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.

Investigation of Temporal Apparent C4 Sugar Change in Manuka Honey.

New Zealand manuka honeys are known for their propensity to increase apparent C4 sugar content during storage. Depending on the particular storage regime and the initial content of dihydroxyacetone (DHA) in honey, the ready-to-market product often fails the C4 sugar test because of the above phenomenon. We have used DHA labeled with a radioactive 14C isotope in a set of honeys subject to an incubation experiment. These honeys were analyzed for DHA, methylglyoxal (MG), hydroxymethylfurfural (HMF), apparent C4 sugars, and 14C scintillation counts over a period of 18 months. The major conclusion of this experiment is that neither DHA nor MG is responsible for the δ13C shift in the honey protein extract. There must be some other yet unknown substance of manuka honey, which binds to the protein and causes negative δ13C shift. One identified candidate for such a binding is carbon dioxide.

Biowastes to augment the essential oil production of Leptospermum scoparium and Kunzea robusta in low-fertility soil.

Biowastes are unwanted materials of biological origin. They include biosolids, dairy shed effluent, and sawdust. When applied to soil, biowastes can provide plant nutrients, but also introduce heavy metals, pathogens, or xenobiotics. Biowastes could improve degraded or low-fertility soils and generate revenue through the production of non-food products such as essential oils. We grew New Zealand native plants, manuka (Leptospermum scoparium J.R. Forst & G. Forst) and kanuka (Kunzea robusta de Lange & Toelken) in series of greenhouse experiments in low-to-medium-fertility soils (Bideford clay loam, Lismore stony silt loam, and Pawson silt loam) amended with either biosolids (up to 13500 kg N ha-1 equiv.), biosolids + sawdust (1:0.5-1250 kg N ha-1 equiv.) and dairy shed effluent (200 kg N ha-1 equiv.). Two types of biosolids from Kaikoura (KB) and Christchurch City Council (CB) were used in the experiments. CB (1500 kg N ha-1 equiv.) and dairy shed effluent (200 kg N ha-1 equiv.) increased the biomass of L. scoparium by up to 120% and 31%, and K. robusta by up to 170% and 34%, respectively. Adding sawdust to KB increased the biomass of L. scoparium and K. robusta although it offset the L. scoparium growth increase in the KB-only treatment. The growth response of K. robusta to biowastes was greater than L. scoparium with oil production in K. robusta increasing by up to 211% when 1500 kg N ha-1 equiv. of CB was applied to Lismore stony silt loam. Generally, the treatments had a negligible effect on oil concentration in all the soil types, except for the KB + sawdust treatment, which increased the oil concentration by 82%. Most of the EOs' major components were unaffected by biowaste addition in the soils, although some components increased in the Bideford clay loam following KB and KB + sawdust application. Biosolids increased foliar concentrations of Zn, Cu, and Cd, but these were below risk-threshold concentrations. Applying CB (up to 1500 kg N ha-1 equiv.) to low-fertility soils is recommended to establish ecosystems dominated by L. scoparium and K. robusta that annually would produce ca. 100 kg ha-1 of EOs worth US$ 26k and 24k, respectively. Adding sawdust to CB could have environmental benefits through reduction of N leaching. Field trials are warranted to elucidate critical ecological variables and production economics in biowaste management.

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

Leptospermum flavescens Sm. (Myrtaceae), locally known as 'Senna makki' is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 µg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death.

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.

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium.

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of beta-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of beta-triketone herbicides. The beta-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified beta-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I50 values of 1.45, 0.96 and 11.5 microg mL(-1), respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O2 consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I50 values 15.0, 4.02, 3.14, 0.22 microg mL(-1) for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe2+ favors the binding of ligands with lipophilic moieties.