HEAT SHOCK FACTOR A8a Modulates Flavonoid Synthesis and Drought Tolerance

Abstract

Drought is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus domestica). Heat shock factors (HSFs) have well-documented functions in stress responses, but their roles in flavonoid synthesis and the flavonoid-mediated drought response mechanism remain elusive. In this study, we demonstrated that a drought-responsive HSF, designated MdHSFA8a, promotes the accumulation of flavonoids, scavenging of reactive oxygen species, and plant survival under drought conditions. A chaperone, HEAT SHOCK PROTEIN90 (HSP90), interacted with MdHSFA8a to inhibit its binding activity and transcriptional activation. However, under drought stress, the MdHSP90-MdHSFA8a complex dissociated and the released MdHSFA8a further interacted with the APETALA2/ETHYLENE RESPONSIVE FACTOR family transcription factor RELATED TO AP2.12 to activate downstream gene activity. In addition, we demonstrated that MdHSFA8a participates in abscisic acid-induced stomatal closure and promotes the expression of abscisic acid signaling-related genes. Collectively, these findings provide insight into the mechanism by which stress-inducible MdHSFA8a modulates flavonoid synthesis to regulate drought tolerance.

Figure 1.

Drought induces flavonoid and anthocyanin accumulation in apple. A, Growth status of apple plants after simulated drought treatment. Different concentrations of PEG were used to simulate drought stress. Bars = 1 cm. B, Anthocyanins extracted in 1% (v/v) HCl-methanol. C, Flavonoids extracted in 1% (v/v) HCl-methanol and colored by NaNO₂, Al(NO₃)₃, and NaOH. D and E, Anthocyanin (D) and flavonoid (E) contents in apple plants under simulated drought treatment. Values are means ± sd of three independent biological replicates. Different lowercase letters indicate significant differences by Tukey’s test using DPS software (P < 0.05). FW, Fresh weight. F, Simplified representation of the flavonoid biosynthetic pathway leading to the three major classes of end products: flavonols, proanthocyanidins, and anthocyanins. EBP, Early biosynthesis pathway; LBP, late biosynthesis pathway. G, Transcript levels of late biosynthesis pathway genes in apple plants under simulated drought treatments. MdActin was used as an internal control gene. Samples treated with 0% PEG were used as an internal standard. Values are means ± sd of three independent biological replicates. Different lowercase letters indicate significant differences by Tukey’s test using DPS software (P < 0.05). H, Wild-type apple ‘GL-3’ plants cultured in pots were subjected to natural drought treatment. Bars = 2 cm for the plants and 1 cm for the leaves. I and J, Anthocyanin (I) and flavonoid (J) contents in apple ‘GL-3’ plants under natural drought treatment. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (**P < 0.01).

Figure 2.

MdHSFA8a is involved in drought-induced accumulation of flavonoids and anthocyanins. A, Wild-type (WT) apple ‘GL-3’ plants, MdHSFA8a-OE (OE), and MdHSFA8a-RNAi (RNAi) transgenic apple plants cultured under simulated drought stress. Bars = 1 cm. B, Detection of MdHSFA8a protein expression in wild-type, OE, and RNAi transgenic apple plants by immunoblotting with specific MdHSFA8a antibody. C, Transcript levels of MdHSFA8a in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatments. MdActin was used as an internal control gene. Wild-type apple plants treated with 0% PEG were used as an internal standard. D, Growth parameters of wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. E and F, Anthocyanin and flavonoid contents in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. FW, Fresh weight. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (*P < 0.05 and **P < 0.01).

Figure 3.

Transcript levels of flavonoid biosynthesis pathway genes in apple plants under simulated drought stress. A, Transcript levels of MdDFR in wild-type (WT), OE, and RNAi transgenic apple plants under simulated drought treatment. B, Transcript levels of MdFLS in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. C, Transcript levels of MdANR in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. ANR, Anthocyanidin reductase. D, Transcript levels of MdLAR in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. LAR, Leucoanthocyanidin reductase. E, Transcript levels of MdANS in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. F, Transcript levels of MdUFGT in wild-type, OE, and RNAi transgenic apple plants under simulated drought treatment. MdActin was used as an internal control gene. Wild-type apple plants treated with 0% PEG were used as an internal standard. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (*P < 0.05 and **P < 0.01).

Figure 4.

MdHSFA8a promotes drought tolerance and stimulates ROS scavenging under natural drought. A, Wild-type (WT), OE, and RNAi apple plants cultured in pots were subjected to natural drought treatment. Plants were photographed 15 d after water was withheld. Arrows indicate the appearance of a drought phenotype, such as leaf curling. Bars = 3 cm. B, DAB staining of wild-type, OE, and RNAi apple plants. Staining with DAB was performed on leaves 15 d after natural water loss. Leaves from normally watered plants served as the control. C, Fluorescence detection of ROS in guard cells of wild-type, OE, and RNAi apple plants using H2DCF-DA after natural drought treatment. Bars = 10 μm. D, Diphenylboric acid 2-aminoethylester (DPBA) staining in wild-type, OE, and RNAi apple leaves. Yellow fluorescence represents the relative flavonol contents in guard cells. Bars = 20 μm. E, H₂O₂ content in wild-type, OE, and RNAi apple plants under drought stress. F to H, CAT, SOD, and POD activities in wild-type, OE, and RNAi apple plants under drought stress. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (*P < 0.05 and **P < 0.01). FW, Fresh weight.

Figure 5.

MdHSFA8a enhances the transcription of MdMYB12, MdANS, and MdFLS by binding to their promoters. A and B, Characteristics of HSEs in the promoters of crucial enzyme genes (A) and MYB TFs (B) associated with flavonoid synthesis. C to E, EMSA showing the binding of MdHSFA8a to the candidate HSE motif in MdANS (C), MdFLS (D), and MdMYB12 (E) promoters. Hot probe was a biotin-labeled fragment containing the HSE motif. Cold probe was a nonlabeled competitive probe (100-fold that of hot probe). Mutant probe contained two nucleotide mutations. F to H, ChIP-qPCR assay showing the binding of MdHSFA8a to the candidate HSE motif in the promoters of MdANS (F), MdFLS (G), and MdMYB12 (H) in vivo. Cross-linked chromatin samples were extracted from MdHSFA8a::GFP transgenic calli and precipitated with anti-GFP. The ChIP assay was repeated three times, and enriched DNA fragments in each ChIP were used as one biological replicate for qPCR. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (**P < 0.01). I to K, Effects of MdHSFA8a on the promoter activities of MdANS (I), MdFLS (J), and MdMYB12 (K) as demonstrated by a dual-luciferase reporter assay in tobacco leaves. Values are means ± sd of three independent biological replicates. Different lowercase letters indicate significant differences by Tukey’s test using DPS software (P < 0.05).

Figure 6.

Interaction of MdHSFA8a with chaperone MdHSP90 and the AP2/ERF TF MdRAP2.12. A, The full-length (FL) CDS of MdHSFA8a was divided into five fragments (F1–F5): AHA, Motif rich in aromatic, hydrophobic, and acidic amino acid residues; DBD, DNA-binding domain; HR-A/B, oligomerization domain; NES, nuclear export; NLS, nuclear import. The amino acid positions of fragments are numbered. B, Conserved sequence analysis of the interaction domain in MdHSFA8a, which is similar to the previously reported temperature-dependent repression (TDR) domain. C to E, Y2H assays showing the interaction of MdHSFA8a with MdHSP90 and MdRAP2.12. The F1 and F3 domains showed no self-activating activity (C). F3 interacted with MdHSP90 (D) and MdRAP2.12 (E), whereas F1 showed no interaction. F, BiFC assay showing the interaction of MdHSFA8a with MdHSP90 and MdRAP2.12 in vivo. The YFP field indicates fluorescence signals; the DAPI (4', 6-diamidino-2-phenylindole) field indicates the locations of nuclei. The right gels show the expression of the fusion protein examined by western blotting with anti-MYC and anti-HA antibodies. Bars = 10 μm. G and H, Pull-down assay was performed by copurifying recombinant HSP90-HIS and RAP2.12-HIS fusion proteins with HSFA8a-GST and GST empty vector. Western blotting with a GST antibody showed that HSFA8a was pulled down by HSP90-HIS and RAP2.12-HIS.

Figure 7.

Effects of drought on MdHSFA8a-MdHSP90 and MdHSFA8a-MdRAP2.12 protein complexes. A, CoIP detection of the interaction between MdHSFA8a and MdHSP90 in vivo under drought treatment. MdHSP90::GFP transgenic calli were used. Immunoprecipitation (IP) samples were assayed using specific anti-HSFA8a. B, Relative quantitative analysis of immunoblotting proteins. Bars 1 to 4 correspond to channels 1 to 4 in A, respectively. Channel 1, MdHSP90::GFP calli cultured under normal conditions; channels 2 to 4, MdHSP90::GFP calli cultured under 10%, 20%, and 40% PEG treatment. C, CoIP detection of the interaction between MdHSFA8a and MdRAP2.12 in vivo under drought treatment. MdRAP2.12::GFP transgenic calli were used. CoIP was conducted as in A. D, Relative quantitative analysis of western-blotting proteins was conducted. Bars 1 to 4 correspond to channels 1 to 4 in C, respectively. Channel 1, MdRAP2.12::GFP calli cultured under normal conditions; channels 2 to 4, MdRAP2.12::GFP calli cultured under 10%, 20%, and 40% PEG treatment. In B and D, values are means ± sd of three independent biological replicates. Different lowercase letters indicate significant differences by Tukey’s test using DPS software (P < 0.05).

Figure 8.

Effects of drought on MdHSFA8a-MdHSP90 and MdHSFA8a-MdRAP2.12 protein complexes. A, EMSA showing that the binding strength of MdHSFA8a to the MdMYB12, MdANS, and MdFLS promoters was significantly inhibited with the addition of MdHSP90. Empty HIS protein was used to ensure that each tube contained the same amount of protein. B, Relative quantitative analysis of proteins bound to the probe. Values are means ± sd of three independent repeated experiments. Asterisks indicate statistical significance by Tukey’s test using DPS software (** P < 0.01). C, EMSA showing the binding strength of MdHSFA8a to the MdMYB12, MdANS, and MdFLS promoters with the addition of MdRAP2.12. EMSA was conducted as in A. D, Relative quantitative analysis of proteins bound to the probe. Values are means ± sd of three independent repeated experiments. E to G, Dual-luciferase reporter assay showing the transcriptional activation of MdHSFA8a to the MdANS (E), MdFLS (F), and MdMYB12 (G) promoters with the addition of MdHSP90. H to J, Dual-luciferase reporter assay showing the transcriptional activation of MdHSFA8a to the MdANS (H), MdFLS (I), and MdMYB12 (J) promoters with the addition of MdRAP2.12. Values are means ± sd of three independent biological replicates. Different lowercase letters indicate significant differences by Tukey’s test using DPS software (P < 0.05). Bars = 1 cm.

Figure 9.

MdHSFA8a participates in ABA-mediated stomatal movement to regulate drought response. A, Detached leaves of wild-type (WT), OE, and RNAi apple plants. Bars = 1 cm. B, Natural water loss assays in detached leaves of wild-type, OE, and RNAi apple plants at 24°C for 12 h. Three leaves from three plants were mixed for each measurement. Values are means ± sd of three independent biological replicates. C, Stomatal apertures of wild-type, MdHSFA8a-OE, and MdHSFA8a-RNAi apple plants in response to exogenous ABA treatment. Stomatal apertures were observed and measured after treatment with 0, 5, or 10 μm ABA for 2 h and were calculated from 100 stomata from leaves of three different apple plants. D, Time-course data for stomatal apertures of different plant leaves treated with 10 μm ABA for 0, 1, and 2 h. The stomatal aperture was calculated for 100 stomata from leaves of three different apple plants. Values are means ± sd of three independent biological replicates. Asterisks indicate statistical significance by Tukey’s test using DPS software (*P < 0.05 and **P < 0.01). Bars = 10 μm. E, Images showing the number of stomata in wild-type, MdHSFA8a-OE, and MdHSFA8a-RNAi apple plants. Bars = 50 μm. F, Stomatal density in the leaves of wild-type, MdHSFA8a-OE, and MdHSFA8a-RNAi apple plants. Stomatal density was calculated from 10 images taken from different parts of the leaf epidermis. Values are means ± sd of three independent biological replicates.

Figure 10.

Proposed model for MdHSFA8a modulation of flavonoid synthesis to regulate drought tolerance in apple. Under normal conditions, MdHSP90 interacts with MdHSFA8a to inhibit the binding activity and transcription activity of MdHSFA8a on downstream target genes. Under drought stress, the MdHSP90-MdHSFA8a complex dissociates and the released MdHSFA8a further interacts with the AP2/ERF family TF MdRAP2.12 to activate downstream gene activity. Solid arrows show positive regulation.