[Show abstract][Hide abstract] ABSTRACT: Arabidopsis VQ motif-containing proteins have recently been demonstrated to interact with several WRKY transcription factors; however, their specific biological functions and the molecular mechanisms underlying their involvement in defense responses remain largely unclear. Here, we showed that two VQ genes, VQ12 and VQ29, were highly responsive to the necrotrophic fungal pathogen Botrytis cinerea. To characterize their roles in plant defense, we generated amiR-vq12 transgenic plants by using an artificial miRNA approach to suppress the expression of VQ12, and isolated a loss-of-function mutant of VQ29. Phenotypic analysis showed that decreasing the expression of VQ12 and VQ29 simultaneously rendered the amiR-vq12 vq29 double mutant plants resistant against B. cinerea. Consistently, the B. cinerea-induced expression of defense-related PLANT DEFENSIN1.2 (PDF1.2) was increased in amiR-vq12 vq29. In contrast, constitutively-expressing VQ12 or VQ29 confered transgenic plants susceptible to B. cinerea. Further investigation revealed that VQ12 and VQ29 physically interacted with themselves and each other to form homodimers and heterodimer. Moreover, expression analysis of VQ12 and VQ29 in defense-signaling mutants suggested that they were partially involved in jasmonate (JA)-signaling pathway. Taken together, our study indicates that VQ12 and VQ29 negatively regulate plant basal resistance against B. cinerea.
[Show abstract][Hide abstract] ABSTRACT: Integrating carbon (C), nitrogen (N), and sulfur (S) metabolism is essential for the growth and development of living organisms. MicroRNAs (miRNAs) play key roles in regulating nutrient metabolism in plants. However, how plant miRNAs mediate crosstalk between different nutrient metabolic pathways is unclear. In this study, deep sequencing of Arabidopsis thaliana small RNAs was used to reveal miRNAs that were differentially expressed in response to C, N, or S deficiency. Comparative analysis revealed that the targets of the differentially expressed miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin signal transduction, nutrient homeostasis, and regulation of development. C, N, and S deficiency specifically induced miR169b/c, miR826 and miR395, respectively. In contrast, miR167, miR172, miR397, miR398, miR399, miR408, miR775, miR827, miR841, miR857, and miR2111 are commonly suppressed by C, N, and S deficiency. In particular, the miRNAs that are induced specifically by a certain nutrient deficiency are often suppressed by other nutrient deficiencies. Further investigation indicated that the modulation of nutrient-responsive miRNA abundance affects the adaptation of plants to nutrient starvation conditions. This study revealed that miRNAs function as important regulatory nodes of different nutrient metabolic pathways.
[Show abstract][Hide abstract] ABSTRACT: Key message
AtWRKY53 is an early factor in drought response and activated expression of AtWRKY53 regulates stomatal movement via reduction of H
content and promotion of starch metabolism in guard cells.
Drought is one of the most serious environmental factors limiting the productivity of agricultural crops worldwide. However, the mechanisms underlying drought tolerance in plants remain unclear. AtWRKY53 belongs to the group III of WRKY transcription factors. In this study, we observed both the mRNA and protein products of this gene are rapidly induced under drought conditions. Phenotypic analysis showed AtWRKY53 overexpression lines were hypersensitive to drought stress compared with Col-0 plants. The results of stomatal movement assays and abscisic acid (ABA) content detection indicated that the impaired stomatal closure of 53OV lines was independent of ABA. Further analysis found that WRKY53 regulated stomatal movement via reducing the H2O2 content and promoting the starch metabolism in guard cells. The results of quantitative real-time reverse transcriptase PCR showed that the expression levels of CAT2, CAT3 and QQS were up-regulated in 53OV lines. Chromatin immunoprecipitation assays demonstrated that AtWRKY53 can directly bind to the QQS promoter sequences, thus led to increased starch metabolism. In summary, our results indicated that the activated expression of AtWRKY53 inhibited stomatal closure by reducing H2O2 content and facilitated stomatal opening by promoting starch degradation.
[Show abstract][Hide abstract] ABSTRACT: Trichome initiation and patterning are controlled by the TTG1-bHLH-MYB regulatory complex. Several MYB transcription factors have been determined to function in trichome development via incorporation into this complex. This study examined the role of MYB82, an R2R3-MYB transcription factor, in Arabidopsis trichome development. MYB82 was revealed to be a nuclear-localized transcription activator. Suppression of MYB82 function by fusion with a dominant repression domain (SRDX) resulted in glabrous leaves, as did overexpression of N-terminal-truncated MYB82. Overexpression of MYB82 genomic sequence, but not its cDNA sequence, led to reduced trichome numbers. Further investigation indicated that at least one of the two introns in MYB82 is essential to the protein's trichome developmental function. An MYB-binding box was identified in the third exon of MYB82, which was inferred to be crucial for MYB82 function because the mutation of this box interfered with the ability of MYB82 to rescue the gl1 mutant. Protein interaction analysis revealed that MYB82 physically interacts with GLABRA3 (GL3). In addition, MYB82 and GL1 can form homodimers and heterodimers at R2R3-MYB domains, which may explain why their overexpression reduces trichome numbers. These results demonstrate the functional diversification of MYB82 and GL1 in trichome development.
[Show abstract][Hide abstract] ABSTRACT: Leaf senescence is regulated by diverse developmental and environmental factors. Exogenous jasmonic acid (JA) can induce leaf senescence, whereas auxin suppresses this physiological process. Crosstalk between JA and auxin signaling has been well studied, but not during JA-induced leaf senescence. Here, we found that upon methyl jasmonate treatment, Arabidopsis thaliana wrky57 mutants produced typical leaf senescence symptoms, such as yellowing leaves, low chlorophyll content, and high cell death rates. Further investigation suggested that senescence-associated genes were upregulated in the wrky57 mutants. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of SENESCENCE4 and SENESCENCE-ASSOCIATED GENE12 and represses their transcription. In vivo and in vitro experiments suggested that WRKY57 interacts with JASMONATE ZIM-DOMAIN4/8 (JAZ4/8) and the AUX/IAA protein IAA29, repressors of the JA and auxin signaling pathways, respectively. Consistent with the opposing functions of JA and auxin in JA-induced leaf senescence, JAZ4/8 and IAA29 also displayed opposite functions in JA-induced leaf senescence and competitively interacted with WRKY57. Our results suggested that the JA-induced leaf senescence process can be antagonized by auxin via WRKY57. Moreover, WRKY57 protein levels were downregulated by JA but upregulated by auxin. Therefore, as a repressor in JA-induced leaf senescence, WRKY57 is a common component of the JA- and auxin-mediated signaling pathways.
The Plant Cell 01/2014; 26(1). DOI:10.1105/tpc.113.117838 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitrogen (N) is an essential macronutrient required for plant growth and development. A number of genes respond to N starvation conditions. However, the functions of most of these N-starvation responsive genes are unclear. Our recent survey suggested that many miRNAs are responsive to N starvation in Arabidopsis thaliana. Here, we identified a new miRNA (miR5090) from the complementary transcript of the MIR826 gene. Further investigation uncovered that both miRNA genes recently evolved from the inverse duplication of their common target gene, AOP2. Similar to miR826, miR5090 is induced by N starvation. In contrast, the AOP2 transcript level was negatively correlated with miR826 and miR5090 under N starvation. GUS-fused AOP2 expression suggested that AOP2 was post-transcriptionally suppressed by miR826 and miR5090. miRNA transgenic plants with significantly low AOP2 expression accumulated much less methionine-derived glucosinolates, phenocopying the aop2 mutants. Most glucosinolate synthesis associated genes were repressed under N starvation conditions. Furthermore, miRNA transgenic plants with less glucosinolate displayed enhanced tolerance to N starvation, such as high biomass, more lateral roots, increased chlorophyll and decreased anthocyanin. Meanwhile, N-starvation-responsive genes were upregulated in transgenic plants, implying improved N uptake activity. Our study reveals a mechanism by which A.thaliana regulates the synthesis of glucosinolates to adapt to environmental changes in N availability.
[Show abstract][Hide abstract] ABSTRACT: The precise control of gene regulation, and hence, correct spatio-temporal tissue patterning, is crucial for plant development. Plant microRNAs can constrain the expression of their target genes at post-transcriptional levels. Recently, miR396 has been characterized to regulated leaf development by mediating cleavage of its GRF targets. miR396 is also preferentially expressed in flowers. However, its function in flower development is unclear. In addition to narrow leaves, pistils with a single carpel were also observed in miR396 over-expression plants. The dramatically reduced expression levels of miR396 targets (GRF1, 2, 3, 4, 7, 8, and 9) caused pistil abnormalities, because the miR396-resistant version of GRF was able to rescue miR396-over-expressing plants. Both GRFs and GIFs are highly expressed in developing pistils, and their expression patterns are negatively correlated with that of miR396. GRF interacted with GIF to form the GRF/GIF complex in plant cell nucleus, and both GRFs and GIFs showed transactivation abilities. miR396 suppressed the expression of GRFs, resulting in reduction of GRF/GIF complex. gif single mutant displayed normal pistils whereas gif triple mutant, gif1/gif2/gif3, produced abnormal pistils, which was a phenocopy of 35S:MIR396a/grf5 plants. GRF and GIF function as co-transcription factors and both are required for pistil development. Our analyses reveal an important role for miR396 in controlling carpel number and pistil development via regulation of the GRF/GIF complex.
[Show abstract][Hide abstract] ABSTRACT: The INDUCER OF CBF EXPRESSION (ICE)-C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance.
The Plant Cell 08/2013; 25(8). DOI:10.1105/tpc.113.112631 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant microRNAs (miRNAs) post-transcriptionally regulate target gene expression to modulate growth and development and biotic and abiotic stress responses. By analyzing small RNA deep sequencing data in combination with the genome sequence, we identified 75 conserved miRNAs and 11 novel miRNAs. Their target genes were also predicted. For most conserved miRNAs, the miRNA-target pairs were conserved across plant species. In addition to these conserved miRNA-target pairs, we also identified some papaya-specific miRNA-target regulatory pathways. Both miR168 and miR530 target the Argonaute 1 gene, indicating a second autoregulatory mechanism for miRNA regulation. A non-conserved miRNA was mapped within an intron of Dicer-like 1 (DCL1), suggesting a conserved homeostatic autoregulatory mechanism for DCL1 expression. A 21-nt miRNA triggers secondary siRNA production from its target genes, nucleotide-binding site leucine-rich repeat protein genes. Certain phased-miRNAs were processed from their conserved miRNA precursors, indicating a putative miRNA evolution mechanism. In addition, we identified a Carica papaya-specific miRNA that targets an ethylene receptor gene, implying its function in the ethylene signaling pathway. This work will also advance our understanding of miRNA functions and evolution in plants.
[Show abstract][Hide abstract] ABSTRACT: WRKY transcription factors are key players in the plant immune response, but less is known about their involvement in antiviral defense than about their roles in defense against bacterial or fungi pathogens. Here, we report that Arabidopsis thaliana WRKY DNA-binding protein 8 (WRKY8) has a role in mediating the long-distance movement of crucifer-infecting tobacco mosaic virus (TMV-cg). The expression of WRKY8 was inhibited by TMV-cg infection, and mutation of WRKY8 accelerated the accumulation of TMV-cg in systemically infected leaves. Quantitative RT-PCR analysis showed that the expression of ABA insensitive 4 (ABI4) was reduced and the expression of 1-aminocyclopropane-1-carboxylic acid synthase 6 (ACS6) and ethylene response factor 104 (ERF104) was enhanced in the systemically infected leaves of wrky8. Immunoprecipitation assays demonstrated that WRKY8 could bind selectively to putative W-boxes of the ABI4, ACS6, and ERF104 promoters. Furthermore, TMV-cg infection enhanced WRKY8 binding to the ABI4 promoter but reduced the binding of WRKY8 to the ACS6 and ERF104 promoters, indicating that regulation of ABI4, ACS6, and ERF104 by WRKY8 is at least partially dependent on TMV-cg. Exogenous applications of abscisic acid (ABA) reduced the systemic accumulation of TMV-cg. Mutations in ABA deficient 1, ABA deficient 2, ABA deficient 3, or abi4 accelerated systemic TMV-cg accumulation. In contrast, exogenous application of aminocyclopropane-1-carboxylic acid enhanced the systemic accumulation of TMV-cg, but mutations in acs6, erf104, or an octuple acs mutant inhibited systemic TMV-cg accumulation. Our results demonstrate that WRKY8 is involved in the defense response against TMV-cg through the direct regulation of the expression of ABI4, ACS6, and ERF104 and may mediate the crosstalk between ABA and ethylene signaling during the TMV-cg-Arabidopsis interaction.
Proceedings of the National Academy of Sciences 05/2013; 110(21). DOI:10.1073/pnas.1221347110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.
PLoS ONE 11/2012; 7(11):e48951. DOI:10.1371/journal.pone.0048951 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drought is one of the most serious environmental factors that limit the productivity of agricultural crops worldwide. However, the mechanism underlying drought tolerance in plants is unclear. WRKY transcription factors are known to function in adaptation to abiotic stresses. By screening a pool of WRKY-associated T-DNA insertion mutants, we isolated a gain-of-function mutant, acquired drought tolerance (adt), showing improved drought tolerance. Under drought stress conditions, adt accumulated higher levels of ABA than wild-type plants. Stomatal aperture analysis indicated that adt was more sensitive to ABA than wild-type plants. Molecular genetic analysis revealed that a T-DNA insertion in adt led to activated expression of a WRKY gene that encodes the WRKR57 protein. Constitutive expression of WRKY57 also conferred similar drought tolerance. Consistently with the high ABA content and enhanced drought tolerance, three stress-responsive genes (RD29A, NCED3, and ABA3) were up-regulated in adt. ChIP assays demonstrated that WRKY57 can directly bind the W-box of RD29A and NCED3 promoter sequences. In addition, during ABA treatment, seed germination and early seedling growth of adt were inhibited, whereas, under high osmotic conditions, adt showed a higher seed germination frequency. In summary, our results suggested that the activated expression of WRKY57 improved drought tolerance of Arabidopsis by elevation of ABA levels. Establishment of the functions of WRKY57 will enable improvement of plant drought tolerance through gene manipulation approaches.
[Show abstract][Hide abstract] ABSTRACT: The WRKY transcription factors are involved in plant resistance against both biotrophic and necrotrophic pathogens. Arabidopsis WRKY46 is specifically induced by salicylic acid (SA) and biotrophic pathogen Pseudomonas syringae infection. To determine its possible roles in plant defense and elucidate potential functional redundancy with structurally related WRKY70 and WRKY53, we examined loss-of-function T-DNA insertion single, double and triple mutants, as well as gain-of-function transgenic WRKY46 over-expressing plants in response to P. syringae. WRKY46 over-expressing plants were more resistant to P. syringae. In contrast, pathogen-infected wrky46wrky70, wrky46wrky53 double mutants and wrky46wrky70wrky53 triple mutants showed increased susceptibility to this pathogen, with increased bacterial growth and more severe disease symptoms. The contrasting responses of gain-of-function plants and loss-of-function mutants were correlated with increased or reduced expression of defense-related PR1 gene. Expression studies of WRKY46, WRKY70, and WRKY53 in various defense-signaling mutants suggested that they are partially involved in SA-signaling pathway. In addition, our findings demonstrated negative cross-regulation among these three genes. These results indicate that WRKY46, WRKY70, and WRKY53 positively regulate basal resistance to P. syringae; and that they play overlapping and synergetic roles in plant basal defense.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are a class of small RNAs that specifically suppress their target genes by transcript cleavage or/and translation repression. Natural miRNA precursors have been used for the backbones of artificial miRNA precursors, which can give rise to expected artificial miRNAs with which to repress specific target genes. Artificial miRNA technology is a powerful tool to silence genes of interest. However, it is costly and time-consuming to construct artificial miRNA precursors by the use of an overlapping PCR method. We describe a new strategy to construct artificial miRNAs. A miRNA gene consists of three components (upstream, stem-loop, and downstream regions). Upstream and downstream regions of a natural miRNA transcript were amplified in conjunction with the introduction of two suitable restriction sites in the amplicons, which were inserted into a plasmid to form a median vector. Production of an artificial miRNA vector was easily achieved by insertion of an artificial stem-loop into the median vector. The artificial miRNAs produced by this method efficiently repressed their target genes in Arabidopsis. In addition, two artificial miRNA constructs were expressed as one polycistron driven by the CaMV 35S promoter and their targets were suppressed simultaneously in Arabidopsis. Thus, artificial miRNAs are a powerful tool with which to analyze rapidly the functions of not only a single gene or multiple homologous genes, but also multiple non-homologous genes.
[Show abstract][Hide abstract] ABSTRACT: The WRKY gene family has been suggested to play important roles in the regulation of transcriptional reprogramming associated with plant stress responses. Modification of the expression patterns of WRKY genes and/or changes in their activity contribute to the elaboration of various signaling pathways and regulatory networks. Furthermore, a single WRKY gene often responds to several stress factors, and then their proteins may participate in the regulation of several seemingly disparate processes as negative or positive regulators. WRKY proteins also function via protein-protein interaction and autoregulation or cross-regulation is extensively recorded among WRKY genes, which help us understand the complex mechanisms of signaling and transcriptional reprogramming controlled by WRKY proteins. Here, we review recent progress made in starting to reveal the role of WRKY transcription factors in plant abiotic stresses. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.
[Show abstract][Hide abstract] ABSTRACT: Arabidopsis WRKY proteins are plant-specific transcription factors, encoded by a large gene family, which contain the highly conserved amino acid sequence WRKYGQK and the zinc-finger-like motifs, Cys(2)His(2) or Cys(2)HisCys. They can recognize and bind the TTGAC(C/T) W-box ciselements found in the promoters of target genes, and are involved in the regulation of gene expression during pathogen defense, wounding, trichome development, and senescence. Here we investigated the physiological function of the Arabidopsis WRKY22 transcription factor during dark-induced senescence. WRKY22 transcription was suppressed by light and promoted by darkness. In addition, AtWRKY22 expression was markedly induced by H(2)O(2). These results indicated that AtWRKY22 was involved in signal pathways in response to abiotic stress. Dark-treated AtWRKY22 over-expression and knockout lines showed accelerated and delayed senescence phenotypes, respectively, and senescence-associated genes exhibited increased and decreased expression levels. Mutual regulation existed between AtWRKY22 and AtWRKY6, AtWRKY53, and AtWRKY70, respectively. Moreover, AtWRKY22 could influence their relative expression levels by feedback regulation or by other, as yet unknown mechanisms in response to dark. These results prove that AtWRKY22 participates in the dark-induced senescence signal transduction pathway.