FIGURE LEGENDS
Figure 1. Endogenous melatonin (MT) play a positive role in thermotolerance of apple plants. (A) MT contents of apple plants under high-temperature stress. Relative expression patterns of MT biosynthetic gene MdTDC1 (B), MdT5H4 (C), MdAANAT2 (D) andMdASMT9 (E) under heat stress. (F) MT contents in MdASMT9overexpressing (OE) apple plants under heat stress. (G) Phenotype, (H) relative electrolyte leakage (REL), and (I) malondialdehyde (MDA) content of WT and MdASMT9 -OE apple plants after 8 h of exposure to 48°C. Asterisks indicated significant differences (*P<0.05, **P<0.01, ***P<0.001 according to Tukey’s test).
Figure 2. Reactive oxygen species (ROS) levels and activities of antioxidant enzymes in WT and MdASMT9 -OE apple plants. (A) Histochemical staining for H2O2 and O2. The (B) H2O2 and (C) O2 contents and (D) SOD, (E) POD, and (F) CAT activities in apple leaves with and without heat stress. Asterisks indicated significant differences (*P<0.05, **P<0.01, ***P<0.001 according to Tukey’s test).
Figure 3. The impact of high temperature on the photosynthetic capacity and stomatal aperture of MdASMT9 -OE apple lines. Changes in (A) the net photosynthesis rate (Pn) and (B) stomatal conductance (GS) after heat treatment. Chlorophyll fluorescence images (C) and FV/FMratios (D) of WT and MdASMT9 -OE plants with and without heat stress. The color in the images represents theFV/FM ratio, with black indicating 0 and red indicating 1. (E) Arrows indicate chloroplasts in mesophyll cells. (F) The scanning electron microscopy (SEM) images of stomata. (G) Stomatal apertures and (H) ABA content of WT andMdASMT9 -OE apple plants under control and heat stress conditions. Asterisks indicated significant differences (*P<0.05 and **P<0.01 according to Tukey’s test).
Figure 4. The levels of soluble sugars (A-C) and amino acids (D-I) in WT and MdASMT9 -OE apple plants under heat stress. Changes in (A) galactose, (B) sorbitol, (C) sucrose, (D) histidine, (E) arginine, (F) aspartate, (G) proline, (H) glutamine, and (I) glycine contents after heat treatment. Asterisks indicated significant differences (*P<0.05, **P<0.01, ***P<0.001 according to Tukey’s test).
Figure 5. Volcano plot showing differentially expressed genes (DEGs) between WT and OE-3 (A), and between WT and OE-4 (B) under heat treatment. (C) Venn diagram showed the number of DEGs in WT andMdASMT9 -OE apple plants. (D) Heatmap showing expression levels of overlapping DEGs in both OE-3 and OE-4 plants (values are presented as log2 fold change). (E) The relative transcripts of heat stress transcription factors (HSFs). Asterisks indicated significant differences (***P<0.001 according to Tukey’s test).
Figure 6. The relative expression levels of (A) MdNCED1 , (B) MdNCED3 , and (C) MdWRKY33 in WT and MdASMT9 -OE apple plants under heat stress. MdWRKY33 binds to the MdNCED1 andMdNCED3 promoters (D-I). EMSA assay of MdWRKY33 binding toMdNCED1 (D) and MdNCED3 (E) promoters. The recombinant proteins were incubated with biotin-labeled P1;2 or mutant oligos P1;2-mut. (F-I) Transiently expressed MdWRKY33 interacts withMdNCED1 and MdNCED3 promoters. The value for the Luc+Empty vector was set as one. Asterisks indicated significant differences (*P<0.05 and ***P<0.001 according to Tukey’s test).
Figure 7. Exogenous MT increased stomatal aperture through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1and MdNCED3 . (A) Phenotype, (B) MdWRKY33 expression, (C) MT contents and (D) REL of TRV-MdWRKY33 plants with or without MT treatment under heat treatment. (E-F) Stomatal apertures and (G) ABA content of TRV-MdWRKY33 plants with or without MT treatment under control and heat stress conditions. Relative expression patterns ofMdNCED1 (H) and MdNCED 3 (I) under heat stress. Asterisks indicated significant differences (*P<0.05, **P<0.01, ***P<0.001 according to Tukey’s test).
Figure 8. Overexpression of MdASMT9 increased stomatal aperture through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3 . (A) Phenotype, (B) MdWRKY33expression, (C) REL of apple plants overexpressing MdASMT9 and suppressing MdWRKY33 under heat treatment. (D-E) Stomatal apertures and (F) ABA content of apple plants overexpressingMdASMT9 and suppressing MdWRKY33 . Relative expression patterns of MdNCED1 (G) and MdNCED 3 (H) under heat stress. Asterisks indicated significant differences (*P<0.05, **P<0.01, ***P<0.001 according to Tukey’s test).
Figure 9. Exogenous MT and overexpression of MdASMT9enhanced autophagic activity through promoting MdWRKY33 -mediated transcriptional enhancement of MdATG18a. (A) Relative expression patterns of MdATG18a in MdASMT9 -OE apple plants under heat stress. (B) TEM images of autophagic structures (arrows indicate autophagosomes in mesophyll cells). (C) Relative autophagic activity in WT and MdASMT9 -OE apple plants. (D) The correlation analysis ofMdWRKY33 and MdATG18a expression levels. (E)MdATG18a expression of TRV-MdWRKY33 plants with or without MT treatment under heat treatment. (F) MdATG18a expression of apple plants overexpressing MdASMT9 and suppressingMdWRKY33 . Asterisks indicated significant differences (**P<0.01 and ***P<0.001 according to Tukey’s test).
Figure 10. Model of MdASMT9 -mediated biosynthesis of MT in apple plants responding to heat stress. The arrows refer to activation, and the blocked arrows refer to inhibition. The dashed arrows refer to indirect effects or unknown effects.