ABA-responsive Element Binding Research Articles

Genome-Wide Analysis of the Phospholipase Ds in Perennial Ryegrass Highlights LpABFs-LpPLDδ3 Cascade Modulated Osmotic and Heat Stress Responses.

The phospholipase Ds (PLDs) are crucial for cellular signalling and play roles in plant abiotic stress response. In this study, we identified 12 PLD genes from the genome data of perennial ryegrass (Lolium perenne), which is widely used as forage and turfgrass. Among them, LpPLDδ3 was significantly repressed by ABA treatment, and induced by drought stress and heat stress treatments. The ectopic overexpression (OE) of LpPLDδ3 in Arabidopsis enhanced plant tolerance to osmotic and heat stress as demonstrated by an increased survival rate and reduced malondialdehyde (MDA) accumulation and electrolyte leakage (EL). Arabidopsis endogenous ABA RESPONSIVE ELEMENT BINDING FACTORs (ABFs) and heat stress responsive genes were elevated in LpPLDδ3 OE lines under osmotic and heat stress treatments. Additionally, overexpression of LpPLDδ3 in perennial ryegrass protoplasts could increase heat stress tolerance and elevate expression level of heat stress responsive genes. Moreover, LpABF2 and LpABF4 depressed the LpPLDδ3 expression by directly binding to its ABRE core-binding motif of promoter region. In summary, LpPLDδ3 was repressed by LpABF2 and LpABF4 and positively involved in perennial ryegrass osmotic and heat stress responses.

Analysis of expression characteristics and identification of interaction proteins of transcription factor BnaABI5 in Brassica napus.

Rapeseed is one important oil crop in China. However, its planting benefit is frequently affected by environmental stresses such as drought in the northwest region of China. The abscisic acid(ABA) signaling pathway plays an important role in plant abiotic stress response and tolerance, and ABFs/AREBs(ABA-responsive element binding factors/ABA-responsive element binding proteins) are the core transcription factors that regulate the expression of ABA-responsive genes. To dissect the key transcription factors mediated abiotic stress, we mainly characterized abscisic acid insensitive 5(BnaABI5) in rapeseed, including its subcellular localization, expression pattern in response to various stress and tissue-specific expression analysis, transcriptional activity analysis as well as interaction screening with BnaMPKs(mitogen-activated protein kinases). Our results showed that the BnaABI5-GFP fusion protein was localized in the nucleus, and its transcript level is induced by drought stress and was mainly expressed in the roots of rapeseed. Furthermore, BnaABI5 showed transcriptional activation activity through a yeast transactivation assay and it also activated the promoter activity of EM6 target gene in the transient expression system in tobacco leaves. Moreover, BnaABI5 interacted with BnaMPK6 and BnaMPK13 through BiFC and Y2H analysis. This study preliminarily explored the expression characteristics of transcription factor BnaABI5 and its interaction with BnaMPKs, which might help us for further understanding the function of BnaABI5.

Melatonin mitigates drought stress by increasing sucrose synthesis and suppressing abscisic acid biosynthesis in tomato seedlings.

The increasing prevalence of drought events poses a major challenge for upcoming crop production. Melatonin is a tiny indolic tonic substance with fascinating regulatory functions in plants. While plants can respond in several ways to alleviate drought stress, the processes underpinning stress sensing and signaling are poorly understood. Hereafter, the objectives of this investigation were to explore the putative functions of melatonin in the regulation of sugar metabolism and abscisic acid biosynthesis in drought-stressed tomato seedlings. Melatonin (100 μM) and/or water were foliar sprayed, followed by the plants being imposed to drought stress for 14 days. Drought stress significantly decreased biomass accumulation, inhibited photosynthetic activity, and stimulated senescence-associated gene 12 (SAG12) expression. Melatonin treatment effectively reversed drought-induced growth retardation as evidenced by increased leaf pigment and water balance and restricted abscisic acid (ABA) accumulation. Sugar accumulation, particularly sucrose content, was higher in drought-imposed seedlings, possibly owing to higher transcription levels of sucrose non-fermenting 1-related protein kinase 2 (SnKR2.2) and ABA-responsive element binding factors 2 (AREB2). Melatonin addition further uplifted the sucrose content, which coincided with increased activity of sucrose synthase (SS, 130%), sucrose phosphate synthase (SPS, 137%), starch degradation encoding enzyme β-amylase (BAM, 40%) and α-amylase (AMY, 59%) activity and upregulated their encoding BAM1(10.3 folds) and AMY3 (8.1 folds) genes expression at day 14 relative to the control. Under water deficit conditions, melatonin supplementation decreased the ABA content (24%) and its biosynthesis gene expressions. Additionally, sugar transporter subfamily genes SUT1 and SUT4 expression were upregulated by the addition of melatonin. Collectively, our findings illustrate that melatonin enhances drought tolerance in tomato seedlings by stimulating sugar metabolism and negatively regulating ABA synthesis.

Genome-wide analysis of ABF gene family in lettuce (Lactuca sativa L.) reveals the negative roles of LsABF1 in thermally induced bolting

ABSTRACT High-temperature-induced early bolting poses a pressing challenge in annual leaf lettuce cultivation. ABF transcription factors, or ABA-responsive element binding factors, are crucial components of the ABA signalling pathway. In this study, we identified 8 members of the ABF gene family in leaf lettuce, categorising them into groups A, B, and C through phylogenetic analysis. All LsABFs exhibited a conserved domain, RMIKNRESAARSRARKQAY (Bzip_plant_BZIP46), as revealed by multiple sequence alignment. A total of 1242 cis-acting elements were detected, with 323 elements screened. Predictive analysis indicated 304 phosphorylation sites. Expression patterns of LsABFs were similar, peaking on the 8th day, implicating their role in bolting. Silencing LsABF1 positively impacted lettuce bolting, signifying its regulatory potential. This study establishes a theoretical foundation for comprehending the molecular mechanisms governing ABF genes in high-temperature-induced bolting in leaf lettuce.

Essential role of ABA signaling and related transcription factors in phenolic acid and lignin synthesis during muskmelon wound healing.

Abscisic acid (ABA) is a key phytohormone involved in wound healing in fruits and vegetables, while fluridone (FLD) is its synthetic inhibitor. However, it is unknown whether ABA signaling and downstream transcription factors are involved in the synthesis of phenolic acids and lignin monomers in muskmelon wounds, and the underlying mechanisms. In our study, exogenous ABA promoted endogenous ABA synthesis by increasing the levels of β-carotenoid and zeaxanthin, activating 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP), facilitated ABA signaling by increasing the expression levels of protein phosphatases type 2C (CmPP2C) and ABA-responsive element binding factors (CmABF), upregulated the expression levels of CmMYB1 and CmWRKY1, and ABA induced phenylpropanoid metabolism by activating phenylalanine ammonia-lyase (PAL), 4-coenzyme A ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD), which further increased the synthesis of phenolic acids and lignin monomers in muskmelon wounds during healing. Taken together, exogenous ABA induced phenylpropanoid metabolism and increased the synthesis of phenolic acid and lignin monomer in muskmelon wounds during healing, and may be involved in endogenous ABA synthesis and signaling and related transcription factors.

Transcription factor ABF3 modulates salinity stress-enhanced jasmonate signaling in Arabidopsis

Salinity is a severe abiotic stress that affects plant growth and yield. Salinity stress activates jasmonate (JA) signaling in Arabidopsis thaliana, but the underlying molecular mechanism remains to be elucidated. In this study, we confirmed the activation of JA signaling under saline conditions and demonstrated the importance of the CORONATINE INSENSITIVE1 (COI1)-mediated signaling for this process. Phenotypic analyses reflected the negative regulation of JASMONATE ZIM-DOMAIN (JAZ) repressors during salinity stress-enhanced JA signaling. Mechanistic analyses revealed that JAZ proteins physically interact with ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTOR1 (ABF1), AREB1/ABF2, ABF3, and AREB2/ABF4, which belong to the basic leucine zipper (bZIP) transcription factor family and respond to salinity stress. Analyses on the ABF3 overexpression plants and ABF mutants indicated the positive role of ABF3 in regulating JA signaling under saline condition. Furthermore, ABF3 overexpression partially recovered the JA-related phenotypes of JAZ1-Δ3A plants. Moreover, ABF3 was observed to indirectly activate ALLENE OXIDE SYNTHASE (AOS) transcription, but this activation was inhibited by JAZ1. In addition, ABF3 competitively bind to JAZ1, thereby decreasing the interaction between JAZ1 and MYC2, which is the master transcription factor controlling JA signaling. Collectively, our findings have clarified the regulatory effects of ABF3 on JA signaling and provide new insights into how JA signaling is enhanced following an exposure to salinity stress.

Genome-Wide Identification and Expression Profiling of the ABF Transcription Factor Family in Wheat (Triticum aestivum L.).

Members of the abscisic acid (ABA)-responsive element (ABRE) binding factor (ABF) and ABA-responsive element binding protein (AREB) families play essential roles in the regulation of ABA signaling pathway activity and shape the ability of plants to adapt to a range of stressful environmental conditions. To date, however, systematic genome-wide analyses focused on the ABF/AREB gene family in wheat are lacking. Here, we identified 35 ABF/AREB genes in the wheat genome, designated TaABF1-TaABF35 according to their chromosomal distribution. These genes were further classified, based on their phylogenetic relationships, into three groups (A-C), with the TaABF genes in a given group exhibiting similar motifs and similar numbers of introns/exons. Cis-element analyses of the promoter regions upstream of these TaABFs revealed large numbers of ABREs, with the other predominant elements that were identified differing across these three groups. Patterns of TaABF gene expansion were primarily characterized by allopolyploidization and fragment duplication, with purifying selection having played a significant role in the evolution of this gene family. Further expression profiling indicated that the majority of the TaABF genes from groups A and B were highly expressed in various tissues and upregulated following abiotic stress exposure such as drought, low temperature, low nitrogen, etc., while some of the TaABF genes in group C were specifically expressed in grain tissues. Regulatory network analyses revealed that four of the group A TaABFs (TaABF2, TaABF7, TaABF13, and TaABF19) were centrally located in protein-protein interaction networks, with 13 of these TaABF genes being regulated by 11 known miRNAs, which play important roles in abiotic stress resistance such as drought and salt stress. The two primary upstream transcription factor types found to regulate TaABF gene expression were BBR/BPC and ERF, which have previously been reported to be important in the context of plant abiotic stress responses. Together, these results offer insight into the role that the ABF/AREB genes play in the responses of wheat to abiotic stressors, providing a robust foundation for future functional studies of these genes.

Identification and Characterization of the AREB/ABF Gene Family in Three Orchid Species and Functional Analysis of DcaABI5 in Arabidopsis.

AREB/ABF (ABA response element binding) proteins in plants are essential for stress responses, while our understanding of AREB/ABFs from orchid species, important traditional medicinal and ornamental plants, is limited. Here, twelve AREB/ABF genes were identified within three orchids' complete genomes and classified into three groups through phylogenetic analysis, which was further supported with a combined analysis of their conserved motifs and gene structures. The cis-element analysis revealed that hormone response elements as well as light and stress response elements were widely rich in the AREB/ABFs. A prediction analysis of the orchid ABRE/ABF-mediated regulatory network was further constructed through cis-regulatory element (CRE) analysis of their promoter regions. And it revealed that several dominant transcriptional factor (TF) gene families were abundant as potential regulators of these orchid AREB/ABFs. Expression profile analysis using public transcriptomic data suggested that most AREB/ABF genes have distinct tissue-specific expression patterns in orchid plants. Additionally, DcaABI5 as a homolog of ABA INSENSITIVE 5 (ABI5) from Arabidopsis was selected for further analysis. The results showed that transgenic Arabidopsis overexpressing DcaABI5 could rescue the ABA-insensitive phenotype in the mutant abi5. Collectively, these findings will provide valuable information on AREB/ABF genes in orchids.

The tree peony DREB transcription factor PrDREB2D regulates seed α-linolenic acid accumulation.

α-Linolenic acid (ALA), an essential fatty acid (FA) for human health, serves as the precursor of 2 nutritional benefits, docosahexaenoic acid and eicosapentaenoic acid, and can only be obtained from plant foods. We previously found that phospholipid:diacylglycerol acyltransferase 2 (PrPDAT2) derived from ALA-rich tree peony (Paeonia rockii) can promote seed ALA accumulation. However, the regulatory mechanism underlying its promoting effect on ALA accumulation remains unknown. Here, we revealed a tree peony dehydration-responsive element binding transcription factor, PrDREB2D, as an upstream regulator of PrPDAT2, which is involved in regulating seed ALA accumulation. Our findings demonstrated that PrDREB2D serves as a nucleus-localized transcriptional activator that directly activates PrPDAT2 expression. PrDREB2D altered the FA composition in transient overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis (Arabidopsis thaliana) seeds. Repressing PrDREB2D expression in P. rockii resulted in decreased PrPDAT2 expression and ALA accumulation. In addition, PrDREB2D strengthened its regulation of ALA accumulation by recruiting the cofactor ABA-response element binding factor PrABF2b. Collectively, the study findings provide insights into the mechanism of seed ALA accumulation and avenues for enhancing ALA yield via biotechnological manipulation.

Comprehensive phytohormone metabolomic and transcriptomic analysis of tobacco (Nicotiana tabacum) infected by tomato spotted wilt virus (TSWV)

Tomato spotted wilt virus (TSWV) is ranked among the top 10 most destructive viruses globally. It results in abnormal leaf growth, stunting, and even death, significantly affecting crop yield and quality. Phytohormones play a crucial role in regulating plant-virus interactions. However, there is still limited research on the effect of TSWV on phytohormone levels, particularly growth hormones and genes involved in the phytohormone pathway. In our study, we combined phytohormone metabolomics and transcriptomics to examine the impact of TSWV infection on phytohormone content and gene expression profile. Metabolomic results showed that 41 metabolites, including major phytohormones and their precursors and derivatives were significantly altered after 14 days of TSWV inoculation tobacco plants cvK326, with 31 being significantly increased and 10 significantly reduced. Specifically, the levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) were significantly reduced. The levels of indole-3-acetic acid (IAA) have remained unchanged. However, the levels of cytokinin isopentenyladenine (iP) and salicylic acid (SA) significantly increased. The transcriptome analysis revealed 2,746 genes with significant changes in expression. Out of these, 1,072 genes were significantly downregulated, while 1,674 genes were significantly upregulated. Among them, genes involved in ABA synthesis and signaling pathways, such as 9-cis-epoxycarotenoid dioxygenase (NCED), protein phosphatase 2C (PP2C), serine/threonine-protein kinase (SnRK2), and abscisic acid responsive element binding factor (ABF), exhibited significant downregulation. Additionally, expression of the lipoxygenase gene LOX, Jasmonate ZIM domain-containing protein gene JAZ, and transcription factor gene MYC were significantly down-regulated. In the cytokinin pathway, while there were no significant changes in the expression of the cytokinin synthesis genes, a significant downregulation of transcriptionally active factor type-B response regulators (type-B RRs) was observed. In terms of SA synthesis and signaling pathways, the isochorismate synthase gene ICS1 and the pathogenesis-related gene PR1 were significantly upregulated. These results can strengthen the theoretical foundation for understanding the interaction between TSWV and tobacco and provide new insights for the future prevention and control of TSWV.

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.

A Cerasus humilis transcription factor, ChDREB2C, enhances salt tolerance in transgenic Arabidopsis.

DREB transcription factors play important roles in plant responses to various abiotic and biotic stresses. We conducted bioinformatics analysis of ChDREB2C, explored subcellular localization, transcription activation activity, and heterologous expression in Arabidopsis, and measured expression of related physiological indicators and genes under salt stress. A transcription factor of the DREB family was cloned and named ChDREB2C. ChDREB2C protein was localized in the nucleus, and its C-terminal domain exhibited transcriptional activation activity. ChDREB2C formed a homologous dimer in yeast. Arabidopsis plants overexpressing ChDREB2C were more tolerant to salt stress than WT plants, through increased scavenging capacity of ROS and accumulation of proline. Overexpression of ChDREB2C resulted in increased expression of AtSOS1, AtNHX1, AtRD29A, AtRD29B, AtKIN1, AtABA4, and AtABF2 genes. The interaction between ChABF2 (ABA response element binding factor 2) and ChDREB2C was verified using yeast two-hybrid and firefly luciferase assays. The results suggest that ChDREB2C could have a positive role in mediating the abiotic response.

Differential response of phenylpropanoid pathway as linked to hormonal change in two Brassica napus cultivars contrasting drought tolerance.

Oilseed rape (Brassica napus L.) is a significant agro-economic crop with a wide range of uses. Drought is the most frequent unfavourable environmental stressor restraining its growth and development worldwide. This study was conducted to characterize the drought-responsive phenylpropanoid pathway and its link to hormonal changes in two cultivars, drought-resistant "Saturnin" and drought-susceptible "Mosa." Drought susceptibility in cv. Mosa was confirmed by its lower water use efficiency and higher lipid peroxidation levels with reactive oxygen species (ROS) accumulation. In cv. Saturnin, higher salicylic acid (SA) levels and expression of dehydration-responsive element binding 2 (DREB2) and non-expressor of pathogenesis-related gene 1 (NPR1) led to an upregulation of production of anthocyanin pigment 1 (PAP1) and phenylpropanoid pathway-related gene (CHS, F5H and COMT1) expression, increasing hydroxycinnamic acid and flavonoid compound concentrations. However, in cv. Mosa, higher increases in the activity of lignifying enzymes (polyphenol oxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, guaiacol peroxidase) and expression of the lignin synthesis-related gene cinnamyl alcohol dehydrogenase 2 (CAD2) were found along with greater increases in abscisic acid (ABA) levels and upregulation of ABA-responsive element binding 2 (AREB2) and basic helix-loop-helix transcription factor MYC2. These results indicate that drought-induced SA-mediated activation of the hydroxycinnamic acid and flavonoid pathways contributes to drought resistance, whereas ABA-mediated lignification contributes to drought susceptibility.

Candidate regulators of drought stress in tomato revealed by comparative transcriptomic and proteomic analyses.

Drought is among the most common abiotic constraints of crop growth, development, and productivity. Integrating different omics approaches offers a possibility for deciphering the metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. Here, we explored the transcriptional and post-transcriptional changes in drought-stressed tomato plants using transcriptomic and proteomic profiles to determine the molecular dynamics of tomato drought stress responses. We identified 22467 genes and 5507 proteins, among which the expression of 3765 genes and 294 proteins was significantly changed under drought stress. Furthermore, the differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) showed a good correlation (0.743). The results indicated that integrating different omics approaches is promising in exploring the multilayered regulatory mechanisms of plant drought resistance. Gene ontology (GO) and pathway analysis identified several GO terms and pathways related to stress resistance, including response to stress, abiotic stimulus, and oxidative stress. The plant hormone abscisic acid (ABA) plays pivotal roles in response to drought stress, ABA-response element binding factor (AREB) is a key positive regulator of ABA signaling. Moreover, our analysis indicated that drought stress increased the abscisic acid (ABA) content, which activated AREB1 expression to regulate the expression of TAS14, GSH-Px-1, and Hsp, ultimately improving tomato drought resistance. In addition, the yeast one-hybrid assay demonstrated that the AREB1 could bind the Hsp promoter to activate Hsp expression. Thus, this study involved a full-scale analysis of gene and protein expression in drought-stressed tomato, deepening the understanding of the regulatory mechanisms of the essential drought-tolerance genes in tomato.

CONSTANS interacts with and antagonizes ABF transcription factors during salt stress under long-day conditions.

CONSTANS (CO) is a critical regulator of flowering that combines photoperiodic and circadian signals in Arabidopsis (Arabidopsis thaliana). CO is expressed in multiple tissues, including seedling roots and young leaves. However, the roles and underlying mechanisms of CO in modulating physiological processes outside of flowering remain obscure. Here, we show that the expression of CO responds to salinity treatment. CO negatively mediated salinity tolerance under long-day (LD) conditions. Seedlings from co-mutants were more tolerant to salinity stress, whereas overexpression of CO resulted in plants with reduced tolerance to salinity stress. Further genetic analyses revealed the negative involvement of GIGANTEA (GI) in salinity tolerance requires a functional CO. Mechanistic analysis demonstrated that CO physically interacts with 4 critical basic leucine zipper (bZIP) transcription factors; ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTOR1 (ABF1), ABF2, ABF3, and ABF4. Disrupting these ABFs made plants hypersensitive to salinity stress, demonstrating that ABFs enhance salinity tolerance. Moreover, ABF mutations largely rescued the salinity-tolerant phenotype of co-mutants. CO suppresses the expression of several salinity-responsive genes and influences the transcriptional regulation function of ABF3. Collectively, our results show that the LD-induced CO works antagonistically with ABFs to modulate salinity responses, thus revealing how CO negatively regulates plant adaptation to salinity stress.

Identification of ABF/AREB gene family in tomato (Solanum lycopersicum L.) and functional analysis of ABF/AREB in response to ABA and abiotic stresses.

Abscisic acid (ABA) is a plant hormone that plays an important regulatory role in plant growth and stress response. The AREB (ABA-responsive element binding protein)/ABF (ABRE-binding factor) are important ABA-signaling components that participate in abiotic stress response. However, genome-scale analysis of ABF/AREB has not been systemically investigated in tomato. This study was conducted to identify tomato ABF/AREB family members and analyze their response to ABA and abiotic stresses. The results show that a total of 10 ABF/AREB members were identified in tomato, which are randomly distributed on five chromosomes. Domain analysis showed that these members exhibit high protein similarity, especially in the basic leucine zipper (bZIP) domain region. Subcellular localization analysis indicated that all 10 ABF/AREB members are localized in the nucleus. Phylogenetic tree analysis showed that tomato ABF/AREB genes are divided into two groups, and they are similar with the orthologs of other plants. The analysis of cis-acting elements showed that most tomato ABF/AREB genes contain a variety of hormones and stress-related elements. Expression profiles of different tissues indicated that SlABF2 and SlABF10 play an important role in fruit ripening. Finally, qRT-PCR analysis revealed that 10 tomato ABF/AREB genes respond to ABA, with SlABF3 being the most sensitive. SlABF3, SlABF5 and SlABF10 positively respond to salt and cold stresses. SlABF1, SlABF3 and SlABF10 are significantly induced under UV radiation treatment. SlABF3 and SlABF5 are significantly induced in osmotic stress. Overall, this study may provide insight into the role of tomato ABF/AREB homologues in plant response to abiotic stresses, which laid a foundation for future functional study of ABF/AREB in tomato.

Overexpression of PtrAREB3 improved cadmium enrichment and tolerance in poplar

Cadmium is a toxic heavy metal that has serious impacts on human and ecosystem health. Poplars play an active role in the phytoremediation of heavy metals in the environment; whereas only a few studies have investigated the molecular mechanisms by which trees absorb heavy metals from the environment. This study identified an abscisic acid-responsive element binding protein, PtrAREB3, in Populus trichocarpa. PtrAREB3 was principally expressed in leaves and markedly induced following the ABA and Cd treatments. Moreover, the poplar PtrAREB3 acts as direct regulator of target genes. Under Cd stress, PtrAREB3 further activates target genes associated with stress response and promotes cadmium tolerance of poplar by reducing the production of oxides, reducing chlorophyll degradation, increasing the synthesis of proline, and activating the antioxidant system. Furthermore, PtrAREB3 might indirectly regulate gene expression relating to Cd uptake, transport, and detoxification; ultimately, this increases Cd accumulation in plants. After 28 days of Cd treatment, the PtrAREB3 over-expression transgenic lines showed 18.2%− 25.7% higher dry biomass accumulation with a net photosynthesis rate than WT. In addition, overexpression of PtrAREB3 increased Cd accumulation in plants by 40.4%− 52.1%. Overall, the overexpression of PtrAREB3 enhanced Cd accumulation and tolerance, which making it an ideal molecular target for genetic improvement in Populus plants grown in metal contaminated areas in the future.

Genome-Wide Association Analysis of Fruit Shape-Related Traits in Areca catechu

The areca palm (Areca catechu L.) is one of the most economically important palm trees in tropical areas. To inform areca breeding programs, it is critical to characterize the genetic bases of the mechanisms that regulate areca fruit shape and to identify candidate genes related to fruit-shape traits. However, few previous studies have mined candidate genes associated with areca fruit shape. Here, the fruits produced by 137 areca germplasms were divided into three categories (spherical, oval, and columnar) based on the fruit shape index. A total of 45,094 high-quality single-nucleotide polymorphisms (SNPs) were identified across the 137 areca cultivars. Phylogenetic analysis clustered the areca cultivars into four subgroups. A genome-wide association study that used a mixed linear model identified the 200 loci that were the most significantly associated with fruit-shape traits in the germplasms. In addition, 86 candidate genes associated with areca fruit-shape traits were further mined. Among the proteins encoded by these candidate genes were UDP-glucosyltransferase 85A2, the ABA-responsive element binding factor GBF4, E3 ubiquitin-protein ligase SIAH1, and LRR receptor-like serine/threonine-protein kinase ERECTA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the gene that encoded UDP-glycosyltransferase, UGT85A2, was significantly upregulated in columnar fruits as compared to spherical and oval fruits. The identification of molecular markers that are closely related to fruit-shape traits not only provides genetic data for areca breeding, but it also provides new insights into the shape formation mechanisms of drupes.

The abscisic acid–responsive element binding factors MAPKKK18 module regulates abscisic acid–induced leaf senescence in Arabidopsis

The mitogen-activated protein kinase kinase kinase 18 (MAPKKK18) has been reported to play a role in abiotic stress priming in long-term abscisic acid (ABA) response including drought tolerance and leaf senescence. However, the upstream transcriptional regulators of MAPKKK18 remain to be determined. Here, we report ABA-responsive element binding factors (ABFs) as upstream transcription factors of MAPKKK18 expression. Mutants of abf2, abf3, abf4, and abf2abf3abf4 dramatically reduced the transcription of MAPKKK18. Our electrophoresis mobility shift assay and dual-luciferase reporter assay demonstrated that ABF2, ABF3, and ABF4 bound to ABA-responsive element cis-elements within the promoter of MAPKKK18 to transactivate its expression. Furthermore, enrichments of the promoter region of MAPKKK18 by ABF2, ABF3, and ABF4 were confirmed by invivo chromatin immunoprecipitation coupled with quantitative PCR. In addition, we found that mutants of mapkkk18 exhibited obvious delayed leaf senescence. Moreover, a genetic study showed that overexpression of ABF2, ABF3, and ABF4 in the background of mapkkk18 mostly phenocopied the stay-green phenotype of mapkkk18 and, expression levels of five target genes of ABFs, that is, NYE1, NYE2, NYC1, PAO, and SAG29, were attenuated as a result of MAPKKK18 mutation. These findings demonstrate that ABF2, ABF3, and ABF4 act as transcription regulators of MAPKKK18 and also suggest that, at least in part, ABA acts in priming leaf senescence via ABF-induced expression of MAPKKK18.

Integrative analysis of transcriptome and metabolome revealed the mechanisms by which flavonoids and phytohormones regulated the adaptation of alfalfa roots to NaCl stress.

Salinity critically affects the growth and development of alfalfa (Medicago sativa), making it necessary to understand the molecular mechanism of alfalfa's adaptation to salt stress. In this study, alfalfa roots were subjected to salt stress and transcriptomics and metabolomics analyses were performed. The results showed that flavonoid synthesis, hormone synthesis, and transduction pathways may be involved in the alfalfa salt stress adaptation reaction, and that they are related. Combined analysis of differential genes and differential metabolites found that dihydroquercetin and beta-ring hydroxylase (LUT5), ABA responsive element binding factor 2 (ABF2), protein phosphatase PP2C (PP2C) and abscisic acid (ABA) receptor PYL2 (PYL), luteolinidin was significantly correlated with PP2C and phytochrome-interacting factor 4 (PIF4) and (+)-7-isomethyl jasmonate were significantly correlated with flavonol synthase (FLS) gene. (+)-7-isomethyl jasmonate and homoeriodictyol chalcone were significantly correlated with peroxidase (POD). POD was significantly up-regulated under NaCl stress for 6 and 24 h. Moreover, flavonoids, gibberellin (GA), jasmonic acid (JA) and ABA were suggested to play an important role in alfalfa's response to salt stress. Further, GA,ABA, and JA may be involved in the regulation of flavonoids to improve alfalfa's salt tolerance, and JA may be a key signal to promote the synthesis of flavonoids. This study revealed the possible molecular mechanism of alfalfa adaptation to salt stress, and identified a number of salt-tolerance candidate genes from the synthesis and signal transduction pathways of flavonoids and plant hormones, providing new insights into the regulatory network of alfalfa response to salt stress.