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Table of Contents
Abstract
1. Introduction
2. Materials And Methods
2.1. Plant Materials And Treatment
2.2. Gene Expression Analysis
2.3. Promoter And Protein Structure Analysis
2.4. Subcellular Localization Analysis
2.5. Virus-Induced Gene Silencing (VIGS)
2.6. Yeast One-Hybrid (Y1H) Assay
2.7. Dual-Luciferase Assay
2.8. Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
2.9. Data Analysis
3. Results
3.1. SlCOL4 Expression Was The Highest In MG Tomato Fruit And Inhibited By ABA Treatment
3.2. SlCOL4 Silencing Might Affect Tomato Fruit Ripening By Inducing Key Genes Involved In Ethylene Synthesis
3.3. TF Characterization And Subcellular Localization Analysis Of SLCOL4
3.4. Interaction Between SLAREB1 And SLCOL4
3.5. ChIP-Seq Identified Downstream Target Genes Of SLCOL4 In Vivo
3.5.1. Overview Of CHIP-Seq Results Of SLCOL4 In Vivo
3.5.2. Potential Target Genes Involved In Ethylene Biosynthesis And Signaling Pathway
4. Discussion
5. Conclusions
Acknowledgements
Abstract
Phytohormones are vital for tomato ripening, and abscisic acid (ABA) is speculated to induce ethylene biosynthesis at the initiate of tomato fruit ripening; however, its interaction sites remain unclear. In the present study, Constans-like 4 (SlCOL4), a CO/COL transcription factor, was cloned from tomato fruit. Amino acid sequence and subcellular location analysis proved that SlCOL4 was a typical transcription factor of the COL family. Gene expression analysis showed SlCOL4 expressed in different tissues of tomato plants and its transcription abundance declined at the onset of fruit ripening, coincidentally it was found to be down-regulated in ABA-treated tomato fruit and responded to exogenous ABA within 6 h after treatment. Meanwhile, yeast one-hybrid and dual-luciferase analysis showed that the ABA response factor SlAREB1 inhibited the transcription of SlCOL4, indicating it was directly regulated by ABA at the mature green stage of tomato fruit. In addition, the pericarp color turned red faster than that of control fruit, and the key ethylene biosynthesis genes, ACS2, ACS4, and ACO1, were up-regulated in SlCOL4 silenced tomato fruit, suggesting that SlCOL4 may be a negative regulator of ripening. Furthermore, several genes associated with the ethylene signaling pathway were identified downstream of SlCOL4 by chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Overall, the transcription factor SlCOL4 may interact with ABA and ethylene signals and negatively regulate tomato fruit ripening. The results in this study provide new insights for elucidating the molecular mechanism of ABA-ethylene interaction in regulating tomato fruit ripening, and provide references for further research on the role of COLs transcription factors in tomato fruit ripening as well.
Transcription factor Constans-like 4 may regulate tomato fruit ripening by interacting with abscisic acid and ethylene signaling Qiong Wu a,*, Xiaoya Tao b, Chunxiao Cui a, Yanan He a, Dongdong Zhang a, Yurong Zhang a, Li Li c,** a School of Food and Strategic Reserves, Grain storage and security engineering research center of education ministry, Collaborative Innovation Center of Henan Grain Crops, Henan university of technology, Zhengzhou, 450001, China b Shenzhen Key Laboratory of Food Nutrition and Health, College of Chemistry and Environmental Engineering and Institute for Innovative Development of Food Industry, Shenzhen University, Shenzhen, 518060, China c College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs, Zhejiang Key Laboratory for Agro-Food Processing, National-Local Joint Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang University, Hangzhou, 310058, China A R T I C L E I N F O Key words: Tomato Fruit ripening SlCOL4 transcription factor Abscisic acid Ethylene interaction A B S T R A C T Phytohormones are vital for tomato ripening, and abscisic acid (ABA) is speculated to induce ethylene biosynthesis at the initiate of tomato fruit ripening; however, its interaction sites remain unclear. In the present study, Constans-like 4 (SlCOL4), a CO/COL transcription factor, was cloned from tomato fruit. Amino acid sequence and subcellular location analysis proved that SlCOL4 was a typical transcription factor of the COL family. Gene expression analysis showed SlCOL4 expressed in different tissues of tomato plants and its transcription abundance declined at the onset of fruit ripening, coincidentally it was found to be down-regulated in ABA-treated tomato fruit and responded to exogenous ABA within 6 h after treatment. Meanwhile, yeast one-hybrid and dual-luciferase analysis showed that the ABA response factor SlAREB1 inhibited the transcription of SlCOL4, indicating it was directly regulated by ABA at the mature green stage of tomato fruit. In addition, the pericarp color turned red faster than that of control fruit, and the key ethylene biosynthesis genes, ACS2, ACS4, and ACO1, were up-regulated in SlCOL4 silenced tomato fruit, suggesting that SlCOL4 may be a negative regulator of ripening. Furthermore, several genes associated with the ethylene signaling pathway were identified downstream of SlCOL4 by chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Overall, the transcription factor SlCOL4 may interact with ABA and ethylene signals and negatively regulate tomato fruit ripening. The results in this study provide new insights for elucidating the molecular mechanism of ABA-ethylene interaction in regulating tomato fruit ripening, and provide references for further research on the role of COLs transcription factors in tomato fruit ripening as well.
1. Introduction
Transcription factors (TFs) are proteins that can specifically bind DNA promoter sequences to activate or inhibit the transcription of target genes (Schwechheimer and Bevan, 1998; Spitz and Furlong, 2012), and they are regarded as the main regulator of gene expression in eukaryotes. In plants, along with growth and development, disease resistance and stress response, TFs have also been demonstrated to play important roles in fruit ripening regulation (Liu et al., 1999; Karlova et al., 2014 Jan et al., 2019). Tomato (Solanum lycopersicum L.) has long been used as a model for fleshy fruit maturation studies owing to its small genome size, ease of transformation and moderate life cycle (Kou et al., 2021). Several TFs have been reported to be implicated in tomato fruit ripening, such as RIN (Vrebalov et al., 2002), FUL1 (Fujisawa et al., 2014), TAGL1 (Vrebalov et al., 2009) and SlbHLH22 (Waseem et al., 2019), which have been shown to be positive regulators, whereas SlMADS1 (Dong et al., 2013), SlAP2a (Chung et al., 2010) and SlEAD1 (Wang et al., 2020) were shown to be negative ones. They were proven to regulate ethylene biosynthesis and ultimately affect fruit ripening via regulating key genes * Corresponding author at: School of Food and Strategic Reserves, Henan university of technology, Zhengzhou, 450001, China. ** Corresponding author at: College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China. E-mail addresses: qiongwu0605@126.com (Q. Wu), lili1984@zju.edu.cn (L. Li). Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti https://doi.org/10.1016/j.scienta.2024.113865 Received 20 September 2023; Received in revised form 29 November 2024; Accepted 30 November 2024 Scientia Horticulturae 339 (2025) 113865 0304-4238/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). in the ethylene synthesis pathway directly or indirectly. The CO/COLs is a group of TFs found throughout plants, which have an N-terminal domain consisting of one or two B-boxes and a C-terminal CCT domain (Sheng et al., 2016). COLs have been reported to regulate plant growth and development. COL3 in Arabidopsis regulates the signal transduction of light and root development (Datta et al., 2006), and COL4 takes part in the abiotic stress response by regulating abscisic acid (ABA) signal transduction (Min et al., 2015). BnCOL2 in canola may regulate the drought stress tolerance by inducing the gene expressions in response to ABA and drought stress (Liu et al., 2020). PhCOL16 in Petunia corollas has been proven to be a positive regulator of chlorophyll biosynthesis (Ohmiya et al., 2019). COLs have also been reported to regulate the flowering process by regulating the photoperiod in several plants (Navarro et al., 2011; Sheng et al., 2016; Liu et al., 2016; Zhang et al., 2020; Gonzalez et al., 2021). In addition, MaCOL1 has been reported to be associated with banana fruit ripening (Chen et al., 2012). However, the function of COLs in tomato fruit needs to be further studied. A complex interaction between phytohormones determines tomato fruit ripening (Depuydt and Hardtke, 2011; McAtee et al., 2013). In phytohormone signaling pathways, phytohormone response factors regulate gene expression by binding with their response elements in the promoters of downstream genes. The ABA response factors ABFs/AREBs bind to downstream genes through ABA response elements (ABRE) (Fujita et al., 2013). For the ethylene response factor (ERF), the binding sites were GCC-box/ERE elements (Ohme-Takagi and Shinshi, 1995). As a typical climacteric fruit, ethylene is indispensable for tomato fruit ripening, while growing evidences have shown that ABA may regulate ethylene biosynthesis in ripening tomato fruit, and TFs such as SlZFP2 and ASR1 have been proven to be their interaction sites (Weng et al., 2015; Breitel et al., 2016). SlAREB1 has been reported to stimulate ethylene biosynthesis by interacting with NOR in ripening tomatoes (Mou et al., 2018). However, further investigations are needed to explore the interactions between ABA and ethylene during tomato ripening. In this study, a CO/COL family gene Constans-like 4 (Solyc12g096500, SlCOL4) was found to be down-regulated by ABA and upregulated by NDGA (the ABA synthesis inhibitor) during tomato fruit ripening. Initial gene expression analysis showed that SlCOL4 was expressed throughout tomato plant tissues, and its expression was the highest in the fruit at the mature green (MG) stage and declined as fruit ripening, and it was also shown to respond to ABA treatment within 6 h, indicating that SlCOL4 may participate in the ripening of tomato fruit and be regulated by ABA. Therefore, the full length of the open reading frame (ORF) of SlCOL4 was cloned, its amino acid sequence was analysed, and the subcellular location analysis was conducted to verify its TF characteristics. In addition, virus-induced gene silencing (VIGS), yeast one-hybrid (Y1H), dual-luciferase and chromatin immunoprecipitation sequencing (ChIP-Seq) analyses were performed to further clarify its role in tomato fruit ripening, so as to provide new insights for both improving the molecular mechanism of ABA regulation and further research on the role of COLs transcription factors in tomato fruit ripening.
2. Materials and methods
2.1. Plant materials and treatment
The MG cherry tomato fruit (Solanum lycopersicum L. cv Xin Taiyang) used in the present study were manually picked about 36 d after anthesis at the second inflorescence (about 0.5 m above the ground) of the tomato plant that cultivated in the standard greenhouse (20–25 ◦C, relative humidity (RH) 70–85 %) of Transfar Agriculture Co. Ltd in Hangzhou, China. To monitor the long-term effect of ABA on SlCOL4 transcription abundance in tomato fruit, ABA and NDGA treatments of tomato fruit were conducted according to the method used in our previous studies (Wu et al., 2018, 2023). Briefly, the collected tomato fruit were sterilized and treated with 1 mmol L− 1 ABA (98 %, HPLC grade, Aladdin; 0.7930 g of ABA was dissolved in 5.00 mL of anhydrous ethanol and sterile water was added to 3.00 L), 1 mmol L− 1 NDGA (an ABA biosynthesis inhibitor, 95 %, HPLC grade, Aladdin; 0.9071 g of NDGA was dissolved in 5.00 mL of anhydrous ethanol and sterile water was added to 3.00 L) or sterile water (control; 5.00 mL of anhydrous ethanol and 2.995 L of sterile water were mixed) under vacuum (60 kPa) for 180 s, then incubated at 20 ◦C and 90 % RH in darkness for 15 d. After removing fruit seeds and sepals, the pericarps from eight tomato fruit were sampled every other day from each treatment group respectively for subsequent analysis. For immediate effect of ABA on SlCOL4 expression, fruit pericarps were soaked in sterilized water or 1.0 mM ABA for 300 s and sampled at 0, 0.25, 0.5, 2, 6, 12, 24, and 48 h respectively. At each sampling point, eight pieces of tomato pericarp were collected as one biological replicate and three replicates were obtained. In addition, roots, stems, leaves, flowers, and fruit at various developmental stages including immature green 1 (IMG1), immature green 2 (IMG2), MG, breaking (Br), turning (Tu), and red ripe (Ri) were obtained for tissue-specific gene expression determination. All samples were immediately frozen in liquid nitrogen after sampling and kept at -80 ◦C until use. Wild-type Nicotiana tabacum plants were used for dual luciferase assay; transgenic Nicotiana benthamiana plants expressing RFP-fused to histone 2B (RFP-H2B, a nuclear marker) (Martin et al., 2009) were used for subcellular localization analysis. Both of the plants were kindly provided by Dr Xiaowei Wang (Zhejiang University, Hangzhou, China) and cultured at 25 ◦C with a 16/8 h photoperiod day/night cycle in a plant cultivation room at Zhejiang University in Hangzhou, China.
2.2. Gene expression analysis
Gene expression analysis was conducted as described in our previous study (Wu et al., 2018), using actin (AK328563.1) as an internal standard. Brifely, the total RNA was isolated and purified with a PureLink® RNA Mini Kit (Invitrogen, Carlsbad, California, USA) in accordance with the manufacturer’s protocol. The RNA quality was evaluated via agarose gel electrophoresis, and the RNA quantity was determined via a spectrophotometer (NanoDrop 2000, Thermo-Fisher Scientific Inc., Wilmington, DE, USA). A total of 1 μg of RNA was reverse-transcribed to cDNA with a PrimeScript® RT Reagent Kit (DRR047A, TaKaRa, Tokyo, Japan) in accordance with the manufacturer’s protocol. Real-time quantitative fluorescence PCR (RT-qPCR) experiments were performed on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA) with SYBR Premix Ex Taq (RR820A, TaKaRa, Tokyo, Japan) with the procedure as follows: 95 ◦C, 30 s and 1 cycle for predenaturation, then keep 95 ◦C for 5 s, 60 ◦C for 30 s and 40 cycles during PCR reaction. The relative expression abundances of genes were normalized in accordance with the 2− ΔΔCT method (Livak and Schmittgen, 2001). Gene sequences were downloaded from the Sol Genomics Network (SGN, https://solgenomics.net/), RT-qPCR primers were designed with Primer 5.0, and all sequences were listed in Supplementary Table S1.
2.3. Promoter and protein structure analysis
For promoter analysis of SlCOL4, 2000 bp base sequences upstream of the ORF of SlCOL4 were downloaded from the Sol Genomics Network (https://solgenomics.net/) and the cis-acting elements within them were analyzed with PlantCARE (Lescot et al., 2002). For protein structure analysis, the amino acid sequences of SlCOL4 were obtained from the Sol Genomics Network (https://solgenomics.net/), and those of AtCOL4 (accession number: NP_197875) in Arabidopsis and OsCOL3 (accession number: O82117.1) in rice were obtain from NCBI (https://www.ncbi.nlm.nih.gov/), then the amino acid sequence alignment was performed with DNAMAN software (Lynnon Biosoft, version 6.0.3.93), and the structure of B-box and CCT was marked according to the structure annotation on NCBI.
2.4. Subcellular localization analysis
Subcellular localization of SlCOL4 was performed as described by Hu et al. (2019). Briefly, the coding region of SlCOL4 was amplified by PCR with the cDNA of MG tomato fruit according to the protocol of PrimeSTAR® Max DNA Polymerase Ver.2 (R047A, TaKaRa, Tokyo, Japan). After digested with XbaI and BamHI (1634/1605, TaKaRa, Tokyo, Japan) and purified with EasyPure® PCR purification kit (EP101–01, TransGen, Beijing, China), it was fused in-frame to the 3′-end of the GFP in the pCAMBIA1305 vector under the control of the full-length CaMV 35S promoter using ClonExpress Ultra One Step Cloning Kit (C116–01, Vazyme, China) according to the manufacturer’s protocol. The pCAMBIA 1305-GFP empty vector was used as the negative control. The recombinant vector with the correct sequence was named pCAMBIA 1305-GFP-SlCOL4 and electroporated into the GV3101 Agrobacterium tumefaciens strain. As described by Espley et al. (2007), the GV3101 strain obtained was cultured and resuspended with infiltration buffer (10 mM MgCl2, 10 mM MES, 150 mM acetosyringone, pH 5.6) to an OD600 of 1.0, then injected into the leaves of transgenic N. benthamiana expressing RFP-H2B with a needleless injector. After 48 h, the tobacco leaf near the injection hole was picked and cut with scissors, and then about 0.5 cm × 0.5 cm leaf tissue was taken and the leaf back side was put up on the slide for GFP fluorescence proteins examination. The microscopy images were collected with a confocal microscope (TCS SP8, Leica, Germany), the excitation wavelength for GFP fluorescence was 488 nm and the fluorescence ranges were detected at 490–520 nm.
2.5. Virus-induced gene silencing (VIGS)
VIGS of SlCOL4 in tomato fruit was conducted according to Fu et al. (2005) and Orzaez et al. (2006) with modifications. Tobacco rattle virus (TRV)-based vectors pTRV1 and pTRV2 were used, and VIGS was carried out on detached MG tomato fruit. According to the analysis result of the SGN VIGS tool (https://vigs.solgenomics.net/), the 300 bp near the 3′-end of the CDS region of SlCOL4, which has good specificity, was selected as the target silenced profile, and it was PCR-amplified (primers shown in Supplementary Table S1) with the cDNA of the MG tomato fruit, then reversely fused into the linearised vector pTRV2 that digested by EcoRI and XhoI (1611/1635, TaKaRa, Tokyo, Japan) with T4 DNA ligase (2011A, TaKaRa, Tokyo, Japan) to form pTRV2-SlCOL4, TRV2 vector that carried the target silenced profiles of phytoene desaturase (SlPDS, Solyc03g123760) in tomato fruit (pTRV2-SlPDS) was obtained in the same manner and used as a positive control. The empty pTRV2 vector was used as a negative control. For VIGS infection operation, pTRV1/pTRV2 and the corresponding derivatives were electroporated into the GV3101 Agrobacterium tumefaciens strain, and the obtained Agrobacterium strains were grown at 28 ◦C in LB medium antibiotics Ampicillin and Kanamycin. After 24 h, Agrobacterium cells were collected and resuspended in the agrobacterium infiltration buffer to a final OD600 of 1.0, and placed for 4–6 h at room temperature before infection. According to the results of our pre-experiments and the description of Orzaez et al. (2006) that half-cut tomato fruit can be effectively infiltrated by agrobacterium cultures, the tomato fruit were cut in half vertically and the seeds were removed and only the pericarp was used. Specifically, one half pericarp was used for control infection and the other half one was used for pTRV2-SlPDS or pTRV2-SlCOL4 infection. The pericarp was fully immersed in the mixture of pTRV1 and pTRV2 or its derivatives (v:v = 1:1) for 5 min, afterwards the infected tomato pericarp was carefully removed from the infection solution using sterile forceps, wiped dry with sterile paper, placed in sterile petri dishes and sealed with the breathable sealing film to avoid interference from external microorganisms, and finally incubated at 20 ◦C and 90 % RH. For each comparison group, 40 individual tomato fruits were used and the whole VIGS infection operation process was carried out on the ultra-clean workbench. After 5 d, the tomato pericarp was imaged and the transcription abundances of SlCOL4 and SlPDS were measured both in control and silenced tomato pericarp using RT-qPCR.
2.6. Yeast one-hybrid (Y1H) assay
The pAbAi and pGADT7 vectors were used for the Y1H assay with the Yeastmaker™ Yeast Transformation System 2 kit (Clontech, Palo Alto, USA), which was performed following the instructions. The full length of the SlAREB1 ORF sequence was obtained through PCR with specific primers (Table S1) from the MG tomato fruit. Then, the fragment was inserted into the pGADT7 vector to obtain pGADT7-SlAREB1, and the promoter sequence (approximately 2000 bp upstream of the ORF) of SlCOL4 was amplified by PCR from the genomic DNA of the MG tomato fruit, and cloned into the pAbAi vector with the KpnI and XhoI restriction enzymes (1618/1635, TaKaRa, Tokyo, Japan) to obtain pAbAiSlCOL4 pro (primers were listed in Supplementary Table S1). The pAbAiSlCOL4 pro1 and pAbAi-SlCOL4 pro2 vectors were generated with the same procedure using two segments of SlCOL4 gene promoter sequence, which was 150 bp in length and contained an AREB cis-elements in the middle position. The pAbAi-SlCOL4 pro vector was digested with BbsI and the linearized plasmids were transformed into the Y1H Gold yeast strain using the protocol for the Yeastmaker™ Yeast transformation System 2 and grown on synthetically defined medium lacking uracil (SD/-Ura) at 30 ◦C. After 3 d, colonies were picked and analyzed by colony PCR. Each positive colony was resuspended in 0.9 % NaCl and the OD600 was adjusted to 0.002, and then 100 μL of the resuspension was plated on SD/-Ura agar plate containing Aureobasidin A (AbA, 500 μg mL− 1) (0, 50, 100, 200, 300, 500, 1000 μg L− 1). After the colonies growing for 3 d at 30 ◦C, the growing situation was screened to determine the minimum inhibitory concentration of AbA in relation to yeast growth. Next, pGADT7 (negative control) and pGADT7-SlAREB1 were cotransformed into yeast strains containing pAbAi-SlCOL4 pro and plated in synthetically defined medium lacking leucine (SD/-Leu) and SD/-Leu agar plates with the minimum inhibitory concentration of AbA (SD/-Leu + AbA), and their growth conditions were monitored after 3 d to determine their interactions. The empty AbAi p53 promoter vector cotransformed with pGADT7-Rec-p53 vector was used as the positive control.
2.7. Dual-luciferase assay
For dual-luciferase assays, the plant expression luciferase reporter pGreen II 62-SK (62-SK) and pGreen II 0800-LUC (0800-LUC) were used as the reporter vectors and the experiment was performed on the leaves of common N. benthamiana plants. The full-length ORF of SlAREB1 was cloned and inserted into a pGreen II 62-SK vector (62-SK-SlAREB1), the promoter sequence of SlCOL4 was cloned and inserted into a pGreen II 0800-LUC vector (0800-LUC-SlCOL4 pro), using the primers described in Supplementary Table S1, and the method of gene sequence amplification and fusion vector construction was the same as 2.4. After transformation into Agrobacterium tumefaciens GV3101, they were cultured and resuspended with infiltration buffer to an OD600 of 1.0–1.5. Then the Agrobacterium culture mixtures of TFs and promoters (10:1) were coinjected into N. benthamiana leaves with needleless syringes according to protocols described in Xu et al. (2018), The mixture of 62-SK+0800-LUC and 62-SK+0800-LUC-SlCOL4 pro was used as the negative and the positive controls respectively, and that of 62-SK-SlAREB1+0800-LUCSlCOL4 pro was the effector and reporter strain. Tobacco leaves were sampled after culturing for 2 d under a 16/8 h day-night rhythm and 25 ◦C, and firefly luciferase (LUC) and renilla luciferase (REN) were measured using Dual-Luciferase Reporter Assay lit (E1960, Promega, USA) with the promega luminescence detector (Promega Glomax 2020, Promega, USA). The binding activity of SlAREB1 to the promoter of SlCOL4 was calculated by finding the LUC to REN ratio. Three independent experiments were performed per transcription factor-promoter interaction analysis.
2.8. Chromatin immunoprecipitation sequencing (ChIP-Seq)
Approximately 4 g of MG tomato pericarp was used for the chromatin immunoprecipitation assay following the protocol of the ChIP kit (Zymo Research Corp., Irvine, CA). For the pretreatment of the ChIP sample, the pericarp was ground in liquid nitrogen and cross-linked with 50 mL of 1 % formaldehyde for 30 min, terminated with 2.5 mL of glycine, and washed three times to remove excess formaldehyde. The obtained samples were then ground again to fine powder with liquid nitrogen, extracted with buffer I to buffer III in turn, and then centrifuged. The sediment obtained was mixed with 0.4 mL of nucleic acid lysis buffer containing protease inhibitors, lysed on ice for 30 min, and sonicated into fragments ranging from 200 to 700 bp with a Bioruptor Plus™ (Diagenode Inc., Denville, NJ) at high power for 40 cycles (30 s on, 30 s off). The broken samples were purified by decross-linking and verified using agarose gel electrophoresis. For the enrichment of DNA fragments bound to SlCOL4 protein on the genome of the MG tomato fruit, 10 μg of SlCOL4 antibody protein, which was obtained by immunising mice multiple times with the SlCOL4 protein and was provided by Abmart Co., Ltd (Shanghai, China) was added to 12 μg of sonicated DNA fragment, and the resulting antibodytarget protein-DNA complex was immunoprecipitated using protein G beads (L00277, Sigma). The complex was eluted with elution buffer and subjected to overnight incubation at 65 ◦C with 20 μL of 5 M NaCl to reverse the cross-linking. Simultaneously, an input sample was mixed with 500 μL of elution buffer and 20 μL of 5 M NaCl to be used as a control. Then, the decross-linking product was mixed with 10 μL of 0.5 M EDTA, 5 μL of RNase, 20 μL of Tris–HCl (pH 7.0), and 2 μL of proteinase K, and then incubated at 45 ◦C for 1 h. The obtained ChIP DNA was purified with ChIP DNA Clean and Concentrator™ (Zymo Research Corp, Irvine, CA) for subsequent sequencing. For the sequencing of the enriched SlCOL4 target DNA profiles, the purified DNA fragments were sequenced with Illumina Genome Analyser IIx and HiSeq 2500 (Illumina, San Diego, CA). The quality of obtained reads was assessed with the fastqc software (version 0.11.5), and filtered using Trimmomatic (version 0.36). The obtained clean reads were mapped to the tomato genome (https://solgenomics, version ITAG 4.0) using the BWA software (version 0.7.15-r1140) allowing at most two mismatches between the resulting reads, and the peak information was analyzed with the MACS software (version 2.1.1.20160309). De novo motif prediction was conducted using the MEME software (http:// meme-suite.org, version 4.11.1) with 100 bp sequences upstream and downstream of top 1000 peaks sorted by -10*log10 (p-value) value from the highest to the lowest. Bioinformatic analysis of sequencing results was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genomen.jp/kegg) and Gene Ontology (GO, http://www. geneontology.org/). The ChIP-Seq experiment was performed by Igenebook Biotechnology Co., Ltd (Wuhan, China).
2.9. Data analysis
Data analysis was performed using SPSS software (version 20.0, IBM Crop, Armonk, USA), and the results were presented as mean values ± standard deviation (SD) values. Significance between different groups was assessed via Student’s t-test and one-way ANOVA with p < 0.05. Three biological replicates were performed for each RT-qPCR analysis.
3. Results
3.1. SlCOL4 expression was the highest in MG tomato fruit and inhibited by ABA treatment
The expression of SlCOL4 in tomato fruit during 15 d after ABA or NDGA treatment was shown in Fig. 1A, which decreased in the first 5 d after ABA treatment and increased after NDGA treatment. In addition, the expression of SlCOL4 responded rapidly to exogenous ABA and showed a significant downregulation within 6 h after ABA treatment and continued to remain at a low level (Fig. 1B). Meanwhile, the tissuespecific transcription abuncances analysis showed different expression levels of SlCOL4 in tomato fruit at different developmental stages, flowers, leaves, stems and roots. The highest expression of SlCOL4 was found in the growing fruit, followed by leaves, flowers, and stems, and the lowest in the root. Specially, during fruit developing period, SlCOL4 expression increased gradually from IMG to MG stage and declined rapidly at the Br stage (Fig. 1C), then a relatively lower level of SlCOL4 expression was observed afterwards (Fig. 1C). The above results indicate that SlCOL4 may play a role in tomato fruit ripening and be regulated by ABA at the onset of fruit ripening.
3.2. SlCOL4 silencing might affect tomato fruit ripening by inducing key genes involved in ethylene synthesis
As shown in Fig. 2A and Supplementary Fig. S1, a striking inhibition of color transition in VIGS fruit infected with the pTRV-SlPDS construct was observed, RT-qPCR results showed that the SlPDS transcripts in the sections of TRV-SlPDS infected pericarp were significantly lower than in the control sections infected by empty TRV vectors alone (Fig. 2B), indicating the VIGS method used in the present study was efficient. In contrast, an obvious orange color (Fig. 2A and Supplementary Fig. S2) and lower SlCOL4 transcripts were observed in TRV-SlCOL4 infected fruit (Fig. 2C), which contrasted with the uniform green phenotype observed in the control section. As the pericarp color is one of the important evaluation indexes of fruit ripeness, the color change in TRVSlCOL4 infected fruit suggesting that SlCOL4 may be involved in regulating tomato fruit ripening. In addition, RT-qPCR results showed that transcription abundances of key genes associated with ethylene biosynthesis, ACO1, ACS2, and ACS4 were higher in SlCOL4 silenced fruit tissue than those in the control, indicating SlCOL4 may affect ethylene biosynthesis.
3.3. TF characterization and subcellular localization analysis of SLCOL4
Bioinformatics analysis showed that SlCOL4 encoded a protein with 358 amino acids and was 39.54 kDa in size, and the ORF of SlCOL4 was 1077 bp. Amino acid sequence alignment results between SlCOL4 and previously reported transcription factors AtCOL4 in Arabidopsis and OsCOL3 in rice showed that two B-box and one CCT structures, which were characteristic conserved region of COL TFs, were located between 21~55, 51~98 and 295~337 amino acids of SlCOL4, respectively (Fig. 3A). Subcellular localization analysis showed that SlCOL4 was localized in the nucleus (Fig. 3B). These results indicate that SlCOL4 is a typical TF that belongs to the COL family.
3.4. Interaction between SLAREB1 and SLCOL4
Analysis of the 2000 bp upstream of the ORF in SlCOL4 showed that two ABRE elements (TACGTG) at -1894–1888 bp (pro1) and -1384–1378 bp (pro2) were observed in the SlCOL4 promoter (Fig. 4A), indicating that it may be regulated by ABA. Y1H assay and dual-luciferase assay were then performed to examine the combination ability and the regulatory effect between SlCOL4 and the known ABA response factor SlAREB1, respectively. As shown in Fig. 4B and C, the results of Y1H assay showed that SlAREB1 could bind to the SlCOL4 promoter by interacting with pro2, and the results of dual-luciferase assay indicated that SlAREB1 had transcriptional inhibition effect on SlCOL4 promoter, and the LUC/REN value was about 65 % of that in the control group.
3.5. ChIP-Seq identified downstream target genes of SLCOL4 in vivo
3.5.1. Overview of CHIP-seq results of SLCOL4 in vivo
A total of 3877 intervals in MG fruit were obtained from the ChIP enrichment, and they were mapped with multiple chromosomes (Fig. 5A). The distribution characteristics of the enrichment intervals in the whole genome and genes were shown in Fig. 5B and C. The identified SlCOL4 target sequences within the genome were 75.8 % for intergenic sequence, 17.92 % for promoter region, 3.73 % for intron sequence, 2.12 % for coding region sequence, 0.25 % for 3′-UTR terminal, and 0.18 % for 5′-UTR terminal (Fig. 5B). Among the target genes, 74.06 % of them were distributed in the promoter region, 15.42 % in the intron region, 8.75 % in the coding region, 1.04 % in the 3′-UTR terminal, and 0.73 % in the 5′-UTR terminal (Fig. 5C). Multiple expression motifs for elicitation (MEME) analysis of ChIP-Seq peak regions revealed the potential SlCOL4 binding motifs was not unique, the first ten prediction motifs were listed in Supplementary Fig. S3. Among the identified target sequences, the promoter information of 640 genes was successfully annotated (Supplementary Table S2), GO annotation showed that they were categorized into 36 functional groups, and including 9 groups in biological process, 11 groups in molecular function, and 16 groups in cell component, and biological processes was the most enriched part (Fig. 6A). KEGG analysis showed that the identified target sequences were distributed in multiple pathways including energy metabolism, lipid metabolism, biosynthesis of secondary metabolism etc., of which the carbohydrate metabolism was the most enriched pathway (Fig. 6B).
3.5.2. Potential target genes involved in ethylene biosynthesis and signaling pathway
According to KEGG analysis results, 20 genes involved in ethylene signal transduction and biosynthesis pathway were identified downstream of SlCOL4 (Table 1), including two ACSs (Solyc02g063540.1.1 and Solyc03g043890.2.1), five ethylene receptors (Solyc06g036450.1.1, Solyc09g089610.2.1, Solyc09g011680.1.1, Solyc09g082910.1.1 and Solyc09g009020.2.1), four AP2 TFs (Solyc03g044300.2.1, Solyc06g066390.1.1, Solyc02g064960.2.1 and Solyc02g093150.2.1), four ERFs (Solyc05g051200.1.1, Solyc03g006320.1.1, Solyc12g038450.1.1 and Solyc05g051180.1.1), two MADS TFs (Solyc00g179240.1.1 and Solyc03g006830.2.1), and three genes involved in the MAPK cascade (Solyc05g041420.2.1, Solyc03g117640.1.1 and Solyc03g123800.1.1), suggesting that SlCOL4 has a potential regulation effect on ethylene signal in MG tomato fruit.
4. Discussion
Plant growth and development are influenced by TFs, and COLs have been reported in a variety of plants (Herrmann et al., 2010; Huang et al., 2011; Zhang et al., 2011; Li et al., 2020). In the COLs family, the C-terminal CCT domain is highly conserved, and the N-terminal B-box domain differs in different species. Accordingly, COLs can be classified into three types depending on their B-box domains: Class I contains two B-box elements; Class II contains a zinc finger area and a CO-like B-box; and Type III contains only one B-box element (Griffiths et al., 2003). In the present study, the results of amino acid sequence and subcellular localization analysis showed that SlCOL4 is a typical COLs TF, indicating it may be functionally similar to COLs in other plant species. COLs have been reported as an important regulatory factor in various life processes in plants, such as flowering time, root and seed development, and other biological activities of different species of plants (Hayama et al., 2003; Datta et al., 2006; Pan et al., 2021; Zhang et al., 2011; Liu et al., 2021). AtCOL3 and AtCOL9 in Arabidopsis were reported to be negative regulators of the flowering process (Datta et al., 2006; Cheng and Wang, 2005). OsCOL4 (Lee et al., 2010), OsCOL3 (Kim et al., 2008), and OsCOL13 (Sheng et al., 2016) in rice were reported to have negative regulatory effects on flowering time. TaCOL-B5 in wheat regulates the formation of tillers and ears (Zhang et al., 2022). However, the role of COLs in tomato fruit development and ripening has not yet been systematically analyzed (Yang et al., 2020). In the present study, SlCOL4 was found expressed in different tissues of tomato plants, which was similar to the expression of MaCOL gene in banana (Sheng et al., 2016). The increased transcription abundance during fruit development and a decline during maturation period, as well as the acceleration of color transition of pTRV-SlCOL4-infected fruit pericarp suggested that SlCOL4 had a negative effect on tomato fruit ripening, suggesting that COLs may be involved in tomato fruit ripening regulation. ABA is considered as an essential phytohormone that initiates the fruit ripening process (Zhang et al., 2009; Sun et al., 2012; Soto et al., 2013). The basic leucine zipper transcription factors AREB/ABFs are mainly responsible for regulating downstream genes via combining with the ABRE element in the promoter (Fujita et al., 2013; Orellana et al., 2010). Two AREB TFs, SlAREB1 and SlAREB2 were reported in tomato fruit, and SlAREB1 was shown to be mainly involved in the regulation of stress responses and nutrition accumulation during the ripening process (Yanez et al., 2009; Orellana et al., 2010; Bastias et al., 2011, 2014). In the present study, two ABRE elements were observed in the promoter of SlCOL4 gene. Y1H and dual-luciferase assays showed that SlAREB1 interacted with the SlCOL4 promoter and inhibited its transcription, and the gene expression results showed that SlCOL4 responded rapidly to exogenous ABA and showed a significant downregulation transcription level within 6 h after exogenous ABA treatment, suggesting the expression of SlCOL4 in tomato fruit can be directly regulated by ABA at the MG stage. Tomato is a typical climacteric fruit, and ethylene plays a key role in its ripening process (Xie et al., 2006). In the present study, the expression levels of key genes ACO1, ACS2 and ACS4, which are known involved in the ethylene synthesis pathway, were up-regulated in pTRV-SlCOL4 infected fruit. Moreover, the results of ChIP-Seq showed that a total of 20 genes involved in ethylene synthesis and signal transduction were preliminarily identified as target downstream genes of SlCOL4. Among them, ETR1 was the earliest identified ethylene receptor (Yang and Hoffman, 1984; Schaller and Bleecker, 1995), ETR3 encoded the Nr (never ripe) gene in tomato (Lashbrook et al., 1998; Hackett et al., 2000), and ETR6 had a negative regulatory effect on ethylene signal transduction and regulated fruit ripening time (Kevany et al., 2007). ACS7 and ACS8 encode the key enzyme ACS in the ethylene synthesis pathway (Yang and Hoffman, 1984); AP2 and ERF are members of the AP2 DNA-binding protein family, which are plant-specific transcription factors that play an important role in plant growth and ethylene response (Dietz et al., 2010). AP2a plays a negative role in ethylene synthesis and tomato fruit ripening (Karlova et al., 2011; Chung et al., 2010), ERF4 and ERF2 are reported to be related to ripening of tomato fruit (Tournier et al., 2003). Therefore, SlCOL4 may participate in ethylene biosynthesis by regulating the expression of ACSs and contribute to ethylene signal transduction pathway by interacting with ethylene receptor ETRs and response factors AP2/ERFs. Overall, SlCOL4 may regulate tomato fruit ripening by interacting with ABA and ethylene signaling at the MG stage (Fig. 7). Specifically, SlCOL4 was directly regulated by ABA response factor SlAREB1, which could respond to exogenous ABA within 6 h. Silencing of SlCOL4 in tomato pericarp could induce the key genes (ACS2, ACS4 and ACO1) involved in ethylene synthesis and accelerate the reddening of tomato pericarp, showing an accelerated ripening phenotype within 5 d. However, the changes of SlCOL4 expression caused by VIGS are transient, not stable and inherited, and the contribution from ChIP-Seq experiments is limited for determining the regulatory model of SlCOL4 on ethylene biosynthesis and signal transduction as it can only enrich the sequences of genes that may interact with SlCOL4, so the regulatory relationship between SlCOL4 and the target sequences can not be confirmed. Further investigations using transgenic or gene-knockout methods are needed to further clarify the function of SlCOL4 and target genes as well as the regulatory effect of SlCOL4 on the expressions of ethylene biosynthetic and signal transduction related genes.
5. Conclusions
In the present study, a COL transcription factor SlCOL4 was identified in tomato fruit. In addition to identifying the characteristics of this transcription factor, we found that it was inhibited by SlAREB1, a critical TF in the ABA signaling pathway, via binding with the ABRE ciselement within its promoter. Using VIGS assay, we found that SlCOL4 may negatively regulated the expression of ethylene biosynthetic genes (SlACS2, SlACS4 and SlACO1) and accelerated the pericarp color transition of pTRV-SlCOL4 infected fruit tissue. In addition, ChIP-Seq analysis showed that SlCOL4 is potential to regulate the expressions of ethylene biosynthesis and signal transduction related genes (such as ETR3 and ERF6, etc.), which may contribute to the enhanced ethylene synthesis. Based on the results, a potential model of the involvement of SlAREB1-SlCOL4 transcriptional regulation in ABA-modulated ethylene biosynthesis during tomato fuit ripening was proposed, which may provide a novel linkage between these two hormonal pathways and contribute to understanding the crosstalk between ABA and ethylene in regulating tomato fruit ripening, and provide references for further research on the role of COLs transcription factors in tomato fruit ripening as well. CRediT authorship contribution statement Qiong Wu: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Xiaoya Tao: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Chunxiao Cui: Writing – original draft, Methodology, Investigation, Data curation. Yanan He: Writing – original draft, Methodology, Investigation, Data curation. Dongdong Zhang: Writing – review & editing, Writing – original draft, Supervision, Software, Methodology. Yurong Zhang: Writing – review & editing, Writing – original draft, Supervision, Software, Methodology. Li Li: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Formal analysis, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The author thanks the grant of the Youth Program of National Natural Science Foundation of China (32402176 and 32001753) and the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.scienta.2024.113865. Data availability The authors are unable or have chosen not to specify which data has been used.
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