Eremane, viscidane and isozizaene diterpenoids from the leaves of Eremophila rigida and their absolute configurations.

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.

Identification of new PTP1B-inhibiting decipiene diterpenoid esters from Eremophila clarkei by high-resolution PTP1B inhibition profiling, enzyme kinetics analysis, and molecular docking.

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 µg/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 µM. 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.

Characterization of Serrulatane Diterpenoids in Eremophila phyllopoda subsp. phyllopoda by Triple High-Resolution α-Glucosidase/PTP1B/Radical Scavenging Profiling, NMR Spectroscopy, DFT-GIAO NMR, and Electronic Circular Dichroism Calculations.

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, 1ß-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.

Serrulatane diterpenoids with unusual side chain modifications from root bark of Eremophila longifolia.

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.

Biodiscoveries within the Australian plant genus Eremophila based on international and interdisciplinary collaboration: results and perspectives on outstanding ethical dilemmas.

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.

Serrulatane diterpenoids from the leaves of Eremophila glabra and their potential as antihyperglycemic drug leads.

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.

Reversal of ABCG2/BCRP-Mediated Multidrug Resistance by 5,3',5'-Trihydroxy-3,6,7,4'-Tetramethoxyflavone Isolated from the Australian Desert Plant Eremophila galeata Chinnock.

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.

Navigating through chemical space and evolutionary time across the Australian continent in plant genus Eremophila.

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.

PTP1B-Inhibiting Branched-Chain Fatty Acid Dimers from Eremophila oppositifolia subsp. angustifolia Identified by High-Resolution PTP1B Inhibition Profiling and HPLC-PDA-HRMS-SPE-NMR Analysis.

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.

Split personality of a Potyvirus: to specialize or not to specialize?

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.

Plant virology and next generation sequencing: experiences with a Potyvirus.

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.

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding.

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.

Development of two sequence-specific PCR markers linked to the le gene that reduces pod shattering in narrow-leafed Lupin (Lupinus angustifolius L.)

Wild types of narrow-leaf lupin (Lupinus angustifolius L.) have seed pods that shatter upon maturity, leading to the loss of their seeds before or during the harvest process. Two recessive genes have been incorporated into domesticated cultivars of this species to maximize harvest-ability of the produce. One of these genes is called lentus (le). Two microsatellite - anchored fragment length polymorphism (MFLP) candidate markers were identified as closely linked to the le gene in a recombinant inbred line (RIL) population derived from a domesticated x wild type cross. The candidate MFLP markers were isolated from the gel, re-amplified by PCR, cloned and sequenced. The MFLP polymorphisms were converted into sequence-specific PCR-based markers. Linkage analysis by MapManager indicated that one of the markers, LeM1, was 2.6 centiMorgans (cM) and the other, LeM2, was 1.3 cM from the gene, with both being on the same side. The correlation between the marker genotype and the plant phenotype for the le gene is 95 percent for the Australian cultivars, and approximately 36 percent on wild types tested. These markers may be useful in marker assisted selection for the le gene when introgressing wild material into lupin breeding programs.

Construction of a genetic linkage map using MFLP and identification of molecular markers linked to domestication genes in narrow-leafed lupin (Lupinus angustifolius L.).

A mapping population of F(8)derived recombinant inbred lines (RILs) was established from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin (Lupinus angustifolius L.). The parents together with the 89 RILs were subjected to DNA fingerprinting using microsatellite-anchored fragment length polymorphism (MFLP) to rapidly generate DNA markers to construct a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 7 or more markers. The total map length was 1543 cM, with an average distance of 3.4 cM between adjacent markers. This is the first published map for a lupin species. The map can be exploited for marker assisted selection for genetic improvement in lupin breeding programs.

A PCR-based molecular marker applicable for marker-assisted selection for anthracnose disease resistance in lupin breeding.

Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F(8)derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program.