Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation.

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea brasiliensis.

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.

Purification and characterization of cassiicolin, the toxin produced by Corynespora cassiicola, causal agent of the leaf fall disease of rubber tree.

Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism.

Molecular characterization of new members of the Hevea brasiliensis hevein multigene family and analysis of their promoter region in rice.

The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.

Differential gene expression in different types of Hevea brasiliensis roots.

Three types of roots (taproots, first order laterals and second order laterals) were functionally characterized on 7-month-old in vitro plantlets regenerated by somatic embryogenesis in Hevea brasiliensis. A histological analysis revealed different levels of differentiation depending on root diameter. A primary structure was found in first and second order lateral roots, while taproots displayed a secondary structure. The expression of 48 genes linked to some of the regulatory pathways acting in roots was compared in leaves, stems and the different types of roots by real-time RT-PCR. Thirteen genes were differentially expressed in the different organs studied in plants grown under control conditions. Nine additional other genes were differentially regulated between organs under water deficit conditions. In addition, 10 genes were significantly regulated in response to water deficit, including 8 regulated mainly in lateral roots types. Our results suggest that the regulation of gene expression in lateral roots is different than that in taproots, which have a main role in nutrient uptake and transport, respectively.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.