Multi-scale comparative transcriptome analysis reveals key genes and metabolic reprogramming processes associated with oil palm fruit abscission.

BACKGROUND: Fruit abscission depends on cell separation that occurs within specialized cell layers that constitute an abscission zone (AZ). To determine the mechanisms of fleshy fruit abscission of the monocot oil palm (Elaeis guineensis Jacq.) compared with other abscission systems, we performed multi-scale comparative transcriptome analyses on fruit targeting the developing primary AZ and adjacent tissues. RESULTS: Combining between-tissue developmental comparisons with exogenous ethylene treatments, and naturally occurring abscission in the field, RNAseq analysis revealed a robust core set of 168 genes with differentially regulated expression, spatially associated with the ripe fruit AZ, and temporally restricted to the abscission timing. The expression of a set of candidate genes was validated by qRT-PCR in the fruit AZ of a natural oil palm variant with blocked fruit abscission, which provides evidence for their functions during abscission. Our results substantiate the conservation of gene function between dicot dry fruit dehiscence and monocot fleshy fruit abscission. The study also revealed major metabolic transitions occur in the AZ during abscission, including key senescence marker genes and transcriptional regulators, in addition to genes involved in nutrient recycling and reallocation, alternative routes for energy supply and adaptation to oxidative stress. CONCLUSIONS: The study provides the first reference transcriptome of a monocot fleshy fruit abscission zone and provides insight into the mechanisms underlying abscission by identifying key genes with functional roles and processes, including metabolic transitions, cell wall modifications, signalling, stress adaptations and transcriptional regulation, that occur during ripe fruit abscission of the monocot oil palm. The transcriptome data comprises an original reference and resource useful towards understanding the evolutionary basis of this fundamental plant process.

Redox-related gene expression and sugar accumulation patterns are altered in the edible inflorescence produced by the cultivated form of pacaya palm (Chamaedorea tepejilote).

BACKGROUND AND AIMS: The pacaya palm is a dioecious neotropical palm species that is exploited in Latin America for its male inflorescence, which is edible when immature. It is cultivated, in a non-intensive manner, in Guatemala, where a morphotype occurs that produces much larger, more highly branched inflorescences compared with wild palms. We sought to identify molecular factors underlying this phenotypic divergence, which is likely to be a product of domestication. METHODS: We performed RNA-seq-based studies on immature pacaya palm male inflorescences in order to identify genes that might be directly or indirectly affected in their expression in relation to domestication. We also measured the accumulation of a range of soluble sugar molecules to provide information on the biochemical status of the two different types of material. KEY RESULTS: A total of 408 genes were found to display significantly different expression levels between the wild and cultivated morphotypes. Three different functional categories were found to be enriched in the gene set that was upregulated in the cultivated morphotype: redox balance; secondary metabolism; and transport. Several sugars were found to accumulate at higher levels in inflorescences of the cultivated morphotype, in particular myo-inositol, fructose and glucose. CONCLUSIONS: The observed upregulation of redox-related genes in the cultivated morphotype is corroborated by the observation of higher myo-inositol accumulation, which has been shown to be associated with enhanced scavenging of reactive oxygen species in other plants and which may affect meristem activity.

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.

Integrative analysis of the late maturation programme and desiccation tolerance mechanisms in intermediate coffee seeds.

The 'intermediate seed' category was defined in the early 1990s using coffee (Coffea arabica) as a model. In contrast to orthodox seeds, intermediate seeds cannot survive complete drying, which is a major constraint for seed storage and has implications for both biodiversity conservation and agricultural purposes. However, intermediate seeds are considerably more tolerant to drying than recalcitrant seeds, which are highly sensitive to desiccation. To gain insight into the mechanisms governing such differences, changes in desiccation tolerance (DT), hormone contents, and the transcriptome were analysed in developing coffee seeds. Acquisition of DT coincided with a dramatic transcriptional switch characterised by the repression of primary metabolism, photosynthesis, and respiration, and the up-regulation of genes coding for late-embryogenesis abundant (LEA) proteins, heat-shock proteins (HSPs), and antioxidant enzymes. Analysis of the heat-stable proteome in mature coffee seeds confirmed the accumulation of LEA proteins identified at the transcript level. Transcriptome analysis also suggested a major role for ABA and for the transcription factors CaHSFA9, CaDREB2G, CaANAC029, CaPLATZ, and CaDOG-like in DT acquisition. The ability of CaHSFA9 and CaDREB2G to trigger HSP gene transcription was validated by Agrobacterium-mediated transformation of coffee somatic embryos.

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.

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.

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

BACKGROUND: The oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the world's 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. RESULTS: In 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. CONCLUSIONS: The 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.

Temporal and spatial expression of polygalacturonase gene family members reveals divergent regulation during fleshy fruit ripening and abscission in the monocot species oil palm.

BACKGROUND: Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. RESULTS: The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700-5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. CONCLUSIONS: The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species.

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.

Micro-RNA-Regulated SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Expression and Cytokinin Accumulation Distinguish Early-Developing Male and Female Inflorescences in Oil Palm (Elaeis guineensis).

Sexual differentiation of inflorescences and flowers is important for reproduction and affects crop plant productivity. We report here on a molecular study of the process of sexual differentiation in the immature inflorescence of oil palm (Elaeis guineensis). This species is monoecious and exhibits gender diphasy, producing male and female inflorescences separately on the same plant in alternation. Three main approaches were used: small RNA-seq to characterise and study the expression of miRNA genes; RNA-seq to monitor mRNA accumulation patterns; hormone quantification to assess the role of cytokinins and auxins in inflorescence differentiation. Our study allowed the characterisation of 30 previously unreported palm MIRNA genes. In differential gene and miRNA expression studies, we identified a number of key developmental genes and miRNA-mRNA target modules previously described in relation to their developmental regulatory role in the cereal panicle, notably the miR156/529/535-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene regulatory module. Gene enrichment analysis highlighted the importance of hormone-related genes, and this observation was corroborated by the detection of much higher levels of cytokinins in the female inflorescence. Our data illustrate the importance of branching regulation within the developmental window studied, during which the female inflorescence, unlike its male counterpart, produces flower clusters on new successive axes by sympodial growth.

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.

A Non-Shedding Fruit Elaeis oleifera Palm Reveals Perturbations to Hormone Signaling, ROS Homeostasis, and Hemicellulose Metabolism.

The developmentally programmed loss of a plant organ is called abscission. This process is characterized by the ultimate separation of adjacent cells in the abscission zone (AZ). The discovery of an American oil palm (Elaeis oleifera) variant that does not shed its has allowed for the study of the mechanisms of ripe fruit abscission in this species. A comparative transcriptome analysis was performed to compare the fruit AZs of the non-shedding E. oleifera variant to an individual of the same progeny that sheds its ripe fruit normally. The study provides evidence for widespread perturbation to gene expression in the AZ of the non-shedding variant, compared to the normal fruit-shedding control, and offers insight into abscission-related functions. Beyond the genes with known or suspected roles during organ abscission or indehiscence that were identified, a list of genes with hormone-related functions, including ethylene, jasmonic acid, abscisic acid, cytokinin and salicylic acid, in addition to reactive oxygen species (ROS) metabolism, transcriptional responses and signaling pathways, was compiled. The results also allowed a comparison between the ripe fruit abscission processes of the African and American oil palm species at the molecular level and revealed commonalities with environmental stress pathways.

Environmental and trophic determinism of fruit abscission and outlook with climate change in tropical regions.

Fruit abscission facilitates the optimal conditions and timing of seed dispersal. Environmental regulation of tropical fruit abscission has received little attention, even though climate change may have its strongest impacts in tropical regions. In this study, oil palm fruit abscission was monitored during multiple years in the Benin Republic to take advantage of the climatic seasonality and the continuous fruit production by this species. An innovative multivariable statistical method was used to identify the best predictors of fruit abscission among a set of climate and ecophysiological variables, and the stage of inflorescence and fruit bunch development when the variables are perceived. The effects of climate scenarios on fruit abscission were then predicted based on the calibrated model. We found complex regulation takes place at specific stages of inflorescence and bunch development, even long before the fruit abscission zone is competent to execute abscission. Among the predictors selected, temperature variations during inflorescence and fruit bunch development are major determinants of the fruit abscission process. Furthermore, the timing of ripe fruit drop is determined by temperature in combination with the trophic status. Finally, climate simulations revealed that the abscission process is robust and is more affected by seasonal variations than by extreme scenarios. Our investigations highlighted the central function of the abscission zone as the sensor of environmental signals during reproductive development. Coupling ecophysiological and statistical modeling was an efficient approach to disentangle this complex environmental regulation.

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.

The PIP Peptide of INFLORESCENCE DEFICIENT IN ABSCISSION Enhances Populus Leaf and Elaeis guineensis Fruit Abscission.

The programmed loss of a plant organ is called abscission, which is an important cell separation process that occurs with different organs throughout the life of a plant. The use of floral organ abscission in Arabidopsis thaliana as a model has allowed greater understanding of the complexities of organ abscission, but whether the regulatory pathways are conserved throughout the plant kingdom and for all organ abscission types is unknown. One important pathway that has attracted much attention involves a peptide ligand-receptor signalling system that consists of the secreted peptide IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and at least two leucine-rich repeat (LRR) receptor-like kinases (RLK), HAESA (HAE) and HAESA-LIKE2 (HSL2). In the current study we examine the bioactive potential of IDA peptides in two different abscission processes, leaf abscission in Populus and ripe fruit abscission in oil palm, and find in both cases treatment with IDA peptides enhances cell separation and abscission of both organ types. Our results provide evidence to suggest that the IDA-HAE-HSL2 pathway is conserved and functions in these phylogenetically divergent dicot and monocot species during both leaf and fruit abscission, respectively.

Transcriptome Analysis of Cell Wall and NAC Domain Transcription Factor Genes during Elaeis guineensis Fruit Ripening: Evidence for Widespread Conservation within Monocot and Eudicot Lineages.

The oil palm (Elaeis guineensis), a monocotyledonous species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene. Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation. The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types. Profiling of the most abundant oil palm polygalacturonase (EgPG4) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation. A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible (EgNAC6 and EgNAC7) phylogenetically similar to the tomato NAC-NOR master-ripening regulator. Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are regulated by evolutionarily conserved molecular physiological processes.

Somaclonal variation in micropropagated oil palm. Characterization of two novel genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.

In vitro micropropagation based on somatic embryogenesis provides an efficient means to multiply selected genotypes of oil palm (Elaeis guineensis Jacq.). Despite its considerable potential, somatic embryogenesis can yield plants bearing a homeotic flowering abnormality known as mantled. Because the mantled abnormality is epigenetic, it cannot be detected with conventional structural molecular markers. Thus, to develop a means of discriminating among callus cultures carrying or lacking the mantled abnormality, we used a gene expression approach. We describe two novel oil palm genes, EgM39A and EgIAA1, both of which display increased transcript accumulation in epigenetically abnormal calli. EgIAA1 codes for an oil palm relative of the Arabidopsis thaliana (L.) Heynh. AXR3/IAA17 protein involved in early auxin response and EgM39A codes for a protein of unknown function sharing sequence similarities with asparagine synthetases. In addition to their enhanced expression in somaclonal variant callus lines, both genes displayed increased transcript accumulation in response to auxin treatment. Normal seed-derived zygotic embryos germinated in the presence of auxin accumulated increased amounts of EgIAA1 transcripts after a few hours of treatment, suggesting a role in auxin response similar to that demonstrated for IAA genes in other species. The EgM39A gene also displayed enhanced transcript accumulation in auxin-treated zygotic embryos. Although only a small increase was seen after 24 h, greater changes were observed after 15 days. Both genes show potential as early markers of clonal conformity and may help to elucidate the nature of the epigenetic changes causing the mantled abnormality.

Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm.

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

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.

Regulation of 7S globulin gene expression in zygotic and somatic embryos of oil palm.

We describe here the characterization and expression analysis of the oil palm GLO7A gene encoding a 7S globulin protein. Previous work carried out in our laboratory showed that 7S globulins accumulate in the oil palm zygotic embryo mostly between the 14 and 17 weeks after pollination. To investigate further the regulation of 7S globulin gene expression in both zygotic and somatic embryos of oil palm, we isolated a cDNA clone, GLO7A, for use as a probe in northern hybridization studies. The nucleotide sequence of the GLO7A cDNA reveals that it encodes a polypeptide of 572 amino acids (66 kDa) sharing significant sequence similarities with various vicilin-like proteins of both dicotyledonous and monocotyledonous plants. Northern hybridization analysis shows that 7S globulin mRNA accumulation in zygotic embryos is temporally regulated with a profile essentially the same as that observed at the protein level. In somatic embryos, 7S globulin proteins were found to occur in amounts approximately 80 times lower than those in zygotic embryos. This lack of 7S globulin protein accumulation in somatic embryos is mirrored by a low accumulation of the GLO7A mRNA. The in vitro production of 7S globulins (and more generally salt-soluble proteins) is improved by the addition to the culture medium of arginine, sucrose and ABA, the effects of these 3 components being additive. To investigate further the action of the 3 molecules of interest, we performed parallel studies on mRNA and protein abundance. Our studies of transcript accumulation suggest that ABA and sucrose act directly on mRNA synthesis or stability; however, it appears that there are also translational or post-translational regulatory factors which act to limit protein accumulation in somatic embryos. The GLO7A gene promoter was cloned and sequenced to assess whether GLO7A gene expression might be modulated by cis-acting promoter elements related to those found in other plants. Two motifs resembling ABREs (ABA-responsive elements) and one motif resembling a seed-specific promoter element were identified within the 5' flanking sequence.