Ulrike Zentgraf

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.

Identification of a transcription factor specifically expressed at the onset of leaf senescence.

Abstract. The differential expression of genes was analyzed during leaf senescence in Arabidopsis thaliana (L.) Heynh., using suppression subtractive hybridization (SSH). In order to characterize the differential expression of regulatory genes, the analysis was performed at a very early time point when leaves first differed in their photochemical efficiency (Fv/Fm) and cab transcript levels, but no visible sign of senescence, and no expression of SAG12 could be determined. After high-throughput screening, we isolated several differentially expressed cDNA clones, including a transcription factor of the WRKY family, WRKY53. All family members contained the WRKY domain, a 60-amino-acid domain with the conserved WRKYGQK motif at the N-terminal end, together with a novel zinc-finger motif. The mRNA level of WRKY53 increased substantially within the rosette leaves of a 6-week-old plant before the expression of SAG12 became detectable, was constant in all leaves of a 7-week-old plant and decreased again in 8-week-old plants. This indicates that WRKY53 is expressed at a very early time point of leaf senescence and might therefore play a regulatory role in the early events of leaf senescence.

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.

Expression of the Apx gene family during leaf senescence of Arabidopsis thaliana

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H2O2 scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.

G-Box Binding Factor1 Reduces CATALASE2 Expression and Regulates the Onset of Leaf Senescence in Arabidopsis

Hydrogen peroxide (H2O2) is discussed as being a signaling molecule in Arabidopsis (Arabidopsis thaliana) leaf senescence. Intracellular H2O2 levels are controlled by the H2O2-scavenging enzyme catalase in concert with other scavenging and producing systems. Catalases are encoded by a small gene family, and the expression of all three Arabidopsis catalase genes is regulated in a senescence-associated manner. CATALASE2 (CAT2) expression is down-regulated during bolting time at the onset of leaf senescence and appears to be involved in the elevation of the H2O2 level at this time point. To understand the role of CAT2 in senescence regulation in more detail, we used CAT2 promoter fragments in a yeast one-hybrid screen to isolate upstream regulatory factors. Among others, we could identify G-Box Binding Factor1 (GBF1) as a DNA-binding protein of the CAT2 promoter. Transient overexpression of GBF1 together with a CAT2:β-glucuronidase construct in tobacco (Nicotiana benthamiana) plants and Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 mutant plants, the CAT2 decrease in expression and activity at bolting time and the increase in H2O2 could no longer be observed. Consequently, the onset of leaf senescence and the expression of senescence-associated genes were delayed in gbf1 plants, clearly indicating a regulatory function of GBF1 in leaf senescence, most likely via regulation of the intracellular H2O2 content.

bZIPs and WRKYs: two large transcription factor families executing two different functional strategies

bZIPs and WRKYs are two important plant transcription factor (TF) families regulating diverse developmental and stress-related processes. Since a partial overlap in these biological processes is obvious, it can be speculated that they fulfill non-redundant functions in a complex regulatory network. Here, we focus on the regulatory mechanisms that are so far described for bZIPs and WRKYs. bZIP factors need to heterodimerize for DNA-binding and regulation of transcription, and based on a bioinformatics approach, bZIPs can build up more than the double of protein interactions than WRKYs. In contrast, an enrichment of the WRKY DNA-binding motifs can be found in WRKY promoters, a phenomenon which is not observed for the bZIP family. Thus, the two TF families follow two different functional strategies in which WRKYs regulate each other’s transcription in a transcriptional network whereas bZIP action relies on intensive heterodimerization.

Loss of stress-induced expression of catalase3 during leaf senescence in Arabidopsis thaliana is restricted to oxidative stress.

Different stress conditions can induce changes in the activity of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6). The enzyme activities of all SOD and APX isoforms detected in young Arabidopsis leaves remained unaffected or slightly decreased after moderate paraquat treatment. While CAT2 activity also remained unaffected under these conditions, CAT3 enzyme activity was enhanced. In contrast to the enzyme activities, mRNA levels of both cat2 and cat3 were enhanced under oxidative stress induced by either paraquat or the fungal toxin cercosporin. This indicates that, with respect to enzyme activity level, CAT3 is the enzyme which is most sensitive to oxidative stress in this developmental stage and that the enzyme activity of CAT2 is possibly regulated at the post-transcriptional level. Interestingly, cat3 mRNA level and CAT3 activity are not elevated by paraquat treatment in senescing leaves. In contrast, the response to other stress conditions, such as water stress induced by flooding of detached leaves and heat stress, is maintained in senescing leaves. Since changes in stress response are not a general phenomenon in leaf senescence but appear to be restricted to oxidative stress, this might be a specific mechanism to promote senescence in Arabidopsis thaliana.

Molecular evolution of the intergenic spacer in the nuclear ribosomal RNA genes of cucurbitaceae.

SummaryThe intergenic spacer (IGS) of a 10-kbp repeat (clone pRZ7D) of the nuclear 18S, 5.8S, and 25S ribosomal RNA genes ofCucurbita pepo (zucchini) was sequenced and compared to the IGS sequences of two other Cucurbitaceae.Cucurbita maxima (squash), andCucumis sativus (cucumber). The nucleotide sequence and the structural organization of the IGS ofC. pepo andC. maxima are rather similar (between 75 and 100% sequence similarity depending on the region compared). The IGS are mainly composed of three different repeated elements interspersed into unique sequences: GC-rich clusters, a 422-bp AT-rich element including the transcription initiation site (TIS) for RNA polymerase I, and 260-bp repeats in the 5′ external transcribed spacer (D repeats). The TIS is duplicated in the 10-kbp repeat class ofC. pepo, as it is also described for the 11.5-kbp rDNA repeat ofC. maxima. The IGS ofCucumis sativus is also composed of different repeated elements; however, obvious sequence identity to theCucurbita species only occurs around the TIS and the preceding AT-rich region. GC-rich clusters with different primary sequences are present in the IGS of all three plants. Remarkably, the repeated elements in the 5′ETS accumulate TpG and TpNpG motifs, whereas CpG and CpNpG motifs less frequently occur. This accumulation might be caused by the transition of methylated cytosines (inmCpG ormCpNpG motifs) into thymidine via deamination in a previously GC-rich ancestor. The following singular region exhibits 50% G + C inC. pepo, 53% G + C inC. maxima, and 63% G + C inC. sativus. A model for a common ancestor of the 3′IGS for these Cucurbitaceae is proposed.