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. 2023 Mar 9;192(3):2067–2080. doi: 10.1093/plphys/kiad151

ETHYLENE-INSENSITIVE 3-LIKE 2 regulates β-carotene and ascorbic acid accumulation in tomatoes during ripening

Chong Chen 1,#, Meng Zhang 2,#, Mingyue Zhang 3,#, Minmin Yang 4, Shanshan Dai 5, Qingwei Meng 6, Wei Lv 7,✉, Kunyang Zhuang 8,✉,e,c,d
PMCID: PMC10315317  PMID: 36891812

Abstract

ETHYLENE-INSENSITIVE 3/ETHYLENE-INSENSITIVE 3-LIKEs (EIN3/EILs) are important ethylene response factors during fruit ripening. Here, we discovered that EIL2 controls carotenoid metabolism and ascorbic acid (AsA) biosynthesis in tomato (Solanum lycopersicum). In contrast to the red fruits presented in the wild type (WT) 45 d after pollination, the fruits of CRISPR/Cas9 eil2 mutants and SlEIL2 RNA interference lines (ERIs) showed yellow or orange fruits. Correlation analysis of transcriptome and metabolome data for the ERI and WT ripe fruits revealed that SlEIL2 is involved in β-carotene and AsA accumulation. ETHYLENE RESPONSE FACTORs (ERFs) are the typical components downstream of EIN3 in the ethylene response pathway. Through a comprehensive screening of ERF family members, we determined that SlEIL2 directly regulates the expression of 4 SlERFs. Two of these, SlERF.H30 and SlERF.G6, encode proteins that participate in the regulation of LYCOPENE-β-CYCLASE 2 (SlLCYB2), encoding an enzyme that mediates the conversion of lycopene to carotene in fruits. In addition, SlEIL2 transcriptionally repressed L-GALACTOSE 1-PHOSPHATE PHOSPHATASE 3 (SlGPP3) and MYO-INOSITOL OXYGENASE 1 (SlMIOX1) expression, which resulted in a 1.62-fold increase of AsA via both the L-galactose and myoinositol pathways. Overall, we demonstrated that SlEIL2 functions in controlling β-carotene and AsA levels, providing a potential strategy for genetic engineering to improve the nutritional value and quality of tomato fruit.

ETHYLENE INSENSITIVE 3-LIKE 2 controls β-carotene and ascorbic acid biosynthesis in tomato fruits via different regulatory pathways.

Introduction

Tomato (Solanum lycopersicum), the second most important vegetable crop worldwide, functions as a classical model system for investigations of fruit development and ripening (Klee and Giovannoni 2011). The major carotenoids in ripe tomato fruit are lycopene (70% to 90%) and β-carotene (5% to 40%), which confer red and orange coloration, respectively; both compounds are also highly beneficial for human health (Ronen et al. 2000; Fraser and Bramley 2004; Perveen et al. 2015). The lycopene and β-carotene biosynthesis and degradation pathway in plants has been clarified at the molecular level, and the genes encoding the key enzymes in this pathway have been identified (Yuan et al. 2015; Llorente et al. 2016). Among these, lycopene β-cyclases (LCYBs) catalyze the conversion of red lycopene to orange α-carotene and β-carotene and play a major role in determining the coloration of various fruits (Ronen et al. 2000; Harjes et al. 2008). In many plants, LCYBs are encoded by 2 genes: one encoding an enzyme that functions in the chloroplasts of photosynthetic tissues and the other encoding an enzyme that functions in the chromoplasts of non-photosynthetic tissues, such as fruits (Alquzar et al. 2009; Ahrazem et al. 2010). Tomato fruits appear deep red due to their massive accumulation of lycopene, mainly resulting from the downregulation of chromoplast-specific LCYB2 (Ronen et al. 2000). This finding points to the crucial roles of LCYB2 in the massive accumulation of downstream carotenoids in fruits. However, the direct regulatory mechanism of LCYB2 remains largely unexplored.

Ascorbic acid (AsA), another abundant metabolite in tomato fruit, is involved in scavenging reactive oxygen species (Yang et al. 2017). Four alternate routes for AsA biosynthesis have been identified in plants: the D-mannose/L-galactose, L-glucose, D-galacturonate, and myoinositol pathways (Wheeler et al. 1998; Agius et al. 2003; Wolucka and Van Montagu 2003; Lorence et al. 2004). Among these, the D-mannose/L-galactose pathway is the dominant and best-understood route (Badejo et al. 2012; Sodeyama et al. 2021), while the role of the myoinositol pathway, which is similar to the AsA biosynthesis pathway in mammals, is controversial (Lorence et al. 2004; Endres and Tenhaken 2009; Ivanov Kavkova et al. 2019; Munir et al. 2020). The myoinositol pathway is divided into 2 parts: myoinositol 1-phosphate synthase (IPS) and inositol monophosphatase (IMP)-mediated myoinositol biosynthesis; and myoinositol oxygenase (MIOX)-mediated AsA biosynthesis (Fleet et al. 2018; Munir et al. 2020). IMP/L-galactose 1-phosphate phosphatase (GPP) was demonstrated to have high affinity for the L-galactose 1-phosphate and myo-inositol 3-phosphate and acted as a bifunctional enzyme in the biosynthesis of AsA through L-galactose and myoinositol pathways (Torabinejad et al. 2009; Zheng et al. 2022). Overexpression of tomato IMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, and delayed fruit softening (Zheng et al. 2022). MIOX as an essential monooxygenase enzyme catalyzes the transferring process of myo-inositol into D-glucuronic acid (D-GlcUA). In Arabidopsis (Arabidopsis thaliana), overexpressing MIOX to upregulate the myoinositol pathway did not lead to an increase in AsA content (Endres and Tenhaken 2009; Ivanov Kavkova et al. 2019). In tomato leaves and fruit, however, overexpressing MIOX4 enhanced AsA accumulation (Munir et al. 2020). MIOX1 was highly expressed from flowering to the fruiting stages (Munir et al. 2020). Exploring the roles and regulatory mechanisms of distinct AsA biosynthetic routes in tomatoes could lay the foundation for improving the quality of this crop.

Ethylene (ET) is essential for the ripening of climacteric fruits; its biosynthesis and signal transduction pathway have been studied intensively (Alexander and Grierson 2002; Liu et al. 2015). Most studies have shown that ET regulates fruit ripening by orchestrating a transcriptional cascade of ET-responsive genes, including transcription factor genes in the ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE 3-LIKE (EIN3/EIL) and ethylene response factor (ERF) families (Yin et al. 2010). EIN3/EILs comprise a small transcription factor gene family whose members directly bind to the conserved EIN3 binding sequence (EBS) A[A/C]G[A/T]A[A/C]CT (An et al. 2018; Huang et al. 2021). Different SlEIL family genes have different transcript levels during tomato fruit development, pointing to distinct roles among SlEILs (Yokotani et al. 2003; Liu et al. 2015). SlEIL1, SlEIL2, and SlEIL3 antisense lines with reduced total SlEIL mRNA levels produce yellow fruit (Tieman et al. 2001). However, the SlEILs that play major roles in tomato fruit development and the molecular regulatory mechanism by which they regulate fruit quality remain to be further explored.

In contrast to the EIN3/EILs, the ERFs constitute one of the largest transcription factor families in plants, with 137 members in tomatoes, each containing a conserved DNA-binding domain (ERF domain). In accordance with the pleiotropic effects of ET in plant physiological responses, ERFs play critical, broad roles in plant stress responses, plant development, hormone signaling, and fruit ripening, depending on their GCC box DNA-binding activity (Hao et al. 1998; Müller and Munné-Bosch 2015; Gao et al. 2020). Among ERF family members, some (such as ERF1, ERF2, and ERF5) activate transcription (Fujimoto et al. 2000). By contrast, ERF3, ERF4, and ERF12, which are categorized as Class II ERFs, are active repressors of transcription, a function dependent on their conserved L/FDLNL/F(x)P motif (Fujimoto et al. 2000; Ohta et al. 2001; Hu et al. 2020). However, ERF4 was also found to activate the expression of downstream genes during fruit ripening (Wang et al. 2021). The specific molecular regulatory mechanisms of ERFs during fruit ripening require further analysis.

In this study, we revealed how EIL2 regulated β-carotene and AsA accumulation in tomato during ripening. Decreased SlEIL2 expression led to the accumulation of β-carotene and AsA. In vitro and in vivo assays confirmed that SlEIL2 directly regulated SlERF.G6 and SlERF.H30 transcription, thereby increasing SlLCYB2 expression to enhance the conversion of red lycopene to orange β-carotene. SlEIL2 also directly binds to the EBS elements in the promoters of SlMIOX1 and SlGPP3 genes, thus repressing their expression to influence AsA biosynthesis.

Results

Decreased SlEIL2 expression results in β-carotene accumulation in tomato fruit at the late ripening stage

SlEIN3/EIL family genes play important roles in tomato fruit ripening. Our RT-qPCR analysis showed that, among the transcripts of these genes, only SlEIL2 transcripts accumulated at the onset of ripening and declined at later stages, suggesting that SlEIL2 has distinct roles in fruit ripening (Supplemental Fig. S1). Indeed, the fruits of SlEIL2 RNA interference (RNAi) lines (ERIs), which had reduced SlEIL2 mRNA and protein levels (Fig. 1, A and B), displayed a substantially delayed onset of fruit ripening compared with the fruits of wild-type (WT) (Fig. 1F and Supplemental Fig. S2). Notably, when the leaves surround ERI fruits died, these fruits remained orange. Maybe these fruits can finally turn red if they continue to develop, but this process was very slow. To further confirm that the delayed ripening phenotype of the ERI lines was caused by the downregulation of SlEIL2, we generated eil2 mutant lines (eil2-1 and eil2-2) using a CRISPR/Cas9 system; these lines were identified by DNA sequencing and immunoblotting (Fig. 1, C and D). Unlike the ERI plants, eil2-1 and eil2-2 plants showed strong leaf epinasty phenotypes, indicating that knockout of SlEIL2 resulted in hypersensitivity to ET (Fig. 1E). The eil2 fruits also ripened slowly and remained orange after anthesis 45 d (45DAA) (Fig. 1F). Then, we detected the absolute values of carotenoids at 30DAA, 35DAA, 39DAA, and 45DAA tomato fruits of ERIs and WT. Compared with WT, the β-carotene content was less in ERI fruits at 35DAA and 39DAA and became more at 45DAA and the lycopene and phytoene content were less in ERI fruits (Fig. 1G). As the downstream metabolite of β-carotene, the β-cryptoxanthin was higher in ERIs than WT only at 45DAA (Fig. 1G). Other carotenoid contents were shown in Supplemental Fig. S3. Furthermore, the fruits of both ERI1 and eil2-1 maintained greater firmness than WT fruit throughout ripening (Fig. 1H).

Figure 1.

Figure 1.

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Phenotypic observation of ERI1 and the eil2 mutants. A) The mRNA levels of SlEIL2 in 3 SlEIL2 RNAi lines (ERI1, ERI2, and ERI3) and the WT. Tomato ACTIN transcript levels were used for normalization. Data represent means ± Sd (n = 3). Statistical analysis was performed by Tukey-test. Columns with different letters are significantly different (P < 0.05). B) Immunoblot analysis of the expression of SlEIL2 in ERI lines. ACTIN was used as the loading control. C) Identification of the CRISPR/Cas9-induced mutations in the eil2 mutant (eil2-1 and eil2-2) by sequencing. In each case, an additional nucleotide was added at position 442 of the SlEIL2 coding sequence, creating a termination codon. D) Immunoblot analysis of the expression of SlEIL2 in 2 eil2 mutant lines. ACTIN was used as the loading control. E) The leaf epinasty phenotype of eil2-1 and eil2-2 plants. Scale bar = 5 cm. F) Ripening stages of fruits between 20 to 45 d after anthesis (DAA) in the WT, ERIs, and eil2-1 lines. ERIs and eil2-1 fruits displayed a significantly delayed onset of fruit ripening compared with the fruits of WT. Scale bar = 2 cm. G) The absolute values of β-carotene, phytoene, lycopene and β-cryptoxanthin in ERIs and WT fruits at 30DAA, 35DAA, 39DAA and 45DAA. Data represent means ± Sd (n = 3). Statistical analysis was performed by Tukey-test. Columns with different letters are significantly different (P < 0.05). H) Fruit firmness of WT and SlEIL2 transgenic fruits at different stages. Fifteen fruits were examined for each measurement; error bars represent ± Sd. P < 0.05 (Tukey-test).

To further investigate the causal factor underlying the β-carotene changes after anthesis 45 d (45DAA) of the ERIs fruits, we performed correlation analysis of the transcriptomes and metabolomes of the ERI and WT ripe fruits (45DAA); the abnormal reproduction of the eil2 mutants made it difficult to obtain enough progeny seeds. Gene ontology (GO) and KEGG analysis were used to cluster the differential expression genes (DEGs) between ERI and WT fruits (Fig. 2, A and B). KEGG analysis showed that the DEGs were more enriched in the carotenoid biosynthesis pathway (Fig. 2B). Correspondingly, ERI fruits contained more β-carotene, neoxanthin, violaxanthin, and ε-carotene and less lycopene, γ-carotene, lutein and phytoene than the WT fruits (Fig. 2C). We further analyzed the expression of important carotenoid biosynthetic genes, including PSY1 (phytoene synthase 1 gene), PDS (phytoene desaturase gene), Z-ISO (15-cis-zeta-carotene isomerase gene), CRTISO (carotenoid isomerase gene), LCYE (lycopene epsilon cyclase gene), LCYB2 (lycopene β-cyclase 2 genes), BCH (β-carotene hydroxylase gene), ZEP (zeaxanthin epoxidase gene), VDE (violaxanthin de-epoxidase gene), CYP97C11/12 (cytochrome P450-type monooxygenase 97C11/12 genes) and NSY (neoxanthin synthase gene) in ERI and WT fruits. The transcriptome data showed that most of these genes expressed higher in ERI fruits than that in WT (Fig. 2C). In contrast, the transcript levels of CRTISO, LCYE, CYP97C11, and VDE were repressed and CYP97C12 did not change substantially in ERI lines (Fig. 2C). Since EIL2 is an important transcription factor in the response to ET during fruit ripening, we performed a yeast 1-hybrid assay to explore the direct regulatory relationship between SlEIL2 and these carotenoid metabolism-related DEGs (Supplemental Fig. S4). However, no positive binding signal was detected, suggesting that SlEIL2 may regulate the expression of these DEGs in an indirect manner to control the carotenoid biosynthesis.

Figure 2.

Figure 2.

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Analysis of carotenoid metabolism in the ERI lines and the WT. A) Gene Ontology (GO) term enrichment analysis of DEGs of ERIs and WT fruits at 45 DAA. B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs of ERIs and WT fruits at 45DAA. C) Transcriptome and metabolome correlation analysis of ERIs and WT fruits at 45DAA. Model of the carotenoid metabolic pathway is shown. Blue heat map indicates the expression of selected carotenoid-associated DEGs from the ERI vs. WT comparison. Red type indicates the carotenoid-associated metabolite levels in ERI and WT fruits. Tomato gene ID and the used data are shown in Supplemental Table S2.

SlEIL2 affects SlLCYB2 expression by regulating SlERF genes expression

ERFs are typical downstream pathway components of EIN3. To explore whether ERFs participate in the regulation of SlEIL2 to affect the expression of carotenoid metabolism-related genes, we analyzed the expression of all tomato ERF family members in the transcriptome data for ERI vs. WT tomato plants (Supplemental Fig. S5A). We chose 8 ERF genes that were significantly downregulated (ERF.C5, ERF.D4, ERF.E1, ERF.F18, ERF.G6, ERF.G7, ERF.H7, and ERF.J1) and 14 that were significantly upregulated (ERF.A1, ERF.B3, ERF.D1, ERF.E2, ERF.E3, ERF.E5, ERF.F7, ERF.F12, ERF.F13, ERF.F16, ERF.G3, ERF.H13, ERF.H27, and ERF.H30) in the ERI lines for further analysis by RT-qPCR, which confirmed their relative expression (Supplemental Fig. S5B). A yeast 1-hybrid assay and an electrophoretic mobility shift assay (EMSA) confirmed that SlEIL2 directly binds to the EBS elements in the promoter regions of SlERF.J1, SlERF.G6, SlERF.F12, and SlERF.H30 among the ERF genes listed above (Fig. 3, A and B). Finally, we performed 2 transient expression assays using a luciferase (LUC) reporter to further confirm the role of SlEIL2 in regulating the expression of those 4 genes in Nicotiana benthamiana leaves. SlEIL2 activated SlERF.J1pro::LUC and SlERF.G6pro::LUC and repressed SlERF.H30pro::LUC and SlERF.F12pro::LUC (Fig. 3, C and D), which is consistent with the RT-qPCR results (Supplemental Fig. S5B). Similar to ERI fruits, SlERF.F12 overexpression lines also displayed a significantly delayed fruit ripening phenotype, which confirmed the regulatory relationship between EIL2 and SlERF.F12 (Deng et al. 2022).

Figure 3.

Figure 3.

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SlEIL2 regulates the expression of 4 ERF genes by binding to the EBS elements in their promoters. A) Diagram of the upstream regions of SlERF.J1, SlERF.G6, SlERF.H30, and SlERF.F12 and results of the yeast 1-hybrid assay. Yeast strain EGY48 was co-transformed with SlEIL2-GAD and SlERF.J1pro-EBS::LacZ, SlERF.G6pro-EBS::LacZ, SlERF.H30pro-EBS::LacZ, or SlERF.F12pro-EBS::LacZ; the corresponding EBS elements are shown in the upper diagram. Blue staining indicates binding. SlERFpro-EBS::LacZ, -GAD, -::LacZ, and SlEIL2-GAD were used as negative controls. B) EMSA. The respective EBS fragments of the SlERF genes were used as the probes (5′ biotin). The same probes without biotin were added as a competitive control (C-probes). Mutant probes, which were shown in the above diagram, were used as negative control. C) LUC reporter assay. Agrobacterium cells containing vectors expressing SlEIL2-FLAG or empty FLAG and Agrobacterium cells containing vectors expressing SlERFpro::LUC (the promoter region of SlERFs containing the SlEIL2 binding element) were co-injected into N. benthamiana leaves in the combinations shown at the bottom. After injection, the plants were cultured for 2 d prior to observation. The color bar indicates the intensity of the signal, with red as the maximum and purple as the minimum. Scale bar = 1 cm. D) pGreen0800 (a double LUC reporter system)-mediated LUC assay. The relative expression of SlERFpro::LUC was normalized to that of 35S::REN (internal control) (LUC/REN, mean ± Sd, n = 3). The combinations of Agrobacterium cells were as described above. **P < 0.01 (Student's t-test).

ERFs are typical transcription factors whose function is dependent on their DNA-binding activity to GCC boxes. We, therefore, analyzed the promotor regions of the carotenoid metabolism-related DEGs listed above. Two GCC-box-enriched regions were identified in the promotor of SlLCYB2 (proSlLCYB2). A yeast 1-hybrid assay and EMSA confirmed that SlERF.G6 and SlERF.H30 bind to the same GCC-box region in proSlLCYB2 (Fig. 4, A and B). In a result similar to that of the LUC reporter assay described above, overexpressing SlERF.G6 inhibited SlLCYB2 expression, whereas overexpressing SlERF.H30 activated its expression (Fig. 4, C and D). We also confirmed that SlEIL2 affected the expression SlLCYB2pro::LUC when co-expressed with SlERF.G6 or SlERF.H30 driven by their own promoters in N. benthamiana leaves (Fig. 4E). These results indicate that SlEIL2 directly regulates the expression of SlERF.G6 and SlERF.H30, thus affecting SlLCYB2 transcript levels, which in turn influences the accumulation of β-carotene in tomato fruit.

Figure 4.

Figure 4.

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SlERF.G6 and SlERF.H30 regulate SlLCYB2 expression. A) Above, diagram of the upstream regions of SlLCYB2, with the GCC element (G-BOX) marked in red; below, results of a yeast 1-hybrid assay to identify the binding of SlERF.G6 and SlERF.H30 to the G-BOX element in the promoter of SlLCYB2. SlERF.G6-GAD or SlERF.H30-GAD was co-transformed into yeast strain EGY48 with SlLCYB2pro-G-BOX::LacZ. Blue staining indicates binding. SlLCYB2pro-G-BOX::LacZ, -GAD, -::LacZ, and SlERF.G6/H30-GAD were used as the negative controls. B) EMSA. The GCC element described above was used as the probe (5′ biotin). The same probe without biotin was added as a competitive control (C-probes). Mutant probe, which were shown in the above diagram, was used as negative control. The left part of the gel shows the binding of SlERF.G6 to the GCC element, and the right part shows the binding of SlERF.H30 to the GCC element. C) LUC reporter assay. Agrobacterium cells containing vectors expressing SlERF.G6-FLAG, SlERF.H30-FLAG, or empty FLAG and Agrobacterium cells containing vectors expressing SlLCYB2pro::LUC (the promoter region of SlLCYB2 containing the GCC element) were co-injected into N. benthamiana leaves in the combinations shown at right. Scale bar = 1 cm. D) pGreen0800 (a double LUC report system)-mediated LUC assay. The relative expression of SlLCYB2pro::LUC was normalized to that of 35S::REN (internal control) (LUC/REN, mean ± Sd, n = 3). The combinations of Agrobacterium cells were as described above. **P < 0.01 (Student's t-test). E) An additional double LUC reporter assay was used to explore the role of SlEIL2 in SlERFs regulation of SlLCYB2. Agrobacterium cells containing vectors expressing SlEIL2-HA, SlERF.G6-FLAG, SlERF.H30-FLAG, or empty FLAG and Agrobacterium cells containing vectors expressing SlLCYB2spro::LUC were co-injected into N. benthamiana leaves. The combinations of Agrobacterium cells are shown at the bottom. The relative expression of SlLCYB2pro::LUC was normalized to that of 35S::REN (internal control) (LUC/REN, mean ± Sd, n = 3). The combinations of Agrobacterium cells were as described above. Columns with different letters are significantly different (P < 0.05, Tukey-test).

SlEIL2 might function in ET and abscisic acid (ABA) biosynthesis

ET and ABA are 2 important phytohormones that function in tomato fruit ripening. Considering the increased contents of neoxanthin (an ABA biosynthesis precursor; Fig. 2B) in the ERI lines, we measured the ABA contents and transcript levels of the major ABA biosynthesis-related genes in these lines. ABA levels were significantly higher at 45DAA in the fruits of ERI vs. the WT (Fig. 5A). Accordingly, the genes involved in ABA biosynthesis (NCED, AAO, and SDR) were all expressed at higher levels in the ERI lines than in the WT (Fig. 5B). These results suggest that the increased β-carotene levels in these fruits led to the activation of various ABA biosynthesis-related genes, thus enhancing ABA accumulation in tomato fruit at 45DAA. Unlike for ABA, less ET was released from the ERI lines than the WT at 33DAA and 37DAA, but it increased to a similar level of WT at 45DAA (Fig. 5C). It should be noted that the content of 1-aminocylopropane-1-carboxylic acid (ACC), the direct precursor of ET biosynthesis, was similar between the ERI lines and the WT at 45DAA but was significantly higher in ERI vs. WT fruits at 37DAA (Fig. 5D). These results might be caused by the differential expression of the SAMS, ACS, and ACO genes in the ET biosynthesis pathway during fruit ripening (Fig. 5E).

Figure 5.

Figure 5.

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SlEIL2 affects the ABA and ET contents in the tomato fruits. A, D) The ABA and ACC contents of WT and ERI fruits at 37DAA and 45DAA. The data are presented as means ± Sd of three biological replicates. P < 0.05 (Tukey-test). Columns with different letters are significantly different. B) Heat map comparisons of selected ABA biosynthesis-associated DEGs from the ERI vs. WT. Blue level indicates expression levels. The tomato gene ID is shown following each gene name. C) The ET contents of WT and ERI fruits at 30DAA, 33DAA, 37DAA and 45DAA. The data are presented as means ± Sd of three biological replicates. P < 0.05 (Tukey-test). Columns with different letters are significantly different. E) Heat map comparisons of selected ET biosynthesis-associated DEGs from the ERI vs. WT. The tomato gene ID is shown following each gene name.

Inhibiting SlEIL2 expression leads to AsA accumulation in tomato fruits

In addition to carotenes, another important metabolite in tomato fruit is AsA, which is synthesized from glucose as the initial substrate. Here, AsA was found to accumulate more strongly in the ERI lines than the WT at 45DAA stage (Fig. 6). L-Galactose pathway is the dominant route of AsA biosynthesis (Badejo et al. 2012). In this route, we found that the ERI fruits contained lower D-glucose-6-P and D-fructose-6-P levels, but higher L-galactose levels than WT (Fig. 6). Correspondingly, some key genes, like GPP, GGP, and GalDH in L-galactose pathway were substantially up-regulated in ERI lines than that in WT (Fig. 6). These results indicated that inhibiting SlEIL2 expression could activate the L-galactose pathway to increase the AsA content. In the KEGG analysis mentioned above, the inositol phosphate metabolism was also a DEGs-enriched pathway, suggesting that myoinositol pathway may also lead to the AsA accumulation in ERI fruits (Fig. 2A). Metabolite analysis showed that the myo-inositol, D-glucoronate and L-gulono-1,4-lactone contents were higher and the myo-inositol-1-P content was lower in ERI lines than that in WT (Fig. 6). Meanwhile, all the important genes (IPS, IMP, MIOX, and GLO) of AsA biosynthesis in myoinositol pathway expressed higher in ERI fruits than that in WT (Fig. 6).

Figure 6.

Figure 6.

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SlEIL2 is involved in the AsA biosynthesis in tomato fruits. Transcriptome and metabolome correlation analysis of ERIs and WT fruits at 45DAA. Model of the L-galactose and myo-inositol pathways of AsA biosynthesis were shown. Blue heat map comparisons indicate the expression of selected AsA biosynthesis-associated DEGs from the ERI vs. WT. Tomato gene ID and the used data are shown in Supplemental Table S2.

To investigate the potential mechanism for this effect of SlEIL2 to AsA biosynthesis, we analyzed the binding of SlEIL2 to the promoters of AsA biosynthesis pathway genes in a yeast 1-hybrid assay. SlEIL2 directly bound to the EBS element in the promotor region of SlMIOX1 (Solyc06g062430) and SlGPP3/IMP3 (Solyc11g012410) (Fig. 7A), which was also confirmed by EMSA (Fig. 7B). A LUC reporter analysis further showed that SlEIL2 repressed the expression of SlMIOX1 and SlGPP3/IMP3 in N. benthamiana leaves (Fig. 7C). In tomato fruits, SlMIOX1 and SlGPP3/IMP3 transcript levels were significantly higher in the ERI lines than in the WT at 45DAA, which supports a role of SlEIL2 in regulating SlMIOX1 and SlGPP3/IMP3 expression (Fig. 7, D and E). These results suggest that the regulation of SlMIOX1 and SlGPP3/IMP3 expression by SlEIL2 affects AsA biosynthesis via the L-galactose and myoinositol pathway.

Figure 7.

Figure 7.

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SlEIL2 regulates the expression of SlGPP3/IMP3 and SlMIOX1. A) Above, diagram of the upstream regions of SlMIOX1 and SlGPP3/IMP3; below, results of a yeast one-hybrid assay to detect SlMIOX1 and SlGPP3/IMP3 regulated by SlEIL2. The EBS element is marked in red. SlEIL2-GAD was co-transformed into yeast strain EGY48 with SlMIOX1pro-EBS::LacZ and SlGPP3/IMP3pro-EBS::LacZ respectively. Blue staining indicates positive binding. SlMIOX1pro/ SlGPP3/IMP3pro -EBS::LacZ, -GAD, -::LacZ, and SlEIL2-GAD were used as the negative controls. B) Electrophoretic mobility shift assay (EMSA). The EBS elements described above were used as the probe (5′ biotin). The same probes without biotin were added as a competitive control (C-probes). Mutant probes, which were shown in the above diagram, was used as negative control. The left part of the gel shows the binding of SlEIL2 to the EBS element of SlMIOX1, and the right part shows the binding of SlEIL2 to the EBS element of SlGPP3/IMP3. C) Luciferase (LUC) reporter assay. Agrobacterium cells containing vectors expressing SlEIL2-FLAG or empty FLAG and Agrobacterium cells containing vectors expressing SlMIOX1pro/ SlGPP3/IMP3pro::LUC (the promoter region of SlMIOX1 or SlGPP3/IMP3 containing the EBS element) were co-injected into N. benthamiana leaves, respectively. After injection, the plants were cultured for 2 days prior to observation. Scale bar =1cm. D, E) mRNA levels of SlMIOX1 in WT and ERI fruits at 45DAA. Tomato ACTIN transcript levels were used for normalization. The data are presented as means ± Sd of three biological replicates. P < 0.05 (Tukey-test). Columns with different letters are significantly different.

Discussion

Reduced expression of SlEIN3/EILs causes strong leaf epinasty and yellow-fruit phenotypes in tomato; SlEIN3 and EIL function redundantly in ET responses (Tieman et al. 2001). Moreover, the F-box protein SlEBF3 interacts with SlEIL1-SlEIL4 and reduces their protein levels, leading to ET response phenotypes similar to those in transgenic tomato with inhibited SlEIL expression (Deng et al. 2018). These findings support the importance of the roles of SlEIL1-4 in leaf epinasty and fruit pigment metabolism. However, the underlying molecular mechanism and the roles of specific SlEIL proteins remain unclear.

Our SlEIL2 RNAi lines showed a delayed onset of fruit ripening phenotype but no obvious leaf epinasty (Fig. 1F and Supplemental Fig. S2). In these lines, SlEIL2 transcript levels were substantially reduced, SlEIL7 was induced, and there were no substantial differences in SlEIL1, SlEIL3, SlEIL4, SlEIL5, SlEIL6, or SlEIL8 expression (Supplemental Fig. S6A). In the SlEIL7 transgenic lines, delayed fruit ripening (orange fruit) was not observed (Supplemental Fig. S7), suggesting that SlEIL2 is the major factor causing the delayed fruit ripening (orange fruit) phenotype. Unlike the SlEIL2 RNAi lines, the eil2 mutant (produced by CRISPR/Cas9 genome editing), with reduced SlEIL1 and SlEIL5 expression and elevated SlEIL7 expression, showed strong leaf epinasty and orange-fruit phenotypes, confirming the important role of SlEIL2 in fruit ripening (Fig. 1 and Supplemental Fig. S6B). There are 2 possible explanations for the leaf epinasty in this mutant. First, the level of SlEIL2 might affect the ET response phenotypes mentioned above; a minimum amount of SlEIL2 can maintain a normal leaf angle and partially restore the fruit color phenotype. Second, knockout of SlEIL2 might cause the other SlEIL family members (such as SlEIL1 and SlEIL5) or other factors to influence leaf epinasty.

It is largely accepted that EIN3/EIL proteins are the initial triggers of the expression of ERF genes, which are responsible for most ET responses (Zhuang et al. 2008). Here, we identified 8 ERF genes significantly downregulated (ERF.C5, ERF.D4, ERF.E1, ERF.F18, ERF.G6, ERF.G7, ERF.H7 and ERF.J1) and 14 ERF genes significantly upregulated (ERF.A1, ERF.B3, ERF.D1, ERF.E2, ERF.E3, ERF.E5, ERF.F7, ERF.F12, ERF.F13, ERF.F16, ERF.G3, ERF.H13, ERF.H27 and ERF.H30) in the SlEIL2 RNAi lines vs. the WT that have functions downstream of SlEIL2 during fruit ripening (Supplemental Fig. S5). Among these, ERF.J1, ERF.H30, ERF.F12, and ERF.G6 are the 4 direct target genes of SlEIL2 (Fig. 3). The function of SlERF.F12 in tomato fruit was revealed. SlERF.F12 overexpression tomato lines displayed a substantially delayed onset of fruit ripening consistent with the ERI fruits, which supports the regulation of SlEIL2 to SlERF.F12 (Deng et al. 2022). While ERF.H30 and ERF.G6 are found to bind to the same G-box element in the promoter of SlLCYB2 (Fig. 4, A and B). How do they coordinate this binding to regulate SlLCYB2 expression? The large size of the ERF gene family and the distinctive expression patterns of its members implies that the temporal and spatial specificity of fruit ET responses arises downstream of EIN3/EILs at the level of ERFs (Sharma et al. 2010; Zhou et al. 2020). We analyzed the expression patterns of ERF.H30 and ERF.G6 starting after 35DAA, finding that ERF.G6 expression gradually increased and ERF.H30 expression gradually decreased over time, leading to progressively increased binding of ERF.G6 (Supplemental Fig. S8). These results suggest that the regulation of SlERFs by SlEIL2 is temporally specific.

Four alternate routes for AsA biosynthesis have been identified in plants. Except for L-galactose pathway, the contribution of other pathways to AsA biosynthesis at different plant species, organs, and developmental stages remains unclear (Badejo et al. 2012). Our results showed that the main metabolites, L-galactose, myo-inositol, D-glucoronate, and L-gulono-1,4-lactone levels in L-galactose and myoinositol pathway were all increased in ERI lines at 45DAA and the corresponding genes, GGP, GPP/IMP, GalDH, MIOX were all expressed higher (Fig. 6). SlGPP3/IMP3 and SlMIOX1 were confirmed as the 2 direct target genes of SlEIL2 (Fig. 7). SlGPP3/IMP3 demonstrated high affinity with the L-Gal 1-P and D-Ins 3-P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol (Torabinejad et al. 2009). Overexpression of SlGPP3/IMP3 not only improved AsA and myoinositol content but also improved fruit firmness and delayed fruit softening (Zheng et al. 2022). This supports the regulatory effect of SlEIL2 to SlGPP3/IMP3 (Fig. 7). We also acquired 3 SlGPP3/IMP3 overexpression lines (OG1-3) and 1 CRISPR/Cas9 system mediated gpp3/imp3 mutant lines and confirmed the positive role of SlGPP3/IMP3 in AsA accumulation of tomato fruits (Supplemental Fig. S9). Then, does the myo-inositol increase cause the AsA accumulation? MIOX is the essential monooxygenase enzyme in this conversion process. Up-regulating MIOX to enhance the myoinositol pathway does not increase the AsA content in Arabidopsis (Endres and Tenhaken 2009; Ivanov Kavkova et al. 2019). However, MIOX4, whose transcript level was higher in ERI fruits, was confirmed to increase the accumulation of AsA in tomato fruit (Munir et al. 2020, Fig. 6). In addition, MIOX1 was highly expressed from flowering to the fruiting stage suggesting its potential roles in fruit ripening (Munir et al. 2020).

A recent study showed that ABA-induced the AsA accumulation, which is mediated by the SlMAPK8-SlARF4-SlMYB11 module in tomato during fruit development and drought stress responses. SlARF4 transcriptionally inhibits the transcription factor gene SlMYB11, thereby modulating AsA accumulation by regulating the transcription of the AsA biosynthesis genes GPP, GaLDH (Xu et al. 2022). In our ERI fruits, the transcript levels of L-galactose pathway genes GPP, GaLDH were higher than WT, which may be caused by the accumulation of ABA (Fig. 6). The above results support that reduced SlEIL2 leads to the activation of AsA biosynthesis through L-galactose and myo-inositol pathway.

ET is one of the most important phytohormones for fruit maturation, which is dependent on its downstream signaling cascade (Lin et al. 2009). In plants, S-adenosylmethionine is a precursor in ET biosynthesis that is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). ACC is then oxidized and converted to ET by ACC oxidase (ACO; Yang and Hoffman 1984; Wang et al. 2002). EIN3/EIL family members play important roles in ET signal transduction pathways and ET biosynthesis (Yin et al. 2010; An et al. 2018; Zhang et al. 2019). The roles may be different at the different fruit ripening stages. In our results, the ACC contents were significantly higher in SlEIL2 RNAi than in WT fruits at 37DAA, but decreased at 45DAA (Fig. 5D). While the ET production increased significantly from 37DAA in ERI fruits suggesting that ACC is converted to ET during this period (Fig. 5C). The higher transcript levels of ACO1, ACO4, ACO6 in 45DAA ERI fruits indicated their roles in this ACC conversion (Fig. 5E). However, the transcript levels of ACO3 and ACO5 were repressed and ACO2 did not change substantially (Fig. 5E). The specific roles of every member of these gene families require further study.

Overall, our results indicate that SlEIL2 plays a prominent role in controlling AsA and β-carotene accumulation in ripe tomato fruits. SlEIL2 regulates the expression of ERFs to affect SlLCYB2 transcript levels, thus controlling the conversion of lycopene to β-carotene and then enhancing the ABA accumulation. Meanwhile, SlEIL2 is involved in the AsA biosynthetic pathway by regulating SlMIOX1 and SlGPP3/IMP3 expression (Fig. 8). No matter whether LCYB overexpression fruits or SlGPP3/IMP3 overexpression fruits all exhibit improved fruit firmness, delayed softening, and extended shelf-life (Diretto et al. 2020; Zheng et al. 2022). SlEIL2 seems like a switch to control fruit softening and shelf-life. Elucidating the regulatory mechanism of the AsA and β-carotene biosynthetic routes will be important for improvement of the nutritional value and the storability of tomato.

Figure 8.

Figure 8.

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Model of the role of SlEIL2 in enhancing β-carotene and AsA accumulation. Model showing the mechanism underlying the role of SlEIL2 in regulating AsA and β-carotene accumulation in ripe tomato fruits. According to the model, SlEIL2 regulates the expression of ERF.H30 and ERF.G6 to influence SlLCYB2 transcript levels, thereby controlling the conversion of lycopene to β-carotene and then enhancing ABA accumulation. SlEIL2 also participates in the 2 AsA biosynthetic pathways by regulating SlMIOX1 and SlGPP3/IMP3 expression.

Materials and methods

Plant materials and fruit sampling

We obtained 3 SlEIL2 RNAi lines (ERI1, ERI2, and ERI3) and 2 eil2 mutant lines (eil2-1 and eil2-2) in the tomato (S. lycopersicum cv. Micro-TOM) background. ERI lines were transformed with PC336 vectors containing specific 224 bp fragments in the SlEIL2 coding sequences. In addition, the eil2 mutant lines were generated using a pYAO-based CRISPR/Cas9 system that was a gift from Xie Qi's laboratory (Yan et al. 2015). The tomato plants were transformed using the Agrobacterium-mediated leaf disk method.

Tomato plants were grown in a greenhouse under the following conditions: 16-h-day/8-h-night cycle, 25 °C/20 °C day/night temperature, 70% relative humidity, and 300 μmol m−2 s−1 light intensity. Fruits were harvested at different ripening stages at 20, 30, 33, 35, 37, 39, and 45 d after anthesis (DAA). At least 9 fruits from 3 different plants (3 biological replicates) were harvested, cut into small pieces, and frozen in liquid nitrogen.

RT-qPCR

A total RNA tissue sample was isolated using the RNAprep Pure kit (DP441) of Tiangen (Beijing, China) and reverse transcribed into cDNA by the MonScript RTIII Super Mix with dsDNase (Two-Step) Kit of Monad (Wuhan, China). RT-qPCR was conducted using MonAmp SYBRGreen qPCR Mix of Monad. Gene expression levels in the raw data were normalized to that of the housekeeping gene ACTIN and to WT values to obtain relative expression levels. For each assay, at least 3 biological replicates were performed. All primers used in this study are listed in Supplemental Table S1.

Protein isolation and immunoblot analysis

The protein extraction of ERI lines, eil2 and WT fruits and further immunoblot analysis were performed as described by Kong et al. (2014). Obtained protein was concentrated with a concentrator plus Eppendorf (Hamburg, Germany). The SlEIL2 antibody and ACTIN antibody were purchased from PhytoAB Inc. (San Jose, CA, USA).

Measurement of the fruit firmness

A firmness tester (GY-2) was used to determine fruit firmness as described by (Ma et al. 2014). A flat probe was placed on the equator of fruit and used at a displacement rate of 1 mm s−1 to press an integrated tomato fruit at a total distance of 3 mm. The maximum force recorded at 3 mm of compression was used as estimated fruit firmness from the averaged value of at least 9 tested fruits and a minimum of 3 compressions per fruit.

Measurements of ET, ABA, and ACC contents

ET measurements were performed as described by Ma et al. (2014) using a gas chromatograph (PerkinElmer Clarus 580, USA). The measurement was performed for 3 biological replicates and each replicate contained 9 fruits. The detection of ABA and ACC contents in tomato fruits was detected by MetWare (http://www.metware.cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. Three replicates of each assay were performed.

Yeast 1-hybrid assay, EMSA, and chromatin immunoprecipitation assay

The yeast 1-hybrid assay was conducted basically as described by Zhuang et al. (2019). The corresponding elements in SlERFs, SlLCYB2, SlMIOX1, and SlGPP3/IMP3 promotor sequences were separately inserted into the KpnI and SalI sites of the pLacZi2u vector. The full-length sequences of SlERF.G6, SlERF.H30, and SlEIL2 were each ligated into the XhoI and EcoRI sites of the GAD vector (Lin et al. 2007).

For the EMSA, the full coding sequences of SlERF.G6 and SlERF.H30, and a partial coding sequence of SlEIL2 (encoding aa 68 to 352) were separately inserted into the pZP211-FLAG plant expressing vector between the BamHI and SalI sites, respectively. SlERF.G6-FLAG, SlERF.H30-FLAG, and SlEIL2-FLAG proteins were purified from crude total protein extracted from N. benthamiana leaves transiently transformed with the respective constructs using a Pierce Classic IP Kit (Thermo Scientific, Waltham, MA, USA) and FLAG antibody and peptides. The proteins were incubated with 0.5 μ M of the corresponding of biotin-labeled ssDNA probes in a total volume of 10 μL for 20 min at 25 °C. Unlabeled probes were added at various concentrations in the competition experiments. A LightShift Chemiluminescent EMSA Kit (Thermo Scientific) was used for chemiluminescence detection. All the used probes were synthesized by BGI (Shenzhen, China).

Transient expression assay in N. benthamiana

For the double-LUC reporter assay, the promoter sequences of 4 SlERFs (SlERF.J1, SlERF.G6, SlERF.H30, and SlERF.F12), SlLCYB2, SlGPP3/IMP3, and SlMIOX1 containing the SlEIL2, SlERF.G6, or SlERF.H30 binding sites were inserted into the LUC vector (Pgreen-0800) via the KpnI and SalI sites. LUC activity was detected as described by Zhuang et al. (2019, 2020).

The analysis of transcriptome data

The fruits (45 d after anthesis) from 3 different plants (3 biological replicates) of ERI lines and WT were harvested, cut into small pieces, and frozen in liquid nitrogen. Total RNA of the samples was extracted with an RNAprep Pure Plant kit (DP441, Tiangen, China). The RNA quality was detected by a NanoPhotometer spectrophotometer (IMPLEN, CA, USA), Qubit 2.0 Fluorometer (Life Technologies, CA, USA), and Agilent Bioanalyzer 2100 system (Agilent Technologies, CA). Illumina RNA-Seq was performed by MetWare Biotechnology (Wuhan, China).

The raw data were processed using the NGS QC Toolkit (Patel and Jain 2012) to remove reads containing poly-N and low-quality reads so as to obtain clean reads; all subsequent analyses were based on clean reads. Next, the clean reads were mapped to the reference tomato transcripts (https://solgenomics.net/organism/Solanum_lycopersicum/genome) and the read counts for each gene were obtained using Kallisto (Bray et al. 2016). Differentially expressed genes (DEGs) were identified using the DESeq2 R package (Love et al. 2014). The threshold for significantly differential expression was defined as follows: adjusted P value < 0.05 and (log2FoldChange>2 or log2FoldChange<−2). DEGs were described using a heatmap analysis with a Z-score using pheatmap R package. The DEGs were analyzed for GO enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment using TBtools (Chen et al. 2020). The used data in Figs. 2 and 6 are listed in Supplemental Table S2.

The analysis of metabolome data and the carotenoids contents

The AsA-related metabolites, α-carotene, β-carotene, γ-carotene, ε-carotene, lutein, violaxanthin, antheraxanthin, neoxanthin, zeaxanthin, β-cryptoxanthin, lycopene and (E/Z)-phytoene contents were detected by MetWare (http://www.metware.cn/) based on the AB Sciex QTRAP6500 LC-MS/MS platform. Each value is the relative content of metabolites, which is obtained by calculating the peak area formed by the characteristic ions of each substance in the detector and can be used to compare the differences of the same metabolite in different samples. The analysis of the absolute levels of carotenoids was also detected by MetWare based on the AB Sciex QTRAP 6500 LC-MS/MS platform. All of the standards were purchased from Sigma-Aldrich (St Louis, MO, USA) and BOC (NY, USA). The peak areas of each sample were used to calculate the absolute carotenoids levels. Three or more replicates of each assay were performed. Different metabolite values were described using a heatmap analysis with a Z-score using pheatmap R package.

Statistical analysis

Data points represent the mean ± Sd of 3 replicates. The statistical significance of differences was tested by the Excel software (Student's t-test) and Tukey-test. Significant differences relative to the control are indicated by *P < 0.05 and **P < 0.01 or different letters (P < 0.05).

Accession numbers

Sequence data from this article can be found in the EMBL data libraries under accession numbers: SlEIL2 (Solyc01g009170); SlEIL7 (Solyc03g096630); SlLCYB2 (Solyc04g040190); SlMIOX1 (Solyc06g062430); SlGPP3 (Solyc11g012410); SlERF.J1 (Solyc02g090770), SlERF.G6 (Solyc03g026270), SlERF.H30 (Solyc12g008350) and SlERF.F12 (Solyc02g093130).

Supplemental data

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

Supplemental Figure S1 . mRNA levels of SlEIL family members ERI fruits at different stages of ripening.

Supplemental Figure S2 . The fruits development of 3 ERI lines and WT.

Supplemental Figure S3 . The detection of other carotenoid contents.

Supplemental Figure S4 . Yeast 1-hybrid assay for the carotenoid metabolic-related genes regulated by SlEIL2.

Supplemental Figure S5 . Analysis of the transcript levels of SlERF family members in WT and ERI1 fruits.

Supplemental Figure S6 . Analysis of the transcript levels of SlEIL family members in ERI1 and eil2 fruits.

Supplemental Figure S7 . The phenotype analysis of SlEIL7 RNAi lines in tomato.

Supplemental Figure S8 . Analysis of the transcript levels of SlERF.H30 and SlERF.G6 in ERI1 and WT fruits at different stages of ripening.

Supplemental Figure S9 . Analysis of the role of SlGPP3/IMP3 in tomato.

Supplemental Table S1 . Primers were used in this manuscript.

Supplemental Table S2 . The metabolome and transcriptome data used in the heatmap of Figs. 2, 5 and 6.

Supplementary Material

kiad151_Supplementary_Data
Click here for additional data file. (1.5MB, zip)

Contributor Information

Chong Chen, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Meng Zhang, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Mingyue Zhang, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Minmin Yang, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Shanshan Dai, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Qingwei Meng, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Wei Lv, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Kunyang Zhuang, State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China.

Funding

This work was supported by National Key Research and Development Program of China (grant number 2020YFA0907600), the China Postdoctoral Foundation (grant number 2021M691974), the National Natural Science Foundation of China (grant number 32100203, 31870239, 31870277), and the Natural Science Foundation of Shandong Province (grant no. ZR202103010453).

Data availability

The data supporting the findings of this study are available from the corresponding author upon request.

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Associated Data

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Supplementary Materials

kiad151_Supplementary_Data
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Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon request.

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