A phosphatase gene is linked to nectar dihydroxyacetone accumulation in manuka (Leptospermum scoparium).

Floral nectar composition beyond common sugars shows great diversity but contributing genetic factors are generally unknown. Manuka (Leptospermum scoparium) is renowned for the antimicrobial compound methylglyoxal in its derived honey, which originates from the precursor, dihydroxyacetone (DHA), accumulating in the nectar. Although this nectar trait is highly variable, genetic contribution to the trait is unclear. Therefore, we investigated key gene(s) and genomic regions underpinning this trait. We used RNAseq analysis to identify nectary-associated genes differentially expressed between high and low nectar DHA genotypes. We also used a manuka high-density linkage map and quantitative trait loci (QTL) mapping population, supported by an improved genome assembly, to reveal genetic regions associated with nectar DHA content. Expression and QTL analyses both pointed to the involvement of a phosphatase gene, LsSgpp2. The expression pattern of LsSgpp2 correlated with nectar DHA accumulation, and it co-located with a QTL on chromosome 4. The identification of three QTLs, some of the first reported for a plant nectar trait, indicates polygenic control of DHA content. We have established plant genetics as a key influence on DHA accumulation. The data suggest the hypothesis of LsSGPP2 releasing DHA from DHA-phosphate and variability in LsSgpp2 gene expression contributing to the trait variability.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

Stress, senescence, and specialized metabolites in bryophytes.

Life on land exposes plants to varied abiotic and biotic environmental stresses. These environmental drivers contributed to a large expansion of metabolic capabilities during land plant evolution and species diversification. In this review we summarize knowledge on how the specialized metabolite pathways of bryophytes may contribute to stress tolerance capabilities. Bryophytes are the non-tracheophyte land plant group (comprising the hornworts, liverworts, and mosses) and rapidly diversified following the colonization of land. Mosses and liverworts have as wide a distribution as flowering plants with regard to available environments, able to grow in polar regions through to hot desert landscapes. Yet in contrast to flowering plants, for which the biosynthetic pathways, transcriptional regulation, and compound function of stress tolerance-related metabolite pathways have been extensively characterized, it is only recently that similar data have become available for bryophytes. The bryophyte data are compared with those available for angiosperms, including examining how the differing plant forms of bryophytes and angiosperms may influence specialized metabolite diversity and function. The involvement of stress-induced specialized metabolites in senescence and nutrient response pathways is also discussed.

Chemical diversity of kanuka: Inter- and intraspecific variation of foliage terpenes and flavanones of Kunzea (Myrtaceae) in Aotearoa/New Zealand.

Kunzea (Myrtaceae) trees and shrubs, generally called kanuka, grow across most of Aotearoa/New Zealand (NZ). With the exception of K. sinclairii, an offshore island endemic, kanuka had been treated as an Australasian species K. ericoides. However, a 2014 taxonomic revision recognized ten species, all endemic to NZ. Kanuka chemistry is less studied than that of its closest relative in NZ, manuka (Leptospermum scoparium), which shows very distinct regional foliage chemotypes. We have used a miniaturized method with GC and 1H NMR to analyze foliage chemistry of voucher specimens from across the geographic ranges of the ten NZ Kunzea species. We found common mono- and sesquiterpenes, with α-pinene dominant in all samples, but only traces of antimicrobial triketones. Two unusual flavanones, with unsubstituted B-rings and known bioactivity against Phytophthora, did distinguish some of the samples. 5,7-Dihydroxy-6,8-dimethyl flavanone was only found at high concentrations in the three K. sinclairii samples in this study's sample set, but this compound has separately been reported in K. robusta samples from a nearby region. Therefore none of the NZ Kunzea species was distinguished by the chemistry analyzed in this study, but there is a possibility of regional flavonoid chemotypes cutting across the species boundaries.

Overexpression of chalcone isomerase in apple reduces phloridzin accumulation and increases susceptibility to herbivory by two-spotted mites.

Apples (Malus spp.) accumulate significant quantities of the dihydrochalcone glycoside, phloridzin, whilst pears (Pyrus spp.) do not. To explain this difference, we hypothesized that a metabolic bottleneck in the phenylpropanoid pathway might exist in apple. Expression analysis indicated that transcript levels of early phenylpropanoid pathway genes in apple and pear leaves were similar, except for chalcone isomerase (CHI), which was much lower in apple. Apples also showed very low CHI activity compared with pear. To relieve the bottleneck at CHI, transgenic apple plants overexpressing the Arabidopsis AtCHI gene were produced. Unlike other transgenic apples where phenylpropanoid flux was manipulated, AtCHI overexpression (CHIox) plants were phenotypically indistinguishable from wild-type, except for an increase in red pigmentation in expanding leaves. CHIox plants accumulated slightly increased levels of flavanols and flavan-3-ols in the leaves, but the major change was a 2.8- to 19-fold drop in phloridzin concentrations compared with wild-type. The impact of these phytochemical changes on insect preference was studied using a two-choice leaf assay with the polyphagous apple pest, the two-spotted spider mite (Tetranychus urticae Koch). Transgenic CHIox leaves were more susceptible to herbivory, an effect that could be reversed (complemented) by application of phloridzin to transgenic leaves. Taken together, these findings shed new light on phenylpropanoid biosynthesis in apple and suggest a new physiological role for phloridzin as an antifeedant in leaves.

Composition and safety evaluation of tea from New Zealand kawakawa (Piper excelsum).

Kawakawa (Piper excelsum) has food, medicinal and cultural importance to the indigenous Maori people of New Zealand, and is being incorporated into a range of commercial food and therapeutic products, including tea. In this study, the chemical compositions of kawakawa fresh leaves, dried leaves for tea, and hot brewed tea, were analysed and compared. The key metabolites were diayangambin, elemicin, myristicin, unidentified lignans and amides. The safety of brewed tea and tea leaves were evaluated in 8 week old Sprague Dawley rats in a 14 day acute study followed by a 28 day subacute study. In the 14 day study, the rats received the equivalent of 1, 2, 3 or 4 cups of kawakawa tea, and the rats in the 28 day study received daily doses that were equivalent to 4 cups per day. There were no adverse effects observed in the rats, and body weights and food intakes were not significantly different between the control and the kawakawa treated animals. There were small differences in organ weights, biochemical and haematology parameters observed in the rats given the kawakawa tea. In conclusion, the consumption of kawakawa tea could be considered safe within the conditions used in this study.

Nortriketones: Antimicrobial Trimethylated Acylphloroglucinols from MaÌ nuka (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 ß-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.

Alkaloid variation in New Zealand kowhai, Sophora species.

Alkaloid contents of leaf and seed samples of eight species of Sophora native to New Zealand, plus Sophora cassioides from Chile are reported. Fifty-six leaf and forty-two seed samples were analysed for alkaloid content by proton nuclear magnetic resonance spectroscopy, which showed major alkaloids as cytisine, N-methyl cytisine and matrine. GC analyses quantified these and identified further alkaloid components. The alkaloids identified were cytisine, sparteine, and matrine-types common to Sophora from other regions of the world. Cytisine, N-methyl cytisine, and matrine were generally the most abundant alkaloids across all species with seeds containing the highest concentrations of alkaloids. However, there was no clear taxonomic grouping based on alkaloid composition. A quantitative analysis of various parts of two Sophora microphylla trees showed that the seeds were the richest source of alkaloids (total 0.4-0.5% DM), followed by leaf and twig (0.1-0.3%) and then bark (0.04-0.06%), with only low amounts (<0.02%) found in the roots. This study represents the most comprehensive phytochemical investigation of New Zealand Sophora species to date and presents data for three species of Sophora for which no prior chemistry has been reported.

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.

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, Cook's 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.

Herbicidal ß-triketones are compartmentalized in leaves of Leptospermum species: localization by Raman microscopy and rapid screening.

The New Zealand manuka shrub, Leptospermum scoparium, and the Australian L. morrisonii produce herbicidal ß-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 ß-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 manuka varieties, revealing high concentrations of bioactive flavonoids localized in these glands. The compartmentalization of ß-triketones within oil glands inside leaves of Leptospermum shrubs may defend the plants against herbicidal activity.

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., ß-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.

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.

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.

Beta-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.

Bullatenone, 1,3-dione and sesquiterpene chemotypes of Lophomyrtus species.

The only known natural source of the volatile bioactive compounds bullatenone 1 and 4-methyl-1-phenylpentane-1,3-dione 2 is the New Zealand endemic shrub Lophomyrtus bullata (Myrtaceae). GC and NMR analyses of essential oils and solvent extracts of L. bullata, L. obcordata and the hybrid L. "ralphii" showed several chemotypes, which did not correlate with species. Levels of 1 and 2 varied from dominant to low/undetectable and the most common chemotype was rich in allo-aromadendrene and other sesquiterpenes. The rare natural product E-4-methyl-1-phenyl-1-penten-3-one 4 was detected for the first time in this genus. The non-volatile cytotoxic compound bullataketal 5 co-occurred with bullatenone 1. An essential oil from the relatively rare bullatenone 1 chemotype showed antifungal activity against Candida albicans and Cladosporium resinae, and an oil from the 4-methyl-1-phenylpentane-1,3-dione 2 chemotype showed antibacterial activity against Bacillus subtilis.

Novel chemotaxonomic markers of the Alternaria infectoria species-group.

Two new structurally related metabolites, novae-zelandin A (1) and novae-zelandin B (2), as well as the novel metabolite 4Z-infectopyrone (3) were purified from extracts of filamentous fungi belonging to the Alternaria infectoria species-group. The structures were elucidated by a combination of 1D and 2D NMR spectroscopic data and MS data. 1-3 are important chemotaxonomic markers of the A. infectoria species-group and exhibit structures similar to those of known biologically active compounds, suggesting that they could be potential phytotoxins.

Triketones active against antibiotic-resistant bacteria: synthesis, structure-activity relationships, and mode of action.

A series of acylated phloroglucinols and triketones was synthesized and tested for activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and multi-drug-resistant Mycobacterium tuberculosis (MDR-TB). A tetra-methylated triketone with a C12 side chain was the most active compound (MIC of around 1.0 microg/ml against MRSA) and was shown to stimulate oxygen consumption by resting cell suspensions, suggesting that the primary target was the cytoplasmic membrane.

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.

Determination of the absolute configuration of Anisotome irregular diterpenes: application of CD and NMR methods.

Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1.