ACBP4-WRKY70-RAP2.12 module positively regulates submergence-induced hypoxia response in Arabidopsis thaliana.

ACYL-CoA-BINDING PROTEINs (ACBPs) play crucial regulatory roles during plant response to hypoxia, but their molecular mechanisms remain poorly understood. Our study reveals that ACBP4 serves as a positive regulator of the plant hypoxia response by interacting with WRKY70, influencing its nucleocytoplasmic shuttling in Arabidopsis thaliana. Furthermore, we demonstrate the direct binding of WRKY70 to the ACBP4 promoter, resulting in its upregulation and suggesting a positive feedback loop. Additionally, we pinpointed a phosphorylation site at Ser638 of ACBP4, which enhances submergence tolerance, potentially by facilitating WRKY70's nuclear shuttling. Surprisingly, a natural variation in this phosphorylation site of ACBP4 allowed A. thaliana to adapt to humid conditions during its historical demographic expansion. We further observed that both phosphorylated ACBP4 and oleoyl-CoA can impede the interaction between ACBP4 and WRKY70, thus promoting WRKY70's nuclear translocation. Finally, we found that the overexpression of orthologous BnaC5.ACBP4 and BnaA7.WRKY70 in Brassica napus increases submergence tolerance, indicating their functional similarity across genera. In summary, our research not only sheds light on the functional significance of the ACBP4 gene in hypoxia response, but also underscores its potential utility in breeding flooding-tolerant oilseed rape varieties.

Coordination between two cis-elements of WRKY33, bound by the same transcription factor, confers humid adaption in Arabidopsis thaliana.

To cope with flooding-induced hypoxia, plants have evolved different strategies. Molecular strategies, such as the N-degron pathway and transcriptional regulation, are known to be crucial for Arabidopsis thaliana's hypoxia response. Our study uncovered a novel molecular strategy that involves a single transcription factor interacting with two identical cis-elements, one located in the promoter region and the other within the intron. This unique double-element adjustment mechanism has seldom been reported in previous studies. In humid areas, WRKY70 plays a crucial role in A. thaliana's adaptation to submergence-induced hypoxia by binding to identical cis-elements in both the promoter and intron regions of WRKY33. This dual binding enhances WRKY33 expression and the activation of hypoxia-related genes. Conversely, in arid regions lacking the promoter cis-element, WRKY70 only binds to the intron cis-element, resulting in limited WRKY33 expression during submergence stress. The presence of a critical promoter cis-element in humid accessions, but not in dry accessions, indicates a coordinated regulation enabling A. thaliana to adapt and thrive in humid habitats.

Exploration of the Regulatory Pathways and Key Genes Involved in the Response to Saline-Alkali Stress in Betula platyphylla via RNA-Seq Analysis.

The pH of saline-alkali soil is high because of carbonate salts, and the deleterious effects of saline-alkali soil on the growth of plants are greater than those of saline soil. Few studies have examined the saline-alkali tolerance of Betula platyphylla at the molecular level. To clarify the regulatory mechanism underlying saline-alkali tolerance in B. platyphylla, RNA sequencing analysis of B. platyphylla seedlings treated with NaHCO3 was conducted. Differences in gene expression in the roots of B. platyphylla seedlings under saline-alkali stress (induced via NaHCO3) for 3 h and 6 h were characterized, and a total of 595 and 607 alkali stress-responsive genes were identified, respectively. Most differentially expressed genes were involved in stress, signal transduction, secondary metabolic process, regulation of jasmonic acid, and the abiotic stimulus signaling pathway. The single nucleotide polymorphism loci in the differentially expressed genes were associated with the alkaline-salt tolerance in birch germplasm. In addition, birch plants overexpressing WRKY70 and NAC9 were obtained using the A. tumefaciens-mediated transient transformation method, and these two genes were found to play key roles in saline-alkali tolerance. Additional study revealed that WRKY70 and NAC9 can increase resistance to saline-alkali stress by enhancing reactive oxygen species scavenging and inhibiting cell death in birch plants. The results of this study enhance our understanding of the saline-alkali stress tolerance of B. platyphylla at the molecular level, and provide several key genes that could be used in the breeding of birch plants in the future.

Brassica napus Transcription Factor Bna.A07.WRKY70 Negatively Regulates Leaf Senescence in Arabidopsis thaliana.

Leaf senescence is the final stage of leaf development and is essential for storage properties and crop productivity. WRKY transcription factors have been revealed to play crucial roles in several biological processes during plant growth and development, especially in leaf senescence. However, the functions of Brassica napus WRKY transcription factors in leaf senescence remain unclear. In the present study, Bna.A07.WRKY70, one paralogue of Brassica napus WRKY70, was cloned from the B. napus cultivar "Zhongshuang11 (ZS11)". We found that Bna.A07.WRKY70 contains a highly conserved WRKY domain and is most closely related to Arabidopsis thaliana WRKY70. The subcellular localization and transcriptional self-activation assays indicated that Bna.A07.WRKY70 functions as a transcription factor. Meanwhile, RT-qPCR and promoter-GUS analysis showed that Bna.A07.WRKY70 is predominantly expressed in the leaves of B. napus and rosette leaves of A. thaliana. In addition, our results demonstrated that ectopic expression of Bna.A07.WRKY70 in A. thaliana wrky70 mutants could restore the senescence phenotypes to wild-type levels. Consistently, the expression levels of three senescence-related marker genes of wrky70 mutants were restored to wild-type levels by ectopic expression of Bna.A07.WRKY70. These findings improve our understanding of the function of Bna.A07.WRKY70 in B. napus and provide a novel strategy for breeding the new stay-green cultivars in rapeseed through genetic manipulation.

Involvement of JMJ15 in the dynamic change of genome-wide H3K4me3 in response to salt stress.

Post-translational histone modifications play important roles in regulating chromatin structure and transcriptional regulation. Histone 3 lysine 4 trimethylation (H3K4me3) is a prominent histone modification mainly associated with gene activation. Here we showed that a histone demethylase, JMJ15, belonging to KDM5/JARID group, is involved in salt stress response in Arabidopsis thaliana. Jmj15 loss-of-function mutants displayed increased sensitivity to salt stress. Moreover, knockout of JMJ15 impaired the salt responsive gene expression program and affected H3K4me3 levels of many stress-related genes under salt-stressed condition. Importantly, we demonstrated that JMJ15 regulated the expression level of two WRKY transcription factors, WRKY46 and WRKY70, which were negatively involved in abiotic stress tolerance. Furthermore, JMJ15 directly bound to and demethylated H3K4me3 mark in the promoter and coding regions of WRKY46 and WRKY70, thereby repressing these two WRKY gene expression under salt stress. Overall, our study revealed a novel molecular function of the histone demethylase JMJ15 under salt stress in plants.

WRKY54 and WRKY70 positively regulate SARD1 and CBP60g expression in plant immunity.

SARD1 and CBP60g are two master regulators in plant immunity. They are required for the constitutive defense responses in the Arabidopsis snc2-1D mutant, which carries a gain-of-function mutation in a receptor-like protein. Here we report that WRKY54 and WRKY70 are required for activation of SARD1 and CBP60g expression and defense responses in snc2-1D. In addition, the induction of SARD1 and CBP60g by the bacterial pathogen Pseudomonas syringae pv. maculicola is significantly reduced in sid2 wrky54 wrky70 triple mutants compared to the sid2 single mutants, suggesting that WRKY54 and WRKY70 positively regulate the SID2-independent expression of SARD1 and CBP60g during pathogen infection. Our study revealed WRKY54 and WRKY70 as positive regulators of SARD1 and CBP60g expression in plant defense.

CHYR1 ubiquitinates the phosphorylated WRKY70 for degradation to balance immunity in Arabidopsis thaliana.

It is critically important for plants to control the trade-off between normal growth and pathogen immunity. However, the underlying molecular mechanism remains largely unknown. Here we report such a mechanism controlled by WRKY70 and its partner CHYR1 in Arabidopsis. We found that both levels of the WRKY70 target gene SARD1 and the phosphorylated forms of WRKY70 were increased in WRKY70OE plants upon Pst DC3000 infection. Mechanistically, phosphorylation of WRKY70 at Thr22 and Ser34 occurs, which then activates SARD1 expression through binding to a WT box. Phosphorylated WRKY70 is degraded by 26S proteasome via CHYR1 when resuming normal growth after infection. In addition, nonphosphorylated WRKY70 represses SARD1 expression by binding to both W (inhibitory activity site) and WT (active activity site) boxes. The binding of WRKY70 to alternative cis-elements of SARD1 through a phosphorylation-mediated switch controlled by CHYR1 contributes to modulating the balance between immunity and growth.

GbMPK3 overexpression increases cotton sensitivity to Verticillium dahliae by regulating salicylic acid signaling.

The soil-born vascular disease Verticillium wilt, which is caused by fungal pathogen Verticillium dahliae, is a devastating disease of cotton worldwide. In the last decade, a large number of genes have been found to participate in cotton-V. dahliae interactions, but the detailed mechanisms of cotton resistance to V. dahliae remain unclear. Here, we functionally characterized MPK3, a MAPK gene from cotton. MPK3 was induced in the roots of both resistant and susceptible cotton cultivars by V. dahliae inoculation. Transgenic cotton and tobacco with constitutively higher GbMPK3 expression conferred higher V. dahliae susceptibility, while MPK3 knockdown in cotton has limited effect on cotton resistance to V. dahliae. Expression profiling revealed that SA-mediated defense pathway genes (WRKY70, PR1, and PR5) accumulated after V. dahliae inoculation in roots of both wild-type and transgenic cotton, and the expression levels of these genes were higher in GbMPK3-overexpressing plants than in wild-type plants, indicating that GbMPK3 upregulation may reduce plant resistance to V. dahliae through regulating salicylic acid signaling transduction.

WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1.

SARD1 is an activator of plant immunity that promotes production of the hormone salicylic acid (SA) and activation of defense gene expression. SARD1 itself is strongly inducible by infection. Here, we investigated the transcriptional control of SARD1. We used yeast one-hybrid assays to identify WRKY70. The WRKY70 binding site was defined using electrophoretic mobility shift assays, and its importance was investigated using an Arabidopsis thaliana protoplast system. The effect of wrky70 mutations was studied by measurements of pathogen growth, SA concentrations, and gene expression by RNA-seq. WRKY70 binds to a GACTTTT motif in the SARD1 promoter in yeast and Arabidopsis protoplasts. Plants with wrky70 mutations have elevated expression of SARD1 in the absence of pathogens, but not when infected. Expression profiling revealed that WRKY70 represses many pathogen-inducible genes in the absence of pathogens, yet is required for activation of many other pathogen-inducible genes in infected plants. The GACTTTT motif is enriched in the promoters of both these gene sets, and conserved in SARD1 orthologs within the Brassicaceae. WRKY70 represses SARD1 by binding the motif GACTTTT in the absence of pathogens. Conservation of the WRKY70 binding among the Brassicaceae suggests that WRKY70 repression of SARD1 is important for fitness.

Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response.

WRKY transcription factors are involved in brassinosteroid signaling and mediate the crosstalk between plant growth and drought tolerance.

Brassinosteroids (BRs) are critical for the plant growth and development. BRs signal through the plasma membrane localized receptor-like kinases to downstream transcription factors BES1/BZR1 to regulate the expression of thousands of genes for various BR responses. In addition to the role in plant growth and development, BRs have been implicated in responses to environmental stresses such as drought. However, the mechanism through which BRs regulate drought have just begun emerging. We have recently found that a group of WRKY transcription factors, WRKY46, WRKY54, WRKY70, which are well known for the function in abiotic and biotic stress, cooperates with BES1 to mediate BR-regulated drought response. The wrky46 wrky54 wrky70 triple mutants showed growth defect, likely due to impaired BR signaling as well as some reduction of endogenous BR level. WRKY46/54/70 cooperates with BES1 to regulate the expression of BR target genes to promote growth. We also found that WRKY46/54/70 negatively modulates drought tolerance by globally repressing drought-inducible gene expression. Thus, our result uncovers a new role for WRKY transcription factors in BR signaling and provides the molecular mechanism for BR-regulated plant growth and drought stress through WRKY46/54/70 and BES1 transcription factors.

Wheat transcription factor TaWRKY70 is positively involved in high-temperature seedling plant resistance to Puccinia striiformis f. sp. tritici.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating disease of wheat (Triticum aestivum) worldwide. Wheat high-temperature seedling plant (HTSP) resistance to Pst is non-race-specific and durable. WRKY transcription factors have been proven to play important roles in plant defence responses to attacks by several pathogens. However, there is no direct evidence as to whether WRKY transcription factors play a role in HTSP resistance to Pst. We isolated a WRKY gene, named TaWRKY70, from wheat cultivar Xiaoyan 6. The expression level of TaWRKY70 was increased significantly when exposed to high temperatures (HTs) during the initial symptom expression stage of Pst infection. The expression of this gene increased in plants treated with ethylene (ET), salicylic acid (SA) and cold (4°C) stresses, but decreased in plants treated with methyl jasmonate (MeJA) and heat (40°C) stresses. Silencing of TaWRKY70 led to greater susceptibility to Pst (in terms of the increase in length of uredinial pustules and the decrease in the number of necrotic cells) compared with non-silenced plants when exposed to HT during the initial symptom expression stage of Pst infection, coinciding with expression changes of the ET- and SA-responsive genes TaPIE1 and TaPR1.1. In contrast, the expression level of the jasmonic acid (JA)-responsive gene TaAOS was not affected by TaWRKY70. These results indicate that TaWRKY70 is positively involved in HTSP resistance, during which SA and ET signalling are probably activated.

Arabidopsis cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Pseudomonas syringae.

Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level.

Direct regulation of WRKY70 by AtMYB44 in plant defense responses.

Cross-talk between hormones is required for plant response to developmental cues and environmental stresses. This cross-talk is achieved through several regulators located in convergence point of distinct hormonal signaling. In plant defense responses, salicylic acid and jasmonic acid affect each other in antagonistic manner. In a recent study we showed that AtMYB44 transcription factor positively regulates SA-mediated defense expression and enhanced resistance to Pst DC3000. On the other hand, AtMYB44 negatively regulates expression of JA-mediated defense gene expression and downregulated resistance to Alternaria brassicicola. Effects of AtMYB44 in SA- and JA-mediated defense responses were achieved through direct regulation of WRKY70 expression which acts as an integrator of cross-talk between SA and JA in plant defense responses. Here we provide further evidence that AtMYB44 regulates defense responses by transcriptional activation of downstream gene, WRKY70. This result shows that AtMYB44 is an integrator of cross-talk between SA and JA in plant defense responses.