The lignin synthesis related genes and lodging resistance of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

Lignin is closely related to the lodging resistance of common buckwheat (Fagopyrum esculentum Moench.). However, the characteristics of lignin synthesis related genes have not yet been reported. We investigated the lignin biosynthesis gene expression, activities of related enzymes, and accumulation of lignin monomers during branching stage, bloom stage, and milky ripe stage by real-time quantitative PCR, UVspectrophotometry, and gas chromatography-mass spectrometry in the 2^nd internode of three common buckwheat cultivars with different lodging resistance. The results showed that lignin content and the activity of phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) were closely related to the lodging resistance of common buckwheat. Further, we studied gene expression of cinnamate 4-hydroxylase (C4H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamoyl-CoA reductase (CCR), and caffeic acid O-methyltransferase (COMT). The lignin biosynthesis genes were divided into three classes according to their expression pattern: 1) expression firstly increasing and then descending (PAL, 4CL, CAD, C4H, CCoAOMT, F5H, and CCR), 2) expression remaining constant during maturation (C3H), and 3) expression decreasing with maturation (COMT). The present study provides preliminary insights into the expression of lignin biosynthesis genes in common buckwheat, laying a foundation for further understanding the lignin biosynthesis.     

Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Cinnamate 4-hydroxylase (C4H) catalyzes the regioselective para-hydroxylation of trans-cinnamic acid to form p-coumaric acid, the biosynthetic precursor of phenylpropanoid-based polymers. These biopolymers play an essential role in plant structure construction, development, and defense. Herein the open reading frame of CaC4H2 was cloned from Camptotheca acuminata, a deciduous camptothecin-producing tree native to China. CaC4H2 showed 94 % amino acid residues identity with those of reported CaC4H, which suggested that CaC4H2 is an isoform of C4Hs presented in C. acuminata. The intact CaC4H2 was overexpressed in Escherichia coli with its functional reaction partner cytochrome P450 reductase, CamCPR, which transfers electrons from NADPH to CaC4H2 to support the catalytic hydroxylation activity of CaC4H2. Upon incubating trans-cinnamic acid with the recombinant CaC4H2 and tCamCPR, the formation of p-coumaric acid was confirmed by the HPLC–DAD and UPLC-DAD-ESIMS analyses, which indicated the catalytic hydroxylation activity of CaC4H2. Quantitative real-time PCR analyses showed that CaC4H2 was expressed in all tissues of C. acuminata seedlings, which is consistent with the well-known conclusion that the C4H-catalyzed hydroxylation reaction is a key step within the biosynthetic pathway of phenylpropanoids. The functional characterization of CaC4H2 will be useful for molecular breeding and sustainable utilization and protection of the camptothecin-producing plant.     

A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

Synthesis and antimicrobial activity of novel 1,4,5-triphenyl-1<Emphasis Type="Italic">H</Emphasis>-imidazol-[1,2,3]-triazole derivatives

Novel 1-phenyl-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole derivatives were synthesized by click chemistry reaction and screened for antimicrobial activity against grampositive and gram-negative bacterial and fungal species. All the compounds were characterized by ¹H and ¹³C NMR, IR, and mass spectral data. The results of antibacterial study indicated that 1-(4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, 1-(4-(4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)ethanone, 1-(2,6-dichloro-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, and 1-(2-methoxy-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole showed appreciable antibacterial activity while 1-(4-fluorophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy) methyl)-1H-1,2,3-triazole, 1-(2,6-dichloro-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, and 1-(4-methoxyphenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole emerged as the most potential antifungal agents.     

<Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strain 06TCa19 suppresses inflammatory chemokine induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in human gastric epithelial cells

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.     

Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank accession no. EU677841) was successfully isolated from young leaves of C. acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp long and contained a 1413-bp open reading frame encoding a polypeptide of 471 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Southern hybridization analysis showed that there were at least two HMGS gene members in the C. acuminata genome. CPT content was found to be much higher in cotyledons and hypocotyls as compared to roots. RT-PCR analysis revealed strong expression in hypocotyls and cotyledons, but no expression in roots, indicating good correlation between CaHMGS expression and CPT content in the tested tissues. The expression of CaHMGS could be regulated by exogenous elicitors, including salicylic acid and methyl jasmonate, suggesting that CaHMGS was elicitor-responsive. This work is a first step to acquire a better understanding on the role of HMGS in CPT biosynthesis.     

Vegetation of the <Emphasis Type="Italic">Hydrochari-Lemnete</Emphasis> and <Emphasis Type="Italic">Potametea</Emphasis> classes in the Danube-Tisza-Danube hydrosystem (Serbia)

Aquatic vegetation of Hydrochari-Lemnetea and Potametea classes in the Danube-Tisza-Danube hydrosystem (Hs DTD) was studied in 2009–2012, by applying the standard Braun-Blanquet method. The canal network vegetation comprises 14 associations, with Trapetum natantis and Ceratophylletum demersi being the most widely distributed. Hs DTD is also a habitat for several important endangered species, which serve as edificators of the following phytocenoses: Nymphaeetum albae, Nymphaeetum albo-luteae, Nymphoidetum peltatae, Trapetum natantis, Lemno-Spirodeletum, Salvinio-Spirodeletum polyrrhizae, Lemno-Utricularietum vulgaris, Potametum nodosi, Myriophyllo-Potametum and Najadetum marinae. In the studied vegetation, we also found an invasive phytocenosis Elodeetum canadensis that did not have an expanding tendency, and Ceratophyllo demersi-Vallisnerietum spiralis that had this tendency, which made monitoring its stands necessary. Physico-chemical analyses of water, conducted at localities in which the studied phytocenoses thrive, revealed that the development and distribution of most phytocenoses is closely linked with specific habitat conditions. Among the studied parameters, the most significant for the phytocenoses differentiation were: pH, alkalinity, COD-MnO₄, BOD₅, NO ₃ ^? , NO ₂ ^? , PO ₄ ^3? and the concentration of total phosphorus.     

Synthesis of functionalized benzo[<Emphasis Type="Italic">f</Emphasis>]2<Emphasis Type="Italic">H</Emphasis>-chromenes and evaluation of their antimicrobial activities

Knoevenagel cyclocondensations of α-hydroxy naphthaldehyde with β-oxodithioesters and ketene dithioacetals yielded 2H-benzo[f]chromene-2-thiones and 2H-benzo[f]chromen-2-ones, respectively, in high yields. The newly synthesized compounds were evaluated for antifungal and antibacterial activities. Among them, compounds (2-furyl)(3-thioxo-3H-benzo[f]chromen-2-yl)methanone and phenyl(3-oxo-3H-benzo[f]chromen-2-yl)methanone exhibited excellent antifungal activity against tested fungi Curvularia lunata and Fusarium moniliforme. The highest antibacterial activity against the tested bacteria Escherichia coli and Staphylococcus aureus was observed for (4-chlorophenyl)(3-oxo-3H-benzo[f]chromen-2-yl)methanone. The results of antimicrobial screening demonstrate that (2-furyl)(3-thioxo-3H-benzo[f]chromen-2-yl)methanone, phenyl(3-oxo-3H-benzo[f]chromen-2-yl)methanone, and (4-chlorophenyl)(3-oxo-3H-benzo[f]chromen-2-yl)methanone are promising as antimicrobial drugs.     

Determining the effects of life stage,shared prey density and host plant on intraguild predation of a native lacewing (<Emphasis Type="Italic">Chrysoperla carnea</Emphasis>) by an invasive coccinellid (<Emphasis Type="Italic">Harmonia axyridis</Emphasis>)

Negative impacts of non-native Harmonia axyridis (Pallas) on members of the native aphid enemy guild have been widely hypothesised but mainly only assessed with other coccinellid species, and mostly in small experimental arenas. Here we investigated the interactions between H. axyridis and Chrysoperla carnea Stephens larvae. In small-scale (Petri dish) arenas 2nd-instar C. carnea were at risk of predation from larval (2nd and 4th-instar) and adult (male and female) H. axyridis while 3rd-instar C. carnea were only at minimal risk from 4th-instar and adult female H. axyridis. Plant species, aphid species and aphid density did not affect intraguild predation of 2nd-instar C. carnea by 4th-instar and adult H. axyridis in mesocosm experiments. Chrysoperla carnea consumed similar numbers of Megoura viciae Buckton, Aphis fabae Scop. and Acyrthosiphon pisum Harris aphids while H. axyridis consumed fewer M. viciae than the other two species. The greatest suppression of A. pisum was achieved in treatments with both C. carnea and H. axyridis. Life stage and the sex of H. axyridis as well as the life stage of C. carnea are important variables affecting intraguild predation and these attributes should be considered when assessing the potential threat of other potentially invasive alien predators.