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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.

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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.

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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.

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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.

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The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

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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.

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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.

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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 H2 O2 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.

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Chloroplasts play a crucial role in plant growth and fruit quality. However, the molecular mechanisms of chloroplast development are still poorly understood in fruits. In this study, we investigated the role of the transcription factor SlBEL2 (BEL1-LIKE HOMEODOMAIN 2) in fruit of Solanum lycopersicum (tomato). Phenotypic analysis of SlBEL2 overexpression (OE-SlBEL2) and SlBEL2 knockout (KO-SlBEL2) plants revealed that SlBEL2 has the function of inhibiting green shoulder formation in tomato fruits by affecting the development of fruit chloroplasts. Transcriptome profiling revealed that the expression of chloroplast-related genes such as SlGLK2 and SlLHCB1 changed significantly in the fruit of OE-SlBEL2 and KO-SlBEL2 plants. Further analysis showed that SlBEL2 could not only bind to the promoter of SlGLK2 to inhibit its transcription, but also interacted with the SlGLK2 protein to inhibit the transcriptional activity of SlGLK2 and its downstream target genes. SlGLK2 knockout (KO-SlGLK2) plants exhibited a complete absence of the green shoulder, which was consistent with the fruit phenotype of OE-SlBEL2 plants. SlBEL2 showed an expression gradient in fruits, in contrast with that reported for SlGLK2. In conclusion, our study reveals that SlBEL2 affects the formation of green shoulder in tomato fruits by negatively regulating the gradient expression of SlGLK2, thus providing new insights into the molecular mechanism of fruit green shoulder formation.

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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.

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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.

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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)-SlWIP2 (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.

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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.

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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.

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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.

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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.

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Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

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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.

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