RIPENING INHIBITOR Research Articles

CRISPR-Cas9: Unraveling Genetic Secrets to Enhance Floral and Fruit Traits in Tomato.

Tomato, a globally consumed vegetable, possesses vast genetic diversity, making it suitable for genetic manipulation using various genetic improvement techniques. Tomatoes are grown extensively for their market value and health benefits, primarily contributed by enhanced yield and nutritional value respectively, influenced by floral and fruit traits. Floral morphology is maintained by genes involved in meristem size control, regulation of inflorescence transition, and pollen development. SP (SELF-PRUNING) and SP5G (SELF-PRUNING 5G) determine growth habit and flowering time. RIN (RIPENING INHIBITOR) and PG (POLYGALACTURONASE) are responsible for the shelf life of fruits. In addition to this, nutrition-enriched tomatoes have been developed in recent times. In this review, we comprehensively discuss the major genes influencing floral morphology, flowering time, fruit size, fruit shape, shelf life, and nutritional value, ultimately resulting in enhanced yield. Additionally, we address the advances in CRISPR/Cas9 applied for the genetic improvement of tomatoes along with prospects of areas in which research development in terms of tomato genetic improvement has to be advanced.

Crossing a CRISPR/Cas9 transgenic tomato plant with a wild-type plant yields diverse mutations in the F1 progeny.

Generating CRISPR/Cas9-mediated mutants in tomato (Solanum lycopersicum L.) involves screening shoots regenerated from cultured cells transformed with a T-DNA harboring sequences encoding Cas9 and single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbers may be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR (RIN) gene, and the resulting F1 seedlings were classified using a kanamycin resistance marker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F1 seedlings, which do carry the T-DNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. This method was also successfully applied to produce mutations in Geranylgeranyl pyrophosphate synthase 2 (GGPS2). Because this method involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants.

Ripening and rot: How ripening processes influence disease susceptibility in fleshy fruits.

Fleshy fruits become more susceptible to pathogen infection when they ripen; for example, changes in cell wall properties related to softening make it easier for pathogens to infect fruits. The need for high-quality fruit has driven extensive research on improving pathogen resistance in important fruit crops such as tomato (Solanum lycopersicum). In this review, we summarize current progress in understanding how changes in fruit properties during ripening affect infection by pathogens. These changes affect physical barriers that limit pathogen entry, such as the fruit epidermis and its cuticle, along with other defenses that limit pathogen growth, such as preformed and induced defense compounds. The plant immune system also protects ripening fruit by recognizing pathogens and initiating defense responses involving reactive oxygen species production, mitogen-activated protein kinase signaling cascades, and jasmonic acid, salicylic acid, ethylene, and abscisic acid signaling. These phytohormones regulate an intricate web of transcription factors (TFs) that activate resistance mechanisms, including the expression of pathogenesis-related genes. In tomato, ripening regulators, such as RIPENING INHIBITOR and NON_RIPENING, not only regulate ripening but also influence fruit defenses against pathogens. Moreover, members of the ETHYLENE RESPONSE FACTOR (ERF) family play pivotal and distinct roles in ripening and defense, with different members being regulated by different phytohormones. We also discuss the interaction of ripening-related and defense-related TFs with the Mediator transcription complex. As the ripening processes in climacteric and non-climacteric fruits share many similarities, these processes have broad applications across fruiting crops. Further research on the individual contributions of ERFs and other TFs will inform efforts to diminish disease susceptibility in ripe fruit, satisfy the growing demand for high-quality fruit and decrease food waste and related economic losses.

A tomato B-box protein regulates plant development and fruit quality through the interaction with PIF4, HY5, and RIN transcription factors.

During the last decade, knowledge about BBX proteins has greatly increased. Genome-wide studies identified the BBX gene family in several ornamental, industry, and food crops; however, reports regarding the role of these genes as regulators of agronomically important traits are scarce. Here, by phenotyping a knockout mutant, we performed a comprehensive functional characterization of the tomato locus Solyc12g089240, hereafter called SlBBX20. The data revealed the encoded protein as a positive regulator of light signaling affecting several physiological processes during the life span of plants. Through inhibition of PHYTOCHROME INTERACTING FACTOR 4 (SlPIF4)-auxin crosstalk, SlBBX20 regulates photomorphogenesis. Later in development, it controls the balance between cell division and expansion to guarantee correct vegetative and reproductive development. In fruits, SlBBX20 is transcriptionally induced by the master transcription factor RIPENING INHIBITOR (SlRIN) and, together with ELONGATED HYPOCOTYL 5 (SlHY5), up-regulates flavonoid biosynthetic genes. Finally, SlBBX20 promotes the accumulation of steroidal glycoalkaloids and attenuates Botrytis cinerea infection. This work clearly demonstrates that BBX proteins are multilayer regulators of plant physiology because they affect not only multiple processes during plant development but they also regulate other genes at the transcriptional and post-translational levels.

Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.

Long-Term Stability of Lavandula x intermedia Essential Oil Nanoemulsions: Can the Addition of the Ripening Inhibitor Impact the Biocidal Activity of the Nanoformulations?

In this work, Lavandula x intermedia essential oil (LEO) was encapsulated in lipid-based nanoemulsions (NanoLEO) using the solvent-displacement technique. In order to preserve the colloidal stability of the formulation, LEO was appropriately doped with the incorporation of different levels of a water-insoluble oil used as a ripening inhibitor. All the nanoemulsion samples were evaluated in terms of the impact of the water-insoluble oil on the nanoemulsion formation, physical-chemical properties, and antibacterial effectiveness against E. coli (Gram-negative) and B. cereus (Gram-positive). The presence of the inert oil added benefits to the formulations in terms of appearance, colloidal stability, and loss of volatile components. However, the antimicrobial activity of the nanoemulsions dramatically decreased with the ripening inhibitor addition, probably because it hampered the internalization of the antimicrobial components of LEO within the bacterial cell membranes, thus nullifying the delivery ability of the nanoemulsion formulation. On the contrary, the undoped NanoLEO formulation showed unaltered antibacterial activity in both E. coli and B. cereus up to 40 weeks from the preparation.

WUSCHEL-related homeobox transcription factor SlWOX13 regulates tomato fruit ripening.

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established importance of WUSCHEL-related homeobox (WOX) TFs in plant development, the involvement of WOX and its underlying mechanism in the regulation of fruit ripening remain unclear. Here, we demonstrate that SlWOX13 regulates fruit ripening in tomato (Solanum lycopersicum). Overexpression of SlWOX13 accelerates fruit ripening, whereas loss-of-function mutation in SlWOX13 delays this process. Moreover, ethylene synthesis and carotenoid accumulation are significantly inhibited in slwox13 mutant fruit but accelerated in SlWOX13 transgenic fruit. Integrated analyses of RNA-seq and chromatin immunoprecipitation (ChIP)-seq identified 422 direct targets of SlWOX13, of which 243 genes are negatively regulated and 179 are positively regulated by SlWOX13. Electrophoretic mobility shift assay, RT-qPCR, dual-luciferase reporter assay, and ChIP-qPCR analyses demonstrated that SlWOX13 directly activates the expression of several genes involved in ethylene synthesis and signaling and carotenoid biosynthesis. Furthermore, SlWOX13 modulates tomato fruit ripening through key ripening-related TFs, such as RIPENING INHIBITOR (RIN), NON-RIPENING (NOR), and NAM, ATAF1, 2, and CUC2 4 (NAC4). Consequently, these effects promote fruit ripening. Taken together, these results demonstrate that SlWOX13 positively regulates tomato fruit ripening via both ethylene synthesis and signaling and by transcriptional regulation of key ripening-related TFs.

RIN enhances plant disease resistance via root exudate-mediated assembly of disease-suppressive rhizosphere microbiota

The RIPENING-INHIBITOR (RIN) transcriptional factor is a key regulator governing fruit ripening. While RIN also affects other physiological processes, its potential roles in triggering interactions with the rhizosphere microbiome and plant health are unknown. Here we show that RIN affects microbiome-mediated disease resistance via root exudation, leading to recruitment of microbiota that suppress the soil-borne, phytopathogenic Ralstonia solanacearum bacterium. Compared with the wild-type (WT) plant, RIN mutants had different root exudate profiles, which were associated with distinct changes in microbiome composition and diversity. Specifically, the relative abundances of antibiosis-associated genes and pathogen-suppressing Actinobacteria (Streptomyces) were clearly lower in the rhizosphere of rin mutants. The composition, diversity, and suppressiveness of rin plant microbiomes could be restored by the application of 3-hydroxyflavone and riboflavin, which were exuded in much lower concentrations by the rin mutant. Interestingly, RIN-mediated effects on root exudates, Actinobacteria, and disease suppression were evident from the seedling stage, indicating that RIN plays a dual role in the early assembly of disease-suppressive microbiota and late fruit development. Collectively, our work suggests that, while plant disease resistance is a complex trait driven by interactions between the plant, rhizosphere microbiome, and the pathogen, it can be indirectly manipulated using “prebiotic” compounds that promote the recruitment of disease-suppressive microbiota.

Lycium RIN negatively modulate the biosynthesis of kukoamine A in hairy roots through decreasing thermospermine synthase expression

Root bark (Lycii cortex) of Lycium contains high contents of characteristic bioactive compounds, including kukoamine A (KuA) and kukoamine B (KuB). RIPENING INHIBITOR (RIN) is well known as a master regulator of Solanaceaous fruit ripening. However, the role of RIN in the biosynthetic pathway of KuA in Lycium remains unclear. In this study, integrated transcriptomic, metabolomic analyses and hairy root system are used to characterize the role of RIN in KuA biosynthesis in Lycium. The ultra performance liquid chromatography electrospray ionization tandem mass spectrometry analysis revealed that KuA was significantly induced in LrRIN1 RNAi lines and not detected in overexpression lines. A total of 20,913 differentially expressed genes (DEGs) and 60 differentially accumulated metabolites (DAMs) were detected in LrRIN1 transgenic hairy roots, which were used for weighted gene co-expression network analysis. Our result reveals a high association between KuA and structural genes in the phenolamide pathway, which shows a negative correlation with LrRIN1. In addition, overexpression of the polyamine pathway gene thermospermine synthase LcTSPMS, a potential target gene of Lycium RIN, increased the contents of both KuA and KuB in L. chinense hairy root, indicating that TSPMS is responsible for KuA biosynthesis and is also the common upstream biosynthetic gene for both KuA and KuB. Our results lay a solid foundation for uncovering the biosynthetic pathway of KuA, which will facilitate the molecular breeding and genetic improvement of Lycium species.

Elevated methylglyoxal levels inhibit tomato fruit ripening by preventing ethylene biosynthesis.

Methylglyoxal (MG), a toxic compound produced as a by-product of several cellular processes, such as respiration and photosynthesis, is well known for its deleterious effects, mainly through glycation of proteins during plant stress responses. However, very little is known about its impact on fruit ripening. Here, we found that MG levels are maintained at high levels in green tomato (Solanum lycopersicum L.) fruits and decline during fruit ripening despite a respiratory burst during this transition. We demonstrate that this decline is mainly mediated through a glutathione-dependent MG detoxification pathway and primarily catalyzed by a Glyoxalase I enzyme encoded by the SlGLYI4 gene. SlGLYI4 is a direct target of the MADS-box transcription factor RIPENING INHIBITOR (RIN), and its expression is induced during fruit ripening. Silencing of SlGLYI4 leads to drastic MG overaccumulation at ripening stages of transgenic fruits and interferes with the ripening process. MG most likely glycates and inhibits key enzymes such as methionine synthase and S-adenosyl methionine synthase in the ethylene biosynthesis pathway, thereby indirectly affecting fruit pigmentation and cell wall metabolism. MG overaccumulation in fruits of several nonripening or ripening-inhibited tomato mutants suggests that the tightly regulated MG detoxification process is crucial for normal ripening progression. Our results underpin a SlGLYI4-mediated regulatory mechanism by which MG detoxification controls fruit ripening in tomato.

Физиологические аспекты созревания и продления срока хранения сочных плодов

This review discusses the physiological aspects of the ripening of juicy fruits in relation to the problem of extending the shelf life of horticultural products. The achievements of molecular biology in the field of genetic regulation of plant quality formation processes are used. Particular attention is paid to the hormonal regulation of the accumulation of nutrients and biologically active substances in fruits. The role of ARF/IAA and DELLA receptor proteins in the interaction of auxin and GA signalling pathways during the growth of tomato, strawberry, and grape fruits is demonstrated. The involvement of DELLA proteins in integrating the function of other phytohormones – cytokinin, ethylene, abscisic acid, brassinosteroids and jasmonic acid – is noted. Evidence is presented for the interaction of cytokinin with auxin and GA in the regulation of early development and fruit size. The combination of transcription factors and epigenetic modifications during fruit development and senescence is considered. The involvement of mechanisms of senescence and loss of fruit shelf life in the absence of external signs is shown. The ripening characteristics of climacteric and nonclimacteric fruits are considered. One of the key regulators of the ripening process in both climacteric and non-climacteric fruits is the MADS domain transcription factor RIPENING INHIBITOR (RIN). The regulation of non-climacteric fruit ripening is reviewed using grape and strawberry as examples. Special attention is paid to growth processes, water exchange, photosynthesis, primary and secondary metabolism of developing and ripening fruits. The formation of integuments and cell walls as a structural basis for the physical properties of products is considered. Promising methods of using regulators of vital processes in the post-harvest period to slow down the fruit senescence are given. Increased knowledge of genetic, hormonal and metabolic networks opens up broad prospects for improving and maintaining the quality of fleshy horticultural products.

Increased ACS Enzyme Dosage Causes Initiation of Climacteric Ethylene Production in Tomato.

Fruits of wild tomato species show different ethylene-dependent ripening characteristics, such as variations in fruit color and whether they exhibit a climacteric or nonclimacteric ripening transition. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are key enzymes in the ethylene biosynthetic pathway encoded by multigene families. Gene duplication is a primary driver of plant diversification and angiosperm evolution. Here, interspecific variations in the molecular regulation of ethylene biosynthesis and perception during fruit ripening in domesticated and wild tomatoes were investigated. Results showed that the activated ACS genes were increased in number in red-ripe tomato fruits than in green-ripe tomato fruits; therefore, elevated dosage of ACS enzyme promoted ripening ethylene production. Results showed that the expression of three ACS isogenes ACS1A, ACS2, and ACS4, which are involved in autocatalytic ethylene production, was higher in red-ripe tomato fruits than in green-ripe tomato fruits. Elevated ACS enzyme dosage promoted ethylene production, which corresponded to the climacteric response of red-ripe tomato fruits. The data suggest that autoinhibitory ethylene production is common to all tomato species, while autocatalytic ethylene production is specific to red-ripe species. The essential regulators Non-ripening (NOR) and Ripening-Inhibitor (RIN) have experienced gene activation and overlapped with increasing ACS enzyme dosage. These complex levels of transcript regulation link higher ethylene production with spatiotemporal modulation of gene expression in red-ripe tomato species. Taken together, this study shows that bursts in ethylene production that accompany fruit color changes in red-ripe tomatoes are likely to be an evolutionary adaptation for seed dispersal.

Fruit ripening specific expression of β-D-N-acetylhexosaminidase (β-Hex) gene in tomato is transcriptionally regulated by ethylene response factor SlERF.E4

N-glycans and N-glycan processing enzymes are key players in regulating the ripening of tomato (Solanum lycopersicum) fruits, a model for fleshy fruit ripening. β-D-N-acetylhexosaminidase (β-Hex) is a N-glycan processing enzyme involved in fruit ripening. The suppression of β-Hex results in enhanced fruit shelf life and firmness in both climacteric and non-climacteric fruits. Previously, we have shown that ripening specific expression of β-Hex is regulated by RIPENING INHIBITOR (RIN), ABSCISIC ACID STRESS RIPENING 1 (SlASR1) and ethylene. However, the precise mechanism of ethylene-mediated regulation of β-Hex remains elusive. To gain insights into this, we have performed 5’ deletion mapping of tomato β-Hex promoter and a shorter promoter fragment (pD-200, 200 bp upstream to translational start site) is identified, which was found critical for spatio-temporal transcriptional regulation of β-Hex. Further, site specific mutagenesis in RIN and ASR1 binding sites in pD-200 provides key insights into ripening specific promoter activity. Furthermore, induction of GUS activity by ethylene, yeast one hybrid assay and EMSA identify Ethylene Response Factor SlERF.E4 as a positive regulator of β-Hex. Taken together, our study suggest that SlERF.E4 together with RIN and SlASR1 transcriptionally regulates β-Hex and all these three proteins are essential for fruit ripening specific expression of β-Hex in tomato.

CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.

A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato.

Although the role of ethylene in tomato (Solanum lycopersicum) fruit ripening has been intensively studied, its role in tomato fruit growth remains poorly understood. In addition, the relationship between ethylene and the developmental factors NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN) during ripening is under debate. Here, we carried out comprehensive genetic analyses of genome-edited mutants of tomato ETHYLENE INSENSITIVE 2 (SlEIN2), four EIN3-like genes (SlEIL1-4), and three EIN3 BINDING F-box protein genes (SlEBF1-3). Both slein2-1 and the high-order sleil mutant (sleil1 sleil2 sleil3/SlEIL3 sleil4) showed reduced fruit size, mainly due to decreased auxin biosynthesis. During fruit maturation, slein2 mutants displayed the complete cessation of ripening, which was partially rescued by slebf1 but not slebf2 or slebf3. We also discovered that ethylene directly activates the expression of the developmental genes NOR, RIN, and FRUITFULL1 (FUL1) via SlEIL proteins. Indeed, overexpressing these genes partially rescued the ripening defects of slein2-1. Finally, the signal intensity of the ethylene burst during fruit maturation was intimately connected with the progression of full ripeness. Collectively, our work uncovers a critical role of ethylene in fruit growth and supports a molecular framework of ripening control in which the developmental factors NOR, RIN, and FUL1 act downstream of ethylene signaling.

A comparative transcriptomics and eQTL approach identifies SlWD40 as a tomato fruit ripening regulator.

Although multiple vital genes with strong effects on the tomato (Solanum lycopersicum) ripening process have been identified via the positional cloning of ripening mutants and cloning of ripening-related transcription factors (TFs), recent studies suggest that it is unlikely that we have fully characterized the gene regulatory networks underpinning this process. Here, combining comparative transcriptomics and expression QTLs, we identified 16 candidate genes involved in tomato fruit ripening and validated them through virus-induced gene silencing analysis. To further confirm the accuracy of the approach, one potential ripening regulator, SlWD40 (WD-40 repeats), was chosen for in-depth analysis. Co-expression network analysis indicated that master regulators such as RIN (ripening inhibitor) and NOR (nonripening) as well as vital TFs including FUL1 (FRUITFUL1), SlNAC4 (NAM, ATAF1,2, and CUC2 4), and AP2a (Activating enhancer binding Protein 2 alpha) strongly co-expressed with SlWD40. Furthermore, SlWD40 overexpression and RNAi lines exhibited substantially accelerated and delayed ripening phenotypes compared with the wild type, respectively. Moreover, transcriptome analysis of these transgenics revealed that expression patterns of ethylene biosynthesis genes, phytoene synthase, pectate lyase, and branched chain amino transferase 2, in SlWD40-RNAi lines were similar to those of rin and nor fruits, which further demonstrated that SlWD40 may act as an important ripening regulator in conjunction with RIN and NOR. These results are discussed in the context of current models of ripening and in terms of the use of comparative genomics and transcriptomics as an effective route for isolating causal genes underlying differences in genotypes.

Transcription Factor RIPENING INHIBITOR and Its Homologs in Regulation of Fleshy Fruit Ripening of Various Plant Species

Transcription Factor RIPENING INHIBITOR and Its Homologs in Regulation of Fleshy Fruit Ripening of Various Plant Species

Postharvest handling induces changes in fruit DNA methylation status and is associated with alterations in fruit quality in tomato (Solanum lycopersicum L.)

Postharvest handling of tomato (Solanum lycopersicum L.), specifically low-temperature storage and early harvest, are used to extend shelf life but often reduce fruit quality. Recent work suggests that DNA methylation dynamics influences fruit ripening through the demethylase SlDML2 gene. However, the influence of postharvest handling on DNA methylation in relation to fruit quality is unclear. This work aimed to clarify these issues by analyzing DNA methylation using methyl-sensitive amplification polymorphism (MSAP), semi-quantitative transcriptional analysis of marker genes for fruit quality (RIN; RIPENING INHIBITOR) and DNA methylation (SlDML2; Solanum lycopersicum L. DNA demethylase 2), and, fruit biochemical quality biomarkers. Multivariate analysis of these data suggested that fruit DNA methylation state was associated with different postharvest handling techniques. Chilled postharvest fruit were distinct in their DNA methylation state and quality characteristics, which implied that these three phenomena i.e., chilling, methylation, and quality are highly connected. In addition, different postharvest handling methods modulated SlDML2 transcript levels but had little effect on the level of RIN transcripts in fruit that reached the Turning stage after early harvest, and cold storage. Although not a comprehensive global assessment, these data collectively helped to advance our interpretation of tomato fruit ripening. In conclusion, our findings revealed that postharvest-induced variation in fruit quality is in relation to DNA methylation. Long-term this work will help better connect physiological changes in tomato fruit to events happening at the molecular level.

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

Light controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement

Abstract Fruits are an essential part of a healthy, balanced diet and it is particularly important for fibre, essential vitamins, and trace elements. Improvement in the quality of fruit and elongation of shelf life are crucial goals for researchers. However, traditional techniques have some drawbacks, such as long period, low efficiency, and difficulty in the modification of target genes, which limit the progress of the study. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique was developed and has become the most popular gene-editing technology with high efficiency, simplicity, and low cost. CRISPR/Cas9 technique is widely accepted to analyse gene function and complete genetic modification. This review introduces the latest progress of CRISPR/Cas9 technology in fruit quality improvement. For example, CRISPR/Cas9-mediated targeted mutagenesis of RIPENING INHIBITOR gene (RIN), Lycopene desaturase (PDS), Pectate lyases (PL), SlMYB12, and CLAVATA3 (CLV3) can affect fruit ripening, fruit bioactive compounds, fruit texture, fruit colouration, and fruit size. CRISPR/Cas9-mediated mutagenesis has become an efficient method to modify target genes and improve fruit quality.