice.

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.

Isolation of three MiDi19-4 genes from mango, the ectopic expression of which confers early flowering and enhances stress tolerance in transgenic Arabidopsis.

Three Di19-4 genes were identified in mango. Overexpression of MiDi19-4B in A. thaliana promoted earlier flowering and enhanced drought, salt, and ABA resistance. Drought-induced protein 19 (Di19) is a drought-induced protein that is mainly involved in multiple stress responses. Here, three Di19-4 genes (MiDi19-4A/B/C) in mango (Mangifera indica L.) were identified, and the coding sequences (CDS) had lengths of 684, 666, and 672bp and encoded proteins with 228, 222, and 224 amino acids, respectively. The promoters of the MiDi19-4 genes contained phytohormone-, light-, and abiotic stress-responsive elements. The MiDi19-4 genes were expressed in every tissue and highly expressed in leaves. Moreover, MiDi19-4 genes were highly correlated with the vegetative growth period and induced by polyethylene glycol (PEG) or salt stress. MiDi19-4B displayed the highest expression during the vegetative growth period and then showed decreased expression, and MiDi19-4B was highly expressed at both the late stage of the vegetative growth period and the initial stage of the flowering induction period. The 35S::GFP-MiDi19-4B fusion protein was located in the cell nucleus. The transgenic plants ectopically expressing MiDi19-4B exhibited earlier flowering and increased expression patterns of FRUITFULL (AtFUL), APETALA1 (AtAP1), and FLOWERING LOCUS T (AtFT). The drought and salt tolerance of MiDi19-4B transgenic plants was significantly increased, and these plants showed decreased sensitivity to abscisic acid (ABA) and considerably increased expression levels of drought- and salt-related genes and ABA signalling pathway genes. Additionally, bimolecular fluorescence complementation (BiFC) experiments revealed that the MiDi19-4B protein interacted with CAULIFLOWER (MiCAL1), MiCAL2, MiAP1-1, and MiAP1-2. Taken together, these results highlighted the important regulatory roles of MiDi19-4B in tolerance to multiple abiotic stresses and in flowering.

Genome-Wide Analysis of Barley bHLH Transcription Factors and the Functional Characterization of HvbHLH56 in Low Nitrogen Tolerance in Arabidopsis.

Improvement of low nitrogen (LN) tolerance or nitrogen use efficiency (NUE) in crops is imperative for environment-friendly agriculture development. The basic helix-loop-helix (bHLH) transcription factors are involved in multiple abiotic stresses and are suitable as candidate genes for improving LN tolerance. Few studies were performed on the characterization of the HvbHLH gene family and their function in response to LN stress in barley. In this study, 103 HvbHLH genes were identified through genome-wide analysis. HvbHLH proteins were classified into 20 subfamilies based on phylogenetic analysis in barley, which was supported by conserved motifs and gene structure analysis. The stress-related cis-element analysis in the promoters showed that HvbHLHs are probably involved in multiple stress responses. By phylogenetic analysis of HvbHLHs and bHLHs in other plants, some HvbHLHs were predicted to play roles in response to nutrition deficiency stress. Furthermore, at least 16 HvbHLHs were differentially expressed in two barley genotypes differing in LN tolerance under LN stress. Finally, overexpression of HvbHLH56 enhanced LN stress tolerance in transgenic Arabidopsis, suggesting it is an important regulator in LN stress response. The differentially expressed HvbHLHs identified herein may be valuable for the breeding of barley cultivars with LN tolerance.