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.

Introduction

Tomato (Solanum lycopersicum), one of the most valuable crops grown worldwide, is an ideal model system for studying fruit development and ripening (Wu et al., 2020). Tomato fruits are a major dietary source of antioxidants such as carotenoids, flavonoids, and ascorbic acid (AsA) (Klee and Giovannoni, 2011). AsA, also known as vitamin C, is a multifunctional phytonutrient that is essential for the human diet and plant development. AsA, which is among the most abundant water-soluble antioxidants, plays an important role in stress tolerance in plants. Extreme abiotic stress induces the accumulation of reactive oxygen species (ROS), leading to oxidative stress in plants. AsA counteracts oxidative stress either directly by detoxifying ROS or via the AsA–glutathione cycle during plant development (Smirnoff and Wheeler, 2000).

To date, four AsA biosynthesis pathways have been proposed in plants: the l-galactose, l-glucose, myo-inositol, and d-galacturonate pathways (Yoshimura and Ishikawa, 2017; Ishikawa et al., 2018). The l-galactose pathway is the dominant pathway in AsA biosynthesis. All enzymatic steps in this pathway have been characterized. The l-galactose pathway is responsible for converting d-glucose-6-P into ascorbate via nine enzymatic steps catalyzed by phosphoglucose isomerase (PGI), phosphomannose isomerase, phosphomannose mutase, GDP-d-mannose pyrophosphorylase (GMP), GDP-d-mannose-35 epimerase, GDP-l-galactose phosphorylase, l-galactose-1-phosphate phosphatase (GPP), l-galactose dehydrogenase, and l-galactono-1,4-lactone dehydrogenase (GLDH) (Yoshimura and Ishikawa, 2017; Ishikawa et al., 2018). The proper AsA concentration is also maintained via the recycling enzymes monodehydroascorbate and dehydroascorbate (DHAR) reductases. The increased expression of AsA biosynthesis and recycling genes improved AsA concentrations in tomato (Mellidou et al., 2021).

In recent years, much has been learned about the regulation of AsA biosynthesis and recycling. AsA biosynthesis and recycling are tightly regulated through transcriptional, translational, and post-translational mechanisms in plants (Bulley and Laing, 2016; Broad et al., 2020). The PbrMYB5 protein from birchleaf pear (Pyrus betulifolia) is an R2R3-type MYB transcription factor that modulates AsA biosynthesis by transcriptionally regulating PbrDHAR2 expression. The exogenous expression of PbrMYB5 in Nicotiana benthamiana increased the AsA concentration and enhanced tolerance to cold stress (Xing et al., 2019). Arabidopsis thaliana (Arabidopsis) ERF98, a member of the AP2/ERF transcription factor family, positively regulates the transcription of the AsA biosynthetic gene GMP. Overexpression (OE) of ERF98 increased the AsA concentration and enhanced tolerance to salt stress in Zhang et al. (2012). Arabidopsis COP9 signalosome subunit 5B (CSN5B), a component of the photomorphogenic COP9 signalosome, interacts with GMP and promotes the ubiquitination-dependent degradation of GMP through the 26S proteasome pathway in the dark (Wang et al., 2013). The tomato C2H2-type zinc finger protein ZF3 interacts with CSN5B and inhibits the ubiquitination-dependent degradation of GMP by CSN5B. Increased expression of the tomato ZF3 gene increased the AsA concentration and enhanced tolerance to salt stress (Li et al., 2018). However, little is known about the phosphorylation of enzymes associated with AsA biosynthesis and recycling.

Auxin is an important phytohormone for fruit setting, fruit initiation, and fruit development (Wang et al., 2005; De Jong et al., 2009). Auxin also plays an essential role in controlling the final fruit size by regulating cell expansion and division. Auxin modulates fruit development by transcriptionally regulating auxin-responsive genes, a process primarily mediated by auxin response factors (ARFs) (Ulmasov et al., 1999; Guilfoyle and Hagen, 2007). ARFs can function as either transcriptional activators or repressors of auxin-responsive genes and play important roles in many developmental processes in plants, such as shoot formation, floral development, trichome formation, and fruit setting and development (De Jong et al., 2009; Krogan et al., 2012; Sagar et al., 2013; Ckurshumova et al., 2014; Liu et al., 2014a, 2014b; Zhang et al., 2015; Yuan et al., 2018, 2019, 2021). SlARF10 and SlARF6A positively modulate chlorophyll and sugar accumulation during tomato fruit development (Yuan et al., 2018, 2019). SlARF4 negatively regulates chlorophyll accumulation and starch biosynthesis in tomato fruit (Sagar et al., 2013). SlARF4 interacts with SlMYB72, which affects the accumulation of chlorophyll, carotenoids, and flavonoids in tomato fruit (Wu et al., 2020). The downregulation or knockout of SlARF4 improved the tolerance of tomato to salinity and osmotic stress (Bouzroud et al., 2020). However, little is known about the relationship between auxin signaling and AsA biosynthesis.

Abscisic acid (ABA) is an important phytohormone that mediates abiotic stress responses in plants. Mitogen-activated protein kinase (MAPK) cascades have been implicated in ABA-mediated abiotic stress responses (Danquah et al., 2015). The ABA-induced transcriptional regulation of genes in the MAPK cascade has been reported in many plant species, such as Arabidopsis, rice (Oryza sativa), and maize (Zea mays) (Xiong and Yang, 2003; Menges et al., 2008; Zhang et al., 2012). ABA induces AsA production and improves drought tolerance by regulating the PTP-like nucleotidase (PTPN) gene in Arabidopsis and maize (Zhang et al., 2020). However, whether the MAPK cascade is involved in ABA-induced AsA biosynthesis remains unknown. In this study, we demonstrate that auxin and ABA affect AsA biosynthesis in tomato, a process mediated by the SlMAPK8–SlARF4–SlMYB11 module. These findings increase our understanding of the regulation of AsA levels by phytohormones and may facilitate the improvement of abiotic stress tolerance in plants.

Results

Auxin-mediated inhibition of AsA biosynthesis is mediated by SlARF4 in tomato

AsA protects cells by detoxifying oxidation products generated by various stresses. To investigate the effects of auxin on AsA accumulation, we measured total and reduced AsA contents at different stages of fruit development (8, 15, 25, 35, 40, and 45 days post-anthesis [DPA]). The AsA content in the fruit pericarp peaked at 25 DPA, decreased gradually, and reached the lowest level at 45 DPA (Figure 1A and Supplemental Figure S1A). We then studied auxin distribution during fruit development and ripening in transgenic tomato expressing the GUS reporter gene driven by the auxin-responsive DR5 promoter. GUS activity in fruits was highest at 8 DPA, decreased gradually, and reached its lowest level at 35 DPA, followed by an increase at 40 and 45 DPA (Figure 1, B and D). We then analyzed the content of indole-3-acetic acid (IAA), the predominant natural auxin, in tomato pericarps at different stages of fruit development. The accumulation of IAA peaked at 8 DPA and then decreased gradually, reaching the lowest level at 35 DPA, followed by an increase at 40 and 45 DPA (Figure 1C). The results showed that the auxin content negatively corresponded with AsA content during fruit development and ripening. We then investigated the effect of auxin on AsA accumulation by treating tomato plants with 10, 20, 30 50, and 100 mg/L IAA. Treatment with all IAA concentrations inhibited AsA accumulation in the leaf and pericarp; 30 mg/L IAA treatment had the strongest effects (Supplemental Figure S2, A–D) and was thus used for further study. These results indicate that auxin inhibits AsA accumulation in tomato plants.

We analyzed the expression patterns of ARF family genes in the pericarps of wild-type (WT) tomato under IAA treatment. The ARF family genes were upregulated after IAA treatment, and among these genes, SlARF4 was induced the most strongly (Supplemental Figure S3). The expression level of SlARF4 was highest at 15 DPA and lowest at 35 DPA (Figure 1E). The expression pattern of SlARF4 is consistent with the auxin concentration pattern but opposite that of AsA in developing fruit. Thus, SlARF4 might be involved in AsA biosynthesis during fruit development and ripening. In a previous study, SlARF4-OE (i.e. SlARF4-OE#28 and SlARF4-OE#31) and downregulation (i.e. SlARF4-RNAi#18) and CRISPR/Cas9 knockout plants (Slarf4) were generated (Yuan et al., 2021). We measured the AsA contents in the fruits of the transgenic plants. The total and reduced AsA contents in the leaves and pericarps of SlARF4-OE plants were lower than those of WT plants but higher than those of Slarf4 and SlARF4-RNAi plants (Figure 1, F–I). To investigate whether SlARF4 is involved in the inhibition of AsA accumulation by auxin, we treated transgenic and mutant plants with IAA. The total and reduced AsA contents in the leaves and pericarps of SlARF4-OE plants treated with IAA were reduced compared with untreated plants. However, IAA treatment did not alter the AsA contents in the leaves or pericarps of Slarf4 or SlARF4-RNAi#18 plants (Figure 1, J–M). These results indicate that the auxin-induced inhibition of AsA accumulation is mediated by SlARF4 in tomato.

Auxin-mediated inhibition of SlMYB11 expression is mediated by SlARF4, and SlARF4 directly targets the SlMYB11 promoter and inhibits its expression

Previous RNA-sequencing data revealed that the ascorbate metabolism pathway is affected in SlARF4-downregulated plants and that the MYB transcription factor gene SlMYB11is upregulated in these plants (Yuan et al., 2021; Figure 2, C and D). The SlMYB11 gene contained an 882-bp open-reading frame encoding a protein with 293 amino acid residues (Supplemental Figure S4 and Supplemental File S1). We conducted phylogenetic analysis using MEGA7 to examine the relationship between SlMYB11 and homologous proteins. SlMYB11, SlMYB12, and SlMYB111 belong to the S2 subfamily of the R2R3-MYB transcription factor family (Supplemental Figure S5 and Supplemental File S2). The SlMYB11 protein fused with green fluorescent protein (GFP) was transiently expressed in N. benthamiana leaf epidermal cells. Fluorescent signals were detected in the nucleus, indicating that SlMYB11 localizes to the nucleus in tomato cells (Figure 2A). The SlMYB11 expression pattern in the pericarp is consistent with the AsA content pattern (Figure 2B), suggesting that SlMYB11 might be involved in AsA biosynthesis.

We evaluated the expression levels of SlMYB11 in the leaves and pericarps of SlARF4 transgenic and mutant plants. SlMYB11 expression increased in SlARF4-downregulated and mutant plants but decreased in SlARF4-OE plants (Figure 2, C and D). The expression level of SlMYB11 in the leaves and pericarps of SlARF4-RNAi and Slarf4 plants treated with IAA did not change compared with the control, whereas SlMYB11 expression decreased in SlARF4-OE plants treated with IAA (Figure 2, E and F). These data indicate that SlMYB11 expression is negatively regulated by SlARF4 and IAA and that the IAA-mediated inhibition of SlMYB11 expression is mediated by SlARF4.

Sequence analysis revealed that the promoter of SlMYB11 contains a TGA (TGTCTC, −576 bp) element, which is an ARF-binding motif. To determine whether SlARF4 physically binds to the promoter of SlMYB11, we performed an electrophoretic mobility shift assay (EMSA) using purified recombinant truncated SlARF4 and glutathione S-transferase (GST) fusion protein (GST-tSlARF4) (Supplemental Figure S6). The GST-tSlARF4 fusion protein bound to biotin-labeled probes from the TGA (TGTCTC) element of the SlMYB11 promoter, resulting in mobility shifts. When the unlabeled promoter fragment was added as a competitor, the mobility shift was eliminated, and no mobility shift was observed when biotin-labeled probes were incubated with GST (Figure 2G). We performed a chromatin immunoprecipitation-qPCR (ChIP-qPCR) assay to verify the binding of SlARF4 with the SlMYB11 promoter in vivo. The promoter sequence of SlMYB11 containing the TGA motif was specifically enriched using the FLAG antibody instead of the nonspecific antibody (Figure 2H and Supplemental Figure S7). SlARF4 (35S:SlARF4) was co-expressed with the reporter SlMYB11:LUC in N. benthamiana, and SlARF6 (35S:SlARF6) and SlARF10 (35S:SlARF10) were co-expressed with the reporter SlMYB11:LUC in N. benthamiana as the negative control. To further test that SlARF4 interacts with the SlMYB11 promoter in vivo, we measured relative luciferase (LUC) activity. OE of SlARF4 decreased LUC activity driven by the SlMYB11 promoter compared with the empty vector control and SlARF6 (35S:SlARF6) and SlARF10 (35S:SlARF10) (Figure 2, I and J). These results indicate that SlARF4 directly targets the SlMYB11 promoter and inhibits its transcription.

SlMYB11 positively affects AsA accumulation via the transcriptional activation of GPP, GLDH, and DHAR in tomato

We generated SlMYB11-RNAi plants to clarify the physiological importance of the SlMYB11 gene in AsA biosynthesis. Compared with WT plants, SlMYB11 was downregulated in the SlMYB11-RNAi#24 and SlMYB11-RNAi#27 lines; these lines were, therefore, used for further study (Supplemental Figure S8). We measured the total and reduced AsA contents in the leaves and pericarps of SlMYB11-RNAi plants, finding that the AsA contents in SlMYB11-RNAi plants were significantly lower than those in WT plants (Figure 3, A–D). The total and reduced AsA contents in the leaves and pericarps of WT plants treated with IAA were reduced compared with untreated plants. However, IAA treatment did not alter the AsA contents in the leaves or pericarps of SlMYB11-RNAi plants (Figure 3, E–H). These data indicate that SlMYB11 positively affects AsA accumulation and that the IAA-induced inhibition of AsA production might be mediated by SlMYB11 in tomato plants.

We analyzed the expression of AsA biosynthesis genes in SlMYB11-RNAi plants to determine whether SlMYB11 directly affects AsA biosynthesis in tomato. GPP, GLDH, and DHAR were distinctly downregulated in SlMYB11-RNAi plants versus WT (Figure 3, I and J), suggesting that SlMYB11 positively regulates the expression of these genes. The GPP, GLDH, and DHAR promoters contain the MYB-binding site CAACCA/TAACCA. The direct binding of SlMYB11 to the GPP, GLDH, and DHAR promoters was verified by EMSA. We then generated recombinant SlMYB11 and GST fusion protein (GST-SlMYB11) (Supplemental Figure S9). The GST-SlMYB11 fusion protein bound to biotin-labeled probes containing the CAACCA/TAACCA element from the GPP, GLDH, and DHAR promoters, resulting in mobility shifts. When unlabeled GPP, GLDH, and DHAR promoter fragments were added as competitors, the mobility shift was effectively eliminated. When biotin-labeled probes were incubated with GST, no mobility shift was observed (Figure 3, K–M). These results indicate that SlMYB11 specifically targets the GPP, GLDH, and DHAR promoters. To analyze the interaction of SlMYB11 with the GPP, GLDH, and DHAR promoters, we performed a dual-LUC reporter assay. The SlMYB11 gene driven by the 35S promoter was co-expressed with the LUC gene driven by the GPP, GLDH, and DHAR promoters in N. benthamiana, and the relative LUC activity was measured. The OE of SlMYB11 significantly enhanced the LUC activity driven by the GPP, GLDH, and DHAR promoters compared with the empty vector control (Figure 3, N and O). These results indicate that SlMYB11 directly targets the GPP, GLDH, and DHAR promoters and promotes their transcription.

Regulation of AsA biosynthesis by SlARF4 and auxin is dependent on SlMYB11

To further investigate the functional relationship between SlARF4 and SlMYB11 in AsA biosynthesis in tomato, we conducted virus-induced gene silencing (VIGS) of SlMYB11 in SlARF4-OE plants. Independent VIGS tomato plants (VIGS-SlMYB11-SlARF4-OE, VIGS-SlMYB11-SlARF4-RNAi, and VIGS-SlMYB11-Slarf4) exhibited decreased expression of SlMYB11 and were chosen for AsA analysis (Figure 4A). The silencing of SlMYB11 decreased the total and reduced AsA contents in the leaves and pericarps of SlARF4-RNAi, SlARF4-OE, Slarf4, and WT plants (Figure 4, B–E). However, the relative reduction in the total and reduced AsA contents in VIGS-SlMYB11-SlARF4-OE plants was less than in SlMYB11-silenced plants in the WT background. The relative reduction in AsA contents in the leaves and pericarps of VIGS-SlMYB11-SlARF4-RNAi and VIGS-SlMYB11-Slarf4 plants was greater than that of WT plants (Figure 4, B–E). Moreover, the total and reduced AsA contents were unchanged in VIGS-SlMYB11-SlARF4-OE, VIGS-SlMYB11-SlARF4-RNAi, and VIGS-SlMYB11-Slarf4 plants treated with IAA compared with untreated plants (Figure 4, F–I). These results indicate that the regulation of AsA biosynthesis by SlARF4 and auxin is at least partially dependent on SlMYB11.

ABA increases AsA accumulation in tomato under drought stress

ABA mediates the drought stress response in plants. Therefore, we studied the role of ABA in AsA accumulation. Drought stress and PEG 6000 treatment increased the ABA content in the leaves and pericarps of tomato compared with the control. However, drought stress and fluridone (an ABA biosynthesis inhibitor) treatment decreased the ABA content in tomato (Figure 5, A and B). We also measured the AsA content in the leaves and pericarps of tomato under ABA treatment. ABA treatment increased the AsA content compared with untreated plants, whereas treatment with ABA and fluridone decreased the AsA content (Figure 5, C–F). Drought stress and PEG 6000 treatment increased the total and reduced AsA contents in the leaves and pericarps of tomato compared with the control. However, drought stress and fluridone treatment decreased the AsA contents in these organs (Figure 5, G–J). These results indicate that drought stress increases ABA accumulation and that ABA increases AsA biosynthesis in tomato.

SlMAPK8 is induced by ABA, and SlMAPK8 phosphorylates SlARF4 and SlMYB11

In Arabidopsis, the mitogen-activated protein kinase AtMPK7 is involved in the drought response induced by ABA (Danquah et al., 2015). SlMAPK8 protein shares highly conserved sequence motifs with AtMPK7 proteins, and RT-qPCR analysis showed that SlMAPK8 was induced by ABA treatment and drought stress (Figure 6, A and B). To investigate whether SlMAPK8 interacts with SlMYB11 and SlARF4, we performed yeast two-hybrid (Y2H) analysis by co-transforming yeast with the SlMAPK8-BD vector and the SlMYB11-AD or SlARF4-AD vector. The transformed yeast grew on medium lacking Leu, Trp, His, and Ade (Figure 6, C and D), indicating that SlMAPK8 interacted with SlMYB11 and SlARF4. We performed a bimolecular fluorescence complementation (BiFC) test to further verify this interaction in vivo by co-transforming N. benthamiana leaf epidermal cells with the SlMAPK8-cYFP plasmid and SlMYB11-nYFP or SlARF4-nYFP. Analysis of fluorescent signals showed that SlMAPK8 interacted with SlMYB11 and SlARF4 in the nucleus (Figure 6, E and F). Finally, we performed co-immunoprecipitation (Co-IP) experiments to verify these interactions. SlMAPK8-FLAG co-precipitated with SlARF4-GFP and SlMYB11-GFP, confirming that SlMAPK8 interacts with SlMYB11 and SlARF4 (Figure 6, G and H).

MAPK family proteins usually phosphorylate serine and threonine residues in the S/T-P motifs of their target proteins. Phosphorylation site analysis showed that SlARF4 contains serine at positions 317 and 355 (Ser³¹⁷ and Ser³⁵⁵) and that SlMYB11 contains threonine at position 167 (Thr¹⁶⁷). To examine whether SlMAPK8 can phosphorylate SlARF4 and SlMYB11, we performed in vitro phosphorylation experiments. We used purified His-SlARF4 and GST-SlMYB11 fusion proteins as substrates and employed the fusion protein GST-SlMAPK8 and His-SlMAPK8, His-SlARF4, and GST-SlMYB11 in an in vitro kinase assay (Supplemental Figure S10). Phosphorylation bands of purified His-SlARF4 and GST-SlMYB11 with GST-SlMAPK8 and His-SlMAPK8, respectively, were detected (Figure 6, I and J). When the Ser³⁵⁵ of SlARF4 was replaced by alanine (SlARF4^355A), the phosphorylation band was not detected (Figure 6I), indicating that SlMAPK8 phosphorylated SlARF4 at the Ser³⁵⁵ position. When the Thr¹⁶⁷ of SlMYB11 was replaced by alanine (SlMYB11^167A), the autophosphorylation band was not detected (Figure 6J), indicating that SlMAPK8 phosphorylated SlMYB11 at the Thr¹⁶⁷ position.

We then performed an in vivo phosphorylation experiment by transiently expressing SlARF4-GFP, SlMAPK8-FLAG, and SlMYB11-GFP in N. benthamiana and detecting phosphorylation bands of SlARF4 and SlMYB11 proteins by immunoblotting. The application of ABA significantly increased the phosphorylation intensity of SlARF4 and SlMYB11. When the Ser³⁵⁵ of SlARF4 and Thr¹⁶⁷ of SlMYB11 were replaced by alanine (SlARF4^355A and SlMYB11^167A), the phosphorylation band was not detected. These data indicate that SlMAPK8 phosphorylates SlARF4 and SlMYB11 and that this phosphorylation is induced by ABA in vivo (Figure 6, K and L).

We performed a dual-LUC reporter assay to analyze the functions of the phosphorylation of SlMYB11 and SlARF4 by SlMAPK8. The transcriptional inhibition of SlMYB11 by SlARF4 was significantly repressed when SlMAPK8 was added to the dual-LUC assay (Figure 7A). The transcriptional activation of GPP, GLDH, and DHAR by SlMYB11 was significantly improved when SlMAPK8 was added to the dual-LUC assay (Figure 7, B–D). However, SlMAPK8 did not affect the transcriptional inhibition of SlMYB11 by SlARF4^355A (Figure 7E). SlMAPK8 did not affect the transcriptional activation of GPP, GLDH, or DHAR by SlMYB11^167A (Figure 7, F–H). These results indicate that SlMAPK8 phosphorylates SlARF4 and inhibits its inhibitory activity and that SlMAPK8 phosphorylates SlMYB11 and enhances its transcriptional activation activity.

SlMAPK8 is involved in ABA-induced AsA accumulation and drought stress tolerance in tomato

To further verify the effect of SlMAPK8 on AsA accumulation under drought stress, we generated SlMAPK8-downregulated plants (RNAi-SlMAPK8) using RNAi (Supplemental Figure S11). We then measured the AsA contents in the leaves and pericarps of the SlMAPK8-RNAi plants. The downregulation of SlMAPK8 reduced the total and reduced AsA contents in the leaves and pericarps of these plants (Figure 8, A–D). We performed ABA and drought-stress treatment and analyzed the AsA contents in SlMAPK8-RNAi plants. ABA treatment increased the AsA contents in the leaves and pericarps of WT plants but did not affect the AsA contents in SlMAPK8-RNAi plants (Figure 8, E–H). Drought stress resulted in responses similar to those induced by ABA treatment (Figure 8, I–L). These results indicate that SlMAPK8 is involved in ABA-induced AsA biosynthesis in the leaves and pericarps of tomato.

We subjected the SlMAPK8-RNAi plants to drought treatment for 20 days to test the tolerance of the plants to drought stress. The SlMAPK8-RNAi plants exhibited more severe wilting than WT plants in response to drought treatment (Figure 8M), indicating that the downregulation of SlMAPK8 decreased the tolerance of plants to drought stress.

ABA antagonizes the effect of auxin on AsA biosynthesis in tomato plants

We analyzed the effects of IAA treatment on the expression levels of SlMAPK8, SlARF4, SlMYB11, GPP, GLDH, and DHAR in the leaves and pericarps of tomato. SlMAPK8 expression was unchanged; SlARF4 expression increased; and SlMYB11, GPP, GLDH, and DHAR expression decreased in treated plants compared with the untreated control (Figure 9, A and B). Under ABA treatment, SlMAPK8, SlMYB11, GPP, GLDH, and DHAR expression increased, whereas SlARF4 expression decreased (Figure 9, C and D). Under the drought stress and PEG 6000 treatment, SlMAPK8, SlMYB11, GPP, GLDH, and DHAR expression increased, whereas SlARF4 expression decreased (Figure 9, E–H). Treatment with fluridone and drought stress inhibited the effect of drought stress on gene expression (Figure 9, E–H).

We treated tomato plants with IAA and ABA to analyze the effects of the two phytohormones on AsA biosynthesis. The expression levels of SlMAPK8, SlMYB11, GPP, GLDH, and DHAR were higher in the leaves and pericarps of plants treated with IAA and ABA compared with plants treated with IAA alone. SlARF4 expression in the leaves and pericarps of plants treated with IAA plus ABA was reduced compared with plants treated with IAA alone (Figure 10, A–D). IAA plus ABA treatment increased the total and reduced AsA contents in leaves and pericarps compared with plants treated with IAA alone (Figure 10, E–H). Finally, we treated SlARF4 and SlMAPK8 transgenic plants with IAA and ABA to further examine the effects of the two phytohormones on AsA biosynthesis. The expression levels of SlMYB11 in the leaves and pericarps were lower in SlARF4-OE and WT plants versus the control when treated with IAA alone (Figure 11, A–D). Treatment with ABA alone, and IAA and ABA in combination, increased the expression of SlMYB11 in the leaves and pericarps of both SlARF4-OE and WT plants (Figure 11, A–D). The expression levels of SlMYB11 in the leaves and pericarps of SlARF4-RNAi, Slarf4, and SlMAPK8-RNAi plants were unchanged compared with the control when treated with IAA, ABA, and both phytohormones combined (Figure 11, A–D). IAA treatment decreased the total and reduced AsA contents in SlARF4-OE and WT leaves and pericarps compared with the control (Figure 11, E–L). ABA alone, and IAA and ABA in combination, increased the total and reduced AsA contents in SlARF4-OE and WT leaves and pericarps (Figure 11, E–L). The total and reduced AsA contents in the leaves and pericarps of SlARF4-RNAi, Slarf4, and SlMAPK8-RNAi plants were unchanged compared with the control under treatment with IAA, ABA, and both phytohormones combined (Figure 11, E–L). These results demonstrate that ABA antagonizes the effect of auxin on AsA accumulation in tomato.

Discussion

Auxin inhibits AsA biosynthesis via the SlARF4–SlMYB11–GPP/GLDH/DHAR transcriptional cascade during tomato development

Little is known about the effects of auxin on AsA biosynthesis. In this study, AsA accumulation negatively corresponded with the IAA concentration during tomato fruit development, indicating that auxin inhibits AsA accumulation in tomato. The results of IAA treatment of tomato fruit and leaves also supported this finding. SlARF4, an ARF gene, is induced by auxin; SlARF4 acts as a transcriptional repressor of auxin-responsive target genes (Sagar et al., 2013). Bouzroud et al. (2020) reported that SlARF4 negatively regulates salinity and osmotic stress responses in tomato. This study showed that SlARF4 negatively modulates AsA accumulation in the pericarps and leaves of tomato plants. The function of SlARF4 in salinity and osmotic stress tolerance might reflect the finding that AsA biosynthesis is affected by SlARF4 in tomato. SlARF4 transcriptionally inhibits the R2R3 MYB factor genes SlMYB75 and SlTHM1, encoding regulators of trichome formation and terpenoid biosynthesis in tomato (Gong et al., 2021; Yuan et al., 2021). In this study, we demonstrated that SlARF4 directly targets SlMYB11 to modulate AsA biosynthesis in tomato. Silencing of SlMYB11 in SlARF4 transgenic plants and mutants by the VIGS method indicated that the regulation of AsA biosynthesis by SlARF4 is at least partially dependent on SlMYB11. Preliminary results showed that SlARF4 regulates an additional gene, SlMYB99, to affect AsA accumulation in tomato. The functionally redundant SlMYBs that are regulated by SlARF4 might be involved in AsA biosynthesis. A study of the interaction between SlARF4 and SlMYBs will be performed in the future to clarify the functional redundancy of SlMYBs in the auxin-mediated regulation of AsA biosynthesis.

PbrMYB5 modulates AsA biosynthesis via the transcriptional activation of PbrDHAR2 expression in P. betulifolia (Xing et al., 2019). In this study, we demonstrated that SlMYB11 transcriptionally activates the expression of GPP, GLDH, and DHAR and positively regulates AsA biosynthesis in tomato. In addition, the effect of SlMYB11 on AsA biosynthesis was inhibited upon IAA treatment, and the inhibition of AsA biosynthesis by SlARF4 was at least partially dependent on SlMYB11. These data demonstrate that the auxin-dependent SlARF4–SlMYB11–GPP/GLDH/DHAR transcriptional cascade modulates AsA biosynthesis, expanding our knowledge of the regulatory mechanism of AsA biosynthesis via plant hormones in tomato.

ABA affects AsA biosynthesis by regulating phosphorylation during drought stress

ABA plays an important role in drought responses in plants (Zhu et al., 2007). Drought stress increases ABA accumulation, which leads to stomatal closure to prevent water loss in plants (Himmelbach et al., 2003). ABA enhances AsA biosynthesis and drought tolerance (Zhang et al., 2020). In this study, we showed that drought stress increases ABA accumulation and that ABA increases AsA biosynthesis in tomato. The increase in AsA production might be one reason for the ABA-mediated drought stress tolerance in plants. Zhang et al. (2020) elucidated one regulatory pathway of ABA-induced AsA production. ABA increases AsA biosynthesis via the regulation of PTPN in Arabidopsis and maize (Zhang et al., 2020). In this study, SlMAPK8 was induced in the leaves and pericarps of tomato under ABA treatment or drought stress. We demonstrated that SlMAPK8 phosphorylates SlARF4 and represses its transcriptional inhibition activity. By contrast, SlMAPK8 phosphorylates SlMYB11 and activates its transcriptional activation activity. SlMAPK8 is involved in ABA-induced AsA biosynthesis in the leaves and pericarps of tomato. The present results reveal an additional regulatory pathway in which ABA-induced AsA production is mediated by SlMAPK8 in tomato. MAPK cascades have been implicated in certain ABA responses, including guard cell signaling, antioxidant defense, and seed germination (Xing et al., 2008; Jammes et al., 2009; Zong et al., 2009; Zhang et al., 2010). The AtMKK1–AtMPK6 module is involved in ABA-induced catalase expression in Arabidopsis (Xing et al., 2008). The present results further support the important role of the MAPK cascade in ABA-induced antioxidant defense. Further studies are needed to identify the MAPK cascade components involved in SlMAPK8-mediated AsA biosynthesis.

The MAPKKK18–MAPKK3 cascade enhances drought tolerance in Arabidopsis (Li et al., 2017a). The Raf-like MAPKKK gene DSM1 positively regulates drought stress tolerance via ROS scavenging in rice (Ning et al., 2010). The MAPKKK15–MAPKK4–MAPK6–WRKY59 pathway regulates the drought response, which is mediated by GhDREB2 in an ABA-independent pathway in cotton (Gossypium hirsutum) (Li et al., 2017b). The MAPKKK14–MAPKK11–MAPK31 pathway modulates drought stress tolerance in cotton (Chen et al., 2020). In this study, the downregulation of SlMAPK8 decreased ABA-dependent tolerance to drought in tomato. These findings show the important roles of the MAPK cascade in ABA-mediated drought tolerance. In the future, the effects of overexpressing SlMAPK8 in plants will be studied to analyze the role of SlMAPK8 in drought tolerance.

ABA promotes AsA production by inhibiting SlARF4 activity and activating SlMYB11 activity induced by auxin

The interaction between ABA and auxin affects various growth and developmental processes in plants (Emenecker and Strader, 2020). Auxin and ABA synergistically inhibit seed germination. Auxin biosynthesis, transport, and signaling are required for the ABA-mediated suppression of seed germination (Rinaldi et al., 2012; Liu et al., 2013; Thole et al., 2014). Auxin and ABA antagonistically affect root hair growth, which is promoted by auxin and inhibited by ABA (Zhang et al., 2016; Rymen et al., 2017). In this study, we demonstrated that auxin and ABA antagonistically modulate AsA production in the leaves and pericarps of tomato. The IAA-induced inhibition of AsA biosynthesis is mediated by the transcriptional regulation of the SlARF4–SlMYB11–GPP/GLDH/DHAR cascade. However, ABA activates AsA production by the phosphorylation of SlARF4 and SlMYB11 via SlMAPK8. These findings demonstrate that the SlMAPK8–SlARF4–SlMYB11 module represents an important integrative hub mediating crosstalk between auxin and ABA during AsA biosynthesis in tomato. The present findings provide insight into the antagonistic effects of auxin and ABA and the transcriptional and post-translational regulation of AsA production in tomato. Treatment with ABA decreased SlARF4 expression but increased SlMYB11, GPP, GLDH, and DHAR expression in tomato. These results suggest that the regulation of AsA biosynthesis by ABA is affected not only by the phosphorylation of SlARF4 and SlMYB11 by SlMAPK8 but also by the transcriptional activation of SlARF4.

In summary, our findings indicate that auxin inhibits AsA accumulation by inducing SlARF4 expression during leaf and pericarp development in tomato. SlARF4 transcriptionally inhibits SlMYB11 expression, and SlMYB11 transcriptionally activates the expression of GPP, GLDH, and DHAR. The auxin-dependent SlARF4–SlMYB11–GPP/GLDH/DHAR transcriptional cascade modulates AsA biosynthesis in tomato. ABA increases AsA accumulation by inducing SlMAPK8 expression under drought stress. SlMAPK8 phosphorylates SlARF4 and inhibits its transcriptional activity, whereas SlMAPK8 phosphorylates SlMYB11 and activates its transcriptional activity (Figure 12). These data demonstrate that auxin and ABA antagonistically regulate AsA accumulation mediated by the SlMAPK8–SlARF4–SlMYB11 module during plant development and drought stress in tomato. These findings expand our knowledge of the regulatory mechanism of AsA biosynthesis in tomato and provide a potential target to improve AsA accumulation and abiotic stress tolerance in horticultural crops.

Materials and methods

Plant material and growth conditions

Seeds of SlARF4-OE, SlARF4-RNAi, and Slarf4 tomato (S. lycopersicum “Micro-Tom”) plants were preserved in our laboratory. The plants were grown in a naturally illuminated greenhouse with a 16-h day (26°C ± 2°C)/8-h night (20°C ± 2°C) cycle, 60% relative humidity, and 250 mol/m²/s light intensity.

IAA treatment

Fifteen-day-old tomato seedlings were sprayed with IAA solution (10, 20, 30, 50, 80, or 100 mg/L) every other day for 30 days. The control group was treated with water containing 0.1% DMSO. Three or four leaves were collected from the upper shoots of the treated plants. The leaves were immediately frozen in liquid nitrogen and stored at −80°C for further analysis. Fruits at 2 days after pollination (DAP) were sprayed with 30 mg/L IAA every other day for 25 days. The control group was treated with water containing 0.1% DMSO. The pericarps of treated fruits were separated from the flesh, immediately frozen in liquid nitrogen, and stored at −80°C for further analysis. All the measurements were performed with at least three biological replicates. The replicate samples were collected from three different plants, and each sample contained three leaves or fruits.

ABA, fluridone, and PEG 6000, and drought stress treatments

Fifteen-day-old tomato plants were sprayed with 100 μM ABA or 30 μM fluridone every other day for 20 days. The leaves were immediately frozen in liquid nitrogen and stored at −80°C for further analysis. Fruits at the 10 DAP stage were sprayed with 100 μM ABA every other day for 10 days. The pericarps were separated from the flesh of fruits, immediately frozen in liquid nitrogen, and stored at −80°C for further analysis. Fifteen-day-old tomato plants were subjected to drought stress by withholding watering or by spraying with 25% (w/v) PEG 6000 for 20 days. Treated leaves or pericarps were immediately frozen in liquid nitrogen and stored at −80°C for further analysis. At least three biological replicates were performed for all measurements. The replicate samples were collected from three different plants, and each sample contained three leaves or fruits.

Analysis of AsA contents

Leaf and pericarp samples from plants under IAA (30 mg/L), ABA (100 µM), and drought treatment and the control were ground in liquid nitrogen. The samples were used for the analysis of total and reduced AsA contents as described by Hu et al. (2016). At least three biological replicates were performed for all measurements. The replicate samples were collected from three different plants, and each sample contained three leaves or fruits.

Quantitative GUS activity assay

GUS activity was determined using 4-methylumbelliferyl-β-d-glucuronide (MUG) as the substrate as described by Jefferson (1987). Samples (0.1 g) of tomato pericarp at various stages of development were ground in liquid nitrogen and homogenized in 1 mL GUS protein extraction buffer (0.1 M sodium phosphate [pH 7.0], 30% sodium lauroyl sarcosine, 0.5 M Na₂EDTA [pH 8.0], 10 mM β-mercaptoethanol, and 10% Triton X-100). The homogenate was centrifuged twice to eliminate the debris at 12,000 g at 4°C for 10 min. The supernatant was used for protein quantification and the fluorometric assay. To quantify GUS activity in the pericarp, 50 μL supernatant was added to 200 μL protein extraction buffer and 250 μL GUS reaction buffer (protein extraction buffer containing 2 mM MUG) and incubated at 37°C for 1 h in a water bath. The reaction was terminated by adding 75 μL of 200 mM Na₂CO₃. The fluorescence intensity was measured using a Hitachi fluorescence spectrophotometer (model F-7100) at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. GUS activity was expressed as nmol MUG/min/mg protein. At least three biological replicates were performed for all measurements. The replicate samples were collected from three different plants, and each sample contained three fruits.

Plasmid construction and plant transformation

To construct the SlMYB11-RNAi and SlMAPK8-RNAi vectors, 200-bp target fragments of SlMYB11 and SlMAPK8 were inserted around a GUS gene spacer in the pCambia2301 vector under the control of the Cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase terminator. The constructs were transformed into Agrobacterium tumefaciens strain GV3101 (WEIDI) following the manufacturer’s instructions, and tomato plants were transformed using the A. tumefaciens-mediated method (Deng et al., 2012). The T₂ and T₃ transgenic seeds were screened on half-strength Murashige and Skoog medium supplemented with 100 mg/L kanamycin. The primer sequences used are listed in Supplemental Data Set S1.

Subcellular localization of SlMYB11

The SlMYB11 full-length cDNA without the stop codon was amplified and inserted into the PCX-DG vector containing the GFP sequence and CaMV 35S promoter. The recombinant vector was transferred into A. tumefaciens strain GV3101 (WEIDI). Agrobacterium cultures were resuspended in infiltration buffer (0.2 M MES [pH 5.6], 1 M MgCl₂, and 200 µM acetosyringone) to a final OD₆₀₀ of 0.5. The vectors were transformed into 4- to 6-week-old N. benthamiana leaves. Fluorescence was observed under a confocal laser scanning microscope (Olympus Fluoview FV1000) after incubation for 48–72 h as described by Yuan et al. (2021). Nuclei were identified by staining with 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, China). The primer sequences used are listed in Supplemental Data Set S1.

RT-qPCR analysis

Total RNA was extracted from the samples using an RNAsimple Total RNA Kit (Tiangen) and reverse-transcribed into cDNA using a Takara PrimeScript Reagent Kit (Takara). RT-qPCR was conducted using 2× TSINGKE Master qPCR Mix (SYBR Green I with UDG) and the CFX96 Real-time PCR Detection System (Bio-Rad) as described by Wu et al. (2020). The relative expression levels of genes were calculated using the 2^−ΔΔCt method and normalized to Actin gene expression. Each experiment was performed using at least three biological replicates. The primer sequences used are listed in Supplemental Data Set S1.

Y2H assays

The Y2H assays were conducted using a GAL4 Yeast Two-Hybrid Media plus Kit following the manufacturer’s instructions (Clontech). The coding sequences of SlMYB11 and SlARF4 were cloned into the pGADT7 vector. The coding sequence of SlMAPK8 was cloned into the pGBKT7 vector. The AD and BD fusion constructs were co-transformed into yeast strain Y2HGold cells (Clontech) and selected on synthetic medium (SD) lacking Trp and Leu, and SD medium lacking Trp, Leu, His, and Ade, supplemented with 0.04 mg/mL X-α-galactose and 100 ng/mL Aureobasidin A. Protein interactions were analyzed based on growth status and α-galactose activity. The primer sequences used are listed in Supplemental Data Set S1.

BiFC and Co-IP assays

For BiFC, the vectors pXY106 and pXY104 carrying the N- and C-terminal halves, respectively, of yellow fluorescent protein were used in the BiFC assay (Wu et al., 2020). The coding sequence of SlMYB11 or SlARF4 was cloned into pXY104, and the coding sequence of SlMAPK8 was cloned into pXY106. Agrobacterium cultures were resuspended in infiltration buffer (0.2 M MES [pH 5.6], 1 M MgCl₂, and 200 µM acetosyringone) to a final OD₆₀₀ of 0.5. The vectors were co-transformed into 4- to 6-week-old N. benthamiana leaves. Fluorescence was observed under a confocal laser scanning microscope (Olympus Fluoview FV1000) after incubation for 48–72 h.

For Co-IP, the coding sequence of SlMYB11 or SlARF4 was cloned into the K303-GFP vector. The SlMAPK8 coding sequence was cloned into the pLP100-35S-FLAG vector. The recombinant vectors were transiently co-expressed in N. benthamiana leaves by Agrobacterium infiltration as previously described. The Co-IP assay was performed as described by Zentella et al. (2016). Briefly, total proteins were extracted from the N. benthamiana leaves in extraction buffer (NP-40 Lysis Buffer [Beyotime] including 1 mM PMSF) and centrifuged at 12,000 g for 10 min. An aliquot (400 μL) of the supernatant was incubated with 2 μL anti-GFP antibody (Proteintech, China, catalog: 66002-1-Ig) and 30 μL protein A + G agarose (Solarbio, catalog: R8281) for 12 h. Beads coated with the protein were washed twice using PBS (0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0) extraction buffer and eluted with 25 µL of 3× SDS loading buffer. Immunoblotting was conducted using anti-GFP (Proteintech, catalog: 66002-1-Ig) and anti-FLAG (Abmart, China, catalog: 334077) antibodies. The primer sequences used are listed in Supplemental Data Set S1.

Dual-LUC transient expression assay

The dual-LUC transient expression assay was performed as described by Yuan et al. (2021). The coding sequences of SlMYB11, SlARF4, and SlMAPK8 were inserted into the pGreenII 62-SK vector. Gene promoter sequences were cloned into the pGreenII 0800-LUC vector. Agrobacterium cultures were resuspended in infiltration buffer (0.2 M MES [pH 5.6], 1 M MgCl₂, and 200 µM acetosyringone) to a final OD₆₀₀ of 0.5–0.8. The ratio of pGreenII 62-SK vector:pGreenII 0800-LUC vector was 9:1 when transformed into 4- to 6-week-old N. benthamiana leaves. The LUC:REN ratio was measured with a Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific) using the Dual-LUC Reporter Assay System (Promega). At least three biological replicates were performed for all measurements. The replicate samples were collected from three different plants, and each sample contained three leaves. The primer sequences used are listed in Supplemental Data Set S1.

EMSA and ChIP-qPCR analysis

EMSA was conducted as described by Yuan et al. (2019). The coding sequence of SlMYB11 or SlARF4 was cloned into the pGEX-4T-1 vector, and the recombinant vector was transformed into Escherichia coli strain BL21 (DE3) (WEIDI) competent cells. Recombinant protein expression was induced using 0.5 mM isopropyl-β-d-thiogalactopyranoside for 16 h at 16°C, and the protein was purified with GSTSep Glutathione Agarose ResinFF (Yeasen). The EMSA probe was labeled with biotin using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific). The ChIP-qPCR assay was conducted as described by Qin et al. (2012). Briefly, leaf tissue from SlARF4-OE was ground in liquid nitrogen and cross-linked in 1% formaldehyde (Sigma-Aldrich) at 4°C for 10 min. Anti-FLAG (Abmart, catalog: 334077) was used for immunoprecipitation. The DNA isolated by ChIP was used for qPCR analysis. The RT-qPCR was conducted using 2× TSINGKE Master qPCR Mix (SYBR Green I with UDG) and a CFX96 Real-time PCR Detection System (Bio-Rad) as described by Wu et al. (2020). The primer sequences used are listed in Supplemental Data Set S1.

In vitro kinase assay

The in vitro kinase assay was performed as previously described (Wang et al., 2020). The recombinant vectors GST-SlMYB11, GST-SlARF4, His-SlMAPK8, GST-SlMYB11 (GST-SlMYB11^167A), and GST-SlARF4 (GST-SlARF4^355A) were transformed into E. coli strain BL21 (DE3) competent cells. Recombinant proteins were purified using a GST-tagged Protein Purification Kit (Clontech) and Profinity IMAC Ni-charged Resin (Bio-Rad) following the manufacturer’s instructions. The purified substrate and kinase proteins were incubated in kinase buffer (25 mM Tris, 2 mM DTT, 10 mM MgCl₂, and 200 μM ATP) at 37°C for 30 min. The samples were subsequently separated in 10% SDS–polyacrylamide gels and 10% Phos-tag acrylamide AAL-107 (NARD Institute). The samples were then transferred to polyvinylidene fluoride membranes, followed by immunoblot analysis with anti-His (Proteintech, China, catalog: 66005-1-Ig) and anti-GST (Proteintech, catalog: 66001-2-Ig) antibodies.

In vivo phosphorylation assay

The coding sequence of SlMYB11^167A or SlARF4^355A was cloned into the K303-GFP vector. The in vivo phosphorylation assay was performed as described by Bi et al. (2018) with some modifications. Nicotiana benthamiana leaves were transfected with the indicated plasmids and treated with ABA. Total proteins were extracted from the N. benthamiana leaves in extraction buffer (NP-40 Lysis Buffer [Beyotime] including 1 mM PMSF) and centrifuged at 12,000 g for 10 min. An aliquot (400 μL) of the supernatant was incubated with 2 μL anti-FLAG antibody (Proteintech, catalog: 66008-4-Ig) and 30 μL protein A + G agarose (Solarbio, catalog: R8281) for 12 h. Beads coated with the protein were washed twice using PBS (0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0) extraction buffer and eluted with 25 µL of 3× SDS loading buffer. The samples were subsequently separated in 10% SDS–polyacrylamide gels and 10% Phos-tag acrylamide AAL-107 (NARD Institute). The samples were then transferred to polyvinylidene fluoride membranes, followed by immunoblot analysis with anti-GFP (Proteintech, catalog: 66002-1-Ig) and anti-Actin (Proteintech, catalog: 66009-1-Ig) antibodies.

VIGS

VIGS was performed using N. benthamiana rattle virus vectors pTRV1 and pTRV2 as described by Fu et al. (2005). The target sequence of SlMYB11 was designed using the VIGS tool (vigs.solgenomics.net). The target sequence of SlMYB11 was amplified and ligated into the pTRV2 vector. The pTRV1 or pTRV2-SlMYB11 vector was introduced into A. tumefaciens strain GV3101 (WEIDI). The expression of the RNA1, RNA2, and CP genes was detected at 15 days after injection of the mixed bacterial solution into tomato seedlings and 10 DPA fruits. The primer sequences used are listed in Supplemental Data Set S1.

Analysis of ABA and IAA contents

Leaf and pericarp samples were ground in liquid nitrogen. Approximately 0.1 g of the powdered sample was added to 9 mL PBS (pH 7.4) buffer for analysis. ABA and IAA were quantified by competitive ELISA using ABA and IAA ELISA kits (Meimian) following the manufacturer’s instructions. The absorbance at 450 nm was measured using a Luminoskan Ascent Microplate Luminometer (Thermo Scientific). At least three biological replicates were performed for all measurements. The replicate samples were collected from three different plants, and each sample contained three leaves or fruits.

Phylogenetic tree construction

Blastp was used to obtain protein sequences from the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were constructed from complete protein sequences using the neighbor-joining method in MEGA 7.0 (Kumar et al., 2016). Branches were compared with bootstrap support values from 500 replicates for each node. An alignment for generating the tree is provided in Supplemental File S1.

Statistical analysis

All experiments were repeated at least three times. Univariate ANOVA and Tukey’s honestly significant difference test were used to analyze the significance of differences between the control and treatment groups. A P-value <0.05 was considered to be significant. Test statistics are shown in Supplemental Data Set S2.

Accession numbers

Sequence data from this article can be found in GenBank/EMBL data libraries under the following accession numbers: Solyc12g049350 (SlMYB11), Solyc02g084870 (SlMAPK8), Solyc05g054760 (DHAR), Solyc11g012410 (GPP), Solyc01g106450 (GLDH), Solyc07g064130 (SlActin), Solyc01g103050 (SlARF1), Solyc03g118290 (SlARF2), Solyc02g077560 (SlARF3), Solyc11g069190 (SlARF4), Solyc04g081240 (SlARF5), Solyc07g043620 (SlARF6), Solyc02g037530 (SlARF8), Solyc11g069500 (SlARF10), Solyc12g042070 (SlARF11), Solyc07g016180 (SlARF19), and Solyc05g056040 (SlARF24).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Reduced AsA content in WT tomato pericarps.

Supplemental Figure S2. Total and reduced AsA contents in WT tomato leaves and pericarps under IAA treatment.

Supplemental Figure S3. RT-qPCR analysis of the expression of ARFs in the leaves of WT plants under IAA treatment.

Supplemental Figure S4. Sequence alignment of SlMYB11 with subgroup members SlMYB111 (Solyc06g009710) and SlMYB12 (Solyc01g079620).

Supplemental Figure S5. Phylogenetic relationship of SlMYB11 with homologous proteins.

Supplemental Figure S6. SDS-PAGE gel stained with Coomassie brilliant blue demonstrating affinity purification of the recombinant GST-tSlARF4 protein used for the EMSA.

Supplemental Figure S7. Schematic diagram of the sequences in the SlMYB11 promoter used for ChIP-qPCR.

Supplemental Figure S8. RT-qPCR analysis of the expression of SlMYB11 in SlMYB11-RNAi plants.

Supplemental Figure S9. SDS-PAGE gel stained with Coomassie brilliant blue demonstrating affinity purification of the recombinant GST-SlMYB11 protein used for the EMSA.

Supplemental Figure S10. SDS-PAGE gels stained with Coomassie brilliant blue demonstrating affinity purification of the recombinant proteins used for the kinase assay.

Supplemental Figure S11. RT-qPCR analysis of the expression of SlMAPK8 in SlMAPK8-RNAi plants.

Supplemental Data Set S1. Primers used in this study.

Supplemental Data Set S2. Statistical analysis using ANOVA.

Supplemental File S1. Sequences of tomato R2R3-MYB S2 subfamily proteins used in phylogenetic tree construction.

Supplemental File S2. Newick format of the phylogenetic tree shown in Supplemental Figure S5.

Acknowledgments

We greatly appreciate Prof. Mondher Bouzayen and Dr. Mohamed Zouine for providing seeds of the Slarf4 CRISPR/Cas9 mutants and the Analytical and Testing Center of Chongqing University for conducting the GUS assays and subcellular localization analysis.

Funding

This work was supported by the National Natural Science Foundation of China (32172596), the Technology Innovation and Application Development Project in Chongqing (cstc2019jscx-gksbX0115), the Graduate Research and Innovation Foundation of Chongqing, China (CYB22048), the Fundamental Research Funds for the Central Universities (2021CDJZYJH-002), and the Graduate Research and Innovation Project of Chongqing, China (CYS21038).

Conflict of interest statement. The authors declare no conflicts of interest.

W.D. and Z.G.L planned and designed the research. X.X., Q.Z., X.G., G.W., M.W., Y.Y., X.Z., X.H., M.G., T.Q., and H.L. performed the experiments. Z.G. and Z.S.L analyzed the data. W.D. and X.X. wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://dbpia.nl.go.kr/plcell) is: Wei Deng ([email protected]).

References