Emeline Teyssier

Structure and Function of a Mitochondrial Late Embryogenesis Abundant Protein Are Revealed by Desiccation

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into α-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.

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.

DNA Methylation and Chromatin Regulation during Fleshy Fruit Development and Ripening

Fruit ripening is a developmental process that results in the leaf-like carpel organ of the flower becoming a mature ovary primed for dispersal of the seeds. Ripening in fleshy fruits involves a profound metabolic phase change that is under strict hormonal and genetic control. This work reviews recent developments in our understanding of the epigenetic regulation of fruit ripening. We start by describing the current state of the art about processes involved in histone post-translational modifications and the remodeling of chromatin structure and their impact on fruit development and ripening. However, the focus of the review is the consequences of changes in DNA methylation levels on the expression of ripening-related genes. This includes those changes that result in heritable phenotypic variation in the absence of DNA sequence alterations, and the mechanisms for their initiation and maintenance. The majority of the studies described in the literature involve work on tomato, but evidence is emerging that ripening in other fruit species may also be under epigenetic control. We discuss how epigenetic differences may provide new targets for breeding and crop improvement.

Functional analysis of SlEZ1 a tomato Enhancer of zeste ( E(z)) gene demonstrates a role in flower development

The Enhancer of Zeste (E(z)) Polycomb group (PcG) proteins, which are encoded by a small gene family in Arabidopsis thaliana, have been shown to participate to the control of flowering and seed development. For the time being, little is known about the function of these proteins in other plants. In tomato E(z) proteins are encoded by at least two genes namely SlEZ1 and SlEZ2 while a third gene, SlEZ3, is likely to encode a truncated non-functional protein. The analysis of the corresponding mRNA demonstrates that these two genes are differentially regulated during plant and fruit development. We also show that SlEZ1 and SlEZ2 are targeted to the nuclei. These results together with protein sequence analysis makes it likely that both proteins are functional E(z) proteins. The characterisation of SlEZ1 RNAi lines suggests that although there might be some functional redundancy between SlEZ1 and SlEZ2 in most plant organs, the former protein is likely to play specific function in flower development.

A CURLY LEAF homologue controls both vegetative and reproductive development of tomato plants

Abstract The Enhancer of Zeste Polycomb group proteins, which are encoded by a small gene family in Arabidopsis thaliana, participate to the control of plant development. In the tomato (Solanum lycopersicum), these proteins are encoded by three genes (SlEZ1,SlEZ2 and SlEZ3) that display specific expression profiles. Using a gene specific RNAi strategy, we demonstrate that repression of SlEZ2 correlates with a general reduction of H3K27me3 levels, indicating that SlEZ2 is part of an active PRC2 complex. Reduction of SlEZ2 gene expression impacts the vegetative development of tomato plants, consistent with SlEZ2 having retained at least some of the functions of the Arabidopsis CURLY LEAF (CLF) protein. Notwithstanding, we observed significant differences between transgenic SlEZ2 RNAi tomato plants and Arabidopsisclf mutants. First, we found that reduced SlEZ2 expression has dramatic effects on tomato fruit development and ripening, functions not described in Arabidopsis for the CLF protein. In addition, repression of SlEZ2 has no significant effect on the flowering time or the control of flower organ identity, in contrast to the Arabidopsisclf mutation. Taken together, our results are consistent with a diversification of the function of CLF orthologues in plants, and indicate that although partly conserved amongst plants, the function of EZ proteins need to be newly investigated for non-model plants because they might have been recruited to specific developmental processes.

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants

Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

Epigenetic regulation during fleshy fruit development and ripening

The development of fleshy fruits is characterized by cell division and expansion events that occur following fertilization. In contrast to dry fruits there is no lignification phase, but fleshy fruits undergo a complex ripening process. Tomato has long been the model for fleshy fruit development and ripening, relying on the establishment and maintenance of differential transcription patterns. A full understanding of fruit development and ripening will not be achieved when it is based only on genetic models. Epigenetic regulations are likely to be essential as well, as recently suggested by the discovery that the tomato Cnr (Colour non-ripening) mutation (characterized by abolishment of fruit ripening) was caused by the hypermethylation of part of the promoter region. Histone marks collaborate with DNA methylation to shape chromatin and suggest a complex interplay between gene transcription and epigenome landscapes. At present, our current understanding of the function of epigenetic marks has been mainly obtained with the model plants Arabidopsis, rice and maize and is by far less advanced in most crop plants. However there is increasing evidence that epigenetic regulations are essential actors that also control traits of agronomical relevance, such as plant adaptation to environmental constraints, and heterosis, a phenomenon extensively used to increase agronomical production, flowering time or fruit quality.

Locus-Specific DNA Methylation Analysis and Applications to Plants

In contrast to mammals where DNA methylation occurs near exclusively at CG dinucleotides, all cytosines of plant genomes can be methylated irrespective to the sequence context, including the symmetrical CG and CHG sequence contexts, and the nonsymmetrical CHH sequence context. Plant genomes do not present CG islands as found in mammalian genomes where a high frequency of CG can be observed at some promoter regions. So far, the methylome of several plants has been described showing variations of both methylation levels between plants, which ranged from 5% in Arabidopsis thaliana to more than 30% in corn, but also of the proportion of methylated cytosines in the CG, CHG, and CHH sequence contexts. DNA methylation was shown to have important roles in the development of many organisms including plants. In this later case, DNA methylation is involved in the regulation of flowering time, flower sex determination, seed development, or fruit ripening. To date, a wide range of low to high resolution methods initially developed for animal genomes allow the analysis of DNA methylation at specific loci or globally of whole genomes. Indeed, part of these methods is not applicable to plants due to the specificities of their methylome; however, some have been successfully modified to overcome the complexity of plant DNA methylation. In this chapter, we describe an exhaustive list of methods devoted to the locus-specific analysis of DNA methylation which have proven to be applicable to plant genomes and their potential use in high throughput screening of plant population.

Tomato fruit quality improvement facing the functional genomics revolution

The process of fruit development has been the object of many studies aimed to investigate genetic as well as environmental factors that control fruit growth, maturation and the biochemical composition. Tomato (Solanum lycopersicum L.) is considered the model plant for fleshy fruit development. For years, genetic engineering of tomato has focused principally on enhancing fruit quality traits (productivity, biotic and abiotic stress tolerance, size, morphology, and more recently taste, aroma, flavour, etc.). Detection of quantitative trait loci (QTL) in tomato breeding populations and interspecific introgression lines offers the advantage that QTL are of direct relevance for the improvement of crops via knowledge-based breeding. With the release of the tomato genome sequence, studies of functional genomics can assist in the identification of gene-to-trait association. Current progress in genetic tools, genomic resources and functional genomics approaches for tomato is summarized, especially devoting particular attention to metabolomics approaches and possible future challenges of fruit breeding assisted by fruit modelling.