Drivers of diversification in Linum (Linaceae) by means of chromosome evolution: correlations with biogeography, breeding system and habit.

BACKGROUND AND AIMS: Chromosome evolution leads to hybrid dysfunction and recombination patterns and has thus been proposed as a major driver of diversification in all branches of the tree of life, including flowering plants. In this study we used the genus Linum (flax species) to evaluate the effects of chromosomal evolution on diversification rates and on traits that are important for sexual reproduction. Linum is a useful study group because it has considerable reproductive polymorphism (heterostyly) and chromosomal variation (n = 6-36) and a complex pattern of biogeographical distribution. METHODS: We tested several traditional hypotheses of chromosomal evolution. We analysed changes in chromosome number across the phylogenetic tree (ChromEvol model) in combination with diversification rates (ChromoSSE model), biogeographical distribution, heterostyly and habit (ChromePlus model). KEY RESULTS: Chromosome number evolved across the Linum phylogeny from an estimated ancestral chromosome number of n = 9. While there were few apparent incidences of cladogenesis through chromosome evolution, we inferred up to five chromosomal speciation events. Chromosome evolution was not related to heterostyly but did show significant relationships with habit and geographical range. Polyploidy was negatively correlated with perennial habit, as expected from the relative commonness of perennial woodiness and absence of perennial clonality in the genus. The colonization of new areas was linked to genome rearrangements (polyploidy and dysploidy), which could be associated with speciation events during the colonization process. CONCLUSIONS: Chromosome evolution is a key trait in some clades of the Linum phylogeny. Chromosome evolution directly impacts speciation and indirectly influences biogeographical processes and important plant traits.

Ancillary polymorphic floral traits between two morphs adaptive to hawkmoth pollination in distylous plant Tirpitzia sinensis (Linaceae).

BACKGROUND: Floral morphs are characterized differentiations in reciprocal positions of sexual organs and ancillary floral traits in heterostylous plants. However, it remains unclear how differential floral morphs ensure reproductive success between morphs using the same pollinator. RESULTS: Measurements of floral traits in white-flowered Tirpitzia sinensis with long corolla tubes indicated that it is typically distylous, long-styled (L-) morph producing more but smaller pollen grains per flower than short-styled (S-) morph. Both morphs secreted more nectar volume at night than in the day and the sugar composition was rich in sucrose, potentially adaptive to pollination by hawkmoths (Macroglossum spp.) which were active at dusk. A bumblebee species functioned as the nectar robber in both morphs and a honeybee as the pollen feeder in the S-morph. The L-morph secreted more nectar volume but relatively lower sucrose/hexose ratio than the S-morph. Floral visitation rate by hawkmoths was higher but its pollination efficiency was lower in the S-morph than the L-morph. Hand pollination treatments indicated self-incompatibility in T. sinensis and seed set of open-pollinated flowers did not differ between morphs. CONCLUSIONS: Our findings suggest that the two morphs differ with respect to traits relevant to pollination. The L-morph, with its exserted stigma, has more pollen grains per anther and a greater volume of nectar, which may prolong the foraging time and increase the pollination efficiency of hawkmoths. The S-morph has a higher sucrose/hexose ratio in its nectar which can be more attractive to hawkmoths and increase the visit rates. Ancillary polymorphic floral traits between two morphs are adaptive to hawkmoth and ensure reproductive success in distylous plant T. sinensis.

Reinwardtia indica: phytochemical screening and evaluation of wound healing activity of the extracts in experimental model rats.

Reinwardtia indica is traditionally used for wound healing. The main aim of this study was to evaluate the wound healing activity of leaves extracts of R. indica using the excision wound model in rats. The leaves of R. indica were collected from Gondrang, Chitwan, Nepal.  Leaves were shade dried, extracted by double maceration and subjected to phytochemical screening. Then, the fusion method was used for the formulation of ointment and evaluated. Rats (n=24) were divided into four groups with 6 in each. Excision wound model was used, 2 cm diameter (314 mm2), 2 mm depth wound was created. The treatment was given daily topically to all groups and the % mean wound contraction rate was calculated on days 4, 8, 12 and 16.  The result was analyzed statistically using Graph pad prism version 5. Phytochemical test revealed the presence of alkaloid, flavonoid, tannin, phenol, terpenoid, carbohydrate, etc.  All the evaluation parameters showed satisfactory results. The extract of R. indica ointment (2% w/w and 5% w/w) increased the wound contraction rate day by day. The % means wound contraction rate, on day 12, (80% and 88%), and on day 16, (97% and 100%) and statistically significant difference was at p<0.0001. The R. indica extract ointment showed an increased wound contraction rate. So, in further R. indica could be used for commercial production of wound healing ointment.

Neuroprotective effect of Reinwardtia indica against scopolamine induced memory-impairment in rat by attenuating oxidative stress.

Reinwardtia indica belongs to Linaceae family and used as a folk medicine in Asian countries. Traditionally, it has been used in the treatment of paralysis and anti-microbial in wound healing, etc. The current study was undertaken in order to investigate the antioxidant and memory protective effect of the alcoholic (99.90%) (AERI) and hydro-alcoholic (70:30) leaves extract (HAERI) of Reinwardtia indica, against scopolamine-induced memory impairment in animals and also tried to determine the possible mechanism of action. In addition, phytochemical profiling of alcoholic leaves extract was also conducted through gas chromatography-mass spectrometry (GC-MS/MS). Rats were pretreated with AERI, HAERI (dose 250 and 500 mg/kg) and Donepezil (standard drug) along with scopolamine (1 mg/kg) for a period of 14 days followed by different test like elevated plus maze, passive avoidance, and Morris water maze to assess learning and memory ability. Acetylcholine levels, acetylcholinesterase (AChE), antioxidant enzymes (SOD, CAT & GSH), histopathology of the brain and biochemical test were also performed at the end of the treatment period. The scopolamine treatment resulted in learning and memory deficits which were partially and significantly ameliorated by the AERI at higher dose among other doses of extracts. The AERI at higher dose also counteracted the scopolamine-induced decrease in acetylcholine levels, increase in AChE activity, and decrease in antioxidant enzymes activities. No significant changes observed in the biochemical estimation of all dose of extracts. Histology of brain tissue showed the marked cellular changes in only scopolamine treated group while the standard, AERI and HAERI treated group were showing less damage at hippocampus region of the brain. The phytochemicals found after chemical profiling through GC-MS also supported the activity because of the presence of chemicals already reported for the neuroprotective, memory-enhancing and antioxidant activity, etc. The results demonstrated that the ability of the AERI at higher dose among all doses of extracts has more potential to revert the scopolamine-induced learning and memory deficits in rats by attenuating the decreased level of acetylcholine and antioxidant enzymes.

Evaluation of antioxidant and antimicrobial potential of a novel Himalayan plant Reinwardtia indica dumort: Scientifically unexplored.

Reinwardtia indica (Lineceae) is a medicinal plant cultivated in the Himalayan region. It is effectively used in folk medicines for the treatment of various health complications. In the present study, the shade dried leaves and stem were extracted in three different solvents such as ethyl acetate, ethanol, and hydro-alcoholic. The antioxidant efficacy of these extracts was confirmed by using different in vitro assays: DPPH free radical scavenging, superoxide radical scavenging, lipid peroxidation, metal ion chelating capability and reducing power determination. Total phenol content was maximum in hydro alcoholic extract of leaf (540.37 mg per g of gallic acid equivalents) and stem (330.51 mg per g of gallic acid equivalents) while flavonoid content was maximum in ethanolic extract of leaf (305 mg per gram of rutin equivalents) and ethyl acetate extract of stem (170.6 mg per gram of rutin equivalents). The antioxidant activity of these extracts was positively correlated with their total phenol and flavonoid content. Among all tested extracts, ethanolic extract of leaf exhibit maximum zone of inhibition against all tested clinical isolates of bacterial (E. coli 11.00 ±â€¯1.73 mm, P. aeurogenosa 11.67 ±â€¯0.58 mm and S. aureus 10.33 ±â€¯1.53 mm) and fungal (C. albicans 11.33 ±â€¯1.10 mm) pathogens, while ethyl acetate extracts of the leaf and stem showed minimum inhibitory concentration against all tested microorganisms. Thus, R. indica extracts can be used as potent natural antioxidant having antifungal and antibacterial action.

Antioxidant, antimicrobial and cytotoxic potential of silver nanoparticles synthesized using flavonoid rich alcoholic leaves extract of Reinwardtia indica.

The present work discusses the establishment of a green route for the rapid synthesis of silver nanoparticles (AgNPs) using an alcoholic extract of Reinwardtia indica (AERI) leaves which act as a reducing as well as a capping agent. The change in color from yellowish green to dark brown confirmed the synthesis of AgNPs. A characteristic surface plasmon resonance (SPR) band at 436 nm advocated the presence of AgNPs. The synthesis process was optimized using one factor at a time approach where 1.0 mM AgNO3 concentration, 5 mL 0.4% (v/v) of AER inoculum dose and 30 min of sunlight exposure were found to be the optimum conditions. The synthesized AgNPs was characterized by several characterizing techniques such as HR- TEM, SAED, HR-SEM, EDX, XRD, FTIR and AFM analysis. For evaluation and comparison of AgNPs with AERI used human pathogen E. coli, P. aeurogenosa, S. aeurus and C. albicans for antimicrobial, for cytotoxicity study SiHa cell line at concentration of (10, 50, 100, 250 and 500 µg mL-1) and for enzymatic assay superoxide dismutase, catalase, malondialdehyde and glutathione peroxidase method were used. The size of nanoparticle in the range of 3-15 nm was confirmed TEM, spherical shape by SEM and crystal lattice nature by XRD. AFM results revealed the 2 D and 3 D pattern of particle scatter nature on the surface. This protocol as simple, rapid, one step, eco-friendly, nontoxic and AgNPs showed strong antimicrobial activity as well as cytotoxic potential in comparison to AERI.

The Linum usitatissimum L. plastome reveals atypical structural evolution, new editing sites, and the phylogenetic position of Linaceae within Malpighiales.

KEY MESSAGE: The plastome of Linum usitatissimum was completely sequenced allowing analyses of evolution of genome structure, RNA editing sites, molecular markers, and indicating the position of Linaceae within Malpighiales. Flax (Linum usitatissimum L.) is an economically important crop used as food, feed, and industrial feedstock. It belongs to the Linaceae family, which is noted by high morphological and ecological diversity. Here, we reported the complete sequence of flax plastome, the first species within Linaceae family to have the plastome sequenced, assembled and characterized in detail. The plastome of flax is a circular DNA molecule of 156,721 bp with a typical quadripartite structure including two IRs of 31,990 bp separating the LSC of 81,767 bp and the SSC of 10,974 bp. It shows two expansion events from IRB to LSC and from IRB to SSC, and a contraction event in the IRA-LSC junction, which changed significantly the size and the gene content of LSC, SSC and IRs. We identified 109 unique genes and 2 pseudogenes (rpl23 and ndhF). The plastome lost the conserved introns of clpP gene and the complete sequence of rps16 gene. The clpP, ycf1, and ycf2 genes show high nucleotide and aminoacid divergence, but they still possibly retain the functionality. Moreover, we also identified 176 SSRs, 20 tandem repeats, and 39 dispersed repeats. We predicted in 18 genes a total of 53 RNA editing sites of which 32 were not found before in other species. The phylogenetic inference based on 63 plastid protein-coding genes of 38 taxa supports three major clades within Malpighiales order. One of these clades has flax (Linaceae) sister to Chrysobalanaceae family, differing from earlier studies that included Linaceae into the euphorbioid clade.

In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

MAIN CONCLUSION: Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72 h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.

Pleistocene radiation of the serpentine-adapted genus Hesperolinon and other divergence times in Linaceae (Malpighiales).

PREMISE OF THE STUDY: Hesperolinon (western flax; Linaceae) is endemic to the western United States, where it is notable for its high and geographically concentrated species diversity on serpentine-derived soils and for its use as a model system in disease ecology. We used a phylogenetic framework to test a long-standing hypothesis that Hesperolinon is a neoendemic radiation. METHODS: Five plastid and two ribosomal nuclear DNA gene regions were sampled from 105 populations of Hesperolinon, including all 13 recently recognized species across their known ranges. We used these data to generate population-level phylogenies of Hesperolinon. We also generated a robustly sampled chronogram of Linaceae using an eight-gene, 100-taxon supermatrix calibrated using fossil Linum pollen and a published chronogram of Malpighiales. KEY RESULTS: Most diversification in Hesperolinon has taken place in the past 1-2 million yr, much more recently than previous estimates. Only the earliest-diverging species, H. drymarioides, was resolved as a clade. Denser taxon and gene sampling generally support previously proposed relationships within Linaceae, but with more recent diversification of key clades. CONCLUSIONS: Hesperolinon is an excellent example of edaphic neoendemism, in support of Raven and Axelrod's hypothesis for the genus. Dense population-level sampling reveals a complex of incipient species, with clades poorly aligned with traditional morphological circumscriptions, likely due in part to continued gene flow. The diversification of Linaceae is more recent than previously estimated, and other recent radiations (e.g., Hugonia) warrant further study.

The diterpenes ovoideal A-G from Tirpitzia ovoidea.

Seven new diterpenes, named ovoideal A (1), B (2), C (3), D (4), E (5), F (6) and G (7), have been isolated along with eleven known diterpenes 8-18 from the petroleum ether soluble fraction of an ethanol extract of the aerial parts of Tirpitzia ovoidea. The structures of the new compounds were elucidated primarily by 1D and 2D NMR spectroscopy, as well as by the HR-ESI-MS spectrometry. All compounds were isolated from the Linaceae family for the first time. The in vitro cytotoxic activity of compounds 1, 3-5, 8-18 was evaluated against the Hela, HepG2 and K562 cell lines. Among them, compounds 3, 9, 11, 12, 13, 14, 15, 17, 18 showed moderate inhibitory activities.

Comprehensive RNA-Seq-based study and metabolite profiling to identify genes involved in podophyllotoxin biosynthesis in Linum album Kotschy ex Boiss. (Linaceae).

Linum album is a well-known rich source of anticancer compounds, i.e., podophyllotoxin (PTOX) and other lignans. These compounds play an important role in the plant's defensive system. The RNA-Seq data of flax (L. usitatissimum) were analyzed under various biotic and abiotic stresses to comprehend better the importance of lignans in plant defense responses. Then, the association between the lignan contents and some related gene expressions was experimented with HPLC and qRT-PCR, respectively. Transcriptomic profiling showed a specific expression pattern in different organs, and just the commonly regulated gene EP3 was detected with a significant increase under all stresses. The in silico analysis of the PTOX biosynthesis pathway identified a list of genes, including laccase (LAC11), lactoperoxidase (POD), 4-coumarate-CoA ligase (4CL), and secoisolariciresinol dehydrogenase (SDH). These genes increased significantly under individual stresses. The HPLC analysis showed that the measured lignan contents generally increased under stress. In contrast, a quantitative expression of the genes involved in this pathway using qRT-PCR showed a different pattern that seems to contribute to regulating PTOX content in response to stress. Identified modifications of critical genes related to PTOX biosynthesis in response to multiple stresses can provide a baseline for improving PTOX content in L. album.

Edaphic quality and plant-pathogen interactions: effects of soil calcium on fungal infection of a serpentine flax.

Spatial variation in the frequency and outcome of interspecific interactions is thought to play a central role in shaping geographic patterns of biodiversity. Previous empirical studies and modeling exercises suggest that negative species interactions should occur with greater frequency and intensity in high-quality/low-stress environments. I tested this hypothesis through a four-year epidemiological study of interactions between the plant Hesperolinon californicum and the pathogenic rust fungus Melampsora lini. By virtue of its association with serpentine soils, H. californicum is exposed to edaphic conditions that are stressful to most plants. Notable among these is the low availability of calcium, an element that plays critically important roles in the immune responses of plants to attacking pathogens. As a serpentine generalist, however, H. californicum grows in soils with a wide range of calcium concentrations, and this should lead to differences in the frequency and/or severity of rust infection among host populations. I investigated geographic variation in disease pressure by conducting annual surveys in 16 populations spanning the host's distribution and representing a range of soil calcium concentrations. Results indicated that plants growing in more stressful low-calcium soils experienced higher rates of rust infection, suggesting that soil calcium may modulate host susceptibility in a manner opposite to that predicted by the a priori hypothesis. Epidemiological surveys further revealed a latitudinal cline in disease prevalence, with high infection rates in northern host populations decreasing gradually toward the south. Studies of the fitness effects of disease demonstrated that rust infection caused significant reductions in host survival and fecundity, and there was evidence of a demographic feedback between infection prevalence and host density across survey years.

Acute and sub-acute toxicity study of hydro-alcoholic leaves extract of Reinwardtia indica in rats.

The present study was to assess the toxicity of hydro alcoholic leaves extract of Reinwardtia indica in Charles foster rats through an acute and sub-acute study. In the acute study, rats were treated orally with single dose and for sub-acute study different doses were given orally for 28 consecutive days. At the dose of 2000 mg/kg satellite group was also used for 6 weeks as per OECD guidelines-407. General behavioral parameters were assessed in acute toxicity and found no mortality or exterior signs of toxicity. While in the sub-acute study; biochemical, hematological and histopathology along with the body weight, food, and water consumptions parameters were screened in the animals after 14 & 28 days. The study reveals the insignificant (P < 0.05) change in treated group in comparison to the control. The hydroalcoholic leaves extract of Reinwardtia indica was found non-toxic up to 5000 mg/kg in acute study whereas up to 2000 mg/kg dose level in the sub-acute study.

Antifungal rosane diterpenes and other constituents of Hugonia castaneifolia.

The rosane diterpenoids hugorosenone [3beta-hydroxyrosa-1(10),15-dien-2-one], 18-hydroxyhugorosenone and 18-hydroxy-3-deoxyhugorosenone, and 12-hydroxy-13-methylpodocarpa-8,11,13-trien-3-one were isolated as antifungal constituents of H. castaneifolia Engl. root bark, together with the previously reported di-podocarpanoids hugonone A and hugonone B that were weakly active, and 1(10),15-rosadiene-2beta,3beta-diol (hugorosenol), 4alpha-methoxyhimachal-10-en-5beta-ol (hugonianene B) and 2-hydroxyhenpentacont-2-enal, and the known compounds tetracosyl-(E)-ferrulate and caryophyllene oxide, all of which were inactive. Hugorosenone also exhibited activity against Anopheles gambiae mosquito larvae. Structural determination was achieved based on spectroscopic data.

Clinal resistance structure and pathogen local adaptation in a serpentine flax-flax rust interaction.

Because disease resistance is a hallmark signature of pathogen-mediated selection pressure on hosts, studies of resistance structure (the spatial distribution of disease resistance genes among conspecific host populations) can provide valuable insights into the influence of pathogens on host evolution and spatial variation in the magnitude of their effects. To date few studies of wild plant-pathogen interactions have characterized resistance structure by sampling across the host's biogeographic range, and only a handful have paired such investigations with studies of disease levels under natural conditions. I used a greenhouse cross-inoculation experiment to characterize genetic resistance of 16 populations of California dwarf flax (Hesperolinon californicum) to attack by multiple samples of the rust fungus Melampsora lini. I documented a latitudinal cline in resistance structure, manifest across the host's biogeographic range, which mirrored almost identically a cline in infection prevalence documented through field surveys of disease in study populations. These results provide empirical evidence for clinal patterns of antagonistic selection pressure, demonstrate that such patterns can be manifest across broad biogeographic scales, and suggest that rates of disease prevalence in wild plant populations may be tightly linked to the distribution of host resistance genes. Tests for local adaptation of the fungus revealed evidence of the phenomenon (significantly greater infection in sympatric plant-fungal pairings) as well as the potential for substantial bias to be introduced into statistical analyses by spatial patterns of host resistance structure.

A new cytotoxic and larvicidal himachalenoid, rosanoids and other constituents of Hugonia busseana.

A new cytotoxic himachalene sesquiterpenoid 4alpha-methoxy-5,9-oxahimachal-9-ene (hugonianene A) which exhibited moderate activity against Anopheles gambiae mosquito larvae after 24 h at a concentration of 0.237 mg mL(-1), and at 48 h and 72 h contact time causing complete larval mortality up to a concentration as low as 0.01369 mg mL(-1), was isolated as the major constituent of the cytotoxic root bark extract of Hugonia busseana. Hugonianene A was obtained together with the hitherto unreported rosane diterpenoid 18-hydroxyrosane, the known rosane diterpenoid hugorosediol, an inseparable mixture of 12-methoxy-13-methylpodocarpa-8,11,13-trien-3,7-dione and 12-methoxy-13-methylpodocarpa-1,8,11,13-tetraen-3,7-dione, and the di-podocarpanoid hugonone B that was previously obtained from H. castaneifolia.

Lignans and diterpenes isolated from Tirpitzia ovoidea and their biological activities.

A new lignan, tirpitzin A (17) together with 20 known compounds (1-16, and 18-21) were isolated from the ethyl acetate soluble fraction of ethanol extract of the aerial parts of Tirpitzia ovoidea. The structure of new compound was elucidated by means of spectroscopic analysis. Of the known compounds, 7-21 were isolated from Linaceae family for the first time. The pharmacological activity of the crude extracts was tested using a mouse inflammation model induced by dimethyl benzene. The results demonstrated that the ethyl acetate soluble fraction had anti-inflammatory activity. Moreover, the cytotoxic and anti-inflammatory activities of some compounds were studied. The new compound 17 showed moderate cytotoxic effect against BxPC-3 cell line (IC50 = 19.51µmol·L-1) and Compound 10 showed significant cytotoxicity against HepG2, HL-60, U87 and BxPC-3 cell lines with IC50 values in the range 4.2-8.3µmol·L-1. Additionally, Compounds 2, 10, 11, and 13 exhibited potent inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages at the concentration of 50µmol·L-1.

Toxicity assessment of the alcoholic leaves extract of Reinwardtia indica

The present study aimed to evaluate the safety of the alcoholic leaves extract of Reinwardtia indica in Charles foster rats through an acute and sub-acute oral administration.For assessment of acute oral toxicity test, ratswere orally treated with single dose of the alcoholic leaves extract of Reinwardtia indica at the doses of 50, 250, 500, 1000 2000 and 5000 mg/kg. In sub-acute toxicity study, using the OECD guidelines no. 407, the extract was administered at the doses of 50, 250, 500, 1000, 2000 mg/kg/day for 28 consecutive days and at the dose of 2000 mg/kg satellite group also used for 6 weeks.In acute toxicity above mentioned doses neither showed mortality nor exterior signs of toxicity. In sub-acute, study no significant changes found in haematological and biochemical level ofthe treated rat after 14 days and 28 days in comparison to control. The histopathology of rat brain, kidney, liver, and heart also showed the no cellular changes after extract treated rat.The alcoholic leaves extract of Reinwardtia indica was found non-toxic in single drug dose administration up to 5000 mg/kg (acute study) and in sub-acute administration up to 2000 mg/kg.

Linseed allergy due to LTP: another food for LTP syndrome

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