Fabienne Morcillo

SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis

BackgroundThe oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the worlds number one source of edible vegetable oil, and the richest dietary source of provitamin A. While new elite genotypes from traditional breeding programs provide steady yield increases, the long selection cycle (10-12 years) and the large areas required to cultivate oil palm make genetic improvement slow and labor intensive. Molecular breeding programs have the potential to make significant impacts on the rate of genetic improvement but the limited molecular resources, in particular the lack of molecular markers for agronomic traits of interest, restrict the application of molecular breeding schemes for oil palm.ResultsIn the current study, 6,103 non-redundant ESTs derived from cDNA libraries of developing vegetative and reproductive tissues were annotated and searched for simple sequence repeats (SSRs). Primer pairs from sequences flanking 289 EST-SSRs were tested to detect polymorphisms in elite breeding parents and their crosses. 230 of these amplified PCR products, 88 of which were polymorphic within the breeding material tested. A detailed analysis and annotation of the EST-SSRs revealed the locations of the polymorphisms within the transcripts, and that the main functional category was related to transcription and post-transcriptional regulation. Indeed, SSR polymorphisms were found in sequences encoding AP2-like, bZIP, zinc finger, MADS-box, and NAC-like transcription factors in addition to other transcriptional regulatory proteins and several RNA interacting proteins.ConclusionsThe identification of new EST-SSRs that detect polymorphisms in elite breeding material provides tools for molecular breeding strategies. The identification of SSRs within transcripts, in particular those that encode proteins involved in transcriptional and post-transcriptional regulation, will allow insight into the functional roles of these proteins by studying the phenotypic traits that cosegregate with these markers. Finally, the oil palm EST-SSRs derived from vegetative and reproductive development will be useful for studies on the evolution of the functional diversity within the palm family.

Regulatory mechanisms underlying oil palm fruit mesocarp maturation, ripening and functional specialization in lipid and carotenoid metabolism

Fruit provide essential nutrients and vitamins for the human diet. Not only is the lipid-rich fleshy mesocarp tissue of the oil palm (Elaeis guineensis) fruit the main source of edible oil for the world, but it is also the richest dietary source of provitamin A. This study examines the transcriptional basis of these two outstanding metabolic characters in the oil palm mesocarp. Morphological, cellular, biochemical, and hormonal features defined key phases of mesocarp development. A 454 pyrosequencing-derived transcriptome was then assembled for the developmental phases preceding and during maturation and ripening, when high rates of lipid and carotenoid biosynthesis occur. A total of 2,629 contigs with differential representation revealed coordination of metabolic and regulatory components. Further analysis focused on the fatty acid and triacylglycerol assembly pathways and during carotenogenesis. Notably, a contig similar to the Arabidopsis (Arabidopsis thaliana) seed oil transcription factor WRINKLED1 was identified with a transcript profile coordinated with those of several fatty acid biosynthetic genes and the high rates of lipid accumulation, suggesting some common regulatory features between seeds and fruits. We also focused on transcriptional regulatory networks of the fruit, in particular those related to ethylene transcriptional and GLOBOSA/PISTILLATA-like proteins in the mesocarp and a central role for ethylene-coordinated transcriptional regulation of type VII ethylene response factors during ripening. Our results suggest that divergence has occurred in the regulatory components in this monocot fruit compared with those identified in the dicot tomato (Solanum lycopersicum) fleshy fruit model.

Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition

Tissue-specific transcriptional regulation determines the differences in oil content and fatty acid composition of oil palm fruit and seed tissues. Oil palm (Elaeis guineensis) produces two oils of major economic importance, commonly referred to as palm oil and palm kernel oil, extracted from the mesocarp and the endosperm, respectively. While lauric acid predominates in endosperm oil, the major fatty acids (FAs) of mesocarp oil are palmitic and oleic acids. The oil palm embryo also stores oil, which contains a significant proportion of linoleic acid. In addition, the three tissues display high variation for oil content at maturity. To gain insight into the mechanisms that govern such differences in oil content and FA composition, tissue transcriptome and lipid composition were compared during development. The contribution of the cytosolic and plastidial glycolytic routes differed markedly between the mesocarp and seed tissues, but transcriptional patterns of genes involved in the conversion of sucrose to pyruvate were not related to variations for oil content. Accumulation of lauric acid relied on the dramatic up-regulation of a specialized acyl-acyl carrier protein thioesterase paralog and the concerted recruitment of specific isoforms of triacylglycerol assembly enzymes. Three paralogs of the WRINKLED1 (WRI1) transcription factor were identified, of which EgWRI1-1 and EgWRI1-2 were massively transcribed during oil deposition in the mesocarp and the endosperm, respectively. None of the three WRI1 paralogs were detected in the embryo. The transcription level of FA synthesis genes correlated with the amount of WRI1 transcripts and oil content. Changes in triacylglycerol content and FA composition of Nicotiana benthamiana leaves infiltrated with various combinations of WRI1 and FatB paralogs from oil palm validated functions inferred from transcriptome analysis.

Transcriptome analysis during somatic embryogenesis of the tropical monocot Elaeis guineensis: evidence for conserved gene functions in early development

With the aim of understanding the molecular mechanisms underlying somatic embryogenesis (SE) in oil palm, we examined transcriptome changes that occur when embryogenic suspension cells are initiated to develop somatic embryos. Two reciprocal suppression subtractive hybridization (SSH) libraries were constructed from oil palm embryogenic cell suspensions: one in which embryo development was blocked by the presence of the synthetic auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-d) in the medium (proliferation library); and another in which cells were stimulated to form embryos by the removal of 2,4-d from the medium (initiation library). A total of 1867 Expressed Sequence Tags (ESTs) consisting of 1567 potential unigenes were assembled from the two libraries. Functional annotation indicated that 928 of the ESTs correspond to proteins that have either no similarity to sequences in public databases or are of unknown function. Gene Ontology (GO) terms assigned to the two EST populations give clues to the underlying molecular functions, biological processes and cellular components involved in the initiation of embryo development. Macroarrays were used for transcript profiling the ESTs during SE. Hierarchical cluster analysis of differential transcript accumulation revealed 4 distinct profiles containing a total of 192 statistically significant developmentally regulated transcripts. Similarities and differences between the global results obtained with in vitro systems from dicots, monocots and gymnosperms will be discussed.

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non‐redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array‐based expression analysis in oil palm.

MADS box genes in oil palm (Elaeis guineensis): Patterns in the evolution of the SQUAMOSA, DEFICIENS, GLOBOSA, AGAMOUS, and SEPALLATA subfamilies

MADS box genes code for a large family of transcription factors which regulate development in higher plants, notably flower formation. We describe here a study of members of the MADS box gene family in oil palm (Elaeis guineensis Jacq.), a representative of the family Arecaceae and order Arecales, a key group of monocotyledons which has been unreported in previous phylogenetic reconstructions of the different recognized clades of MADS box genes. In this study, 13 oil palm MADS box genes were identified and characterized. They were found to belong to five different subfamilies, namely, the previously defined SQUAMOSA, AGAMOUS, AGAMOUS-like2, DEFICIENS, and GLOBOSA groups. Genes belonging to each of these groups play a critical role in the determination of flower structure as defined by the ABCDE model. The in planta expression profiles of the oil palm MADS box genes were studied by RT-PCR and phylogenetic sequence diversity within individual subfamilies was investigated by comparing their deduced protein sequences with those of other angiosperms. Most of the oil palm sequences studied were observed to group with distinct supported clades within their subfamily. Some unexpected groupings were observed between monocot sequences (including oil palm ones) of non-Poaceae origin, probably illustrating the importance of obtaining adequate taxon representation in monocot molecular phylogenies.

EgAP2-1, an AINTEGUMENTA-like (AIL) gene expressed in meristematic and proliferating tissues of embryos in oil palm

In order to better understand the developmental processes that govern the formation of somatic embryos in oil palm (Elaeis guineensis Jacq.), we investigated the transcription factor genes expressed during embryogenesis in this species. The AP2/EREBP transcription factor family includes the AP2 subgroup, which contains several proteins that play important roles in plant development. We identified and characterized EgAP2-1, which codes for a protein that contains two AP2 domains similar to those of the transcription factor BABYBOOM (BBM) and more generally AINTEGUMENTA-like (AIL) proteins of the AP2 subgroup. In a similar way to related genes from eudicots, ectopic expression of EgAP2-1 in transgenic Arabidopsis plants alters leaf morphology and enhances regeneration capacity. In oil palm, EgAP2-1 transcripts accumulate to the greatest extent in zygotic embryos. This expression pattern was investigated in more detail by in-situ hybridization, revealing that in both zygotic and somatic embryos, EgAP2-1 expression is concentrated in proliferating tissues associated with the early development of leaf primordia, root initials and provascular tissues.

Improving palm oil quality through identification and mapping of the lipase gene causing oil deterioration

The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards.

Accumulation of storage protein and 7S globulins during zygotic and somatic embryo development in Elaeis guineensis

The 7S globulins are the predominant storage proteins in oil palm (Elaeis guineensis Jacq.) zygotic embryos. The accumulation of these proteins was studied in embryos during seed development and in single somatic embryos cultured in vitro. Antibodies raised against these proteins were used for their detection by western blot and quantification with ELISA. In zygotic embryos, the 7S globulins were deposited mainly between the 14th and the 17th post-anthesis week, corresponding to the end of embryo growth. This represented 10 % of the dry weight and 50 % of soluble proteins. The amount of soluble proteins and 7S globulins in somatic embryos increased rapidly during the early stage of development, but were almost 80 times lower than in zygotic embryos. In somatic embryos, 7S globulins represented 0.3 % of dry weight and 4 % of soluble proteins. After 22 days development, the protein content declined slowly, suggesting a lack of embryo maturation and an early germination.

Characterization of 2S and 7S storage proteins in embryos of oil palm.

Abstract Storage proteins that accumulated during oil palm embryo development were extracted, purified and characterized. Only water- and low-salt-soluble proteins, with respective sedimentation coefficients of 2S and 7S, were detected in mature embryos. After purification by gel filtration, the various protein classes identified were characterized by electrophoresis and amino acid composition analysis. The 2S proteins comprise polypeptides of 22 kD and 19 kD, which are acidic (pI 9) respectively. The 7S proteins predominate and are heterogeneous oligomers (Mr of 156 and 201 kD), comprising a polypeptide triplet of Mr between 45 and 65 kD with no disulphide bonds. Their amino acid composition is broadly similar to those of the 7S proteins of other monocotyledon embryos, but differs from those of the legume 7S vicilins. Histological examinations and electrophoresis showed that the 2S and 7S proteins appeared at the third month after fertilization, and no qualitative changes were detected up to the sixth month of embryo development. The characterization of the embryo storage proteins of oil palm is discussed with reference to legume, cereal and palm seed storage proteins. This study will enable further investigation of storage protein synthesis during somatic embryogenesis.