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Softening is one of the key fruit quality traits, which results from the selective expression of cell wall metabolism genes during ripening. The identification of transcription factors (TFs) that regulate fruit softening is an important field in order to understand and control fruit softening. In tomato, NAC (NAM, ATAF, and CUC) TFs members have been demonstrated to be involved in fruit ripening regulation, including NAC-NOR (non-ripening), NOR-like1, SlNAC4, SlNAC1. Here, we generated slnac4 mutant knockout (CR-SlNAC4) tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system (CRISPR/Cas9) and SlNAC4 overexpressing (OE-SlNAC4) plant. In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening, we found that SlNAC4 has a strong effect on tomato fruit softening. Compared with the control fruit, fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit. Through RNA-sequencing (RNA-seq) analysis, we found that expression levels of SlEXP1 (expansin) and SlCEL2 (endo-β-1,4 glucanase) genes involved in cell wall metabolism were significantly different in WT (wild type)/slnac4 and WT/OE-SlNAC4 fruit. Further study showed that these genes contained a NAC TF binding domain in their promoter regions. In vitro electrophoretic mobility shift assays (EMSA) and dual-luciferase reporter assays (DLR) demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation. The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.

Ripening of the model fruit tomato (Solanum lycopersicum) is controlled by a transcription factor network including NAC (NAM, ATAF1/2, and CUC2) domain proteins such as No-ripening (NOR), SlNAC1, and SlNAC4, but very little is known about the NAC targets or how they regulate ripening. Here, we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1 (Solyc07g063420) using virus-induced gene silencing (VIGS) inhibited specific aspects of ripening. Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production, retarded softening and chlorophyll loss, and reduced lycopene accumulation. RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis (SlACS2, SlACS4), color formation (SlGgpps2, SlSGR1), and cell wall metabolism (SlPG2a, SlPL, SlCEL2, and SlEXP1) are direct targets of NOR-like1. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assay (DLR) confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo, and activated their expression. Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening, with an important role in the transcriptional regulatory network., Crop genetics: novel gene involved in tomato ripening Chinese researchers have identified a new gene which regulates the ripening of tomatoes. Several genes known to control tomato ripening are members of the NAC family of regulators. To identify others, a team led by Daqi Fu of China Agricultural University blocked the expression of candidate NAC genes. The discovered that silencing NOR-like1 repressed ripening, leaving the tomatoes partially green. The team also engineered plants with defective copies of NOR-like1 and found that this delayed ripening and eventually resulted in partially ripe fruit and impaired seed development. RNA sequencing of these lines revealed that NOR-like1 directly regulates genes involved in ethylene synthesis, carotenoid accumluation, chlorophyll metabolism, and cell wall breakdown. These findings clearly demonstrate a key role for NOR-like1 as a positive regulator of tomato ripening and a potential tool for controlling this important process.

Jasmonates accumulate rapidly and act as key regulators in response to mechanical wounding, but few studies have linked receptor-like cytoplasmic kinases (RLCKs) to wound-induced jasmonic acid (JA) signaling cascades. Here, we identified a novel wounding-induced RLCK-XII-2 subfamily member (SlZRK1) in tomato (Solanum lycopersicum) that was closely related to Arabidopsis HOPZ-ETI-DEFICIENT 1 (ZED1)-related kinases 1 based on phylogenetic analysis. SlZRK1 was targeted to the plasma membrane of tobacco mesophyll protoplasts as determined by transient co-expression with the plasma membrane marker mCherry–H+-ATPase. Catalytic residue sequence analysis and an in vitro kinase assay indicated that SlZRK1 may act as a pseudokinase. To further analyse the function of SlZRK1, we developed two stable knock-out mutants by CRISPR/Cas9. Loss of SlZRK1 significantly altered the expression of genes involved in JA biosynthesis, salicylic acid biosynthesis, and ethylene response. Furthermore, after mechanical wounding treatment, slzrk1 mutants increased transcription of early wound-inducible genes involved in JA biosynthesis and signaling. In addition, JA accumulation after wounding and plant resistance to herbivorous insects also were enhanced. Our findings expand plant regulatory networks in the wound-induced JA production by adding RLCKs as a new component in the wound signal transduction pathway.

With the development of bioinformatics, it is easy to obtain information and data about thousands of genes, but the determination of the functions of these genes depends on methods for rapid and effective functional identification. Virus-induced gene silencing (VIGS) is a mature method of gene functional identification developed over the last 20 years, which has been widely used in many research fields involving many species. Fruit quality formation is a complex biological process, which is closely related to ripening. Here, we review the progress and contribution of VIGS to our understanding of fruit biology and its advantages and disadvantages in determining gene function.

Plant organellar RNA editing is a distinct type of post-transcriptional RNA modification that is critical for plant development. We showed previously that the RNA editing factor SlORRM4 is required for mitochondrial function and fruit ripening in tomato (Solanum lycopersicum). However, a comprehensive atlas of the RNA editing mediated by SlORRM4 is lacking. We observed that SlORRM4 is targeted to both chloroplasts and mitochondria, and its knockout results in pale-green leaves and delayed fruit ripening. Using high-throughput sequencing, we identified 12 chloroplast editing sites and 336 mitochondrial editing sites controlled by SlORRM4, accounting for 23% of chloroplast sites in leaves and 61% of mitochondrial sites in fruits, respectively. Analysis of native RNA immunoprecipitation sequencing revealed that SlORRM4 binds to 31 RNA targets; 19 of these targets contain SlORRM4-dependent editing sites. Large-scale analysis of putative SlORRM4-interacting proteins identified SlRIP1b, a RIP/MORF protein. Moreover, functional characterization demonstrated that SlRIP1b is involved in tomato fruit ripening. Our results indicate that SlORRM4 binds to RNA targets and interacts with SlRIP1b to broadly affect RNA editing in tomato organelles. These results provide insights into the molecular and functional diversity of RNA editing factors in higher plants.

Background and aims In recent years, increasing numbers of long non-coding RNAs (lncRNAs) have been identified in humans, animals and plants, and several of them have been shown to play important roles in diverse biological processes. However, little work has been performed on the regulation mechanism of lncRNA biogenesis and expression, especially in plants. Compared with studies of tomato MADS-box transcription factor RIPENING INHIBITOR (RIN) target coding genes, there are few reports on its relationship to non-coding RNAs. The aim of the present study was to identify and explore the specific role of RIN target lncRNAs in tomato fruit development and ripening. Methods lncRNA targets of RIN were identified by chromatin immunoprecipitation sequencing (ChIP-seq) combined with RNA deep sequencing analysis. Six selected lncRNA targets were validated by quantitative real-time PCR, ChIP and electrophoretic mobility shift assays, and we further confirmed differential expression between wild-type and ripening-deficient mutant fruit, and RIN direct binding in the promoter regions. By means of virus-induced gene silencing (VIGS) assays and a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing strategy, the ripening-related function of a specific target lncRNA (lncRNA2155) was studied. Key results We identified 187 lncRNAs as direct RIN targets, which exhibited RIN binding sites in their promoters and showed different expression between the wild-type and rin mutant. Six target lncRNAs were shown to bind with RIN directly in their promoters in vivo and in vitro. Moreover, using CRISPR/Cas9 technology to knock out the locus of the target lncRNA2155 indicated that it delayed fruit ripening in tomato. Conclusions Collectively, these findings provide new insight into RIN in the transcriptional regulation of lncRNAs and suggest that lncRNAs will contribute to a better understanding of the RIN regulatory network that controls fruit ripening.

Chloroplast development and chlorophyll(Chl)metabolism in unripe tomato contribute to the growth and quality of the fruit, however these mechanisms are poorly understood. In this study, we initially investigated seven homeobox-containing transcription factors (TFs) with specific ripening-associated expression patterns using virus-induced gene silencing (VIGS) technology and found that inhibiting the expression of one of these TFs, BEL1-LIKE HOMEODOMAIN11 (SlBEL11), significantly increased Chl levels in unripe tomato fruit. This enhanced Chl accumulation was further validated by generating stable RNA interference (RNAi) transgenic lines. RNA sequencing (RNA-seq) of RNAi-SlBEL11 fruit at the mature green (MG) stage showed that 48 genes involved in Chl biosynthesis, photosynthesis and chloroplast development were significantly upregulated compared with the wild type (WT) fruit. Genomic global scanning for Homeobox TF binding sites combined with RNA-seq differential gene expression analysis showed that 22 of these 48 genes were potential target genes of SlBEL11 protein. These genes included Chl biosynthesis-related genes encoding for protochlorophyllide reductase (POR), magnesium chelatase H subunit (CHLH) and chlorophyllide a oxygenase (CAO), and chloroplast development-related genes encoding for chlorophyll a/b binding protein (CAB), homeobox protein knotted 2 (TKN2) and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation quantitative polymerase chain reaction (PCR) (ChIP-qPCR) assays were employed to verify that SlBEL11 protein could bind to the promoters for TKN2, CAB and POR. Taken together, our findings demonstrated that SlBEL11 plays an important role in chloroplast development and Chl synthesis in tomato fruit.

Eggplant ( Solanum melongena L.) fruits accumulate flavonoids in their cuticle and epidermal cells during ripening. Although many mutants available in model plant species, such as Arabidopsis thaliana and Medicago truncatula, are enabling the intricacies of flavonoid-related physiology to be deduced, the mechanisms whereby flavonoids influence eggplant fruit physiology are unknown. Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoid function in fruit, and in this study, we successfully applied this technique to downregulate S. melongena chalcone synthase gene ( SmCHS) expression during eggplant fruit ripening. In addition to the expected change in fruit color attributable to a lack of anthocyanins, several other modifications, including differences in epidermal cell size and shape, were observed in the different sectors. We also found that silencing of CHS gene expression was associated with a negative gravitropic response in eggplant fruits. These observations indicate that epidermal cell expansion during ripening is dependent upon CHS expression and that there may be a relationship between CHS expression and gravitropism during eggplant fruit ripening.

Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction, and fruit quality. Tomato (Solanum lycopersicum) ripening inhibitor (rin) mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC. In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network, with hundreds of gene targets controlling changes in color, flavor, texture, and taste during tomato fruit ripening; MC, on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, overexpression of RIN-MC in transgenic wild-type cv Ailsa Craig tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S::RIN-MC RNA interference fruits, a total of 1,168 and 1,234 genes were identified as potential targets of RIN-MC activation and inhibition. Furthermore, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that tomato RIN-MC fusion plays a negative role in ripening and encodes a chimeric transcription factor that modulates the expression of many ripening genes, thereby contributing to the rin mutant phenotype.

With experimental and bioinformatical methods, numerous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), have been found in plants, and they play vital roles in various biological regulation processes. However, most of these small RNAs remain to be functionally characterized. Until now, only several viral vectors were developed to overexpress miRNAs with limited application in plants. In this study, we report a new small RNA overexpression system via viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs in Nicotiana benthamiana First, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely used AtMIR319a backbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (AtMIR319a, AtMIR390a, and SlMIR159) on specificity and significance of generating small RNAs, the AtMIR390a backbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs from AtMIR390a backbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targeted PDS gene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 and athTAS3a 5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, including PDS, Su, and PCNA These results demonstrated that this small RNA overexpression system could contribute to investigating not only the function of endogenous small RNAs, but also the functional genes in plants.

Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels., Fruit ripening: Multiple backup plans The regulatory circuits that govern the expression of genes required for ripening in tomato plants are highly redundant. Fleshy fruits that use the hormone ethylene to regulate ripening have developed independently multiple times in the history of the angiosperms. Guiqin Qu at China Agricultural University in Beijing and colleagues working on the fruitENCODE project are exploring the genetic and epigenetic basis of this convergent evolution. In tomatoes, three transcription factors have been shown to control ethylene production and regulate ripening. However, when gene editing techniques were used to introduce mutations that interfere with the function of these transcription factors, partial ripening or a delay in ripening was observed. The fact that ripening was not abolished indicates that the ripening process is more robust and complex than previously thought.

Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes.

Dicer proteins belong to the RNase III family of proteins, which are key components in small RNA biogenesis. In Solanum lycopersicum, seven Dicer-like (DCL) genes have been identified and have been named SlDCL. In this study, we cloned the full-length sequence of the SlDCL genes including untranslated regions using RNA ligase-mediated rapid amplification of cDNA ends. Our analysis indicates that 7 SlDCLs were located on 5 tomato chromosomes (6, 7, 8, 10, and 11). The gene structure of the SlDCLs covered long genomic regions and contained more than 20 exons. Phylogenetic analysis divided the seven SlDCL members into four subgroups. In general, all seven SlDCLs were expressed in all organs but more in flowers and fruits than in the other parts. Moreover, the expressions of some genes changed slightly after treatment with ethylene or 1-methylcyclopropene suggesting their likely roles in plant responses to ethylene. Our findings provide essential information on SlDCL genes in tomato and will aid in the functional classification of DCL families in plants.

Despite many studies about functions of miR396 were concentrated on cotyledon and leaf growth and development, only few researches were focused on flower and fruit, especially for fleshy fruit, for example, tomato fruit. Here, the roles of miR396 throughout the growth and development of tomato plant were explored with combining bioinformatics and transgene-mediated methods. In tomato, miR396 had two mature types (miR396a and miR396b), and miR396a expressed significantly higher than miR396b in cotyledon, flower, sepal and fruit. Generally, plant growth and development were regulated by miR396 via growth-regulating factors (GRFs). In tomato, all 13 SlGRFs were analyzed comprehensively, including phylogeny, domain and expression patterns. To investigate the roles of miR396 further, STTM396a/396a-88 was over-expressed in tomato, which induced miR396a and miR396b both dramatical down-regulation, and the target GRFs general up-regulation. As a result, the flowers, sepals and fruits all obviously became bigger. Most significantly, the sepal length of transgenic lines #3 and #4 at 39 days post-anthesis was separately increased 75% and 81%, and the fruit weight was added 45% and 39%, respectively. Overall, these results revealed novel roles of miR396 in regulating flower and fruit development, and provided a new potential way for improving tomato fruit yield.

The tomato encode four functional DCL families, of which DCL2 is poorly studied. Here, we generated loss-of-function mutants for a tomato DCL2 gene, dcl2b, and we identified its major role in defending against tomato mosaic virus in relation to both natural and manual infections. Genome-wide small RNA expression profiling revealed that DCL2b was required for the processing 22-nt small RNAs, including a few species of miRNAs. Interestingly, these DCL2b-dependent 22-nt miRNAs functioned similarly to the DCL1-produced 22-nt miRNAs in Arabidopsis and could serve as triggers to generate a class of secondary siRNAs. In particular, the majority of secondary siRNAs were derived from plant defense genes when the plants were challenged with viruses. We also examined differentially expressed genes in dcl2b through RNA-seq and observed that numerous genes were associated with mitochondrial metabolism and hormone signaling under virus-free conditions. Notably, when the loss-of-function dcl2b mutant was challenged with tomato mosaic virus, a group of defense response genes was activated, whereas the genes related to lipid metabolism were suppressed. Together, our findings provided new insights into the roles of tomato DCL2b in small RNA biogenesis and in antiviral defense.

Numerous studies have been focusing on breeding tomato plants with enhanced lycopene accumulation, considering its positive effects of fruits on the visual and functional properties. In this study, we used a bidirectional strategy: promoting the biosynthesis of lycopene, while inhibiting the conversion from lycopene to β- and α-carotene. The accumulation of lycopene was promoted by knocking down some genes associated with the carotenoid metabolic pathway. Finally, five genes were selected to be edited in genome by CRISPR/Cas9 system using Agrobacterium tumefaciens-mediated transformation. Our findings indicated that CRISPR/Cas9 is a site-specific genome editing technology that allows highly efficient target mutagenesis in multiple genes of interest. Surprisingly, the lycopene content in tomato fruit subjected to genome editing was successfully increased to about 5.1-fold. The homozygous mutations were stably transmitted to subsequent generations. Taken together, our results suggest that CRISPR/Cas9 system can be used for significantly improving lycopene content in tomato fruit with advantages such as high efficiency, rare off-target mutations, and stable heredity.

Highlight A relatively reliable list of tomato lncRNAs was provided. Silencing of novel lncRNAs greatly delayed the ripening of tomato fruits, implying that lncRNA might be an essential factor for fruit ripening., Recently, long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in model plants, such as Arabidopsis, rice, and maize. However, the presence of lncRNAs and how they function in fleshy fruit ripening are still largely unknown because fleshy fruit ripening is not present in the above model plants. Tomato is the model system for fruit ripening studies due to its dramatic ripening process. To investigate further the role of lncRNAs in fruit ripening, it is necessary and urgent to discover and identify novel lncRNAs and understand the function of lncRNAs in tomato fruit ripening. Here it is reported that 3679 lncRNAs were discovered from wild-type tomato and ripening mutant fruit. The lncRNAs are transcribed from all tomato chromosomes, 85.1% of which came from intergenic regions. Tomato lncRNAs are shorter and have fewer exons than protein-coding genes, a situation reminiscent of lncRNAs from other model plants. It was also observed that 490 lncRNAs were significantly up-regulated in ripening mutant fruits, and 187 lncRNAs were down-regulated, indicating that lncRNAs could be involved in the regulation of fruit ripening. In line with this, silencing of two novel tomato intergenic lncRNAs, lncRNA1459 and lncRNA1840, resulted in an obvious delay of ripening of wild-type fruit. Overall, the results indicated that lncRNAs might be essential regulators of tomato fruit ripening, which sheds new light on the regulation of fruit ripening.

The development and ripening of tomato fruit are complex processes involving many gene regulatory pathways at the transcriptional and post-transcriptional level. Ripening inhibitor (RIN) is a vital transcription factor, which targets numerous ripening-related genes at the transcriptional level during tomato fruit ripening. MicroRNAs (miRNAs) are a class of short noncoding RNAs that play important roles in post-transcriptional gene regulation. To elucidate the potential regulatory relationship between rin and miRNAs during fruit development and ripening, we identified known miRNAs and profiled their expression in wild-type tomato and rin mutant using a deep sequencing approach combined with quantitative RT-PCR. A total of 33 known miRNA families were identified, of which 14 miRNA families were differently accumulated. Subsequent promoter analysis showed that possible RIN-binding motifs (CArG-box) tended to occur frequently in the promoter regions of partial differently expressed miRNAs. In addition, ethylene may participate in the regulation of miRNAs accumulation during tomato fruit ripening. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay confirmed the direct binding of RIN to the promoter of MIR172a. Collectively, these results showed a close correlation between miRNA expression and RIN as well as ethylene, which further elucidated the regulatory roles of miRNAs during fruit development and ripening and enriched the regulatory network of RIN in tomato fruit.

Glycoalkaloids are toxic compounds that are synthesized by many Solanum species. Glycoalkaloid biosynthesis is influenced by plant genetic and environmental conditions. Although many studies have shown that light is an important factor affecting glycoalkaloid biosynthesis, the specific mechanism is currently unknown. Chlorophyll and carotenoid biosynthesis depend on light signal transduction and share some intermediate metabolites with the glycoalkaloid biosynthetic pathway. Here, we used virus-induced gene silencing to silence genes encoding phytoene desaturase (PDS) and magnesium chelatase (CHLI and CHLH) to reduce chlorophyll and carotenoid levels in eggplant leaves. Quantification of carotenoid and chlorophyll levels is analyzed by LC/PDA/APCI/MS and semipolar metabolite profiling by LC/HESI/MS. Notably, the resulting lines showed decreases in glycoalkaloid production. We further found that the expression of some genes involved in the production of glycoalkaloids and other metabolites were suppressed in these silenced lines. Our results indicate that photosynthetic pigment accumulation affects steroidal glycoalkaloid biosynthesis in eggplant leaves. This finding lays the foundation for reducing the levels of endogenous antinutritional compounds in crops.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced by Solanaceous plant species. They contribute to pathogen defense but are considered as anti-nutritional compounds and toxic to humans. Although the genes involved in the SGA biosynthetic pathway have been successfully cloned and identified, transcription factors regulating this pathway are still poorly understood. We report that silencing tomato light signal transduction transcription factors ELONGATED HYPOCOTYL 5 (SlHY5) and PHYTOCHROME INTERACTING FACTOR3 (SlPIF3), by virus-induced gene silencing (VIGS), altered glycoalkaloids levels in tomato leaves compared to control plant. Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) analysis confirmed that SlHY5 and SlPIF3 bind to the promoter of target genes of GLYCOALKALOID METABOLISM (GAME1, GAME4, GAME17), affecting the steady-state concentrations of transcripts coding for SGA pathway enzymes. The results indicate that light-signaling transcription factors HY5 and PIF3 regulate the abundance of SGAs by modulating the transcript levels of these GAME genes. This insight into the regulation of SGA biosynthesis can be used for manipulating the level of these metabolites in crops.

With the development of high-throughput sequencing, many long non-coding RNAs (lncRNAs) have been found to play important roles in diverse biological processes. However, the biological functions of most plant lncRNAs are still unknown. We have previously discovered a tomato ripening-related lncRNA, lncRNA1459. Here, we cloned the full-length lncRNA1459, giving two transcript isoforms. In addition, lncRNA1459 exhibited a specific location in the nucleus. Furthermore, in order to fully identify the function of lncRNA1459 in tomato ripening, loss-of-function mutants of lncRNA1459 were developed using clustered regularly interspaced short palindromic repeats (CRISPR)/-associated protein 9 (Cas9)-induced genome editing technology. Compared with wild-type fruits, the tomato ripening process was significantly repressed in lncRNA1459 mutants. Ethylene production and lycopene accumulation were largely repressed in lncRNA1459 mutants. Additionally, genes related to ethylene and carotenoid biosynthesis were distinctly downregulated in lncRNA1459 mutants compared with wild-type fruits. Moreover, expression of numerous ripening-related genes was changed significantly when lncRNA1459 was knocked out. Expression of potential tomato ripening-related lncRNAs was also specifically changed after knocking out lncRNA1459. Taken together, these results provide insight into the role of lncRNA1459 in tomato fruit ripening.

RNA editing plays a key posttranscriptional role in gene expression. Existing studies on cytidine-to-uridine RNA editing in plants have focused on maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana). However, the importance and regulation of RNA editing in several critical agronomic processes are not well understood, a notable example of which is fruit ripening. Here, we analyzed the expression profile of 33 RNA editing factors and identified 11 putative tomato (Solanum lycopersicum) fruit ripening-related factors. A rapid virus-induced gene silencing assay indicated that the organelle RNA recognition motif-containing protein SlORRM4 affected tomato fruit ripening. Knocking out SlORRM4 expression using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing strategy delayed tomato fruit ripening by lowering respiratory rate and ethylene production. Additionally, the expression of numerous genes associated with fruit ripening and mitochondrial functions changed significantly when SlORRM4 was knocked out. Moreover, the loss of SlORRM4 function significantly reduced RNA editing of many mitochondrial transcripts, leading to low-level expression of some core subunits that are critical for mitochondrial complex assembly (i.e. Nad3, Cytc1, and COX II). Taken together, these results indicate that SlORRM4 is involved in RNA editing of transcripts in ripening fruit that influence mitochondrial function and key aspects of fruit ripening.

Fruit ripening is a complex biological process affecting fruit quality. In tomato the fruit ripening process is delicately regulated by transcription factors (TFs). Among these, the TOMATO AGAMOUS-LIKE 1 (TAGL1) gene plays an important role in both the development and ripening of fruit. In this study, the TAGL1 gene was successfully silenced by virus-induced gene silencing technology (VIGS), and the global gene expression and metabolites profiles of TAGL1-silenced fruits were analyzed by RNA-sequence analysis (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS/MS). The TAGL1-silenced fruits phenotypically displayed an orange pericarp, which was in accordance with the results expected from the down-regulation of genes associated with carotenoid synthesis. Levels of several amino acids and organic acids were lower in the TAGL1-silenced fruits than in the wild-type fruits, whereas, α-tomatine content was greatly increased (more than 10-fold) in the TAGL1-silenced fruits compared to wild-type fruits. The findings of this study showed that TAGL1 not only regulates the ripening of tomato fruits, but also affects the synthesis and levels of nutrients in the fruit.

Summary In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.

Green-ripe (Gr) tomato carries a dominant mutation and yields a nonripening fruit phenotype. The mutation results from a 334 bp deletion in a gene of unknown function at the Gr locus. The putative influence of Gr gene-deletion mutation on biochemical changes underlying the nonripening phenotype remains largely unknown. Respiration of Gr fruit was found to be reduced at mature green and breaker stage of ripening, while the fruit softening was dramatically prolonged. We studied the proteome of Gr mutant fruit using high-throughput iTRAQ and high-resolution mass spectrometry and identified 43 proteins representing 43 individual genes as potential influence-targets of Gr mutated fruit. The identified proteins are involved in several ripening-related pathways including cell-wall metabolism, photosynthesis, oxidative phosphorylation, carbohydrate and fatty acid metabolism, protein synthesis, and processing. Affected protein levels are correlated with the corresponding gene transcript levels. The modulation in the accumulation levels of PI(U1)P, PGIP, and PG2 supported the delayed softening phenotype of the Gr fruit. Further investigation in GR gene-silencing fruit ascertained the doubtless modulation of these targets by the deletion mutation of GR gene.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.

The tomato SlERF1 gene belongs to a distinct subfamily of the large ERF gene family. It is a downstream component in the ethylene signaling pathway in tomato, and is involved in plant defense and stress responses. Overexpression of the SlERF1 gene (SlERF1-OEs) in tomato dramatically enhanced the resistance of fruit to Rhizopus nigricans. In attempt to elucidate the mechanism basis of this observation, our study revealed a dual modulation. Firstly, SlERF1-OEs fruit showed a remarkable transcript accumulation of PR5 and PAL genes before infection with R. nigricans, which was not observed in wild-type (WT) fruit. Secondly, SlERF1-OEs fruit had higher activitiers of phenylalanine ammonia lyase (PAL) and chitinase (CHI) in response to the fungal stress. These results show that SlERF1 positively modulated the ethylene-dependent pathogenesis defense pathway in tomato. That may be one of the mechanisms by which ERFs enhance plant tolerance to fungal invasion.

Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

MicroRNAs (miRNAs) are a specialized class of small silencing RNAs that regulate gene expression in eukaryotes. In plants, miRNAs negatively regulate target mRNAs containing a highly complementary sequence by either mRNA cleavage or translational repression. As a model plant to study fleshy fruit ripening, miRNA studies in tomato have made great progress recently. MiRNAs were predicted to be involved in nearly all biological processes in tomato, particularly development, differentiation, and biotic and abiotic stress responses. Surprisingly, several miRNAs were verified to be involved in tomato fruit ripening and senescence. Recent studies suggest that miRNAs are related to host-virus interactions, which raises the possibility that miRNAs can be used as diagnostic markers for response to virus infection in tomato plants. In this review, we summarize our current knowledge systematically and advance future directions for miRNA research in tomato.

Regardless of whether a leaf is simple or compound, the mechanism underlying its development will give rise to a full comprehension of plant morphogenesis. The role of Argonaute1 (AGO1) in the development of simple leaves has been established, but its role in the development of compound leaves remains to be characterized. In this paper, a virus-induced gene silencing (VIGS) strategy was used to dramatically down-regulate the expression of AGO1 ortholog in tomatoes, a model plant for research into compound leaves. AGO1-silenced tomato compound leaves exhibited morphological defects of leaf adaxial-abaxial and trichome development. Analysis of global gene expression profiles indicated that the silencing of AGO1 in tomato compound leaf caused significant changes in the expression of several critical genes, including Auxin Response Factor 4 (ARF4) and Non-expressor of PR5 (NPR5), which were involved in adaxial-abaxial formation and IAA15 that was found to contribute to growth of trichomes as well as Gibberellic Acid Insensitive (GAI) which participated in hormone regulation. Collectively, these results shed light on the complicated mechanism by which AGO1 regulates compound leaf development.

Plant virus-induced gene silencing (VIGS) is currently a powerful tool for the study of gene function in plants. Here we report the silencing of LeACS2 by vacuum-infiltration and the tobacco rattle virus (TRV)-based VIGS method, which leads to a significant delay of the postharvest ripening and senescence of tomato fruit. Harvested mature green tomato fruit were vacuum-infiltrated with Agrobacterium strain GV3101 containing pTRV1 and pTRV2-LeACS2. Because of the silencing of LeACS2, the ethylene climacteric and pigment changes were clearly delayed. The onset of fruit ripening and senescence was significantly postponed, and transcription of LeACS2 and ACC synthase activity were also suppressed in treated tomato fruit during storage. The silencing of LeACS2 by vacuum infiltration, however, did not alter the contents assayed at the end of storage. Our results indicate that vacuum infiltration is a highly efficient TRV-based VIGS method to silence LeACS2 in harvested tomato fruit. It can obviously delay ripening and senescence, and is a potential method for postharvest preservation of tomato fruit.

During the past decade, fruit aroma biosynthetic pathways were established in some climacteric fruits, such as tomato, apple, and melon. Inhibition of ethylene biosynthesis or its action in these fruits can reduce the production of fruit volatiles. Furthermore, ethylene partially regulates expression of a few important enzyme genes in fruit volatile biosynthetic pathways. The aim of this review is to bring together recent advances for understanding the regulatory role of ethylene in the biosynthesis of aroma volatiles in some fruits.

Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.

Recent studies have reported that decreased level of DNA cytosine methylation in the global genome was closely related to the initiation of tomato (Solanum lycopersicum) fruit ripening. However, genome-scale analysis of cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in tomato has not been engaged. In this study, 7 C5-MTases and 3 demethylases were identified in tomato genome, which probably contributed to DNA cytosine methylation level in tomato. The 7 C5-MTases were categorized into 4 subgroups, and the 3 demethylases were classified into 2 subgroups based on phylogenetic analyses. Comprehensive analysis of their structure and genomic localization was also performed in this paper. According to online RNA-seq data, 4 S. lycopersicum C5-MTase (SlC5-MTase) genes (SlMET, SlDRM1L1, SlDRM5, SlMET3L) were expressed higher than others, and one DNA demethylase gene (SlDML) was significantly changed during tomato fruit development and ripening. Furthermore, all these five gene expressions at breaker (BK) stage changed with 1-methylcyclopropene (1-MCP) treatment, indicating that they were regulated by ethylene directly or indirectly in tomato fruit. In addition, subcellular localization analysis indicated that SlDRM1L1 and SlDRM5 located in the nucleus might have responsibility for RNA-directed DNA methylation (RdDM). Collectively, this paper provided a framework for gene discovery and functional characterization of C5-MTases and DNA demethylases in other Solanaceae species.

Plants are endowed with a sophisticated defense mechanism that gives signals to plant cells about the immediate danger from surroundings and protects them from pathogen invasion. In the search for the particular proteins involved in fruit defense responses, we report here a comparative analysis of tomato fruit (Solanum lycopersicum cv. Ailsa Craig) infected by Rhizopus nigricans Ehrenb, which is a significant contributor to postharvest rot disease in fresh tomato fruits. In total, four hundred forty-five tomato proteins were detected in common between the non-infected group and infected tomato fruit of mature green. Forty-nine differentially expressed spots in 2-D gels were identified, and were sorted into fifteen functional groups. Most of these proteins participate directly in the stress response process, while others were found to be involved in several equally important biological processes: protein metabolic process, carbohydrate metabolic process, ethylene biosynthesis, and cell death and so on. These responses occur in different cellular components, both intra- and extracellular spaces. The differentially expressed proteins were integrated into several pathways to show the regulation style existing in tomato fruit host. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their roles in pathogen-plant interactions. Collectively results provide evidence that several regulatory pathways contribute to the resistance of tomato fruit to pathogen.

Natural mutants of the Non-ripening (Nor) gene repress the normal ripening of tomato fruit. The molecular mechanism of fruit ripening regulation by the Nor gene is unclear. To elucidate how the Nor gene can affect ripening and fruit quality at the protein level, we used the fruits of Nor mutants and wild-type Ailsa Craig (AC) to perform iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The Nor mutation altered tomato fruit ripening and affected quality in various respects, including ethylene biosynthesis by down-regulating the abundance of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), pigment biosynthesis by repressing phytoene synthase 1 (PSY1), ζ-carotene isomerase (Z-ISO), chalcone synthase 1 (CHS1) and other proteins, enhancing fruit firmness by increasing the abundance of cellulose synthase protein, while reducing those of polygalacturonase 2 (PG2) and pectate lyase (PL), altering biosynthesis of nutrients such as carbohydrates, amino acids, and anthocyanins. Conversely, Nor mutation also enhanced the fruit's resistance to some pathogens by up-regulating the expression of several genes associated with stress and defense. Therefore, the Nor gene is involved in the regulation of fruit ripening and quality. It is useful in the future as a means to improve fruit quality in tomato.

Small RNAs have emerged as critical regulators in the expression and function of eukaryotic genomes at the post-transcriptional level. To elucidate the functions of microRNA (miRNAs) and endogenous small-interfering RNAs (siRNAs) in tomato fruit ripening process, the deep sequencing and bioinformatics methods were combined to parse the small RNAs landscape in three fruit-ripening stages (mature green, breaker and red-ripe) on a whole genome. Two species-specific miRNAs and two members of TAS3 family were identified, 590 putative phased small RNAs and 125 cis-natural antisense (nat-siRNAs) were also found in our results which enriched the tomato small RNAs repository and all of them showed differential expression patterns during fruit ripening. A large amount of the targets of the small RNAs were predicted to be involved in fruit ripening and ethylene pathway. Furthermore, the promoters of the conserved and novel miRNAs were found to contain the conserved motifs of TATA-box and CT microsatellites which were also found in Arabidopsis and rice, and several species-specific motifs were found in parallel.

Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing—sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. Key message SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.

BACKGROUND: The transcription factor RIN (Ripening Inhibitor) belongs to the MADS box family and regulates tomato ripening. Virus-induced gene silencing (VIGS) is a powerful tool for studying gene function and down-regulation of specific gene expression in plants. In this study, VIGS was employed in silencing the LeRIN gene to determine whether and how RIN transcription factor regulated gene expression of ethylene biosynthesis. RESULTS: Tobacco rattle virus (TRV)-LeRIN (LeACS2, LeACS4, LeACO1) VIGS fruits significantly developed green colour phenotypes, whereas control fruits turned red normally. Strikingly, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) activities in the green sections of VIGS fruits were significantly lower than those in control fruits. Dissection of the silenced areas for analysis showed that silencing of LeRIN represses LeACS2, LeACS4 and LeACO1 expression, which consequently leads to inhibited ripening. In particular, the results indicated that RIN transcription factor binds to the promoter of LeACS4 in vitro and in vivo. CONCLUSIONS: The syringe infiltration method of VIGS was successfully applied to silence the LeRIN, LeACS2, LeACS4 and LeACO1 genes in tomato fruits. In addition, the target genes of RIN transcription factor in ethylene biosynthesis were identified. The results are critical for understanding the mechanism of ethylene biosynthesis in tomato fruit ripening. Copyright © 2011 Society of Chemical Industry

BACKGROUND: LE-RIN encodes the RIN (Ripening Inhibitor) transcription factor belonging to the MADS (Mcm1, Agamous, Deficiens, Srf) box family and regulates tomato ripening. The RIN transcription factor plays an indispensable role in ethylene biosynthesis. In the study, we respectively evaluated gene expressions and enzymatic activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) and ethylene production to understand the transcriptional regulation of RIN transcription factor in ethylene biosynthesis. RESULTS: RIN transcription factor generates strong or weak induction of the expression of LE-ACS2, LE-ACS4 and LE-ACO1 associated with ethylene biosynthesis. Exogenous ethylene could positively affect gene expression of mature green AC tomato fruit compared with the control, including LE-ACS2, LE-ACO1 and LE-ACO4. However, LE-ACS6 expression was negatively affected by RIN transcription factor and high ethylene production. Accordingly, the activities of ACC synthase and ACC oxidase and ethylene production were also changed. CONCLUSION: RIN transcription factor has an impact upon ethylene biosynthesis via regulating transcription of LE-ACS2, LE-ACS4, LE-ACO1 and LE-ACS6. These results will help to further clarify the mechanism of tomato fruit development and ripening, and to maintain quality and prolong shelf life of tomato fruit.

Background Fruit ripening is a complex developmental process that depends on a coordinated regulation of numerous genes, including ripening-related transcription factors (TFs), fruit-related microRNAs, DNA methylation and chromatin remodeling. It is known that various TFs, such as MADS-domain, MYB, AP2/ERF and SBP/SPL family proteins play key roles in modulating ripening. However, little attention has been given to members of the large NF-Y TF family in this regard, although genes in this family are known to have important functions in regulating plant growth, development, and abiotic or biotic stress responses. Results In this study, the evolutionary relationship between Arabidopsis thaliana and tomato (Solanum lycopersicum) NF-Y genes was examined to predict similarities in function. Furthermore, through gene expression analysis, 13 tomato NF-Y genes were identified as candidate regulators of fruit ripening. Functional studies involving suppression of NF-Y gene expression using virus induced gene silencing (VIGS) indicated that five NF-Y genes, including two members of the NF-YB subgroup (Solyc06g069310, Solyc07g065500) and three members of the NF-YA subgroup (Solyc01g087240, Solyc08g062210, Solyc11g065700), influence ripening. In addition, subcellular localization analyses using NF-Y proteins fused to a green fluorescent protein (GFP) reporter showed that the three NF-YA proteins accumulated in the nucleus, while the two NF-YB proteins were observed in both the nucleus and cytoplasm. Conclusions In this study, we identified tomato NF-Y genes by analyzing the tomato genome sequence using bioinformatics approaches, and characterized their chromosomal distribution, gene structures, phylogenetic relationship and expression patterns. We also examined their biological functions in regulating tomato fruit via VIGS and subcellular localization analyses. The results indicated that five NF-Y transcription factors play roles in tomato fruit ripening. This information provides a platform for further investigation of their biological functions. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2334-2) contains supplementary material, which is available to authorized users.

Background MicroRNAs (miRNAs), a ubiquitous class of short RNAs, play vital roles in physiological and biochemical processes in plants by mediating gene silencing at post-transcriptional (PTGS) level. Tomato is a model system to study molecular basis of fleshy fruit ripening and senescence, ethylene biosynthesis and signal transduction owing to its genetic and molecular tractability. To study the functions of miRNAs in tomato fruit ripening and senescence, and their possible roles in ethylene response, the next generation sequencing method was employed to identify miRNAs in tomato fruit. Bioinformatics and molecular biology approaches were combined to profile the miRNAs expression patterns at three different fruit ripening stages and by exogenous ethylene treatment. Results In addition to 7 novel miRNA families, 103 conserved miRNAs belonging to 24 families and 10 non-conserved miRNAs matching 9 families were identified in our libraries. The targets of many these miRNAs were predicted to be transcriptional factors. Other targets are known to play roles in the regulation of metabolic processes. Interestingly, some targets were predicted to be involved in fruit ripening and softening, such as Pectate Lyase, beta-galactosidase, while a few others were predicted to be involved in ethylene biosynthesis and signaling pathway, such as ACS, EIN2 and CTR1. The expression patterns of a number of such miRNAs at three ripening stages were confirmed by stem-loop RT-PCR, which showed a strong negative correlation with that of their targets. The regulation of exogenous ethylene on miRNAs expression profiles were analyzed simultaneously, and 3 down-regulated, 5 up-regulated miRNAs were found in this study. Conclusions A combination of high throughput sequencing and molecular biology approaches was used to explore the involvement of miRNAs during fruit ripening. Several miRNAs showed differential expression profiles during fruit ripening, and a number of miRNAs were influenced by ethylene treatment. The results suggest the importance of miRNAs in fruit ripening and ethylene response.