Arabidopsis Cys2/His2 Zinc Finger Transcription Factor ZAT18 Modulates the Plant Growth-Defense Tradeoff

Abstract

Plant defense responses under unfavorable conditions are often associated with reduced growth. However, the mechanisms underlying the growth-defense tradeoff remain to be fully elucidated, especially at the transcriptional level. Here, we revealed a Cys2/His2-type zinc finger transcription factor, namely, ZAT18, which played dual roles in plant immunity and growth by oppositely regulating the signaling of defense- and growth-related hormones. ZAT18 was first identified as a salicylic acid (SA)-inducible gene and was required for plant responses to SA in this study. In addition, we observed that ZAT18 enhanced the plant immunity with growth penalties that may have been achieved by activating SA signaling and repressing auxin signaling. Further transcriptome analysis of the zat18 mutant showed that the biological pathways of defense-related hormones, including SA, ethylene and abscisic acid, were repressed and that the biological pathways of auxin and cytokinin, which are growth-related hormones, were activated by abolishing the function of ZAT18. The ZAT18-mediated regulation of hormone signaling was further confirmed using qRT-PCR. Our results explored a mechanism by which plants handle defense and growth at the transcriptional level under stress conditions.

Keywords: ZAT18; auxin; hormone signaling; salicylic acid; the growth-defense tradeoff; transcription factor.

Figure 1

ZAT18 is an SA-induced gene that encodes a protein that is important for SA responses. (A) Two-week-old wild-type (WT) seedlings were dipped with 1 mM SA, and samples were collected at the indicated time points to examine the expression levels of ZAT18 with UBQ5 as a reference. Significant differences were detected using Student’s t-test. Data are shown as mean ± s.d. (n = 3, n indicates the number of biological replicates). (B) The expression levels of ZAT18 in the WT and npr1-2 and tga2/5/6 mutants after sterile dH₂O (CK) or 1 mM SA treatment for 1 h. Experiments were performed as described in (A). Significant differences were detected using two-way ANOVA. Data are shown as mean ± s.d. (n = 3, n indicates the number of biological replicates). (C) The expression levels of WRKY54 and WRKY70 in the WT and zat18-1 mutant after sterile dH₂O (CK) or 1 mM SA treatment for 1 h. Experiments were performed as described in (A). n = 3, n indicates the number of biological replicates. (D) Three-week-old WT and zat18-1 and npr1-2 mutants were pre-treated with dH₂O (CK) or 120 μM BTH for 1 day before infiltration with Psm ES4326 (OD_600nm = 0.001). The bacterial titer was checked at 3 days post-inoculation (dpi). cfu, colony forming unit. n = 8, n indicates the number of biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significant difference.

Figure 2

Generation of the zat18cr mutant and its SA-related phenotypes. (A) The Sanger sequencing chromatograms of WT and zat18cr are shown and the inserted A is marked. (B) The protein length encoded by ZAT18 in the WT and zat18cr. The light blue bar indicates the WT sequence of ZAT18 and the red bar represents the unmatched sequence. The bar graph of 50 AA represents the length of 50 amino acids (AAs) in the ZAT18 and zat18cr structures. (C) The expression levels of WRKY54 and WRKY70 in the WT and zat18cr mutant after sterile dH₂O (CK) or 1 mM SA treatment for 1 h. Experiments were performed as described in Figure 1A. n = 3, n indicates the number of biological replicates. (D) BTH-induced resistance in the WT and zat18cr and npr1-2 mutants. Experiments were performed as described in Figure 1D. n = 8, n indicates the number of biological replicates.**** p < 0.0001.

Figure 3

ZAT18 enhances the plant defense against Psm ES4326. (A) Three-week-old wild-type (WT) seedlings were infiltrated with Psm ES4326 (OD_600nm = 0.001) and samples were collected at indicated time points to examine the expression levels of ZAT18 with UBQ5 as a reference. n = 3, n indicates the number of biological replicates. (B) Three-week-old WT and npr1-2 and zat18 mutants were sprayed with Psm ES4326 (OD_600nm = 0.01) and the third to sixth true leaves were collected to measure the bacterial titer at 3 dpi. cfu, colony forming unit. n = 8, n indicates the number of biological replicates. (C,D) The expression levels of ZAT18 (C) and WRKY54 and WRKY70 (D) in three-week-old WT and ZAT18-OE seedlings without any treatment. n = 3, n indicates the number of biological replicates. (E) Three-week-old WT and ZAT18-OE lines were sprayed with Psm ES4326 (OD_600nm = 0.1) and the third to sixth true leaves were collected to measure the bacterial titer at 3 dpi. cfu, colony forming unit. n = 8, n indicates the number of biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

ZAT18 overexpression resulted in growth inhibition. (A–C) Representative plants of ten-day-old (A), three-week-old (B) and eight-week-old (C) WT and ZAT18-OE lines. (D) Ten-day-old WT and ZAT18-OE seedlings were collected and weighed. n = 10, n indicates the number of biological replicates. (E) Mesophyll cells of the WT and ZAT18-OE lines were observed from a paradermal view. The fifth and sixth true leaves from the three-week-old plants were collected, fixed and cleared for microscopic analysis. Bar: 20 μm. (F) The areas of mesophyll cells in (E) were measured using ImageJ. n = 8, n indicates the number of biological replicates. (G) The numbers of mesophyll cells per leaf were determined via dividing the leaf size by the average cell area of the corresponding leaf. n = 8, n indicates the number of biological replicates. ** p < 0.01; **** p < 0.0001.

Figure 5

ZAT18 overexpression lines showed auxin-deficient phenotypes. (A) Representative seedlings of two-week-old WT and ZAT18-OE lines to show the growth status of their roots. (B) The primary root lengths of the two-week-old WT and ZAT18-OE lines were measured using ImageJ. n = 15, n indicates the number of biological replicates. (C) The numbers of visible lateral roots of the two-week-old WT and ZAT18-OE lines. n = 15, n indicates the number of biological replicates. (D) The expression levels of IAA4 and YUC2 in the two-week-old WT and ZAT18-OE seedlings without any treatment. n = 3, n indicates the number of biological replicates. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 6

Transcriptome changes in the zat18-1 mutant. Dozens of three-week-old WT and zat18-1 mutant leaves were collected and RNA was extracted for sequencing. (A) The number of DEGs (differently expressed genes) up/downregulated in the zat18-1 mutant. (B) Gene Ontology (GO) term enrichment (p < 0.05) of the genes that were specifically upregulated in the zat18-1 mutant. (C) The relative expression levels of DEGs involved in hormone pathways. (D) GO term enrichment (p < 0.05) of the genes that were specifically downregulated in the zat18-1 mutant. Hormone-related pathways are indicated by lines with different colors.

Figure 7

Validation of the expression levels of hormone-related DEGs in the zat18 mutant and ZAT18-OE lines using qRT-PCR. (A) The expression levels of representative hormone-related genes identified using RNA sequencing of the zat18 mutants. (B) The expression levels of representative hormone-related genes identified using RNA sequencing of the ZAT18-OE lines. n = 3, n indicates the number of biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significant difference.

Figure 8

The working model of ZAT18. Under stress conditions, the expression of ZAT18 is induced. As a transcription factor, ZAT18 induces plant defense by upregulating the SA, ET and ABA pathways. Meanwhile, the auxin and CK pathways are repressed by ZAT18 to inhibit plant growth.