Occurrence of the cyanogen linustatin in Hevea brasiliensis

During seedling development ofHevea brasiliensis the cyanogenic diglucoside linustatin is exuded from the endosperm. These data support the hypothesis, that the stored cyanogenic monoglucoside linamarin is glucosylated to linustatin during mobilization of the cyanogenic glucosides.     

Hevea brasiliensis and Urtica dioica impact the in vitro mycorrhization of neighbouring Medicago truncatula seedlings

Hevea brasiliensis is a mycotrophic tree for which root colonization by arbuscular mycorrhizal fungi (AMF) under in vitro or pot culture conditions can take several weeks. The reason for this slow colonization is still unknown, but the exudation of antifungal compounds such as hevein by the roots may be one of the causes. Here, the root colonization of Medicago truncatula, a highly mycotrophic plant, was assessed after 12 days of growth in the extraradical mycelium network of the AMF Rhizophagus irregularis in close vicinity of H. brasiliensis plantlets or Urtica dioica seedlings (also known to synthesize antifungal compounds of the hevein family). We hypothesized that a negative impact on the root colonization of a M. truncatula seedling developing close to H. brasiliensis and U. dioica may give indirect proofs for the exudation of inhibitory molecules. The percentages of total root colonization of M. truncatula were 30.1 % lower in the presence of H. brasiliensis than in the control plants, and 29.1 % lower in presence of U. dioica. The abundance of arbuscules in the roots of M. truncatula was also lower in plants grown in presence of H. brasiliensis plantlets than in the control plants. Similarly, the succinate dehydrogenase and the phosphatase activities measured in the extraradical mycelium of R. irregularis were significantly lower in the presence of both plants, compared with the controls. No root colonization was observed in H. brasiliensis and U. dioica within the time-frame of the experiments. The low root colonization of M. truncatula when grown in the presence of rubber or stinging nettle suggested the exudation of diffusible molecules which could also explain the delayed root colonization of H. brasiliensis and the absence of colonization of U. dioica.     

Mobilization and utilization of cyanogenic glycosides: the linustatin pathway

In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2-β-d-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-β-d-glucosyl-β-d-glucopyranosyloxy]-2- methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the β-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the following pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)—glucosylation—transport of the diglucoside (out of the endosperm into the seedling)—cleavage by diglucosidase—reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect.     

Hevea Linamarase-A Nonspecific beta-Glycosidase

In the leaf tissue of the cyanogenic plant Hevea brasiliensis, which contains large amounts of linamarin, there is no specific linamarase. In Hevea leaves only one β-glucosidase is detectable. It is responsible for the cleavage of all β-glucosides and β-galactosides occurring in Hevea leaf tissue, including the cyanogenic glucoside linamarin. Therefore, the enzyme is referred to as a β-glycosidase instead of the term β-glucosidase. This β-glycosidase has a broad substrate spectrum and occurs in multiple forms. These homo-oligomeric forms are interconvertible by dissociation-association processes. The monomer is a single protein of 64 kilodaltons.     

Compartmentation of cyanogenic glucosides and their degrading enzymes

Whereas high activities of β-glucosidase occur in homogenates of leaves of Hevea brasiliensis Muell.-Arg., this enzyme, which is capable of splitting the cyanogenic monoglucoside linamarin (linamarase), is not present in intact protoplasts prepared from the corresponding leaves. Thus, in leaves of H. brasiliensis the entire linamarase is located in the apoplasmic space. By analyzing the vacuoles obtained from leaf protoplasts isolated from mesophyll and epidermal layers of H. brasiliensis leaves, it was shown that the cyanogenic glucoside linamarin is localized exclusively in the central vacuole. Analyses of apoplasmic fluids from leaves of six other cyanogenic species showed that significant linamarase activity is present in the apoplasm of all plants tested. In contrast, no activity of any diglucosidase capable of hydrolyzing the cyanogenic diglucoside linustatin (linustatinase) could be detected in these apoplasmic fluids. As described earlier, any translocation of cyanogenic glucosides involves the interaction of monoglucosidic and diglucosidic cyanogens with the corresponding glycosidases (Selmar, 1993a, Planta 191, 191–199). Based on this, the data on the compartmentation of cyanogenic glucosides and their degrading enzymes in Hevea are discussed with respect to the complex metabolism and the transport of cyanogenic glucosides.     

Transport of cyanogenic glucosides: linustatin uptake by Hevea cotyledons

The ¹⁴C-labelled cyanogenic glucosides linustatin (diglucoside of acetone cyanohydrin) and linamarin (monoglucoside of acetone cyanohydrin), prepared by feeding [¹⁴C]valine to plants of Linum usitatissimum L., were applied to cotyledons of Hevea brasiliensis Muell.-Arg. in order to study their transport. Both [¹⁴C]-linustatin and [¹⁴C]linamarin were efficiently taken up by the cotyledons. Whereas ¹⁴C was recovered completely when [¹⁴C]linustatin was applied to the seedling, only about one-half of the radioactivity fed as [¹⁴C]linamarin could be accounted for after incubation. This observation is in agreement with the finding that apoplasmic linamarase hydrolyzes linamarin but not the related diglucoside linustatin. These data prove that, in vivo, linamarin does not occur apoplasmically and that linustatin, which is exuded from the endosperm, is taken up by the cotyledons very efficiently. Thus, these findings confirm the linustatin pathway (Selmar et al. 1988, Plant Physiol. 86, 711–716), which describes mobilization and transport of the cyanogenic glucoside linamarin, initiated by the glucosylation of linamarin to yield linustatin. When linustatin is metabolized to non-cyanogenic compounds, in Hevea this cyanogenic diglucoside is hydrolyzed by a diglucosidase which splits off both glucose molecules simultaneously as one gentiobiose moiety (Selmar et al. 1988). In contrast, [¹⁴C]linustatin, which is taken up by the cotyledon, is not metabolized but is reconverted in high amounts to the monoglucosidic [¹⁴C]linamarin, which then is temporarily stored in the cotyledons. These data demonstrate that in Hevea, besides the simultaneous diglucosidase, there must be present a further diglucosidase which is able to hydrolyze cyanogenic diglucosides sequentially by splitting off only the terminal glucose moiety from linustatin to yield linamarin. From this, it is deduced that the metabolic fate of linustatin, which is transported into the source tissues, depends on the activities of the different diglucosidases. Whereas sequential cleavage — producing linamarin — is purely a part of the process of linamarin translocation (using linustatin as the transport vehicle), simultaneous cleavage, producing acetone cyanohydrin, is part of the process of linamarin metabolization in which the nitrogen from cyanogenic glucosides is used to synthesize non-cyanogenic compounds.     

Cyanogenic Lipids: Utilization during Seedling Development of Ungnadia speciosa

Large amounts of cyanogenic lipids (esters of 1 cyano-2-methylprop-2-ene-1-ol with C:20 fatty acids) are stored in the seeds of Ungnadia speciosa. During seedling development, these lipids are completely consumed without liberation of free HCN to the atmosphere. At the same time, cyanogenic glycosides are synthesized, but the total amount is much lower (about 26%) than the quantity of cyanogenic lipids formerly present in the seeds. This large decrease in the total content of cyanogens (HCN-potential) demonstrates that at least 74% of cyanogenic lipids are converted to noncyanogenic compounds. Whether the newly synthesized cyanogenic glycosides are derived directly from cyanogenic lipids or produced by de novo synthesis is still unknown. Based on the utilization of cyanogenic lipids for the synthesis of noncyanogenic compounds, it is concluded that these cyanogens serve as storage for reduced nitrogen. The ecophysiological significance of cyanolipids based on multifunctional aspects is discussed.     

Relationship of Cyanogenic Capacity (HCN-c) of the Rubber Tree Hevea brasiliensis to Susceptibility to Microcyclus ulei, the Agent Causing South American Leaf Blight

Liberation of HCN from cyanogenic plant tissue depends on cyanogen content (HCN-potential), cyanogen splitting enzymes, cyanohydrin cleaving activity (hydroxynitrile lyase) and nonenzymatic cyanide detoxifying compounds. The maximal amount of HCN potentially set free is governed by the total cyanogen content, whereas the velocity of HCN liberation depends on enzymatic activities (β-glucosidase [β-G], hydroxynitrile lyase [HN]). Plants revealing a high HCN-potential and a high β-glucosidase activity generally are susceptible to infection with Microcyclus ulei. Based on the data of HCN-p and β-G activity of different Hevea species a proposal for future screening work in Hevea resistance selection is given.     

Ethylene stimulation of latex production in Hevea brasiliensis

Rubber tree (Hevea brasiliensis) is an important industrial crop for natural rubber production. Ethylene, as a stimulant of latex production in H. brasiliensis, has been widely used in commercial latex production. However, the mechanism of ethylene action are not completely elucidated, especially in molecular aspect. Here, we focus on the molecular biological progression of ethylene stimulation of latex production. Our data and all previous information showed ethylene had little direct effect on accelerating rubber biosynthesis. The prolonged latex flow and acceleration of sucrose metabolism by ethylene may be the main reasons for the stimulation of latex yield by ethylene.Key words: Hevea brasiliensis, ethylene, rubber production, gene, sucrose     

The leaf, inner bark and latex cyanide potential of Hevea brasiliensis: Evidence for involvement of cyanogenic glucosides in rubber yield

The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells in the inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limiting factors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanide is released when the plant is damaged providing an efficient defense mechanism against herbivores. In H. brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in the leaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) were studied using various parameters. Results showed that the younger the leaf, the higher the CNp. Leaf CNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to determine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNp was found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highest leaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk inner bark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itself exhibited very low (if any) CNp, while harboring all the enzymes (β-d-diglucosidase, linamarase and β-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic compounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be a source of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.     

Light affects in vitro organogenesis of Linum usitatissimum L. and its cyanogenic potential

The relationships between organogenesis of oil flax (Linum usitatissimum L., cv. ‘Szafir’) in vitro, cyanogenic potential (HCN-p) of these tissues and light were investigated. Shoot multiplication obtained on Murashige and Skoog medium containing 0.05 mg L^?1 2,4-dichloro-phenoxyacetic acid and 1 mg L^?1 6-benzyladenine (BA), was about twice higher in light-grown cultures than those in darkness. Light-grown explants showed also higher rate of roots regeneration (in medium containing 1 mg L^?1 α-naphtaleneacetic acid and 0.05 mg L^-1 BA) than dark-grown ones. The cyanogenic potential (expressed both as linamarin and lotaustralin content and linamarase activity) of flax cultured in vitro was tissue-specific and generally was higher under light conditions than in darkness. The highest concentration of linamarin and lotaustralin was detected in light-regenerated shoots, and its amount was twice as high as in roots, and about threefold higher than in callus tissue. The activities of linamarase and β-cyanoalanine synthase in light-regenerated organs were also higher than those in darkness. Thus, higher frequency of regeneration of light-grown cultures than dark-grown ones seems to be correlated with higher HCN-p of these tissues. We suggest that free HCN, released from cyanoglucosides potentially at higher level under light conditions, may be involved in some organogenetic processes which improve regeneration efficiency.     

Glycolipid composition of Hevea brasiliensis latex

Glycolipids of fresh latex from three clones of Hevea brasiliensis were characterized and quantified by HPLC/ESI-MS. Their fatty acyl and sterol components were further confirmed by GC/MS after saponification. The four detected glycolipid classes were steryl glucosides (SG), esterified steryl glucosides (ESG), monogalactosyl diacylglycerols (MGDG) and digalactosyl diacylglycerols (DGDG). Sterols in SG, ESG and total latex unsaponifiable were stigmasterol, β-sitosterol and Δ⁵-avenasterol. The latter was found instead of fucosterol formerly described. Galactolipids were mainly DGDG and had a fatty acid composition different from that of plant leaves as they contained less than 5% C18:3. Glycolipids, which represented 27–37% of total lipids, displayed important clonal variations in the proportions of the different fatty acids. ESG, MGDG and DGDG from clone PB235 differed notably by their higher content in furan fatty acid, which accounted for more than 40% of total fatty acids. Clonal variation was also observed in the relative proportions of glycolipid classes except MGDG (8%), with 43–51% DGDG, 30–34% SG and 7–19% ESG. When compared with other plant cell content, the unusual glycolipid composition of H. brasiliensis latex may be linked to the peculiar nature of this specialized cytoplasm expelled from laticiferous system, especially in terms of functional and structural properties.     

Species Diversity of Arbuscular Mycorrhizal Fungi in the Rhizosphere of <i>Hevea brasiliensis</i> in Hainan Island,China

Hevea brasiliensis is one of the important economic trees with a great economic value for natural rubber production. Symbiosis between roots of H. brasiliensis and arbuscular mycorrhizal fungi (AMF) is widely recognized, and can provide a range of benefits for both of them. Hainan Island harbors is one of the largest plantations of H. brasiliensis in China, whereas the information regarding the diversity of AMF in the rhizosphere of H. brasiliensis on this island is scarce. The diversity of AMF species in the rhizosphere of rubber tree plantations in Hainan was investigated in this study. A total of 72 soil samples from the rhizosphere of H. brasiliensis RY7-33-97 were collected. These included 48 samples from plantations in 11 cities or counties that had been planted for 15–25 years, and 24 samples from a demonstrating plantation site of the China National Rubber Tree Germplasm Repository representing plantations with tree plantation ages from one to 40 year-old. Collectively, a total of 68 morphotypes of AMF, belonging to the genera of Archaeospora (1), Glomus (43), Acaulospora (18), Entrophospora (3), Scutellospora (2), and Gigaspora (1) were isolated and identified, as per morphological characteristics of spores presented in the collected soil samples. Glomus (Frequency, F = 100%) and Acaulospora (F = 100%) were the predominant genera, and A. mellea (F = 63.9%) and A. scrobiculata (F = 63.9%) were the predominant species. AMF species differed significantly among collected sites in spore density (SD, 290.7–2,186.7 spores per 100 g dry soil), species richness (SR, 4.3–12.3), and Shannon-Weiner index of diversity (H, 1.24–2.24). SD was negatively correlated with available phosphorus level in the soil; SR was positively correlated with soil total phosphorus content; and H was positively correlated with levels of soil organic matter and total phosphorus. Similarly, SD, SR, and H were also correlated with H. brasiliensis plantation age, and an increasing trend was observed up to 40 years. These results suggest that the AMF community was complex and ubiquitous in the island plantation ecosystems of H. brasiliensis, with high species abundance and diversity. Soil factors and plantation age dramatically affected AMF diversity at species level.     

Field nursery inoculation of Hevea brasiliensis Muell. Arg. seedling rootstock with vesicular-arbuscular mycorrhizal (VAM) fungi

The growth response of Hevea brasiliensis to vesicular-arbuscular mycorrhizal (VAM) fungi inoculation was assessed in two field nursery sites containing indigenous mycorrhizal fungi (IMF). Seedling rootstocks were inoculated with mixed VAM-fungal species in a factorial combination with phosphorus (P) fertilizer application, and planted in randomised blocks on sandy (site 1) and clayey (site 2) soils. Plants were harvested after 26 weeks for measurements of shoot dry weight (DW), stem diameter, height, mycorrhizal root colonization and leaf nutrient contents. At site 1, VAM increased shoot DW, stem diameter and plant height only in treatments without P applied. Increases in shoot DW due to VAM were 70% greater than the uninoculated controls although this was reduced to 5% when P was applied. At site 2, VAM inoculation also increased shoot DW and stem diameter but the magnitude of the increases was smaller. Shoot DW response due to VAM was only 29%. At this second site, applying phosphate to uninoculated plants did not increase shoot yields further. Leaf concentrations of all nutrients were unaffected by VAM at both sites, except for copper (Cu) which was increased by VAM in treatments where P was not applied. However, leaf contents of P, potassium (K), magnesium (Mg) and Cu were increased by VAM at site 1, and of leaf nitrogen (N) and K at site 2. These experiments demonstrate that VAM-fungi could be introduced into field nursery sites to improve growth and P uptake by H. brasiliensis. The relevance of VAM-fungi to H. brasiliensis seedling rootstock development and the influence of IMF in determining field responses is discussed.     

Novel bacterial endophytes from Hevea brasiliensis as biocontrol agent against Phytophthora leaf fall disease

Bacterial endophytes offer control against many diseases of crop plants as potential biocontrol agents. Antagonistic bacterial endophytes acting against Phytophthora meadii have been screened from leaf, petiole and root tissues of Hevea brasiliensis. Six bacterial endophytes could exhibit more than 50 % inhibition of P. meadii, among which EIL-2, from disease-free zones showed a maximum of 62.5 % inhibition. The isolate EIL-2 was characterized as Alcaligenes sp. and the other isolates were identified as Pseudomonas aeruginosa. 16S rDNA sequence analysis showed that there existed genetic variation among the five isolates of P. aeruginosa from different tissues of the plant indicating the tissue type adaptation of the isolates. Dual culture technique with endophyte EIL-2 completely arrested the growth of P. meadii when inoculated prior to pathogen. The bioassay with EIL-2 in H. brasiliensis clones, RRII 105 showed 43 % reduction of lesion size on infected leaves whereas in RRIM 600 it was only 30 %.