Wild-type Fruit Research Articles

Loss-of-Function Mutations in the Fruit Softening Gene POLYGALACTURONASE1 Doubled Fruit Firmness in Strawberry

Abstract Wildtype fruit of cultivated strawberry (Fragaria × ananassa) are typically soft and highly perishable when fully ripe. The development of firm-fruited cultivars by phenotypic selection has greatly increased shelf life, decreased post-harvest perishability, and driven the expansion of strawberry production worldwide. Hypotheses for the firm-fruited phenotype include mutations affecting the expression of genes encoding polygalacturonases that soften fruit by degrading cell wall pectins. Here we show that loss-of-function mutations in the fruit softening gene POLYGALACTURONASE1 (FaPG1; PG1-6A1) double fruit firmness in strawberry. PG1-6A1 was one of three tandemly duplicated polygalacturonase genes found to be in linkage disequilibrium with a quantitative trait locus affecting fruit firmness on chromosome 6A. PG1-6A1 was strongly expressed in soft-fruited (wildtype) homozygotes and weakly expressed in firm-fruited (mutant) homozygotes. Genome-wide association, quantitative trait transcript, DNA sequence, and expression-QTL analyses identified genetic variants in linkage disequilibrium with PG1-6A1 that were positively correlated with fruit firmness and negatively correlated with PG1-6A1 expression. An Enhancer/Suppressor-mutator (En/Spm) transposable element insertion was discovered upstream of PG1-6A1 in mutant homozygotes that we hypothesize transcriptionally downegulates the expression of PG1-6A1. The PG1-6A1 locus was incompletely dominant and explained 26-76% of the genetic variance for fruit firmness among phenotypically diverse individuals. Additional loci are hypothesized to underlie the missing heritability. Highly accurate codominant genotyping assays were developed for modifying fruit firmness by marker-assisted selection of the En/Spm insertion and SNPs associated with the PG1-6A1 locus.

Overexpression of the persimmon ABA receptor DkPYL3 gene alters fruit development and ripening in transgenic tomato

Abscisic acid (ABA) is a crucial plant hormone that regulates various aspects of plant development. However, the specific function of the ABA receptor PYL in fruit development has not been fully understood. In this study, we focused on DkPYL3, a member of the ABA receptor subfamily Ⅰ in persimmon, which exhibited high expression levels in fruit, particularly during the young fruit and turning stages. Through yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), protein inhibition assays, and RNA-seq techniques, we identified and characterized the DkPYL3 protein, which was found to inhibit the activity of protein phosphatase type 2 C (PP2C). By heterologous overexpressing (OE) persimmon DkPYL3 in tomatoes, we investigated the impact of the DkPYL3 gene on fruit development and ripening. DkPYL3-OE upregulated the expression of genes related to chlorophyll synthesis and development, leading to a significant increase in chlorophyll content in young fruit. Several fruit quality parameters were also affected by DkPYL3 expression, including sugar content, single fruit weight, and photosynthesis rate. Additionally, fruits overexpressing DkPYL3 exhibited earlier ripening and higher levels of carotenoids and flavonoids compared to wild-type fruits. These results demonstrate the pivotal role of DkPYL3 in ABA-mediated young fruit development, ripening onset, and fruit quality in transgenic tomatoes.

Loss-of-function mutation in anthocyanidin reductase activates the anthocyanin synthesis pathway in strawberry

Fruit color substantially affects consumer preferences, with darker red strawberries being economically more valuable due to their higher anthocyanin content. However, the molecular basis for the dark red coloration remains unclear. Through screening of an ethyl methanesulfonate mutant library, we identified a rg418 mutant, that demonstrated anthocyanin accumulation during early fruit development stages. Furthermore, the ripening fruits of this mutant had higher anthocyanin content than wild-type (WT) fruits. An analysis of flavonoid content in WT and rg418 mutant fruits revealed substantial changes in metabolic fluxes, with the mutant exhibiting increased levels of anthocyanins and flavonols and decreased levels of proanthocyanidins. Bulked sergeant analysis sequencing indicated that the mutant gene was anthocyanidin reductase (ANR), a key gene in the proanthocyanidin synthesis pathway. Furthermore, transcriptome sequencing revealed the increased expression of MYB105 during the early development stage of mutant fruits, which promoted the expression of UFGT (UDP-glucose flavonoid 3-O-glucosyltransferase), a key gene involved in anthocyanin synthesis, thus substantially enhancing the anthocyanin content in the mutant fruits. Additionally, mutating ANR in a white-fruited strawberry variant (myb10 mutant) resulted in appealing pink-colored fruits, suggesting the diverse roles of ANR in fruit color regulation. Our study provides valuable theoretical insights for improving strawberry fruit color.

Mutation of tomato xyloglucan transglucosylase/hydrolase5 increases fruit firmness and contributes to prolonged shelf life

Fruit ripening in tomato is a highly coordinated developmental process accompanied with fruit softening, which is closely associated with cell wall degradation and remodeling. Xyloglucan endotransglucosylase/hydrolases (XTHs) are known to play an essential role in cell wall xyloglucan metabolism. Tomato XTH5 exhibits xyloglucan endotransglucosylase (XET) activity in vitro, but the understanding of its biological role in fruit ripening remains unclear. In this study, we revealed that SlXTH5 is highly expressed in mature fruits. Knockout mutant plants of SlXTH5 were generated by CRISPR/Cas9 gene editing strategy in tomato cultivar Micro-Tom. The mutant fruits showed accelerated transition from unripe to ripe process and earlier ethylene accumulation compared to wild type fruits. Although the mutation of SlXTH5 did not affect the size, weight and number of fruits, it indeed increased fruit firmness and extended shelf life, which is probably attributed to the increased cell layer and cell wall thickness of pericarp tissue. Pathogen infection experiment showed the enhanced resistance of mutant fruits to Botrytis cinerea. These results revealed the role of SlXTH5 in fruit ripening process, and provide new insight into how cell wall metabolism and remodeling regulate fruit softening and shelf life.

Glycoside hydrolase PpGH28BG1 modulates benzaldehyde metabolism and enhances fruit aroma and immune responses in peach.

Benzaldehyde (BAld) is one of the most widely distributed volatiles that contributes to flavor and defense in plants. Plants regulate BAld levels through various pathways, including biosynthesis from trans-cinnamic acid (free BAld), release from hydrolysis of glycoside precursors (BAld-H) via multiple enzymatic action steps, and conversion into downstream chemicals. Here, we show that BAld-H content in peach (Prunus persica) fruit is up to 100-fold higher than that of free BAld. By integrating transcriptome, metabolomic and biochemical approaches, we identified glycoside hydrolase PpGH28BG1 as being involved in the production of BAld-H through the hydrolysis of glycoside precursors. Overexpressing and silencing of PpGH28BG1 significantly altered BAld-H content in peach fruit. Transgenic tomatoes heterologously expressing PpGH28BG1 exhibited a decrease in BAld-H content and an increase in SA accumulation, while maintaining fruit weight, pigmentation, and ethylene production. These transgenic tomato fruits displayed enhanced immunity against Botrytis cinerea compared to wild type (WT). Induced expression of PpGH28BG1 and increased SA content were also observed in peach fruit when exposed to Monilinia fructicola infection. Additionally, elevated expression of PpGH28BG1 promoted fruit softening in transgenic tomatoes, resulting in a significantly increased emission of BAld compared to WT. Most untrained taste panelists preferred the transgenic tomatoes over WT fruit. Our study suggests that it is feasible to enhance aroma and immunity in fruit through metabolic engineering of PpGH28BG1 without causing visible changes in the fruit ripening process.

Ectopic overexpression of ShCBF1 and SlCBF1 in tomato suggests an alternative view of fruit responses to chilling stress postharvest.

Postharvest chilling injury (PCI) is a physiological disorder that often impairs tomato fruit ripening; this reduces fruit quality and shelf-life, and even accelerates spoilage at low temperatures. The CBF gene family confers cold tolerance in Arabidopsis thaliana, and constitutive overexpression of CBF in tomato increases vegetative chilling tolerance, in part by retarding growth, but, whether CBF increases PCI tolerance in fruit is unknown. We hypothesized that CBF1 overexpression (OE) would be induced in the cold and increase resistance to PCI. We induced high levels of CBF1 in fruit undergoing postharvest chilling by cloning it from S. lycopersicum and S. habrochaites, using the stress-inducible RD29A promoter. Harvested fruit were cold-stored (2.5°C) for up to three weeks, then rewarmed at 20°C for three days. Transgene upregulation was triggered during cold storage from 8.6- to 28.6-fold in SlCBF1-OE, and between 3.1- to 8.3-fold in ShCBF1-OE fruit, but developmental abnormalities in the absence of cold induction were visible. Remarkably, transgenic fruit displayed worsening of PCI symptoms, i.e., failure to ripen after rewarming, comparatively higher susceptibility to decay relative to wild-type (WT) fruit, lower total soluble solids, and the accumulation of volatile compounds responsible for off-odors. These symptoms correlated with CBF1 overexpression levels. Transcriptomic analysis revealed that the ripening and biotic and abiotic stress responses were altered in the cold-stored transgenic fruit. Seedlings grown from 'chilled' and 'non-chilled' WT fruit, in addition to 'non-chilled' transgenic fruit were also exposed to 0°C to test their photosynthetic response to chilling injury. Chilled WT seedlings adjusted their photosynthetic rates to reduce oxidative damage; 'non-chilled' WT seedlings did not. Photosynthetic parameters between transgenic seedlings were similar at 0°C, but SlCBF1-OE showed more severe photoinhibition than ShCBF1-OE, mirroring phenotypic observations. These results suggest that 1) CBF1 overexpression accelerated fruit deterioration in response to cold storage, and 2) Chilling acclimation in fructus can increase chilling tolerance in seedling progeny of WT tomato.

Overexpression of Sly-miR172a improved quality of tomato fruit by regulating MADS-box family

Fruit development is the key link in the reproductive cycle of plants, which determines the quality, yield and economic value of fruit. The present study investigated the effect of Sly-miR172a on the quality of the tomato fruit. The research results showed that overexpression of Sly-miR172a (OE-miR172a) in tomato fruit efficaciously increased the transverse and longitudinal diameter, weight, pericarp thickness, cell number and cell area. Compared with the wild-type (WT) fruit and the silencing miR172a by short tandem target mimic (STTM-miR172a), the relative expression levels of the MADS-box and the flavonoid biosynthesis synthesis pathway genes were higher in OE-miR172a. The expression level of AP2 and Cytokinin oxidase/dehydrogenase (CKX) family genes in OE-miR172a fruit was decreased, and accumulated more content of soluble sugars, lycopene, carotenoid, total phenol, anthocyanin and total flavonoid. The present study showed that overexpression of Sly-miR172 promoted the development and improved quality of tomato fruit.

Effect of SlSAHH2 on metabolites in over-expressed and wild-type tomato fruit.

Tomato (Solanum lycopersicum) is an annual or perennial herb that occupies an important position in daily agricultural production. It is an essential food crop for humans and its ripening process is regulated by a number of genes. S-adenosyl-l-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) is widespread in organisms and plays an important role in regulating biological methylation reactions. Previous studies have revealed that transgenic tomato that over-express SlSAHH2 ripen earlier than the wild-type (WT). However, the differences in metabolites and the mechanisms driving how these differences affect the ripening cycle are unclear. To investigate the effects of SlSAHH2 on metabolites in over-expressed tomato and WT tomato. SlSAHH2 over-expressed tomato fruit (OE-5# and OE-6#) and WT tomato fruit at the breaker stage (Br) were selected for non-targeted metabolome analysis. A total of 733 metabolites were identified by mass spectrometry using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Human Metabolome database (HMDB). The metabolites were divided into 12 categories based on the superclass results and a comparison with the HMDB. The differences between the two databases were analyzed by PLS-DA. Based on a variable important in projection value >1 and P < 0.05, 103 differential metabolites were found between tomato variety OE-5# and WT and 63 differential metabolites were found between OE-6# and WT. These included dehydrotomatine, L-serine, and gallic acid amongst others. Many metabolites are associated with fruit ripening and eight common metabolites were found between the OE-5# vs. WT and OE-6# vs. WT comparison groups. The low L-tryptophan expression in OE-5# and OE-6# is consistent with previous reports that its content decreases with fruit ripening. A KEGG pathway enrichment analysis of the significantly different metabolites revealed that in the OE-5# and WT groups, up-regulated metabolites were enriched in 23 metabolic pathways and down-regulated metabolites were enriched in 11 metabolic pathways. In the OE-6# and WT groups, up-regulated metabolites were enriched in 29 pathways and down-regulated metabolites were enriched in six metabolic pathways. In addition, the differential metabolite changes in the L-serine to flavonoid transformation metabolic pathway also provide evidence that there is a phenotypic explanation for the changes in transgenic tomato. The metabolomic mechanism controlling SlSAHH2 promotion of tomato fruit ripening has been further elucidated.

Visualization of mitochondrial dynamics in tomato based on green fluorescent protein

This study aimed to visualize the morphological features and dynamic changes of tomato mitochondria to provide a basis for the study of its mitochondrial functions. In this study, transgenic tomatoes expressing mitochondria-localized green fluorescent protein (mitochondria-GFP, Mt-GFP) were obtained by Agrobacterium-mediated genetic transformation. The color, hardness, soluble solids, acidity content, respiration rate, and ethylene production of the transgenic Mt-GFP tomato fruits were determined at the stage of mature green, breaker, and 3, 6, 9 days after breaker, while the wild-type tomato fruits were used as a control. As expected, Mt-GFP recombinant protein did not affect the ripening process, but induced the increased acidity of tomato fruits. The accumulations of Mt-GFP protein in tomato leaves and fruits were successfully verified by Western blotting. The morphological characteristics of mitochondria in flower, leaf and fruit cells as well as the dynamic changes of mitochondria in flower cells were clearly observed and studied under confocal laser microscope. The development of transgenic Mt-GFP tomato plants helps the visualization of tomato mitochondria and provides good research materials for the study of mitochondrial function during tomato development and fruit ripening.

Characterization of a New Citrus Mutant Induced by Gamma Irradiation with a Unique Fruit Shape, Gwonje-Early, and Determination of Specific Selection Markers Using Allele-Specific PCR

Gamma-ray irradiation is one of the most widely used mutagens worldwide. We previously conducted mutation breeding using gamma irradiation to develop new Citrus unshiu varieties. Among these mutants, Gwonje-early had an ovate shape, a protrusion of the upper part of the fruit, and a large fruit size compared with wild-type (WT) fruits. We investigated the external/internal morphological characteristics and fruit sugar/acid content of Gwonje-early. Additionally, we investigated genome-wide single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variants in Gwonje-early using whole-genome re-sequencing. Functional annotation by Gene Ontology analysis confirmed that InDels were more commonly annotated than SNPs. To identify specific molecular markers for Gwonje-early, allele-specific PCR was performed using homozygous SNPs detected via Gwonje-early genome re-sequencing. The GJ-SNP1 and GJ-SNP4 primer sets were effectively able to distinguish Gwonje-early from the WT and other commercial citrus varieties, demonstrating their use as specific molecular markers for Gwonje-early. These findings also have important implications in terms of intellectual property rights and the variety protection of Gwonje-early. Our results may provide insights into the understanding of morphological traits and the molecular breeding mechanisms of citrus species.

Dissection of mRNA ac4C acetylation modifications in AC and Nr fruits: insights into the regulation of fruit ripening by ethylene

N4-acetylcytidine (ac4C) modification of mRNA has been shown to be present in plant RNAs, but its regulatory function in plant remains largely unexplored. In this study, we investigated the differentially expressed mRNAs, lncRNAs and acetylation modifications of mRNAs in tomato fruits from both genotypes. By comparing wild-type (AC) tomato and the ethylene receptor-mutant (Nr) tomato from mature green (MG) to six days after the breaker (Br6) stage, we identified differences in numerous key genes related to fruit ripening and observed the corresponding lncRNAs positively regulated the target genes expression. At the post-transcriptional level, the acetylation level decreased and increased in AC and Nr tomatoes from MG to Br6 stage, respectively. The integrated analysis of RNA-seq and ac4C-seq data revealed the potential positive role of acetylation modification in regulating gene expression. Furthermore, we found differential acetylation modifications of certain transcripts (ACO, ETR, ERF, PG, CesA, β-Gal, GAD, AMY, and SUS) in AC and Nr fruits which may explain the differences in ethylene production, fruit texture, and flavor during their ripening processes. The present study provides new insights into the molecular mechanisms by which acetylation modification differentially regulates the ripening process of wild-type and mutant tomato fruits deficient in ethylene signaling.Graphical

Metabolomics Study of the Effect of Transcription Factor NOR-like1 on Flavonoids in Tomato at Different Stages of Maturity Using UPLC-MS/MS.

Tomato fruits are rich in flavonoids. This study explores the effect of transcription factor SlNOR-like1 on the accumulation of flavonoids in tomato fruits at different ripening stages. We used ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze wild-type (WT) and NOR-like1 CRISPR/Cas9-edited (NOR-like1) tomato fruits. A total of 50 flavonoid metabolites were accurately identified and determined in tomatoes. The flavonoid metabolic differences were observed among the different tomato sample groups using PCA and OPLS-DA analysis. There were 16 differential flavonoids (13 upregulated and 3 downregulated) identified between WT-GR (WT tomato at the green-ripening stage) and NOR-like1-GR (NOR-like1 tomato at the green-ripening stage), 9 differential flavonoids (six upregulated and three downregulated) identified between WT-BR3 (WT tomato at the color-breaking stage) and NOR-like1-BR3 (NOR-like1 tomato at the color-breaking stage), and 12 differential flavonoids (11 upregulated and 1 downregulated) identified between WT-BR9 (WT tomato at the red-ripening stage) and NOR-like1-BR9 (NOR-like1 tomato at the red-ripening stage). Rutin, nicotiflorin, naringenin chalcone, eriodictyol, and naringenin-7-glucoside were the five flavonoids with the highest content in the ripening stages (BR3 and BR9) in both WT and NOR-like1 tomato fruits. The overall flavonoid contents in WT tomato fruits changed little from GR to BR3 and decreased from BR3 to BR9; meanwhile, in the NOR-like1 tomato fruits, the total amounts of the flavonoids exhibited an increasing trend during all three ripening stages. The accumulation pattern of flavonoid metabolites in NOR-like1 tomato fruits differed from that in WT tomato fruits, especially in the later ripening process of BR9. The transcription factor SlNOR-like1 has an impact on the accumulation of flavonoids in tomato fruits. The results provide a preliminary basis for subsequent research into its regulatory mechanism and will be helpful for attaining future improvements in the nutritional quality and postharvest treatment of tomato fruits.

Effect of oxalic acid on postharvest life of tomato modified with the TomLoxB gene in anti-sense

Oxalic acid is an organic compound found in green leafy vegetables, which has proven to be effective in delaying ripening by inhibiting ethylene synthesis in fruits such as banana, mango, peach, tomato, plum, and others. In this study, the response of oxalic acid application on postharvest physiology was evaluated in transgenic tomatoes (Solanum lycopersicum) variety TA234 with the TomLoxB gene insertion in antisense, at two concentrations of oxalic acid: 3 and 10 mM, during 30 d of storage at 25 ± 1 ºC and a relative humidity of 65 - 70 %. The fruits were harvested at break stage and immersed for 10 minutes in the oxalic acid solution, which was maintained at 25 ºC. Upon treatment, less weight loss, better retention of lightness, delay in the decrease of firmness and hue angle, decrease in lipoxygenase activity, low electrolyte leakage and increase in total phenolics content were observed. The most effective oxalic acid concentration was 3 mM, that extended postharvest life by up to 30 d and reduced deterioration of the genetically modified (GM) tomatoes. In turn, the untreated GM tomatoes showed an acceptable appearance up to day 24 of storage, while the wild type fruits were kept satisfactorily for 15 d.

Integrated transcriptomic and metabolomic analyses revealed the role of SlMYC2 in tomato (Solanum lycopersicum L.) fruit development and ripening

Transcription factor (TF) MYC2 is a fundamental regulator of the jasmonic acid (JA) signaling pathway and has a role in plant development and stress regulation. However, a comprehensive understanding of MYC2 regulatory role in fruit development and ripening is still lacking. In this study, the integrative and comparative analysis was performed on the transcriptome and metabolome of wild-type (WT) and SlMYC2 knockout tomato fruit at mature green and red ripening stages to uncover the function of SlMYC2. A total of 764 metabolites and 30,280 annotated unigenes were identified. The differential accumulated metabolites (DAMs) and differentially expressed genes (DEGs) caused by SlMYC2 mutation were 32.3 % and 159.8 % higher in red ripening fruit than in mature green fruit, respectively. From the mature green to red ripening stages of tomato fruit, KEGG enrichment analysis revealed that the DEGs affected by SlMYC2 were primarily involved in photosynthesis, carbohydrate metabolism, phenylpropanoid pathway, carotenoid biosynthesis, and plant hormone signal transduction. In addition, there are 36 TFs families in these DEGs, mainly including MYB_related, MYB, bHLH, B3, ERF, NAC, HB-other, WRKY and bZIP. Furthermore, the co-expression and promoter analysis indicated that MYB44, PLA1, WRKY17, LOX2, PPA1-LIKE, JAZ, ACSL1 and PYL4 may play an important role in the aforementioned metabolic pathways regulated by SlMYC2. Overall, these findings indicate that many key metabolic pathways, transcription factors, and genes may be governed by SlMYC2, potentially being related to fruit development, ripening, quality and stress resistance mechanisms. This study provides new insights into the function of MYC2 and lays a foundation for further research on the molecular mechanism underlying fruit development and ripening.

ABC transporter SlABCG23 regulates chilling resistance of tomato fruit by affecting JA signaling pathway

To explore the effect and mechanism of an ATP-binding cassette transporter, SlABCG23, on the chilling resistance of tomato fruit during storage, we generated mutant using CRISPR/Cas9 technology for further investigation. This research showed under cold stress, the slabcg23 mutant fruit exhibited severe chilling injury (CI), lower jasmonic acid (JA) contents, and higher expression levels of JA signaling inhibitor, SlJAZ6, than wild-type (WT). The mutation of SlABCG23 caused a decrease in the thickness of the fruit pericarp. The expression levels of SlCBF1 and SlICE1 in slabcg23 mutant fruit were lower than those in WT fruit under cold stress. Moreover, the slabcg23 mutant fruit exhibited reduced activities of antioxidant enzymes, including superoxide dismutase, catalase and peroxidase, and increased reactive oxygen species and malondialdehyde contents. These findings indicated that SlABCG23 affects fruit resistance to CI by influencing the JA signaling pathway, fruit pericarp development, cold response signal and antioxidant capacity.

Ethylene perception influences the chilling injury and SlCBF1 gene expression during cold storage of tomato fruit

Endogenous and exogenous ethylene were reported to positively regulate the cold tolerance, and mitigate the chilling injury (CI) damage of stored tomato fruit. However, the involvement of ethylene perception in cold resistance is still unclear and needs further investigation in postharvest fruit. Here, we used Never ripen (Nr) and wild-type (WT) tomato fruit as inhibited- and normal-ethylene perception models to investigate how inhibiting ethylene perception affected fruit quality and cold responses of stored tomato fruit. Results showed that inhibiting ethylene perception deteriorated fruit quality and aggravated cell membrane damage of tomato fruit during cold storage. In comparison with WT fruit, Nr fruit accumulated higher relative oxygen species but maintained lower activities of antioxidant enzymes. Besides, endogenous abscisic acid, methyl jasmonate, and gibberellin levels in Nr fruit were lower, whereas brassinolide contents were higher than those in WT fruit. Moreover, a lack of ethylene perception was preceded by the decrease in cold-induced upregulation of SlCBF1 gene expression, while no significant difference in the transcription of SlICE1 gene was observed between WT and Nr fruit. This study demonstrated that inhibiting ethylene perception increased the cold susceptibility of tomato fruit, suggesting that ethylene perception played a crucial role in maintaining fruit quality during cold storage.

Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon.

The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (Cucumis melo var. reticulatus, 'Harukei-3') via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five CmACOs and the gene CmACO1 predominantly expressed in harvested fruits. From this information, CmACO1 was expected to be a key gene for shelf-life in melons. Based on this information, the CmACO1 was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T2 generation, the fruit phenotypes 14days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that CmACO1 knockout via CRISPR/Cas9 extended the melon's shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security.

SlMAPK3 Positively Regulates the Ethylene Production of Postharvest Tomato Fruits and Is Involved in Ethylene-Mediated Cold Tolerance.

Mitogen-activated protein kinase (MAPK) cascades and ethylene are crucial for plant growth, development, and stress responses, but their potential mechanisms in cold resistance remain unclear. We revealed that SlMAPK3 transcript levels were dramatically induced by cold treatment in an ethylene-dependent manner. Under cold stress, the proline content of SlMAPK3-overexpression fruit was 96.5 and 115.9% higher than that of wild-type fruit (WT), respectively, while the ion leakage was 37.3 and 32.5% lower than that of WT. RNA sequencing revealed that overexpression of SlMAPK3 caused upregulation of genes that are enriched in the ethylene-activated signaling pathway (GO:0009873), cold signaling pathway (GO:0009409), and heat signaling pathway (GO:0009408). RT-qPCR demonstrated that the expression levels of SlACS2, SlACS4, SlSAHH, SlCBF1, SlDREB, SlGolS1, and SlHSP17.7 in the OE.MAPK3 fruits were consistent with the RNA sequencing results. Meanwhile, the knockout of SlMAPK3 reduced the ethylene content, ACC content, and ACS activity. Moreover, the knockout of SlMAPK3 reduced the positive effect of ethylene in cold stress, while suppressing the expression of SlICE1 and SlCBF1. In conclusion, our study demonstrated a novel mechanism by which SlMAPK3 positively regulates the ethylene production of postharvest tomato fruits and is involved in ethylene-mediated cold tolerance.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

A D-cysteine desulfhydrase, SlDCD2, participates in tomato fruit ripening by modulating ROS homoeostasis and ethylene biosynthesis.

Hydrogen sulfide (H2S) is involved in multiple processes during plant growth and development. D-cysteine desulfhydrase (DCD) can produce H2S with D-cysteine as the substrate; however, the potential developmental roles of DCD have not been explored during the tomato lifecycle. In the present study, SlDCD2 showed increasing expression during fruit ripening. Compared with the control fruits, the silencing of SlDCD2 by pTRV2-SlDCD2 accelerated fruit ripening. A SlDCD2 gene-edited mutant was constructed by CRISPR/Cas9 transformation, and the mutant exhibited accelerated fruit ripening, decreased H2S release, higher totalcysteine and ethylene contents, enhanced chlorophyll degradation and increased carotenoid accumulation. Additionally, the expression of multiple ripening-related genes, including NYC1, PAO, SGR1, PDS, PSY1, ACO1, ACS2, E4, CEL2, and EXP was enhanced during the dcd2 mutant tomato fruit ripening. Compared with the wild-type fruits, SlDCD2 mutation induced H2O2 and malondialdehyde (MDA) accumulation in fruits, which led to an imbalance in reactive oxygen species (ROS) metabolism. A correlation analysis indicated that H2O2 content was strongly positively correlated with carotenoids content, ethylene content and ripening-related gene expression and negatively correlated with the chlorophyll content. Additionally, the dcd2 mutant showed earlier leaf senescence, which may be due to disturbed ROS homeostasis. In short, our findings show that SlDCD2 is involved in H2S generation and that the reduction in endogenous H2S production in the dcd2 mutant causes accelerated fruit ripening and premature leaf senescence. Additionally, decreased H2S in the dcd2 mutant causes excessive H2O2 accumulation and increased ethylene release, suggesting a role of H2S and SlDCD2 in modulating ROS homeostasis and ethylene biosynthesis.