Dual-Localized WHIRLY1 Affects Salicylic Acid Biosynthesis via Coordination of ISOCHORISMATE SYNTHASE1, PHENYLALANINE AMMONIA LYASE1, and S-ADENOSYL-L-METHIONINE-DEPENDENT METHYLTRANSFERASE1

Abstract

Salicylic acid (SA) influences developmental senescence and is spatiotemporally controlled by various mechanisms, including biosynthesis, transport, and conjugate formation. Altered localization of Arabidopsis WHIRLY1 (WHY1), a repressor of leaf natural senescence, in the nucleus or chloroplast causes a perturbation in SA homeostasis, resulting in adverse plant senescence phenotypes. WHY1 loss-of-function mutation resulted in SA peaking 5 d earlier compared to wild-type plants, which accumulated SA at 42 d after germination. SA accumulation coincided with an early leaf-senescence phenotype, which could be prevented by ectopic expression of the nuclear WHY1 isoform (nWHY1). However, expressing the plastid WHY1 isoform (pWHY1) greatly enhanced cellular SA levels. Transcriptome analysis in the WHY1 loss-of-function mutant background following expression of either pWHY1 or nWHY1 indicated that hormone metabolism-related genes were most significantly altered. The pWHY1 isoform predominantly affected stress-related gene expression, whereas nWHY1 primarily controlled developmental gene expression. Chromatin immunoprecipitation-quantitative PCR assays indicated that nWHY1 directly binds to the promoter region of isochorismate synthase1 (ICS1), thus activating its expression at later developmental stages, but that it indirectly activates S-adenosyl- l -Met-dependent methyltransferase1 (BSMT1) expression via ethylene response factor 109 (ERF109). Moreover, nWHY1 repressed expression of Phe ammonia lyase-encoding gene (PAL1) via R2R3-MYB member 15 (MYB15) during the early stages of development. Interestingly, rising SA levels exerted a feedback effect by inducing nWHY1 modification and pWHY1 accumulation. Thus, the alteration of WHY1 organelle isoforms and the feedback of SA are involved in a circularly integrated regulatory network during developmental or stress-induced senescence in Arabidopsis.

Figure 1.

Transcript levels of genes encoding key enzymes related to the SA metabolism pathway and SA content in the why1 line during plant development. A, The SA metabolism pathway in the cell. B, Transcript levels of genes encoding key enzymes related to SA metabolism in the why1 line during plant development. C, Content of conjugated (C-SA) and free (F-SA) SA in the wild-type (WT) and why1 mutant plants during the period 28 to 55 dag. D, Changes of conjugated and free SA content in a series of double mutants at 37 and 42 dag. Error bars represent standard deviation of triplicate experiments and statistical significance was checked by a two-way ANOVA. The different lowercase letters indicated the significance. E, Senescence phenotype of 37-d-old double mutants. The relative expression level was normalized to GAPC, and the wild type at 28 dag (B) was set as 1. The error bars represent the mean ± sd of triplicate biological replicates and triplicate technical replicates. Asterisks in B and C indicate significant differences relative to the wild-type line according to pair-wide multiple t tests (*P < 0.05 and **P < 0.01).

Figure 2.

Transcript level analysis of genes encoding key enzymes related to SA and SA content in various WHY1 mutants during plant development. Genes analyzed were related to the SA metabolism pathway (A), SA content C-SA (B). and SA content F-SA (C) during plant development in pWHY1/why1, nWHY1/why1, and pnWHY1/why1 transgenic plants compared to the wild type over the period from 28 to 42 dag. The data represent triplicate biological replicates, with values shown as the means ± se. Asterisks indicate significant differences from the wild type within the respective conditions based on Student’s t test (*P < 0.05 and **P < 0.01).

Figure 3.

The VEX:pWHY1, VEX:nWHY1, and why1 plants exhibit a complex nuclear genetic reprogramming. A, MapMan analysis for gene ontology term enrichment of the entire VEX:pWHY1, VEX:nWHY1, and why1 nuclear transcriptome. B, Histogram representing the ratio of differentially expressed genes enrichment changes of selected biological process of the VEX:pWHY1, VEX:nWHY1, and why1 transcriptome. C, Heatmap of SA metabolism-related gene expression levels of pWHY1/why1, nWHY1/why1, pnWHY1/why1, and why1 plants. VEX:pWHY1, VEX:pWHY1/why1 plants; VEX:nWHY1, VEX:nWHY1/why1 plants.

Figure 4.

WHY1 activates/represses target gene expression. A, Enrichment profiles of WHY1 protein in five target genes, ERF109, MYB15, WRKY33, ICS1, and WRKY53, by ChIP-seq. B, Position of promoter motifs (GTNNNNAAT plus AT-rich) of WHY1-target genes. C, Enrichment folds of WHY1 at the promoters of target genes by ChIP-qPCR at 37 and 42 dag. D, Expression levels of target genes at 37 and 42 dag in the why1 mutant compared to the wild type (WT). Error bars represent the mean ± sd of triplicate biological replicates. Asterisks indicate a significant difference from ACTIN as determined by two-tailed Student’s t test (*P < 0.05 and **P < 0.01).

Figure 5.

Promoter activation assays using the LUC/REN system. A, Structures of activator and reporter constructs. B, Promoters of ICS1, MYB15, ERF109, WRKY53, and WRKY33 genes were coinfiltrated with a vector containing WHY1 under regulation of the ACTIN promoter. C, Coinfiltration of MYB15 and ERF109 with the PAL1, PAL2, ICS1, and BSMT1 promoters. Background promoter activity was assayed by coinfiltration with an empty vector of the same type. Shown are the means ± se of six biological replicates. Asterisks denote statistically significant differences from the empty vector, calculated by Student’s t test (***P = 0.001).

Figure 6.

Phenotyping of WHY1 loss-of-function mutants and mutants of WHY1 downstream target genes. A, Phenotypes of PAL1, ICS1, MYB15, and BSMT1 loss-of-function mutants at 37 dag compared to why1 mutants. Whole rosettes are shown at top, with a graph of the senescent leaf ratios in five plants. B, ROS accumulation of PAL1, ICS1, MYB15, and BSMT1 loss-of-function mutants compared to why1 mutants at 37 dag as determined by NBT and DAB staining. C, Transcript levels of SAG genes in the PAL1 and BSMT1 loss- or gain-of-function mutants and the MBY15, ERF109, and ICS1 loss-of-function mutants at 37 dag determined by RT-qPCR. The data represent triplicate biological replicates. Values are shown as means ± se, with the wild type set to 1 in the heatmap.

Figure 7.

Changes in plastid and nuclear isoform WHY1 protein levels determined by immunodetection in the sid2, pal1, or sid2 pal1 mutants compared to the wild type. A, Expression level of WHY1 in wild-type plants after MeSA treatment for 1, 2, 4, 6, and 8 h. B, WHY1 immunodetection in nuclear extracts after MeSA treatment for 1, 3, and 6 h. C, WHY1 immunodetection in plastid extracts after treatment with MeSA for 1, 3, and 6 h. D and E, WHY1 immunodetection in nuclear and plastid extracts after MeSA treatment for 4 h (D) and in the sid2, pal1, or sid2 pal1 mutants compared to the wild type (WT; E). Coomassie and silver staining are shown as the protein amount loading controls. nWHY1-l, large size (37 kD) of nWHY1; nWHY1-S, small size (29 kD) of nWHY1. The antibody against peptide WHY1 was a commercial product. F and G, Quantification of the alteration of pWHY1 and nWHY1 after MeSA treatment for 4 h (F) and in the sid2, pal1, or sid2 pal1 mutants compared to the wild type (G). The protein band signals were captured and calculated by Image J software (http://www.di.uq.edu.au/sparqimagejblots). Data show the ratio of means of three replicates normalized to histone or PSII. Asterisks indicate significant differences from the water treatment (F) and the wild type (G) as determined by Student’s t test (*P < 0.05 and **P < 0.01).

Figure 8.

Working model of the senescence pathway for dual-localized WHY1 in response to SA. The nuclear isoforms of WHY1 are represented as both a large (37 kD [larger letters]) and a small molecular mass (29 kD [smaller letters]). WHY1 has a dual function in plastids and the nucleus. WHY1 loss-of-function mutation increases SA accumulation at an early developmental stage (37 dag) through increased PAL1 and repressed BSMT1 expression; elevated SA promotes nuclear WHY1 de-modification and promotes ICS1 and BSMT1 expression, thereby balancing SA homeostasis in the cells. High SA levels by ICS1 cause feedback enhancing ROS accumulation, thus promoting senescence. pWHY1 stimulates PAL1/ICS1 expression but represses BSMT1, allowing high levels of SA, also leading to early senescence. Thus, distribution of WHY1 organelle isoforms and the putative feedback of SA form a circularly integrated regulatory network during plant senescence in a developmental-dependent manner. Plastid (Chl) is shown as a green ovary and nucleus (Nuc) as a gray ovary. Lines indicate regulation, wide arrows indicate transfer or translocation, and broken lines indicate uncertainty.