John William van Klink

Mānuka ( Leptospermum scoparium Myrtaceae), a shrub that grows throughout Aotearoa New Zealand, is highly valued for the medicinal properties of its bioactive natural products. This first comprehensive review of mānuka chemistry examines traditional uses and cultural significance to Māori; botany and genetics showing its distinctness from Australian Leptospermum species and natural products chemistry and bioactivity. Mānuka nectar is a rich source of dihydroxyacetone (DHA), a precursor to the potent antibacterial methylglyoxal that is generated in mānuka honey. DHA has only been found in the nectar of mānuka and of some Australian Myrtaceae. The worldwide popularity of mānuka honey has led to high prices and honey fraud, so analytical markers for authentication and adulteration are discussed. Mānuka leaf oils have different bioactives from nectar and honey, with some rich in rare triketones active against antibiotic‐resistant Gram‐positive bacteria. These volatile triketones, which are also herbicidal, are high only in one regional chemotype. Unique nortriketones and unusual flavonoids with relaxant activity are found in other mānuka, all encapsulated within leaf oil glands. Advances in the knowledge of the genetics and biosynthesis of the valuable mānuka molecules in this ‘chemical crop' are summarised, and future research directions are proposed.

Summary: Pathogenesis‐related (PR) proteins are diverse stress‐ or pathogen‐induced proteins. Some are associated with specialised metabolism, including proposed functions for anthocyanin biosynthesis. However, data are limited to a few angiosperm species, and the mode(s) of action are uncertain. Using the liverwort Marchantia polymorpha (Marchantia), we examined whether pathogenesis‐related 10 (PR10) contributes to flavonoid biosynthesis in other land plant lineages and investigated its mode of action. Marchantia produces two major flavonoid types: flavones and the pigment auronidin. MpPR10.5 is a target of the auronidin regulator MpMYB14; therefore, Mppr10.5 mutants were generated using CRISPR/Cas9 and analysed for transcript abundance (via RNA sequencing) and for metabolite content. Recombinant MpPR10.5 protein was used for metabolite binding and stabilisation assays. Mppr10.5 mutants had reduced auronidin and flavone content, demonstrating that MpPR10.5 promotes flavonoid biosynthesis. Flavone and auronidin biosynthesis share a single flavonoid intermediate, naringenin chalcone (NC), suggesting MpPR10.5 acts on this compound. MpPR10.5 protein binds NC strongly (micromolar affinity), preventing spontaneous self‐cyclisation in vitro. Several phenylpropanoid and flavonoid genes were downregulated in Mppr10.5 and Mpchalcone isomerase‐like plants. This suggests PR10 proteins promote flavonoid biosynthesis by selectively binding unstable intermediates (NC), protecting them from degradation or undesirable nonenzymatic conversions and facilitating their transport to subsequent pathway steps.

Summary: Floral nectar composition beyond common sugars shows great diversity but contributing genetic factors are generally unknown. Mānuka ( 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 mānuka 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.

Plant & Food Research (PFR) is a biologically orientated multi-disciplinary research institute with the goal of improving the value of New Zealand’s horticultural products. Within PFR, the practice of chemistry is both widespread and the focus of specialist chemists. To improve safety in the handling of laboratory chemicals, we sought to understand the state of chemical practice and knowledge, the awareness of risk, and how chemical knowledge was acquired and authenticated. A collaborative inquiry approach was chosen so that the social interactions and practice of the research process would itself serve to exchange knowledge and improve communication and laboratory safety. A “survey─small group meeting” model for interaction with users of chemicals was developed and rolled out across PFR. Survey responses were received from 346 respondents who reported their usage of laboratory chemicals, and this was followed by interactive small group meetings with 38 biology-focused research teams located across 12 sites. While the survey provided a generally positive view of practice and perceptions of risk, the team visits provided more site-specific, granular, and informative insights into the handling of chemicals and functioning of laboratories. Textual analysis of contemporaneous notes identified six interconnecting themes associated with risk in the handling of chemicals: lack of knowledge resulting in uncertainty and unrecognized risks; practical difficulties in effecting structural changes to laboratories; poor engagement by a few key individuals; feelings of lack of support by safety managers; the presence of students, visitors, and new graduates needing extensive training; and work pressures resulting in poor decision-making. This research demonstrates the use of collaborative inquiry to provide the granular knowledge needed to improve safety in handling chemicals in biological laboratories.

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.

Taramea is the prized resinous exudate obtained from native Aciphylla plants (speargrass) identified as a taonga by Ngāi Tahu Māori in their Treaty of Waitangi tribunal claim Settlement. Ngāi Tahu recognised two types of Aciphylla , the larger was known as taramea and used as a fragrance, while the tap root of the smaller type, called papaī, was eaten but not used as kakara (fragrance). The gum of the taramea is called ‘ware’ or ‘wai‐whenua’, and was often spoken as ‘ware‐o‐te‐taramea’. Plants were traditionally tapped in the evening by cutting or using fire. In the morning the exuded resin was gathered and processed. To preserve the aroma it was saturated in hinu‐weka (woodhen fat) or the fat of other native bird or animal species (tui, kiore) and worn in a pouch (hei‐taramea) close to the body. Taramea was used to dress the hair and rub on the body and became a sought after trade item with northern tribes. Scientific studies on taramea plants include those relating to plant morphology, taxonomy, genetics, ecology and phytochemistry. A resurgence of interest in taramea is supporting further scientific studies to define the chemical composition of this taonga plant.

Summary: 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.

Leptospermum scoparium J. R. et G. Forst. (Myrtaceae), called mānuka or kahikātoa by Māori, shows distinct foliage chemotypes in different regions of Aotearoa New Zealand, including one with rare antimicrobial triketones. To extend knowledge of mānuka chemotypes geographically and historically, we have developed a miniaturised method for analyses of herbarium samples needing just a few small leaves analysed by headspace solid phase microextraction gas chromatography (HS‐SPME‐GC). This method was validated on herbarium samples of known chemotypes. Geographical knowledge was extended by analysing herbarium samples collected on New Zealand's offshore islands, ranging from the Manawa/Three Kings in the North to Rakiura/Stewart Island in the South. The chemotypes were generally similar to the nearest populations on the main North or South islands. Historically, volatiles were identified from herbarium samples collected by the first botanists from Europe in 1769, showing that the oil glands in mānuka leaves act as ‘chemical time capsules’.

Chemotaxonomic analysis of kōwhai leaf extracts in New Zealand are limited with previous reports of Sophora tetraptera having a flavonoid profile distinct from S. microphylla sensu lato and S. prostrata . Eight Sophora species are now recognised in New Zealand: S. chathamica , S. fulvida , S. godleyi , S. longicarinata , S. microphylla , S. molloyi , S. prostrata and S. tetraptera . We now report liquid chromatography‐ultraviolet‐mass spectrometry (LC‐UV‐MS) analyses of leaf and seed extracts of individual plants (2–16) from each of these eight species, plus the Chilean S. cassioides . All of the S. tetraptera leaf extracts had similar LC‐UV‐MS profiles, different from all of the other Sophora samples, consisting of four predominant compounds, characterised by MS and nuclear magnetic resonance (NMR) spectroscopy as: luteolin‐7‐O‐rutinoside 1 , luteolin‐7‐O‐glucoside 2 , apigenin‐7‐O‐rutinoside 3 , and apigenin‐7‐O‐glucoside 4 . The other Sophora leaf extracts showed complex flavonoid compositions, with no clear distinction between species. Most of the Sophora seed samples, including those of S. tetraptera , showed one major phenolic compound, but a few had a related compound. These were purified and characterised by MS and NMR spectroscopy as 3′,4′,7‐trihydroxyisoflavone 5 (in most seeds) and its 7‐O‐glucoside 6 (in a few seeds), neither of which has been previously reported from these Sophora species.