Natalie H. Chapman

Fruit development and ripening.

Fruiting structures in the angiosperms range from completely dry to highly fleshy organs and provide many of our major crop products, including grains. In the model plant Arabidopsis, which has dry fruits, a high-level regulatory network of transcription factors controlling fruit development has been revealed. Studies on rare nonripening mutations in tomato, a model for fleshy fruits, have provided new insights into the networks responsible for the control of ripening. It is apparent that there are strong similarities between dry and fleshy fruits in the molecular circuits governing development and maturation. Translation of information from tomato to other fleshy-fruited species indicates that regulatory networks are conserved across a wide spectrum of angiosperm fruit morphologies. Fruits are an essential part of the human diet, and recent developments in the sequencing of angiosperm genomes have provided the foundation for a step change in crop improvement through the understanding and harnessing of genome-wide genetic and epigenetic variation.

Transcriptional control of fleshy fruit development and ripening

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits. The model for the study of fleshy fruit development is tomato, because of the availability of single gene mutants and transgenic knock-down lines. In other species, our knowledge is often incomplete or absent. Tomato fruit size and shape are co-determined by transcription factors acting during formation of the ovary. Other transcription factors play a role in fruit chloroplast formation, and upon ripening impact quality aspects such as secondary metabolite content. In tomato, the transcription factors NON-RIPENING (NOR), COLORLESS NON-RIPENING (CNR), and RIPENING INHIBITOR (MADS-RIN) in concert with ethylene signalling regulate ripening, possibly in response to a developmental switch. Additional components include TOMATO AGAMOUS-LIKE1 (TAGL1), APETALA2a (AP2a), and FRUITFULL (FUL1 and FUL2). The links between this highly connected regulatory network and downstream effectors modulating colour, texture, and flavour are still relatively poorly understood. Intertwined with this network is post-transcriptional regulation by fruit-expressed microRNAs targeting several of these transcription factors. This important developmental process is also governed by changes in DNA methylation levels and possibly chromatin remodelling.

Regulation of ripening and opportunities for control in tomato and other fruits

Fruits are an important part of a healthy diet. They provide essential vitamins and minerals, and their consumption is associated with a reduced risk of heart disease and certain cancers. These important plant products can, however, be expensive to purchase, may be of disappointing quality and often have a short shelf life. A major challenge for crop improvement in fleshy fruit species is the enhancement of their health-promoting attributes while improving quality and reducing postharvest waste. To achieve these aims, a sound mechanistic understanding of the processes involved in fruit development and ripening is needed. In recent years, substantial insights have been made into the mechanistic basis of ethylene biosynthesis, perception and signalling and the identity of master regulators of ripening that operate upstream of, or in concert with a regulatory pathway mediated by this plant hormone. The role of other plant hormones in the ripening process has, however, remained elusive, and the links between regulators and downstream processes are still poorly understood. In this review, we focus on tomato as a model for fleshy fruit and provide an overview of the molecular circuits known to be involved in ripening, especially those controlling pigment accumulation and texture changes. We then discuss how this information can be used to understand ripening in other fleshy fruit-bearing species. Recent developments in comparative genomics and systems biology approaches are discussed. The potential role of epigenetic changes in generating useful variation is highlighted along with opportunities for enhancing the level of metabolites that have a beneficial effect on human health.

A DEMETER-like DNA demethylase governs tomato fruit ripening

Significance This work shows that active DNA demethylation governs ripening, an important plant developmental process. Our work defines a molecular mechanism, which has until now been missing, to explain the correlation between genomic DNA demethylation and fruit ripening. It demonstrates a direct cause-and-effect relationship between active DNA demethylation and induction of gene expression in fruits. The importance of these findings goes far beyond understanding the developmental biology of ripening and provides an innovative strategy for its fine control through fine modulation of epimarks in the promoters of ripening related genes. Our results have significant application for plant breeding especially in species with limited available genetic variation. In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits

A likely regulator of tomato ripening is identified from a gene network, its function is validated in transgenic plants, and an orthologous gene is shown to play a similar role in pepper. Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

High-Resolution Mapping of a Fruit Firmness-Related Quantitative Trait Locus in Tomato Reveals Epistatic Interactions Associated with a Complex Combinatorial Locus

Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Firs.p.QTL2.1 to Firs.p.QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Firs.p.QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Firs.p.QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

The development of PCR-based markers for the selection of eyespot resistance genes Pch1 and Pch2.

Two eyespot resistance genes (Pch1 and Pch2) have been characterised in wheat. The potent resistance gene Pch1, transferred from Aegilops ventricosa, is located on the distal end of the long arm of chromosome 7D (7DL). Pch2 derives from the variety Cappelle Desprez and is located at the distal end of chromosome 7AL. The RFLP marker Xpsr121 and the endopeptidase isozyme allele Ep-D1b, are very closely linked to Pch1, probably due to reduced recombination in the region of the introgressed A. ventricosa segment. Pch2 is less closely linked to these markers but is thought to be closer to Xpsr121 than to Ep-A1b. In the present study simple sequence repeat (SSR) markers were integrated into the genetic map of a single chromosome (7D) recombinant (RVPM) population segregating for Pch1. Sequence-tagged-site (STS)-based assays were developed for Xpsp121 and a 7DL wheat EST containing a SSR. SSR markers Xwmc14 and Xbarc97 and the Xpsr121-derived marker co-segregated with Pch1 in the RVPM population. A single chromosome (7A) recombinant population segregating for Pch2 was screened for eyespot resistance and mapped using SSRs. QTL interval mapping closely associated Pch2 with the SSR marker Xwmc525.

The identification of candidate genes associated with Pch2 eyespot resistance in wheat using cDNA-AFLP

Eyespot is a fungal disease of the stem base of cereal crops and causes lodging and the premature ripening of grain. Wheat cultivar Cappelle Desprez contains a highly durable eyespot resistance gene, Pch2 on the long arm of chromosome 7A. A cDNA-amplified fragment length polymorphism (AFLP) platform was used to identify genes differentially expressed between the eyespot susceptible variety Chinese Spring (CS) and the CS chromosome substitution line Cappelle Desprez 7A (CS/CD7A) which contains Pch2. Induced and constitutive gene expression was examined to compare differences between non-infected and plants infected with Oculimacula acuformis. Only 34 of approximately 4,700 cDNA-AFLP fragments were differentially expressed between CS and CS/CD7A. Clones were obtained for 29 fragments, of which four had homology to proteins involved with plant defence responses. Fourteen clones mapped to chromosome 7A and three of these mapped in the region of Pch2 making them putative candidates for involvement in eyespot resistance. Of particular importance are two fragments; 4CD7A8 and 19CD7A4, which have homology to an Oryza sativa putative callose synthase protein and a putative cereal cyst nematode NBS-LRR disease resistance protein (RCCN) respectively. Differential expression associated with Pch2 was examined by semi-quantitative RT-PCR. Of those genes tested, only four were differentially expressed at 14 days post inoculation. We therefore suggest that a majority of the differences in the cDNA-AFLP profiles are due to allelic polymorphisms between CS and CD alleles rather than differences in expression.

Homoeologous gene silencing in tissue cultured wheat callus

BackgroundIn contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.ResultsEvidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.ConclusionOur results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

Corrigendum: Genetic improvement of tomato by targeted control of fruit softening

Nat. Biotechnol. 34, 950–952 (2016); published online 25 July 2016; corrected after print 14 September 2016 In the version of this article initially published, the volume and page numbers for reference 46 were incorrect. The error has been corrected in the HTML and PDF versions of the article