MbEOMT1 regulates methyleugenol biosynthesis in Melaleuca bracteata F. Muell.

Methyleugenol, a bioactive compound in the phenylpropene family, undergoes its final and crucial biosynthetic transformation when eugenol O-methyltransferase (EOMT) converts eugenol into methyleugenol. While Melaleuca bracteata F. Muell essential oil is particularly rich in methyleugenol, it contains only trace amounts of its precursor, eugenol. This suggests that the EOMT enzyme in M. bracteata is highly efficient, although it has not yet been characterized. In this study, we isolated and identified an EOMT gene from M. bracteata, termed MbEOMT1, which is primarily expressed in the flowers and leaves and is inducible by methyl jasmonate (MeJA). Subcellular localization of MbEOMT1 in the cytoplasm was detected. Through transient overexpression experiments, we found that MbEOMT1 significantly elevates the concentration of methyleugenol in M. bracteata leaves. Conversely, silencing of MbEOMT1 via virus-induced gene silencing led to a marked reduction in methyleugenol levels. Our in vitro enzymatic assays further confirmed that MbEOMT1 specifically catalyzes the methylation of eugenol. Collectively, these findings establish that the MbEOMT1 gene is critical for methyleugenol biosynthesis in M. bracteata. This study enriches the understanding of phenylpropene biosynthesis and suggests that MbEOMT1 could serve as a valuable catalyst for generating bioactive compounds in the future.

Bark-dwelling methanotrophic bacteria decrease methane emissions from trees.

Tree stems are an important and unconstrained source of methane, yet it is uncertain whether internal microbial controls (i.e. methanotrophy) within tree bark may reduce methane emissions. Here we demonstrate that unique microbial communities dominated by methane-oxidising bacteria (MOB) dwell within bark of Melaleuca quinquenervia, a common, invasive and globally distributed lowland species. In laboratory incubations, methane-inoculated M. quinquenervia bark mediated methane consumption (up to 96.3 µmol m-2 bark d-1) and reveal distinct isotopic δ13C-CH4 enrichment characteristic of MOB. Molecular analysis indicates unique microbial communities reside within the bark, with MOB primarily from the genus Methylomonas comprising up to 25 % of the total microbial community. Methanotroph abundance was linearly correlated to methane uptake rates (R2 = 0.76, p = 0.006). Finally, field-based methane oxidation inhibition experiments demonstrate that bark-dwelling MOB reduce methane emissions by 36 ± 5 %. These multiple complementary lines of evidence indicate that bark-dwelling MOB represent a potentially significant methane sink, and an important frontier for further research.

Cajuputones A-C, ß-Triketone Flavanone Hybrids from the Branches and Leaves of Melaleuca cajuputi.

Three new ß-triketone flavanone hybrids, cajuputones A-C were obtained from Melaleuca cajuputi (the Australian 'tea tree'). The structures of cajuputones A-C were elucidated by 1D/2D NMR spectroscopy and HR-ESI-MS analyses; and their absolute configurations were established by electric circular dichroism (ECD) calculations using TDDFT method. Structurally, cajuputones A-C feature a rare 6/6/6/6 oxatetracyclic ring system fused between an acylphloroglucinol-derived ß-triketone and a pinocembrin or strobopinin moiety via an angle-type pyran-like motif. DFT-based conformational optimization in chloroform explained the similarity of the 1D NMR data of cajuputones B and C (C-2 epimers).

Metabolomics approach for determining potential metabolites correlated with sensory attributes of Melaleuca cajuputi essential oil, a promising flavor ingredient.

Melaleuca cajuputi subsp. cajuputi is one of the Australian Melaleuca species commonly found in Pulau Buru (Maluku, Indonesia). Its oil, the M. cajuputi essential oil (MCEO), has been utilized as the main flavor of the Indonesian functional food, Cajuputs Candy. However, the availability of MCEO is becoming limited. On the other hand, Indonesia has many other potential MCEO sources which can be developed as flavor ingredient. Thus, it is noteworthy to explore these new MCEO sources by studying their sensory characteristics and metabolite profiles. This study was conducted to identify potential metabolites that are correlated to sensory attributes of MCEO by using the metabolomics approach. The metabolite profiles of thirteen MCEOs from different origins were analyzed by gas chromatography-mass spectrometry while sensory analyses on Cajuputs Candy were conducted by difference-from-control and rate-all-that-apply tests. Sixty metabolites from the MCEO were annotated that includes 1,8-cineole, α-terpineol, caryophyllene, α-pinene, and γ-terpinene. Sensory analysis revealed cooling aftertaste and sweet taste as favorable attributes. Further analysis using Orthogonal Partial Least Square indicated that 1,8-cineole and γ-terpinene were correlated with cooling aftertaste, while 1,8-cineole and caryophyllene were also correlated with sweet taste. In contrast, linalool and nerolidol were associated with the feature of the most characteristic manufacturer's products which have unfavorable attributes such as floral, iodophor-like, metallic, and soapy attributes. The identification of these metabolites will be useful for the selection of MCEOs that can potentially be used as flavor.

Ecotype Variation of Methyl Eugenol Content in Tea Tree (Melaleuca alternifolia and Melaleuca linariifolia).

Methyl eugenol is a natural phenylpropanoid compound found in a wide range of plants used for food, flavouring, cosmetics, and health-care. As a suspected rodent carcinogen, methyl eugenol may also be harmful to humans when present in significant concentrations. Consequently, its level has been restricted in some foodstuffs and cosmetics for some markets. In order to assess the potential to breed uniformly low methyl eugenol cultivars for an essential oil crop, tea tree, the source of 'Oil of Melaleuca, terpinene-4-ol type', we examine levels in individual trees (n = 30) from two geographic regions and six terpene chemotypes. Overall, methyl eugenol levels were low in this species (Mean [SD] 354 [239] ppm, n = 30), much lower than levels predicted to be of toxicological concern. Within each chemotype, there was a lack of evidence for correlations between terpenoid constituents and methyl eugenol levels. Further support for the independence of methyl eugenol and terpene biosynthesis was evident from similar mean levels in selected (Mean [SD] 586 [339] ppm, n = 12) and undomesticated Melaleuca alternifolia trees (Mean [SD] 480 [299] ppm, n = 5) with terpinen-4-ol type oils. By contrast, methyl eugenol level varied by geographic origin and chemotype. Trees from the upland region, where there is a prevalence of terpinolene type trees, had lower average methyl eugenol levels than trees from the coastal region, where there is a prevalence of terpinen-4-ol and 1,8-cineole type trees.

An investigation into the effectiveness of sand media amended with biochar to remove BOD5, suspended solids and coliforms using wetland mesocosms.

Constructed wetland ecotechnologies (CWEs) are a promising solution to effectively treat domestic wastewater in developing countries at low cost. This paper reports the findings of the effectiveness of sand media amended with woody biochar and two plants species (Melaleuca quinquenervia and Cymbopogon citratus) in removing biological oxygen demand (BOD5), suspended solids and coliforms. The experimental design consisted of 21 vertical flow (VF) mesocosms. There were seven media treatments using sand amended with varying proportions of biochar. During the first 8 months, the mesocosms were loaded with secondary clarified wastewater (SCW) then septage. The influent had a 4-day hydraulic retention time. Samples were monitored for BOD5, total suspended solids (TSS), total volatile solids (TVS), total coliforms and faecal coliforms. In the first 8 months, there were no significant performance differences between media treatments in the outflow concentrations of BOD5, TSS and TVS. The significant differences occurred during the last 3 months; using septage with biochar additions performed better than pure sand. For coliforms, the significant differences occurred after 6 months. In conclusion, the addition of biochar was not effective for SCW. The VF mesocosms system proved to be more effective in removing BOD5, TSS, TVS and coliforms when septage was loaded into the media.

Proportion of phospholipids in the plasma membrane is an important factor in Al tolerance.

The negative charge on the plasma membrane (PM) is mainly derived from the phosphate group of phospholipids. One of the mechanisms of aluminum (Al) toxicity is to increase the PM permeability of root cells by binding to the negative sites on the PM. Thus, PM with a higher proportion of phospholipids could be more susceptible to Al toxicity. In our previous study, we showed that tolerance to Al and low-calcium in rice was enhanced by decreasing the proportion of phospholipids in root cells. Both Melastoma malabathricum L. and Melaleuca cajuputi Powell are dominant woody species that grow in tropical acid sulfate soils, and have been reported to be more tolerant to Al than rice. Surprisingly, the proportion of PM phospholipids in root cells of M. malabathricum and M. cajuputi was considerably low. Our present findings suggest that PM lipid composition plays an important role in Al tolerance mechanisms in various plant species.

Impact of pretreatments on morphology and enzymatic saccharification of shedding bark of Melaleuca leucadendron.

The effects of subcritical water (SCW) and dilute acid pretreatments on the shedding bark of Melaleuca leucadendron (paper bark tree, PBT) biomass morphology, crystallinity index (CrI) and enzymatic saccharification were studied. The morphology of PBT bark was characterized by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. SCW pretreatment mainly extracted amorphous parts of the biomass hence its CrI increased, partial decrystallization of cellulose and exposing of intact nanofibers of cellulose were observed for SCW pretreatment at 180°C. On the other hand, dilute acid pretreatment at 160°C exhibited a large decrease in CrI, an increase in surface area, a decrease in lignin content and decrystallization of cellulose as well as the peel-off and degradation of some nanofiber bundles. Dilute acid and SCW pretreatments of PBT biomass resulted in about 4.5 fold enhancement in glucose release relative to the untreated one.

The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway.

Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway.

Bioethanol production from pretreated Melaleuca leucadendron shedding bark--simultaneous saccharification and fermentation at high solid loading.

Bioethanol production from the shedding bark of Melaleuca leucadendron (Paper-bark Tree, PBT) was studied using subcritical water (SCW) pretreatment at various severities (So). High ethanol production was attained by implementing a factorial design on three parameters (So, solid loading and enzyme loading) in simultaneous saccharification and fermentation (SSF) mode. Ethanol concentration of 63.2 g L(-1) corresponding to ethanol yield of 80.9% were achieved from pretreated biomass (So=2.37) at 0.25 g mL(-1) solid and 16 FPU g(-1) glucan enzyme loadings. Similarly at 0.15 g mL(-1) solid loadings both high ethanol concentration (43.7 g L(-1)) and high ethanol yield (91.25%) were achieved. Regression analysis of experimental results shows that all process parameters had significant role on maximum ethanol production, glucose solubility, ethanol yield and ethanol volumetric productivity. SSF of SCW treated PBT biomass is economically feasible for production of bioethanol.

Carbon and nitrogen supply to the underground orchid, Rhizanthella gardneri.

*Rhizanthella gardneri is a rare and fully subterranean orchid that is presumably obligately mycoheterotrophic. R. gardneri is thought to be linked via a common mycorrhizal fungus to co-occurring autotrophic shrubs, but there is no experimental evidence to support this supposition. *We used compartmentalized microcosms to investigate the R. gardneri tripartite relationship. (13)CO(2) was applied to foliage of Melaleuca scalena plants and [(13)C-(15)N]glycine was fed to the common mycorrhizal fungus, and both sources traced to R. gardneri plants. *In our microcosm trial, up to 5% of carbon (C) fed as (13)CO(2) to the autotrophic shrub was transferred to R. gardneri. R. gardneri also readily acquired soil C and nitrogen (N), where up to 6.2% of C and 22.5% of N fed as labelled glycine to soil was transferred via the fungus to R. gardneri after 240 h. *Our study confirms that R. gardneri is mycoheterotrophic and acquires nutrients via mycorrhizal fungus connections from an ectomycorrhizal autotrophic shrub and directly from the soil via the same fungus. This connection with a specific fungus is key to explaining why R. gardneri occurs exclusively under certain Melaleuca species at a very limited number of sites in Western Australia.

Nutrient and sediment removal by stormwater biofilters: a large-scale design optimisation study.

A large-scale column study was conducted in Melbourne, Australia, to test the performance of stormwater biofilters for the removal of sediment, nitrogen and phosphorus. The aim of the study was to provide guidance on the optimal design for reliable treatment performance. A variety of factors were tested, using 125 large columns: plant species, filter media, filter depth, filter area and pollutant inflow concentration. The results demonstrate that vegetation selection is critical to performance for nitrogen removal (e.g. Carex appressa and Melaleuca ericifolia performed significantly better than other tested species). Whilst phosphorus removal was consistently very high (typically around 85%), biofilter soil media with added organic matter reduced the phosphorus treatment effectiveness. Biofilters built according to observed 'optimal specifications' can reliably remove both nutrients (up to 70% for nitrogen and 85% for phosphorus) and suspended solids (consistently over 95%). The optimally designed biofilter is at least 2% of its catchment area and possesses a sandy loam filter media, planted with C. appressa or M. ericifolia. Further trials will be required to test a wider range of vegetation, and to examine performance over the longer term. Future work will also examine biofilter effectiveness for treatment of heavy metals and pathogens.

Flavonol glucuronides and C-glucosidic ellagitannins from Melaleuca squarrosa.

Two flavonoids and three ellagitannins, squarrosanins A, B, and C, were isolated from the leaves of Melaleuca squarrosa. The flavonoids were characterized structurally as kaempferol-3-O-(2''-O-galloyl)-glucuronide and herbacetin-3-O-glucuronide, while the ellagitannins were characterized as monomeric and dimeric C-glucosidic ellagitannins by application of spectroscopic and chemical methods. The antioxidant effect of the polyphenolic constituents of the M. squarrosa leaves was also examined in vitro, and C-glucosidic tannins including oligomers were shown to be more effective radical scavengers against 1,1-diphenyl-2-picrylhydrazyl (DPPH) than flavonoids and ordinary ellagitannins.

C-Glucosidic ellagitannin oligomers from Melaleuca squarrosa Donn ex Sm., Myrtaceae.

C-glucosidic ellagitannin dimers were classified as types A-C according to a putative biogenetic oligomerization mode. They were characterized by different positions of the C-C bond between the phenolic acyl unit in one monomer and the benzylic C-1 of the open-chain glucose core in the other monomer. In recent years, four C-glucosidic tannins, melasquanins A-D (18-21), have been found in the leaves of Melaleuca squarrosa Donn ex Sm. (Myrtaceae). These are characterized as a dimer (melasquanin A) of a dimerization mode (type D), and trimers (melasquanins B-D) based on spectroscopic analysis including various two-dimensional nuclear magnetic resonance (2D NMR) experiments. Melasquanins B (19) and D (21) are C-glucosidic tannin trimers with a structure containing, non-repeating condensation modes, which was hitherto unknown.

Photosynthesis and photoassimilate transport during root hypoxia in Melaleuca cajuputi, a flood-tolerant species, and in Eucalyptus camaldulensis, a moderately flood-tolerant species.

We compared the photosynthetic and photoassimilate transport responses of Melaleuca cajuputi Powell seedlings to root hypoxia with those of Eucalyptus camaldulensis Dehnh. Control and hypoxia treated roots were maintained in a nutrient solution through which air or nitrogen was bubbled. Under root hypoxic conditions, seedlings of M. cajuputi, a flood-tolerant species, maintained height growth, whereas seedlings of E. camaldulensis, a moderately flood-tolerant species, showed markedly decreased height growth compared with control seedlings. Root hypoxia caused decreases in whole-plant biomass, photosynthetic rate and stomatal conductance in E. camaldulensis, but not in M. cajuputi. Photoassimilate transport to roots decreased significantly in E. camaldulensis seedlings 4 days after treatment and starch accumulated in mature leaves. Photoassimilate supply to hypoxic roots of E. camaldulensis seedlings was, thus, limited by reduced photoassimilate transport rather than by reduced photosynthesis. In contrast, M. cajuputi seedlings showed sustained photoassimilate transport to hypoxic roots and persistent photosynthesis, which together provided a substantial photoassimilate supply to the roots. Sucrose accumulated in hypoxic E. camaldulensis roots, but not in hypoxic M. cajuputi roots. A stable, low sucrose concentration in hypoxic roots would let M. cajuputi seedlings prolong photoassimilate transport to the roots. Photoassimilate partitioning among the water-soluble carbohydrates, starch and structural carbohydrates within the roots was unaffected by root hypoxia in E. camaldulensis, but in M. cajuputi, partitioning was shifted somewhat from structural carbohydrates to water-soluble carbohydrates. This suggests that M. cajuputi seedlings are able to increase photoassimilate utilization in metabolism and sustain energy production under root hypoxic conditions.

Variable tolerance of wetland tree species to combined salinity and waterlogging is related to regulation of ion uptake and production of organic solutes.

Melaleuca cuticularis and Casuarina obesa occur in wetlands, whereas Banksia attenuata occurs in adjacent well-drained sandy soils. Salt and waterlogging tolerances in these tree species were studied, as the levels of these stresses have increased in south-western Australia. Seedlings were exposed to 0.01, 200 or 400 mm NaCl, with or without waterlogging, in a sand culture with nutrient solution for 22 d in a glasshouse. Melaleuca cuticularis and C. obesa survived all treatments, and generally maintained high rates of net photosynthesis. Banksia attenuata tolerated neither waterlogging nor salinity. Salt tolerance of M. cuticularis and C. obesa was associated with the regulation of foliar sodium (Na+), chloride (Cl-) and potassium (K+) concentrations. Under saline-waterlogged conditions, this regulation was maintained in M. cuticularis, but was reduced in C. obesa. Foliage of these two species also contained appreciable levels of compatible organic solutes: methyl proline in M. cuticularis and proline in C. obesa; in both cases the concentrations increased at higher salinity. Melaleuca cuticularis formed a higher proportion of aerenchyma in adventitious roots than C. obesa, so enhanced internal root aeration in M. cuticularis might contribute to its higher tolerance of combined salinity and waterlogging.

Comparative water use by the riparian trees Melaleuca argentea and Corymbia bella in the wet-dry tropics of northern Australia.

We examined sources of water and daily and seasonal water use patterns in two riparian tree species occupying contrasting niches within riparian zones throughout the wet-dry tropics of northern Australia: Corymbia bella Hill and Johnson is found along the top of the levee banks and Melaleuca argentea W. Fitzg. is restricted to riversides. Patterns of tree water use (sap flow) and leaf water potential were examined in four trees of each species at three locations along the Daly River in the Northern Territory. Predawn leaf water potential was higher than -0.5 MPa throughout the dry season in both species, but was lower at the end of the dry season than at the beginning of the dry season. Contrary to expectations, predawn leaf water potential was lower in M. argentea trees along the river than in C. bella trees along the levees. In contrast, midday leaf water potential was lower in the C. bella trees than in M. argentea trees. There were no seasonal differences in tree water use in either species. Daily water use was lower in M. argentea trees than in C. bella trees. Whole-tree hydraulic conductance, estimated from the slope of the relationship between leaf water potential and sap flow, did not differ between species. Xylem deuterium concentrations indicated that M. argentea trees along the riverbank were principally reliant on river water or shallow groundwater, whereas C. bella trees along the levee were reliant solely on soil water reserves. This study demonstrated strong gradients of tree water use within tropical riparian communities, with implications for estimating riparian water use requirements and for the management of groundwater resources.

Monoterpenoid accumulation in 1,8-cineole, terpinolene and terpinen-4-ol chemotypes of Melaleuca alternifolia seedlings.

Individual leaves of the three most common chemotypes of Melaleuca alternifolia were examined both quantitatively and qualitatively for volatile constituents from the emergence of the first true leaves, through to 6-week-old tenth leaf set material. The 1,8-cineole and terpinolene chemotypes were investigated and compared with the recently reported commercial terpinen-4-ol chemotype. The 1,8-cineole chemotype was found to accumulate 1,8-cineole and associated p-menthanes limonene, terpinen-4-ol and alpha-terpineol gradually with increasing leaf set number. As with the terpinen-4-ol variety, higher than expected concentrations of the pinenes and terpinolene were found only in the early leaf sets. The terpinolene variety showed two stages of terpinolene accumulation, the first at leaf sets 2-3 similar to the unexpected biosynthesis of terpinolene in the terpinen-4-ol chemotype and the second at leaf sets 8-9 which is characteristic of the terpinolene variety.