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Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.

Transcription factors (TFs) are crucial for regulating fruit ripening in tomato (Solanum lycopersicum). The GRAS (GAI, RGA, and SCR) TFs are involved in various physiological processes, but their role in fruit ripening has seldom been reported. We have previously identified a gene encoding GRAS protein named SlFSR (F ruit S helf-life R egulator), which is implicated in fruit ripening by regulating cell wall metabolism; however, the underlying mechanism remains unclear. Here, we demonstrate that SlFSR proteins are localized to the nucleus, where they could bind to specific DNA sequences. SlFSR acts downstream of the master ripening regulator RIN and could collaborate with RIN to control the ripening process by regulating expression of ethylene biosynthesis genes. In SlFSR -CR (CRISPR/Cas9) mutants, the initiation of fruit ripening was not affected but the reduced ethylene production and a delayed coloring process occurred. RNA-sequencing (RNA-seq) and promoter analysis reveal that SlFSR directly binds to the promoters of two key ethylene biosynthesis genes (SlACO1 and SlACO3) and activates their expression. However, SlFSR -CR fruits displayed a significant down-regulation of key rate-limiting genes (SlDXS1 and SlGGPPS2) in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which may account for the impaired lycopene synthesis. Altogether, we propose that SlFSR positively regulates ethylene biosynthesis and lycopene accumulation, providing valuable insights into the molecular mechanisms underlying fruit ripening. • SlFSR regulates ET biosynthesis by activating the transcription of SlACO1 and SlACO3. • SlFSR regulates carotenoids accumulation by influencing the MEP pathway. • SlFSR and RIN cooperate to regulate the ripening processes in tomato. [ABSTRACT FROM AUTHOR]

Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr , nor , and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr , nor , and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality. [ABSTRACT FROM AUTHOR]

Highlights • Ethanol stimulated expression of LeACS2 and LeACS4. • Ethanol treatment might induce genes encoding ripening-specific transcription factor. • Expression of RIN -dependent ripening-related genes was inhibited by ethanol. Abstract Postharvest ethanol vapor treatment suppresses tomato fruit ripening. The aim of this study was to clarify the mechanism of suppression at the molecular level. Tomato (Solanum lycopersicum cv. 'Micro Tom') fruit harvested at mature green and breaker stages was continuously treated with ethanol using an ethanol pad at 20 °C in the dark. Ethanol treatment stimulated ethylene production and increased gene expression of LeACS2 and LeACS4 , which are related to ethylene-related ripening. The expression of genes encoding ripening-specific transcription factors acting upstream of ethylene was also induced, when the fruit was treated with ethanol. In the ethylene-dependent ripening process, ethanol inhibited the expression of RIN -dependent and ethylene-dependent ripening-related genes, suggesting that ethanol could inhibit some processes downstream of ethylene perception. In the ethylene-independent ripening process, expression of RIN -dependent and ethylene-independent ripening related genes was inhibited by ethanol, suggesting that ethanol could inhibit some processes downstream of RIN. Inhibition of each process by ethanol could be a mechanism by which ethanol inhibits ripening, although it stimulates ethylene production and factors upstream of the ripening process. [ABSTRACT FROM AUTHOR]

Summary: Ethylene is the main hormone controlling climacteric fruit ripening; however, the mechanisms underlying the developmental transition leading to the initiation of the ripening process remain elusive, although the presumed role of active hormone interplay has often been postulated.To unravel the putative role of auxin in the unripe‐to‐ripe transition, we investigated the dynamics of auxin activity in tomato fruit and addressed the physiological significance of Sl‐SAUR69, previously identified as a RIN target gene, using reverse genetics approaches.Auxin signalling undergoes dramatic decline at the onset of ripening in wild‐type fruit, but not in the nonripening rin mutant. Sl‐SAUR69 exhibits reduced expression in rin and its up‐regulation results in premature initiation of ripening, whereas its down‐regulation extends the time to ripening. Overexpression of Sl‐SAUR69 reduces proton pump activity and polar auxin transport, and ectopic expression in Arabidopsis alters auxin transporter abundance, further arguing for its active role in the regulation of auxin transport.The data support a model in which Sl‐SAUR69 represses auxin transport, thus generating auxin minima, which results in enhanced ethylene sensitivity. This defines a regulation loop, fed by ethylene and auxin as the main hormonal signals and by RIN and Sl‐SAUR69 as modulators of the balance between the two hormones. [ABSTRACT FROM AUTHOR]

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. • A shorter fragment of tomato β-Hex promoter (pD-200) is sufficient for ripening specific expression. • Ethylene induces pD-200 promoter activity and SlERF.E4 binds to pD-200 promoter. • RIN, SlASR1 and SlERF.E4 are transcriptional activators of β-Hex and work in a complementary manner. • pD-200 promoter can be used for fruit specific gene expression studies and improving fruit quality traits. [ABSTRACT FROM AUTHOR]

Climacteric fruit ripening, as it occurs in many fruit crops, depends on a rapid, autocatalytic increase in ethylene production. This agriculturally important process has been studied extensively, with tomato simultaneously acting both as a model species and target crop for modification. In tomato, the ethylene biosynthetic genes ACC SYNTHASE2 (ACS2) and ACS4 are highly expressed during fruit ripening, with a combined loss of both ACS2 and ACS4 activity preventing generation of the ethylene burst necessary for fruit ripening. However, the individual roles and importance of ACS2 and ACS4 have not been determined. In this study, we examined specifically the role of ACS4 by comparing the phenotype of an acs4 mutant firstly with that of the wild-type, and secondly with two novel ripening-inhibitor (rin) mutants. Ethylene production during ripening was significantly reduced in both acs4-1, and rin lines, with rin genotypes showing the weaker ethylene burst. Also i) the time between anthesis and the start of fruit ripening and ii) the time required to progress through ripening were significantly longer in acs4-1 than in the wild type, but shorter than in the strongest rin mutant. The delay in ripening was reflected in the lower expression of ripening-related transcripts during the mature green and light red ripening stages. Furthermore, expression of ACS2 and ACS4 was strongly dependent on a functional RIN gene, while ACS2 expression was largely independent of ACS4. Altogether, we show that ACS4 is necessary for normal progression of tomato fruit ripening and that mutation of this gene may provide a useful means for altering ripening traits.

Background: Tomato fruit ripening is controlled by ethylene and is characterized by a shift in color from green to red, a strong accumulation of lycopene, and a decrease in β-xanthophylls and chlorophylls. The role of other hormones, such as auxin, has been less studied. Auxin is retarding the fruit ripening. In tomato, there is no study of the carotenoid content and related transcript after treatment with auxin. Results: We followed the effects of application of various hormone-like substances to "Mature-Green" fruits. Application of an ethylene precursor (ACC) or of an auxin antagonist (PCIB) to tomato fruits accelerated the color shift, the accumulation of lycopene, α-, β-, and δ-carotenes and the disappearance of β-xanthophylls and chlorophyll b. By contrast, application of auxin (IAA) delayed the color shift, the lycopene accumulation and the decrease of chlorophyll a. Combined application of IAA + ACC led to an intermediate phenotype. The levels of transcripts coding for carotenoid biosynthesis enzymes, for the ripening regulator Rin, for chlorophyllase, and the levels of ethylene and abscisic acid (ABA) were monitored in the treated fruits. Correlation network analyses suggest that ABA, may also be a key regulator of several responses to auxin and ethylene treatments. Conclusions: The results suggest that IAA retards tomato ripening by affecting a set of (i) key regulators, such as Rin, ethylene and ABA, and (ii) key effectors, such as genes for lycopene and β-xanthophyll biosynthesis and for chlorophyll degradation. [ABSTRACT FROM AUTHOR]

The development and ripening of tomato fruit are complex processes involving many gene regulatory pathways at the transcriptional and post-transcriptional level. Ripening inhibitor ( RIN) is a vital transcription factor, which targets numerous ripening-related genes at the transcriptional level during tomato fruit ripening. Micro RNAs (mi RNAs) are a class of short noncoding RNAs that play important roles in post-transcriptional gene regulation. To elucidate the potential regulatory relationship between rin and mi RNAs during fruit development and ripening, we identified known mi RNAs and profiled their expression in wild-type tomato and rin mutant using a deep sequencing approach combined with quantitative RT- PCR. A total of 33 known mi RNA families were identified, of which 14 mi RNA families were differently accumulated. Subsequent promoter analysis showed that possible RIN-binding motifs ( CAr G-box) tended to occur frequently in the promoter regions of partial differently expressed mi RNAs. In addition, ethylene may participate in the regulation of mi RNAs accumulation during tomato fruit ripening. Chromatin immunoprecipitation ( Ch IP) and electrophoretic mobility shift assay confirmed the direct binding of RIN to the promoter of MIR172a. Collectively, these results showed a close correlation between mi RNA expression and RIN as well as ethylene, which further elucidated the regulatory roles of mi RNAs during fruit development and ripening and enriched the regulatory network of RIN in tomato fruit. [ABSTRACT FROM AUTHOR]

Climacteric fruit ripening, as it occurs in many fruit crops, depends on a rapid, autocatalytic increase in ethylene production. This agriculturally important process has been studied extensively, with tomato simultaneously acting both as a model species and target crop for modification. In tomato, the ethylene biosynthetic genes ACC SYNTHASE2 (ACS2) and ACS4 are highly expressed during fruit ripening, with a combined loss of both ACS2 and ACS4 activity preventing generation of the ethylene burst necessary for fruit ripening. However, the individual roles and importance of ACS2 and ACS4 have not been determined. In this study, we examined specifically the role of ACS4 by comparing the phenotype of an acs4 mutant firstly with that of the wild-type, and secondly with two novel ripening-inhibitor (rin) mutants. Ethylene production during ripening was significantly reduced in both acs4-1, and rin lines, with rin genotypes showing the weaker ethylene burst. Also i) the time between anthesis and the start of fruit ripening and ii) the time required to progress through ripening were significantly longer in acs4-1 than in the wild type, but shorter than in the strongest rin mutant. The delay in ripening was reflected in the lower expression of ripening-related transcripts during the mature green and light red ripening stages. Furthermore, expression of ACS2 and ACS4 was strongly dependent on a functional RIN gene, while ACS2 expression was largely independent of ACS4. Altogether, we show that ACS4 is necessary for normal progression of tomato fruit ripening and that mutation of this gene may provide a useful means for altering ripening traits. [ABSTRACT FROM AUTHOR]

Highlights: [•] In tomato fruit, we identified 419 ripening-associated genes, with 224 upregulated and 195 downregulated. [•] Dependence of the ripening-associated genes with ethylene, RIN and NOR was determined. [•] Several new transcription and signaling factors associated with ripening were identified. [Copyright &y& Elsevier]

A thorough understanding of molecular mechanisms underlying ripening is the prerequisite for genetic manipulation of fruits for better shelf-life and nutritional quality. Mutation in LeMADS- RIN, a MADS-box gene, leads to non-ripening phenotype of rin fruits in tomato. Characterization of ripening- inhibitor ( rin) mutant has elucidated important role of ethylene in the regulation of climacteric fruit ripening. A complete understanding of this mutation will unravel novel genetic regulatory mechanisms involved in fruit ripening. In this study, fruit transcriptomes of two genotypes, including a cultivated Indian cultivar Solanum lycopersicum cv. Pusa Ruby and a homozygous line harboring the rin mutation (LA1795) were compared to get better insight into RIN-regulated ethylene-dependent and ethylene-independent events during ripening. Cluster analysis of ripening-related genes indicated a major shift in their expression profiles in rin mutant fruit. A total of 112 genes, exhibiting expression patterns similar to that of LeMADS- RIN in wild-type fruits, showed down regulation of expression in the rin mutant. In silico analysis of putative promoters of these genes for the presence of CArG box along with ERE and ethylene inducibility of these genes revealed that genes lacking CArG box in their regulatory regions could be indirectly regulated by LeMADS-RIN. New regulators of ethylene-dependent aspect of ripening were also identified. In this study, we have made an attempt to distinguish between ethylene-dependent and ethylene-independent aspects of ripening, which will be useful for developing strategies to improve fruit-related agronomic traits in tomato and other crops. [ABSTRACT FROM AUTHOR]