Two FT orthologs from <Emphasis Type="Italic">Populus simonii</Emphasis> Carrière induce early flowering in <Emphasis Type="Italic">Arabidopsis</Emphasis> and poplar trees

Genes in the FLOWERING LOCUS T (FT) family have been shown to be important in the control of the switch between vegetative and reproductive growth in several plant species. In this study, two FT orthologs were isolated from Populus simonii, a species indigenous to China and planted in North China as a shelterbelt. The orthologs were named PsFT1 and PsFT2. Phylogenetic analysis revealed that the two genes belonged to the FT gene family. PsFT1 and PsFT2 control flowering initiation in response to day-length when ectopically expressed in transgenic Arabidopsis and transgenic juvenile poplar clone T89 plants. Moreover, when poplar T89 was transformed with PsFT1 or PsFT2, flowering was induced within 40 days. Subsequently, floral meristems emerged on the flanks of the axillary inflorescence shoots. These findings suggest that PsFT1 and PsFT2 are involved in the control of the timing of flowering in Populus simonii.     

Changes in the pollen seasons of the early flowering trees <Emphasis Type="Italic">Alnus</Emphasis> spp. and <Emphasis Type="Italic">Corylus</Emphasis> spp. in Worcester,United Kingdom, 1996–2005

Previous work on Betula spp. (birch) in the UK and at five sites in Europe has shown that pollen seasons for this taxon have tended to become earlier by about 5–10 days per decade in most regions investigated over the last 30 years. This pattern has been linked to the trend to warmer winters and springs in recent years. However, little work has been done to investigate the changes in the pollen seasons for the early flowering trees. Several of these, such as Alnus spp. and Corylus spp., have allergens, which cross-react with those of Betula spp., and so have a priming effect on allergic people. This paper investigates pollen seasons for Alnus spp. and Corylus spp. for the years 1996–2005 at Worcester, in the West Midlands, United Kingdom. Pollen data for daily average counts were collected using a Burkard volumetric trap sited on the exposed roof of a three-storey building. The climate is western maritime. Meteorological data for daily temperatures (maximum and minimum) and rainfall were obtained from the local monitoring sites. The local area up to approximately 10 km surrounding the site is mostly level terrain with some undulating hills and valleys. The local vegetation is mixed farmland and deciduous woodland. The pollen seasons for the two taxa investigated are typically late December or early January to late March. Various ways of defining the start and end of the pollen seasons were considered for these taxa, but the most useful was the 1% method whereby the season is deemed to have started when 1% of the total catch is achieved and to have ended when 99% is reached. The cumulative catches (in grains/m³) for Alnus spp. varied from 698 (2001) to 3,467 (2004). For Corylus spp., they varied from 65 (2001) to 4,933 (2004). The start dates for Alnus spp. showed 39 days difference in the 10 years (earliest 2000 day 21, latest 1996 day 60). The end dates differed by 26 days and the length of season differed by 15 days. The last 4 years in the set had notably higher cumulative counts than the first 2, but there was no trend towards earlier starts. For Corylus spp. start days also differed by 39 days (earliest 1999 day 5, latest 1996 day 44). The end date differed by 35 days and length of season by 26 days. Cumulative counts and lengths of season showed a distinct pattern of alternative high (long) and low (short) years. There is some evidence of a synchronous pattern for Alnus spp.. These patterns show some significant correlations with temperature and rainfall through the autumn, winter and early spring, and some relationships with growth degree 4s and chill units, but the series is too short to discern trends. The analysis has provided insight to the variation in the seasons for these early flowering trees and will form a basis for future work on building predictive models for these taxa.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Invasibility of reservoirs in the Paraná Basin,Brazil, to <Emphasis Type="Italic">Cichla</Emphasis><Emphasis Type="Italic">kelberi</Emphasis> Kullander and Ferreira, 2006

The invasion process comprises not only the characteristics of nonindigenous species but also the attributes of the invaded environment which make it susceptible to the establishment of nonindigenous species. Habitat attributes operate like filters in determining the establishment of introduced species and the invasibility of a region. In the Upper Paraná River Basin, Brazil, the practice of introducing species was quite common and frequently carried out by hydroelectric companies. The target species of the present study, the peacock-bass Cichla kelberi, is native to the Amazon Basin. This species was introduced into several reservoirs of the Upper Paraná River Basin and is dispersing rapidly throughout the system. This study shows which characteristics of the reservoirs facilitate their invasibility after testing for the effect of propagule pressure. We conclude that a set of abiotic factors favors the invasibility of these reservoirs. To be more precise, the largest, deepest, most transparent and warmest reservoirs are the most likely to be colonized by Cichla kelberi. It is possible that other environments with similar characteristics to these reservoirs, such as the lagoons from the Upper Paraná River Basin floodplain, can also be colonized by Cichla kelberi.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Prevalence of Community-Occurring Extended Spectrum β-Lactamase-Producing <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> in Brazil

The occurrence of extended-spectrum-β-lactamase (ESBL)-producing strains in the community was investigated in a private laboratory located in Juiz de Fora, Brazil. All enterobacterial isolates analysed were collected from urine of human patients between the years 2000 and 2002. ESBL production was confirmed by double disk screening, combination disk method, and Etest ESBL strip. The isoelectric point of each β-lactamase was determined in the crude extracts from each isolate. Detection of ESBL genes was performed by polymerase chain reaction and the genetic relatedness of the isolates determined by pulsed-field gel electrophoresis (PFGE). Of the 1,481 isolates, 22 (12 Klebsiella pneumoniae, 7 Escherichia coli, 1 Providencia stuartii, 1 Citrobacter freundii, and 1 Serratia marcescens) were identified as ESBL producers. The frequency of ESBL producers in the community was 1.48%. TEM-type enzymes were identified in 95.4% of the isolates, followed by the SHV type. Seven strains produced CTX-M–type enzymes. This study showed that strains producing multiple β-lactamases are also present in community-acquired bacterial isolates. Multiple strains exhibiting identical PFGE genotypes were found in individual patients indicating a common source of acquisition.     

Patterns of <Emphasis Type="Italic">Azteca</Emphasis> ants’ defence of <Emphasis Type="Italic">Cecropia</Emphasis> trees in a tropical rainforest: support for optimal defence theory

Optimal defence theory (ODT) predicts that, whereas high risk of herbivory should select for high constitutive levels of defence, induced defences should be more advantageous in environments with a low probability of herbivory. In the present field study, conducted on the Azteca–Cecropia ant–plant system in a Neotropical rainforest, we evaluated whether the constitutive and induced ant defence of leaves are directly and inversely related to an estimate of herbivory risk, respectively. To assess the constitutive level of Azteca defence in Cecropia obtusifolia trees, we recorded the number of ants patrolling undamaged leaves. To evaluate the induced level of Azteca defence, the same leaves were subjected to simulated herbivory by punching circular holes in them. We recorded the maximum number of ants patrolling the damaged leaves from 2 to 15 min after damage. Past herbivory (% defoliation of old leaves) was assumed to indicate a risk of herbivory. Regression analyses showed that, whereas the constitutive level of ant patrolling was positively associated with the magnitude of herbivory on old leaves, there was a negative association between the magnitude of induced ant defence and past herbivory. These preliminary results lend support to ODT.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Expression of β-carotene hydroxylase gene (<Emphasis Type="Italic">crtR-B</Emphasis>) from the green alga <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> in chloroplasts of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding β-carotene hydroxylase that was able to catalyze the conversion of β-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RT-PCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.     

Carbon isotope fractionation during <Emphasis Type="Italic">cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> isomerization of unsaturated fatty acids in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cis–trans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cis–trans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cis–trans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp² carbon linkage of the cis-double bond to an intermediate sp³ within an enzyme–substrate complex. The sp² linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe³⁺), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp² linkage into sp³.     

Characterization of the complete sequences and stability of plasmids carrying the genes <Emphasis Type="Italic">aac(6′)-Ib-cr</Emphasis> or <Emphasis Type="Italic">qnrS</Emphasis> in <Emphasis Type="Italic">Shigella flexneri</Emphasis> in the Hangzhou area of China

The aim of this study was to explore the fluoroquinolone resistance mechanism of aac (6′)-Ib-cr and qnrS gene by comparing complete sequences and stability of the aac(6′)-Ib-cr- and qnrS-positive plasmids from Shigella isolates in the Hangzhou area of China. The complete sequences of four newly acquired plasmids carrying aac(6′)-Ib-cr or qnrS were compared with those of two plasmids obtained previously and two similar reference Escherichia coli plasmids. The results showed that the length, antibiotic resistance genes and genetic environment were different among the plasmids. Moreover, the plasmid stability of three wild-type isolates and five plasmid transformants carrying aac(6′)-Ib-cr and/or qnrS was measured in vitro, and all eight isolates were found to have lost their aac(6′)-Ib-cr- or qnrS-positive plasmids to a different extent at different stages. When the plasmids were electroporated into Shigella flexneri or they lost positive plasmids, the MICs of ciprofloxacin increased or decreased two- to eightfold for aac(6′)-Ib-cr-positive plasmids and 16- to 32-fold for qnrS-positive plasmids. To our knowledge, this is the first report comparing the complete sequences and describing stability for the aac(6′)-Ib-cr- and qnrS-positive plasmids from Shigella isolates.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.