Relationships between genome methylation, levels of non-coding RNAs, mRNAs and metabolites in ripening tomato fruit.

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.

Noncoding RNAs: functional regulatory factors in tomato fruit ripening.

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.

An untargeted metabolomic approach reveals significant postharvest alterations in vitamin metabolism in response to LED irradiation in pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee).

The main objective of this study was to investigate the effect of low-level light emitting diode (LED) irradiation on the metabolite profile of pak-choi. A total of 633 different molecular features (MFs) were identified among sample groups (initial, dark-treated, light-treated) using an untargeted metabolomic approach. The identified metabolites were associated with 24 different metabolic pathways. Four of the pathways including carbon pool by folate, folate biosynthesis, thiamine metabolism, and glutathione metabolism, all of which are associated with vitamin biosynthesis, changed significantly. Metabolites in four of the pathways exhibited significant differences from the control in response to LED irradiation. Additionally, porphyrin and chlorophyll metabolism, as well as glucosinolate biosynthesis, riboflavin metabolism, and carotenoid biosynthesis were positively induced by LED irradiation. These results indicate that postharvest LED illumination represents a potential tool for modifying the metabolic profile of pak-choi to maintain quality and nutritional levels.

Effect of low-temperature conditioning combined with methyl jasmonate treatment on the chilling resistance of eggplant (Solanum melongena L.) fruit.

Chilling injury (CI) can injure harvested eggplants and lead to a reduction in postharvest quality. The present study examined the effect of low-temperature conditioning (LTC) combined with a methyl jasmonate (MeJA) treatment on CI by analyzing the visual appearance and physiology of eggplants stored at 4 °C. Results indicated that treatment of eggplants with LTC + MeJA effectively maintained the visual quality of eggplants, inhibited a decline in chlorophyll and total phenolics, delayed the accumulation of malondialdehyde (MDA), decreased polyphenol oxidase (PPO) gene expression and enzyme activity, and enhanced the activity of the antioxidant enzymes, catalase (CAT) and peroxidase (POD), as well as the expression of their corresponding genes. Collectively, the data indicate that LTC combined with MeJA effectively improves the CI tolerance of postharvest eggplant fruit stored at 4 °C, by enhancing the activity and relative gene expression of antioxidant enzymes.

Analysis of the Coding and Non-Coding RNA Transcriptomes in Response to Bell Pepper Chilling.

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.

Integrative analysis of circRNAs acting as ceRNAs involved in ethylene pathway in tomato.

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.

SRNAome and degradome sequencing analysis reveals specific regulation of sRNA in response to chilling injury in tomato fruit.

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.

Deciphering the roles of circRNAs on chilling injury in tomato.

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.

Whole-transcriptome RNA sequencing reveals changes in amino acid metabolism induced in harvested broccoli by red LED irradiation.

Whole-transcriptomic profiling combined with amino acid analysis were conducted in order to gain a better understanding of global changes in amino acid metabolism induced in broccoli by red LED irradiation. The results showed that the contents of almost all 16 amino acids in postharvest broccoli were maintained under red LED illumination. The red LED irradiation enhanced the anabolism of amino acid, including the biosynthesis of aromatic amino acids by upregulating the genes' expression in the shikimate pathway, as well as by upregulating the genes' expression which encoding biosynthetic enzymes in the branched-chain amino acid biosynthesis pathway. Red LED irradiation induced the expression of genes encoding aspartate aminotransferase, which plays a role in Asp synthesis, aspartate kinase, which functions in aspartate metabolism, and a cytoplasmic aspartate aminotransferase that converts 2-Oxoglutarate into Glu. Genes encoding imidazole glycerol-phosphate synthase and histidinol-phosphatase, which function in the His biosynthesis pathway, were also upregulated. According to our results, red LED irradiation delays broccoli's yellowing and senescence by regulating amino acid metabolism. These results enhance our understanding of the role of amino acid metabolism in the senescence of broccoli and the mechanism of red LED irradiation to alter amino acid metabolism in harvested broccoli.

The Microbial Metagenome of Eluates Obtained From the Surface of Broccoli Heads Subjected to Different Light Treatments.

Foodborne illnesses present a major threat to public health and are frequently attributed to foodborne pathogens present on fresh produce. Some opportunistic pathogens of broccoli are also responsible for causing head rot. Three different light treatments, UV-C, red LED (50 µml/m2/s), and UV-C + LED were used to treat broccoli prior to or during storage. Following the light treatments, microorganisms present in eluates obtained from the surface of broccoli heads were characterized using a metagenomic approach. Metagenomic DNA libraries were subjected to high-throughput sequencing on an Illumina Hiseq platform. Results indicated that the combined treatment of LED red light and UV-C provided the best sensory preservation of broccoli, followed by LED red light and then UV-C. The bacterial communities in the eluates obtained from the surface of broccoli heads in all three light treatments were primarily represented at the phylum level by Proteobacteria and Firmicutes, while fungal communities were primarily represented by Ascomycota and Basidiomycota. Further analysis indicated that the all three light treatments reduced the presence of foodborne pathogens and bacterial taxa responsible for broccoli spoilage. While UV-C had a significant inhibitory effect on Botrytis cinerea, the light treatments increased the relative abundance of Pseudomonas fluorescens. Results indicate that a metagenomic approach can be used to detect pathogenic bacteria and fungi on fresh vegetables and assess the impact of management practices, such as light treatments, designed to maintain postharvest quality, on the composition of the microbiome present on the surface of harvested produce.

Comparative proteomic analysis of wild-type and a SlETR-3 (Nr) mutant reveals an ethylene-induced physiological regulatory network in fresh-cut tomatoes.

Ethylene plays a crucial role in regulating fruit ripening, quality, and defense response. However, the mechanism(s) responsible for wound-induced ethylene regulation of fruit physiology at a network level is unclear. We used mass spectrometry (MS) to identify differences in the physiological response between fresh-cut fruits of wild-type (WT) tomato and an ethylene receptor mutant (SlETR-3) (also referred to as Nr) during storage. We found that Nr mutants exhibited better appearance and quality, as well as higher ethylene levels during the first 3 d of storage at 4 °C. Thirty-seven (0 h), eighty-two (12 h) and twelve (24 h) differentially abundant proteins were identified between the fresh-cut slices of the two genotypes during storage at the designated timepoints. In particular, antioxidant enzymes, such as ascorbate peroxidase, glutathione S-transferase, and peroxiredoxin were highly expressed in WT fruit, which was associated with higher H2O2 production, and high levels of transcription of cell-wall degrading enzymes. Leucine aminopeptidase, a marker enzyme for response to wounding exhibited higher levels in the Nr mutant, which is consistent with its higher production of ethylene. Collectively, our results provide a deeper insight into the ethylene-induced physiological regulatory network that is activated in fresh-cut tomatoes.

Cucurbitaceae genome evolution, gene function and molecular breeding.

The Cucurbitaceae is one of the most genetically diverse plant families in the world. Many of them are important vegetables or medicinal plants and are widely distributed worldwide. The rapid development of sequencing technologies and bioinformatic algorithms has enabled the generation of genome sequences of numerous important Cucurbitaceae species. This has greatly facilitated research on gene identification, genome evolution, genetic variation and molecular breeding of cucurbit crops. So far, genome sequences of 18 different cucurbit species belonging to tribes Benincaseae, Cucurbiteae, Sicyoeae, Momordiceae and Siraitieae have been deciphered. This review summarizes the genome sequence information, evolutionary relationship, and functional genes associated with important agronomic traits (e.g., fruit quality). The progress of molecular breeding in cucurbit crops and prospects for future applications of Cucurbitaceae genome information are also discussed.

Effect of folic acid on the postharvest physiology of broccoli during storage.

The objective of the present study was to explore the effect of folic acid on the postharvest physiology of broccoli placed in storage. Broccoli heads were immersed in 5 mg L-1 folic acid for 10 min, then stored at 20 ± 1 °C for 4 days. Results indicated that the postharvest treatment of broccoli with folic acid decreased the rate of flower opening and yellowing, inhibited weight loss, reduced the level of respiration, as well as ethylene generation. Folic acid-treated broccoli maintained their level of chlorophyll, total soluble solids, vitamin C, total phenolics, flavonoids, glucosinolate, and folic acid. Treated broccoli also exhibited reduced accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS). Concomitantly, antioxidant enzyme activity and corresponding gene expression were also enhanced. In contrast, chlorophyll-degrading enzyme gene expression was suppressed. These results indicated that folic acid treatment of broccoli could be used to prolong shelf-life.

Combined genomic, transcriptomic, and metabolomic analyses provide insights into chayote (Sechium edule) evolution and fruit development.

Chayote (Sechium edule) is an agricultural crop in the Cucurbitaceae family that is rich in bioactive components. To enhance genetic research on chayote, we used Nanopore third-generation sequencing combined with Hi-C data to assemble a draft chayote genome. A chromosome-level assembly anchored on 14 chromosomes (N50 contig and scaffold sizes of 8.40 and 46.56 Mb, respectively) estimated the genome size as 606.42 Mb, which is large for the Cucurbitaceae, with 65.94% (401.08 Mb) of the genome comprising repetitive sequences; 28,237 protein-coding genes were predicted. Comparative genome analysis indicated that chayote and snake gourd diverged from sponge gourd and that a whole-genome duplication (WGD) event occurred in chayote at 25 ± 4 Mya. Transcriptional and metabolic analysis revealed genes involved in fruit texture, pigment, flavor, flavonoids, antioxidants, and plant hormones during chayote fruit development. The analysis of the genome, transcriptome, and metabolome provides insights into chayote evolution and lays the groundwork for future research on fruit and tuber development and genetic improvements in chayote.

Revealing the Specific Regulations of Brassinolide on Tomato Fruit Chilling Injury by Integrated Multi-Omics.

Tomato fruit is susceptible to chilling injury (CI) when stored at low temperatures, limiting its storage potential, and resulting in economic loss if inappropriate temperatures are used. Brassinolide (BR) is a plant growth regulator that is known to decrease the susceptibility of fruit to CI. In this study, transcriptome, metabolome, and proteome analysis revealed the regulation mechanism of BR treatment in alleviating tomato fruit CI. The results showed that the differentially expressed metabolites mainly included amino acids, organic acids, carbohydrates, and lipids. Differentially expressed genes (DEGs) were involved in plant cold stress response (HSFA3, SHSP, and TPR), fruit redox process (POD, PAL, and LOX), related to the fruit texture (CESA, ß-Gal, and PAE), plant hormone signal transduction (ACS3, ARF, and ERF,), transcription factors (TCP, bHLH, GATA). Moreover, differentially expressed proteins were associated with fruit texture (CESA, PE, PL, and CHI), plant oxidation processes (LOX, GPX, CAT, and POD), plant cold stress response (HSF, HSP20, HSP70, and HSP90B), plant hormone signal transduction (BSK1 and JAR1) and transcription factors (WRKY and MYB). Our study showed that BR alleviates CI symptoms of tomato fruit by regulating LOX in the α-linolenic acid metabolism pathway, enhancing jasmonic acid-CoA (JA-CoA) synthesis, inhibiting cell wall and membrane lipid damage. The results provided a theoretical basis for further study on the CI mechanism of tomato fruit.

Effect of methyl jasmonate on the quality of harvested broccoli after simulated transport.

The effect of simulated transport vibration on the quality of broccoli and the ability of methyl jasmonate (MeJA) to ameliorate vibration damage in broccoli were investigated. Results indicated that transport injury, simulated by vibrational stress, promoted the deterioration in broccoli quality during subsequent storage. Treatment of broccoli with methyl jasmonate (MeJA), however, effectively ameliorated the impact of vibrational injury, maintained the appearance quality and delayed the yellowing and senescence of florets after simulated transportation stress. The effect of the MeJA may be related to of its ability to suppress the accumulation of reactive oxygen species, enhance vitamin C content, and induce antioxidant gene expression and enzyme activity, as well as suppress chlorophyll-degrading enzyme activity and gene expression. Overall, the MeJA treatment inhibited the adverse physiological changes that occur in broccoli as a result of vibrational and mechanical injury. Thus, MeJA has the potential to be used to decrease stress-induced reductions in the postharvest quality of horticultural crops that occur during transport and storage, thus, prolonging their shelf life.

The genome and transcriptome analysis of snake gourd provide insights into its evolution and fruit development and ripening.

Snake gourd (Trichosanthes anguina L.), which belongs to the Cucurbitaceae family, is a popular ornamental and food crop species with medicinal value and is grown in many parts of the world. Although progress has been made in its genetic improvement, the organization, composition, and evolution of the snake gourd genome remain largely unknown. Here, we report a high-quality genome assembly for snake gourd, comprising 202 contigs, with a total size of 919.8 Mb and an N50 size of 20.1 Mb. These findings indicate that snake gourd has one of the largest genomes of Cucurbitaceae species sequenced to date. The snake gourd genome assembly harbors 22,874 protein-coding genes and 80.0% of the genome consists of repetitive sequences. Phylogenetic analysis reveals that snake gourd is closely related to sponge gourd but diverged from their common ancestor ~33-47 million years ago. The genome sequence reported here serves as a valuable resource for snake gourd genetic research and comparative genomic studies in Cucurbitaceae and other plant species. In addition, fruit transcriptome analysis reveals the candidate genes related to quality traits during snake gourd fruit development and provides a basis for future research on snake gourd fruit development and ripening at the transcript level.

Effects of putrescine on the postharvest physiology characteristics in cowpea.

The effects of putrescine (Put) treatment on postharvest physiology characteristics in cowpea during cold storage have been investigated. The results indicated that Put with 8 mmol/L treatment greatly delayed aging of the cowpea; the sensory quality of cowpea was well maintained; the increase in weight loss was also inhibited, and the decrease in the content of ascorbic acid, chlorophyll, and total phenol was reduced efficiently. Antioxidant enzyme activities containing POD, CAT, and APX were preserved at higher levels in treated groups than the control during cold storage. In addition, the activity of PPO was restrained with Put. Overall, the quality of cowpea was maintained by 8 mmol/L Put treatment during cold storage.

Network analysis of noncoding RNAs in pepper provides insights into fruit ripening control.

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.

Comparative Analysis of DNA Methylation Reveals Specific Regulations on Ethylene Pathway in Tomato Fruit.

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.