John W. van Klink

Red leaf margins indicate increased polygodial content and function as visual signals to reduce herbivory in Pseudowintera colorata

Red-pigmented leaf margins are common, but their functional significance is unknown. We hypothesized that red leaf margins reduce leaf herbivory by signalling to herbivorous insects the presence of increased chemical defences. Leaves were collected from a natural population of Pseudowintera colorata. Margin size, herbivory damage, anthocyanin content and concentrations of polygodial, a sesquiterpene dialdehyde with antifeedant properties, were quantified. Feeding trials involving larvae of Ctenopseustis obliquana, a generalist herbivore, were conducted on red- and green-margined P. colorata leaves in darkness, or under white, green or red light. Leaves with wider red margins contained higher concentrations of polygodial and anthocyanins, and incurred less natural herbivory. In trials under white light, C. obliquana consumed disproportionately more green- than red-margined leaf laminae. Larvae exhibited no feeding preference when light was manipulated such that leaf colour discrimination was impaired. Red leaf margins provide a reliable and effective visual signal of chemical defence in P. colorata. Ctenopseustis obliquana larvae perceive and respond to the colour of the leaf margins, rather than to olfactory signals. Our study provides direct experimental evidence for aposematic coloration in red leaves.

β-Triketone inhibitors of plant p-hydroxyphenylpyruvate dioxygenase: modeling and comparative molecular field analysis of their interactions.

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is the target site of beta-triketone herbicides in current use. Nineteen beta-triketones and analogues, including the naturally occurring leptospermone and grandiflorone, were synthesized and tested as inhibitors of purified Arabidopsis thaliana HPPD. The most active compound was a beta-triketone with a C(9) alkyl side chain, not reported as natural, which inhibited HPPD with an I(50) of 19 +/- 1 nM. This is significantly more active than sulcotrione, which had an I(50) of 250 +/- 21 nM in this assay system. The most active naturally occurring beta-triketone was grandiflorone, which had an I(50) of 750 +/- 70 nM. This compound is of potential interest as a natural herbicide because it can be extracted with good yield and purity from some Leptospermum shrubs. Analogues without the 1,3-diketone group needed to interact with Fe(2+) at the HPPD active site were inactive (I(50)s > 50 microM), as were analogues with prenyl or ethyl groups on the triketone ring. Modeling of the binding of the triketones to HPPD, three-dimensional QSAR analysis using CoMFA (comparative molecular field analysis), and evaluation of the hydrophobic contribution with HINT (hydropathic interactions) provided a structural basis to describe the ligand/receptor interactions.

Fast Phenotyping of LFS-Silenced (Tearless) Onions by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS)

Fast MS techniques have been applied to the analysis of sulfur volatiles in Allium species and varieties to distinguish phenotypes. Headspace sampling by proton transfer reaction (PTR) MS and surface sampling by desorption electrospray ionization (DESI) MS were used to distinguish lachrymatory factor synthase (LFS)-silenced (tearless; LFS-) onions from normal, LFS-active (tear-inducing; LFS+), onions. PTR-MS showed lower concentrations of the lachrymatory factor (LF, 3) and dipropyl disulfide 12 from tearless onions. DESI-MS of the tearless onions confirmed the decreased LF 3 and revealed much higher concentrations of the sulfenic acid condensates. Using DESI-MS with MS(2) could distinguish zwiebelane ions from thiosulfinate ions. DESI-MS gave reliable fast phenotyping of LFS+ versus LFS- onions by simply scratching leaves and recording the extractable ions for <0.5 min. DESI-MS leaf compound profiles also allowed the rapid distinction of a variety of Allium cultivars to aid plant breeding selections.

Herbicidal β‐triketones are compartmentalized in leaves of Leptospermum species: localization by Raman microscopy and rapid screening

The New Zealand mānuka shrub, Leptospermum scoparium, and the Australian L. morrisonii produce herbicidal β-triketones in their leaves. The localization of these potential self-toxicants has not been proven. We investigated the localization of these compounds in leaves using Raman microscopy. The results are presented as heat maps derived from principal component analysis (PCA) of the Raman spectra from sampling grids of leaf sections. This approach used undirected, data-driven analysis to qualitatively distinguish localized plant chemistry. The presence of β-triketones and lipophilic flavonoids was confirmed by GC-MS and (1) H NMR spectroscopy. Grandiflorone was compartmentalized within the leaf oil glands of L. morrisonii. Leptospermum scoparium also contained high concentrations of grandiflorone, previously reported as only a trace component in essential oils, localized in the oil glands in the leaves of varieties from diverse geographical locations. Raman microscopy was used to probe the chemistry of oil glands in several ornamental mānuka varieties, revealing high concentrations of bioactive flavonoids localized in these glands. The compartmentalization of β-triketones within oil glands inside leaves of Leptospermum shrubs may defend the plants against herbicidal activity.

Nortriketones: Antimicrobial Trimethylated Acylphloroglucinols from Ma̅nuka (Leptospermum scoparium)

Four trimethylated acylphloroglucinols (5-8) have been isolated from ma̅nuka (Leptospermum scoparium) foliage. Apart from myrigalone A (8), which has previously been isolated from European bog myrtle (Myrica gale), these compounds have not been characterized before. The nortriketones are structurally similar to the bioactive tetramethylated β-triketones from ma̅nuka, but have one less ring methyl group. Two oxidized trimethylated compounds, 9 and 10, were also isolated, but these are likely isolation artifacts. When evaluated for antibacterial activity against Gram-positive bacteria, myrigalone A (8) was slightly less potent (MIC 64 μg/mL) than the corresponding tetramethylated compound, grandiflorone (4) (MIC 16-32 μg/mL). Unlike their tetramethylated analogues, the nortriketones were inactive against the herbicide target enzyme p-hydroxyphenylpyruvate dioxygenase. The Raman spectra of leaf oil glands in different ma̅nuka varieties can be used to distinguish plants that contain nortriketones from those that accumulate triketones.

Chemistry of New Zealand Apiaceae: New irregular diterpenes from Anisotome flexuosa and A. haastii showing conformational exchange

Two new derivatives of a recently-reported class of irregular diterpene have been discovered in New Zealand species of the Apiaceae family. 16-Acetoxyanisotomenoic acid (8) (3,3a,4,4-tetramethyl-3-{(Z)-5-acetoxy-4-methyl-3-pentenyl}-2,3,3a,4,5,6-hexahydro-1H-2-indenecarboxylic acid) and anisotomene-1,12-diol (11) (3,3a,4,4-tetramethyl-3-(2-hydroxy-4-methyl-3-pentenyl)-2,3,3a,4,5,6-hexahydro-1H-2-indenyl) methanol) have been obtained from the sub-alpine plants, Anisotome flexuosa and A. haastii respectively. 13 C nuclear magnetic esonance (n.m.r.) spectra of the anisotomene derivatives are complicated by line broadening. Molecular modelling of anisotomenoic acid (1) and anisotomenol (2) suggests that this is due to conformational exchange in the highly substituted bicyclo [4,3,0] non-1-ene ring system.

Flavor, Glucosinolates, and Isothiocyanates of Nau (Cook’s Scurvy Grass, Lepidium oleraceum) and Other Rare New Zealand Lepidium Species

The traditionally consumed New Zealand native plant nau, Cooks scurvy grass, Lepidium oleraceum, has a pungent wasabi-like taste, with potential for development as a flavor ingredient. The main glucosinolate in this Brassicaceae was identified by LC-MS and NMR spectroscopy as 3-butenyl glucosinolate (gluconapin, 7-22 mg/g DM in leaves). The leaves were treated to mimic chewing, and the headspace was analyzed by solid-phase microextraction and GC-MS. This showed that 3-butenyl isothiocyanate, with a wasabi-like flavor, was produced by the endogenous myrosinase. Different postharvest treatments were used to create leaf powders as potential flavor products, which were tasted and analyzed for gluconapin and release of 3-butenyl isothiocyanate. A high drying temperature (75 °C) did not give major glucosinolate degradation, but did largely inactivate the myrosinase, resulting in no wasabi-like flavor release. Drying at 45 °C produced more pungent flavor than freeze-drying. Seven other Lepidium species endemic to New Zealand were also analyzed to determine their flavor potential and also whether glucosinolates were taxonomic markers. Six contained mostly gluconapin, but the critically endangered Lepidium banksii had a distinct composition including isopropyl glucosinolate, not detected in the other species.

Chemosystematic analyses of Gingidia volatiles.

The leave volatiles of six Gingidia species from New Zealand and Australia and the seed volatiles of G. grisea were characterized by solid‐phase microextraction (SPME)‐GC/MS analysis. This technique, using a small quantity of samples and automated extraction, gave repeatable results, with maximum sensitivity for medium volatility compounds. The major monoterpenes among the volatiles, i.e., β‐phellandrene (4), limonene (6), and γ‐terpinene (5), and phenylpropanoids, i.e., estragole (3), (E)‐anethole (7), and myristicin (1), showed to be useful chemotaxonomic markers. For G. grisea leaves and seeds, similar compositions were detected, characterized by high contents of 4. As leaves were more readily available for study than seeds, they were used for further investigations. The G. grisea leaf volatiles showed infraspecific variation in the ratio of 4/5 between and within sites of collection. The G. montana leaf volatiles also showed infraspecific variation, with high contents of 3 at one site and high contents of 7 at another. The SPME‐GC/MS analysis of G. montana herbarium voucher specimens resulted in the identification of further chemotypes for this species. The volatiles of the G. amphistoma samples were all dominated by 7 and those of the G. haematitica samples were rich in 5. Moreover, single plants of two Australian Gingidia species were analyzed; the volatiles of G. harveyana showed high concentrations of 5 and 7, whereas those of G. rupicola were dominated by 5 and 1.

Biosynthesis of irregular diterpenes in Anisotome lyallii by head-to-head coupling of geranyl diphosphate

Anisotomenes, bicyclic irregular diterpenes found in the genus Anisotome(family Apiaceae), are shown to be products of a unique biosynthetic pathway, involving head-to-head coupling of two geranyl diphosphate units. (13)C labelling studies with [1-(13)C]-glucose on plantlets of A. lyallii also revealed that the isoprene subunits were formed via the MEP pathway. The in vitro-cultured plant material also yielded a new irregular, linear diterpene alcohol, that shares the same biosynthetic pathway.

Cytotoxic Amides from Fruits of Kawakawa, Macropiper excelsum.

Cytotoxic amides have been isolated from the fruits of the endemic New Zealand medicinal plant kawakawa, Macropiper excelsum (Piperaceae). The main amide was piperchabamide A and this is the first report of this rare compound outside the genus Piper. Eleven other amides were purified including two new compounds with the unusual 3,4-dihydro-1(2H)-pyridinyl group. The new compounds were fully characterized by 2D NMR spectroscopy, which showed a slow exchange between two rotamers about the amide bond, and they were chemically synthesized. In view of the antitumor activity of the related piperlongumine, all of these amides plus four synthetic analogs were tested for cytotoxicity. The most active was the piperine homolog piperdardine, with an IC50 of 14 µM against HT 29 colon cancer cells.