Rubber Tree (Hevea brasiliensis) Bark Necrosis Syndrome III: A Physiological Disease Linked to Impaired Cyanide Metabolism.

First attempts to discriminate between tapping panel dryness (TPD) and bark necrosis (BN), two Hevea sp. bark diseases leading to the cessation of latex production, showed differences in latex biochemical characteristics (1). Further, contrary to TPD, BN is characterized by inner phloem necrosis starting at the rootstock/scion junction (RS/S) and spreading upward to the tapping cut. Recent etiological (3) and epidemiological studies did not provide evidence of a causative pathogen for BN, but showed that BN is favored by a combination of various stresses (2). Searching for molecular markers of BN using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses highlighted differential expression of some proteins in the latex and bark, especially a 67-kDa protein, which accumulates in the inner phloem of the BN trees. This protein was identified by peptide microsequencing as a linamarase (cyanogenic ß-glucosidase). This led to the suspicion of the involvement of cyanogenesis in the spread of the syndrome inside the inner bark. The cDNAs of enzymes involved in cyanide (CN) metabolism (linamarase, hydroxynitrile lyase, and cyanoalanine synthase) were cloned from our Hevea sp. phloem specific cDNA library. In addition, the most BN-susceptible rubber clones were shown to exhibit higher cyanide potentions in the leaves and bark, together with low cyanoalanine synthase (CAS) gene expression and activity. Furthermore, linamarine (the cyanogene glucoside substrate of linamarase) was shown to accumulate in the phloem at the base of the trunk, especially above the rootstock/scion junction. The results of biochemical and gene expression studies associated with recent ecophysiological advances (2) strongly suggest a possible cell decompartmentalization near the RS/S junction, resulting in a local release of toxic concentration of highly diffusive CN. This, combined with a lethal imbalance between cyanogenic and CN-detoxifying activities (CAS) in the phloem of BN trees, could lead to poisoning of neighboring cells and to the spread of tissue necrosis toward the tapping cut. In conclusion, after providing evidence of exogenous factors favoring BN (2), this report highlights endogenous disorders that may be at the origin of this physiological disease leading to BN. References: (1) D. Nandris et al. Eur. J. For. Pathol. 21:325, 1991. (2) D. Nandris et al. Plant Dis. 88:1047, 2004.

Genetic linkage map of Coffea canephora: effect of segregation distortion and analysis of recombination rate in male and female meioses.

Two complementary segregating plant populations of Coffea canephora were produced from the same clone. One population (DH) comprised 92 doubled haploids derived from female gametes, while the other population (TC) was a test cross consisting of 44 individuals derived from male gametes. Based on the DH population, a genetic linkage map comprising 160 loci was constructed. Eleven linkage groups that putatively correspond to the 11 gametic chromosomes of C. canephora were identified. The mapped loci included more than 40 specific sequence-tagged site markers, either single-copy RFLP probes or microsatellites, that could serve as standard landmarks in coffee-genome analyses. Furthermore, comparisons for segregation distortion and recombination frequency between the two populations were performed. Although segregation distortions were observed in both populations, the frequency of loci exhibiting a very pronounced degree of distortion was especially high in the DH population. This observation is consistent with the hypothesis of strong zygotic selection among the DH population. The recombination frequencies in both populations were found to be almost indistinguishable. These results offer evidence in favour of the lack of significant sex differences in recombination in C. canephora.

Single-locus inheritance in the allotetraploid Coffea arabica L. and interspecific hybrid C. arabica x C. canephora.

Molecular cytogenetic analysis has indicated that Coffea arabica is an amphidiploid formed from the hybridization between two closely related diploid progenitor species, C. canephora and C. eugenioides. Our aim was to determine the mode of inheritance in C. arabica and in a tetraploid interspecific hybrid (called arabusta) between C. arabica and C. canephora as revealed by segregation analyses of restriction fragment length polymorphism (RFLP) loci markers. The observed RFLP allele segregations in an F(2) progeny of C. arabica conform to disomic inheritance as expected, with regular bivalent pairing of homologous chromosomes in the F1 hybrid. In contrast, RFLP loci followed tetrasomic inheritance in the arabusta interspecific hybrid, although bivalents have been reported to predominate greatly at meiosis in its hybrid. These results suggest that homologous chromosomes do not pair in C. arabica, not as a consequence of structural differentiation, but because of the functioning of pairing regulating factors. Moreover, the arabusta hybrid seems to offer the possibility of gene exchange between the homologous genomes.

Chloroplast DNA extraction from herbaceous and woody plants for direct restriction fragment length polymorphism analysis.

The technique described here is a fast and simple method of extracting chloroplast DNA (cpDNA). It overcomes the need for differential centrifugation using density gradients. The leaves do not have to be kept in the dark and lyophilized before extraction, but lyophilization is still possible. The chloroplasts are specifically lysed in a cell extract of leaves, using a non-ionic detergent. After isolation by centrifugation, the cpDNA is purified by the combined action of proteolytic enzymes and detergents, followed by the elimination of proteins using a mixture of chloroform and isoamyl alcohol. This method provided good quality restriction profiles for all species analyzed.

Molecular characterisation and origin of the Coffea arabica L. genome.

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period.

Phylogenetic analysis of chloroplast DNA variation in Coffea L.

The trnL-trnF intergenic spacer of cpDNA has been sequenced from 38 tree samples representing 23 Coffea taxa and the related genus Psilanthus. These sequences were used for phylogenetic reconstruction using parsimony analyses. The results suggest a radial mode of speciation and a recent origin in Africa for the genus Coffea. Phylogenetic relationships inferred from the cpDNA analysis suggest several major clades, which present a strong geographical correspondence (i.e., west Africa, central Africa, east Africa, and Madagascar). The overall results agree well with the phylogeny previously inferred from nuclear genome data. However, several inconsistencies are observed among taxa endemic to west Africa, suggesting the occurrence of introgressive hybridization. Evidence is also obtained for the genetic origin of the allotetraploid species C. arabica.

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L.

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The results were in contradiction with the present classification into two genera. Additionally, cpDNA inheritance was studied in interspecific hybrids between C. arabica and C. canephora, and in an intraspecific progeny of C. canephora, using PCR-based markers. Both studies showed exclusively maternal inheritance of cpDNA.

Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex.

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100: 255-260) was solubilized by octylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters Km and Vmax. The Km value for Mg2+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the Vmax value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport systems, the latter being eliminated during the solubilization/reconstitution of lutoid membrane proteins.

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis.

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg(2+) diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg(2+). These results suggest the presence of a Mg(2+)/H(+) antiporter. The maximum Mg(2+)/H(+) exchange rate was observed at pH 8.5. The K(m) values for Mg(2+) (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K(+) and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg(2+)/H(+) exchange was electroneutral. Mg(2+)/H(+) exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K(m) for Mg(2+) are similar to those reported for the Mg(2+)/2Na(+) antiporter in animals cell. These data are consistent with the existence of a Mg(2+)/2H(+) antiporter in a plant tonoplast.

The occurence of ribonucleic acid in the lutoïd fraction (lysosomal compartment) from Hevea brasiliensis Künth. (Müll.-Arg.) latex.

The lutoïds from Hevea brasiliensis latex represent a polydisperse lysosomal compartment. They contain RNA which is resistant to RNase in conditions which maintain the integrity of the lutoïds but is hydrolyzed when these organelles are destabilized. This RNA appears to be a structural component of the lutoïds.

Some evidence for the occurrence of ribonucleic acid in the lutoid fraction (lysosomal compartment) from Hevea brasiliensis latex.

Some results are reported for the presence of some RNA species in the lutoid fraction from Hevea brasiliensis latex. The origin of this RNA is discussed.