Phenylalanine derived cyanogenic diglucosides from Eucalyptus camphora and their abundances in relation to ontogeny and tissue type

The cyanogenic glucoside profile of Eucalyptus camphora was investigated in the course of plant ontogeny. In addition to amygdalin, three phenylalanine-derived cyanogenic diglucosides characterized by unique linkage positions between the two glucose moieties were identified in E. camphora tissues. This is the first time that multiple cyanogenic diglucosides have been shown to co-occur in any plant species. Two of these cyanogenic glucosides have not previously been reported and are named eucalyptosin B and eucalyptosin C. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora produce only the cyanogenic monoglucoside prunasin, and genetically based variation was observed in the age at which seedlings initiate prunasin biosynthesis. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. The role of multiple cyanogenic glucosides in E. camphora is unknown, but may include enhanced plant defense and/or a primary role in nitrogen storage and transport.     

Enhanced CO2 alters the relationship between photosynthesis and defence in cyanogenic Eucalyptus cladocalyx F. Muell.

The effect of elevated CO₂ and different levels of nitrogen on the partitioning of nitrogen between photosynthesis and a constitutive nitrogen-based secondary metabolite (the cyanogenic glycoside prunasin) was examined in Eucalyptus cladocalyx . Our hypothesis was that the expected increase in photosynthetic nitrogen-use efficiency of plants grown at elevated CO₂ concentrations would lead to an effective reallocation of available nitrogen from photosynthesis to prunasin. Seedlings were grown at two concentrations of CO₂ and nitrogen, and the proportion of leaf nitrogen allocated to photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), protein and prunasin compared. Up to 20% of leaf nitrogen was allocated to the cyanogenic glycoside, although this proportion varied with leaf age, position and growth conditions. Leaf prunasin concentration was strongly affected by nitrogen supply, but did not increase, on a dry weight basis, in the leaves from the elevated CO₂ treatments. However, the proportion of nitrogen allocated to prunasin increased significantly, in spite of a decreasing pool of leaf nitrogen, in the plants grown at elevated concentrations of CO₂. There was less protein in leaves of plants grown at elevated CO₂ in both nitrogen treatments, while the concentration of active sites of Rubisco only decreased in plants from the low-nitrogen treatment. These changes in leaf chemistry may have significant implications in terms of the palatability of foliage and defence against herbivores.     

Ontogenetic and temporal trajectories of chemical defence in a cyanogenic eucalypt

Many studies have shown that similarly aged plants within a species or population can vary markedly in the concentration of defence compounds they deploy to protect themselves from herbivores. Some studies have also shown that the concentration of these compounds can change with development, but no empirical research has mapped such an ontogenetic trajectory in detail. To do this, we grew cyanogenic Eucalyptus yarraensis seedlings from three half-sibling families under constant glasshouse conditions, and followed their foliar cyanogenic glycoside (prunasin) concentration over time for 338 days after sowing (DAS). Plants in all families followed a similar temporal pattern. Plants increased in foliar prunasin concentration from a very low level (10 μg cyanide (CN) equivalents g⁻¹) in their first leaves, to a maximum of, on average, 1.2 mg CN g⁻¹ at about 240 DAS. From 240 to 338 DAS, prunasin concentration gradually decreased to around 0.7 mg CN g⁻¹. Significant differences between families in maximum prunasin concentration were detected, but none were detected in the time at which this maximum occurred. In parallel with these changes in prunasin concentration, we detected an approximately linear increase in leaf mass per unit leaf area (LMA) with time, which reflected a change from juvenile to adult-like leaf anatomy. When ontogenetic trajectories of prunasin against LMA were constructed, we failed to detect a significant difference between families in the LMA at which maximum prunasin concentration occurred. This remarkable similarity in the temporal and ontogenetic trajectories between individuals, even from geographically remote families, is discussed in relation to a theoretical model for ontogenetic changes in plant defence. Our results show that ontogeny can constrain the expression of plant chemical defense and that chemical defense changes in a nonlinear fashion with ontogeny.     

Growth and defence in young pine and spruce and the expression of resistance to a stem-feeding weevil

Defence in young trees has been much less studied than defence in older ones. In conifers, resin within ducts in bark is an important quantitative defence, but its expression in young trees may be influenced by developmental or physical constraints on the absolute size of the resin ducts as well as by differential allocation of resources to growth and resin synthesis. To examine these relationships, we used nitrogen fertilisation of 1- and 2-year-old pine and spruce to produce trees of different sizes and measured the effect on the number and size of resin ducts and the amount of resin they contained. All of these variables tended to increase with stem diameter, indicating a positive relationship between resin-based defence and growth of 1- and 2-year-old trees. In pine, however, the mass of resin flowing from severed ducts was much lower relative to duct area in 1- than in 2-year-old trees, suggesting that the older trees allocated a higher proportion of the carbon budget to resin synthesis. Resin-based defence in 1-year-old pines appears to be both positively related to growth and resource limited. In spruce, resin production was generally lower, and age-related differences were not observed, suggesting that resin-based defence is less important in this species. Bio-assays of 2-year-old trees with the pine weevil, Hylobius abietis, emphasised the importance of resin as a defence against this bark feeding insect. Nitrogen fertilisation had a limited influence on resistance expression. One-year-old trees remained susceptible because of their small size, low resin production and limited response to fertilisation. The strong growth response of 2-year-old trees to fertilisation increased resin-based defence, but most spruce trees remained susceptible, while most pines were resistant at all levels of fertilisation.     

Population divergence in the ontogenetic trajectories of foliar terpenes of a Eucalyptus species

Background and Aims The development of plant secondary metabolites during early life stages can have significant ecological and evolutionary implications for plant–herbivore interactions. Foliar terpenes influence a broad range of ecological interactions, including plant defence, and their expression may be influenced by ontogenetic and genetic factors. This study investigates the role of these factors in the expression of foliar terpene compounds in Eucalyptus globulus seedlings.Methods Seedlings were sourced from ten families each from three genetically distinct populations, representing relatively high and low chemical resistance to mammalian herbivory. Cotyledon-stage seedlings and consecutive leaf pairs of true leaves were harvested separately across an 8-month period, and analysed for eight monoterpene compounds and six sesquiterpene compounds.Key Results Foliar terpenes showed a series of dynamic changes with ontogenetic trajectories differing between populations and families, as well as between and within the two major terpene classes. Sesquiterpenes changed rapidly through ontogeny and expressed opposing trajectories between compounds, but showed consistency in pattern between populations. Conversely, changed expression in monoterpene trajectories was population- and compound-specific.Conclusions The results suggest that adaptive opportunities exist for changing levels of terpene content through ontogeny, and evolution may exploit the ontogenetic patterns of change in these compounds to create a diverse ontogenetic chemical mosaic with which to defend the plant. It is hypothesized that the observed genetically based patterns in terpene ontogenetic trajectories reflect multiple changes in the regulation of genes throughout different terpene biosynthetic pathways.     

Gynocardin from Baileyoxylon lanceolatum and a revision of cyanogenic glycosides in Achariaceae

The cyclopentenone cyanhydrin glycoside gynocardin was the only cyanogen isolated from foliage of monotypic Australian rainforest tree, Baileyoxylon lanceolatum (Achariaceae). The presence of cyanogenic compounds in plants can have considerable taxonomic utility. A review of previous reports of cyanogenesis in the recently revised Achariaceae revealed distinct taxonomic patterns as well as inconsistencies in the reporting of cyanogenic compounds. This variation appears to be due to tissue level localisation of cyanogenic compounds as well as discrepancies in results obtained from different detection methods. Recommendations are made for future investigations.     

Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific beta-glucosidases

Cyanogenesis (i.e. the evolution of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one controlling synthesis of the cyanogenic glycoside and the other controlling the production of a specific degradative beta-glucosidase. The sole cyanogenic glycoside in Eucalyptus nobilis was identified as prunasin (D-mandelonitrile beta-D-glucoside) using HPLC and GC-MS. Seedlings from three populations of E. nobilis were grown under controlled conditions and 38% were found to be acyanogenic, a proportion far greater than reported for any other cyanogenic eucalypt. A detailed study of the acyanogenic progeny from a single open-pollinated parent found that 23% lacked a cyanogenic beta-glucosidase, 32% lacked prunasin and 9% lacked both. Of the remaining seedlings initially identified as acyanogenics, 27% contained either trace amounts of beta-glucosidase or prunasin, while 9% contained trace amounts of both. Results support the hypothesis that the two components necessary for cyanogenesis are inherited independently. Trace amounts are likely to result from the presence of non-specific beta-glucosidases or the glycosylation of the cyanohydrin intermediate by non-specific UDP glycosyl transferases.     

夏季自然高温对桉树光合速率和暗呼吸速率的影响

在湖南夏季的自然高温下,所测7种桉树的日平均净光合速率在12.56～27.85 mg*dm-2*h-1之间,日平均暗呼吸速率在6.06～22.39 mg*dm-2*h-1之间。高温对桉树净光合速率的影响依品种而异。樟脑桉和蓝桉的净光合速率最高峰值出现在35 ℃的气温下,而邓恩桉的净光合速率却受35 ℃气温的显著抑制。桉树暗呼吸速率并不随气温的升高而增大。     

夏季自然高温对桉树光合速率和暗呼吸速率的影响

在湖南夏季的自然高温下,所测7种桉树的日平均净光合速率在12.56～27.85mg·dm     

Salt tolerance in Eucalyptus spp.: identity and response of putative osmolytes

In four species of salt-tolerant eucalypts (Eucalyptus raveretiana, E. spathulata, E. sargentii and E. loxophleba), we found substantial concentrations of quercitol – a cyclitol known for its accumulation in seeds of Quercus. Quercitol was absent in old foliage of E. globulus, a species noted for greater susceptibility to salinity, and also absent in the moderately tolerant E. camaldulensis, but, relative to other species, both had higher foliar concentrations of inositol. Simple sugars and cyclitols accumulated to osmotically significant concentrations in all species. The osmotic potential of expressed sap was always less than that of the external ‘soil’ solution and increasing salinity produced predictable reductions in growth and increases in ion concentrations in foliage of saplings of four eucalypt species. The more salt-tolerant species, E. spathulata, E. loxophleba and E. sargentii, were able to maintain well-regulated leaf Na⁺ concentrations even at 300 mol m⁻³ NaCl. These more salt-tolerant species also showed an apparent increase in net selectivity for K⁺ over Na⁺ as salinity increased, irrespective of the Na⁺ : Ca²⁺ ratio of the external medium (range 25 : 1 to 75 : 1; Ca²⁺ always ≥ 4.0 mol m⁻³). By contrast, E. globulus was unable to exclude Na⁺ when exposed to higher NaCl concentrations (e.g. 200 and 300 mol m⁻³). Carbon isotope signatures of foliage reflected imposed salinity but were not strongly enough correlated with growth to support previous suggestions that isotope discrimination be a means of evaluating salt tolerance. On the other hand, patterns of sugar and cyclitol accumulation should be further explored in eucalypts as traits contributing to salt tolerance, and with potential use as markers in breeding programmes.     

Daily stem growth patterns in irrigated Eucalyptus globulus and E. nitens in relation to climate

Daily increments in stem radius were determined from hourly dendrometer measurements in each of three irrigated Eucalyptus nitens and E. globulus trees. Multiple regressions determined from daily weather variables accounted for 40–50% of the variance in increment. The use of weather variables lagged by 1–2 days increased the variance explained. The diurnal variation in stem radius was resolved into three mathematically defined phases: shrinkage, recovery and increment. The positive daily net increment in stem radius, by definition, occurred in the increment phase. Average weather conditions during this phase (predominantly night-time) did not explain any more variance in increment than the average daily conditions, determined over a 24 h period. Daily increment was resolved into a rate of stem radius increase during the increment phase and the duration (hours) of that phase. Significant species by month interactions were evident with growth in summer characterised by faster rates of stem expansion over shorter time periods within each diurnal cycle. E. nitens tended to have longer increment phases in spring and autumn, and faster phase rates in autumn than E. globulus. Interactions between weather variables and cambial growth were complicated and varied over the year. The correlation between temperature and stem growth varied from positive in spring to zero or negative during summer. The data indicate a need to understand weather-by- climate interactions at the level of whole tree physiology in order to fully understand the effect of weather on cambial activity and therefore stem increment and wood properties. Received: 12 April 1999 / Accepted: 6 July 1999     

Growth,reproduction and defence in nettles: responses to herbivory modified by competition and fertilization

We studied the effects of environmental factors on the ability to compensate for simulated herbivory in an annual (Urtica urens) and two perennial (U. dioica subspp. dioica and sondenii) nettle species in order to test the compensatory continuum hypothesis. Further, we studied the expression of costs of structural defence in different environmental conditions. Herbivory interacted significantly with density and fertilization for height growth, branching and reproductive traits. The negative effects of simulated herbivory on relative height growth were less detrimental for plants grown in low density and high fertilization level, which supports the compensatory continuum hypothesis. However, with respect to other traits measured, our results do not support the idea of compensatory continuum. The negative effects of apical excision on inflorescence biomass were relatively more prominent in plants growing at low density and receiving more fertilization than on those growing in worse conditions. In addition, branching was reduced by apical excision regardless of resource levels. The lack of compensation for herbivory is explained by the role which the damaged or removed tissue plays in plant development and function. In the perennial U. dioica, defensive responses to herbivory, measured as changes in trichome density, were stronger than in the annual U. urens. In the southern subspecies dioica, trichome density increased in newly emerged leaves after leaf clipping. In the northern subspecies sondenii, trichome density increased after apical excision. The differences in the defensive responses between the two subspecies of U. dioica may be due to differential natural herbivory pressures on subspecies inhabiting different geographical regions. We observed negative phenotypic correlations between structural defence and other plant traits, which suggests the existence of costs of defence. In addition to differences among the species studied, the expression of costs of defence was dependent on the resource levels.     

Early events in the elicitation of plant defence

Plants successfully use inducible defence mechanisms to combat potential pathogens. Elicitors are signaling compounds that stimulate any of such defence responses. Recent progress in the isolation of pure elicitors has made possible investigations on elicitor-binding proteins which might function as receptors in signal transduction pathways that ultimately activate the defences. The elicitor-binding sites studied so far show a high degree of ligand specificity, as do the candidate binding proteins identified for some of the ligands. Following elicitor perception, a number of rapid reactions are detectable in plant cells, including enhanced ion fluxes across the plasma membrane, formation of reactive oxygen intermediates, changes in protein phosphorylation, and lipid oxidation. Intriguing questions arising from these observations are whether the elicitor-binding proteins constitute receptors in plant defence signaling and whether any of the rapid events participate in signal transduction during defence activation. Received: 17 November 1997 / Accepted: 22 April 1998     

Plant structural traits and their role in anti-herbivore defence

We consider the role that key structural traits, such as spinescence, pubescence, sclerophylly and raphides, play in protecting plants from herbivore attack. Despite the likelihood that many of these morphological characteristics may have evolved as responses to other environmental stimuli, we show that each provides an important defence against herbivore attack in both terrestrial and aquatic ecosystems. We conclude that leaf-mass–area is a robust index of sclerophylly as a surrogate for more rigorous mechanical properties used in herbivory studies. We also examine herbivore counter-adaptations to plant structural defence and illustrate how herbivore attack can induce the deployment of intensified defensive measures. Although there have been few studies detailing how plant defences vary with age, we show that allocation to structural defences is related to plant ontogeny. Age-related changes in the deployment of structural defences plus a paucity of appropriate studies are two reasons why relationships with other plant fitness characteristics may be obscured, although we describe studies where trade-offs between structural defence and plant growth, reproduction, and chemical defences have been demonstrated. We also show how resource availability influences the expression of structural defences and demonstrate how poorly our understanding of plant structural defence fits into contemporary plant defence theory. Finally, we suggest how a better understanding of plant structural defence, particularly within the context of plant defence syndromes, would not only improve our understanding of plant defence theory, but enable us to predict how plant morphological responses to climate change might influence interactions at the individual (plant growth trade-offs), species (competition), and ecosystem (pollination and herbivory) levels.     

Genetic covariation between effectiveness and cost of defence in aphids

Ecological immunology distinguishes between the long-term evolutionary costs of possessing defences against parasites and the short-term costs of using them. Evolutionary biologists have typically focused on the former in the search for constraints on the evolution of resistance. Here, we show in the peach-potato aphid, Myzus persicae, that short-term costs may be of equal evolutionary importance. Survivors of more resistant aphid clones suffered a higher reduction of fecundity upon parasitoid attack than survivors of more susceptible clones. This genetically based trade-off between benefits and costs of defence may limit the evolution of increased resistance and explain the maintenance of genetic variation for resistance under environmental variation in parasitism risk.     

Metabolism,excretion and avoidance of cyanogenic glucosides in insects with different feeding specialisations

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds releasing toxic hydrogen cyanide when hydrolysed by specific β-glucosidases after plant tissue damage. In contrast to specialist herbivores that have mechanisms to avoid toxicity from CNglcs, it is generally assumed that non-adapted herbivores are negatively affected by CNglcs. Recent evidence, however, implies that the defence potential of CNglcs towards herbivores may not be as effective as previously anticipated. Here, performance, metabolism and excretion products of insects not adapted to CNglcs were analysed, including species with different degrees of dietary specialisation (generalists, specialists) and different feeding modes (leaf-snipping lepidopterans, piercing-sucking aphids). Insects were reared either on cyanogenic or acyanogenic plants or on an artificial cyanogenic diet. Lepidopteran generalists (Spodoptera littoralis, Spodoptera exigua, Mamestra brassicae) were compared to lepidopteran glucosinolate-specialists (Pieris rapae, Pieris brassicae, Plutella xylostella), and a generalist aphid (Myzus persicae) was compared to an aphid glucosinolate-specialist (Lipaphis erysimi). All insects were tolerant to cyanogenic plants; in lepidopterans tolerance was mainly due to excretion of intact CNglcs. The two Pieris species furthermore metabolized aromatic CNglcs to amino acid conjugates (Cys, Gly, Ser) and derivatives of these, which is similar to the metabolism of benzylglucosinolates in these species. Aphid species avoided uptake of CNglcs during feeding. Our results imply that non-adapted insects tolerate plant CNglcs either by keeping them intact for excretion, metabolizing them, or avoiding uptake.     

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).