Jean D'Auzac

Effects of polyamine biosynthetic inhibitors on somatic embryogenesis and cellular polyamines in Hevea brasiliensis

Summary Hevea brasiliensis , like other ligneous plants, has been considered for a long time to be a recalcitrant embryogenic species. Nevertheless, exogenous polyamines (PA) added individually or together to the culture medium constitute one among other factors, favorizing the calli embryogenic capacity. Inhibitors of enzymes involved in polyamine biosynthesis like DL-difluoromethylarginine (DFMA), DL-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) are usually used to demonstrate that PA are required for normal growth and development. In Hevea , application of these inhibitors, at concentrations that did not affect callogenesis, lowered somatic embryo formation. In a few cases, the inhibition was partially reversed by addition of spermidine (Spd) (50 µM). The effect of inhibitors on PA levels was opposite to actual knowledge of the pathway for PA synthesis. MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), largely decreases the putrescine (Put) level; however, synthesis of this diamine should have been increased by this treatment. DFMO, an inhibitor specific for ornithine decarboxylase (ODC), paradoxically increases Put. The accumulation of Spd and spermine (Spm) by DFMA also appears paradoxical because DFMA, known as a specific inhibitor of arginine decarboxylase (ADC), should have produced a decrease in both PA. The specificity of the classical polyamine biosynthesis inhibitors is discussed.

The composition of natural latex from Hevea brasiliensis.

Hevea brasiliensis latex collected by tapping can be considered as cytoplasm, in spite of the absence of nucleus and mitochondrial material. It possesses the complexity of cytoplasm both in its subcellular organization and the diversity of the constituent molecules. Its essential characteristic is the high rubber content. Rubber is a very long chaincis-polyisoprene polymer synthesized in the cytosol compartment. Rubber forms 90% of latex dry weight and 40% of fresh weight, which underlines the astonishing dominance of this anabolic pathway in the laticiferous metabolism. However, the preparation of dry rubber or concentrated latex as used in industry removes a fairly large proportion of the mineral and organic solutes from fresh latex. Latex processing conditions in plantation factories can considerably modify the technological properties of rubber after vulcanization through the influence of the nature and proportion of the residual nonrubber substances. Some of these give natural rubber properties that are still better than those of syntheticcis-polyisoprene.In contrast, it appears to have been clearly demonstrated that the proteins extracted from latex or from rubber tree leaves containing latex display allergenic properties (46,47). Turjanmaa et al. (48) observed that allergenic proteins extracted from latex gloves have molecular weights of between 2 and 30 kDa and are thus similar to various allergens, including those of pollen. It has been mentioned that large quantities of proteins with low molecular weights are found in latex.

Role of the Lutoidic Tonoplast in the Senescence and Degeneration of the Laticifers of Hevea brasiliensis

Summary An enzymatic NAD(P)H oxidase activity that generates species of toxic oxygen was located at the level of the lutoidic tonoplast. The partial characterization of this enzyme shows that it is insensible towards classical inhibitors of respiratory chains, and still functions at very low oxygen concentrations. It is greatly activated by physiological concentrations of metallic cations (Fe3+ and Cu++), and by quinone-like compounds, among which naphtoquinones and ubiquinones may act as physiological activators or electron carriers. The diverse species of toxic oxygen emitted, lead to the peroxidatic degradation of the unsaturated lipids of the membrane and then to destabilisation and lysis of the organelles, in a way similar to that described for the NAD(P)H induced O2- production and consecutive membrane alteration by mammalian microsomes or granulocytes. Intensive hormonal treatment of bark («over-stimulation» with ethrel, an ethylene generator), or too frequent bark wounding (i.e. over-tapping) increase the O2- generating activity, and simultaneously decrease the level of cytosolic scavengers. This leads to lysis of the latex organelles and especially of the lutoids (vacuolysosomes), and to subsequent liberation into the latex of the «coagulating factors» which they normally compartmentalize. It results in latex coagulation within the bark of the stressed trees: the so-called «dry-cuts syndrome». However, the NAD(P)H oxidase activity remains very low and the scavenging activities (superoxyde-dismutase, catalase, glutathion reductase) and chemical scavengers (e.g. reduced thiols) fully efficient, in the latex from healthy non-stressed Heveas (stable latex). Computer data analysis allowed us to draw a model describing the biochemical events leading to this type of cell degeneration.

Role of the Lutoidic Tonoplast in the Control of the Cytosolic Homeostasis within the Laticiferous Cells of Hevea

Summary Lutoids, the vacuo-lysosomes of the Hevea latex cells, compartmentalize, in vivo , numerous ions such as H + , Mg ++ , Ca ++ , Pi, citrate, some of them strongly toxic for the cytosolic metabolism. Evidence is given for the correlation of the in vivo compartimentation of some of these ions inside the lutoids with the latex production by Hevea . Two opposing H + pumps were localized on the lutoidic tonoplast; the one is a Mg ++ -dependent ATPase, the other a NADH-consuming redox system (cytochrome c: artificial acceptor). The functioning of these H + pumps may account for a major part of the transtonoplastic ΔpH variations, and therefore the cytosolic pH control, which probably regulates the highly pH-dependent latex metabolism. The resulting proton-motive force energizes the accumulation and compartmentation of the inhibiting ions inside the lutoids, and ensures the control of the «detoxification» and ionic equilibrium of the cytoplasm of the laticiferous cells. Treatment of Hevea bark with ethrel, an ethylene generator which «stimulates» latex production, induces an increase in the H + -pumping ATPase activity, resulting in the activation of the metabolism in the latex cells. All the results reviewed lead us to propose that the lutoids play a double role as a «biophysical pH-STAT and a «detoxicating trap», thus controlling the cytosolic homeostasis.

Scopoletin production and degradation in relation to resistance of Hevea brasiliensis to Corynespora cassiicola

Summary Inoculation of leaves of resistant and susceptible Hevea brasiliensis clones with Corynespora cassiicola induced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Hevea phytoalexin. Foliar symptoms appeared, as soon as 24 h after infection, and precede Scp accumulation in inoculum droplets (peak: 48 h after inoculation). Scp concentration was 5-fold higher in the susceptible than in the resistant clone 48 h after infection. Nevertheless, a fungitoxic effect of Scp on spore germination and on mycelium growth was shown in bioassays, but the efficient concentrations were greater than those found with another foliar pathogenic fungus of rubber tree such as Microcyclus ulei and Colletotrichum gloeosporioides. Bioassay showed that the low Scp fungitoxicity to C. cassiicola could also be related to the ability of fungus to detoxify Sep. Isoelectric focusing analysis of peroxidase activity in Hevea infected leaflets has shown an increase in acidic and basic isoperoxidases that are able to use Scp as substrate. The rapid increase of Scp-oxidase activity (as soon as 16 h after inoculation) was higher in the resistant than in the susceptible clone. In vitro testing of Scp peroxidation products has shown a toxic effect on C. cassiicola (conidia germination and mycelium growth), but not significantly higher than Scp. It appears that the level of Sep accumulation was a balance between its synthesis and its degradation by the pathogen or/and by foliar Scp-peroxidases. In conclusion, these results lead to the fact that Scp cannot be considered as a major defense mechanism of Hevea towards C. cassiicola disease.

Effects of Gelling Agents and Liquid Medium on Embryogenic Potential, Polyamines and Enzymatic Factors in Browning in Hevea brasiliensis Calli

Summary The effects of gelling agents and liquid medium on the embryogenic potential of Hevea brasiliensis calli were investigated in three types of medium: Difco agar (A), Gelrite (G) and Sorbarod cellulose blocks in liquid medium (S). Cellulose blocks, as compared with the solid media (A and G), induced early callogenesis but reduced, even obliterated, somatic embryo formation. Embryogenic potential and callus polyamine contents were increased while browning potential was decreased by Gelrite treatment. In addition, while phenol levels and bound polyphenoloxidase activity were lowered, superoxide dismutase (SOD) activity was maintained higher after day 20 in Gelrite medium. These physiological situations could be related to differences in the mineral supply and water potential between the three types of support medium and could be responsible for the calli embryogenic capacity. It can be suggested that SOD activity and cellular polyamine levels playa key role in somatic embryogenesis in Hevea .

Physiological activators of invertase from Hevea brasiliensis latex

Abstract Potassium or sodium nitrates or phosphates, and thiols such as reduced glutathione or cysteine, stimulate the activity of invertase from Hevea brasiliensis latex. These activators raise the V max but do not affect the K m of the enzyme for sucrose. The action of these effectors is additive. Their efficiency is pH dependent, being higher below pH 7.0 and markedly decreasing above it.

Characterization of Hevea brasiliensis rubber from virgin trees : A possible role of cis-polyisoprene in unexploited tree

Abstract The role of rubber in Hevea brasiliensis was analyzed based on the structural analysis of rubber in rubber trees as latex. The rubber obtained from a mature-tree which has never tapped before, so-called “virgin mature-tree” (Vir-NR), contained the gel fraction higher than 80%, while that from a regularly tapped mature-tree (Reg-NR) was less than 3%. The gel showed almost the same structure as a crosslinked rubber prepared from fresh latex in the presence of peroxide, with the molecular weight between crosslinks (Mc) of 3×103. This value is extremely low comparing with that of 7×105 observed for the soft-gel in rubber from high-ammonia latex and Pale crepe. The sol fraction from Vir-NR was an oxidative degraded product containing aldehyde and epoxide groups. Aging properties of Vir-NR are much worse than those of Reg-NR. This indicates that rubber accepts free-radicals to form C-C crosslinking and partly oxidative scission of main-chains during storage in laticiferous tubes of Hevea trees. This su...