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Long non-coding RNAs (lncRNAs) play crucial roles in various biological processes in plants. However, the functional mechanism of lncRNAs in fruit ripening, particularly the transition from unripe to ripe stages, remains elusive. One such lncRNA1840, reported by our group, was found to have important role in tomato fruit ripening. In the present study, we gain insight into its functional role in fruit ripening. CRISPR-Cas9 mediated lncRNA1840 mutants caused the delayed tomato fruit ripening. Notably, loss function of lncRNA1840 did not directly impact ethylene signaling but rather delay ethylene synthesis. Transcriptomic analysis revealed differences in the expression of ripening related genes in lncRNA1840 mutants, suggesting that it is involved in gene regulation of fruit ripening. We used Chromatin Isolation by RNA Purification (ChIRP)-Seq to identify lncRNA1840 binding sites on chromatin. ChIRP-seq suggested that lncRNA1840 had occupancy on 40 genes, but none of them is differentially expressed genes in transcriptomic analysis, which indicated lncRNA1840 might indirectly modulate the gene expression. ChIRP-mass spectrometry analysis identified potential protein interactors of lncRNA1840, Pre-mRNA processing splicing factor 8, highlighting its involvement in post-transcriptional regulatory pathways. In summary, lncRNA1840 is key player in tomato plant growth and fruit ripening, with multifaceted roles in gene expression and regulatory networks., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Domestication of tomato (Solanum lycopersicum) has led to large variation in fruit size and morphology. The development of the distal end of the fruit is a critical factor in determining its overall shape. However, the intricate mechanisms underlying distal fruit development require further exploration. This study aimed to investigate the regulatory role of an organelle RNA recognition motif (RRM)-containing protein SlORRM2 in tomato fruit morphology development. Mutant plants lacking SlORRM2 exhibited fruits with pointed tips at the distal end. However, this phenotype could be successfully restored through the implementation of a "functional complementation" strategy. Our findings suggest that the formation of pointed tips in the fruits of the CR-slorrm2 mutants is linked to alterations in the development of the ovary and style. We observed a substantial decrease in the levels of indole-3-acetic acid (IAA) and altered expression of IAA-related response genes in the ovary and style tissues of CR-slorrm2. Moreover, our data demonstrated that SlORRM2 plays a role in regulating mitochondrial RNA editing sites, particularly within genes encoding various respiratory chain subunits. Additionally, the CR-slorrm2 mutants exhibited modified organellar morphology and increased levels of reactive oxygen species. These findings provide valuable insights into the mechanisms underlying the formation of fruit pointed tips in tomato and offer genetic resources for tomato breeding., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Main Conclusion: Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases. Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ascorbic acid (AsA) is an important nutrient for human health and disease cures, and it is also a crucial indicator for the quality of fruit and vegetables. As a reductant, AsA plays a pivotal role in maintaining the intracellular redox balance throughout all the stages of plant growth and development, fruit ripening, and abiotic stress responses. In recent years, the de novo synthesis and regulation at the transcriptional level and post-transcriptional level of AsA in plants have been studied relatively thoroughly. However, a comprehensive and systematic summary about AsA-involved biochemical pathways, as well as AsA's physiological functions in plants, is still lacking. In this review, we summarize and discuss the multiple physiological and biochemical functions of AsA in plants, including its involvement as a cofactor, substrate, antioxidant, and pro-oxidant. This review will help to facilitate a better understanding of the multiple functions of AsA in plant cells, as well as provide information on how to utilize AsA more efficiently by using modern molecular biology methods.

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Glycosylation is a widespread glycosyl modification that regulates gene expression and metabolite bioactivity in all life processes of plants. Phosphoribosylation is a special glycosyl modification catalyzed by phosphoribosyltransferase (PRTase), which functions as a key step in the biosynthesis pathway of purine and pyrimidine nucleotides, histidine, tryptophan, and coenzyme NAD(P) + to control the production of these essential metabolites. Studies in the past decades have reported that PRTases are indispensable for plant survival and thriving, whereas the complicated physiological role of PRTases in plant life and their crosstalk is not well understood. Here, we comprehensively overview and critically discuss the recent findings on PRTases, including their classification, as well as the function and crosstalk in regulating plant development, abiotic stress response, and the balance of growth and stress responses. This review aims to increase the understanding of the role of plant PRTase and also contribute to future research on the trade-off between plant growth and stress response.

RNA editing in plant organelles involves numerous C-U conversions, which often restore evolutionarily conserved codons and may generate new translation initiation and termination codons. These RNA maturation events rely on a subset of nuclear-encoded protein cofactors. Here, we provide evidence of the role of SlRIP1b on RNA editing of mitochondrial transcripts in tomato (Solanum lycopersicum) plants. SlRIP1b is a RIP/MORF protein that was originally identified as an interacting partner of the organellar editing factor SlORRM4. Mutants of SlRIP1b, obtained by CRISPR/Cas9 strategy, exhibited abnormal carpel development and grew into fruit with more locules. RNA-sequencing revealed that SlRIP1b affects the C-U editing of numerous mitochondrial pre-RNA transcripts and in particular altered RNA editing of various cytochrome c maturation (CCM)-related genes. The slrip1b mutants display increased H 2 O 2 and aberrant mitochondrial morphologies, which are associated with defects in cytochrome c biosynthesis and assembly of respiratory complex III. Taken together, our results indicate that SlRIP1b is a global editing factor that plays a key role in CCM and oxidative phosphorylation system biogenesis during fruit development in tomato plants. These data provide important insights into the molecular roles of organellar RNA editing factors during fruit development., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

In flowering plants, sepals play important roles in the development of flowers and fruit, and both processes are regulated by MADS-box (MADS) transcription factors (TFs). SlMADS1 was previously reported to act as a negative regulator of fruit ripening. In this study, expression analysis shown that its transcripts were very highly expressed during the development of sepals. To test the role of SlMADS1, we generated KO-SlMADS1 (knock-out) tomato mutants by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology and over-expression of SlMADS1 (OE-SlMADS1). The sepals and individual cells of KO-SlMADS1 mutants were significantly elongated, compared with the wild type (WT), whereas the sepals of OE-SlMADS1 tomatoes were significantly shorter and their cells were wider. RNA-seq (RNA-sequencing) of sepal samples showed that ethylene-, gibberellin-, auxin-, cytokinin- and cell wall metabolism-related genes were significantly affected in both KO-SlMADS1 and OE-SlMADS1 plants with altered sepal size. Since SlMACROCALYX (MC) is known to regulate the development of tomato sepals, we also studied the relationship between SlMC and SlMADS1 and the result showed that SlMADS1 interacts directly with SlMC. In addition, we also found that manipulating SlMADS1 expression alters the development of tomato plant leaves, roots and plant height. These results enrich our understanding of sepal development and the function of SlMADS1 throughout the plant., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tomato ripening is a complex and dynamic process coordinated by many regulatory elements, including plant hormones, transcription factors, and numerous ripening-related RNAs and proteins. Although recent studies have shown that some RNA-binding proteins are involved in the regulation of the ripening process, understanding of how RNA-binding proteins affect fruit ripening is still limited. Here, we report the analysis of a glycine-rich RNA-binding protein, RZ1A-Like (RZ1AL), which plays an important role in tomato ripening, especially fruit coloring. To analyze the functions of RZ1AL in fruit development and ripening, we generated knockout cr-rz1al mutant lines via the CRISPR/Cas9 gene-editing system. Knockout of RZ1AL reduced fruit lycopene content and weight in the cr-rz1al mutant plants. RZ1AL encodes a nucleus-localized protein that is associated with Cajal-related bodies. RNA-seq data demonstrated that the expression levels of genes that encode several key enzymes associated with carotenoid biosynthesis and metabolism were notably downregulated in cr-rz1al fruits. Proteomic analysis revealed that the levels of various ribosomal subunit proteins were reduced. This could affect the translation of ripening-related proteins such as ZDS. Collectively, our findings demonstrate that RZ1AL may participate in the regulation of carotenoid biosynthesis and metabolism and affect tomato development and fruit ripening., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Many glycine-rich RNA-binding proteins (GR-RBPs) have critical functions in RNA processing and metabolism. Here, we describe a role for the tomato (Solanum lycopersicum) GR-RBP SlRBP1 in regulating mRNA translation. We found that SlRBP1 knockdown mutants (slrbp1) displayed reduced accumulation of total chlorophyll and impaired chloroplast ultrastructure. These phenotypes were accompanied by deregulation of the levels of numerous key transcripts associated with chloroplast functions in slrbp1. Furthermore, native RNA immunoprecipitation-sequencing (nRIP-seq) recovered 61 SlRBP1-associated RNAs, most of which are involved in photosynthesis. SlRBP1 binding to selected target RNAs was validated by nRIP-qPCR. Intriguingly, the accumulation of proteins encoded by SlRBP1-bound transcripts, but not the mRNAs themselves, was reduced in slrbp1 mutants. Polysome profiling followed by RT-qPCR assays indicated that the polysome occupancy of target RNAs was lower in slrbp1 plants than in wild-type. Furthermore, SlRBP1 interacted with the eukaryotic translation initiation factor SleIF4A2. Silencing of SlRBP1 significantly reduced SleIF4A2 binding to SlRBP1-target RNAs. Taking these observations together, we propose that SlRBP1 binds to and channels RNAs onto the SleIF4A2 translation initiation complex and promotes the translation of its target RNAs to regulate chloroplast functions., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Postharvest deterioration is among the major challenges for the fruit industry. Regulation of the fruit softening rate is an effective strategy for extending shelf-life and reducing the economic losses due postharvest deterioration. The tomato myoinositol monophosphatase 3 gene SlIMP3, which showed highest expression level in fruit, was expressed and purified. SlIMP3 demonstrated high affinity with the L-Gal 1-P and D-Ins 3-P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol. Overexpression of SlIMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, delayed fruit softening, decreased water loss, and extended shelf-life. Overexpression of SlIMP3 also increased uronic acid, rhamnose, xylose, mannose, and galactose content in cell wall of fruit. Treating fruit with myoinositol obtained similar fruit phenotypes of SlIMP3-overexpressed fruit, with increased cell wall thickness and delayed fruit softening. Meanwhile, overexpression of SlIMP3 conferred tomato fruit tolerance to Botrytis cinerea. The function of SlIMP3 in cell wall biogenesis and fruit softening were also verified using another tomato species, Ailsa Craig (AC). Overexpression of SlDHAR in fruit increased AsA content, but did not affect the cell wall thickness or fruit firmness and softening. The results support a critical role for SlIMP3 in AsA biosynthesis and cell wall biogenesis, and provide a new method of delaying tomato fruit softening, and insight into the link between AsA and cell wall metabolism., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Functional gene transcription mainly occurs in the nucleus and has a significant role in plant physiology. The isolation of nuclei tagged in specific cell type (INTACT) technique provides an efficient and stable nucleus purification method to investigate the dynamic changes of nuclear gene transcriptional expression. However, the application of traditional INTACT in plants is still limited to seedlings or root cells because of severe chloroplast pollution. In this study, we proposed a newly designed and simplified INTACT based on mas-enhanced GFP (eGFP)- Sl WIP2 (gwINTACT) for nuclear purification in tomato ( Solanum lycopersicum ) leaves, flowers, and fruits for the first time. The yield of the nucleus purified using gwINTACT from transgenic tomato leaves was doubled compared with using a traditional INTACT procedure, accompanied by more than 95% removal of chloroplasts. Relative gene expression of ethylene-related genes with ethylene treatment was reevaluated in gwINTACT leaves to reveal more different results from the traditional gene expression assay based on total RNA. Therefore, establishing the gwINTACT system in this study facilitates the precise deciphering of the transcriptional status in various tomato tissues, which lays the foundation for the further experimental study of nucleus-related molecular regulation on fruit ripening, such as ChIP-seq and ATAC-seq., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chu, Gong, Wu, Liu, Du, Ma, Fu, Zhu, Qu and Zhu.)

Plant sterols are important components of the cell membrane and lipid rafts, which play a crucial role in various physiological and biochemical processes during development and stress resistance in plants. In recent years, many studies in higher plants have been reported in the biosynthesis pathway of plant sterols, whereas the knowledge about the regulation and accumulation of sterols is not well understood. In this review, we summarize and discuss the recent findings in the field of plant sterols, including their biosynthesis, regulation, functions, as well as the mechanism involved in abiotic stress responses. These studies provide better knowledge on the synthesis and regulation of sterols, and the review also aimed to provide new insights for the global role of sterols, which is liable to benefit future research on the development and abiotic stress tolerance in plant.

This study compares the characteristics of a self-report questionnaire (SRQ) and eye tracking (ET) based on a simple human-beverage visual cognition model. The young participants were mainly defined by their gender and body mass index (BMI). The beverage samples consisted of milk, coffee, cup, and coaster. SRQs allow the participants to clearly express their overall cognition of the samples in the form of vocabulary, while ET captures their hidden thinking process. The analysis, using a random forest (RF) classifier, found that participant parameters (gender and BMI) played a more important role for SRQ, while ET was related to beverage parameters (color and shape). This work reiterates that these two methods have their advantages and complement each other in food sensory analysis.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Patients with renal failure suffer from symptoms caused by uraemic toxins, possibly of gut microbial origin, as deduced from studies in animals. The aim of the study is to characterise relationships between the intestinal microbiome composition, uraemic toxins and renal failure symptoms in human end-stage renal disease (ESRD)., Design: Characterisation of gut microbiome, serum and faecal metabolome and human phenotypes in a cohort of 223 patients with ESRD and 69 healthy controls. Multidimensional data integration to reveal links between these datasets and the use of chronic kidney disease (CKD) rodent models to test the effects of intestinal microbiome on toxin accumulation and disease severity., Results: A group of microbial species enriched in ESRD correlates tightly to patient clinical variables and encode functions involved in toxin and secondary bile acids synthesis; the relative abundance of the microbial functions correlates with the serum or faecal concentrations of these metabolites. Microbiota from patients transplanted to renal injured germ-free mice or antibiotic-treated rats induce higher production of serum uraemic toxins and aggravated renal fibrosis and oxidative stress more than microbiota from controls. Two of the species, Eggerthella lenta and Fusobacterium nucleatum , increase uraemic toxins production and promote renal disease development in a CKD rat model. A probiotic Bifidobacterium animalis decreases abundance of these species, reduces levels of toxins and the severity of the disease in rats., Conclusion: Aberrant gut microbiota in patients with ESRD sculpts a detrimental metabolome aggravating clinical outcomes, suggesting that the gut microbiota will be a promising target for diminishing uraemic toxicity in those patients., Trial Registration Number: This study was registered at ClinicalTrials.gov (NCT03010696)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Ripening of tomato fruit is a complex tightly orchestrated developmental process that involves multiple physiological and metabolic changes that render fruit attractive, palatable and nutritious. Ripening requires initiation, activation and coordination of key pathways at the transcriptional and post-transcriptional levels that lead to ethylene synthesis and downstream ripening events determining quality. We studied wild-type, Gr and r mutant fruits at the coding and non-coding transcriptomic, metabolomic and genome methylation levels. Numerous differentially expressed non-coding RNAs were identified and quantified and potential competing endogenous RNA regulation models were constructed. Multiple changes in gene methylation were linked to the ethylene pathway and ripening processes. A combined analysis of changes in genome methylation, long non-coding RNAs, circular RNAs, micro-RNAs and fruit metabolites revealed many differentially expressed genes (DEGs) with differentially methylated regions encoding transcription factors and key enzymes related to ethylene or carotenoid pathways potentially targeted by differentially expressed non-coding RNAs. These included ACO2 (targeted by MSTRG.59396.1 and miR396b), CTR1 (targeted by MSTRG.43594.1 and miR171b), ERF2 (targeted by MSTRG.183681.1), ERF5 (targeted by miR9470-3p), PSY1 (targeted by MSTRG.95226.7), ZISO (targeted by 12:66127788|66128276) and NCED (targeted by MSTRG.181568.2). Understanding the functioning of this intricate genetic regulatory network provides new insights into the underlying integration and relationships between the multiple events that collectively determine the ripe phenotype., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The tomato non-ripening (nor) mutant generates a truncated 186-amino-acid protein (NOR186) and has been demonstrated previously to be a gain-of-function mutant. Here, we provide more evidence to support this view and answer the open question of whether the NAC-NOR gene is important in fruit ripening. Overexpression of NAC-NOR in the nor mutant did not restore the full ripening phenotype. Further analysis showed that the truncated NOR186 protein is located in the nucleus and binds to but does not activate the promoters of 1-aminocyclopropane-1-carboxylic acid synthase2 (SlACS2), geranylgeranyl diphosphate synthase2 (SlGgpps2), and pectate lyase (SlPL), which are involved in ethylene biosynthesis, carotenoid accumulation, and fruit softening, respectively. The activation of the promoters by the wild-type NOR protein can be inhibited by the mutant NOR186 protein. On the other hand, ethylene synthesis, carotenoid accumulation, and fruit softening were significantly inhibited in CR-NOR (CRISPR/Cas9-edited NAC-NOR) fruit compared with the wild-type, but much less severely affected than in the nor mutant, while they were accelerated in OE-NOR (overexpressed NAC-NOR) fruit. These data further indicated that nor is a gain-of-function mutation and NAC-NOR plays a significant role in ripening of wild-type fruit., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Tomato has emerged as the model system for investigations into the regulation of fleshy-fruit ripening and senescence, and the ripening process involving the coordinated regulation at the gene/chromatin/epigenetic, transcriptional, post-transcriptional and protein levels. Noncoding RNAs play important roles in fruit ripening as important transcriptional and post-transcriptional regulatory factors. In this review, we systematically summarize the recent advances in the regulation of tomato fruit ripening involved in ethylene biosynthesis and signal transduction, fruit pigment accumulation, fruit flavor and aroma, fruit texture by noncoding RNAs and their coordinate regulatory network model were set up and also suggest future directions for the functional regulations of noncoding RNAs on tomato fruit ripening.

RIPENING INHIBITOR (RIN)-deficient fruits generated by CRISPR/Cas9 initiated partial ripening at a similar time to wild-type (WT) fruits but only 10% WT concentrations of carotenoids and ethylene (ET) were synthesized. RIN-deficient fruit never ripened completely, even when supplied with exogenous ET. The low amount of endogenous ET that they did produce was sufficient to enable ripening initiation and this could be suppressed by the ET perception inhibitor 1-MCP. The reduced ET production by RIN-deficient tomatoes was due to an inability to induce autocatalytic system-2 ET synthesis, a characteristic feature of climacteric ripening. Production of volatiles and transcripts of key volatile biosynthetic genes also were greatly reduced in the absence of RIN. By contrast, the initial extent and rates of softening in the absence of RIN were similar to WT fruits, although detailed analysis showed that the expression of some cell wall-modifying enzymes was delayed and others increased in the absence of RIN. These results support a model where RIN and ET, via ERFs, are required for full expression of ripening genes. Ethylene initiates ripening of mature green fruit, upregulates RIN expression and other changes, including system-2 ET production. RIN, ET and other factors are required for completion of the full fruit-ripening programme., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Pepper is an important vegetable worldwide and is a model plant for nonclimacteric fleshy fruit ripening. Drastic visual changes and internal biochemical alterations are involved in fruit coloration, flavor, texture, aroma, and palatability to animals during the pepper fruit ripening process. To explore the regulation of bell pepper fruit ripening by noncoding RNAs (ncRNAs), we examined their expression profiles; 43 microRNAs (miRNAs), 125 circular RNAs (circRNAs), 366 long noncoding RNAs (lncRNAs), and 3266 messenger RNAs (mRNAs) were differentially expressed (DE) in mature green and red ripe fruit. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the targets of the DE ncRNAs and DE mRNAs included several kinds of transcription factors (TFs) (ERF, bHLH, WRKY, MYB, NAC, bZIP, and ARF), enzymes involved in cell wall metabolism (beta-galactosidase, beta-glucosidase, beta-amylase, chitinase, pectate lyase (PL), pectinesterase (PE) and polygalacturonase (PG)), enzymes involved in fruit color accumulation (bifunctional 15-cis-phytoene synthase, 9-cis-epoxycarotenoid dioxygenase, beta-carotene hydroxylase and carotene epsilon-monooxygenase), enzymes associated with fruit flavor and aroma (glutamate-1-semialdehyde 2,1-aminomutase, anthocyanin 5-aromatic acyltransferase, and eugenol synthase 1) and enzymes involved in the production of ethylene (ET) (ACO1/ACO4) as well as other plant hormones such as abscisic acid (ABA), auxin (IAA), and gibberellic acid (GA). Based on accumulation profiles, a network of ncRNAs and mRNAs associated with bell pepper fruit ripening was developed that provides a foundation for further developing a more refined understanding of the molecular biology of fruit ripening.

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., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that they have no conflict of interest.

Long-Non-Coding RNAs (LncRNAs) are a class of non-coding endogenous RNAs contributing to numerous biological processes. LeERF1 is a tomato ethylene response factor (ERF) near the end of the ethylene signal transduction pathway. To identify lncRNAs in tomato and elucidate their roles in ethylene signaling, deep sequencing was deployed in over-expression and repression LeERF1 transgenic and control tomato fruits. A total of 397 lncRNAs were identified, including 169 tomato lncRNAs that had not previously been identified. Among these, 12 were differentially expressed between the transgenic and control tomato fruits. Numerous lncRNA target genes were identified including many associated with ethylene signaling including auxin response factors and auxin-induced proteins, F-box proteins, ERFs and MADS-box proteins. In addition, two lncRNAs were found to be the precursor of three miRNAs and four lncRNAs could be targeted by five miRNAs. We propose a regulatory model highlighting the relationships between lncRNAs and their targets involved in ethylene signal transduction which establishes a foundation for addressing the role of LncRNAs in ethylene response., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cowpea is an important horticultural crop in tropical and subtropical areas of Asia, Africa, and Latin America, as well as parts of southern Europe and Central and South America. Chilling injury is a common physiological hazard of cowpea in cold chain logistics which reduce the cowpea pods nutritional quality and product value. However, the molecular mechanism involved in chilling injury remains unclear in cowpea pods. RNA-Seq and sRNA-Seq technologies were employed to decipher the miRNAs and mRNAs expression profiles and their regulatory networks in cowpea pods involved in chilling stress. Differentially expressed miRNAs and mRNA profiles were obtained based on cluster analysis, miRNAs and target genes were found to show coherent relationships in the regulatory networks of chilling injury. Furthermore, we found that numerous miRNAs and nat-siRNAs' targets were predicted to be key enzymes involved in the redox reactions such as POD, CAT, AO and LOX, energy metabolism such as ATPase, FAD and NAD related enzymes and different transcription factors such as WRKY, bHLH, MYB, ERF and NAC which play important roles in chilling injury., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that they have no conflict of interest.

Long-non-coding RNA (LncRNA) is a kind of non-coding endogenous RNA that plays essential roles in diverse biological processes and various stress responses. To identify and elucidate the intricate regulatory roles of lncRNAs in chilling injury in tomato fruit, deep sequencing and bioinformatics methods were performed here. After strict screening, a total of 1411 lncRNAs were identified. Among these lncRNAs, 239 of them were significantly differentially expressed. A large amount of target genes were identified and many of them were found to code chilling stress related proteins, including redox reaction related enzyme, important enzymes about cell wall degradation, membrane lipid peroxidation related enzymes, heat and cold shock protein, energy metabolism related enzymes, salicylic acid and abscisic acid metabolism related genes. Interestingly, 41 lncRNAs were found to be the precursor of 33 miRNAs, and 186 lncRNAs were targets of 45 miRNAs. These lncRNAs targeted by miRNAs might be potential ceRNAs. Particularly, a sophisticated regulatory model including miRNAs, lncRNAs and their targets was set up. This model revealed that some miRNAs and lncRNAs may be involved in chilling injury, which provided a new perspective of lncRNAs role., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Increasing evidence suggests that long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) have roles during biotic and abiotic stress, though their exact contributions remain unclear. To explore their biological functions in response to chilling in bell pepper, we examined their accumulation profiles by deep sequencing and identified 380 lncRNAs, 36 circRNAs, 18 miRNAs, and 4128 differentially expressed mRNAs in the chilled versus the non-chilled fruit. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed differentially expressed genes and putative ncRNA targets, including transcription factors of multiple classes, such as myeloblastosis (MYB), basic helix-loop-helix (bHLH), and ethylene response factor (ERF) transcription factors (TFs), enzymes involved in bio-oxidation and oxidative phosphorylation (serine/threonine-protein kinase, polyphenol oxidase, catalase, peroxidase, lipoxygenase, and ATPase), and cell wall metabolism-related enzymes (beta-galactosidase, pectate lyase, pectinesterase, and polygalacturonase). On the basis of the accumulation profiles, a network of putatively interacting RNAs associated with bell pepper chilling was developed, which pointed to ncRNAs that could provide the foundation for further developing a more refined understanding of the molecular response to chilling injury.

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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

DNA methylation is an essential feature of epigenetic regulation and plays a role in various physiological and biochemical processes at CG, CHG, and CHH sites in plants. LeERF1 is an ethylene response factor (ERF) found in tomatoes which plays an important role in ethylene signal transduction. To explore the characteristics of DNA methylation in the ethylene pathway, sense-/antisense-LeERF1 transgenic tomato fruit were chosen for deep sequencing and bioinformatics parsing. The methylation type with the greatest distribution was CG, (71.60⁻72.80%) and CHH was found least frequently (10.70⁻12.50%). The level of DNA methylation was different among different tomato genomic regions. The differentially methylated regions (DMRs) and the differentially expressed genes (DEGs) were conjointly analyzed and 3030 different expressed genes were found, of which several are involved in ethylene synthesis and signaling transduction (such as ACS , ACO , MADS-Box , ERFs , and F-box ). Furthermore, the relationships between DNA methylation and microRNAs (miRNAs) were also deciphered, providing basic information for the further study of DNA methylation and small RNAs involved in the ethylene pathway., Competing Interests: The authors declare no conflict of interest.

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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

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.

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., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Circular RNAs (circRNAs) are a large class of non-coding endogenous RNAs that could act as competing endogenous RNAs (ceRNAs) to terminate the mRNA targets' suppression of miRNAs. To elucidate the intricate regulatory roles of circRNAs in the ethylene pathway in tomato fruit, deep sequencing and bioinformatics methods were performed. After strict screening, a total of 318 circRNAs were identified. Among these circRNAs, 282 were significantly differentially expressed among wild-type and sense-/antisense-LeERF1 transgenic tomato fruits. Besides, 1254 target genes were identified and a large amount of them were found to be involved in ethylene pathway. In addition, a sophisticated regulatory model consisting of circRNAs, target genes and ethylene was set up. Importantly, 61 circRNAs were found to be potential ceRNAs to combine with miRNAs and some of the miRNAs had been revealed to participate in the ethylene signaling pathway. This research further raised the possibility that the ethylene pathway in tomato fruit may be under the regulation of various circRNAs and provided a new perspective of the roles of circRNAs., (© 2017 Scandinavian Plant Physiology Society.)

Plant genomes encode diverse small RNA classes that function in distinct gene-silencing pathways. To elucidate the intricate regulation of microRNAs (miRNAs) and endogenous small-interfering RNAs (siRNAs) in response to chilling injury in tomato fruit, the deep sequencing and bioinformatic methods were combined to decipher the small RNAs landscape in the control and chilling-injured groups. Except for the known miRNAs and ta-siRNAs, 85 novel miRNAs and 5 ta-siRNAs members belonging to 3 TAS families (TAS5, TAS9 and TAS10) were identified, 34 putative phased small RNAs and 740 cis/trans-natural antisense small-interfering RNAs (nat-siRNAs) were also found in our results which enriched the tomato small RNAs repository. A large number of genes targeted by those miRNAs and siRNAs were predicted to be involved in the chilling injury responsive process and five of them were verified via degradome sequencing. Based on the above results, a regulatory model that comprehensively reveals the relationships between the small RNAs and their targets was set up. This work provides a foundation for further study of the regulation of miRNAs and siRNAs in the plant in response to chilling injury., (© 2016 Scandinavian Plant Physiology Society.)

Small RNAs are a class of short non-coding endogenous RNAs that play essential roles in many biological processes. Recent studies have reported that microRNAs (miRNAs) are also involved in ethylene signaling in plants. LeERF1 is one of the ethylene response factors (ERFs) in tomato that locates in the downstream of ethylene signal transduction pathway. To elucidate the intricate regulatory roles of small RNAs in ethylene signaling pathway in tomato, the deep sequencing and bioinformatics methods were combined to decipher the small RNAs landscape in wild and sense-/antisense- LeERF1 transgenic tomato fruits. Except for the known miRNAs, 36 putative novel miRNAs, 6 trans-acting short interfering RNAs (ta-siRNAs), and 958 natural antisense small interfering RNAs (nat-siRNAs) were also found in our results, which enriched the tomato small RNAs repository. Among these small RNAs, 9 miRNAs, and 12 nat-siRNAs were differentially expressed between the wild and transgenic tomato fruits significantly. A large amount of target genes of the small RNAs were identified and some of them were involved in ethylene pathway, including AP2 TFs, auxin response factors, F-box proteins, ERF TFs, APETALA2-like protein, and MADS-box TFs. Degradome sequencing further confirmed the targets of miRNAs and six novel targets were also discovered. Furthermore, a regulatory model which reveals the regulation relationships between the small RNAs and their targets involved in ethylene signaling was set up. This work provides basic information for further investigation of the function of small RNAs in ethylene pathway and fruit ripening.

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Various circular RNAs (circRNAs) are newly identified in animals and plants through high-throughput deep sequencing which play important roles in miRNA function and transcriptional controlling. However, little is known regarding circRNAs in tomato fruit. In this study, we systematically parse the circRNAs in the whole genome using the combination methods of deep sequencing and bioinformatics. In all, 854 circRNAs were identified in our results, among them, 163 circRNAs exhibit chilling responsive expression. Intriguingly, several circRNAs were predicted to involved in chilling responsive process, such as redox reaction, cell wall degradation, Arginine and polyamine metabolism, heat and cold shock protein, energy metabolism, Jasmonic acid and abscisic acid metabolism, low temperature and salt responsive protein and low temperature-induced transcription factors(CBF and WRKY). Furthermore, 102 circRNAs were found to act as the corresponding 24 miRNAs sponges in tomato. These discoveries deciphered the unexpected complexity of the regulatory circRNAs and may open a window for understanding the functions of circRNAs in plants., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

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 Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)