TY - JOUR
AU - Hu,, Zongli
AB - Abstract Ethylene signaling pathways regulate several physiological alterations that occur during tomato fruit ripening, such as changes in colour and flavour. The mechanisms underlying the transcriptional regulation of genes in these pathways remain unclear, although the role of the MADS-box transcription factor RIN has been widely reported. Here, we describe a bHLH transcription factor, SlbHLH95, whose transcripts accumulated abundantly in breaker+4 and breaker+7 fruits compared with rin (ripening inhibitor) and Nr (never ripe) mutants. Moreover, the promoter activity of SlbHLH95 was regulated by RIN in vivo. Suppression of SlbHLH95 resulted in reduced sensitivity to ethylene, decreased accumulation of total carotenoids, and lowered glutathione content, and inhibited the expression of fruit ripening- and glutathione metabolism-related genes. Conversely, up-regulation of SlbHLH95 in wild-type tomato resulted in higher sensitivity to ethylene, increased accumulation of total carotenoids, slightly premature ripening, and elevated accumulation of glutathione, soluble sugar, and starch. Notably, overexpression of SlbHLH95 in rin led to the up-regulated expression of fruit ripening-related genes (FUL1, FUL2, SAUR69, ERF4, and CNR) and multiple glutathione metabolism-related genes (GSH1, GSH2, GSTF1, and GSTF5). These results clarified that SlbHLH95 participates in the regulation of fruit ripening and affects ethylene sensitivity and multiple metabolisms targeted by RIN in tomato. bHLH transcription factor, fruit ripening, overexpression, rin, RNAi, SlbHLH95, tomato Introduction Tomato fruit ripening consists of complex physiological and biochemical processes, which include changes in colour, flavour, aroma, texture, and nutritional content that make tomato fruit an important and nutritious fruit in the human diet (Alba et al., 2005; Osorio et al., 2011). Tomato fruit is a climacteric fruit that is typically characterized by autocatalytic ethylene production and respiration burst (Matas et al., 2009). Therefore, the regulatory mechanism of tomato fruit ripening mostly involves the ethylene signalling pathway. Recently, ethylene synthesis, ethylene receptors, and other ripening-related genes have been functionally identified in tomato (Alexander and Grierson, 2002; Mata et al., 2018; Li et al., 2019). For example, the SlACO1, SlACO3, and SlACS2 genes control ethylene synthesis, and reducing their expression can produce unripened fruits. Carotenoid biosynthesis has been well characterized, and PSY1, a tomato phytoene synthase gene, controls metabolic flow into the carotenoid biosynthesis pathway (Enfissi et al., 2017). FUL1 and FUL2 are key lycopene accumulation-related genes that encode transcription factors (Bemer et al., 2012). It was also reported that SlERF.B3/ERF4, a member of the tomato ERF family gene, plays a key role in regulating ethylene responses and fruit ripening (Liu et al., 2014; Liu et al., 2018). Recently, an auxin-responsive gene, SlSAUR69, was reported to affect fruit ripening and ethylene sensitivity (Shin et al., 2019). Moreover, the rin (ripening inhibitor) mutant was thoroughly characterized: it shows abnormal fruit ripening, including a lack of accumulation of lycopene and slower softening, resulting in non-commercial value. Studies have shown that the RIN gene regulates ethylene-dependent/independent ripening processes (Vrebalov et al., 2002). Therefore, the normal expression of the RIN gene also seems to be closely related to the colour, flavour, aroma, and nutritious value of tomato fruit. Ripening can be considered a stressful process, and exhibits a progressive increase in oxidation in tomato and peach (Camejo et al., 2010). Gamma-glutamylcysteinyl glycine (glutathione; GSH) is a non-enzymatic antioxidant that plays an important role in plant growth and various developmental processes ranging from developmental aspects to various stress responses (Camejo et al., 2010). Recently, a well-recognized body of research has established the involvement of GSH with ethylene in combating environmental stress (Pieterse et al., 2009; Caarls et al., 2015). In further experiments involving the ectopic expression of the tomato glutathione biosynthesis gene LeGSH1 in Nicotiana tabacum, ACO and ERF4 were up-regulated (Ghanta et al., 2014). Interestingly, GSH also induced the transcription of ACS2 and ACS6 in a WRKY33-mediated manner. On the other hand, GSH also improved the stability of ACO1 mRNA (Datta et al., 2015). However, the mechanism of GSH–ethylene interplay has not been elucidated in tomato. The basic helix-loop-helix (bHLH) proteins are transcription factors that perform a variety of functions; they consist of 60 amino acids with highly conserved domains. In plants, bHLHs play an important role in numerous developmental and biological processes. For example, the bHLH member MdbHLH3 regulates anthocyanin accumulation at low temperatures in apple (Malus domestica) (Xie et al., 2012), while expression of another bHLH gene, TabHLH060, increases resistance against pathogens in wheat (Wang et al., 2015). Similarly, the bHLH genes AtPIF3/4, HFR1, and FIT control light signalling, cause heavy metal detoxification, and increase resistance to iron acquisition in Arabidopsis (Fairchild et al., 2000; Yuan et al., 2008; Dong et al., 2017). The bHLH transcription factor AtSPT (SPATULA) controls the final leaf size in Arabidopsis through reduced cell proliferation in the leaf primordial meristematic region (Ichihashi et al., 2010). Suppression of PhFBH4 in Petunia hybrida enhances flower longevity by controlling the expression of ethylene biosynthesis genes (ACO1 or ACS1) (Yin et al., 2015). The bHLH transcription factor family ScFBHs control ACC synthase expression to regulate ethylene biosynthesis in internode maturation of sugarcane (Alessio et al., 2018). Recently, approximately 158 bHLHs have been identified in tomato (Sun et al., 2015), but most of their functions have not been investigated in detail. Previous studies have shown that SlbHLH95 (Solyc10g079050) is a direct RIN target confirmed by chromatin immunoprecipitation–quantitative PCR (qChIP–PCR), which displayed a high fold change (FC) value in wild-type (WT) tomato fruits (FCWT; 23.8) during the ripening stage relevant to the pre-ripening stage, while a lower fold change value in rin mutant fruits (FCrin; 1.3) at a similar stage was obtained. Moreover, the highest expression change score (ECS; 17.7) ratio of FCWT to FCrin showed the gene expression dependency on RIN (Fujisawa et al., 2013). Therefore, we speculate that SlbHLH95 may be involved in the regulation of tomato fruit ripening. In this study, we isolated the SlbHLH95 gene from tomato fruits and found that its transcripts notably accumulate at the breaker (B)+4 and B+7 stages, but are significantly reduced in rin and Nr (Never ripe) mutants, especially in rin mutants. Moreover, down-regulation of SlbHLH95 resulted in reduced sensitivity to ethylene, decreased the accumulation of total carotenoids, and suppressed the expression of fruit ripening-related genes. Conversely, up-regulation of SlbHLH95 resulted in higher sensitivity to ethylene, increased accumulation of total carotenoids, and caused slightly premature ripening. Our results suggest that SlbHLH95 plays an essential role in the regulation of tomato fruit ripening. Materials and methods Plant materials and growth conditions The WT tomato (Solanum lycopersicum Mill. cv. Ailsa Craig), Nr and rin mutants, transgenic lines (SlbHLH95-RNAi in WT, SlbHLH95-OE in WT, and SlbHLH95-OE-rin in rin) were grown in the same greenhouse under the following conditions: 16 h day (27 °C) and 8 h night (19 °C) at 80% relative humidity. The flowers, leaves, roots, sepals, and fruits at different periods were collected and used for expression analysis of SlbHLH95. Flowers at the anthesis stage were marked. Fruit developmental stages were recorded as days post-anthesis (DPA). Thereafter, fruits at 20 DPA were defined as IMG (immature green); fruits at 35 DPA were defined as MG (mature green) and were regarded as having full fruit growth but with no clear ripe fruit colour evident. B (breaker) fruit was identified as fruit with the first appearance of orange colour. The ripening periods were distinguished as 4 days after breaker (B+4) and 7 days after breaker (B+7). Fruits of the rin and Nr mutants were collected at IMG, MG, B, B+4, and B+7 stages corresponding to WT tomato. All tissues were collected at the same time on each day, frozen instantly in liquid nitrogen, and transferred to a −80 °C freezer for storage until necessary. Construction of RNAi and overexpression vectors and plant transformation For SlbHLH95-RNAi construction, a 426 bp specific fragment was amplified with SlbHLH95-RNAi-F/R primers (see Supplementary Table S1 at JXB online for details of primers). The amplified products were digested with the restriction enzymes ClaI/XbaI and KpnI/XhoI and inserted into the pHANNIBAL plasmid. The hairpin expression unit was digested with the restriction enzymes SacI/SpeI and linked to the plant binary vector pBIN19 via SacI/XbaI restriction sites to form the SlbHLH95-RNAi vector. For SlbHLH95-OE construction, the full-length cDNA of SlbHLH95 was amplified with SlbHLH95-over-F/R primers (Supplementary Table S1). The amplified products were digested with the restriction enzymes XbaI/SacI and inserted into the pBI121 vector to form the SlbHLH95-OE vector. These recombinant plasmids were transformed into Agrobacterium tumefaciens (strain LBA4404) by the freeze–thaw method (An, 1987). The WT tomato was used to create SlbHLH95-RNAi and SlbHLH95-OE transgenic lines, and the rin mutant was used to produce SlbHLH95-OE-rin transgenic lines. The cotyledons of 5-day-old seedlings were used for plant transformation. The transgenic lines were subsequently selected on Murashige and Skoog (MS) medium with kanamycin (50 mg l–1). Total genomic DNA of the transgenic lines, rin, and WT were extracted, and the existence of T-DNA was further confirmed by PCR using NPTII-F/R primers (Supplementary Table S1). The T2 transgenic lines were used for all experiments. Ethylene and 1-methylcyclopropene treatments For ethylene treatment, three fruits at the MG stage from the WT, rin, and each transgenic line were harvested and placed into a glass container with 500 μl l–1 ethephon (ET) solution (Xu et al., 2012). For the treatment with 1-methylcyclopropene (1-MCP), an ethylene receptor inhibitor, three fruits at the MG stage from the WT, rin, and each transgenic line were harvested and placed into a glass bottle with 5 ppm of 1-MCP, which was prepared by dissolving 24 mg of 1-MCP-releasing powder (SmartFresh, 0.14% of active ingredient by weight; Rohm and Haas) in 25 ml of water (Fujisawa et al., 2013). For the control treatments, an equal volume of double-distilled water was used instead of ethylene or 1-MCP. All treatments were incubated for 24 h at room temperature and then transferred to open air for 4 d or 15 d. After treatment, a 5 mm wide strip of the pericarp was cut from around the equator of the fruits and quickly frozen in liquid nitrogen and then stored in a −80 °C freezer. The treatments were repeated at least three times for each sample. Pigment extraction and quantification The pigments of pericarp tissue from fruit at B, B+4, and B+7 stages were extracted using an updated protocol based on Forth and Pyke (2006). Pericarp tissue sections were cut from around the equator of fruits in a 5 mm wide strip. Samples were weighed and then crushed into powder with liquid nitrogen using a mortar and pestle. Total carotenoids were extracted from the samples by adding hexane:acetone (6:4, v/v). Then, the samples were centrifuged at 4000 g for 5 min, and the supernatant was transferred into a new tube after centrifugation. The deposits were then extracted with hexane:acetone (6:4, v/v) until they were colourless. The absorbance of the supernatant at 450 nm was measured immediately using a spectrophotometer, and the total carotenoid content was quantified by using the following equation: total carotenoids (mg ml–1)=(OD450)/0.25. Lycopene (a major carotenoid) was extracted from B+4 and B+7 fruit by the addition of hexane:ethanol:acetone (2:1:1, v/v/v), with 0.05% butylated hydroxytoluene previously added to the acetone. The absorbance of the supernatant at 503 nm was measured by a spectrophotometer. The lycopene content of each sample was calculated as follows: lycopene (mg g–1)=(A503×31.2) g–1 fresh weight. For pigment extraction, three fruits at each stage from each line were used. Each experiment was conducted three times for each sample. Total RNA extraction and gene expression analysis Frozen tomato tissues were ground into powder with liquid nitrogen using a mortar and pestle. Then, 0.2 g powdered tissue was placed in a 1.5 ml centrifuge tube with 1 ml TRIzol. Subsequent steps were performed as described in a kit (Invitrogen, Shanghai, China). The quality and quantity of total RNA were assessed by absorbance ratios (OD260/280 and OD260/230) and by 1% agarose gel electrophoresis. RNA quality was also confirmed using a 2100 Bioanalyzer (Agilent, USA); the RNA integrity number range of parts of RNA is shown in Supplementary Table S2. The first cDNA strand was synthesized using M-MLV reverse transcriptase (Promega, Beijing, China). Quantitative reverse transcription–PCR (qRT−PCR) was performed by using a CFX96™ Real-Time System (Bio-Rad, USA). The composition of the reaction mixture and conditions were as described in Zhang et al. (2018). The tomato gene SlCAC (clathrin adaptor complex medium subunit) was used as an internal control for expression analysis (Nicot et al., 2005; Expósito-Rodríguez et al., 2008). All expression analysis experiments were performed three times and the primers used in this study are listed in Supplementary Table S1. Metabolite analysis The GSH contents were assessed as described by Anderson et al. (1992). In brief, 0.5 g of fresh pericarp tissues (B+4 stage) were ground into powder with liquid nitrogen, transferred into a tube containing 1 ml 6% (w/v) trichloroacetic acid, and homogenized. The mixture was centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was transferred to a new tube as the crude extract. A 100 μl volume of supernatant was added to 600 μl of reaction buffer (0.1 M phosphate-buffered saline, pH 7.0, 1 mM EDTA), 40 μl of 0.15% 5,5′-dithiobis-(2-nitrobenzoic acid), and 400 μl of water. A series of GSH concentrations (0.5, 1, 2, 5, 10, and 20 μM) was used to generate a standard curve. The GSH content was determined at 412 nm after 5 min. Three independent experiments were performed for each sample. The soluble starch and sugar contents were assessed by using the anthrone–sulfuric acid method. Briefly, 0.2 g fresh pericarp tissues (B+4 stage) were ground into powder with liquid nitrogen, incubated in a water bath at 80 °C for 30 min with 5 ml 80% ethanol, and then centrifuged at 3500 g for 10 min at room temperature. The supernatant was used to measure soluble sugar content, and the residue was used to measure starch content. For soluble sugar, a mixture of 2 ml supernatant and 5 ml anthrone–sulfuric acid reagent (0.2% anthrone, 1% thiourea, and 100 ml H2SO4) was incubated in a water bath at 100 °C for 10 min. The absorbance was measured at 620 nm by a spectrophotometer. For starch, 2 ml distilled water was added to the residue and then incubated in a water bath at 100 °C for 15 min. After cooling at room temperature, the solution was shaken with 2 ml HClO4 (9.2 M) for 15 min at 200 rpm, mixed with 4 ml distilled water and centrifuged at 4000 g for 10 min, and the supernatant was transferred to a new centrifuge tube. The residue was shaken with 2 ml HClO4 (4.6 M) for 15 min at 200 rpm, mixed with 4 ml distilled water, and centrifuged again at 4000 g for 10 min. The supernatants were combined and used to measure starch content by spectrophotometry at 540 nm. A series of glucose concentrations (20, 40, 60, 80 and 100 μg ml–1) was used to generate a standard curve to calculate soluble sugar and starch contents. Three replicate experiments were performed. Ethylene measurement Fruits of the WT, rin, and transgenic lines (at least six fruits for each line) were collected at the B+4 stage and stored in open 250 ml glass bottles for 3 h to reduce the wound ethylene effect due to harvesting the fruits. These fruits were then stored for 3 h at room temperature in glass jars sealed with a plastic membrane and ethylene production was measured as described by Chung et al. (2010). Ethylene triple response assay Seeds of the WT, rin, and transgenic lines were washed gently and then dipped in distilled water and shaken for up to 3 days at 4 °C in the dark until germination. The WT seeds were grown on MS medium with 0, 1.0, 2.0, 5.0, 10, and 20 µM 1-aminocyclopropane-1-carboxylate (ACC). The seeds of SlbHLH95-RNAi, rin, and SlbHLH95-OE-rin were grown on MS medium with 0, 5.0, and 10 µM ACC. Seedlings were then grown at 25±1 °C in the dark conditions for 7 days, and their hypocotyls and root lengths were then measured. Approximately 30 seedlings of each of the WT, rin, and transgenic lines were assessed and the transcript levels of ethylene sensitivity-related genes in each line were analyzed. The expression of SlbHLH95 was also examined in WT seedlings exposed to various concentrations of ACC. Transient expression assay in tobacco leaves The entire RIN coding sequence was amplified using PrimerSTAR Max DNA Polymerase (Takara) from tomato fruit cDNA with gene-specific primers (see Supplementary Table S1) and cloned into pGreen II 62-SK driven by the cauliflower mosaic virus (CaMV) 35S promoter, used as an effector vector (Hellens et al., 2005). Different promoter fragments of SlbHLH95 were amplified and cloned into pGreen II 0800-LUC and pGreenII 0800-GUS used as a reporter vector (Hellens et al., 2005; Xu et al., 2018). Firefly luciferase and Renilla luciferase were measured using a dual luciferase assay kit (Promega, USA) according to the manufacturer’s instructions. Transient expression assays of the reporter GUS gene were performed; the effector was SlbHLH95 driven by the CaMV 35S promoter. The promoter sequences of SAUR69 and CNR, which included the E-box cis-element, were fused to the GUS gene and used as the reporter, respectively. The effector vector and reporter vector were co-transformed into young tobacco (Nicotiana benthamiana) leaves by A. tumefaciens (strain GV3101). GUS histochemical staining was detected as described by Yan et al. (2014). Three replicate experiments were performed. RNA-seq analysis Samples of B+4 fruit from at least six plants of each line were harvested and pooled. Total RNA was extracted using TRIzol reagent. The quality and quantity of total RNA were assessed by absorbance ratios (OD260/280 and OD260/230) and by 1% agarose gel electrophoresis. RNA quality was also confirmed using a 2100 Bioanalyzer (Agilent); the RNA integrity number range of parts of RNA is shown in Supplementary Table S3. Strand-specific RNA-seq libraries were constructed according to Zhong et al. (2011) and sequenced on an Illumina HiSeq2000 system (Sangon Biotech, Shanghai, China). After sequencing, the raw reads were trimmed by removing adapter sequences and low-quality sequences. The reads were checked for quality with a Q-value <20 bases. Next, the unigenes were generated by using Trinity (http://trinityrnaseq.sourceforge.net/) software. The unigenes were further analyzed against the Solanaceae Genomics Network (https://solgenomics.net/) and Eukaryotic Orthologous Groups (KOG; https://www.ncbi.nlm.nih.gov/COG/) databases (E-value_1e-5) (Liu et al., 2012). Functional annotation by Gene Ontology (GO) was analyzed using the Blast2GO program, which mapped each of the differentially expressed genes (DEGs) to one GO term (Conesa et al., 2005). The GO terms in DEGs were considered to be significantly enriched with a Q-value ≤0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was also used to predict and classify possible functions. The DEGs were functionally annotated and KEGG pathways were automatically generated with KEGG Automatic Annotation Server by BLAST comparisons against the KEGG GENES database. Statistical analysis Data presented are the mean ±SE of three independent experiments. The statistical significance of differences in the measured parameters was tested by Student’s t test in Excel (Microsoft). Significance was accepted at P<0.05. Results Transcriptome analysis of rin mutant fruit and WT fruit Tomato fruits at the B+4 stage were harvested from WT and rin mutant plants, and RNA-seq analyses were performed. A total of 14 244 genes were identified in both of the groups of samples, 647 genes occurred only in the WT, and 465 genes occurred only in rin (Fig. 1A). Of these genes, 5648 differentially occurred in the libraries of these two groups of samples (P<0.05): 2318 genes were up-regulated in rin and 3330 genes were down-regulated in WT fruits (Fig. 1B). To determine the exact functions of the DEGs, GO functional enrichment analysis was performed to categorize these genes based on their roles in biological process (BP), molecular function (MF), and cellular component (CC). The results suggested that these DEGs were enriched in 51 GO terms (22 BP, 15 MF, and 12 CC; Q-value≤1). For BP, exhaustive analysis showed that many DEGs were metabolic and cellular process-related. For MF, many DEGs were involved in catalytic activity. For CC, the majority of DEGs were enriched in membrane, cell, and cell part (Fig. 1C). In addition, KEGG analysis showed that the dominant DEGs were involved in carbon metabolism, biosynthesis of amino acids, photosynthesis, carotenoid biosynthesis, and GSH metabolism (Fig. 1D). Collectively, the results indicated that the expression of genes related to various pathways differed between WT and rin fruits. Fig. 1. Open in new tabDownload slide RNA-seq analysis of tomato fruit at the B+4 stage in the WT and rin mutant. (A) Venn diagram indicating the differential gene expression in B+4 fruit of the WT and rin. (B) Differentially expressed genes (DEGs) of the WT and rin. The scale bar represents the value of log2(TPM). TPM, Transcripts per million. (C) Gene Ontology (GO) classification analysis of the main up- and down-regulated DEGs. (D) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs. ‘Rich factor’ indicates the ratio of the number of DEGs associated with one KEGG pathway to the total number of all DEGs. (This figure is available in colour at JXB online.) Fig. 1. Open in new tabDownload slide RNA-seq analysis of tomato fruit at the B+4 stage in the WT and rin mutant. (A) Venn diagram indicating the differential gene expression in B+4 fruit of the WT and rin. (B) Differentially expressed genes (DEGs) of the WT and rin. The scale bar represents the value of log2(TPM). TPM, Transcripts per million. (C) Gene Ontology (GO) classification analysis of the main up- and down-regulated DEGs. (D) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs. ‘Rich factor’ indicates the ratio of the number of DEGs associated with one KEGG pathway to the total number of all DEGs. (This figure is available in colour at JXB online.) Expression profiles of SlbHLH95 in WT tomato and Nr and rin mutants Through the analysis of transcriptome data from WT and rin fruits, we noticed that the expression of SlbHLH95 was up-regulated by ~6.8-fold (log2FC) in the WT, similar to that of SlSAUR69 and FUL1, which act as ethylene sensitivity and fruit ripening regulators (Supplementary Table S4). We therefore selected SlbHLH95 for further investigation. First, we investigated the expression pattern of SlbHLH95 in different tissues of WT tomato by qRT−PCR. The transcripts of SlbHLH95 were low in stems, young leaves, and IMG fruits, moderate in roots, sepals, and MG fruits, and high in mature and senescent leaves, flowers, and ripening fruits (from B to B+7 stage). Most noticeably, SlbHLH95 transcripts increased rapidly during fruit ripening (Fig. 2A). In addition, the expression of SlbHLH95 was significantly down-regulated at the B+4 and B+7 stages in mutant fruits, especially in rin mutants (Fig. 2B), suggesting a possible regulation of SlbHLH95 by RIN. Furthermore, we observed significantly reduced transcripts of SlbHLH95 in the MG fruits of WT tomato treated with 1-MCP (Fig. 2C), which indicated that SlbHLH95 is involved in ethylene signalling. Fig. 2. Open in new tabDownload slide Expression of SlbHLH95 in WT and ripening mutant tomato. (A) Relative expression of SlbHLH95 in different tissues in WT tomato. B, Breaker stage; B+4, 4 days after breaker stage; B+7, 7 days after breaker stage; FL, flower; IMG, immature green; MG, mature green; ML, mature leaf; RT, root; SE, sepal; SL, senescent leaf; ST, stem; YL, young leaf. (B) Relative expression of SlbHLH95 in the WT and mutant fruits at different stages of ripening. (C) Relative expression levels of SlbHLH95 in MG fruit treated with 1-MCP. (D) pSlbHLH95 reporter and p35S::RIN effector constructs for a transactivation assay in tobacco (Nicotiana benthamiana) leaves co-transfected with the two constructs. LUC, Firefly luciferase; REN, Renilla luciferase; Nos, NOS terminator. (E) Results of the transactivation activity of tomato RIN protein in the transient expression system in tobacco leaves, using the double-reporter plasmid containing the promoters of SlbHLH95 fused to LUC and REN driven by CaMV 35S. The control assay was performed with an empty vector as the effector construct. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (F) Schematic diagrams of the effector and reporter used for the transient transactivation assay. (G) Histochemical analysis of GUS activity in N. benthamiana leaves transiently expressing the constructs shown in (F). Bar=0.5 cm. (This figure is available in colour at JXB online.) Fig. 2. Open in new tabDownload slide Expression of SlbHLH95 in WT and ripening mutant tomato. (A) Relative expression of SlbHLH95 in different tissues in WT tomato. B, Breaker stage; B+4, 4 days after breaker stage; B+7, 7 days after breaker stage; FL, flower; IMG, immature green; MG, mature green; ML, mature leaf; RT, root; SE, sepal; SL, senescent leaf; ST, stem; YL, young leaf. (B) Relative expression of SlbHLH95 in the WT and mutant fruits at different stages of ripening. (C) Relative expression levels of SlbHLH95 in MG fruit treated with 1-MCP. (D) pSlbHLH95 reporter and p35S::RIN effector constructs for a transactivation assay in tobacco (Nicotiana benthamiana) leaves co-transfected with the two constructs. LUC, Firefly luciferase; REN, Renilla luciferase; Nos, NOS terminator. (E) Results of the transactivation activity of tomato RIN protein in the transient expression system in tobacco leaves, using the double-reporter plasmid containing the promoters of SlbHLH95 fused to LUC and REN driven by CaMV 35S. The control assay was performed with an empty vector as the effector construct. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (F) Schematic diagrams of the effector and reporter used for the transient transactivation assay. (G) Histochemical analysis of GUS activity in N. benthamiana leaves transiently expressing the constructs shown in (F). Bar=0.5 cm. (This figure is available in colour at JXB online.) RIN directly activates the transcription of SlbHLH95 in tobacco Most direct targets of the RIN protein have the CArG-box motif [C(A/T)8G] in their promoter region (Fujisawa et al., 2013). We noticed that the upstream promoter (–2450 bp to the start codon ATG) of SlbHLH95 contains five CArG-box motif sequences (Supplementary Fig. S1). Previous qChIP–PCR studies have identified SlbHLH95 as one of the direct targets of RIN (Fujisawa et al., 2013; Zhong et al., 2013). To study whether RIN can regulate the activity of the SlbHLH95 promoter, a transient transactivation assay in tobacco (N. benthamiana) leaves was performed. The double-reporter plasmid contained the promoter of SlbHLH95 fused to LUC luciferase and REN luciferase. The effector was RIN driven by CaMV 35S; the control construct lacked RIN (Fig. 2D). As shown in Fig. 2E, the LUC/REN ratio was approximately 2-fold higher in the presence of RIN compared with the control (empty vector). In addition, the GUS reporter gene was activated by co-expressing RIN with the sequence of the SlbHLH95 promoter (Fig. 2F, G). These results indicated that the activity of the SlbHLH95 promoter was regulated positively by RIN in vivo. SlbHLH95 affects ripening of tomato fruit To understand the role of SlbHLH95 in more detail, the SlbHLH95-RNAi vector was transformed into WT tomato, and three independent SlbHLH95-RNAi lines (RNAi11, RNAi13, and RNAi15) with significantly decreased expression of SlbHLH95 in the fruits were obtained (Fig. 3B). Suppression of the expression of SlbHLH95 delayed fruit ripening (Supplementary Table S5) and decreased the accumulation of total carotenoids and lycopene (Fig. 3A, D, E). Moreover, lower ethylene production was observed in the RNAi11 and RNAi13 lines (Fig. 3C). Correspondingly, fruit ripening-related genes were also suppressed in SlbHLH95-RNAi fruits, specifically in the B+4 stage (Fig. 3F−O). The expression levels of two ethylene receptors (ETR3 and ETR4) were also down-regulated in SlbHLH95-RNAi fruits (Fig. 3P, Q). However, the transcript levels of EIN2 (ETHYLENE INSENSITIVE 2) were up-regulated in SlbHLH95-RNAi fruits at the B+4 and B+7 stages (Fig. 3R). Fig. 3. Open in new tabDownload slide Phenotypic, ethylene production, and fruit ripening-related gene expression changes in the fruits from WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (A) Phenotypes of fruit from the WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. Bars=1 cm. (B) Expression analysis of SlbHLH95 in the WT, RNAi, and OE lines. (C) Production of ethylene in the WT, RNAi, and OE fruits at B+4 stage. (D, E) Total carotenoids and lycopene content in the WT, RNAi, and OE fruits. (F–R) Expression levels of the fruit ripening-related genes PSY1, FUL1, FUL2, SAUR69, ACS2, ERF4, CNR, RIN, ACO1, ACS6, ETR3, ETR4, and EIN2. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) Fig. 3. Open in new tabDownload slide Phenotypic, ethylene production, and fruit ripening-related gene expression changes in the fruits from WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (A) Phenotypes of fruit from the WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. Bars=1 cm. (B) Expression analysis of SlbHLH95 in the WT, RNAi, and OE lines. (C) Production of ethylene in the WT, RNAi, and OE fruits at B+4 stage. (D, E) Total carotenoids and lycopene content in the WT, RNAi, and OE fruits. (F–R) Expression levels of the fruit ripening-related genes PSY1, FUL1, FUL2, SAUR69, ACS2, ERF4, CNR, RIN, ACO1, ACS6, ETR3, ETR4, and EIN2. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) Three independent SlbHLH95-OE lines (OE2, OE13, and OE34) were also obtained by transforming the SlbHLH95-OE vector into WT tomato. These overexpression lines exhibited dramatically increased expression of SlbHLH95 in the fruits (Fig. 3B). As expected, the up-regulation of SlbHLH95 resulted in a slight increase in the accumulation of total carotenoids and lycopene (Fig. 3D, E), and ethylene production was also increased in lines OE13 and OE34 (Fig. 3C). However, there were no significant increases in the expression of ripening-related genes at the B+4 and B+7 stages, except for the expression of ACS2 and ACS6 in line OE13 at the B+4 stage (Fig. 3F−O). The expression of ERF4 and CNR was also up-regulated at the B stage in SlbHLH95-OE lines (Fig. 3K, L). Notably, the expression levels of ETR3, ETR4, and EIN2 were also up-regulated in SlbHLH95-OE fruits at the B+4 and B+7 stages (Fig. 3P−R). SlbHLH95 affects ethylene sensitivity of tomato To further study the relationship between SlbHLH95 and ethylene, the fruits (at MG stage) of WT and SlbHLH95-RNAi lines were treated with ET, air, and 1-MCP. The results showed that SlbHLH95-RNAi fruits had lower accumulations of total carotenoids and lycopene after treatment with ET and air (Fig. 4A−C, Supplementary Fig. S2). In addition, the expression of SlbHLH95, PSY1, ETR3, ETR4, and SAUR69 was significantly decreased compared with the WT (Fig. 4D−I), which indicated that the SlbHLH95-RNAi fruits were less sensitive to ethylene. No difference was observed between the WT and SlbHLH95-RNAi lines after treatment with 1-MCP. In contrast to the phenotype of SlbHLH95-RNAi lines, SlbHLH95-OE fruits treated with ET had higher accumulations of total carotenoids and lycopene compared with the WT. This suggested that SlbHLH95-OE fruits are more responsive to ethylene, although no difference was observed between WT and SlbHLH95-OE fruits after treatment with air and 1-MCP (Fig. 4A−C) (Supplementary Fig. S2). The expression of SlbHLH95 was greatly up-regulated in the pericarp tissues of SlbHLH95-OE fruits compared with the WT (Fig. 4D). However, the transcripts of PSY1, ETR3, ETR4, and EIN2 (Fig. 4E−H) showed no clear difference between SlbHLH95-OE and WT fruits. Notably, the expression level of SAUR69 was up-regulated in SlbHLH95-OE fruits (Fig. 4I). Fig. 4. Open in new tabDownload slide Ethylene sensitivity of fruits and seedlings from WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (A) Phenotype of MG fruits after exposure to air, ET, and 1-MCP for 24 h at room temperature and then moved to open air for 4 days. Bar=1 cm. (B, C) Total carotenoids and lycopene content in MG fruits after exposure to air, ET, and 1-MCP. (D–I) Expression of SlbHLH95, PSY1, ETR3, ETR4, EIN2, and SAUR69 in MG fruits after exposure to air, ET, and 1-MCP. (J) Seedling phenotypes of the RNAi and OE lines after treatment with 5 μM and 10 μM ACC. Bar=1 cm. (K, L) Seedling hypocotyl and root lengths after treatment with ACC. (M, N) Expression of SlbHLH95 in WT seedlings after treatment with different concentrations of ACC (0–20 μM). (O, P) Expression of ACS2, ACS6, ACO1, ETR3, ETR4, EIN2, and SAUR69 after treatment with 10 μM ACC. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) Fig. 4. Open in new tabDownload slide Ethylene sensitivity of fruits and seedlings from WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (A) Phenotype of MG fruits after exposure to air, ET, and 1-MCP for 24 h at room temperature and then moved to open air for 4 days. Bar=1 cm. (B, C) Total carotenoids and lycopene content in MG fruits after exposure to air, ET, and 1-MCP. (D–I) Expression of SlbHLH95, PSY1, ETR3, ETR4, EIN2, and SAUR69 in MG fruits after exposure to air, ET, and 1-MCP. (J) Seedling phenotypes of the RNAi and OE lines after treatment with 5 μM and 10 μM ACC. Bar=1 cm. (K, L) Seedling hypocotyl and root lengths after treatment with ACC. (M, N) Expression of SlbHLH95 in WT seedlings after treatment with different concentrations of ACC (0–20 μM). (O, P) Expression of ACS2, ACS6, ACO1, ETR3, ETR4, EIN2, and SAUR69 after treatment with 10 μM ACC. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) In addition, WT and transgenic seedlings were subjected to ACC in ethylene triple response assays. The seedlings of SlbHLH95-RNAi lines showed a slight increase in hypocotyl length with ACC treatment (5 μM and 10 μM) compared with the WT, and no obvious changes were observed in root length (Fig. 4J−L). By contrast, the seedlings of SlbHLH95-OE lines showed shorter hypocotyl and root lengths than the WT (Fig. 4K, L). We also observed that the expression level of SlbHLH95 was slightly decreased in SlbHLH95-RNAi seedlings compared with WT, but was strongly up-regulated in SlbHLH95-OE seedlings with or without ACC treatment (Fig. 4M). Moreover, the expression of SlbHLH95 was induced by high concentrations of ACC (5 μM and 10 μM) in WT seedlings (Fig. 4N). Additionally, there was no change in the transcripts of ACS2, ACS6, ETR3, ETR4, EIN2, and SAUR69 in SlbHLH95-RNAi lines treated with ACC, but ACO1 expression was increased. However, the transcription of all of these genes was up-regulated in SlbHLH95-OE lines (Fig. 4O, P), suggesting that overexpression of SlbHLH95 enhances ethylene sensitivity by activating the expression of these ethylene-related genes in tomato. In addition, the apical hook of SlbHLH95-OE seedlings showed exaggerated curvature after treatment with ACC (Supplementary Fig. S3). Together, these results indicated that SlbHLH95 is closely related to ethylene sensitivity in tomato. SlbHLH95 affects GSH, sugar, and starch metabolism To further study biological changes in transgenic plants, the GSH, soluble sugar, and starch contents were measured in B+4 fruits. SlbHLH95-RNAi fruits displayed a significant reduction in GSH content compared with the WT. In contrast, a significant increase was observed in SlbHLH95-OE lines (Fig. 5A). In addition, the expression of the GSH metabolism-related genes GSH1 (gamma-glutamylcysteine synthetase) (Cairns et al., 2006), GSH2 (glutathione synthetase) (Pasternak et al., 2008), GPX (glutathione peroxidase) (Passaia and Margis-Pinheiro, 2015), GSTU18 (glutathione-S-transferase U18) (Le Martret et al., 2011), GSTF1 (glutathione-S-transferase F1), and GSTF5 (glutathione-S-transferase F5) (Islam et al., 2017), was significantly reduced in SlbHLH95-RNAi fruits, whereas there were no changes in SlbHLH95-OE fruits, except for GSTU18 and GSTF1, which showed higher expression levels than the WT (Fig. 5B). The soluble sugar and starch contents were higher in SlbHLH95-OE fruits than in WT fruits, but there were no clear changes in SlbHLH95-RNAi lines (Fig. 5C, D). These results indicated that SlbHLH95 may affect GSH, sugar, and starch metabolism during fruit ripening. Fig. 5. Open in new tabDownload slide Altered GSH, soluble sugar, and starch contents of transgenic fruits. (A) GSH contents of fruits at B+4 stage in WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (B) Expression of the GSH metabolism-related genes GSH1, GSH2, GPX, GSTU18, GSTF1, and GSTF5 in B+4 stage fruits of the WT, RNAi, and OE lines. (C, D) Soluble sugar and starch contents of B+4 stage fruits of the WT, RNAi, and OE lines. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) Fig. 5. Open in new tabDownload slide Altered GSH, soluble sugar, and starch contents of transgenic fruits. (A) GSH contents of fruits at B+4 stage in WT, SlbHLH95-RNAi, and SlbHLH95-OE lines. (B) Expression of the GSH metabolism-related genes GSH1, GSH2, GPX, GSTU18, GSTF1, and GSTF5 in B+4 stage fruits of the WT, RNAi, and OE lines. (C, D) Soluble sugar and starch contents of B+4 stage fruits of the WT, RNAi, and OE lines. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences from the WT (P<0.05). (This figure is available in colour at JXB online.) Overexpression of SlbHLH95 in the rin mutant induces the expression of multiple ripening-related genes The SlbHLH95-OE vector was transformed into the tomato mutant rin and three independent lines (OE3, OE6, and OE8) were selected (Fig. 6). It was clear that the up-regulation of SlbHLH95 activated the expression of the ripening-related genes PSY1, FUL1, FUL2, SAUR69, ACS2, ERF4, and CNR, although it could not restore some of their transcripts to the level of WT (Fig. 6F−L). Overexpression of SlbHLH95 did not generally activate the expression of ETR3, ETR4 and EIN2 in SlbHLH95-OE-rin fruits, except for OE3 fruits, in which a dramatic increase in transcripts of ETR3 and ETR4 was observed (Fig. 6M−O). There was no clear difference in fruit colour, total carotenoid and lycopene accumulation, or ethylene production between SlbHLH95-OE-rin and rin fruits (Fig. 6A, C−E). In addition, there were no significant differences in the accumulation of total carotenoids and lycopene between SlbHLH95-OE-rin and rin fruits after treatment with ET, air, and 1-MCP (Fig. 7A−C), although the expression of PSY1 showed slight differences, and SlbHLH95 was significantly up-regulated in SlbHLH95-OE-rin compared with rin (Fig. 7D, E). However, the expression levels of ETR4 and SAUR69 were up-regulated, while the expression of ETR3 and EIN2 had no clear change (Fig. 7F–I). Fig. 6. Open in new tabDownload slide Comparison of fruits between rin and SlbHLH95-OE lines (in the rin background). (A) Fruit phenotype. Bar=1 cm. (B) Relative expression of SlbHLH95 in the fruits. (C) Ethylene production in B+4 fruits. (D, E) Total carotenoids and lycopene content in the fruits. (F–O) Expression levels of the fruit ripening-related genes PSY1, FUL1, FUL2, SAUR69, ACS2, ERF4, CNR, ETR3, ETR4, and EIN2. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. (This figure is available in colour at JXB online.) Fig. 6. Open in new tabDownload slide Comparison of fruits between rin and SlbHLH95-OE lines (in the rin background). (A) Fruit phenotype. Bar=1 cm. (B) Relative expression of SlbHLH95 in the fruits. (C) Ethylene production in B+4 fruits. (D, E) Total carotenoids and lycopene content in the fruits. (F–O) Expression levels of the fruit ripening-related genes PSY1, FUL1, FUL2, SAUR69, ACS2, ERF4, CNR, ETR3, ETR4, and EIN2. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. (This figure is available in colour at JXB online.) Fig. 7. Open in new tabDownload slide Differences in sensitivity to ethylene between rin and SlbHLH95-OE lines (in the rin background). (A) Phenotype of MG fruit after treatment with 1-MCP, ET, and air for 24 h at room temperature and then moved to open air for 4 days. Bar=1 cm. (B, C) Total carotenoids and lycopene content in MG fruits after treatment with 1-MCP, ET, and air. (D–I) Expression of SlbHLH95, PSY1, ETR3, ETR4, EIN2, and SAUR69 in MG fruits after exposure to air, ET, and 1-MCP. (J, K) Seedling hypocotyl and root lengths of the rin and OE lines after treatment with 0, 5, and 10 μM ACC. (L) Expression of SlbHLH95 after treatment with 0, 5, and 10 μM ACC. (M, N) Expression of ACS2, ACS6, ACO1, ETR3, ETR4, EIN2 and SAUR69 after treatment with 10 μM ACC. (O) Seedling phenotypes of the rin and OE lines after treatment with 0, 5, and 10 μM ACC. Bar=1 cm. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. (This figure is available in colour at JXB online.) Fig. 7. Open in new tabDownload slide Differences in sensitivity to ethylene between rin and SlbHLH95-OE lines (in the rin background). (A) Phenotype of MG fruit after treatment with 1-MCP, ET, and air for 24 h at room temperature and then moved to open air for 4 days. Bar=1 cm. (B, C) Total carotenoids and lycopene content in MG fruits after treatment with 1-MCP, ET, and air. (D–I) Expression of SlbHLH95, PSY1, ETR3, ETR4, EIN2, and SAUR69 in MG fruits after exposure to air, ET, and 1-MCP. (J, K) Seedling hypocotyl and root lengths of the rin and OE lines after treatment with 0, 5, and 10 μM ACC. (L) Expression of SlbHLH95 after treatment with 0, 5, and 10 μM ACC. (M, N) Expression of ACS2, ACS6, ACO1, ETR3, ETR4, EIN2 and SAUR69 after treatment with 10 μM ACC. (O) Seedling phenotypes of the rin and OE lines after treatment with 0, 5, and 10 μM ACC. Bar=1 cm. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. (This figure is available in colour at JXB online.) Overexpression of SlbHLH95 in the rin mutant enhances ethylene sensitivity Although up-regulation of SlbHLH95 did not restore fruit ripening in the rin mutant, the hypocotyl and root lengths of the SlbHLH95-OE-rin lines were shorter than those of the WT in the presence of ACC (5.0 μM and 10.0 μM) (Fig. 7J, K, O), and the apical hook of SlbHLH95-OE-rin seedlings showed an exaggerated curvature (Supplementary Fig. S3). In addition, SlbHLH95-OE-rin seedlings showed higher expression levels of SlbHLH95 than the rin mutant in either the absence or the presence of ACC (Fig. 7L). Moreover, ACS6, ACO1, ETR4, EIN2 and SAUR69 were significantly up-regulated in SlbHLH95-OE-rin seedlings after treatment with ACC, whereas the expression of ACS2 and ETR3 showed no clear change (Fig. 7M, N), suggesting that up-regulation of SlbHLH95 in the rin mutant enhances ethylene sensitivity by increasing the expression of some ethylene-related genes. Overexpression of SlbHLH95 in the rin mutant affects GSH, sugar, and starch metabolism The GSH content was also measured in SlbHLH95-OE-rin and rin mutant fruits. A significantly higher GSH content was observed in SlbHLH95-OE-rin B+4 fruits compared with rin fruits (Fig. 8A). Consequently, the transcript accumulation of GSH metabolism-related genes (GSH1, GSH2, GPX, GSTF1, GSTF5, and GSTU18) was significantly increased in SlbHLH95-OE-rin fruits, except for GSTU18 (Fig. 8B). The soluble sugar and starch contents were also measured in SlbHLH95-OE-rin and rin mutant fruits, and significantly higher levels were observed in the SlbHLH95-OE-rin lines (Fig. 8C, D). These results suggest that the up-regulated expression of SlbHLH95 may affect multiple metabolic pathways. Fig. 8. Open in new tabDownload slide Altered GSH, soluble sugar, and starch contents of SlbHLH95-OE fruits (in the rin background). (A) GSH contents of fruits at B+4 stage in the rin and OE lines. (B) Expression of the GSH metabolism-related genes GSH1, GSH2, GPX, GSTU18, GSTF1, and GSTF5 in B+4 stage fruits of the rin and OE lines. (C, D) Soluble sugar and starch contents of t B+4 stage fruits of the rin and OE lines. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. Fig. 8. Open in new tabDownload slide Altered GSH, soluble sugar, and starch contents of SlbHLH95-OE fruits (in the rin background). (A) GSH contents of fruits at B+4 stage in the rin and OE lines. (B) Expression of the GSH metabolism-related genes GSH1, GSH2, GPX, GSTU18, GSTF1, and GSTF5 in B+4 stage fruits of the rin and OE lines. (C, D) Soluble sugar and starch contents of t B+4 stage fruits of the rin and OE lines. Data are the mean ±SE of three independent experiments. Asterisks indicate significant differences (P<0.05) from rin. RNA-seq analysis of WT, SlbHLH95-RNAi, rin, and SlbHLH95-OE-rin fruits RNA-seq analyses were performed with WT, SlbHLH95-RNAi, rin, and SlbHLH95-OE-rin fruits at the B+4 stage. A summary of the RNA-seq data from a total of six samples is shown in Supplementary Fig. S4. A total of 4909 down-regulated and 1531 up-regulated DEGs occurred in the RNAi lines, and 749 down-regulated and 595 up-regulated DEGs occurred in the SlbHLH95-OE-rin lines (Fig. 9A). Among the DEGs, 167 genes were down-regulated in the SlbHLH95-RNAi lines and up-regulated in the SlbHLH95-OE-rin lines (Fig. 9A). In contrast, 68 genes were up-regulated in the SlbHLH95-RNAi lines and down-regulated in the SlbHLH95-OE-rin lines (Fig. 9A). Hierarchical clustering analysis of those common DEGs was performed, and a heat map was drawn showing the potential induced and repressed genes regulated by SlbHLH95 (Fig. 9B). To study the detailed function of DEGs, we used GO analysis. This analysis clarified that multiple metabolic pathways, including 22 BP, 14 CC, and 15 MF terms, were affected in the SlbHLH95-RNAi lines. For BP, many DEGs were observed to be involved in metabolic, cellular, and single-organism processes. For MF, the DEGs were primarily enriched in catalytic activity and binding. For CC, cell and cell part were the most enriched terms (Supplementary Fig. S5). In addition, KEGG studies showed that most of the DEGs in SlbHLH95-RNAi11 were significantly enriched in pathways related to ribosome and GSH metabolism (Fig. 9C). Similarly, the pathways of plant hormone signal transduction, MAPK signalling pathway, and metabolism of xenobiotics were significantly enriched in SlbHLH95-OE3-rin by KEGG analysis (Fig. 9D). As expected, the GSH metabolism pathway was also significantly enriched in SlbHLH95-OE3-rin (Fig. 9D). Additionally, GSH1, GSH2, and multiple GSTs were down- or up-regulated in the SlbHLH95-RNAi and OE lines, respectively (Supplementary Fig. S6A). Interestingly, several DEGs participating in different pathways, such as xenobiotics, and drug and GSH metabolism, were identified as the same genes (Supplementary Fig. S6B). To confirm the results of the transcriptome data, 12 genes related to fruit ripening and GSH metabolism were selected for qRT–PCR verification of the RNA-seq data. As shown in Supplementary Fig. S7, the qRT–PCR results were consistent with the transcriptome data. Fig. 9. Open in new tabDownload slide RNA-seq analysis of B+4 stage fruits in WT and rin transgenic lines. (A) Venn diagram indicating the commonly expressed genes in the RNAi and OE lines. (B) Heat map analysis of DEGs of the RNAi and OE lines. (C) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs in line RNAi11. (D) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs in line OE3. ‘Rich factor’ indicates the ratio of the number of DEGs associated with one KEGG pathway to the total number of all DEGs. (This figure is available in colour at JXB online.) Fig. 9. Open in new tabDownload slide RNA-seq analysis of B+4 stage fruits in WT and rin transgenic lines. (A) Venn diagram indicating the commonly expressed genes in the RNAi and OE lines. (B) Heat map analysis of DEGs of the RNAi and OE lines. (C) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs in line RNAi11. (D) KEGG pathway enrichment analysis of the main up- and down-regulated DEGs in line OE3. ‘Rich factor’ indicates the ratio of the number of DEGs associated with one KEGG pathway to the total number of all DEGs. (This figure is available in colour at JXB online.) SlbHLH95 directly regulates the transcription of SAUR69 and CNR by binding to their promoters SlbHLH95 affects the expression of fruit ripening-related genes, motivating us to investigate the possible regulation of their transcription. We found that the promoter sequences of SAUR69 and CNR contain conserved E-box (CANNTG) cis-elements known to be putative targets of bHLH transcription factors (Supplementary Table S6). To examine whether SlbHLH95 could regulate the activity of the promoters of SAUR69 and CNR, transient expression assays of the reporter GUS gene was performed in N. benthamiana leaves. GUS staining showed that SlbHLH95 regulates the expression of the GUS reporter gene driven by the SAUR69 and CNR promoters (Fig. 10), indicating that SlbHLH95 positively regulates the transcription of SAUR69 and CNR. Fig. 10. Open in new tabDownload slide SlbHLH95 regulates the activity of the SAUR69 and CNR promoters in N. benthamiana leaves. (A) Schematic diagrams of the effector and reporter constructs used in transient trans-regulation assays. (B, C) Histochemical analysis of GUS activity in N. benthamiana leaves. Bar=0.5 cm. (This figure is available in colour at JXB online.) Fig. 10. Open in new tabDownload slide SlbHLH95 regulates the activity of the SAUR69 and CNR promoters in N. benthamiana leaves. (A) Schematic diagrams of the effector and reporter constructs used in transient trans-regulation assays. (B, C) Histochemical analysis of GUS activity in N. benthamiana leaves. Bar=0.5 cm. (This figure is available in colour at JXB online.) Discussion SlbHLH95 functions downstream of RIN Tomato is an ideal model plant for the study of ripening of climacteric fruits, and a number of studies have attempted to elucidate the molecular basis of fruit ripening. RIN has been known as an indispensable factor for initiating tomato ripening since the mutant rin was discovered (Vrebalov et al., 2002). Here, we cloned the transcription factor SlbHLH95, known as a RIN target gene (Fujisawa et al., 2013), and checked its expression levels in WT tomato. The qRT−PCR results showed that SlbHLH95 was highly expressed in ripening fruits (especially at the B+4 and B+7 stages), consistent with previous reports (Fujisawa et al., 2013), while low expression was observed in immature and mature green fruits (Fig. 2A), indicating its potential function in fruit ripening. It is well known that the rin mutant shows no increase in ethylene production, and its fruit ripening is suppressed by the fusion protein RIN-MC, indicating that RIN is a key regulator of fruit ripening and modulates the expression of fruit ripening-related genes (Li et al., 2018). The Nr mutant, which fails to fully ripen, is ethylene insensitive (Wilkinson et al., 1995). In this study, we found that the expression of SlbHLH95 was not induced in either rin or Nr during the fruit ripening process (Fig. 2B) and was also significantly down-regulated by treatment with 1-MCP (Fig. 2C), suggesting that SlbHLH95 is closely involved with RIN and ethylene. Moreover, the SlbHLH95 promoter contained five CArG-box motif sequences and was shown to be directly regulated by RIN in a transient transactivation assay in tobacco (N. benthamiana) leaves (Fig. 2D−G), which confirmed previous reports using qChIP–PCR technology (Fujisawa et al., 2013; Zhong et al., 2013). Based on these results, we speculated that SlbHLH95 may play a role in tomato fruit ripening and functions downstream of RIN. SlbHLH95 is closely related to tomato fruit ripening During tomato fruit ripening, a colour change from green to red is the distinctive characteristic, and the accumulation of total carotenoids and lycopene is associated with the colour transition. The down-regulation of SlbHLH95 resulted in lower total carotenoid and lycopene accumulation in SlbHLH95-RNAi fruits (Fig. 3A, D, E). In contrast, the up-regulation of SlbHLH95 resulted in slightly higher accumulations of total carotenoids and lycopene (Fig. 3A, D, E). Correspondingly, ethylene contents were changed in the two types of transgenic lines (Fig. 3C). Ethylene biosynthesis begins with S-adenosylmethionine and includes two key steps catalyzed by the enzymes ACS and ACO (Adams and Yang, 1977; Van de Poel et al., 2012). In our study, a lower expression of ACS2 was detected in SlbHLH95-RNAi fruits at the B+4 stage (Fig. 3J). However, the transcripts of ACS6 and ACO1 were not significantly changed in SlbHLH95 transgenic fruits. It has been reported that ACS6 belongs to the Type I genes, which do not have high expression in fruit ripening (Van de Poel et al., 2012), and the mRNA stability of ACO1 is often changed by GSH, while ACO1 transcription remained unaffected (Datta et al., 2015). Based on these results, we speculated that the changes in ACS2 expression may be the main reason for the change in ethylene content in transgenic fruits. In addition, the expression of some fruit ripening-related genes (PSY1, FUL1, FUL2, SAUR69, ERF4, CNR, and RIN) was significantly down-regulated in SlbHLH95-RNAi fruits at the B+4 stage (Fig. 3F−M). These results indicated that SlbHLH95 may be involved in ethylene signalling in tomato. On the other hand, the ethylene triple response assay and ethylene treatment experiments demonstrated that SlbHLH95 could affect the ethylene sensitivity of tomato and may be reduced by ethylene, indicating that SlbHLH95 is associated with ethylene. However, the accumulation of total carotenoids and lycopene was not restored by increasing the amount of transcripts of SlbHLH95 in the rin mutant (Fig. 6). It has been shown that a large amount of ethylene is required for total carotenoid and lycopene accumulation at the beginning of fruit ripening (Fray and Grierson, 1993). In our study, there was no clear difference in ethylene production between SlbHLH95-OE-rin and rin fruits, although ACS2 transcripts were slightly higher in SlbHLH95-OE-rin fruits but considerably less than in the WT (Fig. 6), indicating that the overexpression of SlbHLH95 in the rin mutant could not activate ethylene biosynthesis and did not restore normal ripening in rin fruit. SlbHLH95 influences ethylene sensitivity and regulates GSH metabolism GSH has been reported to play an important role in stress responses and various developmental processes (Noctor et al., 2012; Cheng et al., 2015; Schippers et al., 2016). Silencing of GSH1 also causes lethality at the embryo stage in Arabidopsis (Cairns et al., 2006), and the GSH2-knockout mutant shows a reduction in growth related to death at the seedling stage (Pasternak et al., 2008). GSH is considered to be one of the most important cellular antioxidants, and brings about changes in the intercellular redox state during development or the defense response in plants via reactive oxygen species-dependent signalling pathways (Foyer and Noctor, 2005, 2016). Moreover, considerable evidence has indicated that plant development is regulated by extensive interactions among different hormones, such as ethylene and GSH (Loake and Grant, 2007; Datta et al., 2015), but a comprehensive mechanism governing their interactions has not been elucidated to date. In our study, the RNA-seq data showed that many GSH-related genes were down-regulated in SlbHLH95-RNAi lines and up-regulated in SlbHLH95-OE-rin lines (Fig. 9, Supplementary Fig. S6), which was confirmed by qRT−PCR (Figs 5, 8). Overexpression of SlbHLH95 in the WT and rin mutant increased the GSH content, and lower GSH content was observed in SlbHLH95-RNAi lines (Figs 5A, 8A). Decreased expression of ACS2 was observed in SlbHLH95-RNAi fruits at the B+4 stage; conversely, SlbHLH95-OE (in the WT and rin background, respectively) lines showed increased expression of ACS2 (Figs 3J, 6J). Previous studies reported that a strong up-regulation of ACO and ERF4 at the transcript level was observed in transgenic N. tabacum plants ectopically expressing LeGSH1 (Ghanta et al., 2014). In addition, GSH also induced the transcription of ACS2 and ACS6 via WRKY33-mediated transcriptional induction in Arabidopsis thaliana (Datta et al., 2015). Therefore, we speculated that GSH metabolism is related to the ethylene signalling pathway in tomato. Moreover, we found that SlbHLH95 affected the expression of ETR3, ETR4, EIN2, and SAUR69 in tomato fruit (Figs 3, 6). In addition, SlbHLH95 directly regulates the transcription of SAUR69 and CNR by binding to their promoters (Fig. 10). These results suggested that SlbHLH95 could regulate GSH metabolism and influence ethylene sensitivity, even affecting fruit ripening. Conclusion We identified a novel tomato bHLH transcription factor, SlbHLH95. Based on the results of down-regulation of SlbHLH95 in WT and overexpression of SlbHLH95 in WT and the rin mutant, we speculate that SlbHLH95 affects the accumulation of total carotenoids and lycopene, affects ethylene sensitivity, and influences multiple metabolisms. We propose a model to elucidate the potential function of SlbHLH95 in tomato fruit ripening and multiple metabolic pathways (Fig. 11). In brief, our study helps to elucidate the genetic mechanism responsible for tomato agronomic traits and benefits, and will help to facilitate future molecular breeding efforts to provide better-quality tomato fruit. Fig. 11. Open in new tabDownload slide Proposed model depicting the regulation of SlbHLH95 and its role in controlling tomato fruit ripening. Fig. 11. Open in new tabDownload slide Proposed model depicting the regulation of SlbHLH95 and its role in controlling tomato fruit ripening. Supplementary data The following supplementary data are available at JXB online. Fig. S1. RIN binding motifs in promoters of SlbHLH95. Fig. S2. Phenotype of MG fruits after exposure to air, ET, and 1-MCP. Fig. S3. Seedling phenotypes after treatment with ACC. Fig. S4. RNA-seq analysis of the genes regulated by SlbHLH95 in B+4 fruit. Fig. S5. Gene Ontology (GO) classification. Fig. S6. Analysis of GSH-related differentially expressed genes (DEGs) in RNAi and OE lines. Fig. S7. qRT–PCR and RNA-seq analysis of the expression of 12 genes in tomato fruits at B+4 stage. Table S1. Specific primers used in this study. Table S2. RNA integrity number range of parts of RNA used in qRT–PCR. Table S3. RNA integrity number range of RNA used in RNA-seq analysis. Table S4. Expression of SlbHLH95 and other genes in wild-type and rin mutant fruit by RNA-seq or ChIP–seq. Table S5. Days from anthesis to breaker stage for WT and SlbHLH95-transgenic lines. Table S6. Putative bHLH-binding cis-elements present in the promoter regions of SAUR69 and CNR. Acknowledgements This work was supported by the Natural Science Foundation of China (grant no.31872121) and the Natural Science Foundation of Chongqing of China (cstc2018jcyjAX0458). Author contributions LZ, JK, and JG performed the experiments and data analysis; LZ, XQ, HS, and YC wrote the manuscript; GC and ZH conceived and directed the project and improved the manuscript. 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TI - The basic helix-loop-helix transcription factor bHLH95 affects fruit ripening and multiple metabolisms in tomato
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eraa363
DA - 2020-10-22
UR - https://www.deepdyve.com/lp/oxford-university-press/the-basic-helix-loop-helix-transcription-factor-bhlh95-affects-fruit-RzdguUlXix
SP - 6311
EP - 6327
VL - 71
IS - 20
DP - DeepDyve
ER -