Heat shock factor ZmHsf17 positively regulates phosphatidic acid phosphohydrolase ZmPAH1 and enhances maize thermotolerance

Abstract

Heat stress adversely impacts plant growth, development, and grain yield. Heat shock factors (Hsf), especially the HsfA2 subclass, play a pivotal role in the transcriptional regulation of genes in response to heat stress. In this study, the coding sequence of maize ZmHsf17 was cloned. ZmHsf17 contained conserved domains including a DNA binding domain, oligomerization domain, and transcriptional activation domain. The protein was nuclear localized and had transcription activation activity. Yeast two-hybrid and split luciferase complementation assays confirmed the interaction of ZmHsf17 with members of the maize HsfA2 subclass. Overexpression of ZmHsf17 in maize significantly increased chlorophyll content and net photosynthetic rate, and enhanced the stability of cellular membranes. Through integrative analysis of ChIP-seq and RNA-seq datasets, ZmPAH1, encoding phosphatidic acid phosphohydrolase of lipid metabolic pathways, was identified as a target gene of ZmHsf17. The promoter fragment of ZmPAH1 was bound by ZmHsf17 in protein-DNA interaction experiments in vivo and in vitro. Lipidomic data also indicated that the overexpression of ZmHsf17 increased levels of some critical membrane lipid components of maize leaves under heat stress. This research provides new insights into the role of the ZmHsf17-ZmPAH1 module in regulating thermotolerance in maize.

Keywords: Heat shock factor; ZmPAH1; maize; membrane lipid component; thermotolerance; transcriptional regulation.

Fig. 1.

Amino acid sequence alignment and phylogenetic analysis of ZmHsf17. (A) ZmHsf17 was predicted to be a HsfA2 subclass member in the HEATSTER platform. AHA, aromatic hydrophobic acidic domain; DBD, DNA-binding domain; NES, nuclear export signal; NLS, nuclear localization signal; OD, oligomerization domain. (B) Phylogenetic analysis of ZmHsf17 (Zea mays, QEQ56175) with SbHsfA2b (Sorghum bicolor, XP_002459419), SiHsfA2b (Setaria italica, XP_004955620), DoHsfA2b (Dichanthelium oligosanthes, OEL199-86), PhHsfA2b (Panicum hallii, XP_025799458), OsHsfA2b (Oryza sativa, XP_015645272), HvHsfA2b (Hordeum vulgare, BAJ90333), TaHsfA2b (Triticum aestivum, AHZ44765), and AtHsfA2b (Arabidopsis, AEC07800). (C) Alignment of the amino acid sequence of ZmHsf17 with its homologous proteins in other species.

Fig. 2.

Subcellular localization of ZmHsf17–GFP fusion protein in N. benthamiana epidermal cells. Green fluorescent protein (GFP) exhibited green fluorescence field, 4ʹ,6-diamidino-2-phenylindole (DAPI) exhibited the blue fluorescence of nucleus, chlorophyll fluorescence (CHI) exhibited chloroplast auto-fluorescence field, differential interference contrast (DIC) exhibited bright field. The transfected N. benthamiana plants were cultured at optimal temperature (OT), or subjected to heat stress (HS). Bars in the lower right corner represent 10 μm.

Fig. 3.

Transcriptional activation of ZmHsf17 and protein interaction between ZmHsf17 and HsfA2 members of maize. (A) Transcriptional activation of ZmHsf17 in yeast. The full length of ZmHsf17 and the truncated fragment without transcriptional activating domain (AHA domain) were constructed in pGBKDT-7 (BD). The gradient diluted yeast cells transformed with recombinants were sampled on triple-dropout SD medium containing 1 mM X-α-Gal (TDO, −Trp/−His/−Ade+X-α-Gal). Positive control: pGBKDT7-p53+pGADT7-T; negative control: pGBKT7-p53+pGADT7-Lam. (B) Yeast two-hybrid assays showing growth and staining of the yeast strain co-transformed by ZmHsf17 and HsfA2 members of maize on double-dropout SD medium (DDO, −Trp/−Leu) and quadruple-dropout SD medium containing X-α-Gal (QDO, −Trp/−Leu/−His/−Ade+X-α-Gal). Positive control: pGBKDT7-p53+pGADT7-T; negative control: pGBKT7-p53+pGADT7-Lam. (C) Split luciferase complementation assays showed the interaction of ZmHsf17 with other HsfA2 members of maize in N. benthamiana leaves.

Fig. 4.

Relative expression levels of ZmHsf17 in maize, as determined by qRT-PCR. (A) Tissue specific expression of ZmHsf17 in young root (YR), young stem (YS), young leaf (YL), mature leaf (ML), mature pollen (MP), mature ear (ME), and immature embryos (IE). (B–H) The relative expression levels of ZmHsf17 in maize leaves under different treatment: (B) 42 °C heat stress, (C) 4 °C cold stress, (D) 20% polyethylene glycol (PEG)-simulated drought stress, (E) 10 mM H₂O₂ stress, (F) exogenous 0.8 mM salicylic acid (SA) treatment, (G) exogenous 200 μM abscisic acid (ABA) treatment, (H) exogenous 100 μM methyl jasmonate (MeJA) treatment. The data were normalized to the expression of β-actin and Ubiquitin. The mean values were determined from three independent biological replicates, and error bars represent the standard error of the mean (SEM).

Fig. 5.

Overexpression of ZmHsf17 in maize improved the tolerance to heat stress. (A) The phenotype of wild-type Zheng58 and ZmHsf17 overexpression lines 17_68, 17_88, and 17_89 at optimum temperature (OT). (B) The phenotype of the plants treated at 55 °C HS for 13 h and then recovered at optimum temperature for 1 week (HS). (C) The ZmHsf17–Flag fusion protein detected in all plants by western blot. (D) The content of chlorophyll a and b measured after HS and growth recovery. (E) The net photosynthetic rate (Pn) of leaves measured after HS and growth recovery. (F) The relative electric conductivity (REC) of leaves measured after HS and growth recovery. Data are means ±SD of three independent sample replicates. Significant differences were determined using one-way ANOVA (*P<0.05, **P<0.01).

Fig. 6.

Integrated analysis of RNA-seq and ChIP-seq and identification of downstream target genes of ZmHsf17. (A) Volcano plot of differentially expressed genes (DEG1) in Zheng58 before and after 42 °C heat stress for 40 min. (B) Volcano plot of differentially expressed genes (DEG2) in ZmHsf17 overexpression lines before and after HS. (C) Volcano plot of differentially expressed genes (DEG3) in ZmHsf17 overexpression line under HS compared with Zheng58. (D) Venn diagram showing the overlap among the up-regulated genes in DEG1, DEG2, DEG3 and 1159 enriched target genes from ChIP-seq sets. (E) Gene ontology (GO) enrichment analysis of 3398 up-regulated genes from the DEG3 set. (F) GO enrichment analysis of 1159 enriched target genes from ChIP-seq assay. Biological process (BP), molecular function (MF), and cellular component (CC) are three pivotal aspects of gene function. FDR, false discovery rate.

Fig. 7.

Transcription levels of potential target genes of ZmHsf17 and chromatin binding peak plots of ZmPAH1. (A) Heatmap of the transcription levels of the 14 potential target genes of ZmHsf17. (B) The chromatin binding peak (Peak_611) plots of ZmPAH1/Zm00001d038389 in Input and IP-ZmHsf17 group. (C) The expression levels of ZmPAH1 relative to the expression of two reference genes, β-actin and Ubiquitin, in Zheng58 and three ZmHsf17 overexpression lines (17_68, 17_88, and 17_89) under HS.

Fig. 8.

ZmHsf17 directly bound to the promoter of ZmPAH1 and activated the transcription of luciferase reporter gene. (A) The HSE motifs were found in the promoter regions (−2 kb) of ZmPAH1. Blue dashed lines were used to indicate the nucleotide sequence of typical HSE motifs (5ʹ-nGAAnnTTCn-3ʹ). (B) The promoter of ZmPAH1 was confirmed to be bound by ZmHsf17 through ChIP-qPCR analysis. The IgG-bound target gene levels were used as control to calculate the fold enrichment of the IP-Flag-bound gene. The experiment was based on three biological replicates. Significant differences were determined using Student’s t-test (*P<0.05). (C) Yeast one-hybrid assays show growth of the co-transformed yeast strain by ZmHsf17 and ZmPAH1 promoter on single-dropout SD medium containing 200 mM AbA [DO, −Leu+200 mM aureobasidin A (AbA)]. pGADT7 (AD) is the empty vector control. (D) EMSA assays validated the ZmHsf17 interaction with the HSE motif of ZmPAH1 promoter in vitro. Both the biotin probe (PAH1 probe) and mutated probe (mPAH1 probe) are shown. (E) A dual luciferase reporter assay affirmed that ZmHsf17 activated the transcription of luciferase reporter gene through binding to the ZmPAH1 promoter in N. benthamiana leaves. The experimental group (PAH1pro: LUC+35S: ZmHsf17) and control group (PAH1pro: LUC+35S: null) were tested on the same leaves to ensure the consistency of the experimental data. The bar represents fluorescence intensity. (F) Relative luciferase activity in dual luciferase reporter assay was calculated as LUC/REN. Data are means ±SD of three independent sample replicates. Significant differences were determined using Student’s t-test (**P<0.01).

Fig. 9.

Quantitative lipidomic analysis on leaves from both the ZmHsf17 overexpression line and Zheng58 maize plants subjected to 55 °C heat stress for 13 h. (A) Principal component analysis revealed the clear separation in the major lipid components between Zheng58 and ZmHsf17 overexpression line. (B) Total content of all detected lipid components in Zheng58 and ZmHsf17 overexpression line. Significant differences were determined using Student’s t-test (**P<0.01). (C) The total content of all components in each lipid subclass related to the ZmPAH1-associated lipid metabolism pathway. DG, diacylglycerol; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; SQDG, sulfoquinoxayldiacylglycerol; TG, triacylglycerol. Significant differences were determined using Student’s t-test (*P<0.05, ***P<0.001, ****P<0.0001).

Fig. 10.

Differential analysis of lipid components between Zheng58 and ZmHsf17 overexpression line and KEGG enrichment analysis. (A) Volcano plot of significantly up-regulated and down-regulated lipid components in the ZmHsf17 overexpression line compared with Zheng58. (B) Scatter plot of differential components in 26 lipid subclasses. (C) Heatmap depicting the content levels of 37 significantly up-regulated lipid components in ZmHsf17 overexpression line compared with Zheng58. (D) KEGG enrichment analysis of 62 differentially expressed lipid components between Zheng58 and ZmHsf17 overexpression line.