Trihelix proteins are plant-specific transcription factors that play crucial roles in plant development and stress responses. However, the involvement of trihelix proteins in fruit ripening and transcriptional regulatory mechanisms remains largely unclear. In this study, we cloned a trihelix gene SlGT31, whose relative expression was significantly induced by the application of exogenous ethylene but repressed by 1-methylcyclopropene (1-MCP). Suppression of SlGT31 resulted in delayed fruit ripening, decreased accumulation of total carotenoids and ethylene content, and inhibition of relative expression of genes related to ethylene and fruit ripening. Conversely, the opposite results were observed in SlGT31-overexpression lines. Yeast one-hybrid and dual-luciferase assays suggested that SlGT31 could bind to the promoters of two key ethylene biosynthesis genes ACO1 and ACS4. These results indicate that SlGT31 may act as a positive modulator during fruit ripening.

Plant Materials and Growth Conditions

Tomato (Solanum lycopersicum Mill.var. Ailsa Craig) was used as the wild type (WT) in this study. WT and transgenic lines were grown in a glasshouse under controlled conditions with 16-h-light/8-h-dark cycles, 25°C-day/18°C-night temperatures, 80% relative humidity, and 250 μmol m⁻² sec⁻¹ luminous intensity. Flowers were tagged at the anthesis stage, immature green was defined as 20 DPA (days post-anthesis), and mature green was defined as 35 DPA. Breaker fruits were defined as fruits of 38 DPA with the color starting to generate a slight yellow color. Other fruits from the 4th and 7th days after breaker were also used. Fruits at different ripening stages were collected, frozen immediately in liquid nitrogen, and stored at -80°C until use.

Sequence analysis and subcellular localization

For SlGT31-GFP construction, the cDNA with the termination codon removed from SlGT31 was amplified with SlGT31-GFP-F/R primers (Supplementary Table.S1). The amplified products were digested with the restriction enzymes Pst I/Sal I and inserted into the pHB vector to form the SlGT31-GFP vector (Supplementary Fig.S2). The SlGT31-GFP fusion construct and the control vector GFP were transformed into Agrobacterium tumefaciens strain GV3101 separately, and then infiltrated into tobacco (Nicotiana benthamiana) leaves about 5 weeks old. Studies have shown that the transcription factor HY5 is localized in the nucleus (Burman et al., 2018), so the subcellular localization of SlGT31 was studied using HY5-RFP localization. Tobacco was cultured in darkness for 24 hours, then cultured under normal conditions for 2–3 days. The fluorescence images of localization samples were acquired on a confocal laser scanning microscope (Leica TCS SP8) after 72h of infiltration. Excitation wavelengths used were 488 nm for GFP and 563 nm for RFP, emission wavelengths used were 507 nm for GFP and 582 nm for RFP. All the primers used in this experiment were listed in Supplementary Table.S1.

Construction of RNAi and overexpression vectors and plant transformation

For overexpressing SlGT31, the open reading frame sequence of SlGT31 was introduced into the pBI121 expression vector under cauliflower mosaic virus (CaMV) 35S promoters. In order to down-regulate the relative expression of the SlGT31 gene, an RNAi vector was constructed (Supplementary Fig.S3). A 382 bp specific DNA fragment of SlGT31 was amplified, which had been tailed with Kpn I/Cla I restriction sites at the 5^’ end and Xba I /Xho I restriction sites at the 3^’ end. Then, the amplified products were digested with Cla I/Xba I and Kpn I/Xho I and linked into the pHANNIBAL plasmid at the Cla I/Xba I restriction site in the sense orientation and at the Kpn I/Xho I restriction site in the antisense orientation. Finally, the double-stranded RNA expression unit was purified and inserted into the plant binary vector pBIN19 with Sac I and Xba I restriction sites. The two vectors were sequenced, checked, and transformed into Agrobacterium tumefaciens LBA4404 strain (An, 1987), and then transformed into Solanum Lycopersicum cv. Ailsa Craig as described in the previous study (Chen et al., 2004). We obtained SlGT31-OE (overexpression) and SlGT31-RNAi (RNA interference) transgenic plants.

Total RNA extraction and quantitative reverse-transcription PCR analysis

Total RNA was extracted from samples using Trizol reagent (Invitrogen, Shanghai, China). Quantitative reverse-transcription PCR (qRT-PCR) was performed by using a CFX96™ Real-Time System (Bio-Rad, USA). The RNA extraction method and PCR reaction system were based on previous reports (Zhang et al., 2018). The tomato SlCAC gene was used as an internal control for expression analysis (Nicot et al., 2005; Exposito-Rodriguez et al., 2008), the relative expression levels of the genes were analyzed using the 2^−ΔΔCT method (Livak et al., 2001), and all experiments were repeated three biological replicates.

Ethylene content determination and ethylene triple response experiment

The fruits at different ripening stages were picked and placed in jars for 3 hours to eliminate the effect of wound-induced ethylene caused by fruit picking (Zhu et al., 2014). The ethylene content was determined after being sealed for 24 hours (Chung et al., 2010). The seeds of wild type and transgenic lines were sown on MS medium supplemented with 0 and 5.0 µM ACC (1-aminocyclopropane-1-carboxylicacid) and then cultured in the dark at 25°C. Hypocotyl and root elongation were measured at 6 days after sowing, and at least 30 seedlings were measured for each line. The relative expression levels of ACS4 in treated wild-type and transgenic seedlings were determined by qRT-PCR. All experiments were repeated three biological replicates.

Pigment extraction

A 0.5 g sample of each line was cut from the pericarp in a 5-mm-wide strip around the equator of B+4 (4 days after breaker fruits) and B+7 (7 days after breaker fruits) fruits. Then, 5 mL of 60:40 (v/v) hexane: acetone was added, and total carotenoids of wild-type, SlGT31-RNAi fruits, and SlGT31-OE fruits were extracted. The extract was centrifuged at 4,000g for 5 min, and the absorbance of the supernatant was measured at 450 nm. Carotenoid content was calculated with the following equation: total carotenoid (mg mL⁻¹) = 4×(OD₄₅₀ nm) × 5 mL/0.5 g (Fray et al., 1993; Forth et al., 2006). All experiments were repeated three biological replicates.

The lycopene extraction was performed according to the previous method (Fish et al., 2002). A 0.5 g sample of each line was cut from the same area of pericarp around the equator of fruits at the breaker, B+4, and B+7 stages. Firstly, the samples were triturated in the liquid nitrogen, then 20 mL 0.05% (w/v) BHT in acetone: 95% ethanol: hexane (1:1:2, v/v) was added to the 50 mL centrifuge tube with samples. After shaking for 15 min at 180 rpm, the ice-deionized water (3 mL) was added into each tube, the tubes were shaken for 5 min and kept at room temperature for 5 min to allow for phase separation. The supernatant (hexane layer) was used to measure the absorbance at 503 nm. Carotenoid content was calculated with the following equation: lycopene (mg/kg) = (OD₅₀₃nm×31.2)/g tissue. All experiments were repeated in three biological replicates.

Fruit Storage Assay

Fruits of WT and transgenic lines were harvested at the B+4 stage. All fruits were disinfected with 10% bleach for 10 minutes, followed by rinsing with sterilized water and air-drying (Zhang et al., 2018). The fruits of WT and transgenic lines were stored at room temperature for 30 days. The chromatic fruit tissue disruption of the fruits was evaluated by observing and taking photographs at the end of the storage period. Water loss per unit fruit weight was calculated after recording the weight decrease over time. The weight loss of WT, SlGT31-OE, and SlGT31-RNAi fruits was measured at 5, 10, 15, 20, and 25 days, respectively.

Ethephon (ET) and 1-MCP treatment

To understand if exogenous ethylene accelerates fruit ripening, WT tomato fruits and transgenic fruits at the mature green stage were disinfected with 10% bleach for 10 mins. Ethylene gas was released by dissolving ethephon powder (Solarbio, E8021) in a small amount of water, and the fruit was treated and sealed at 25°C for 24 hours at a concentration of 10ppm. After treatment, fruits were left at room temperature for two days (Breitel et al., 2016; Zhang et al., 2020). 1-MCP gas was released by dissolving 1-MCP powder (Macklin, M875517) in a small amount of water, and the fruit was treated sealed at 25°C for 16 hours at a concentration of 1 mg/L, which was then dried and placed at room temperature for two days (Hao et al., 2015; Liu et al., 2021). After treatment, the pericarp tissues of the tomato fruits were sliced, frozen, and then used for RNA isolation and qRT-PCR. For each treatment, at least three biological replicates from independent samples were included.

Yeast one-hybrid assay

The open reading frame sequence of SlGT31 was amplified and introduced into pGADT7 to construct a prey vector. The promoter fragments of ACO1 and ACS4 containing the GT2-binding motif were inserted into pAbAi to construct a bait vector. The pAbAi–ACO1 and pAbAi–ACS4 plasmids were linearized and then transformed into Y1H Gold yeast strains. The minimal inhibitory concentration of aureobasidin A (AbA) was screened to avoid self-activation. The prey vector was introduced into the bait yeast strains and grown on SD/-Ura/-Leu/AbA culture medium at 30°C for 2–3 days. The pAbAi-p53 and pGADT7-p53 plasmids were used as positive controls.

Dual-luciferase reporter gene assay

We constructed the coding region of SlGT31 into the pGreenII 62-SK vector as effector. The promoter sequences of ACO1 and ACS4 were constructed into the pGreenII 800-LUC vector as reporters, respectively. The recombinant vector was introduced into Agrobacterium tumefaciens strain GV3101, then the effector and reporter were mixed and injected into the leaves of 5-week-old tobacco when the OD₆₀₀nm values of the culture were 0.8. After 24 hours of cultivation in darkness, the injected tobacco plants were restored to normal conditions for 2–3 days. The activities of LUC and REN luciferase were measured using the Dual-Luciferase Assay Kit (Biyuntian, RG027) according to the instruction manual. At least six transient assay measurements were performed for each assay.

Statistical analysis

All data are means ± standard deviation of at least three independent experiments. Significance in a difference between the two groups was assessed by Student’s t-test (*P < 0.05). A single asterisk (*) and double asterisks (**) in the figures indicate significant differences of P < 0.05 and significant differences of P < 0.01, respectively.