CLASS-II KNOX genes coordinate spatial and temporal patterns of the tomato ripening

Ripening is a complex developmental change of a mature organ, the fruit. In plants like a tomato, it involves softening, pigmentation, and biosynthesis of metabolites beneficial for the human diet. Examination of the transcriptional changes towards ripening suggests that redundant uncharacterized factors may be involved in the coordination of the ripening switch. Previous studies have demonstrated that Arabidopsis CLASS-II KNOX genes play a significant role in controlling the maturation of siliques and their transition to senescence. Here we examined the combined role of all four tomato CLASS-II KNOX genes in the maturation and ripening of fleshy fruits using an artificial microRNA targeting them simultaneously. As expected, the knockdown plants (35S::amiR-TKN-CL-II) exhibited leaves with increased complexity, reminiscent of the leaf phenotype of plants overexpressing CLASS- I KNOX, which antagonize CLASS-II KNOX gene functions. The fruits of 35S::amiR-TKN-CL-II plants were notably smaller than the control. While their internal gel/placenta tissue softened and accumulated the typical pigmentation, the pericarp color break took place ten days later than control, and eventually, it turned yellow instead of red. Additionally, the pericarp of 35S::amiR-TKN-CL-II fruits remained significantly firmer than control even after three weeks of shelf storage. Strikingly, the 35S::amiR-TKN-CL-II fruits showed early ethylene release and respiration peak, but these were correlated only with liquefaction and pigmentation of the internal tissues. Our findings suggest that CLASS-II KNOX genes are required to coordinate the spatial and temporal patterns of tomato fruit ripening.

Rosmarinic Acid Delays Tomato Fruit Ripening by Regulating Ripening-Associated Traits

Fruits are excellent sources of essential vitamins and health-boosting minerals. Recently, regulation of fruit ripening by both internal and external cues for the improvement of fruit quality and shelf life has received considerable attention. Rosmarinic acid (RA) is a kind of natural plant-derived polyphenol, widely used in the drug therapy and food industry due to its distinct physiological functions. However, the role of RA in plant growth and development, especially at the postharvest period of fruits, remains largely unknown. Here, we demonstrated that postharvest RA treatment delayed the ripening in tomato fruits. Exogenous application of RA decreased ripening-associated ethylene production and inhibited the fruit color change from green to red based on the decline in lycopene accumulation. We also found that the degradation of sucrose and malic acid during ripening was significantly suppressed in RA-treated tomato fruits. The results of metabolite profiling showed that RA application promoted the accumulation of multiple amino acids in tomato fruits, such as aspartic acid, serine, tyrosine, and proline. Meanwhile, RA application also strengthened the antioxidant system by increasing both the activity of antioxidant enzymes and the contents of reduced forms of antioxidants. These findings not only unveiled a novel function of RA in fruit ripening, but also indicated an attractive strategy to manage and improve shelf life, flavor, and sensory evolution of tomato fruits.

Novel Translational and Phosphorylation Modification Regulation Mechanisms of Tomato (Solanum lycopersicum) Fruit Ripening Revealed by Integrative Proteomics and Phosphoproteomics

The tomato is a research model for fruit-ripening, however, its fruit-ripening mechanism still needs more extensive and in-depth exploration. Here, using TMT and LC-MS, the proteome and phosphoproteome of AC++ (wild type) and rin (ripening-inhibitor) mutant fruits were studied to investigate the translation and post-translational regulation mechanisms of tomato fruit-ripening. A total of 6141 proteins and 4011 phosphorylation sites contained quantitative information. One-hundred proteins were identified in both omics’ profiles, which were mainly found in ethylene biosynthesis and signal transduction, photosynthesis regulation, carotenoid and flavonoid biosynthesis, chlorophyll degradation, ribosomal subunit expression changes, MAPK pathway, transcription factors and kinases. The affected protein levels were correlated with their corresponding gene transcript levels, such as NAC-NOR, MADS-RIN, IMA, TAGL1, MADS-MC and TDR4. Changes in the phosphorylation levels of NAC-NOR and IMA were involved in the regulation of tomato fruit-ripening. Although photosynthesis was inhibited, there were diverse primary and secondary metabolic pathways, such as glycolysis, fatty acid metabolism, vitamin metabolism and isoprenoid biosynthesis, regulated by phosphorylation. These data constitute a map of protein—protein phosphorylation in the regulation of tomato fruit-ripening, which lays the foundation for future in-depth study of the sophisticated molecular mechanisms of fruit-ripening and provide guidance for molecular breeding.

Role of the tomato fruit ripening regulator MADS-RIN in resistance to Botrytis cinerea infection

Tomato MADS-RIN (RIN) transcription factor has been shown to be a master activator regulating fruit ripening. Recent studies have revealed that in addition to activating many other cell wall genes, it also represses expression of XTH5, XTH8 and MAN4a, which are positively related to excess flesh softening and cell wall degradation, which might indicate it has a potential role in pathogen resistance of ripening fruit. In this study, both wild type (WT) and RIN-knockout (RIN-KO) mutant tomato fruit were infected with Botrytis cinerea, to investigate the function of RIN in defence against pathogen infection during ripening. The results showed that RIN-KO fruit were much more sensitive to B.cinerea infection with larger lesion sizes. Transcriptiome data and qRT-PCR assay indicate genes of phenylalanine ammonialyase (PAL) and chitinase (CHI) in RIN-KO fruit were reduced and their corresponding enzyme activities were decreased. Transcripts of genes encoding pathogenesis-related proteins (PRs), including PR1a, PRSTH2 and APETALA2/Ethylene Response Factor (AP2/ERF) including ERF.A1, Pti5, Pti6, ERF.A4 were reduced in RIN-KO fruit comparing to WT fruit. Moreover, in the absence of RIN the expression of genes encoding cell wall modifying enzymes XTH5, XTH8, MAN4a has been reported to be elevated, which is potentially correlated with cell wall properties. When present, RIN represses transcription of XTH5 by activating ERF.F4 a class II (repressor class) ERF gene family member and ERF.F5. These results support the conclusion that RIN enhances ripening-related resistance to grey mould infection by upregulating pathogen-resistance genes and defense enzyme activies as well as reducing accumulation of transcripts encoding some cell wall enzymes.

Transcriptomic analysis of a wild and a cultivated varieties of Capsicum annuum over fruit development and ripening

Chili pepper (Capsicum annuum) is one of the most important crops worldwide. Its fruits contain metabolites produced over the maturation process like capsaicinoids and carotenoids. This metabolic process produces internal changes in flavor, color, texture, and aroma in fruits to make them more attractive for seed dispersal organisms. The chiltepin (C. annuum L. var. glabriusculum) is a wild variety of the C. annuum L. species that is considered a source of genetic resources that could be used to improve the current chili crops. In this study, we performed a transcriptomic analysis on two fruit maturation stages: immature stage (green fruit) and mature stage (red fruit) of a wild and a cultivated pepper variety. We found 19,811 genes expressed, and 1,008 genes differentially expressed (DEGs) in at least one of the five contrast used; 730 DEGs were found only in one contrast, and most DEGs in all contrasts were downregulated. GO enrichment analysis showed that the majority of DEGs are related to stress responses. KEGG enrichment analysis detected differences in expression patterns in metabolic pathways related to phenylpropanoid biosynthesis, secondary metabolites, plant hormone signal transduction, carotenoid biosynthesis and sesquiterpenoid and triterpenoid biosynthesis. We selected 105 tomato fruit ripening-related genes, and found 53 pepper homologs differentially expressed related to shape, size, and secondary metabolite biosynthesis. According to the transcriptome analysis, the two peppers showed very similar gene expression patterns; differences in expression patterns of genes related to shape, size, ethylene and secondary metabolites biosynthesis suggest that changes produced by domestication of chilli pepper could be very specific to the expression of genes related to traits desired in commercial fruits.

A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening–specific LATERAL ORGAN BOUNDRIES (LOB) TF, SlLOB1, up-regulates a suite of cell wall–associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 (EXP1) are strongly suppressed in SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.