A tomato NAC transcription factor, SlNAP1, directly regulates gibberellin-dependent fruit ripening

Abstract

In tomato (Solanum lycopersicum), the ripening of fruit is regulated by the selective expression of ripening-related genes, and this procedure is controlled by transcription factors (TFs). In the various plant-specific TF families, the no apical meristem (NAM), Arabidopsis thaliana activating factor 1/2 (ATAF1/2), and cup-shaped cotyledon 2 (CUC2; NAC) TF family stands out and plays a significant function in plant physiological activities, such as fruit ripening (FR). Despite the numerous genes of NAC found in the tomato genome, limited information is available on the effects of NAC members on FR, and there is also a lack of studies on their target genes. In this research, we focus on SlNAP1, which is a NAC TF that positively influences the FR of tomato. By employing CRISPR/Cas9 technology, compared with the wild type (WT), we generated slnap1 mutants and observed a delay in the ethylene production and color change of fruits. We employed the yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) assays to confirm that SlNAP1 directly binds to the promoters of two crucial genes involved in gibberellin (GA) degradation, namely SlGA2ox1 and SlGA2ox5, thus activating their expression. Furthermore, through a yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BIFC) and luciferase (LUC) assays, we established an interaction between SlNAP1 and SlGID1. Hence, our findings suggest that SlNAP1 regulates FR positively by activating the GA degradation genes directly. Additionally, the interaction between SlNAP1 and SlGID1 may play a role in SlNAP1-induced FR. Overall, our study provides important insights into the molecular mechanisms through which NAC TFs regulate tomato FR via the GA pathway.

Keywords: Fruit ripening; Gibberellin; NAC; Protein–protein interaction; Tomato; Transcription factor.

Fig. 1

SlNAP1 transcripts in different organs of tomato plants and after the treatment of wild-type (WT) fruit with gibberellin (GA) and paclobutrazol (PAC). A Quantitative real-time polymerase chain reaction (qRT-PCR) of SlNAP1 in different tomato organs (root, stem, leaf, and flower) and fruit ripening stages (MG, Br, and Br + 10). B Transcriptional level of SlNAP1 in WT fruit after treatment with GA. C Transcriptional level of SlNAP1 in WT fruit after treatment PAC. The Slactin gene was used as the internal control. Bars indicate mean ± SD of three independent replicates. Asterisks indicate significant differences determined by Student’s t-test (*P < 0.05). qRT-PCR quantitative real-time PCR, MG, mature green, Br breaker, Br + 10 10 days after the breaker, SD standard error

Fig. 2

Tomato fruit ripening phenotype of wild-type (WT) and CR-SlNAP1. A Gene editing analysis of slnap1-5 and slnap1-16 homozygous mutants. B Phenotype of tomato fruit in WT, slnap1-5, and slnap1-16. WT, Wild-type. C Value of tomato fruit in WT, slnap1-5, and slnap1-16 at MG, Br, Br + 4 and Br + 7 stages. D b Value of tomato fruit in WT, slnap1-5, and slnap1-16 at MG, Br, Br + 4, and Br + 7 stages. E Lycopene content of tomato fruit in WT, slnap1-5, and slnap1-16 at MG, Br, Br + 4, and Br + 7 stages. Bars represent mean ± SD of three biological replicates. Asterisks indicate significant differences determined by Student’s t-test (*P < 0.05). MG mature green, Br breaker, Br + 4 4 days after the breaker, Br + 7 7 days after the breaker, SD standard error

Fig. 3

Endogenous gibberellin (GA₃) content and related genes expression in wild-type (WT), slnap1-5, and slnap1-16 tomato fruits at mature green (MG), breaker (Br), 4 days after breaker (Br + 4), and 7 days after breaker (Br + 7) stages. A GA3 content in WT, slnap1-5, and slnap1-16 tomato fruits at MG, Br, Br + 4, and Br + 7 stages; B relative expression of SlGA2ox1 gene in WT, slnap1-5, and slnap1-16 tomato fruits at MG, Br, Br + 4, and Br + 7 stages; C relative expression of SlGA2ox5 gene in WT, slnap1-5, and slnap1-16 tomato fruits at MG, Br, Br + 4, and Br + 7 stages

Fig. 4

SlNAP1 directly binds to the promoters of SlGA2ox1 and SlGA2ox5. A Y1H assays. The CDS of SlNAP1 was fused to the pGADT7 vector, and the promoter fragments of SlGA2ox1 and SlGA2ox5 corresponding to the regions −1500 to −1 were fused to pAbAi vector. B DLR assays. The CDS of SlNAP1 was cloned into the pGreenII 62-SK vector to generate the SlNAP1-62SK effector. The promoters of SlGA2ox1 and SlGA2ox5 (1500-bp upstream of the start codon) were introduced into the pGreenII 0800-LUC vector to generate the ProSlGA2ox1:LUC and ProSlGA2ox5:LUC, reporter constructs, respectively. The constructs were transformed into Agrobacterium tumefaciens strain GV3101. A. tumefaciens was mixed and coinfiltrated into tobacco leaves for transient expression. A DLR assay system was used to measure the ratio of luminescence of firefly LUC to Renilla LUC. Each value represents the means of six biological replicates. *P < 0.05 (Student’s t-test). Y1H yeast one-hybrid, DLR dual-luciferase reporter, CDS coding sequence

Fig. 5

SlNAP1 interacts with SlGID1 in vitro and in vivo. A BiFC assays. Full-length SlNAP1 and SlGID1 were fused to the N-terminal part and the C-terminal part of YFP, respectively. Constructs were transformed to Agrobacterium tumefaciens strain GV3101, and were then injected into 4-week-old tobacco leaves. The YFP fluorescence was observed under a confocal laser scanning microscope after incubating at 22 °C for 24–48 h. Bars = 50 μm. B Y2H assays. The full-length SlGID4 was fused with the activation domain (pGADT7-SlGID4) and the full-length SlNAP1 was fused with the binding domain (pGBK7-SlNAP1). Transformed yeast cells were grown on SD-Leu-Trp, or SD- Leu-Trp-His-Ade media. These experiments were performed three times with similar results, and a representative picture was shown. C and D LUC assays. CDS of SlNAP1 (with no stop codon) was cloned into pCAMBIA1300-nLUC, and the CDS of SlGID1 was cloned into the pCAMBIA1300-cLUC vector. The constructs were transformed into Agrobacterium tumefaciens strain GV3101, and A. tumefaciens was mixed (1:1, v/v) and coinfiltrated into tobacco, and luminescence was observed in optical in vivo imaging and was analyzed by PlantView. BiFC, bimolecular fluorescence complementation; DAPI 4, 6-diamidino-2-phenylindole, Y2H yeast two-hybrid, LUC Luciferase