Elizabeth H. Neilson

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.

Utilization of a high-throughput shoot imaging system to examine the dynamic phenotypic responses of a C4 cereal crop plant to nitrogen and water deficiency over time

Highlight A high-throughput imaging system was used to compare growth and architectural traits of sorghum grown under different environmental conditions, and validated against traditional measures of plant performance and composition.

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

BackgroundThe 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.ResultsLeaves 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.ConclusionsThe 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.

Novel aspects of cyanogenesis in Eucalyptus camphora subsp. humeana

Cyanogenesis is the release of cyanide from certain organisms upon tissue disruption. Tissue disruption, such as that caused by folivory, brings cyanogenic glycosides into contact with catabolic enzymes and toxic HCN is subsequently released. The process provides a measure of defence against generalist herbivores. Within the genus Eucalyptus, several species have been identified as cyanogenic and all of these store cyanide exclusively in the form of the cyanogenic glycoside prunasin. Here we report for the first time cyanogenesis in Eucalyptus camphora subsp. humeana L.A.S. Johnson & K.D. Hill. We found that foliage contains at least five different cyanogenic glycosides, three of which were purified and identified (prunasin, sambunigrin and amygdalin). Two natural populations of E. camphora trees were screened for cyanogenesis, and quantitative polymorphism was measured at both sites. Trees varied in their capacity for cyanogenesis from 0.014 to 0.543 mg CN g-1 DW in one population and from 0.011 to 0.371 mg CN g-1 DW in the other. A progeny trial, testing both cyanogenesis and carbon-based defence (namely total phenolics and condensed tannins), was performed with seed sourced from two cyanogenic, open-pollinated maternal trees. Interestingly, the seedlings exhibited markedly lower levels of cyanogenesis and condensed tannins than the adult population, with some individuals completely lacking one or both of the chemical defences. Total phenolic concentrations, however, were significantly higher in the seedlings than in the parental population from which the seed was sourced. Eucalyptus camphora is relatively unique among cyanogenic trees having multiple foliar cyanogenic glycosides and an apparently marked ontogenetic regulation of cyanogenic capacity.

Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis)

Two missing enzyme activities in the amygdalan pathway result in sweet versus formation of bitter almonds. Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a β(1→6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana. Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.

Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55

Cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx requires an additional pathway step, involving three CYPs from the CYP79, CYP706, and CYP71 families, and a glucosyltransferase UGT85. Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx. This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.