Philippe Gallusci

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.

Effects of long-term cadmium exposure on growth and metabolomic profile of tomato plants

The response of tomato plants to long-term cadmium exposure was evaluated after a 90-days long culture in hydroponic conditions (0, 20, and 100 μM CdCl(2)). Cadmium preferentially accumulated in roots, and to a lower extent in upper parts of plants. Absolute quantification of 28 metabolites was obtained through (1)H NMR, HPLC-PDA, and colorimetric methods. The principal component analysis showed a clear separation between control and Cd treated samples. Proline and total ascorbate amounts were reduced in Cd-treated leaves, whereas α-tocopherol, asparagine, and tyrosine accumulation increased, principally in 100 μM Cd treated leaves. Carotenoid and chlorophyll contents decreased only in 100 μM Cd-mature-leaves, which correlate with a reduced expression of genes essential for isoprenoid and carotenoid accumulations. Our results show that tomato plants acclimatize during long-term exposure to 20 μM Cd. On the contrary, 100μM Cd treatment results in drastic physiological and metabolic perturbations leading to plant growth limitation and fruit set abortion.

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.

Prolonged root hypoxia induces ammonium accumulation and decreases the nutritional quality of tomato fruits

Here we examined the effects of root hypoxia (1-2% oxygen) on the physiology of the plant and on the biochemical composition of fruits in tomato (Solanum lycopersicum cv. Micro-Tom) plants submitted to gradual root hypoxia at first flower anthesis. Root hypoxia enhanced nitrate absorption with a concomitant release of nitrite and ammonium into the medium, a reduction of leaf photosynthetic activity and chlorophyll content, and an acceleration of fruit maturation, but did not affect final fruit size. Quantitative metabolic profiling of mature pericarp extracts by (1)H NMR showed that levels of major metabolites including sugars, organic acids and amino acids were not modified. However, ammonium concentration increased dramatically in fruit flesh, and ascorbate and lycopene concentrations decreased. Our data indicate that the unfavorable effects of root hypoxia on fruit quality cannot be explained by two of the well-known effects of root hypoxia on the plant, namely a decrease in photosynthesis or an excess in ethylene production, but may instead result from disturbances in the supply of either growth regulators or ammonium, by the roots.

Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening.

In plants, microRNAs (miRNAs) play essential roles in growth, development, yield, stress response and interactions with pathogens. However no miRNA has been experimentally documented to be functionally involved in fruit ripening although many miRNAs have been profiled in fruits. Here we show that SlymiR157 and SlymiR156 differentially modulate ripening and softening in tomato (Solanum lycopersicum). SlymiR157 is expressed and developmentally regulated in normal tomato fruits and in those of the Colourless non-ripening (Cnr) epimutant. It regulates expression of the key ripening gene LeSPL-CNR in a likely dose-dependent manner through miRNA-induced mRNA degradation and translation repression. Viral delivery of either pre-SlymiR157 or mature SlymiR157 results in delayed ripening. Furthermore, qRT-PCR profiling of key ripening regulatory genes indicates that the SlymiR157-target LeSPL-CNR may affect expression of LeMADS-RIN, LeHB1, SlAP2a and SlTAGL1. However SlymiR156 does not affect the onset of ripening, but it impacts fruit softening after the red ripe stage. Our findings reveal that working together with a ripening network of transcription factors, SlymiR157 and SlymiR156 form a critical additional layer of regulatory control over the fruit ripening process 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.

Requirement of CHROMOMETHYLASE3 for somatic inheritance of the spontaneous tomato epimutation Colourless non-ripening

Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter, and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.

Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening.

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato.

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.

Effects of exogenous glucose on carotenoid accumulation in tomato leaves

To investigate the effect of carbohydrate on carotenoid accumulation in leaves, excised plants of tomato (Lycopersicum esculentum var. cerasiformae, wva 106) were supplied with glucose through the transpiration stream for 48 h. We report here that sugar accumulation in leaves led to a decrease of carotenoid content, which was related to the reduction of Chl. The decrease in carotenoid amount correlated with a sugar-induced repression of genes encoding enzymes of the carotenoid and of the Rohmer pathways. The lower 1-deoxy-D-xylulose-5-phosphate synthase transcript level probably leads to a decreased metabolic flux through the methylerythritol pathway and subsequently to a lower amount of substrate available for plastidic isoprenoid synthesis. Differences between responses of young (sink) and mature (source) leaves to carbohydrate accumulation are discussed.