Does sequence polymorphism of <Emphasis Type="Italic">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Photoinhibition in seedlings of <Emphasis Type="Italic">Fraxinus</Emphasis> and <Emphasis Type="Italic">Fagus</Emphasis> under natural light conditions: implications for forest regeneration?

Ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.) seedlings were grown in the field under three levels of natural light: (1) open, (2) gap and (3) shade. Light acclimation of photosynthesis was characterized by means of modulated chlorophyll a fluorescence of intact leaves and growth parameters were measured at the end of the growing season. Measurements of maximum photochemical efficiency (F_v/F_m) of dark-adapted leaves at intervals through the day showed that ash had a higher F_v/F_m than beech in open and gap plots but not in shade plots. This indicated a larger build-up of photoinhibition in beech under gap and open conditions. Steady-state light response curves of the operating efficiency of PSII (F_q/F_m), the electron transport rate (ETR) and the photochemical efficiency factor (F_q/F_v) showed greater variability across light treatments in ash than in beech. Both species exhibited similar responses of non-photochemical quenching (NPQ) to light. When the data were normalized to the mean maximum irradiance in the growth environment, all photochemical parameters showed a reduction in variation across treatments, indicating that light acclimation in the two species occurred primarily through adjustments in rates of photochemistry. Adjustments in thermal heat dissipation were small in both species. This pattern was stronger in ash, suggesting a greater degree of phenotypic plasticity in photosynthetic capacity in this earlier successional species. Contrary to our expectations, the build-up of photoinhibition in beech did not appear to have a negative effect on total biomass accumulation relative to ash.Abbreviations ETR Electron transport rate - F_m Maximal fluorescence in the dark-adapted state - F_o Minimal fluorescence in the dark-adapted state - F_s Steady-state fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in the dark-adapted state - F_v/F_m Maximum photochemical efficiency of photosystem II in the dark-adapted state - F_m Maximal fluorescence in actinic light - F_o Minimal fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in actinic light - F_q=F_m–F_s; F_q/F_m Operating efficiency of photosystem II in actinic light - F_q/F_v Efficiency factor of PSII photochemistry (also referred to as qP—photochemical quenching) - F_v/F_m Maximum efficiency of PSII under actinic light if all reaction centres were open - NPQ Stern-Volmer non-photochemical quenching - PPFD Photosynthetic photon flux density (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a cosine-corrected quantum sensor - PPFFR Photosynthetic photon flux fluence rate (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a spherical quantum sensor. Fluorescence nomenclature follows Oxborough and Baker (2000).     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

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.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Pollinator community structure and sources of spatial variation in plant–pollinator interactions in <Emphasis Type="Italic">Clarkia xantiana</Emphasis> ssp. <Emphasis Type="Italic">xantiana</Emphasis>

The structure of diverse floral visitor assemblages and the nature of spatial variation in plant–pollinator interactions have important consequences for floral evolution and reproductive interactions among pollinator-sharing plant species. In this study, I use surveys of floral visitor communities across the geographic range of Clarkia xantiana ssp. xantiana (hereafter C. x. xantiana) (Onagraceae) to examine the structure of visitor communities, the specificity of the pollination system, and the role of variation in the abiotic vs. biotic environment in contributing to spatial variation in pollinator abundance and community composition. Although the assemblage of bee visitors to C. x. xantiana is very diverse (49 species), few were regular visitors and likely to act as pollinators. Seventy-four percent of visitor species accounted for only 11% of total visitor abundance and 69% were collected in three or fewer plant populations (of ten). Of the few reliable visitors, Clarkia pollen specialist bees were the most frequent visitors, carried more Clarkia pollen compared to generalist foragers, and were less likely to harbor foreign pollen. Overall, the core group of pollinators was obscured by high numbers of incidental visitors that are unlikely to contribute to pollination. In a geographic context, the composition of specialist pollinator assemblages varied considerably along the abiotic gradient spanning the subspecies range. However, the overall abundance of specialist pollinators in plant populations was not influenced by the broad-scale abiotic gradient but strongly affected by local plant community associations. C. x. xantiana populations sympatric with pollinator-sharing congeners were visited twice as often by specialists compared to populations occurring alone. These positive indirect interactions among plant species may promote population persistence and species coexistence by enhancing individual reproductive success.Electronic Supplementary Material Supplementary material is available for this article at     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

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.     

Assessment of conditions affecting <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">rhizogenes</Emphasis>-mediated transformation of soybean

Agrobacterium rhizogenes-mediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. This transformation method is particularly suitable for those plants, including legumes, whose transformation using Agrobacterium tumefaciens has been challenging. Although there are some reports on A. rhizogenes-mediated transformation of legumes to produce ‘composite’ plants, conditions influencing A. rhizogenes-mediated transformation of soybean [Glycine max (L.) Merr.] have not been yet fully investigated. To better understand A. rhizogenes-mediated root transformation in soybean, we have evaluated the impact of genotype, plant age for infection, bacterial inoculating concentration, inoculation temperature, and other factors on transformation of soybean. The results have shown that there are significant differences among soybean genotypes in their susceptibility to A. rhizogenes. Soybean cv. Zigongdongdou is the most susceptible to A. rhizogenes strain K599 among 10 genotypes tested. The effects of seedling age have been evaluated, and 1-day-old plantlets are found to be optimal for hairy root induction. There are no significant differences in hairy root induction for bacterial suspension from OD₆₀₀ = 0.2 to OD₆₀₀ = 1.2. Under 16 h photoperiod, hairy roots can be induced both at 23°C/20°C and 28°C/25°C, but not at 33°C/30°C as day/night temperature regimes. Using this transformation protocol, almost 100% of the composite plants formed hairy roots within 2 weeks, and based on GUS histochemical analysis, 94.2% transformation frequency is obtained. Transgene integration has been also confirmed by Southern blot analysis. D. Cao and W. Hou contributed equally to this work.     

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.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

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.     

Distribution of <Emphasis Type="Italic">S</Emphasis> haplotypes and its relationship with restorer–maintainers of self-incompatibility in cultivated <Emphasis Type="Italic">Brassica napus</Emphasis>

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

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.