Manipulation of ß-carotene levels in tomato fruits results in increased ABA content and extended shelf life.

Tomato fruit ripening is controlled by the hormone ethylene and by a group of transcription factors, acting upstream of ethylene. During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE ß-CYCLASE (LCYb) resulted in increased ß-carotene (provitamin A) content. Unexpectedly, LCYb-overexpressing fruits also exhibited a diverse array of ripening phenotypes, including delayed softening and extended shelf life. These phenotypes were accompanied, at the biochemical level, by an increase in abscisic acid (ABA) content, decreased ethylene production, increased density of cell wall material containing linear pectins with a low degree of methylation, and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoid compounds were also altered in the transgenic fruits, which could be attributed to delayed fruit ripening and/or to ABA. Network correlation analysis and pharmacological experiments with the ABA biosynthesis inhibitor, abamine, indicated that altered ABA levels were a direct effect of the increased ß-carotene content and were in turn responsible for the extended shelf life phenotype. Thus, manipulation of ß-carotene levels results in an improvement not only of the nutritional value of tomato fruits, but also of their shelf life.

Ubiquitin-conjugated degradation of golden 2-like transcription factor is mediated by CUL4-DDB1-based E3 ligase complex in tomato.

CULLIN4-RING ubiquitin ligases (CRL4s) as well as their targets are fundamental regulators functioning in many key developmental and stress responses in eukaryotes. In tomato (Solanum lycopersicum), molecular cloning has revealed that the underlying genes of natural spontaneous mutations high pigment 1 (hp1), high pigment 2 (hp2) and uniform ripening (u) encode UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), DE-ETIOLATED 1 (DET1) and GOLDEN 2-LIKE (GLK2), respectively. However, the molecular basis of the opposite actions of tomato GLK2 vs CUL4-DDB1-DET1 complex on regulating plastid level and fruit quality remains unknown. Here, we provide molecular evidence showing that the tomato GLK2 protein is a substrate of the CUL4-DDB1-DET1 ubiquitin ligase complex for the proteasome degradation. SlGLK2 is degraded by the ubiquitin-proteasome system, which is mainly determined by two lysine residues (K11 and K253). SlGLK2 associates with the CUL4-DDB1-DET1 E3 complex in plant cells. Genetically impairing CUL4, DDB1 or DET1 results in a retardation of SlGLK2 degradation by the 26S proteasome. These findings are relevant to the potential of nutrient accumulation in tomato fruit by mediating the plastid level and contribute to a deeper understanding of an important regulatory loop, linking protein turnover to gene regulation.

Transcriptomic events associated with internal browning of apple during postharvest storage.

BACKGROUND: Postharvest ripening of apple (Malus x domestica) can be slowed down by low temperatures, and a combination of low O2 and high CO2 levels. While this maintains the quality of most fruit, occasionally storage disorders such as flesh browning can occur. This study aimed to explore changes in the apple transcriptome associated with a flesh browning disorder related to controlled atmosphere storage using RNA-sequencing techniques. Samples from a browning-susceptible cultivar ('Braeburn') were stored for four months under controlled atmosphere. Based on a visual browning index, the inner and outer cortex of the stored apples was classified as healthy or affected tissue. RESULTS: Over 600 million short single-end reads were mapped onto the Malus consensus coding sequence set, and differences in the expression profiles between healthy and affected tissues were assessed to identify candidate genes associated with internal browning in a tissue-specific manner. Genes involved in lipid metabolism, secondary metabolism, and cell wall modifications were highly modified in the affected inner cortex, while energy-related and stress-related genes were mostly altered in the outer cortex. The expression levels of several of them were confirmed using qRT-PCR. Additionally, a set of novel browning-specific differentially expressed genes, including pyruvate dehydrogenase and 1-aminocyclopropane-1-carboxylate oxidase, was validated in apples stored for various periods at different controlled atmosphere conditions, giving rise to potential biomarkers associated with high risk of browning development. CONCLUSIONS: The gene expression data presented in this study will help elucidate the molecular mechanism of browning development in apples at controlled atmosphere storage. A conceptual model, including energy-related (linked to the tricarboxylic acid cycle and the electron transport chain) and lipid-related genes (related to membrane alterations, and fatty acid oxidation), for browning development in apple is proposed, which may be relevant for future studies towards improving the postharvest life of apple.

Functional characterization of a tomato COBRA-like gene functioning in fruit development and ripening.

BACKGROUND: Extensive studies have demonstrated that the COBRA gene is critical for biosynthesis of cell wall constituents comprising structural tissues of roots, stalks, leaves and other vegetative organs, however, its role in fruit development and ripening remains largely unknown. RESULTS: We identified a tomato gene (SlCOBRA-like) homologous to Arabidopsis COBRA, and determined its role in fleshy fruit biology. The SlCOBRA-like gene is highly expressed in vegetative organs and in early fruit development, but its expression in fruit declines dramatically during ripening stages, implying a primary role in early fruit development. Fruit-specific suppression of SlCOBRA-like resulted in impaired cell wall integrity and up-regulation of genes encoding proteins involved in cell wall degradation during early fruit development. In contrast, fruit-specific overexpression of SlCOBRA-like resulted in increased wall thickness of fruit epidermal cells, more collenchymatous cells beneath the epidermis, elevated levels of cellulose and reduced pectin solubilization in the pericarp cells of red ripe fruits. Moreover, transgenic tomato fruits overexpressing SlCOBRA-like exhibited desirable early development phenotypes including enhanced firmness and a prolonged shelf life. CONCLUSIONS: Our results suggest that SlCOBRA-like plays an important role in fruit cell wall architecture and provides a potential genetic tool for extending the shelf life of tomato and potentially additional fruits.

Limited conservation in cross-species comparison of GLK transcription factor binding suggested wide-spread cistrome divergence.

Non-coding cis-regulatory variants in animal genomes are an important driving force in the evolution of transcription regulation and phenotype diversity. However, cistrome dynamics in plants remain largely underexplored. Here, we compare the binding of GOLDEN2-LIKE (GLK) transcription factors in tomato, tobacco, Arabidopsis, maize and rice. Although the function of GLKs is conserved, most of their binding sites are species-specific. Conserved binding sites are often found near photosynthetic genes dependent on GLK for expression, but sites near non-differentially expressed genes in the glk mutant are nevertheless under purifying selection. The binding sites' regulatory potential can be predicted by machine learning model using quantitative genome features and TF co-binding information. Our study show that genome cis-variation caused wide-spread TF binding divergence, and most of the TF binding sites are genetically redundant. This poses a major challenge for interpreting the effect of individual sites and highlights the importance of quantitatively measuring TF occupancy.

Metabolism of soluble sugars in developing melon fruit: a global transcriptional view of the metabolic transition to sucrose accumulation.

The sweet melon fruit is characterized by a metabolic transition during its development that leads to extensive accumulation of the disaccharide sucrose in the mature fruit. While the biochemistry of the sugar metabolism pathway of the cucurbits has been well studied, a comprehensive analysis of the pathway at the transcriptional level allows for a global genomic view of sugar metabolism during fruit sink development. We identified 42 genes encoding the enzymatic reactions of the sugar metabolism pathway in melon. The expression pattern of the 42 genes during fruit development of the sweet melon cv Dulce was determined from a deep sequencing analysis performed by 454 pyrosequencing technology, comprising over 350,000 transcripts from four stages of developing melon fruit flesh, allowing for digital expression of the complete metabolic pathway. The results shed light on the transcriptional control of sugar metabolism in the developing sweet melon fruit, particularly the metabolic transition to sucrose accumulation, and point to a concerted metabolic transition that occurs during fruit development.

Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4.

Fruits are a major source of nutrition in human diets, providing carbohydrates, fiber, vitamins and phytonutrients. Carotenoids are a principal class of compounds found in many fruits, providing nutritional benefits both as precursors to essential vitamins and as antioxidants. Molecular characterization revealed that the tomato high pigment mutant genes (hp1 and hp2) encode UV-DAMAGED DNA BINDING PROTEIN-1 (DDB1) and DE-ETIOLATED-1 (DET1) homologs, respectively, and both are essential components of the recently identified CUL4-based E3 ligase complex. Here we have isolated a tomato CUL4 homolog and performed yeast two-hybrid assays to suggest possible association of tomato DDB1 with CUL4 and DET1. Real-time RT-PCR analysis indicated that both HP1 and CUL4 are expressed constitutively. Abscisic acid is implicated in plastid division control and its application substantially enhances HP1/DDB1 mRNA accumulation. Transformation of constructs expressing CUL4-YFP and DDB1-YFP fusion proteins driven by the CaMV 35S promoter reveals that both CUL4 and DDB1 are targeted to tomato plastids and nuclei simultaneously. Using fruit-specific promoters combined with RNAi technology, we show that downregulated DDB1 expression in transgenic fruits results in a significant increase in the number of plastids and corresponding enhanced pigment accumulation. CUL4-RNAi repression lines provide insight regarding CUL4 function during tomato development, and reveal that this tomato cullin is important in the regulation of plastid number and pigmentation, which in turn have a direct impact on fruit nutrient quality.

Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome.

Eleven sequenced BACs were annotated and localized via FISH to tomato pachytene chromosomes providing the first global insights into the compositional differences of euchromatin and pericentromeric heterochromatin in this model dicot species. The results indicate that tomato euchromatin has a gene density (6.7 kb/gene) similar to that of Arabidopsis and rice. Thus, while the euchromatin comprises only 25% of the tomato nuclear DNA, it is sufficient to account for approximately 90% of the estimated 38,000 nontransposon genes that compose the tomato genome. Moreover, euchromatic BACs were largely devoid of transposons or other repetitive elements. In contrast, BACs assigned to the pericentromeric heterochromatin had a gene density 10-100 times lower than that of the euchromatin and are heavily populated by retrotransposons preferential to the heterochromatin-the most abundant transposons belonging to the Jinling Ty3/gypsy-like retrotransposon family. Jinling elements are highly methylated and rarely transcribed. Nonetheless, they have spread throughout the pericentromeric heterochromatin in tomato and wild tomato species fairly recently-well after tomato diverged from potato and other related solanaceous species. The implications of these findings on evolution and on sequencing the genomes of tomato and other solanaceous species are discussed.

Signal transduction systems regulating fruit ripening.

Fruit ripening is a unique aspect of plant development with direct implications for a large component of the food supply and related areas of human health and nutrition. Recent advances in ripening research have given insights into the molecular basis of conserved developmental signals coordinating the ripening process and suggest that sequences related to floral development genes might be logical targets for additional discovery. Recent characterization of hormonal and environmental signal transduction components active in tomato fruit ripening (particularly ethylene and light) show conservation of signaling components yet novel gene family size and expression motifs that might facilitate complete and timely manifestation of ripening phenotypes. Emerging genomics tools and approaches are rapidly providing new clues and candidate genes that are expanding the known regulatory circuitry of ripening.

The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.