Investigating the role of DNA methyltransferases in the mantled somaclonal variation of oil palm

DNA methyltransferase (DMTases) genes have emerged as targets of interest in the exploration of epigenetic mechanisms underlying the mantled variant phenotype in oil palm. Indeed, reduction in global DNA methylation rates and perturbations of floral phenotype could be due to underexpresion of these genes. Therefore, our research efforts focused on the isolation of members of all three families of DNA-methyltranferases identified in higher plants, namely MET, CMT (chromomethylase) and DRM (domain-rearranged). Using both a library screening and a PCR-based approach involving degenerated primers, we successfully cloned partial cDNA sequences belonging to all three families. The respective full-length cDNAs were subsequently obtained through the RACE (Rapid Amplification of cDNA Ends) method. For each DMTase type, we then determined the size of the family in the oil palm genome, and we examined the overall transcription level of each family member individually. The comparative transcription pattern of each class of DMTase was studied in normal and variant tissues through semi-quantitative Reverse-Transcription-PCR (sqRT-PCR) and Real Time quantitative PCR. Our work shows for the first time the characterization of the three DNA methyltransferase gene families in oil palm, and addresses the hypothesis of their role in the determinism of the mantled variant phenotype. (Résumé d'auteur

Epigenetic Regulation of Flower Development in the Oil Palm. W490

The mantled floral phenotype which occurs in somatic embryo-derived oil palms (Elaeis guineensis Jacq.) shows detrimental consequences on the large scale production of clonal planting material for this strategic oil crop. Indeed, mantled somaclonal variants display a feminization of male floral organs which bears a major threat on the formation of oil-producing fruits. The unpredictability of such variation warrants the search for molecular markers for an early detection. In parallel, the mantled phenotype provides a challenging puzzle to the researcher since it is both reminiscent of floral abnormalities governed by MADS Box genes in model plants and susceptible to provide insights into the particulars of sex differentiation and flower organogenesis in Palms species. Since a genome-wide deficit in DNA methylation has been demonstrated in mantled tissues and since gene expression differs substantially with respect to true-to-type material, it is now widely accepted that this unstable variant phenotype is correlated with (and likely caused by) the disturbance of epigenetic mechanisms during the in vitro micropropagation process, which involves a series of differentiation/dedifferentiation phases. Our talk provides an update on ongoing research work aimed at deciphering the role of epigenetic regulations in the floral development of the oil palm. (Texte integral

A marker-based strategy for the assessment of epigenetic instability in oil palm

Plant tissues from the mantled somaclonal variant of oil palm have been found to display both large-scale perturbations of genomic methylation levels and sequence-specific DNA methylation pattern changes when compared to their true-to-type counterparts. Concomitantly, several markers showing phenotype-specific transcription patterns have been identified in our group. Some of them are likely to be indicators of the altered physiological state of abnormal cultures, others are possible targets of epigenetic misregulations of flower development. We now aim to establish the exact link between the expression of these candidate marker genes and the mantled somaclonal variation state, through the exploration of their global epigenetic behaviour. This will be achieved using three complementary approaches applied to normal and abnormal tissues: i) the evaluation of transcription levels through quantitative or semi-quantitative techniques (semi-quantitative RT-PCR, real-time PCR); ii) the determination of sequence-wide DNA methylation patterns (bisulfite sequencing) and iii) the identification of the chromatin regulatory environment of these genes (Chromatin Immuno-Precipitation: ChIP). These combined strategies will ultimately help to elucidate the epigenetic mechanisms which give rise to the mantled somaclonal variant. Also, we will be able to identify which of the candidate markers which are indeed consistently linked with the abnormal state. This work will provide the basis for a molecular detection test of the variant phenotype. (Résumé d'auteur

Identification of early molecular markers associated with the mantled phenotype in micropropagated oil palms by subtractive PCR and cDNA array analysis

Tissue culture of oil palm induces somaclonal variation. The main one, known as the mantled abnormality, displays homeotic floral modifications and is associated with a lower oil yield. To detect abnormal cell lines as early as possible during the tissue culture process, studies to identify molecular markers have been carried out. Given the epigenetic nature of the mantled phenotype, differential gene expression has been focused upon rather than genome structure. We investigated gene expression patterns at the transcriptome level for three developmental stages: embryogenic cell suspensions, somatic embryos and leafy shoots. In the first step, the technique of subtractive PCR (SSH) was used to construct six cDNA libraries from variant and normal plant materials allowing enrichment for cDNA corresponding to genes which are up- or down-regulated in association with clonal conformity. In the second step, transcript abundance was monitored by macroarray hybridization. A range of genes altered in their expression in abnormal tissues has been identified and a number of which have been validated by RT-PCR. This approach will provide insights into the biological processes involved in the generation of the mantle phenotype and the identified markers should lead to the development of a clonal conformity test. (Résumé d'auteur

A transcriptome approach to study epigentic somaclonal variation in oil palm (Elaeis guineeensis Jacq.)

Micropropagation by somatic embryogenesis in oil palm produces a small but significant proportion of individuals displaying homeotic floral modifications known as the "mantled" phenotype. The identification of molecular markers has been undertaken in order to discriminate as early as possible during the in vitro process those cell lines which are variant and also to better understand the mechanism leading to abnormal flower development. Since a DNA sequence mutation does not appear to be involved, but rather an epigenetic alteration, we chose to analyse differential gene expression patterns at the transcriptome level between variant and normal material. Four SSH libraries were constructed from oil palm cell suspensions and male inflorescences. Studies of transcript abundance by macroarray hybridization allowed the identification of a range of genes altered in their expression in abnormal tissues, several of which were subsequently validated by RT-PCR analysis. The relevance of the genes identified and the biological processes in which they may be involved will be discussed. (Texte intégral

Investigating the epigenetic regulation of reproductive development in the oil palm

The oil palm (Elaeis guineensis Jacq.) is the first world source of vegetable fats, and as such it is generating a great amount of research efforts. In particular, understanding the molecular mechanisms underlying the setting up of the reproductive organs and their consecutive development in flowers and fruits appears essential in the perspective of improving oil production. The complexes formed by Polycomb group (PcG) proteins are involved in an epigenetic memory system which represses the expression of developmental regulator genes in both animals and plants and thereby promotes phase changes. Among the PcG proteins, those involved in the Polycomb Repressive Complex 2 (PCR2), i.e. the members of the ENHANCER OF ZESTE [E(Z)], SUPPRESSOR OF ZESTE 12 [SU(Z)12] and EXTRA SEX COMBS [ESC] superfamilies are known to act on the MADS-box genes controlling floral organogenesis through the formation of heterochromatin. The aim of the present project is 1) to identify the oil palm orthologues of the PcG genes and 2) to characterize their expression during inflorescence and fruit development. (Résumé d'auteur