Diterpenes of Coffea seeds show antifungal and anti-insect activities and are transferred from the endosperm to the seedling after germination.

Species of the genus Coffea accumulate diterpenes of the ent-kaurane family in the endosperm of their seeds, of which cafestol and kahweol are the most abundant. The diterpenes are mainly stored in esterified form with fatty acids, mostly palmitate. In contrast to the numerous studies on their effects on human health and therapeutic applications, nothing was previously known about their biological and ecological role in planta. The antifungal and anti-insect activities of cafestol and cafestol palmitate were thus investigated in this study. Cafestol significantly affected the mycelial growth of five of the six phytopathogenic fungi tested. It also greatly reduced the percentage of pupation of larvae and the pupae and adult masses of one of the two fruit flies tested. By contrast, cafestol palmitate had no significant effect against any of the fungi and insects studied. Using confocal imaging and oil body isolation and analysis, we showed that diterpenes are localized in endosperm oil bodies, suggesting that esterification with fatty acids enables the accumulation of large amounts of diterpenes in a non-toxic form. Diterpene measurements in all organs of seedlings recovered from whole seed germination or embryos isolated from the endosperm showed that diterpenes are transferred from the endosperm to the cotyledons during seedling growth and then distributed to all organs, including the hypocotyl and the root. Collectively, our findings show that coffee diterpenes are broad-spectrum defence compounds that protect not only the seed on the mother plant and in the soil, but also the seedling after germination.

Natural diversity in the carotene, tocochromanol and fatty acid composition of crude palm oil.

Crude palm oil (CPO) is extracted from the mesocarp of oil palm (Elaeis guineensis) fruits. CPO is widely consumed in many African countries. Due to its high provitamin A carotenoid content, it is also widely used in programmes designed to prevent vitamin A deficiency. Elaeis guineensis occurs naturally across a wide geographical range in Africa. We investigated the carotene, tocochromanol (vitamin E) and fatty acid composition of a large set of genotypes representative of this genetic and geographic diversity. We found considerable intraspecific diversity in most lipid traits. Populations from Côte d'Ivoire were distinguished from other origins by their very low palmitate content and high tocochromanol content. Genotypes from Benin, Côte d'Ivoire and Nigeria were characterized by high carotene contents. Finally, hybrids of crosses between genotypes from Côte d'Ivoire and Nigeria produce CPO with exceptionally high provitamin A and vitamin E contents together with low palmitate content.

Palm seed and fruit lipid composition: phylogenetic and ecological perspectives.

BACKGROUND AND AIMS: Palms are vital to worldwide human nutrition, in particular as major sources of vegetable oils. However, our knowledge of seed and fruit lipid diversity in the family Arecaceae is limited. We therefore aimed to explore relationships between seed and fruit lipid content, fatty acid composition in the respective tissues, phylogenetic factors and biogeographical parameters. METHODS: Oil content and fatty acid composition were characterized in seeds and fruits of 174 and 144 palm species respectively. Distribution, linear regression and multivariate analyses allowed an evaluation of the chemotaxonomic value of these traits and their potential relationship with ecological factors. KEY RESULTS: A considerable intra-family diversity for lipid traits was revealed. Species with the most lipid-rich seeds belonged to the tribe Cocoseae, while species accumulating oil in the mesocarp occurred in all subfamilies and two-thirds of the tribes studied. Seed and fruit lipid contents were not correlated. Fatty acid composition of mesocarp oil was highly variable within tribes. By contrast, within-tribe diversity for seed lipid traits was low, whereas between-tribe variability was high. Consequently, multivariate analyses of seed lipid traits produced groupings of species belonging to the same tribe. Medium-chain fatty acids predominated in seeds of most palm species, but they were also accumulated in the mesocarp in some cases. Seed unsaturated fatty acid content correlated with temperature at the coldest latitude of natural occurrence. CONCLUSION: Several previously uncharacterized palms were identified as potential new sources of vegetable oils for comestible or non-food use. Seed lipid traits reflect genetic drift that occurred during the radiation of the family and therefore are highly relevant to palm chemotaxonomy. Our data also suggest that seed unsaturated fatty acids may provide an adaptive advantage in the coldest environments colonized by palms by maintaining storage lipids in liquid form for efficient mobilization during germination.

Seed comparative genomics in three coffee species identify desiccation tolerance mechanisms in intermediate seeds.

In contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses.

Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for ß-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

Genome-wide analysis of LTR-retrotransposons in oil palm.

BACKGROUND: The oil palm (Elaeis guineensis Jacq.) is a major cultivated crop and the world's largest source of edible vegetable oil. The genus Elaeis comprises two species E. guineensis, the commercial African oil palm and E. oleifera, which is used in oil palm genetic breeding. The recent publication of both the African oil palm genome assembly and the first draft sequence of its Latin American relative now allows us to tackle the challenge of understanding the genome composition, structure and evolution of these palm genomes through the annotation of their repeated sequences. METHODS: In this study, we identified, annotated and compared Transposable Elements (TE) from the African and Latin American oil palms. In a first step, Transposable Element databases were built through de novo detection in both genome sequences then the TE content of both genomes was estimated. Then putative full-length retrotransposons with Long Terminal Repeats (LTRs) were further identified in the E. guineensis genome for characterization of their structural diversity, copy number and chromosomal distribution. Finally, their relative expression in several tissues was determined through in silico analysis of publicly available transcriptome data. RESULTS: Our results reveal a congruence in the transpositional history of LTR retrotransposons between E. oleifera and E. guineensis, especially the Sto-4 family. Also, we have identified and described 583 full-length LTR-retrotransposons in the Elaeis guineensis genome. Our work shows that these elements are most likely no longer mobile and that no recent insertion event has occurred. Moreover, the analysis of chromosomal distribution suggests a preferential insertion of Copia elements in gene-rich regions, whereas Gypsy elements appear to be evenly distributed throughout the genome. CONCLUSIONS: Considering the high proportion of LTR retrotransposon in the oil palm genome, our work will contribute to a greater understanding of their impact on genome organization and evolution. Moreover, the knowledge gained from this study constitutes a valuable resource for both the improvement of genome annotation and the investigation of the evolutionary history of palms.

The embryo and the endosperm contribute equally to argan seed oil yield but confer distinct lipid features to argan oil.

In the perspective of studying lipid biosynthesis in the argan seed, the anatomy, ploidy level and lipid composition of mature seed tissues were investigated using an experimental design including two locations in Algeria and four years of study. Using flow cytometry, we determined that mature argan seeds consist of two well-developed tissues, the embryo and the endosperm. The lipid content of the embryo was higher than that of the endosperm, but the dry weight of the endosperm was higher. Consequently, both tissues contribute equally to seed oil yield. Considerable differences in fatty acid composition were observed between the two tissues. In particular, the endosperm 18:2 percentage was twofold higher than that of the embryo. The tocopherol content of the endosperm was also markedly higher than that of the embryo. In contrast, the endosperm and the embryo had similar sterol and triterpene alcohol contents and compositions.

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

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.

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.

The 'PUCE CAFE' Project: the first 15K coffee microarray, a new tool for discovering candidate genes correlated to agronomic and quality traits.

BACKGROUND: Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. RESULTS: The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. CONCLUSION: We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Metabolic pathways in tropical dicotyledonous albuminous seeds: Coffea arabica as a case study.

* The genomic era facilitates the understanding of how transcriptional networks are interconnected to program seed development and filling. However, to date, little information is available regarding dicot seeds with a transient perisperm and a persistent, copious endosperm. Coffea arabica is the subject of increasing genomic research and is a model for nonorthodox albuminous dicot seeds of tropical origin. * The aim of this study was to reconstruct the metabolic pathways involved in the biosynthesis of the main coffee seed storage compounds, namely cell wall polysaccharides, triacylglycerols, sucrose, and chlorogenic acids. For this purpose, we integrated transcriptomic and metabolite analyses, combining real-time RT-PCR performed on 137 selected genes (of which 79 were uncharacterized in Coffea) and metabolite profiling. * Our map-drawing approach derived from model plants enabled us to propose a rationale for the peculiar traits of the coffee endosperm, such as its unusual fatty acid composition, remarkable accumulation of chlorogenic acid and cell wall polysaccharides. * Comparison with the developmental features of exalbuminous seeds described in the literature revealed that the two seed types share important regulatory mechanisms for reserve biosynthesis, independent of the origin and ploidy level of the storage tissue.

Development of solid-phase extraction and methylation procedures to analyse free fatty acids in lipid-rich seeds.

In order to develop a sensitive and reliable method for FFA quantification in lipid matrices of seeds, two SPE procedures employed in meat and dairy chemistry were compared using a 100/1 mixture of triolein/heptadecanoic acid. The overall efficiency of the SPE procedure retained was satisfactory since it allowed removal of 99.8% of triacylglycerols (TAG) and recovery of 99.2% of FFA as quantified by gas chromatography of fatty acid methyl esters (FAME). However, the low amount of TAG eluted in the FFA fraction represented a non-negligible percentage (17%) of FAME and the procedure thus required further improvement. TAG pollution was successively decreased to 12%, 8% and finally 1.5% by: i) modifying the volume of elution of TAG; ii) removing the saponification step initially performed according to the standard FAME procedure; and iii) reducing the duration of the BF(3)-catalyzed methylation reaction to 1 min. The new SPE/methylation procedure described here was then compared to the most widely used method for FFA measurement in plants which is based on thin-layer chromatography (TLC). Both procedures were applied to coffee seeds stored for 0-18 months at 15 degrees C under 62% relative humidity and provided consistent results. A very clear negative correlation was observed between the loss of seed viability and the accumulation of FFA in seeds during the course of storage independent of the method employed for FFA quantification. However, we demonstrated that the TLC/on-silica methylation procedure underestimates FFA contents in comparison with the new SPE/methylation procedure because of a selective loss of unsaturated FA.