Previously undescribed eremane, viscidane, and isozizaene diterpenoids, eremorigidanes A-F, along with six known O-methylated flavonoids and three known triterpenoids were isolated and identified from the leaves of Eremophila rigida Chinnock by combined use of high-resolution PTP1B inhibition profiling, semipreparative- and analytical-scale HPLC separations, HPLC-PDA-HRMS analysis, and NMR spectroscopy. The absolute configuration of the unreported diterpenoids were determined by comparison of their experimental and calculated ECD spectra as well as by biosynthetic arguments. All isolates were evaluated for their PTP1B inhibitory activities, which revealed the flavonoid penduletin (3) to show inhibition with an IC50 value of 18.3 μM, and the triterpenoids 3,4-seco-olean-12-ene-3,28-dioic acid (15), oleanolic acid (16), and 3-oxo-oleanolic acid (17) to show inhibition with IC50 values of 55.7, 9.9, and 6.3 μM, respectively. The preliminary structure-activity relationship (SAR) of isolated flavonoids and triterpenoids is discussed. Plausible biosynthetic steps involved in eremane and isozizaene metabolism are presented and discussed.

Extracts of Eremophila phyllopoda subsp. phyllopoda showed α-glucosidase and PTP1B inhibitory activity with IC50 values of 19.6 and 13.6 μg/mL, respectively. High-resolution α-glucosidase/PTP1B/radical scavenging profiling was performed to establish a triple high-resolution inhibition profile that allowed direct pinpointing of the constituents responsible for one or more of the observed bioactivities. Subsequent targeted isolation and purification by analytical-scale HPLC led to the identification of 21 previously undescribed serrulatane diterpenoids, eremophyllanes A-U, as well as two known serrulatane diterpenoids, 1β-trihydroxyserrulatane (8) and 1α-trihydroxyserrulatane (10d), and five known furofuran lignans, (+)-piperitol (6), horsfieldin (7e), (-)-sesamin (9), (+)-sesamin (10h), and asarinin (10i). Their structures were elucidated by extensive analysis of HRMS and 1D and 2D NMR spectroscopic data. The relative configurations of the previously undescribed compounds were established by analysis of ROESY spectra as well as by DFT-GIAO NMR calculations followed by DP4+ probability analysis. The absolute configurations were determined by comparison of experimental and calculated ECD spectra. Serrulatane diterpenoids 7b and 14 exhibited α-glucosidase inhibitory activity with IC50 values of 28.4 and 64.2 μM, respectively, while 11, 12, 14, and 15 exhibited PTP1B inhibitory activity with IC50 values ranging from 16.6 to 104.6 μM. Hypothetical routes for formation of all identified serrulatane diterpenoids are proposed.

In this study, an extract of the leaves of Eremophila clarkei Oldfield & F.Muell. showed protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 33.0 mu g/mL. The extract was therefore investigated by high-resolution PTP1B inhibition profiling to pinpoint the constituents responsible for the activity. Subsequent isolation and purification using analytical-scale HPLC led to identification of eight previously undescribed decipiene diterpenoids, eremoclarkanes A-H, as well as eremoclarkic acid, a biogenetically related new phenolic acid. In addition, one known decipiene diterpenoid and ten known O-methylated flavonoids were isolated. The structures of the isolated compounds were elucidated by extensive analysis of their HRMS and 1D and 2D NMR spectra. The absolute configuration of decipiene diterpenoids was determined by comparison of experimental and calculated ECD spectra. The flavonoid hispidulin (2b) and the four decipiene diterpenoids 13a, 13b, 13f, and 14b exhibited PTP1B inhibitory activity with IC50 values ranging from 22.8 to 33.6 mu M. This is the first report of PTP1B inhibitory activity of decipienes, and enzyme kinetics revealed that 13a and 13b are competitive inhibitors of PTP1B, whereas 13f and 14b displayed mixed-type-mode inhibition of PTP1B. Finally, molecular docking indicated that 13a, 13b, 13f, and 14b showed comparable binding affinity towards the active and/or allosteric site of PTP1B enzyme. Structure-activity relationship (SAR) of the identified O-methylated flavonoids and decipiene diterpenoids towards PTP1B is discussed. Plausible enzymatic and photochemically driven routes for the formation of the decipienes and conversion products thereof are presented and discussed.

Crude extract of Eremophila spathulata leaves was investigated by semi-preparative scale high-performance liquid chromatography (HPLC), analytical scale HPLC, and hyphenated high-performance liquid chromatography-photodiode array-high-resolution mass spectrometry-nuclear magnetic resonance (HPLC-PDA-HRMS-SPE-NMR), which afforded seven previously unreported caryophyllane sesquiterpenoids. Semi-preparative scale separation of the crude extract afforded (1R*,4R*,9S*,E)-8-formyl-11,11-dimethylbicyclo[7.2.0]undec-7-ene-4-carboxylic acid (5) and analytical-scale HPLC separation afforded (1R*,4S*,7S*,9S*)-7-hydroxy-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid (1), (1S*,6R*,9R*,E)-10,10-dimethylbicyclo[7.2.0]undec-2-ene-2,6-dicarboxylic acid (2), (1R*,4S*,9S*)-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid (3), and (1R*,4R*,9S*)-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid (4). HPLC-PDA-HRMS-SPE-NMR afforded (1R*,4R*,9S*)-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid (6) and (1R*,4S*,9S*)-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid (7). The structures of all isolated compounds were established based on HRMS as well as extensive 1D and 2D NMR analysis. Relative configurations were determined by correlations in spectra from rotational Overhauser effect spectroscopy.

Eremophila (Scrophulariaceae) is a genus of Australian desert plants, which have been used by Australian Aboriginal people for various medicinal purposes. Crude extracts of the leaf resin of Eremophila glabra (R.Br.) Ostenf. showed α-glucosidase and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC50 values of 19.3 ± 1.2 μg/mL and 11.8 ± 2.1 μg/mL, respectively. Dual α-glucosidase/PTP1B high-resolution inhibition profiling combined with HPLC-PDA-HRMS and NMR were used to isolate and identify the compounds providing these activities. This resulted in isolation of seven undescribed serrulatane diterpenoids, eremoglabrane A-G, together with nine previously identified serrulatane diterpenoids and flavonoids. Three of the serrulatane diterpenoids showed PTP1B inhibitory activities with IC50 values from 63.8 ± 5.8 μM to 104.5 ± 25.9 μM.

SUMMARY In a cross‐continental research initiative, including researchers working in Australia and Denmark, and based on joint external funding by a 3‐year grant from the Novo Nordisk Foundation, we have used DNA sequencing, extensive chemical profiling and molecular networking analyses across the entire Eremophila genus to provide new knowledge on the presence of natural products and their bioactivities using polypharmocological screens. Sesquiterpenoids, diterpenoids and dimers of branched‐chain fatty acids with previously unknown chemical structures were identified. The collection of plant material from the Eremophila genus was carried out according to a ‘bioprospecting agreement’ with the Government of Western Australia. We recognize that several Eremophila species hold immense cultural significance to Australia's First Peoples. In spite of our best intentions to ensure that new knowledge gained about the genus Eremophila and any potential future benefits are shared in an equitable manner, in accordance with the Nagoya Protocol, we encounter serious dilemmas and potential conflicts in making benefit sharing with Australia's First Peoples a reality.

The plant genus Eremophila is endemic to Australia and widespread in arid regions. Root bark extract of Eremophila longifolia (R.Br.) F.Muell. (Scrophulariaceae) was investigated by LC-PDA-HRMS, and dereplication suggested the presence of a series of diterpenoids. Using a combination of preparative- and analytical-scale HPLC separation as well as extensive 1D and 2D NMR analysis, the structures of 12 hitherto unreported serrulatane diterpenoids, eremolongine A-L, were established. These structures included serrulatanes with unusual side chain modifications to form hitherto unseen skeletons with, e.g., cyclopentane, oxepane, and bicyclic hexahydro-1H-cyclopenta[c]furan moieties. Serrulatane diterpenoids in Eremophila have recently been shown to originate from a common biosynthetic precursor with conserved stereochemical configuration, and this was used for tentative assignment of the relative and absolute configuration of the isolated compounds. Triple high-resolution α-glucosidase/α-amylase/PTP1B inhibition profiling demonstrated that several of the eremolongines had weak inhibitory activity towards targets important for management of type 2 diabetes.

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone (2), which at a concentration of 25 μg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 μM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

Eremophila is the largest genus in the plant tribe Myoporeae (Scrophulariaceae) and exhibits incredible morphological diversity across the Australian continent. The Australian Aboriginal Peoples recognize many Eremophila species as important sources of traditional medicine, the most frequently used plant parts being the leaves. Recent phylogenetic studies have revealed complex evolutionary relationships between Eremophila and related genera in the tribe. Unique and structurally diverse metabolites, particularly diterpenoids, are also a feature of plants in this group. To assess the full dimension of the chemical space of the tribe Myoporeae, we investigated the metabolite diversity in a chemo-evolutionary framework applying a combination of molecular phylogenetic and state-of-the-art computational metabolomics tools to build a dataset involving leaf samples from a total of 291 specimens of Eremophila and allied genera. The chemo-evolutionary relationships are expounded into a systematic context by integration of information about leaf morphology (resin and hairiness), environmental factors (pollination and geographical distribution), and medicinal properties (traditional medicinal uses and antibacterial studies), augmenting our understanding of complex interactions in biological systems.

Eremophila is a significant component of the Australian arid zone flora, but its generic limits and relationships to the other six genera of tribe Myoporeae remain largely untested. In this study, we assembled a dataset of the nuclear ribosomal cistron (ca. 6000 bases including ITS1+2, ETS regions, non-transcribed spacer and associated genes) for all genera in tribe Myoporeae with a particular focus on Eremophila (205 of total 233 formally described species of Eremophila, and 28 species of related genera sampled). Phylogenetic analyses were performed using maximum likelihood and Bayesian inference methods. These resolved four major clades that were well supported. Eremophila was paraphyletic, with all other genera of tribe Myoporeae nested in it. We discuss two taxonomic options for addressing the paraphyly of Eremophila. As an immediate step, we propose that the small genera, Calamphoreus and Diocirea, should be placed in synonymy under Eremophila, and herein make the necessary new nomenclatural combinations. Additional phylogenetic data are needed, ideally in the form of multiple independent nuclear loci, to clarify the positions of Bontia, Glycocystis and Myoporum relative to Eremophila before further taxonomic changes are proposed.

Ten new branched-chain fatty acid (BCFA) dimers with a substituted cyclohexene structure, five new monomers, and two known monomers, (2E,4Z,6E)-5-(acetoxymethyl)tetradeca-2,4,6-trienoic acid and its 5-hydroxymethyl analogue, were identified in the leaf extract of Eremophila oppositifolia subsp. angustifolia using a combination of HPLC-PDA-HRMS-SPE-NMR analysis and semipreparative-scale HPLC. The dimers could be classified as three types of Diels-Alder reaction products formed between monomers at two different sites of unsaturation of the dienophile. Two of the monomers represent potential biosynthetic intermediates of branched-chain fatty acids. Several compounds were found by high-resolution bioactivity profiling to inhibit PTP1B and were purified subsequently by semipreparative-scale HPLC. The dimers were generally more potent than the monomers with IC50 values ranging from 2 to 66 μM, compared to 38-484 μM for the monomers. The ten fatty acid dimers represent both a novel class of compounds and a novel class of PTP1B inhibitors.

This study presents an analysis of plastomes to assess relationships among the seven genera of tribe Myoporeae (Scrophulariaceae). Sampling included individuals from type species for all seven genera included in Myoporeae (Bontia, Calamphoreus, Diocirea, Eremophila, Glycocystis, Myoporum, Pentacoelium), one additional species of the large genus Eremophila, one representative of the sister tribe Leucophylleae (Leucophyllum), and the previously published plastome of Scrophularia takesimensis as an outgroup. Phylogenetic analyses were performed using maximum parsimony and Bayesian inference analyses based on > 7000 variable nucleotide positions. Maximum parsimony analysis produced a single tree with the same topology as the Bayesian consensus tree, and all nodes were fully resolved with 100% bootstrap support and 1.0 Bayesian posterior probability. Tribe Myoporeae was supported as monophyletic and sister to Leucophylleae. Two lineages are identified within Myoporeae; one containing the Australian endemic genera Calamphoreus, Diocirea, Glycocystis and the type species of Eremophila (E. oppositifolia), the second containing species with distributions outside of Australia (Bontia, Myoporum, Pentacoelium) and a species of Eremophila (E. gibbifolia). Eremophila is thus supported as polyphyletic. The application of genome skimming and assembly of plastomes has resolved generic relationships in tribe Myoporeae and revealed variable regions of the genome that will be useful in further genetic study of the group.

yinnetharrensis Buirchell & A.P.Br

Bean yellow mosaic virus (BYMV), genus Potyvirus, has an extensive natural host range encompassing both dicots and monocots. Its phylogenetic groups were considered to consist of an ancestral generalist group and six specialist groups derived from this generalist group during plant domestication. Recombination was suggested to be playing a role in BYMV's evolution towards host specialization. However, in subsequent phylogenetic analysis of whole genomes, group names based on the original hosts of isolates within each of them were no longer supported. Also, nine groups were found and designated I-IX. Recombination analysis was conducted on the complete coding regions of 33 BYMV genomes and two genomes of the related Clover yellow vein virus (CYVV). This analysis found evidence for 12 firm recombination events within BYMV phylogenetic groups I-VI, but none within groups VII-IX or CYVV. The greatest numbers of recombination events within a sequence (two or three each) occurred in four groups, three which formerly constituted the single ancestral generalist group (I, II and IV), and group VI. The individual sequences in groups III and V had one event each. These findings with whole genomes are consistent with recombination being associated with expanding host ranges, and call into question the proposed role of recombination in the evolution of BYMV, where it was previously suggested to play a role in host specialization. Instead, they (i) indicate that recombination explains the very broad natural host ranges of the three BYMV groups which infect both monocots and dicots (I, II, IV), and (ii) suggest that the three groups with narrow natural host ranges (III, V, VI) which also showed recombination now have the potential to reduce host specificity and broaden their natural host ranges.

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

Hardenbergia mosaic virus (HarMV), genus Potyvirus, belongs to the bean common mosaic virus (BCMV) potyvirus lineage found only in Australia. The original host of HarMV, Hardenbergia corn ptoniana, family Fabaceae, is indigenous to the South-West Australian Floristic Region (SWAFR), where Lupinus spp. are grown as introduced grain legume crops, and exist as naturalised weeds. Two plants of H. comptoniana and one of Lupinus cosentinii, each with mosaic and leaf deformation symptoms, were sampled from a small patch of disturbed vegetation at an ancient ecosystem recent agroecosystem interface. Potyvirus infection was detected in all three samples by ELISA and RT-PCR. After sequencing on an Illumina HiSeq 2000, three complete and two nearly complete HarMV genomes from H. comptoniana and one complete HarMV genome from L. cosentinii were obtained. Phylogenetic analysis which compared (i) the four new complete genomes with the three HarMV genomes on Genbank (two of which were identical), and (ii) coat protein (CP) genes from the six new genomes with the 38 HarMV CP sequences already on Genbank, revealed that three of the complete and one of the nearly complete new genomes were in HarMV clade I, one of the complete genomes in clade V and one nearly complete genome in clade VI. The complete HarMV genome from L cosendnii differed by only eight nucleotides from one of the HarMV clade I genomes from a nearby H. comptoniana plant, with only one of these nucleotide changes being non-synonymous. Pairwise comparison between all the complete HarMV genomes revealed nucleotide identities ranging between 82.2% and 100%. Recombination analysis revealed evidence of two recombination events amongst the six complete genomes. This study provides the first report of HarMV naturally infecting L cosentinii and the first example for the SWAFR of virus emergence from a native plant species to invade an introduced plant species. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Black pod syndrome (BPS) causes devastating losses in Lupinus angustifolius (narrow-leafed lupin) crops in Australia, and infection with Bean yellow mosaic virus (BYMV) was suggested as a possible cause. In 2011, an end-of-growing-season survey in which L. angustifolius plants with BPS were collected from six locations in southwestern Australia was done. Tissue samples from different positions on each of these symptomatic plants were tested for BYMV and generic potyvirus by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR). Detection was most reliable when RT-PCR with generic potyvirus primers was used on tissue taken from the main stem of the plant just below the black pods. Partial coat protein nucleotide sequences from eight isolates from BPS-symptomatic L. angustifolius plants all belonged to the BYMV general phylogenetic group. An initial glasshouse experiment revealed that mechanical inoculation of L. angustifolius plants with BYMV after pods had formed caused pods to turn black. This did not occur when the plants were inoculated before this growth stage (at first flowering) because BYMV infection caused plant death. A subsequent experiment in which plants were inoculated at eight different growth stages confirmed that BPS was only induced when L. angustifolius plants were inoculated after first flowering, when pods had formed. Thus, BYMV was isolated from symptomatic L. angustifolius survey samples, inoculated to and maintained in culture hosts, inoculated to healthy L. angustifolius test plants inducing BPS, and then successfully reisolated from them. As such, Koch's postulates were fulfilled for the hypothesis that late infection with BYMV causes BPS in L. angustifolius plants.

Root system architecture influences nutrient and water uptake efficiency and thus plant growth and productivity. Root architecture traits conferring efficiency in capturing resources from soil are considered a key factor in crop breeding for enhanced water and nutrient uptake. Mechanical impedance such as soil compaction is common in the Western Australian wheatbelt, limiting root growth and crop productivity. The present study investigated root responses to subsoil compaction in two field trials at Wongan Hills (central wheatbelt) and Buntine (northern wheatbelt) in Western Australia. Substantial alteration to rooting patterns was observed in the commercial wheat cultivar Wyalkatchem and in narrow-leafed lupin (Lupinus angustifolius) grown in sandy soils where compaction is common. The root systems of narrow-leafed lupin plants were dominated by a short and thickened taproot (10-26 cm depth, 7-14 mm root-collar diameter) and horizontally distributed primary lateral roots when compared to previous observations of the same genotypes under non-compacted soil conditions. Genotypic variability in root architecture traits among four wild genotypes and four commercial cultivars (Mandelup, Merrit, Quilinock and Tanjil) of narrow-leafed lupin was demonstrated. Taproot length, total root length, root surface area, root mass and root collar diameter were the most important root traits correlated to shoot yield (P< 0.001). Deep ripping resulted in significantly improved rooting depth (up to 100 mm) and root distribution in wheat in the soil profile compared to non-ripped soil, where roots were restrained mainly in the top 0-30 cm layer. Root number, root length, root length density, root mass and grain yield of plants grown in deep-ripped soil were increased by 38, 36, 27, 24 and 19%, respectively, compared to those of non-ripped treatment. The data from this study form the basis for future research leading to selection and breeding for suitable root traits for soil constraints and provides information for alleviating management of compacted soil in deep sandy soils. (C) 2014 Elsevier By. All rights reserved.