Auxin and abscisic acid antagonistically regulate ascorbic acid production via the SlMAPK8-SlARF4-SlMYB11 module in tomato

Abstract

Ascorbic acid (AsA) is a multifunctional phytonutrient that is essential for the human diet as well as plant development. While much is known about AsA biosynthesis in plants, how this process is regulated in tomato (Solanum lycopersicum) fruits remains unclear. Here, we found that auxin treatment inhibited AsA accumulation in the leaves and pericarps of tomato. The auxin response factor gene SlARF4 is induced by auxin to mediate auxin-induced inhibition of AsA accumulation. Specifically, SlARF4 transcriptionally inhibits the transcription factor gene SlMYB11, thereby modulating AsA accumulation by regulating the transcription of the AsA biosynthesis genes l-galactose-1-phosphate phosphatase, l-galactono-1,4-lactone dehydrogenase, and dehydroascorbate. By contrast, abscisic acid (ABA) treatment increased AsA accumulation in tomato under drought stress. ABA induced the expression of the mitogen-activated protein kinase gene SlMAPK8. We demonstrate that SlMAPK8 phosphorylates SlARF4 and inhibits its transcriptional activity, whereas SlMAPK8 phosphorylates SlMYB11 and activates its transcriptional activity. SlMAPK8 functions in ABA-induced AsA accumulation and drought stress tolerance. Moreover, ABA antagonizes the effects of auxin on AsA biosynthesis. Therefore, auxin- and ABA-induced regulation of AsA accumulation is mediated by the SlMAPK8-SlARF4-SlMYB11 module in tomato during fruit development and drought stress responses, shedding light on the roles of phytohormones in regulating AsA accumulation to mediate stress tolerance.

Figure 1

Auxin-mediated inhibition of AsA accumulation is mediated by SlARF4 in tomato. A, Total AsA content in WT tomato pericarps. B, GUS activity measured with a MUG assay. GUS activity is expressed as nmol/mg protein/min. C, Concentration of IAA in tomato pericarps at various developmental stages. D, DR5:GUS activity during fruit development and ripening. Bars = 50 μm. E, SlARF4 expression pattern during fruit development and ripening. F–I, Total and reduced AsA contents in tomato leaves (F, G) and pericarps (H, I) of SlARF4 transgenic plants. J–M, Total and reduced AsA contents in tomato leaves (J, K) and pericarps (L, M) of SlARF4 transgenic plants under IAA (30 mg/L) treatment. SlARF4-OE, SlARF4 overexpression plants; SlARF4-RNAi, SlARF4 RNAi plants; Slarf4, SlARF4 CRISPR/Cas9 mutant. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 2

Auxin-mediated inhibition of SlMYB11 expression is mediated by SlARF4, and SlARF4 directly targets the SlMYB11 promoter and inhibits its expression. A, Subcellular localization analysis of SlMYB11. SlMYB11-GFP was transiently expressed in N. benthamiana leaves. Nuclei were identified by DAPI staining. Bars = 20 μm. B, RT-qPCR analysis of SlMYB11 expression in tomato fruit. C and D, Relative expression level of SlMYB11 in leaves (C) and pericarps (D) of SlARF4 transgenic plants. E and F, Relative expression level of SlMYB11 in leaves (E) and pericarps (F) of SlARF4 transgenic plants under IAA treatment. G, EMSA showing the binding of SlARF4 to the promoter of SlMYB11. The nonlabeled fragment was used as a competitor, −: absence; +: presence. H, ChIP-qPCR showing the binding of SlARF4 to the SlMYB11 promoter containing the TAG element. SlMYB11 (TGA), promoter containing the TAG element. SlMYB11-1/2, negative control (without the TAG element). Values are percentages of DNA fragments that coimmunoprecipitated with anti-FLAG antibodies or nonspecific antibodies (anti-IgG) relative to the input DNA. I, Schematic diagrams of the reporter and effector vectors used in the dual-LUC reporter assay. J, Dual-LUC assays showing the inhibition of SlMYB11 expression by SlARF4 in N. benthamiana. SlARF10 and SlARF6 were used as negative controls. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 3

SlMYB11 positively affects AsA accumulation via the transcriptional activation of GPP, GLDH, and DHAR in tomato. A–D, Total and reduced AsA contents in leaves (A, B) and pericarps (C, D) of SlMYB11 transgenic plants. E–H, Total and reduced AsA contents in leaves (E, F) and pericarps (G, H) of SlMYB11 transgenic plants under IAA (30 mg/L) treatment. I and J, RT-qPCR analysis of the expression levels of AsA biosynthesis genes in leaves (I) and pericarps (J) of SlMYB11-RNAi transgenic plants. K–M, EMSA showing the binding of SlMYB11 to the binding motif of the GPP, GLDH, and DHAR promoters. The non-labeled fragment was used as a competitor, −: absence; +: presence. N, Schematic diagrams of the reporter and effector vectors used in the dual-LUC reporter assay. O, LUC assay showing the activation of GPP, GLDH, and DHAR expression by SlMYB11. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 4

SlARF4-regulated AsA accumulation partially depends on SlMYB11 in tomato. A, RT-qPCR analysis of SlMYB11 expression in VIGS-SlMYB11/SlARF4 transgenic plants. B–E, Total and reduced AsA contents in leaves (B, C) and pericarps (D, E) of VIGS-SlMYB11/SlARF4 transgenic plants. F–I, Total and reduced AsA contents in leaves (F, G) and pericarps (H, I) of VIGS-SlMYB11/SlARF4 transgenic plants under IAA (30 mg/L) treatment. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 5

ABA increases AsA accumulation in tomato under drought stress. A and B, ABA contents in leaves (A) and pericarps (B) of WT plants after drought, PEG 6000, and fluridone (30 μM) treatment. D + Flu, drought and fluridone treatment. C–F, Total and reduced AsA contents in leaves (C, D) and pericarps (E, F) of WT plants under ABA and fluridone treatment. ABA + Flu, ABA, and fluridone treatment. G–J, Total and reduced AsA contents in leaves (G, H) and pericarps (I, J) of WT plants under drought, PEG 6000, and fluridone (30 μM) treatment. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 6

SlMAPK8 is induced by ABA and drought stress, and SlMAPK8 phosphorylates SlARF4 and SlMYB11. A, RT-qPCR analysis of SlMAPK8 expression in tomato plants under ABA treatment. B, RT-qPCR analysis of SlMAPK8 expression in tomato plants under drought and PEG 6000 treatment. C and D, Y2H assay of the interaction of SlMAPK8 with SlARF4 (C) and SlMAPK8 with SlMYB11 (D). Yeast cells co-transformed with pGBKT7-SlMAPK8 and pGADT7-SlARF4 or pGBKT7-SlMAPK8 and pGADT7-SlMYB11 were grown on medium without Leu and Trp or medium without Leu, Trp, and His. Negative control, pGBKT7-SlMAPK8 + pGADT7; positive control, pGADT7-T + pGBKT7-53. E and F, BiFC analysis of the interaction of SlMAPK8 with SlARF4 (E) and SlMAPK8 with SlMYB11 (F). Nuclei were identified by DAPI staining. Bars = 100 μm. G and H, Coimmunoprecipitation assay of the interaction of SlMAPK8 with SlARF4 (G) and SlMAPK8 with SlMYB11 (H). Precipitates were detected with anti-GFP and anti-FLAG antibodies. I and J, SlMAPK8 phosphorylates SlARF4 and SlMYB11 in vitro. SlMAPK8 phosphorylates SlARF4 at Ser³⁵⁵ (I) and SlMAPK8 phosphorylates SlMYB11 at Thr¹⁶⁷ (J). Equal amounts of GST-SlARF4 and GST-SlMYB11, and mutated versions (GST-SlARF4^355A and GST-SlMYB11^167A) were detected with anti-GST antibodies and the phosphorylated proteins were detected with Phos-tag SDS-PAGE (arrows). GST purified protein and His purified protein were used as negative controls. K and L, SlMAPK8 phosphorylates SlARF4 and SlMYB11 in vivo. SlARF4/SlMYB11-GFP, SlARF4^355A/SlMYB11^167A-GFP, and SlMAPK8-FLAG were transiently expressed in N. benthamiana leaves. SlARF4/SlMYB11-GFP, SlARF4^355A/SlMYB11^167A-GFP, and SlMAPK8-FLAG proteins were purified by immunoprecipitation using anti-FLAG antibodies. Precipitates were detected with anti-GFP antibodies and the phosphorylated proteins were detected with Phos-tag SDS-PAGE (arrows). FLAG purified protein was used as a negative control. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 7

Effects of SlMAPK8 on the transcriptional activities of SlARF4 and SlMYB11. A, SlMAPK8 represses the transcriptional inhibition of SlMYB11 by SlARF4. SlARF4 and SlMAPK8 were used as effectors, SlMYB11 Pro was used as a reporter. B–D, SlMAPK8 increases the transcriptional activation activity of SlMYB11 on GLDH (B), GPP (C), and DHAR (D). SlMYB11 and SlMAPK8 were used as effectors, GLDH Pro, GPP Pro, and DHAR Pro were used as reporters. E, SlMAPK8 does not repress the transcriptional inhibition of SlMYB11 by SlARF4^355A. SlARF4^355A and SlMAPK8 were used as effectors, SlMYB11 Pro was used as a reporter. F–H, SlMAPK8 does not increase the transcriptional activation activity of SlMYB11^167A on GLDH (F), GPP (G), or DHAR (H). SlMYB11^167A and SlMAPK8 were used as effectors, GLDH Pro, GPP Pro, and DHAR Pro were used as reporters. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 8

SlMAPK8 is involved in ABA-induced AsA accumulation and drought stress tolerance in tomato. A–D, Total and reduced AsA contents in leaves (A, B) and pericarps (C, D) of SlMAPK8-RNAi plants. E–H, Total and reduced AsA contents in leaves (E, F) and pericarps (G, H) of SlMAPK8-RNAi plants under ABA (100 μM) treatment. I–L, Total and reduced AsA contents in leaves (I, J) and pericarps (K, L) of SlMAPK8-RNAi plants under drought treatment. Data are the mean ± se of at least three biological replicates. M, Drought tolerance of SlMAPK8-RNAi plants. RNAi-18: SlMAPK8-RNAi#18, RNAi-5: SlMAPK8-RNAi#5. Plants were treated with drought for 20 days. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 9

Effects of auxin, ABA, drought, and fluridone on the expression of genes involved in AsA biosynthesis in tomato. A and B, RT-qPCR analysis of the expression of SlARF4, SlMYB11, SlMAPK8, GPP, GLDH, and DHAR in leaves (A) and pericarps (B) of WT plants under IAA (30 μM) treatment. C and D, RT-qPCR analysis of the expression of SlARF4, SlMYB11, SlMAPK8, GPP, GLDH, and DHAR in leaves (C) and pericarps (D) of WT plants under ABA (100 μM) treatment. E–H, RT-qPCR analysis of the expression of SlARF4, SlMYB11, SlMAPK8, GPP, GLDH, and DHAR in leaves (E, F) and pericarps (G, H) of WT plants under drought, PEG 6000, and fluridone (30 μM) treatment. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 10

Auxin and ABA treatment affect AsA accumulation and the expression of AsA biosynthesis genes in tomato. A–D, RT-qPCR analysis of the expression of SlARF4, SlMYB11, SlMAPK8, GPP, GLDH, and DHAR in leaves (A, B) and pericarps (C, D) of WT plants under IAA and ABA treatment. E–H, Total and reduced AsA contents in leaves (E, F) and pericarps (G, H) of WT plants under IAA (30 mg/L) and ABA (100 μM) treatment. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 11

ABA antagonizes the effect of auxin on AsA accumulation in tomato. A–D, RT-qPCR analysis of the expression of SlMYB11 in leaves (A, B) and pericarps (C, D) of SlARF4 and SlMAPK8 transgenic plants under IAA and ABA treatment. E–L, Total and reduced AsA contents in leaves (E–H) and pericarps (I–L) of SlARF4 and SlMAPK8 transgenic plants under IAA (30 mg/L) and ABA (100 μM) treatments. Data are the mean ± se of at least three biological replicates. Significant differences (Tukey’s multiple range test, P < 0.05) are indicated by different lowercase letters.

Figure 12

Working model. Auxin and ABA antagonistically regulate the AsA accumulation mediated by the SlMAPK8–SlARF4–SlMYB11 module during plant development and drought tolerance in tomato.