Increasing flavonoid contents of tomato fruits through disruption of the SlSPL-CNR, a suppressor of SlMYB12 transcription activity

Fruits of tomato (Solanum lycopersicum) are rich in nutrients and generally served as critical resources for human diet. In fruit pericarps, the peel tissues adhering to the flesh produce functional components including flavonoids that are indeed mixed polyphenolic compounds. Flavonoids play an essential role in determining peel colour and are proven to be functionally important for human health as favourable hydrophilic antioxidants (Ballester et al., 2010). However, the content of endogenous flavonoids in numerous fruits including tomato tends to be inadequate and fails to meet the requirement of health benefits. Therefore, digging in essential flavonoid-regulated genes and further breeding of tomato fruits rich in flavonoids is a significant target for tomato improvement in quality. Flavonoid biosynthesis is derived from the phenylpropanoid pathway and undergoes combinatorial transformations by a series of enzymes, which are mainly controlled by flavonoid biosynthetic genes. SlMYB12, a R2R3-MYB transcription factor, was uncovered to be a master regulator in flavonoid biosynthesis by extensively targeting and activating the transcription of flavonoid biosynthetic genes (Adato et al., 2009). Disruption of SlMYB12 gene resulted in the decreased accumulation of naringenin chalcone and a pink-coloured tomato fruit (Ballester et al., 2010), indicating that modulation of SlMYB12 could be an efficient strategy in controlling flavonoid biosynthesis in tomato peel. Apart from its significant role in mediating flavonoid content, the molecular basis underlying the transcriptional activity of SlMYB12 remains largely unknown. The tomato SBP-box protein Colorless Non-ripening (SlSPL-CNR, referred as to SlCNR) was early cloned and studied as a master ripening transcription factor. While we recently found that fruits of the slcnr mutants generated by CRIPSR/Cas9 system can turn to full red, which are distinct from the original Cnr mutant that fails to ripen (Gao et al., 2019). Herein, we further investigated the maturity characteristics in fruits of the slcnr mutants (slcnr-16, slcnr-22 and slcnr-23) with the complete deficiency of SlCNR protein (Figure S1) and found that they only exhibited an approximately 3-day delay in ripening initiation and a slight decreased lycopene content (Figure S2). These results demonstrated that SlCNR is not a master regulator of fruit ripening, and its mutation does not change the overall ripening process. Western blot analysis using the anti-SlCNR polyclonal antibody revealed that SlCNR protein dominantly expresses in fruits (Figure 1a). Moreover, the peel tissues harbour obviously more abundant SlCNR protein than the flesh (Figure 1b), indicating that SlCNR may be functionally important in modulating the development of fruit peel. Intriguingly, fruits of the slcnr mutants exhibited a yellow-orange colour appearance during the ripening, while the wild-type appeared to red rapidly, and the former harboured a deeper yellow-coloured peel tissues than the later (Figure 1c). To further investigate the regulation role of SlCNR on fruit colour, a comparative transcriptomic analysis was performed with high Pearson correlation coefficients (Figure S3). A total of 2165 differentially expressed genes (DEGs; fold change ≥2 and P value < 0.05) were identified between Br and Br + 7 wild-type fruits, of which 8% (187) and 92% (1978) were up-regulated and down-regulated in Br + 7 fruits, respectively (Figure S4a, Table S1). In slcnr-23 mutant, 1603 DEGs were identified between Br and Br + 7 fruits, of which 21% (341) were up-regulated and 78% (1162) were down-regulated in Br + 7 fruits (Figure S4b, Table S2). Compared with the wild-type, 362 and 573 DEGs were identified in the slcnr-23 fruits at Br and Br + 7 stages, respectively (Figure S4c,d, Tables S3 and S4). Accordingly, the percentage of higher expressed genes in slcnr-23 increased from 34% of Br stage to 82% of Br + 7 stage. Together, these data indicate a potential negative role of SlCNR in modulating gene expression during fruit ripening. KEGG analysis showed that DEGs in the Br + 7 slcnr-23 fruits compared with those of the wild-type were highly enriched in multiple flavonoid-related pathways (Figure 1d). Furthermore, by overlapping those DEGs with the 49 SlMYB12-targeted genes identified by Zhu et al. (2018), we found that 20 genes were overlapped (Figure 1e), which all exhibited higher expression level in the slcnr-23 mutant fruits at Br + 7 stage than the wild-type (Figure 1f; Table S5). These results indicate that SlCNR might negatively regulate the transcription of SlMYB12-targeted genes. Several key flavonoid biosynthetic genes were included in the overlapped genes mentioned above, that is CHS1, CHS2, CHI3, F3H, FLS and 3GT (Figure S5a,b; Table S5), which also exhibited increased mRNA levels in peel tissues of the slcnr mutants (slcnr-16, slcnr-22 and slcnr-23) compared with the wild-type as determined by quantitative RT-PCR (Figure 1g). We then measured the accumulation of total flavonoids and four essential flavonoid monomers including naringenin chalcone, naringenin, kaempferol-3-rutinoside and rutin (Figure S5a), and found that the pericarps of slcnr-23 fruits at B + 7 stage harboured higher flavonoids and the four monomers than the wild-type (Figure S5c). Furthermore, the content of total flavonoids in peel tissues of the slcnr mutants at Br + 7 stage increased significantly to approximately 26 mg·g−1 while that in wild-type was only 15 mg·g−1, and the contents of four flavonoid monomers elevated dramatically to a 2–5-fold of the wild-type (Figure 1h). These results revealed an obvious increase in flavonoid biosynthesis in fruits of the slcnr mutants. By contrast, the expression of those flavonoid biosynthetic genes was significantly repressed in the peel tissues of SlCNR overexpression fruits (OE-SlCNR-21 and OE-SlCNR-24). Consistently, fruits of OE-SlCNR-21 and OE-SlCNR-24 at Br + 7 stage harboured a lighter yellow-coloured peel tissues and lower contents of total flavonoids, kaempferol-3-rutinoside and rutin compared with the wild-type (Figure S5d–g). Thus, SlCNR negatively regulates flavonoid biosynthesis, and its disruption by CRISPR/Cas9 gene editing will endow tomato fruits with increased flavonoids. In addition, fruits of slcnr mutants did not exhibit obvious difference in contents of several important quality traits, including total carotenoid, total ascorbate and total soluble solids, compared with those of the wild-type, implicating that SlCNR deficiency has no significant effect on the main quality traits of tomato fruit (Figure S6). To gain insights into the molecular basis underlying SlCNR-mediated flavonoid biosynthesis, we next investigated the regulation role of SlCNR on the master transcription factor SlMYB12. Dual-luciferase reporter assay revealed that SlCNR does not directly modulate the transcription of SlMYB12 (Figure S7). Interestingly, co-immunoprecipitation (Co-IP) assay showed that SlCNR interacts with SlMYB12 in vivo (Figure 1i), and this interaction was further revealed to occur in cell nucleus by bimolecular fluorescence complementation (BiFC) assay (Figure 1j). Furthermore, transcription activity assay in Nicotiana benthamiana leaves showed that SlCNR co-expression significantly reduced the SlMYB12-activated FLUC transcription under the driving of CHS2, F3H, or 3GT promoter, while individual SlCNR expression did not cause effects on the transcriptional activity of CHS2, F3H, or 3GT promoter (Figure 1k). These results suggest that SlCNR negatively regulates the expression of flavonoid biosynthetic genes as a suppressor of SlMYB12 transcription activity. In conclusion, herein, we uncovered that SlSPL-CNR negatively regulates flavonoid biosynthesis by repressing SlMYB12 transcription activity and successfully increased flavonoid contents in tomato fruits through SlSPL-CNR knockout using the CRISPR/Cas9 editing system (Figure S8). Our study provides important gene resource and molecular basis for tomato breeding in quality improvement. This work was supported by the National Key R&D Program of China (2022YFD2100101) and the National Natural Science Foundation of China (31972128). The authors declare no conflicts of interest. GQ, HZ and LZ designed the experiments. LZ, ZS, TH, DC, XC and QZ performed the experiments. LZ, ZS, TH and XC analysed the data. JC, BZ and DF provided important discussions. GQ, LZ, HZ and ZS wrote the article. The data used to support the findings of this study are available in the main text and Supporting Information of this article. The full legend of Figure 1 is provided in Data S1. Materials and methods used in this study are provided in Data S2. All primer sequences are listed in Table S6. Data S1 Supplementary Figure legend. Data S2 Supplementary Materials and Methods. Figure S1 SlCNR protein is completely deficient in slcnr mutants generated by CRISPR/Cas9 editing system. Figure S2 Fruits of the slcnr mutants generated by CRISPR/Cas9 editing system exhibit a slight ripening delay. Figure S3 Pearson correlation analysis between RNA-seq samples. Figure S4 Transcriptome reprogramming induced by SlCNR mutation during fruit ripening. Figure S5 SlCNR negatively regulates the accumulation of flavonoids and the transcription of flavonoid biosynthetic genes. Figure S6 The contents of several important quality traits in fruits of the wild-type and slcnr mutants. Figure S7 Transcription activity assay in N. benthamiana leaves. Figure S8 Model for SlCNR-mediated regulation of flavonoid biosynthesis and the generation of tomato fruits with increased flavonoids by CRISPR/Cas9 editing system. Table S1 Differentially expressed genes in fruits of the wild-type at Br+7 stage compared with those at Br stage. Table S2 Differentially expressed genes in fruits of the slcnr-23 mutant at Br+7 stage compared with those at Br stage. Table S3 Differentially expressed genes in fruits of the slcnr-23 mutant at Br stage compared with those of the wild-type. Table S4 Differentially expressed genes in fruits of the slcnr-23 mutant at Br+7 stage compared with those of the wild-type. Table S5 Overlapping of the differentially expressed genes in the slcnr-23 mutant fruits at Br+7 stage compared to those of wild-type and the reported SlMYB12-targeted genes. Table S6 A summary of primer informations. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.