Thierry Beulé

Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis

Elaeis guineensis Jacq.) currently hampers the scaling-up of clonal plant production. In order to investigate the relationship between the “mantled” somaclonal variant and possible alterations in genomic DNA methylation rate, two complementary approaches have been used. HPLC quantification of relative amounts of 5-methyl-deoxycytidine has shown that global methylation in leaf DNA of abnormal regenerants is 0.5–2.5% lower than in their normal counterparts (20.8% vs 22%, respectively). When comparing nodular compact calli and fast growing calli, yielding respectively 5% and 100% of “mantled” plantlets, this decrease was up to 4.5% (from 23.2 to 18.7%). An alternative method, the SssI-methylase accepting assay, based on the enzymatic saturation of CG sites with methyl groups, gave convergent results. This work demonstrates that a correlation exists between DNA hypomethylation and the “mantled” somaclonal variation in oil palm.

Epigenetic imbalance and the floral developmental abnormality of the in vitro-regenerated oil palm Elaeis guineensis

BACKGROUND The large-scale clonal propagation of oil palm (Elaeis guineensis) is being stalled by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. SCOPE In the last 15 years, the prevailing point of view on the origin of the mantled floral phenotype has evolved from a random mutation event triggered by in vitro culture to a hormone-dependent dysfunction of gene regulation processes. In this review, we retrace the history of the research on the mantled variation in the light of the parallel advances made in the understanding of plant development regulation in model systems and more specifically in the role of epigenetic mechanisms. An overview of the current state of oil palm genomic and transcriptomic resources, which are key to any comparison with model organisms, is given. We show that, while displaying original characteristics, the mantled phenotype of oil palm is morphologically, and possibly molecularly, related to MADS-box genes mutants described in model plants. We also discuss the occurrence of comparable floral phenotypes in other palm species. CONCLUSIONS Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical palm. On the basis of recent results, we propose that future efforts should concentrate on the epigenetic regulation targeting MADS-box genes and transposable elements of oil palm, since both types of sequences are most likely to be involved in the mantled variant phenotype.

Development of photosynthetic characteristics in oil palm during in vitro micropropagation

Summary Various photosynthetic parameters (photochemical activities, CO 2 exchange and carboxylase enzymatic activities) were studied during four characteristic stages of oil palm ( Elaeis guineensis Jacq.) in vitro micropropagation through somatic embryogenesis: proliferating somatic embryos, shootlets (1st and 2nd caulogenesis cycles) and rooted plantlets. In vivo chlorophyll fluorescence measurements indicated that the maximum photochemical activity of photosystem II (PSII) was very low in proliferating embryos and strongly increased in later development stages, finally reaching an activity very close to that measured in acclimatized plants. The quantum yield of photosynthetic electron transport followed the same trend except that a marked depression of electron transport activity was observed in the rooted plandets. CO 2 exchange measurements showed that absolute levels of in vitro photosynthesis were low, but measurable. Photosynthetic activity was also investigated by focusing on certain aspects of carbon metabolism. The activities of two of the primary enzymes of CO 2 fixation, namely PEPC (EC 4.1.1.31) and RuBisCO (EC 4.1.1.39) were measured throughout the micropropagation process. The PEPC:RuBisCO ratio progressively decreased, due to a substantial depletion of PEPC activity. Specific RuBisCO activity did not show any significant alteration, except a transient increase during the first caulogenesis stage on gelified medium. Quantitation of RuBisCO was carried out via rocket immunoelectrophoresis, using a polyclonal antibody raised against RuBisCO from green tobacco leaves. The relative amount of RuBisCO increased during somatic embryo development (from 3.2 % in proliferating embryos to 38.8 % in 2nd-cycle-shoot-lets), then it decreased during the rooting treatment (26.4%). The impact of sucrose enriched media on photosynthetic activity at this stage is discussed.

Photosynthetic ability of in vitro grown coconut (Cocos nucifera L.) plantlets derived from zygotic embryos

Photosynthetic parameters have been investigated using complementary approaches throughout the in vitro development of coconut zygotic embryos into plantlets. Patterns of chlorophyll fluorescence were comparable in in vitro grown coconut plantlets (OFAx = 0.72 and O, = 0.45) and in autotrophic adult palms (OpAX = 0.76 and Bp=0.5O). Chlorophyll content was lower in in vitro-cultured plantlets (0.92 mg/g fresh weight (FW)) than in autotrophic plants (2.43 mg/g FW). The photosynthetic rate (1.14 pmol CO,/m’ per s) of autotrophic palms was half that of in vitro grown plantlets, while transpiration rates were similar in both. Changes in the PEPC:RubisCO ratio during the development of in vitro grown plantlets (from 89.17 to 0.04 pmol COJh per mg total soluble protein (TSP)) reflected a transition from a heterotrophic towards a RubisCO-mediated mode of CO, fixation. The RubisCO enzyme capacity (2.83 pmol CO,/h per mg TSP) and content (172.8 mg/g TSP) measured in in vitro-cultured plantlets were lower than those measured in autotrophic palms (6.60 pmol CO,/h per mg TSP and 217.6 mg/g TSP respectively). Transmission electronic microscopy (TEM) observations showed a complete ultrastructural organisation of chloroplasts in plantlets at the end of the in vitro culture process (6 weeks under light). All the studied parametersj Abbreoiatiotis: Chl a, Chlorophyll a; Chl b, Chlorophyll b; FW, Fresh weight; PAR, Photosynthesis active radiations; PEPC, Phosphoenolpyruvate carboxylase; PVP, Polyvinyl pyrrolidone; RH, Relative humidity; RubisCO, Ribulose 1,5-bisphosphate carboxylase oxygenase; To, Temperature; TCA cycle, Tricarboxylic acid or Krebs cycle; TEM, Transmission electronic microscopy; TSP, Total soluble proteins.

Transcriptome analysis reveals differentially expressed genes associated with the mantled homeotic flowering abnormality in oil palm (Elaeis guineensis).

Micropropagation of oil palm by somatic embryogenesis produces a proportion of off-type individuals (approximately 5% overall) displaying a homeotically modified flower structure known as mantled. Transformation of the fertile or sterile androecium into carpel-like structures is observed in staminate and pistillate mantled flowers, respectively, resulting in lower oil yields in affected plantations. Given the epigenetic nature of the mantled condition, a gene expression-based approach was used rather than a genetic one to investigate its molecular basis. Suppression subtractive hybridisation (SSH) and macroarray hybridisation were used to compare transcriptome patterns between normal and mantled inflorescences. Two SSH libraries, enriched for complementary deoxyribonucleic acids (cDNAs) of either true-to-type or somaclonal variant material, were generated. Bioinformatic analysis of these two libraries allowed the identification of 1,350 unique sequences and their annotation by a gene ontology-based approach. Macroarray hybridisation was used to compare gene expression between normal and mantled inflorescences, and 32 genes were found to be differentially expressed. The temporal expression patterns of six genes were further investigated in more detail in relation to male and female inflorescence development. Full-length cDNAs were isolated and characterised for two of these genes, EgFB1 and EgRING1, both of which are down-regulated in the mantled inflorescences and both of which encode proteins associated with proteolytic signalling complexes. Our data shed light on gene expression changes associated with the mantled phenotype and have provided novel transcriptome markers which can help to distinguish the abnormal and wild-type inflorescences.

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 β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

DNA methylation and expression of the EgDEF1 gene and neighboring retrotransposons in mantled somaclonal variants of oil palm.

The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm.

Genome-wide analysis of LTR-retrotransposons in oil palm

BackgroundThe 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.MethodsIn 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.ResultsOur 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.ConclusionsConsidering 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.

Specific nutritional requirements of coconut calli (Cocos nucifera L.) during somatic embryogenesis induction

Summary Coconut calli were cultivated on two somatic embryogenesis induction media (SEIMs), differing in their 2,4-D content. Gain in dry matter weight, composition of soluble sugars within calli, but also pH and contents of glucose and macroelements in media were analysed at 0, 15, 28, and 60 days of culture. Relationships between contents of endogenous sugars, on the one hand, and between contents of media macroelements, on the other hand, were analysed. Comparison was made with calli maintained on a control multiplication medium. Traits could be classified into 3 types of response with regard to condition of somatic embryogenesis induction (SEI condition). The first correspond to traits that were modified by the SEI condition and varying over time. Two phases were determined. During the first phase (T0–T15), soluble sugar contents within calli decreased over time. The higher the 2,4-D content in SEIMs, the higher the sugar contents. Consumption of glucose and macroelements in media was negligible. However, strong relationships in the contents of chloride, nitrate, phosphate, and sulfate were modified in the SEI condition. During the second phase (T15–T60, growth became lower in the SEI condition. Requirements for glucose, nitrate and phosphate and acidification of media were higher. The relationship, determined by changes in nitrate and phosphate (R>0.98), was modified by the SEI conditions, showing a preferential consumption for nitrate in this case. Endogenous sucrose content decreased to become lower in the SEI condition. The higher the 2,4-D content in SEIMs, the higher the requirements for media compounds, the higher the contents of sugars within calli, but the lower the growth. The second type of response corresponded to traits modified by the SEI condition, but constant over time. It concerned relationships between contents of some cations in the media. The third type of response corresponded to traits unchanges by the SEI condition and over time. It concerned the high relationship contents of endogenous glucose and fructose (R = 0.88), and between contents of chloride, ammonium, calcium, magnesium, and potassium.

Developmental changes in carboxylase activities in in vitro cultured coconut zygotic embryos : comparison with corresponding activities in seedlings

Phosphoenolpyruvate Carboxylase (PEPC; EC: 4.1.1.31) and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (RubisCO; EC: 4.1.1.39) enzyme specific activities were measured during the in vitro development of coconut (Cocos nucifera L.) zygotic mature embryos into plantlets and compared with those of palms produced by conventional seed germination. At the time of initiation of germination, high PEPC and low RubisCO activities were measured in both cultured and conventionally germinated embryos, thus indicating an anaplerotic CO2 fixation. During both in vitro and in planta development, RubisCO progressively took over and became the main route for inorganic carbon fixation. The in vitro-grown coconut plantlets showed a faster decrease in their PEPC:RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the in vitro-derived material. Just before acclimatization, the RubisCO activity in in vitro-derived plantlets (2.83 µmol CO2h−1mg−1TSP) was lower than that in seedlings (6.98 µmol CO2h−1mg−1TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material