Ying Miao

Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis

Arabidopsis WRKY proteins comprise a family of plant specific zinc-finger-type transcription factors involved in the regulation of gene expression during pathogen defense, wounding, trichome development, and senescence. To understand the regulatory role of the senescence-related WRKY53 factor, we identified target genes of this transcription factor by a pull down assay using genomic DNA and recombinant WRKY53 protein. We isolated a number of candidate target genes including other transcription factors, also of the WRKY family, stress- and defence related genes, and senescence-associated genes (SAGs). WRKY53 protein could bind to these different promoters in vitro and in vivo and it could act either as transcriptional activator or transcriptional repressor depending on the sequences surrounding the W-boxes. Overexpression, RNAi and knock-out lines showed accelerated and delayed senescence phenotypes, respectively, and exhibited altered expression levels of the target genes. WRKY53 can be induced by H2O2 and can regulate its own expression in a negative feed back loop. Our results suggest that WRKY53 acts in a complex transcription factor signalling network regulating senescence specific gene expression and that hydrogen peroxide might be involved in signal transduction.

The antagonist function of Arabidopsis WRKY53 and ESR/ESP in leaf senescence is modulated by the jasmonic and salicylic acid equilibrium.

Crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling is well-studied but not during leaf senescence. We found that the senescence-specific WRKY53 transcription factor interacts with the JA-inducible protein EPITHIOSPECIFYING SENESCENCE REGULATOR (ESR/ESP). The expression of these genes is antagonistically regulated in response to JA and SA, respectively, and each negatively influences the other. Leaf senescence is accelerated in ESR knockout plants (ESR-KO) but retarded in ESR overexpressors (ESR-OE), with the reverse true for WRKY53. ESR-OE showed higher resistance than ESR-KO to bacterial and fungal pathogens. However, pathogen resistance was not altered in WRKY53 overexpressors or knockouts (W53-KO), suggesting that ESR has a greater impact on WRKY53 function in senescence than WRKY53 on ESR function in pathogen resistance. ESR inhibits WRKY53 DNA binding in vitro, and their interaction is localized to the nucleus in vivo; however, ESR is exclusively in the cytoplasm in W53-KO cells, indicating that ESR is brought to the nucleus by the interaction. Therefore, ESR has dual functions: as cytoplasmic epithiospecifier and as negative regulator of WRKY53 in the nucleus. These results suggest that WRKY53 and ESR mediate negative crosstalk between pathogen resistance and senescence, which is most likely governed by the JA and SA equilibrium.

A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53

WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member, WRKY53, is tightly regulated by various mechanisms, and is a convergence node between senescence and pathogen responses. Using WRKY53 in a yeast two-hybrid screen, we isolated the HECT domain E3 ubiquitin ligase UPL5. In contrast to mammals, Arabidopsis contains only seven HECT E3 ubiquitin ligases, whose targets and functions are largely unknown. In yeast cells, UPL5 interacts with WRKY53 via its leucine zipper domain, and this interaction was confirmed in the cytoplasm of plant cells by a bimolecular fluorescence complementation assay. UPL5 was able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol-inducible system in upl5 plants led to degradation of the WRKY53 protein. Expression of both genes is regulated antagonistically in response to hydrogen peroxide, jasmonic acid and plant development. Two T-DNA insertion lines (upl5-1 and upl5-2) showed the same senescence phenotype as WRKY53 over-expressers. Over-expression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 over-expressers. Therefore, we conclude that UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame.

The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana

Many different agriculturally important traits, e.g. number and quality of seeds, timing of seed set, fruit ripening, are affected by senescence. Despite the importance of the senescence processes in plants, our knowledge on regulatory mechanisms of senescence is still poor. A central step is a massive reprogramming of the transcriptome, implying an important role of transcription factors. In Arabidopsis 12-16% of all genes are up- or down-regulated. WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member of this family, WRKY53, is tightly regulated by different unexpected mechanisms and is a convergence node between senescence and biotic and abiotic stress responses.

Single-Stranded DNA-Binding Protein Whirly1 in Barley Leaves Is Located in Plastids and the Nucleus of the Same Cell

This article concerns the intriguing protein Whirly1 (Why1) that belongs to a small family of single-stranded DNA-binding proteins and has been described to have functions in the nucleus ([Desveaux et al., 2002][1]; [Yoo et al., 2007][2]). In contrast, in vitro import assays with isolated organelles

A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thaliana

Arabidopsis WRKY proteins comprise a family of zinc finger-type transcription factors involved in the regulation of gene expression during pathogen defence, wounding, trichome development and senescence. To better understand the regulatory role of the senescence-related WRKY53 factor, we identified upstream regulatory factors using the yeast one-hybrid system. Among others, we identified a DNA-binding protein with a so far unknown function that contains a transcriptional activation domain and a kinase domain with similarities to HPT kinases. In vitro studies revealed that this activation domain protein (AD protein) can phosphorylate itself and that phosphorylation increases its DNA-binding activity to the WRKY53 promoter region. Using the yeast two-hybrid system, an interaction with proteins that were previously shown to bind to the WRKY53 promoter was tested. The AD protein interacted with MEKK1. The interaction with MEKK1 was confirmed in vivo by bimolecular fluorescence complementation (BiFC); however, the AD protein was not phosphorylated by MEKK1 in vitro and vice versa. This indicates that there may be competition between WRKY53 and AD protein for binding of MEKK1 at the WRKY53 promoter. Overexpression and knockout of the respective gene resulted in changes in transcription levels of WRKY53, indicating that AD protein is a positive regulator of WRKY53 expression. Expression of the AD protein gene can be induced by hydrogen peroxide treatment and reduced by jasmonic acid treatment, as previously shown for WRKY53.

The Single-Stranded DNA-Binding Protein WHIRLY1 Represses WRKY53 Expression and Delays Leaf Senescence in a Developmental Stage-Dependent Manner in Arabidopsis

A single-stranded DNA-binding protein represses expression and delays leaf senescence in a developmental stage-dependent manner in Arabidopsis. Leaf senescence in plants involves both positive and negative transcriptional regulation. In this work, we show evidence for the single-stranded DNA-binding protein WHIRLY1 (WHY1) that functions as an upstream suppressor of WRKY53 in a developmental stage-dependent manner during leaf senescence in Arabidopsis (Arabidopsis thaliana). The why1 mutant displayed an early-senescence phenotype. In this background, the expression levels of both WRKY53 and the senescence-associated protease gene SAG12 increased. WHY1 bound to the sequence region that contains an elicitor response element motif-like sequence, GNNNAAATT, plus an AT-rich telomeric repeat-like sequence in the WRKY53 promoter in in vivo and in vitro mutagenesis assays as well as in a chromatin immunoprecipitation assay. This binding to the promoter of WRKY53 was regulated in a developmental stage-dependent manner, as verified by chromatin immunoprecipitation-polymerase chain reaction assay. This direct interaction was further determined by a transient expression assay in which WHY1 repressed β-GLUCURONIDASE gene expression driven by the WRKY53 promoter. Genetic analysis of double mutant transgenic plants revealed that WHY1 overexpression in the wrky53 mutant (oeWHY1wrky53) had no effect on the stay-green phenotype of the wrky53 mutant, while a WHY1 knockout mutant in the wrky53 mutant background (why1wrky53) generated subtle change in the leaf yellow/green phenotype. These results suggest that WHY1 was an upstream regulator of WRKY53 during leaf senescence.

Nuclear targeted AtS40 modulates senescence associated gene expression in Arabidopsis thaliana during natural development and in darkness

The Arabidopsis thaliana AtS40-3 gene belongs to a group of genes sharing the conserved DUF548 sequence motif with up to now unknown function. One member of this group, the barley HvS40, was shown before to play a role in regulation of leaf senescence. Similar as the barley gene, AtS40-3 is induced during senescence and is also regulated in response to dark treatment, ABA, salicylic acid and pathogen attack. By localization of the GUS fusion protein, the AtS40-3 gene was shown to encode a nucleus targeted protein. The s40-3a mutant with a T-DNA insertion in the promoter region of the gene was observed to have a staygreen phenotype. By quantitative real-time PCR analyses expression of the AtS40-3 gene in this mutant was observed to be constitutive and not induced during senescence. This coincided with WRKY53 gene expression in nonsenescent leaves and lowered expression levels of WRKY53 and SAG12 at later stages of development. While in the wildtype expression of AtS40-3 was activated by darkness, in the s40-3a mutant the expression of AtS40-3 stayed at a low level. This coincided with lower expression of the SEN1 and SAG12 genes. In another promoter mutant with a T-DNA insertion further upstream of the coding sequence the levels of AtS40-3 and SAG12 transcripts increased in parallel both in a natural light–dark regime and in darkness. The data on gene expression in promoter T-DNA insertion mutants of the s40-3 gene indicate that AtS40 regulates senescence either by modulation of WRKY53 or by activation of SAG12 independent of WRKY53.