WRKY47 transcription factor modulates leaf senescence through regulating PCD-associated genes in Arabidopsis.

Transcription factors play crucial roles in almost all physiological processes including leaf senescence. Cell death is a typical symptom appearing in senescing leaves, which is also classified as developmental programmed cell death (PCD). However, the link between PCD and leaf senescence still remains unclear. Here, we found a WRKY transcription factor WRKY47 positively modulates age-dependent leaf senescence in Arabidopsis (Arabidopsis thaliana). WRKY47 was expressed preferentially in senescing leaves. A subcellular localization assay indicated that WRKY47 was exclusively localized in nuclei. Overexpression of WRKY47 showed precocious leaf senescence, with less chlorophyll content and higher electrolyte leakage, but loss-of-function mutants of WRKY47 delayed this biological process. Through qRT-PCR and dual luciferase reporter assays, we found that WRKY47 could activate the expression of senescence-associated genes (SAGs) and PCD-associated genes to regulate leaf senescence. Furthermore, through electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR, WRKY47 was found to bind to W-box fragments in promoter regions of BFN1 (Bifunctional Nuclease 1) and MC6 (Metacaspase 6) directly. In general, our research revealed that WRKY47 regulates age-dependent leaf senescence by activating the transcription of two PCD-associated genes.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Rapeseed PP2C37 Interacts with PYR/PYL Abscisic Acid Receptors and Negatively Regulates Drought Tolerance.

Global water deficit is a severe abiotic stress threatening the yielding and quality of crops. Abscisic acid (ABA) is a phytohormone that mediates drought tolerance. Protein kinases and phosphatases function as molecular switches in eukaryotes. Protein phosphatases type 2C (PP2Cs) are a major family that play essential roles in ABA signaling and stress responses. However, the role and underlying mechanism of PP2C in rapeseed (Brassica napus L.) mediating drought response has not been reported yet. Here, we characterized a PP2C family member, BnaPP2C37, and its expression level was highly induced by ABA and dehydration treatments. It negatively regulates drought tolerance in rapeseed. We further identified that BnaPP2C37 interacted with multiple PYR/PYL receptors and a drought regulator BnaCPK5 (calcium-dependent protein kinase 5) through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Specifically, BnaPYL1 and BnaPYL9 repress BnaPP2C37 phosphatase activity. Moreover, the pull-down assay and phosphatase assays show BnaPP2C37 interacts with BnaCPK5 to dephosphorylate BnaCPK5 and its downstream BnaABF3. Furthermore, a dual-luciferase assay revealed BnaPP2C37 transcript level was enhanced by BnaABF3 and BnaABF4, forming a negative feedback regulation to ABA response. In summary, we identified that BnaPP2C37 functions negatively in drought tolerance of rapeseed, and its phosphatase activity is repressed by BnaPYL1/9 whereas its transcriptional level is upregulated by BnaABF3/4.

Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

WRKY6 transcription factor modulates root potassium acquisition through promoting expression of AKT1 in Arabidopsis.

Potassium (K+), being an essential macronutrient in plants, plays a central role in many aspects. Root growth is highly plastic and is affected by many different abiotic stresses including nutrient deficiency. The Shaker-type K+ channel Arabidopsis (Arabidopsis thaliana) K+ Transporter 1 (AKT1) is responsible for K+ uptake under both low and high external K+ conditions. However, the upstream transcription factor of AKT1 is not clear. Here, we demonstrated that the WRKY6 transcription factor modulates root growth to low potassium (LK) stress in Arabidopsis. WRKY6 showed a quick response to LK stress and also to many other abiotic stress treatments. The two wrky6 T-DNA insertion mutants were highly sensitive to LK treatment, whose primary root lengths were much shorter, less biomass and lower K+ content in roots than those of wild-type plants, while WRKY6-overexpression lines showed opposite phenotypes. A further investigation showed that WRKY6 regulated the expression of the AKT1 gene via directly binding to the W-box elements in its promoter through EMSA and ChIP-qPCR assays. A dual luciferase reporter analysis further demonstrated that WRKY6 enhanced the transcription of AKT1. Genetic analysis further revealed that the overexpression of AKT1 greatly rescued the short root phenotype of the wrky6 mutant under LK stress, suggesting AKT1 is epistatic to WRKY6 in the control of LK response. Further transcriptome profiling suggested that WRKY6 modulates LK response through a complex regulatory network. Thus, this study unveils a transcription factor that modulates root growth under potassium deficiency conditions by affecting the potassium channel gene AKT1 expression.

Advances in membrane-tethered NAC transcription factors in plants.

Transcription factors are central components in cell signal transduction networks and are critical regulators for gene expression. It is estimated that approximately 10% of all transcription factors are membrane-tethered. MTFs (membrane-bound transcription factors) are latent transcription factors that are inherently anchored in the cellular membrane in a dormant form. When plants encounter environmental stimuli, they will be released from the membrane by intramembrane proteases or by the ubiquitin proteasome pathway and then were translocated to the nucleus. The capacity to instantly activate dormant transcription factors is a critical strategy for modulating diverse cellular functions in response to external or internal signals, which provides an important transcriptional regulatory network in response to sudden stimulus and improves plant survival. NTLs (NTM1-like) are a small subset of NAC (NAM, ATAF1/2, CUC2) transcription factors, which contain a conserved NAC domain at the N-terminus and a transmembrane domain at the C-terminus. In the past two decades, several NTLs have been identified from several species, and most of them are involved in both development and stress response. In this review, we review the reports and findings on NTLs in plants and highlight the mechanism of their nuclear import as well as their functions in regulating plant growth and stress response.

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 in vivo 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.

ANAC087 transcription factor positively regulates age-dependent leaf senescence through modulating the expression of multiple target genes in Arabidopsis.

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness. Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis, there is still a gap to understanding the complex regulatory network. In this study, we discovered that Arabidopsis ANAC087 transcription factor (TF) positively modulated leaf senescence. Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator. Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants, whereas T-DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) profiling showed that the expression of an array of senescence-associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1, NYE1, CEP1, RbohD, SAG13, SAG15, and VPEs, which are involved in programmed cell death (PCD), chlorophyll degradation and reactive oxygen species (ROS) accumulation. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence (NACRS) motif in promoters of its target genes. Moreover, mutation of two representative target genes, BFN1 or NYE1 alleviated the senescence rate of ANAC087-overexpression plants, suggesting their genetic regulatory relationship. Taken together, this study indicates that ANAC087 serves as an important regulator linking PCD, ROS, and chlorophyll degradation to leaf senescence.

WRKY29 transcription factor regulates ethylene biosynthesis and response in arabidopsis.

The gaseous phytohormone ethylene participates in a lot of physiological processes in plants. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and the ACC oxidase (ACO, EC 1.14.17.4) are key enzymes in ethylene biosynthesis. However, how ACSs and ACOs are regulated at the transcriptional level is largely unknown. In the present study, we showed that an Arabidopsis (Arabidopsis thaliana) WRKY-type transcription factor (TF), WRKY29 positively regulated the expression of ACS5, ACS6, ACS8, ACS11 and ACO5 genes and thus promoted basal ethylene production. WRKY29 protein was localized in nuclei and was a transcriptional activator. Overexpression of WRKY29 caused pleiotropic effect on plant growth, development and showed obvious response even without ACC treatment. Inducible overexpression of WRKY29 also reduced primary root elongation and lateral root growth. A triple response assay of overexpression and mutant seedlings of WRKY29 showed that overexpression seedlings had shorter hypocotyls than the transgenic GFP (Green Fluorescence Protein) control, while mutants had no difference from wild-type. A qRT-PCR assay demonstrated that expression of multiple ACSs and ACO5 was up-regulated in WRKY29 overexpression plants. A transactivation assay through dual luciferase reporter system confirmed the regulation of promoters of ACS5, ACS6, ACS8, ACS11 and ACO5 by WRKY29. Both in vivo chromatin immunoprecipitation (ChIP)- quantitative PCR and in vitro electrophoretic mobility shift assay (EMSA) revealed that WRKY29 directly bound to the promoter regions of its target genes. Taken together, these results suggest that WRKY29 is a novel TF positively regulating ethylene production by modulating the expression of ACS and ACO genes.

Rapeseed NAM transcription factor positively regulates leaf senescence via controlling senescence-associated gene expression.

Leaf senescence is one of the most visible forms of programmed cell death in plants. It can be a seasonal adaptation in trees or the final stage in crops ensuring efficient translocation of nutrients to seeds. Along with developmental cues, various environmental factors could also trigger the onset of senescence through transcriptional cascades. Rapeseed (Brassica napus L.) is an important oil crop with its yielding affected by significant falling leaves as a result of leaf senescence, compared to many other crops. Therefore, a better understanding of leaf senescence and developing strategies controlling the progress of leaf senescence in rapeseed is necessary for warranting vegetable oil security. Here we functionally characterized the gene BnaNAM encoding No Apical Meristem (NAM) homologue to identify transcriptional regulation of leaf senescence in rapeseed. A combination of transient and stable expression techniques revealed overexpression of BnaNAM induced ROS production and leaf chlorosis. Quantitative evaluation of up-regulated genes in BnaNAM overexpression lines identified genes related to ROS production (RbohD, RbohF), proteases (ßVPE, γVPE, SAG12, SAG15), chlorophyll catabolism (PaO, PPH) and nucleic acid degradation (BFN1) as the putative downstream targets. A dual luciferase-based transcriptional activation assay of selected promoters further confirmed BnaNAM mediated transactivation of promoters of the downstream genes. Finally, an electrophoretic mobility shift assay further confirmed direct binding of BnaNAM to promoters of ßVPE, γVPE, SAG12, SAG15 and BFN1. Our results therefore demonstrate a novel role of BnaNAM in leaf senescence.

Calcium-dependent Protein Kinase 5 (CPK5) positively modulates drought tolerance through phosphorylating ABA-Responsive Element Binding Factors in oilseed rape (Brassica napus L.).

Drought is an environmental stress that causes severe crop loss. Drought stress can induce abscisic acid (ABA) accumulation and cytoplasmic calcium oscillation. Calcium-dependent protein kinases (CPKs) constitute a group of Ser/Thr protein kinases decoding calcium signals. However, the function and molecular mechanisms of most CPKs in oilseed rape (Brassica napus) remain unknown. Here, we report the functional characterization of BnaCPK5 in drought stress tolerance. BnaCPK5 belongs to Group I of the CPK family and was localized at the plasma membrane and nuclei. Overexpression of BnaCPK5 enhanced drought stress tolerance compared with the control. A screening of interacting proteins identified that BnaCPK5 interacted strongly with two ABA-Responsive Element Binding Factors (ABF/AREBs), BnaABF3 and BnaABF4. BnaCPK5 was shown to phosphorylate both BnaABF3 and BnaABF4 in a kinase assay. Further, it was found that the phosphorylation of BnaABF3 and BnaABF4 by BnaCPK5 increased their transcriptional activities against the famous drought stress marker gene, Responsive to Dehydration (RD) 29B and protein stability. Taken together, these data demonstrate that BnaCPK5 acts as a positive regulator of drought tolerance by, at least in part, phosphorylating two core ABA-signaling components to modulate Late-Embryogenesis Abundant (LEA)-like RD29B expression.

Membrane-Bound Transcriptional Activator NTL1 from Rapeseed Positively Modulates Leaf Senescence through Targeting Genes Involved in Reactive Oxygen Species Production and Programmed Cell Death.

Leaf senescence is the last stage of leaf development and is determined by various environmental and endogenous signals. Leaf senescence can determine plant productivity and fitness. Transcription factors (TFs) with the transmembrane domain constitute a special group of regulatory proteins that can translocate from the membrane system into nuclei to exert the transcriptional function upon endogenous or exogenous stimuli. Reactive oxygen species (ROSs) play an important role in numerous processes throughout the life cycle of plants including leaf senescence. Leaf senescence is characterized by massive programmed cell death (PCD) and is a type of developmental PCD. The transcriptional regulatory relationships between membrane-bound TFs and leaf senescence remain largely uncharacterized, especially in rapeseed (Brassica napus L.), an important oil crop. Here, we show that BnaNTL1 is a membrane-bound NAC (NAM, ATAF, and CUC) TF, which is predominantly expressed in senescent leaves. Expression of BnaNTL1ΔTM, a form of BnaNTL1 devoid of the transmembrane domain, can induce serious HR-like cell death symptoms and ROS accumulation in cells. Plants overexpressing BnaNTL1ΔTM show earlier leaf senescence compared with the control, accompanied by chlorophyll degradation and electrolyte leakage. Genes involved in ROS production (RbohD), PCD (VPEs and CEP1), and leaf senescence (BFN1) are significantly induced and activated by BnaNTL1ΔTM according to the quantitative reverse transcription PCR (qRT-PCR) analysis and dual luciferase reporter (Dual-LUC) assay. Moreover, electrophoretic mobility shift assay revealed that BnaNTL1 directly bound to the NTLBS elements in promoters of RbohD, γVPE, and BFN1. In conclusion, these results demonstrate that BnaNTL1 positively modulates ROS production and HR-like cell death to induce leaf senescence.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

WRKY55 transcription factor positively regulates leaf senescence and the defense response by modulating the transcription of genes implicated in the biosynthesis of reactive oxygen species and salicylic acid in Arabidopsis.

Reactive oxygen species (ROS) and salicylic acid (SA) are two factors regulating leaf senescence and defense against pathogens. However, how a single gene integrates both ROS and SA pathways remains poorly understood. Here, we show that Arabidopsis WRKY55 transcription factor positively regulates ROS and SA accumulation, and thus leaf senescence and resistance against the bacterial pathogen Pseudomonas syringaeWRKY55 is predominantly expressed in senescent leaves and encodes a transcriptional activator localized to nuclei. Both inducible and constitutive overexpression of WRKY55 accelerates leaf senescence, whereas mutants delay it. Transcriptomic sequencing identified 1448 differentially expressed genes, of which 1157 genes are upregulated by WRKY55 expression. Accordingly, the ROS and SA contents in WRKY55-overexpressing plants are higher than those in control plants, whereas the opposite occurs in mutants. Moreover, WRKY55 positively regulates defense against P. syringae Finally, we show that WRKY55 activates the expression of RbohD, ICS1, PBS3 and SAG13 by binding directly to the W-box-containing fragments. Taken together, our work has identified a new WRKY transcription factor that integrates both ROS and SA pathways to regulate leaf senescence and pathogen resistance.

WRKY42 transcription factor positively regulates leaf senescence through modulating SA and ROS synthesis in Arabidopsis thaliana.

Leaf senescence represents the final stage of leaf growth and development, and its onset and progression are strictly regulated; however, the underlying regulatory mechanisms remain largely unknown. In this study we found that WRKY42 was highly induced during leaf senescence. Loss-of-function wrky42 mutants showed delayed leaf senescence whereas the overexpression of WRKY42 accelerated senescence. Transcriptome analysis revealed 2721 differentially expressed genes between wild-type and WRKY42-overexpressing plants, including genes involved in salicylic acid (SA) and reactive oxygen species (ROS) synthesis as well as several senescence-associated genes (SAGs). Moreover, WRKY42 activated the transcription of isochorismate synthase 1 (ICS1), respiratory burst oxidase homolog F (RbohF) and a few SAG genes. Consistently, the expression of these genes was reduced in wrky42 mutants but was markedly increased in transgenic Arabidopsis overexpressing WRKY42. Both in vitro electrophoretic mobility shift assays (EMSAs) and in vivo chromatin immunoprecipitation and dual luciferase assays demonstrated that WRKY42 directly bound to the promoters of ICS1 and RbohF, as well as a few SAGs, to activate their expression. Genetic analysis further showed that mutations of ICS1 and RbohF suppressed the early senescence phenotype evoked by WRKY42 overexpression. Thus, we have identified WRKY42 as a novel transcription factor positively regulating leaf senescence by directly activating the transcription of ICS1, RbohF and SAGs, without any seed yield penalty.

A Rapeseed WRKY Transcription Factor Phosphorylated by CPK Modulates Cell Death and Leaf Senescence by Regulating the Expression of ROS and SA-Synthesis-Related Genes.

Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence. Here, we identified a novel WRKY transcription factor gene WSR1 (WRKY regulating SA and ROS 1) in Brassica napus (rapeseed) in promoting SA and ROS production, which eventually led to leaf senescence thereafter. Its expression increased in senescing leaves. Ca2+-dependent protein kinase (CPK) 5 and -6 interacted with and phosphorylated BnaWSR1. Overexpression of phosphomimic BnaWSR1 (BnaWSR1ca) in rapeseed protoplasts elicited ROS production and cell death while its ectopic expression in Arabidopsis enhanced SA and ROS levels and, hence, accelerated leaf senescence. Furthermore, BnaWSR1ca activated the expression of Isochorismate Synthase 1 (ICS1), Respiratory Burst Oxidase Homologue (Rboh) D, and Senescence-Associated Gene 14 (SAG14). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated that BnaWSR1ca directly bound to promoter regions of ICS1, RbohD, and SAG14. These data have identified a CPK-WSR1 module that integrates SA and ROS to control cell death and leaf senescence.

The R2R3-MYB transcription factor BnaMYB111L from rapeseed modulates reactive oxygen species accumulation and hypersensitive-like cell death.

As one of the largest families of transcription factors in plants, the R2R3-MYB proteins play important roles in diverse biological processes including growth and development, primary and secondary metabolism such as flavonoid and anthocyanin biosynthesis as well as abiotic and biotic stress responses. However, functions of R2R3-MYB genes in rapeseed (Brassica napus L.) remain elusive. Here, we characterized BnaMYB111L, which is homologous to Arabidopsis MYB111 and encodes an R2R3-MYB protein in rapeseed. BnaMYB111L is responsive to abscisic acid (ABA), heat, cold, hydrogen peroxide and fungal pathogen Sclerotinia scelerotiorum treatments through quantitative RT-PCR assay. BnaMYB111L encodes a transcriptional activator and is localized exclusively to nuclei. Interestingly, overexpression of BnaMYB111L in tobacco (Nicotiana benthamiana) and rapeseed protoplasts promoted reactive oxygen species (ROS) production and hypersensitive response-like cell death, accumulation of malondialdehyde (MDA) as well as degradation of chlorophyll. Furthermore, BnaMYB111L expression evoked the alterations of transcript levels of genes encoding ROS-producing enzyme, vacuolar processing enzymes and genes implicated in defense responses. A further dual luciferase reporter assay indicated that BnaMYB111L activated the expression of RbohB, MC4 and ACRE132, which are involved in ROS generation, cell death as well as defense responses. Taken together, this study characterized the function of rapeseed MYB111L and identified its putative target genes involved in ROS production and cell death.

Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors.

Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.

Rapeseed calcium-dependent protein kinase CPK6L modulates reactive oxygen species and cell death through interacting and phosphorylating RBOHD.

Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD.

An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF.

MAIN CONCLUSION: Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression. Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK-WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.