WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana

Abstract

The plant-specific WRKY transcription factor (TF) family with 74 members in Arabidopsis thaliana appears to be involved in the regulation of various physiological processes including plant defence and senescence. WRKY53 and WRKY70 were previously implicated as positive and negative regulators of senescence, respectively. Here the putative function of other WRKY group III proteins in Arabidopsis leaf senescence has been explored and the results suggest the involvement of two additional WRKY TFs, WRKY 54 and WRKY30, in this process. The structurally related WRKY54 and WRKY70 exhibit a similar expression pattern during leaf development and appear to have co-operative and partly redundant functions in senescence, as revealed by single and double mutant studies. These two negative senescence regulators and the positive regulator WRKY53 were shown by yeast two-hydrid analysis to interact independently with WRKY30. WRKY30 was expressed during developmental leaf senescence and consequently it is hypothesized that the corresponding protein could participate in a senescence regulatory network with the other WRKYs. Expression in wild-type and salicylic acid-deficient mutants suggests a common but not exclusive role for SA in induction of WRKY30, 53, 54, and 70 during senescence. WRKY30 and WRKY53 but not WRKY54 and WRKY70 are also responsive to additional signals such as reactive oxygen species. The results suggest that WRKY53, WRKY54, and WRKY70 may participate in a regulatory network that integrates internal and environmental cues to modulate the onset and the progression of leaf senescence, possibly through an interaction with WRKY30.

Fig. 1.

Arabidopsis WRKY group III transcription factor family. RT-qPCR time course study of WRKY group IIIa (A) and IIIb (B) gene expression in wild-type leaves treated with 5 mM salicylic acid (SA). Phylogenetic relationships between these WRKY group III transcription factors are indicated below the expression data. Protein alignment was carried out with ClustalX and the trees were constructed by Neighbor–Joining distance analysis. Line lengths indicate the relative distances between nodes. (C) Protein sequence alignment of WRKY54 and WRKY70. The WRKY domain is underlined, with the consensus motif WRKYGQK and the zinc-finger motif C2-HC in boxes. Symbols on the consensus lines represent amino acid positions: ‘*’ fully conserved, ‘:’ one of the strong amino acids group is conserved, and ’.’ one of the weak amino acid groups is conserved.

Fig. 2.

Identification of WRKY group III transcription factor interactions with yeast two-hybrid analysis. A split-ubiquitin system was used to screen interactions. Yeast strain NMY51 was co-transformed with various bait and prey constructs as indicated and plated on SD medium without Leu and Trp (line 1: all transformed yeast grown with red/white colonies depending on protein interactions) and without Leu, Trp, His, and Ade (line 2: transformed yeast grown depending on protein interactions). Each transformed yeast line was used to perform X-gal assays on the pellet (line 3). The largeT gene was used as bait control. Vectors carrying NubI or NubG were used as a prey control for negative and positive interactions, respectively.

Fig. 3.

Time course of WRKY30 and WRKY54 expression during developmental leaf senescence. (A) WRKY expression was measured by RT-qPCR on RNA isolated from wild-type leaves 5 and 6 of different developmental stages. RNA samples were collected each week, from 3-week-old plants. (B) Expression of the senescence-related genes CAB and SAG12 was measured by RT-qPCR from the same samples to monitor progress of senescence. (C) Chlorophyll content in wild-type leaves 4 and 5 at each senescence stage. (D) Picture of leaf number 5 at each time point of collection.

Fig. 4.

Characterization of WRKY transgenic lines. (A) Schematic representation of WRKY54 and WRKY70 gene structure indicating the location of T-DNA insertions. Exons are shown as dark boxes. The grey part indicates the region encoding the WRKY domain. (B) RT-qPCR analysis of WRKY54 and WRKY70 transcript levels in wrky54/wrky70 single and double mutants sprayed with 5 mM SA, compared with wild-type plants. Measurements were done 5 h after treatment. (C) RNA gel blot analysis of the WRKY30 transcript level in two independent miRNA-WRKY30 lines sprayed with 5 mM SA compared with wild-type plants. Measurments were done 5 h after treatment. EtBr (ethidium bromide) staining of the gel was used as loading control.

Fig. 5.

Early senescence phenotype of the wrky54/wrky70 double mutant compared with single mutants and wild-type plants. (A) Phenotype of rosette leaves in 5.5-week-old plants: whole plants and excised leaves are arranged according to their age from older to younger. (B) Distribution of leaf senescence stages in 5.5-week-old plants. Leaves were classified into three groups according to their colour: brown/dry, yellow, and green. Seven plants of each line were used. (C) RT-qPCR analysis of expression of senescence-related genes (WRKY53, CAB, SEN1, and SAG12) during developmental leaf senescence in the wrky54/wrky70 double mutant.

Fig. 6.

Expression of WRKY30, WRKY53, WRKY54, and WRKY70 under oxidative stress. WRKY expression was measured by RT-qPCR. (A) RNA samples were isolated from 2-week-old wild-type seedlings submerged in liquid MS medium with 10 mM H₂O₂. (B) RNA samples were extracted from 3-week-old wild-type plants treated with 250 ppb ozone.