Nigel E. Gapper

Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening

Ripening of tomato fruits is triggered by the plant hormone ethylene, but its effect is restricted by an unknown developmental cue to mature fruits containing viable seeds. To determine whether this cue involves epigenetic remodeling, we expose tomatoes to the methyltransferase inhibitor 5-azacytidine and find that they ripen prematurely. We performed whole-genome bisulfite sequencing on fruit in four stages of development, from immature to ripe. We identified 52,095 differentially methylated regions (representing 1% of the genome) in the 90% of the genome covered by our analysis. Furthermore, binding sites for RIN, one of the main ripening transcription factors, are frequently localized in the demethylated regions of the promoters of numerous ripening genes, and binding occurs in concert with demethylation. Our data show that the epigenome is not static during development and may have been selected to ensure the fidelity of developmental processes such as ripening. Crop-improvement strategies could benefit by taking into account not only DNA sequence variation among plant lines, but also the information encoded in the epigenome.

Molecular and genetic regulation of fruit ripening

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.

Biology and genetic engineering of fruit maturation for enhanced quality and shelf-life.

Commercial regulation of ripening is currently achieved through early harvest, by controlling the postharvest storage atmosphere and genetic selection for slow or late ripening varieties. Although these approaches are often effective, they are not universally applicable and often result in acceptable, but poor quality, products. With increased understanding of the molecular biology underlying ripening and the advent of genetic engineering technologies, researchers have pursued new strategies to address problems in fruit shelf-life and quality. These have been guided by recent insights into mechanisms by which ethylene and a complex network of transcription factors regulate ripening, and by an increased appreciation of factors that contribute to shelf-life, such as the fruit cuticle.

Tomato GOLDEN2-LIKE Transcription Factors Reveal Molecular Gradients That Function during Fruit Development and Ripening

Many fruits develop as chloroplast-rich organs that transition to ripening, where photosynthesis wanes and flavor, aroma, and nutritional metabolites predominate. Regulatory genes are shown to influence the numbers and patterns of chloroplast distribution in fruit and leaves and that chloroplast abundance in green fruit is related to the nutrition and quality attributes of ripe fruit. Fruit ripening is the summation of changes rendering fleshy fruit tissues attractive and palatable to seed dispersing organisms. For example, sugar content is influenced by plastid numbers and photosynthetic activity in unripe fruit and later by starch and sugar catabolism during ripening. Tomato fruit are sinks of photosynthate, yet unripe green fruit contribute significantly to the sugars that ultimately accumulate in the ripe fruit. Plastid numbers and chlorophyll content are influenced by numerous environmental and genetic factors and are positively correlated with photosynthesis and photosynthate accumulation. GOLDEN2-LIKE (GLK) transcription factors regulate plastid and chlorophyll levels. Tomato (Solanum lycopersicum), like most plants, contains two GLKs (i.e., GLK1 and GLK2/UNIFORM). Mutant and transgene analysis demonstrated that these genes encode functionally similar peptides, though differential expression renders GLK1 more important in leaves, while GLK2 is predominant in fruit. A latitudinal gradient of GLK2 expression influences the typical uneven coloration of green and ripe wild-type fruit. Transcriptome profiling revealed a broader fruit gene expression gradient throughout development. The gradient influenced general ripening activities beyond plastid development and was consistent with the easily observed yet poorly studied ripening gradient present in tomato and many fleshy fruits.

Biomarker development for external CO2 injury prediction in apples through exploration of both transcriptome and DNA methylation changes

Apple is a unique horticultural crop that is available to consumers year round, though harvested just once annually. A year-long supply is reliant on current postharvest practices such as refrigeration, controlled atmosphere and chemical treatment. However, disorders can develop during storage leading to loss of the crop at great cost to orchardists and storage facilities. The goal of this work is to develop predictive biomarkers the apple industry can use to market apples susceptible to disorders early, consequently reducing postharvest losses. This article outlines the genomics based approach we are taking to develop such tools, and presents our first list of putative predictive biomarkers.

Transcriptomic events associated with internal browning of apple during postharvest storage

BackgroundPostharvest 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.ResultsOver 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.ConclusionsThe 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.

Understanding development and ripening of fruit crops in an 'omics' era

Next generation sequencing has revolutionized plant biology. Not only has our understanding of plant metabolism advanced using model systems and modern chromatography, but application of ‘omics’-based technology has been widely extended to non-model systems as costs have plummeted and efficiency increased. As a result, important fundamental questions relating to important horticultural crops are being answered, and novel approaches with application to industry are in progress. Here we review recent research advances on development and ripening of fruit crops, how next generation sequencing approaches are driving this advance and the emerging future landscape.

Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.