Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity.

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we isolated an evolved strain derived from CGA009 capable of growing on 3-CBA. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed both strains contained large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When the RCB100 aliA allele and a deletion in badM were introduced into R. palustris CGA009, the resulting strain grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution in which regulatory constraints were overcome to enable the selection of a variant of a promiscuous enzyme with enhanced substrate specificity.IMPORTANCEBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found that a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required. This work provides insight into how an environmental isolate evolved to use a halogenated aromatic compound.

Overexpression of RuBisCO form I and II genes in Rhodopseudomonas palustris TIE-1 augments polyhydroxyalkanoate production heterotrophically and autotrophically.

With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1's genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH4Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH4Cl, and two times under photoelectrotrophic growth with N2 . In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganism Rhodopseudomonas palustris TIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO2, and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of the phaR or phaZ genes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic modifications in the environmental microbe TIE-1 can improve PHA production. When combined with other approaches (such as reactor design, use of microbial consortia, and different feedstocks), genetic and metabolic manipulations of purple nonsulfur bacteria like TIE-1 are essential for replacing petroleum-derived plastics with biodegradable plastics like PHA.

Solibacillus palustris sp. nov., isolated from wetland soil of ecology park.

A Gram-staining-positive, motile, aerobic and rod-shaped bacterium, designated strain MA9T was isolated from wetland soil of ecology park, in Seoul, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. Strain MA9T grew at 10-37 °C and at pH 6.0-9.5 on TSB. Menaquinone MK-7 was the predominant respiratory quinone and iso-C15 : 0, iso-C16 : 0 and C16 : 1 ω7c alcohol were the major fatty acids. The main polar lipids were phosphatidylethanolamine (PE), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). The peptidoglycan type of the cell wall was A4α l-Lys-d-Glu. Based on 16S rRNA gene sequencing, strain MA9T clustered with species of the genus Solibacillus and appeared closely related to S. silvestris DSM 12223T (97.8 % sequence similarity), S. cecembensis DSM 21993T (97.6 %), S. isronensis DSM 21046T (97.6 %) and S. kalamii DSM 101595T (96.6 %). The G+C content of the genomic DNA was 37.0 mol%. Digital DNA-DNA hybridization between strain MA9T and type strains of S. silvestris, S. isronensis, S. cecembensis and S. kalamii resulted in values below 70 %. Strain MA9T could be differentiated genotypically and phenotypically from the recognized species of the genus Solibacillus. The isolate therefore represents a novel species, for which the name Solibacillus palustris sp. nov. is proposed, with the type strain MA9T (=KACC 22212T = LMG 32188T).

Full quantitative resource utilization of raw mustard waste through integrating a comprehensive approach for producing hydrogen and soil amendments.

BACKGROUND: Pickled mustard, the largest cultivated vegetable in China, generates substantial waste annually, leading to significant environmental pollution due to challenges in timely disposal, leading to decomposition and sewage issues. Consequently, the imperative to address this concern centers on the reduction and comprehensive resource utilization of raw mustard waste (RMW). To achieve complete and quantitative resource utilization of RMW, this study employs novel technology integration for optimizing its higher-value applications. RESULTS: Initially, subcritical hydrothermal technology was applied for rapid decomposition, with subsequent ammonia nitrogen removal via zeolite. Thereafter, photosynthetic bacteria, Rhodopseudomonas palustris, were employed to maximize hydrogen and methane gas production using various fermentation enhancement agents. Subsequent solid-liquid separation yielded liquid fertilizer from the fermented liquid and soil amendment from solid fermentation remnants. Results indicate that the highest glucose yield (29.6 ± 0.14) was achieved at 165-173℃, with a total sugar content of 50.2 g/L and 64% glucose proportion. Optimal ammonia nitrogen removal occurred with 8 g/L zeolite and strain stable growth at 32℃, with the highest OD600 reaching 2.7. Several fermentation promoters, including FeSO4, Neutral red, Na2S, flavin mononucleotide, Nickel titanate, Nickel oxide, and Mixture C, were evaluated for hydrogen production. Notably, Mixture C resulted in the maximum hydrogen production (756 mL), a production rate of 14 mL/h, and a 5-day stable hydrogen production period. Composting experiments enhanced humic acid content and organic matter (OM) by 17% and 15%, respectively. CONCLUSIONS: This innovative technology not only expedites RMW treatment and hydrogen yield but also substantially enriches soil fertility. Consequently, it offers a novel approach for low-carbon, zero-pollution RMW management. The study's double outcomes extend to large-scale RMW treatment based on the aim of full quantitative resource utilization of RMW. Our method provides a valuable reference for waste management in similar perishable vegetable plantations.

Nutrient enrichment and probiotics for sea urchin Anthocidaris crassipina larvae in captivity to promote large-scale aquaculture.

Sea urchin contains physiologically active substances, such as amino acids and unsaturated fatty acids, and an important aquatic organism. Purple sea urchin, one of the common edible sea urchins, is an important aquatic product. In order to supply the vast seafood market, large-scale aquaculture of sea urchins is very important. The aim of this study was to optimize the rearing of the Anthocidaris crassipina larvae enhancing the nutrition by mixing feed to improve their growth and survival. The survival rate of Chaetoceros muelleri feeding alone is only 40%. If the survival rate is improved through nutrient enrichment, the large-scale aquaculture of larvae can be promoted. The experiment was divided into two parts. Experiment 1: Two types of commonly used microalgae, Isochrysis galbana tml (I), C. muelleri (C) and two types of probiotics, Rhodopseudomonas palustris (R), and Saccharomyces cerevisiae (S) were used in the. Feeding amounts are 5000, 10,000, and 20,000 cell mL-1, and the control group (N) did not eat. Experiment 2: C. muelleri 20,000 cell mL-1 was mixed with I. galbana tml, R. palustris (R) and S. cerevisiae (S) at 5000 and 10,000 cell mL-1. After the experiment, body length, body width, stomach length, rudiment length, rudiment length, body composition, digestive enzymes and survival rate were measured to evaluate the best feed. The results showed that the mixed feeding of C. muelleri 20,000 cell mL-1 and R. palustris 5000 cell mL-1 can achieve the best development and survival of larval embryos and can promote metamorphosis into juveniles in the shortest time. The research results will be applied to the large-scale aquaculture of A. crassipina larvae to promote the diversity of aquaculture.

α-glucosidase and α-amylase inhibitory activity of flavonols from Stenochlaena palustris (Burm.f.) Bedd.

Stenochlaena palustris (Burm.f.) Bedd., a fern species native to India, Southeast Asia, Polynesia, and Australia, has a long history of medical including as a diabetic therapy. This study aimed to isolate bioactive compounds from S. palustris ethyl acetate extract and assess their in vitro and in silico inhibitory activities against α-glucosidase and α-amylase. The successful separation of five flavonols, namely stenopalustroside A (1), tiliroside (2), kaempferol (3), quercetin (4), and rutin (5), was achieved through phytochemical analysis. The compounds exhibited a range of inhibitory activities against α-glucosidase and α-amylase, with IC50 values ranging from 40 to 250 µg/mL. Notably, the biological activities of compound 1 have been reported for the first time. Compound 4 was the most effective inhibitor of both enzymes among the isolated compounds. Studies performed in silico reveal that the interactions between amino acids in compounds 4 and 5 are remarkably comparable to those observed in the positive control. These compounds share this commonality, and as a result, they both have the potential to be active agents.

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

The effect of light emission spectrum on biohydrogen production by Rhodopseudomonas palustris.

Photofermentative hydrogen production has gained increasing attention as a source of green energy. To make such photofermentation processes economically competitive, operating costs need to be reduced, possibly through outdoor operation. Because photofermentation processes are light dependent, the emission spectrum and intensity of light both have a significant influence on the hydrogen production and merit investigation. This study investigates the effect of light sources on the hydrogen production and growth of Rhodopseudomonas palustris, comparing the organism's productivity under longer-wavelength light and light mimicking sunlight. Hydrogen production is enhanced under longer-wavelength light, producing 26.8% (± 7.3%) more hydrogen as compared to under light mimicking that of sunlight; however, R. palustris is still able to produce a considerable volume of hydrogen under light with a spectrum mimicking that of sunlight, providing a promising avenue for future research.

Characterization of the Pyrroloquinoline Quinone Producing Rhodopseudomonas palustris as a Plant Growth-Promoting Bacterium under Photoautotrophic and Photoheterotrophic Culture Conditions.

Rhodopseudomonas palustris is a purple non-sulfide bacterium (PNSB), and some strains have been proven to promote plant growth. However, the mechanism underlying the effect of these PNSBs remains limited. Based on genetic information, R. palustris possesses the ability to produce pyrroloquinoline quinone (PQQ). PQQ is known to play a crucial role in stimulating plant growth, facilitating phosphorous solubilization, and acting as a reactive oxygen species scavenger. However, it is still uncertain whether growth conditions influence R. palustris's production of PQQ and other characteristics. In the present study, it was found that R. palustris exhibited a higher expression of genes related to PQQ synthesis under autotrophic culture conditions as compared to acetate culture conditions. Moreover, similar patterns were observed for phosphorous solubilization and siderophore activity, both of which are recognized to contribute to plant-growth benefits. However, these PNSB culture conditions did not show differences in Arabidopsis growth experiments, indicating that there may be other factors influencing plant growth in addition to PQQ content. Furthermore, the endophytic bacterial strains isolated from Arabidopsis exhibited differences according to the PNSB culture conditions. These findings imply that, depending on the PNSB's growing conditions, it may interact with various soil bacteria and facilitate their infiltration into plants.

Whole-genome assembly and annotation of northern wild rice, Zizania palustris L., supports a whole-genome duplication in the Zizania genus.

Zizania palustris L. (northern wild rice, NWR) is an aquatic grass native to North America that is notable for its nutritious grain. This is an important species with ecological, cultural and agricultural significance, specifically in the Great Lakes region of the USA. Using flow cytometry, we first estimated the NWR genome size to be 1.8 Gb. Using long- and short-range sequencing, Hi-C scaffolding and RNA-seq data from eight tissues, we generated an annotated whole-genome de novo assembly of NWR. The assembly was 1.29 Gb in length, highly repetitive (approx. 76.0%) and contained 46 421 putative protein-coding genes. The expansion of retrotransposons within the genome and a whole-genome duplication (WGD) after the Zizania-Oryza speciation event have both led to an increase in the genome size of NWR in comparison with Oryza sativa L. and Zizania latifolia. Both events depict a genome rapidly undergoing change over a short evolutionary time. Comparative analyses revealed the conservation of large syntenic blocks between NWR and O. sativa, which were used to identify putative seed-shattering genes. Estimates of divergence times revealed that the Zizania genus diverged from Oryza approximately 26-30 million years ago (26-30 MYA), whereas NWR and Z. latifolia diverged from one another approximately 6-8 MYA. Comparative genomics confirmed evidence of a WGD in the Zizania genus and provided support that the event occurred prior to the NWR-Z. latifolia speciation event. This genome assembly and annotation provides a valuable resource for comparative genomics in the Oryzeae tribe and provides an important resource for future conservation and breeding efforts of NWR.

Interacting global change drivers suppress a foundation tree species.

Ecological stress caused by climate change, invasive species and anthropogenic disturbance is driving global environmental change, but how these stressors interact to impact native species are poorly understood. We used a field experiment to test how two stressors (drought and plant invasion by Imperata cylindrica) interacted to determine the effects of a third stressor (fire) on a foundation tree species (Pinus palustris). The invasion combined with prolonged drought resulted in shorter trees than invasion alone. The invasion also resulted in 65% greater fuel loads, four times taller flames, greater maximum temperatures and longer heating duration. Consequently, nearly all tree mortality occurred due to a synergistic interaction between the drought + invasion treatment and fire, where invasion caused taller flames that impacted trees that were shorter due to drought. These findings demonstrate that synergy amongst ecological stressors can dramatically impact native species, with significant implications for forecasting the effects of multiple stressors under global change.

PioABC-Dependent Fe(II) Oxidation during Photoheterotrophic Growth on an Oxidized Carbon Substrate Increases Growth Yield.

Microorganisms that carry out Fe(II) oxidation play a major role in biogeochemical cycling of iron in environments with low oxygen. Fe(II) oxidation has been largely studied in the context of autotrophy. Here, we show that the anoxygenic phototroph, Rhodopseudomonas palustris CGA010, carries out Fe(II) oxidation during photoheterotrophic growth with an oxidized carbon source, malate, leading to an increase in cell yield and allowing more carbon to be directed to cell biomass. We probed the regulatory basis for this by transcriptome sequencing (RNA-seq) and found that the expression levels of the known pioABC Fe(II) oxidation genes in R. palustris depended on the redox-sensing two-component system, RegSR, and the oxidation state of the carbon source provided to cells. This provides the first mechanistic demonstration of mixotrophic growth involving reducing power generated from both Fe(II) oxidation and carbon assimilation. IMPORTANCE The simultaneous use of carbon and reduced metals such as Fe(II) by bacteria is thought to be widespread in aquatic environments, and a mechanistic description of this process could improve our understanding of biogeochemical cycles. Anoxygenic phototrophic bacteria like Rhodopseudomonas palustris typically use light for energy and organic compounds as both a carbon and an electron source. They can also use CO2 for carbon by carbon dioxide fixation when electron-rich compounds like H2, thiosulfate, and Fe(II) are provided as electron donors. Here, we show that Fe(II) oxidation can be used in another context to promote higher growth yields of R. palustris when the oxidized carbon compound malate is provided. We further established the regulatory mechanism underpinning this observation.

Investigating and modeling the effect of light intensity on Rhodopseudomonas palustris growth.

Photosynthetic bacteria can be useful biotechnological tools-they produce a variety of valuable products, including high purity hydrogen, and can simultaneously treat recalcitrant wastewaters. However, while photobioreactors have been designed and modeled for photosynthetic algae and cyanobacteria, there has been less work on understanding the effect of light in photosynthetic bacterial fermentations. To design photobioreactors, and processes using these organisms, robust models of light penetration, utilization, and conversion are needed. This stydy uses experimental data from a tubular photobioreactor designed to focus in on light intensity effects, to model the effect of light intensity on the growth of Rhodopseudomonas palustris, a model photosynthetic bacterium. The work demonstrates that growth is controlled by light intensity, and that this organism does experience photolimitation below 200 W/m2 and photoinhibition above 600 W/m2 . This has implications for outdoor applications, as there will be low growth during the periods of limited light, and growth may be inhibited during the light intensive hours of mid-day. Further, the work presents a model for light penetration in cylindrical photobioreactors, which tends to be the most common geometry. The model developed showed good fit to the experimental data for each light intensity investigated, with high R2 values and NRMSE values all below 20%. The work extends the modeling tools for these organisms, and will allow for better photobioreactor design, and the integration of modeling tools in designing processes which use photosynthetic bacteria.

Growth and tissue nutrient responses of adults of Sarracenia alata to prey exclusion, nutrient addition, and neighbor reduction.

PREMISE: Carnivorous plants are often associated with nutrient-poor soils and fires. Fire can decrease available soil nitrogen (N) and increase light availability, thus potentially favoring carnivory if prey provide N. Prey can also be a source of phosphorus (P), however, and soil P-availability often increases and competition for prey can decrease following fire. Carnivory thus might be more advantageous before fire when prey and/or soil P are more limiting. METHODS: We examined nutrient limitation of growth in a carnivorous plant, Sarracenia alata, in a wet pine savanna in southeastern Mississippi, USA. We measured growth and N:P tissue concentration responses of adult plants to a factorial arrangement of prey capture, neighbor reduction, and addition of N, P, and ash to the soil. We tested two hypotheses: (1) Prey provide N, and neighbor reduction and ash addition increase light and soil P and thus the benefit of carnivory; and (2) Prey provide P, neighbor reduction increases prey and/or P, and prey exclusion reduces growth the most when neighbors are not reduced. RESULTS: The exclusion of prey reduced growth more when neighbors were not reduced, an effect that was ameliorated slightly by the addition of P to the soil (the P-limitation hypothesis). Prey exclusion caused a decrease in tissue P when N was added to the soil. CONCLUSIONS: The results of this study with adult plants differed from those of a previous study using small juvenile plants, suggesting a shift from light limitation to P and prey limitation with increasing size.

Culture optimization to enhance carotenoid production of a selected purple nonsulfur bacterium and its activity against acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus.

Purple nonsulfur bacteria (PNSB) were investigated for their carotenoid production and anti-vibrio activity against acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus. To test carotenoid production, selected strains were cultivated in basic isolation medium (BIM), glutamate acetate medium, G5 medium and artificial acetic acid wastewater (AAW) medium. From 144 PNSB, Rhodopseudomonas palustris KTSSG46 was selected to produce carotenoids under microaerobic light conditions in BIM. When the culture medium was optimized, strain KTSSG46 grown in BIM modified with l-glutamate at 1 g/L more effectively inhibited AHPND-causing V. parahaemolyticus strains than standard BIM with 1 g/L (NH4 )2 SO4 . BIM was further modified with 1.23 g/L MgSO4 ·7H2 O and carotenoid production increased 40.22%. Carotenoid production at day 2 by strain KTSSG46 grown in BIM modified with l-glutamate at 1 and 1.23 g/L MgSO4 ·7H2 O was the same as production in BIM modified with monosodium glutamate (MSG). Culture supernatants from all BIM formulations showed similar activity against the resistant AHPND strain SR2. Based on high-performance liquid chromatography, carotenoids of strain KTSSG46 might be canthaxanthin. Grown in BIM modified with MSG, strain KTSSG46 could produce inexpensive carotenoids and release anti-vibrio compounds that, applied as shrimp feed additive, would prevent AHPND strains.

New Polymethoxyflavones from Hottonia palustris Evoke DNA Biosynthesis-Inhibitory Activity in An Oral Squamous Carcinoma (SCC-25) Cell Line.

Four new compounds, 5-hydroxy-2',6'-dimethoxyflavone (4), 5-hydroxy-2',3',6'-trimethoxyflavone (5), 5-dihydroxy-6-methoxyflavone (6), and 5,6'-dihydroxy-2',3'-dimethoxyflavone (7), and three known compounds, 1,3-diphenylpropane-1,3-dione (1), 5-hydroxyflavone (2), and 5-hydroxy-2'-methoxyflavone (3), were isolated from the aerial parts of Hottonia palustris. Their chemical structures were determined through the use of spectral, spectroscopic and crystallographic methods. The quantitative analysis of the compounds (1-7) and the zapotin (ZAP) in methanol (HP1), petroleum (HP6), and two chloroform extracts (HP7 and HP8) were also determined using HPLC-PDA. The biological activity of these compounds and extracts on the oral squamous carcinoma cell (SCC-25) line was investigated by considering their cytotoxic effects using the MTT assay. Subsequently, the most active compounds and extracts were assessed for their effect on DNA biosynthesis. It was found that all tested samples during 48 h treatment of SCC-25 cells induced the DNA biosynthesis-inhibitory activity: compound 1 (IC50, 29.10 ± 1.45 µM), compound 7 (IC50, 40.60 ± 1.65 µM) and extracts ZAP (IC50, 20.33 ± 1.01 µM), HP6 (IC50, 14.90 ± 0.74 µg), HP7 (IC50, 16.70 ± 0.83 µg), and HP1 (IC50, 30.30 ± 1.15 µg). The data suggest that the novel polymethoxyflavones isolated from Hottonia palustris evoke potent DNA biosynthesis inhibitory activity that may be considered in further studies on experimental pharmacotherapy of oral squamous cell carcinoma.

Growth Inhibition and Apoptotic Effect of Pine Extract and Abietic Acid on MCF-7 Breast Cancer Cells via Alteration of Multiple Gene Expressions Using In Vitro Approach.

In vitro anti-proliferative activity of Pinus palustris extract and its purified abietic acid was assessed against different human cancer cell lines (HepG-2, MCF-7 and HCT-116) compared to normal WI-38 cell line. Abietic acid showed more promising IC50 values against MCF-7 cells than pine extract (0.06 µg/mL and 0.11 µM, respectively), with insignificant cytotoxicity toward normal fibroblast WI-38 cells. Abietic acid triggered both G2/M cell arrest and subG0-G1 subpopulation in MCF-7, compared to SubG0-G1 subpopulation arrest only for the extract. It also induced overexpression of key apoptotic genes (Fas, FasL, Casp3, Casp8, Cyt-C and Bax) and downregulation of both proliferation (VEGF, IGFR1, TGF-ß) and oncogenic (C-myc and NF-κB) genes. Additionally, abietic acid induced overexpression of cytochrome-C protein. Furthermore, it increased levels of total antioxidants to diminish carcinogenesis and chemotherapy resistance. P. palustris is a valuable source of active abietic acid, an antiproliferative agent to MCF-7 cells through induction of apoptosis with promising future anticancer agency in breast cancer therapy.

In Vitro Screening for Anti-Acetylcholinesterase and Antioxidant Activities of Hottonia palustris L. Extracts and Their Unusual Flavonoids.

Hottonia palustris L. is from the genus Hottonia (Primulaceae), and the understanding of its phytochemical and pharmacological properties is limited. In this study, the use of chromatographic techniques led to the isolation of a further eleven compounds, including three new flavonoids: 2',5-dihydroxyflavone 2'-O-ß-glucopyranoside, 5,6-dihydroxyflavone 6-O-(6"-O-glucopyranosyl)-ß-glucopyranoside (hottonioside A), and 4',5,7-trihydroxyflavone 7-O-(2"-O-ß-glucuronide)-ß-glucopyranoside. Their structures were determined using extensive 1D and 2D NMR data and mass spectrometry (HRMS). The qualitative assessment of the chemical composition of the investigated extracts and fractions was performed using the LC-HRMS technique. Furthermore, the antioxidant potential of extracts, fractions, and compounds and their ability to inhibit acetylcholinesterase were also evaluated. Thus, we may conclude that the observed biological effects are the result of the presence of many biologically active compounds, of which dibenzoylmethane is the most active. Therefore, H. palustris is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.

Intraspecific trait variability shapes leaf trait response to altered fire regimes.

BACKGROUND AND AIMS: Understanding impacts of altered disturbance regimes on community structure and function is a key goal for community ecology. Functional traits link species composition to ecosystem functioning. Changes in the distribution of functional traits at community scales in response to disturbance can be driven not only by shifts in species composition, but also by shifts in intraspecific trait values. Understanding the relative importance of these two processes has important implications for predicting community responses to altered disturbance regimes. METHODS: We experimentally manipulated fire return intervals in replicated blocks of a fire-adapted, longleaf pine (Pinus palustris) ecosystem in North Carolina, USA and measured specific leaf area (SLA), leaf dry matter content (LDMC) and compositional responses along a lowland to upland gradient over a 4 year period. Plots were burned between zero and four times. Using a trait-based approach, we simulate hypothetical scenarios which allow species presence, abundance or trait values to vary over time and compare these with observed traits to understand the relative contributions of each of these three processes to observed trait patterns at the study site. We addressed the following questions. (1) How do changes in the fire regime affect community composition, structure and community-level trait responses? (2) Are these effects consistent across a gradient of fire intensity? (3) What are the relative contributions of species turnover, changes in abundance and changes in intraspecific trait values to observed changes in community-weighted mean (CWM) traits in response to altered fire regime? KEY RESULTS: We found strong evidence that altered fire return interval impacted understorey plant communities. The number of fires a plot experienced significantly affected the magnitude of its compositional change and shifted the ecotone boundary separating shrub-dominated lowland areas from grass-dominated upland areas, with suppression sites (0 burns) experiencing an upland shift and annual burn sites a lowland shift. We found significant effects of burn regimes on the CWM of SLA, and that observed shifts in both SLA and LDMC were driven primarily by intraspecific changes in trait values. CONCLUSIONS: In a fire-adapted ecosystem, increased fire frequency altered community composition and structure of the ecosystem through changes in the position of the shrub line. We also found that plant traits responded directionally to increased fire frequency, with SLA decreasing in response to fire frequency across the environmental gradient. For both SLA and LDMC, nearly all of the observed changes in CWM traits were driven by intraspecific variation.