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The widely held explanation for photoperiod-controlled flowering in long-day plants is largely embodied in the External Coincidence Hypothesis which posits that flowering is induced when activity of a rhythmic gene that regulates it (a putative “flowering gene”) occurs in the presence of light. Nevertheless, re-examination of the Arabidopsis flowering data from non 24-hour cycles of Roden et al. suggests that External Coincidence is not tenable if the circadian rhythm of the “flowering gene” were entrained to sunrise as commonly accepted. On the other hand, the hypothesis is supported if circadian cycling of the gene conforms to a solar rhythm, and its entrainment is to midnight on the solar clock. Data available point to flowering being induced by the gene which peaks in its expression between 16 to 19 h after midnight. In the normal 24 h cycle, that would be between 4 p.m. and 7 p.m., regardless of the photoperiod. Such timing of the “flowering gene” expression allows for variable coincidence between gene activity and light, depending on the photoperiod and cycle period. A correlation is found between earliness of flowering and the degree of coincidence of “flowering gene” expression with light (r = 0.88, p

The pathogenic fungus Corynespora cassiicola a serious threat to rubber trees (Hevea brasiliensis); the infected leaves develop necrotic lesions and abscise, leaving the tree unproductive. The destructiveness of C. cassiicola has been largely attributed to cassiicolin, a secreted protein toxin of the fungus. Recombinant antibody technology coupled to genetic transformation offer hope to curtail the disease in Hevea. Single chain variable fragment (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the functional antibody gene. A scFv phage library was constructed from cassiicolin immunized Balb/C mice spleen IgG heavy and light variable chains. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. Nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. The hemagglutinin (HA) tagged scFv expressed in Escherichia coli is discerned as a band at circa 30 kDa on SDS-PAGE. The corresponding band was detected by anti-HA IgG on a Western immunoblot of the gel. Homology-based modelling was employed to create three-dimensional structures of the scFv antibody and cassiicolin, and binding (docking) of the antibody to the toxin antigen was simulated. Furthermore, deactivation of cassiicolin by the scFv was demonstrated by detached leaf bio-assay on selected susceptible Hevea clones. Efforts are underway to genetically transform Hevea clones with the cassiicolin-specific scFv gene to enhance resistance to Corynespora leaf disease. (Texte intégral)

Corynespora cassiicola leaf disease is a serious threat to rubber trees; the fungus infected leaves develop necrotic lesions and abscind, leaving the tree unproductive. The destructiveness of C. cassiicola has been largely attributed to cassiicolin, a host-selective protein toxin secreted by the fungus. Recombinant antibody technology offers hope to curtail the disease; single chain variable fragment (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the antibody gene. Anti-cassiicolin scFv phage library was constructed from cassiicolin immunized Balb/C mice spleen IgG heavy and light variable chains. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. The nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. The hemagglutinin (HA) tagged scFv expressed in Escherichia coli is discerned as a band at circa 30 kDa on SDS-PAGE. The corresponding band was detected by anti-HA IgG on a Western immunoblot of the gel. Deactivation of cassiicolin by the scFv was demonstrated by leaf bio-assay on PB 260, a susceptible clone. Leaf bioassay is in progress to evaluate deactivation of cassiicolin on the other susceptible clones that are also amenable for genetic modification i.e. GL 1, RRIM 600 and BRIM 2020.

Latex allergy affects only about 0.1% of the general population in Europe1-3, although healthcare workers in the Europe and America who frequently use latex examination and surgical gloves are more likely to be allergic to latex. Reports from Europe and the USA give prevalence figures between 2.4 to 17%4,5. Hundreds of proteins are found in natural rubber latex. Of these, only a small handful are allergenic. There has been much interest in identifying the proteins in Hevea latex responsible for latex allergy and much effort was expended on isolating and purifying the allergenic proteins from Hevea latex or latex products6. Also of considerable interest to researchers is the cloning of the cDNA that encode the protein allergens7. Successful DNA cloning would enable production of the recombinant protein that can then be used in immunoassays and in various immunological investigations such as epitope mapping. The International Union of Immunological Societies (IUIS) recognises eleven allergenic proteins (Hev b 1 to Hev b 11) of natural rubber latex from Hevea brasiliensis. Among these, Hev b 28 is a major allergen that has been detected in latex gloves9. This paper reports the cloning of Hev b 2 and the production of the recombinant protein. Some of its enzymic and immunological characterisatics are described.

Natural rubber from Hevea brasiliensis latex consists of a high molecular weight polymer composed of isopentenyl diphosphate (IPP) molecules. Latex is the cytoplasmic fluid contained in the vessel tubes or laticifers which are located in the bark of the Hevea tree. As cytoplasm, latex would be expected to contain general cellular processes carried out by eukaryotic cells and more specifically, rubber biosynthesis. In this project, we are interested in building a profile of genes expressed in latex. These expressed genes or Expressed Sequence Tags (ESTs) are obtained by sequencing random cDNA clones which represent the genes transcribed in latex. Our objective is to generate a pool of Hevea ESTs which would serve as a molecular genetics resource to facilitate various aspects of rubber research. Furthermore, an EST library will also create the way for microarray-based studies of Hevea gene expression. In this study, we report the current status of EST sequencing of clones from a latex cDNA library.

Hevea brasiliensis produces voluminous latex which can be extracted non-destructively through the process of tapping. By transforming Hevea with genes that control the production of high-value proteins, transgenic rubber plants could serve as efficient, low cost, low maintenance and environment-friendly production lines for the production of the targeted protein. The transgenic rubber tree then becomes essentially a living ¿factory¿. The system would enable continual harvesting of the protein exuded in the latex that is free of bacteria and animal viruses. As Hevea is amenable to vegetative propagation, large numbers of genetically identical plants can be multiplied from a single selected transformant. Examples of commercially valuable proteins are those employed in pharmacology and personal care products. One of the genes that has been transformed into Hevea encoded an antibody single chain variable fragment (scFv) against the coat protein of the bacterium Streptococcus sanguis. This recombinant antibody fragment has been successfully synthesised in plants, but extraction and recovery of the target protein remained a problem because of the low yield and the fact that destructive harvesting was involved1.

Latex from the rubber tree (Hevea brasiliensis) is separated into the rubber cream, the clear aqueous phase (C-serum), and the sediment, which consists mainly of vacuole-like organelles called lutoids by high-speed centrifugation. The fluid content of the latter, known as B-serum has drawn attention for many years, owing to its hydrolytic enzymes and pathogenesis-related proteins. A number of these proteins were also pursued in latex allergy studies. [bêta]-glucosidase is among the first hydrolases detected in the H. brasiliensis latex B-serum. It is in the leaf tissue, however, that a [bêta]-glucosidase was first isolated and purified. Besides [bêta]-monoglucosides, this enzyme from Hevea leaves cleaves a wide range of indigenous heteroglycosides including coniferin and cyanoglucosides (linamarin and lataustralin) indicating a possible role in lignification and cyanogenesis. Accordingly it is given the name cyanogenic glycosidase (linamarase). In cassava, hydrolysis of cyanoglucosides by linamarase produces cyanohydrins and glucose. Subsequent breakdown of cyanohydrins either spontaneous or by an á-hydroxynitrile lyase releases a ketone and hydrogen cyanide (HCN), which among others has a role in plant defense. Likewise, the H. brasiliensis linamarase has been implicated in the resistance against South American Leaf Blight, a devastating disease caused by the pathogenic fungi Microcyclus ulei. The present study is aimed at isolating, identifying and characterising a cyanogenic [bêta]-glucosidase in the B-serum, as well as cloning and sequencing of its transcript in H. brasiliensis latex.

One of the goals in the Hevea genetic transformation programme at the RRIM is the production of recombinant proteins in the latex of transgenic Hevea, so that it can be extracted continually and nondestructively by tapping the rubber tree. Gene transfer in Hevea has become routine, facilitated by Agrobacterium. Recombinant proteins produced from plants is one of the most exciting and fastest developing areas in biology. This aspect becomes increasingly important in cases where large amounts of protein need to be produced. Hevea trees with their large volume of latex production capability, and easy scale-up of transgenics through vegetative propagation could be the choice. As the transgenic Hevea system does not depend on repeated success with genetic transformation, clonal copies could be vegetatively propagated form high expressing transformants. From a commercial standpoint, transgenic rubber trees have the highest potential as a safe production system for heterologous proteins in bulk quantities. The probability of health risks because of contamination with potential human pathogens and toxins in recombinant products derived from Hevea in minimized. To explore the potential to produce commercially interesting proteins, we set out to express the 68 kDa protein human serum albumin (HSA) in transgenic Hevea. HSA is initially synthesised in human liver as prepro-albumin and released after cleavage of the aminoterminal prepeptide consisting of 18 amino acids from the endoplasmic reticulum. The proalbumin is processed further in the Golgi complex by the removal of the propeptide (6 residues) by a serine proteinase. The mature polypeptide (585 amino acids) is thus secreted.