The MdHSC70-MdWRKY75 module mediates basal apple thermotolerance by regulating the expression of heat shock factor genes

Abstract

Heat stress severely restricts the growth and fruit development of apple (Malus domestica). Little is known about the involvement of WRKY proteins in the heat tolerance mechanism in apple. In this study, we found that the apple transcription factor (TF) MdWRKY75 responds to heat and positively regulates basal thermotolerance. Apple plants that overexpressed MdWRKY75 were more tolerant to heat stress while silencing MdWRKY75 caused the opposite phenotype. RNA-seq and reverse transcription quantitative PCR showed that heat shock factor genes (MdHsfs) could be the potential targets of MdWRKY75. Electrophoretic mobility shift, yeast one-hybrid, β-glucuronidase, and dual-luciferase assays showed that MdWRKY75 can bind to the promoters of MdHsf4, MdHsfB2a, and MdHsfA1d and activate their expression. Apple plants that overexpressed MdHsf4, MdHsfB2a, and MdHsfA1d exhibited heat tolerance and rescued the heat-sensitive phenotype of MdWRKY75-Ri3. In addition, apple heat shock cognate 70 (MdHSC70) interacts with MdWRKY75, as shown by yeast two-hybrid, split luciferase, bimolecular fluorescence complementation, and pull-down assays. MdHSC70 acts as a negative regulator of the heat stress response. Apple plants that overexpressed MdHSC70 were sensitive to heat, while virus-induced gene silencing of MdHSC70 enhanced heat tolerance. Additional research showed that MdHSC70 exhibits heat sensitivity by interacting with MdWRKY75 and inhibiting MdHsfs expression. In summary, we proposed a mechanism for the response of apple to heat that is mediated by the "MdHSC70/MdWRKY75-MdHsfs" molecular module, which enhances our understanding of apple thermotolerance regulated by WRKY TFs.

Figure 1.

MdWRKY75 positively regulates the heat tolerance of apple. A) Phenotypes of MdWRKY75 transgenic apple plants at 0, 4, 8, and 14 h of heat stress. Bars = 10 cm. B) Phenotypes of MdWRKY75 transgenic apple leaves at 0, 4, 8, and 14 h of heat stress. Bars = 3 cm. C) to F) Total chlorophyll content C), REL D), MDA content E), and proline content F) of MdWRKY75 transgenic apple plants under heat stress. Control, 23 °C for 8 h; Heat, 48 °C for 8 h. The selected plants were “GL-3” and MdWRKY75 transgenic apples at approximately 60 d after transplantation. WT, wild type. OE1-3 represent MdWRKY75 overexpression lines 1 to 3, respectively. Ri1-3 represent MdWRKY75 RNAi lines 1 to 3, respectively. All the indices were determined at 8 h after treatment. Values with different letters are significantly different according to a one-way ANOVA followed by a Duncan's test (P < 0.05). Data are the means ± SD (n = 3 for C to F). ANOVA, analysis of variance; FW, fresh weight; MDA, malondialdehyde; REL, relative electrolyte leakage; RNAi, RNA interference; SD, standard deviation.

Figure 2.

The ROS and photosynthetic capacity of MdWRKY75 transgenic apple under heat stress. A) DAB staining, bars = 3 cm; B) NBT staining, bars = 3 cm; C)Fv/Fm images; D)Fv/Fm ratio; and E) net photosynthesis (Pn). Control, 23 °C for 8 h; Heat, 48 °C for 8 h. The selected plants were “GL-3” and MdWRKY75 transgenic apples at approximately 60 d after transplantation. WT, wild type. OE1-3 represent MdWRKY75 overexpression lines 1 to 3, respectively. Ri1-3 represent MdWRKY75 RNAi lines 1 to 3, respectively. A) to B) were determined at 8 h, and C) to E) were determined at 6 h after treatment, respectively. Values with different letters are significantly different according to a one-way ANOVA followed by Duncan's test (P < 0.05). Data are the means ± SD (n = 3 for D) and E)). ANOVA, analysis of variance; Fv/Fm, maximum photosynthetic efficiency of photosystem II; DAB, 3,3′-diaminobenzidine; NBT, nitroblue tetrazolium; ROS, reactive oxygen species; SD, standard deviation.

Figure 3.

KEGG enrichment analysis of DEGs in the MdWRKY75 transgenic apple leaves under heat stress. A), C), and E) KEGG enrichment analyses between the WT and MdWRKY75-OE3 after 0, 4, and 8 h of heat stress, respectively. B), D), and F) KEGG enrichment analyses between the WT and MdWRKY75-Ri3 after 0, 4, and 8 h of heat stress, respectively. WT, wild type. OE3 and Ri3 represent MdWRKY75 overexpression line 3 and RNAi line 3, respectively. The vertical and horizontal axes represent the pathway and enrichment factor, respectively. The size of dots represents the number of genes and the color of dots represents the q-value.

Figure 4.

MdWRKY75 binds directly to the promoters of MdHsfs. A) Distribution of the W-boxes (5′-AGTCAA-3′) in the promoters of 5 candidate MdHsfs. B) EMSAs of the interaction between MdWRKY75 and 5′-biotin labeled probes. C) Competing EMSAs of the interaction between MdWRKY75 and 5′-biotin labeled probes. Competitor-Probe, without biotin labeling. Mut, mutant probes in which the 5′-AGTCAA-3′ motifs were replaced by 5′-ACTCAA-3′. D) to H) Y1H assays between the interaction of MdWRKY75 and the promoters of MdHsf4D), MdHsfA2E), MdHsfB2aF), MdHsfA3G), and MdHsfA1dH). -T/-L, -Trp-Leu; -T/-L/-H, -Trp-Leu-His; 3-AT, 3-amino-1,2,4-triazole; EMSA, electrophoretic mobility shift assay; Y1H, yeast one-hybrid.

Figure 5.

MdWRKY75 positively regulates the levels of expression of the MdHsfs. A) Schematic diagram of reporter and effector vectors for the GUS assays. B) to F) The staining and activities of GUS between the interaction of MdWRKY75 and the promoters of MdHsf4B), MdHsfA2C), MdHsfB2aD), MdHsfA3E), and MdHsfA1dF). Darker blue indicates stronger GUS activity. Bars = 2 cm. Data are means ± SD (n = 3). G) Schematic diagram of the reporter and effector vectors for dual-luciferase assays. H) to L) The images of luciferase and relative LUC/REN activities between the interaction of MdWRKY75 and the promoters of MdHsf4H), MdHsfA2I), MdHsfB2aJ), MdHsfA3K), and MdHsfA1dL). Bars = 2 cm. Data are means ± SD (n = 3). Statistical significance was analyzed using a Student's t-test (*P < 0.05, **P < 0.01, and ***P < 0.001). GUS, β-glucuronidase; LUC/REN, luciferase/Renilla; n.s., no significance; SD, standard deviation.

Figure 6.

MdHsfs positively regulate apple heat tolerance. A) Phenotypes of apple plants that overexpressed the MdHsfs mediated by transient transformation under heat stress. Bars = 2 cm. WT-2300GFP and MdHsfs-OE represent “GL-3” plants transformed with pCambia2300-GFP and MdHsfs-pCambia2300-GFP, respectively. B) The relative level of expression of MdHsf4, MdHsfB2a, and MdHsfA1d in MdHsfs transgenic apple plants. C) DAB and NBT staining of the leaves in MdHsfs transgenic apple plants after 3 h of heat stress. D) Chlorophyll fluorescence images and Fv/Fm values of leaves in the MdHsfs transgenic apple plants after 1 h of heat stress. E) Phenotypes of MdWRKY75-Ri3 + MdHsfs-OE mediated by transient transformation under heat stress. Bars = 2 cm. WT, “GL-3” plants. F) The relative level of expression of MdHsf4, MdHsfB2a, and MdHsfA1d in MdWRKY75-Ri3 + MdHsfs-OE transgenic apple plants. G) DAB and NBT staining of the leaves in MdWRKY75-Ri3 + MdHsfs-OE transgenic apple plants after 3 h of heat stress. H) Chlorophyll fluorescence images and Fv/Fm values of MdWRKY75-Ri3 + MdHsfs-OE transgenic apple plants after 1 h of heat stress. Control, 23 °C for 3 h; Heat, 48 °C for 3 h. The selected “GL-3” plants were cultured on media for 30 d. Values with different letters are significantly different according to a one-way ANOVA followed by a Duncan's test (P < 0.05). Data are the means ± SD (n = 3). ANOVA, analysis of variance; DAB, 3,3′-diaminobenzidine; Fv/Fm, maximum photosynthetic efficiency of photosystem II; NBT, nitroblue tetrazolium; SD, standard deviation.

Figure 7.

MdWRKY75 interacts directly with MdHSC70. A) A Y2H assay indicates the interaction of MdWRKY75 and MdHSC70. -T/-L, -Trp-Leu; -T/-L/-H/-A, -Trp-Leu-His-Ade; X-α-gal, 5-bromo-4-chloro-3-indolyl-α-D-galactoside; pGAD, pGADT7 vector; pGBD, pGBKT7 vector. B) A Split-LUC assay indicates the interaction of MdWRKY75 and MdHSC70. C) A BiFC assay indicates the interaction of MdWRKY75 and MdHSC70. Bars = 30 μm. MdWRKY75*, the form with a small deletion of the C-terminus of MdWRKY75, were used for the negative control. BF, bright field; BiFC, bimolecular fluorescence complementation. D) A pull-down assay indicates the interaction of MdWRKY75 and MdHSC70. Split-LUC, split luciferase complementation assay; Y2H; yeast two-hybrid; YFP, yellow fluorescent protein.

Figure 8.

MdHSC70 plays a negative role in the response to heat stress. A) Phenotypes of MdHSC70 transgenic apple plants mediated by transient transformation under heat stress. WT-2300GFP, “GL-3” plants transformed with pCambia2300-GFP. Bars = 2 cm. B) RT-qPCR identification of MdHSC70 transgenic plants. C) The relative level of expression of MdHsf4, MdHsfB2a, and MdHsfA1d in MdHSC70 overexpression apple plants after 3 h of heat stress. D) DAB and NBT staining of the leaves in MdHSC70 overexpression apple plants after 3 h of heat stress. E) Chlorophyll fluorescence images and Fv/Fm values of the leaves of MdHSC70 overexpression apple plants after 1 h of heat stress. F) Phenotypes of apple plants silenced with MdHSC70 mediated by VIGS under heat stress. WT, “GL-3” transformed with VIGS empty vector. Bars = 2 cm. G) RT-qPCR identification of the silencing of MdHSC70 in apple plants. H) The relative level of expression of MdHsf4, MdHsfB2a, and MdHsfA1d in apple plants with the silencing of MdHSC70 after 3 h of heat stress. I) DAB and NBT staining of the leaves in apple plants with silenced MdHSC70 after 3 h of heat stress. J) Chlorophyll fluorescence images and Fv/Fm values in apple plants with silenced MdHSC70 after 1 h of heat stress. Control, 23 °C for 3 h; Heat, 48 °C for 3 h. The selected “GL-3” plants were cultured on media for 30 d. Values with different letters are significantly different according to a one-way ANOVA followed by a Duncan's test (P < 0.05). Data are the means ± SD (n = 3). ANOVA, analysis of variance; DAB, 3,3′-diaminobenzidine; Fv/Fm, maximum photosynthetic efficiency of photosystem II; NBT, nitroblue tetrazolium; RT-qPCR, reverse transcription quantitative PCR; SD, standard deviation; VIGS, virus-induced gene silencing; WT, wild type.

Figure 9.

MdHSC70 inhibits the ability of MdWRKY75 to bind the MdHsfs. A) to C) GUS assays show that MdHSC70 inhibits the activation of MdWRKY75 to the promoters of MdHsf4A), MdHsfB2aB), and MdHsfA1dC). D) to F) Dual-luciferase assays show that MdHSC70 inhibits the activation of MdWRKY75 to the promoters of MdHsf4D), MdHsfB2aE), and MdHsfA1dF). G) EMSAs show MdHSC70 inhibits the binding of MdWRKY75 to the 5′ biotin probes. Values with different letters are significantly different according to a one-way ANOVA followed by a Duncan's test (P < 0.05). Bars = 2 cm. Data are the means ± SD (n = 3). ANOVA, analysis of variance; SD, standard deviation; EMSAs, electrophoretic mobility shift assays; GUS, β-glucuronidase; LUC/REN, luciferase/Renilla.

Figure 10.

MdHSC70 inhibits the heat stress tolerance of MdWRKY75. A) Phenotypes of MdWRKY75-OE3 apple plants with overexpressed and silenced MdHSC70 mediated by transient transformation under heat stress. WT, “GL-3” apple plants. Bars = 2 cm. B) RT-qPCR identification of MdWRKY75-OE3 apple plants with overexpressed and silenced MdHSC70. C) The relative level of expression of MdHsf4, MdHsfB2a, and MdHsfA1d in MdWRKY75-OE3 apple plants with overexpressed and silenced MdHSC70 under heat stress. D) DAB and NBT staining of leaves in the MdWRKY75-OE3 apple plants with overexpressed and silenced MdHSC70 after 3 h of heat stress. E) Chlorophyll fluorescence images and Fv/Fm values in the MdWRKY75-OE3 apple plants with overexpressed and silenced MdHSC70 after 1 h of heat stress. Control, 23 °C for 3 h; Heat, 48 °C for 3 h. The selected “GL-3” plants were cultured on media for 30 d. Values with different letters are significantly different according to a one-way ANOVA followed by a Duncan's test (P < 0.05). Data are the means ± SD (n = 3). ANOVA, analysis of variance; DAB, 3,3′-diaminobenzidine; Fv/Fm, maximum photosynthetic efficiency of photosystem II; NBT, nitroblue tetrazolium; RT-qPCR, reverse transcription quantitative PCR; SD, standard deviation; WT, wild type.

Figure 11.

A working model of the regulation of apple MdWRKY75 under heat tolerance. When apple is subjected to heat stress, the expression of MdWRKY75 is activated, which, in turn, activates MdHsfs expression and confers tolerance to heat. MdHSC70 negatively regulates the tolerance of apple to heat by interacting with MdWRKY75 to inhibit its activation of MdHsfs.