Recruitment of distinct UDP-glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.

Molecular Characterisation of Flavanone O-methylation in Eucalyptus.

Flavonoids are ubiquitous polyphenolic compounds in plants, long recognised for their health-promoting properties in humans. Methylated flavonoids have received increasing attention due to the potential of methylation to enhance medicinal efficacy. Recently, Eucalyptus species with high levels of the O-methylated flavanone pinostrobin have been identified. Pinostrobin has potential commercial value due to its numerous pharmacological and functional food benefits. Little is known about the identity or mode of action of the enzymes involved in methylating flavanones. This study aimed to identify and characterise the methyltransferase(s) involved in the regiospecific methylation of pinostrobin in Eucalyptus and thereby add to our limited understanding of flavanone biosynthesis in plants. RNA-seq analysis of leaf tips enabled the isolation of a gene encoding a flavanone 7-O-methyltransferase (EnOMT1) in Eucalyptus. Biochemical characterisation of its in vitro activity revealed a range of substrates upon which EnOMT1 acts in a regiospecific manner. Comparison to a homologous sequence from a Eucalyptus species lacking O-methylated flavonoids identified critical catalytic amino acid residues within EnOMT1 responsible for its activity. This detailed molecular characterisation identified a methyltransferase responsible for chemical ornamentation of the core flavanone structure of pinocembrin and helps shed light on the mechanism of flavanone biosynthesis in Eucalyptus.

The efficacy and safety of pinocembrin in a sheep model of bleomycin-induced pulmonary fibrosis.

The primary flavonoid, pinocembrin, is thought to have a variety of medical uses which relate to its reported anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer properties. Some studies have reported that this flavonoid has anti-fibrotic activities. In this study, we investigated whether pinocembrin would impede fibrosis, dampen inflammation and improve lung function in a large animal model of pulmonary fibrosis. Fibrosis was induced in two localized lung segments in each of the 10 sheep participating in the study. This was achieved via two infusions of bleomycin delivered bronchoscopically at a two-week interval. Another lung segment in the same sheep was left untreated, and was used as a healthy control. The animals were kept for a little over 5 weeks after the final infusion of bleomycin. Pinocembrin, isolated from Eucalyptus leaves, was administered to one of the two bleomycin damaged lung segments at a dose of 7 mg. This dose was given once-weekly over 4-weeks, starting one week after the final bleomycin infusion. Lung compliance (as a measure of stiffness) was significantly improved after four weekly administrations of pinocembrin to bleomycin-damaged lung segments. There were significantly lower numbers of neutrophils and inflammatory cells in the bronchoalveolar lavage of bleomycin-infused lung segments that were treated with pinocembrin. Compared to bleomycin damaged lung segments without drug treatment, pinocembrin administration was associated with significantly lower numbers of immuno-positive CD8+ and CD4+ T cells in the lung parenchyma. Histopathology scoring data showed that pinocembrin treatment was associated with significant improvement in inflammation and overall pathology scores. Hydroxy proline analysis showed that the administration of pinocembrin did not reduce the increased collagen content that was induced by bleomycin in this model. Analyses of Masson's Trichrome stained sections showed that pinocembrin treatment significantly reduced the connective tissue content in lung segments exposed to bleomycin when compared to bleomycin-infused lungs that did not receive pinocembrin. The striking anti-inflammatory and modest anti-fibrotic remodelling effects of pinocembrin administration were likely linked to the compound's ability to improve lung pathology and functional compliance in this animal model of pulmonary fibrosis.

Monoterpene synthases responsible for the terpene profile of anther glands in Eucalyptus polybractea R.T. Baker (Myrtaceae).

Research on terpene biosynthesis in the genus Eucalyptus (Myrtaceae) is poorly developed, but recently large numbers of terpene synthase (TPS) genes have been identified. Few of these have been characterized or their expression localized to specific tissues. A prime candidate for detailed examination of TPS gene expression is the bisexual eucalypt flower-composed of male and female reproductive organs, and vegetative tissues that may express different TPS genes. We aimed to characterize and compare the terpene profile and TPS genes expressed in anthers and gynoecia in the high oil-yielding Eucalyptus polybractea R.T. Baker. We hypothesized that gynoecia will produce greater amounts of defensive terpenes, whereas anthers will have a terpene profile that is biased towards a role in pollination. Microscopy of isolated anthers showed them to possess a single, prominent oil gland. Chemical analysis of whole floral structures at different stages of development showed total oil per unit dry mass increased as flower buds expanded, with highest concentrations in mature flower buds just prior to flower opening. The oil profile of gynoecia was dominated by the monoterpene 1,8-cineole, whereas that of isolated anthers were enriched with the monoterpene α-pinene. Through transcriptomic analysis and recombinant protein expression, we were able to identify monoterpene synthases responsible for the different profiles. Synthases for α-pinene and 1,8-cineole were expressed in each tissue type, but the relative expression of the former was higher in anthers. Sequence comparison and site-directed mutagenesis of the α-pinene synthase allowed us to identify amino acids that influence the α-pinene to ß-pinene ratio of the product profile. We suggest the terpene constituents of anthers may have multiple roles including attracting pollinators through emission of volatile α-pinene, deterrence of palynivores through emission of volatile 1,8-cineole and adhesion of pollen to pollinators via the release of sticky α-pinene onto the anther surface.

Differential metabolic specialization of foliar oil glands in Eucalyptus brevistylis Brooker (Myrtaceae).

Trees and shrubs from the genus Eucalyptus are characterized by the presence of numerous foliar oil glands that generally house mono- and sesquiterpenes. In some species, glands are also known to house substantial quantities of unrelated secondary metabolites such as volatile, aromatic ß-triketones. It is not known if these compounds are co-housed with terpenes or if they are produced in distinct, metabolically specialized glands. We showed that Eucalyptus brevistylis-a species with appreciable foliar quantities of both ß-triketones and terpenes-contains two visually distinct gland types in leaves, one that is translucent and the other golden-brown. Gas chromatographic analyses of solvent extracts of the two gland types showed that the translucent glands contain sesquiterpene alcohol cubenols and cubebols (termed 'sesquiterpene glands'), whereas the golden-brown glands contain predominantly the ß-triketone conglomerone with lesser amounts of sesquiterpene hydrocarbon caryophyllenes (termed 'triketone glands'). Analysis of leaves from trees of different ages, from young saplings through to advanced age trees, showed a gradual increase in the abundance of sesquiterpene glands relative to triketone glands as plants aged. Such ontogenetic regulation of foliar secondary metabolite concentration appears to be a common feature of Eucalyptus species, albeit at different temporal scales. A similar ontogenetic pattern was observed in ageing leaves, with mature leaves having a higher proportion of sesquiterpene glands than young leaf tips. It is concluded that regulation of the relative abundances of the two gland types with ontogeny likely reflects the different herbivores present at the different life stages of leaves and whole plants. In particular, leaf tips and young plants may be advantaged by deploying higher amounts of insecticidal ß-triketones. The concurrent deployment of two metabolically distinct gland types in leaves is a rare phenomenon and a novel finding for myrtaceous trees.

Correction: Foliar Essential Oil Glands of Eucalyptus Subgenus Eucalyptus (Myrtaceae) Are a Rich Source of Flavonoids and Related Non-Volatile Constituents.

[This corrects the article DOI: 10.1371/journal.pone.0151432.].

Location-specific cuticular hydrocarbon signals in a social insect.

Social insects use cuticular hydrocarbons (CHCs) to convey different social signals, including colony or nest identity. Despite extensive investigations, the exact source and identity of CHCs that act as nest-specific identification signals remain largely unknown. Perhaps this is because studies that identify CHC signals typically use organic solvents to extract a single sample from the entire animal, thereby analysing a cocktail of chemicals that may serve several signal functions. We took a novel approach by first identifying CHC profiles from different body parts of ants (Iridomyrmex purpureus), then used behavioural bioassays to reveal the location of specific social signals. The CHC profiles of both workers and alates varied between different body parts, and workers paid more attention to the antennae of non-nest-mate and the legs of nest-mate workers. Workers responded less aggressively to non-nest-mate workers if the CHCs on the antennae of their opponents were removed with a solvent. These data indicate that CHCs located on the antennae reveal nest-mate identity and, remarkably, that antennae both convey and receive social signals. Our approach and findings could be valuably applied to chemical signalling in other behavioural contexts, and provide insights that were otherwise obscured by including chemicals that either have no signal function or may be used in other contexts.

Foliar Essential Oil Glands of Eucalyptus Subgenus Eucalyptus (Myrtaceae) Are a Rich Source of Flavonoids and Related Non-Volatile Constituents.

The sub-dermal secretory cavities (glands) embedded within the leaves of Eucalyptus (Myrtaceae) were once thought to be the exclusive repositories of monoterpene and sesquiterpene oils. Recent research has debunked this theory and shown that abundant non-volatile compounds also occur within foliar glands. In particular, glands of four species in subgenus Eucalyptus contain the biologically active flavanone pinocembrin. Pinocembrin shows great promise as a pharmaceutical and is predominantly plant-sourced, so Eucalyptus could be a potential commercial source of such compounds. To explore this we quantified and assessed the purity of pinocembrin in glands of 11 species of E. subg. Eucalyptus using Electro-Spray Ionisation Liquid Chromatography Mass Spectrometry of acetonitrile extracts and Gas Chromatography Mass Spectrometry analyses of hexane extracts of isolated glands which were free from other leaf tissues. Our results showed that the glands of subgenus Eucalyptus contain numerous flavanones that are structurally related to pinocembrin and often present in much greater abundance. The maximum concentration of pinocembrin was 2 mg g-1 dry leaf found in E. stellulata, whereas that of dimethylpinocembrin (5,7-dimethoxyflavanone) was 10 mg g-1 in E. oreades and that of pinostrobin (5-hydroxy-7-methoxyflavanone) was 12 mg g-1 in E. nitida. We also found that the flavanones are exclusively located within the foliar glands rather than distributed throughout leaf tissues. The flavanones differ from the non-methylated pinocembrin in the degree and positions of methylation. This finding is particularly important given the attractiveness of methylated flavonoids as pharmaceuticals and therapeutics. Another important finding was that glands of some members of the subgenus also contain flavanone O-glucosides and flavanone-ß-triketone conjugates. In addition, glands contain free ß-triketones, ß-triketone heterodimers and chromone C-glucosides. Therefore, the foliar glands of this taxonomically distinct group of plants are a rich source of a range of flavonoids and other biologically active compounds with great commercial potential.

Chloroplast genome analysis of Australian eucalypts--Eucalyptus, Corymbia, Angophora, Allosyncarpia and Stockwellia (Myrtaceae).

We present a phylogenetic analysis and comparison of structural features of chloroplast genomes for 39 species of the eucalypt group (genera Eucalyptus, Corymbia, Angophora, and outgroups Allosyncarpia and Stockwellia). We use 41 complete chloroplast genome sequences, adding 39 finished-quality chloroplast genomes to two previously published genomes. Maximum parsimony and Bayesian analyses, based on >7000 variable nucleotide positions, produced one fully resolved phylogenetic tree (35 supported nodes, 27 with 100% bootstrap support). Eucalyptus and its sister lineage Angophora+Corymbia show a deep divergence. Within Eucalyptus, three lineages are resolved: the 'eudesmid', 'symphyomyrt' and 'monocalypt' groups. Corymbia is paraphyletic with respect to Angophora. Gene content and order do not vary among eucalypt chloroplasts; length mutations, especially frame shifts, are uncommon in protein-coding genes. Some non-synonymous mutations are highly incongruent with the overall phylogenetic signal, notably in rbcL, and may be adaptive. Application of custom informatics pipelines (GYDLE Inc.) enabled direct chloroplast genome assembly, resolving each genome to finished-quality with no need for PCR gap-filling or contig order resolution. Analysis of whole chloroplast genomes resolved major eucalypt clades and revealed variable regions of the genome that will be useful in lower-level genetic studies (including phylogeography and geneflow).

Contrasting ontogenetic trajectories for phenolic and terpenoid defences in Eucalyptus froggattii.

BACKGROUND AND AIMS: Plant defence metabolites are considered costly due to diversion of energy and nutrients away from growth. These costs combined with changes in resource availability and herbivory throughout plant ontogeny are likely to promote changes in defence metabolites. A comprehensive understanding of plant defence strategy requires measurement of lifetime ontogenetic trajectories--a dynamic component largely overlooked in plant defence theories. This study aimed to compare ontogenetic trajectories of foliar phenolics and terpenoids. Phenolics are predicted to be inexpensive to biosynthesize, whereas expensive terpenoids also require specialized, non-photosynthetic secretory structures to avoid autotoxicity. Based on these predicted costs, it is hypothesized that phenolics would be maximally deployed early in ontogeny, whereas terpenoids would be maximally deployed later, once the costs of biosynthesis and foregone photosynthesis could be overcome by enhanced resource acquisition. METHODS: Leaves were harvested from a family of glasshouse-grown Eucalyptus froggattii seedlings, field-grown saplings and the maternal parent tree, and analysed for total terpenoids and phenolics. KEY RESULTS: Foliar phenolics were highest in young seedlings and lowest in the adult tree. Indeed the ratio of total phenolics to total terpenoids decreased in a significantly exponential manner with plant ontogeny. Most individual terpene constituents increased with plant ontogeny, but some mono- and sesquiterpenes remained relatively constant or even decreased in concentration as plants aged. CONCLUSIONS: Plant ontogeny can influence different foliar defence metabolites in directionally opposite ways, and the contrasting trajectories support our hypothesis that phenolics would be maximally deployed earlier than terpenoids. The results highlight the importance of examining ontogenetic trajectories of defence traits when developing and testing theories of plant defence, and illustrate an advantage of concurrently studying multiple defences.

Plant chemical defense: at what cost?

Plants are sessile organisms and dependent on deployment of secondary metabolites for their response to biotic and abiotic challenges. A trade-off is envisioned between resources allocated to growth, development, and reproduction and to the biosynthesis, storage, and maintenance of secondary metabolites. However, increasing evidence suggests that secondary metabolites serve auxiliary roles, including functions associated with primary metabolism. In this opinion article, we examine how the costs of plant chemical defense can be offset by multifunctional biosynthesis and the optimization of primary metabolism. These additional benefits may negate the trade-off between primary and secondary metabolism, and provide plants with an innate plasticity required for growth, development, and interactions with their environment.

Localization of oleuropeyl glucose esters and a flavanone to secretory cavities of Myrtaceae.

We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.

Plant homeostasis of foliar manganese sinks: specific variation in hyperaccumulators.

Plant manganese (Mn) hyperaccumulation provides unusual insight into homeostasis of this essential micronutrient, in particular its excessive storage in shoot tissues. The compartmentation of hyperaccumulated foliar Mn appears exceptional among metal hyperaccumulators, since it occurs via specific microdistribution patterns. Here, three associated Mn hyperaccumulators, Virotia neurophylla, Maytenus fournieri, and Garcinia amplexicaulis exhibiting distinctly different Mn detoxification strategies were examined. Non-invasive sample preparation in conjunction with cryo scanning electron microscopy (SEM) was used to obtain in vivo quantitative microprobe X-ray and anatomical data from fully hydrated cells. Highly vacuolated large palisade mesophyll cells in V. neurophylla leaves were found to contain around 650 mM Mn. The large non-photosynthetic hypodermal cells of M. fournieri leaves, also with high vacuolar content, and the main site for Mn disposal, had an estimated mean vacuolar Mn concentration of around 600 mM. Previous qualitative X-ray mapping had shown Mn to be almost evenly sequestered across the entire leaf cross section of G. amplexicaulis. However, quantitative data obtained here showed a marked variation in localised concentrations that ranged between ~15 and >800 mM. Notable among these were mean values of >600 mM in spongy mesophyll cells, and ~800 mM within cells of a narrow sub epidermal layer preceding the palisade mesophyll. This study demonstrated the extraordinary Mn carrying capacities of different types of leaf cell vacuoles.

α,ß-Unsaturated monoterpene acid glucose esters: structural diversity, bioactivities and functional roles.

The glycosylation of lipophilic small molecules produces many important plant secondary metabolites. The majority of these are O-glycosides with relatively fewer occurring as glucose esters of aromatic or aliphatic acids. In particular, monoterpene acid glucose esters have much lower structural diversity and distribution compared to monoterpene glycosides. Nevertheless, there have been over 20 monoterpene acid glucose esters described from trees in the genus Eucalyptus (Myrtaceae) in recent years, all based on oleuropeic acid, menthiafolic acid or both. Here we review all of the glucose esters containing these monoterpenoids identified in plants to date. Many of the compounds contain phenolic aglycones and all contain at least one α,ß-unsaturated carbonyl, affording a number of important potential therapeutic reactivities such as anti-tumor promotion, carcinogenesis suppression, and anti-oxidant and anti-inflammatory activities. Additional properties such as cytotoxicity, bitterness, and repellency are suggestive of a role in plant defence, but we also discuss their localization to the exterior of foliar secretory cavity lumina, and suggest they may also protect secretory cells from toxic terpenes housed within these structures. Finally we discuss how the use of a recently developed protocol to isolate secretory cavities in a functional state could be used in conjunction with systems biology approaches to help characterize their biosynthesis and roles in plants.

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.

Isolation of intact sub-dermal secretory cavities from Eucalyptus.

BACKGROUND: The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. RESULTS: Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. CONCLUSIONS: The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.

Huperzine alkaloids from Australasian and southeast Asian Huperzia.

CONTEXT: The pharmaceutical alkaloid huperzine A (HupA), currently used in herbal supplements and medicines worldwide, is predominantly sourced from the Chinese lycopod Huperzia serrata (Thunb. ex Murray) Trev. (Lycopodiaceae), which on average contains only 0.08 mg HupA g(-1) dry weight, and is experiencing a rapid decline in China due to over-harvesting. OBJECTIVE: To find a high-yielding, natural source of HupA and/or the related huperzine B (HupB) that could potentially be used as the starting material in a commercial propagation program. MATERIALS AND METHODS: We surveyed 17 Huperzia species (15 indigenous to Australia and southeast Asia) for their foliar HupA and HupB concentrations. We also studied intra-specific variation for the huperzines in four species that were available in sufficient numbers, and determined tissue-specific accumulation in larger specimens. RESULTS: HupA was detected in 11 Australasian and southeast Asian species, with eight also containing HupB, albeit at much lower concentrations. A H. elmeri (Herter) Holub plant from the Philippines had one of the highest HupA concentrations recorded (1.01 mg g(-1) dry wt) and it also had the highest HupB content of all plants surveyed (0.34 mg g(-1) dry wt). Intra-specific HupA and HupB concentrations were extremely variable, and at the intra-plant level, reproductive strobili were found to accumulate the highest HupA concentrations. DISCUSSION AND CONCLUSION: Select Huperzia species from Australia and southeast Asia have potential as the starting material for establishing commercial HupA plantations, but the high intra-specific variability observed suggests that detailed screening is needed to isolate high huperzine-yielding individuals.

Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus.

Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K(1) and K(2). Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.

Allocation of nitrogen to chemical defence and plant functional traits is constrained by soil N.

Plants have evolved a vast array of defence mechanisms to avoid or minimize damage caused by herbivores and pathogens. The costs and benefits of defences are thought to vary with the availability of resources, herbivore pressure and plant functional traits. We investigated the resource (nitrogen) and growth cost of deploying cyanogenic glycosides in seedlings of Eucalyptus cladocalyx (Myrtaceae). To do this, we grew the plants under a range of soil N conditions, from levels that were limiting for growth to those that were saturating for growth, and we measured correlations between foliar chemical and performance attributes. Within each N treatment, we found evidence that, for every N invested in cyanogenic glycosides, additional N is added to the leaf. For the lowest N treatment, the additional N was less than one per cyanogenic glycoside, rising to some two Ns for the other treatments. The interaction between cyanogenic glycosides and both condensed tannins and total phenolic compounds was also examined, but we did not detect correlations between these compounds under constant leaf N concentrations. Finally, we did not detect a correlation between net assimilation rate, relative growth rate and cyanogenic glycoside concentrations under any soil N treatment. We conclude that the growth cost of cyanogenic glycosides was likely too low to detect and that it was offset to some degree by additional N that was allocated alongside the cyanogenic glycosides.

Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.).

Cyanide assimilation by the beta-cyanoalanine pathway produces asparagine, aspartate and ammonium, allowing cyanide to serve as alternate or supplemental source of nitrogen. Experiments with wheat and sorghum examined the enrichment of (15)N from cyanide as a function of external cyanide concentration in the presence or absence of nitrate and/or ammonium. Cyanogenic nitrogen became enriched in plant tissues following exposure to (15)N-cyanide concentrations from 5 to 200 microm, but when exposure occurred in the absence of nitrate and ammonium, (15)N enrichment increased significantly in sorghum shoots at solution cyanide concentrations of > or =50 microm and in wheat roots at 200 microm cyanide. In an experiment with sorghum using (13)C(15)N, there was also a significant difference in the tissue (13)C:(15)N ratio, suggestive of differential metabolism and transport of carbon and nitrogen under nitrogen-free conditions. A reciprocal (15)N labelling study using KC(15)N and (15)NH(4)(+) and wheat demonstrated an interaction between cyanide and ammonium in roots in which increasing solution ammonium concentrations decreased the enrichment from 100 microm cyanide. In contrast, with increasing solution cyanide concentrations there was an increase in the enrichment from ammonium. The results suggest increased transport and assimilation of cyanide in response to decreased nitrogen supply and perhaps to ammonium supply.