Genetic transformation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> with <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A₄GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B₅ medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 ( and ) and the GM2 ( and DNA) type clones in considerably higher amount whether as two but callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B₅ vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B₅ media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the T_L-DNA and T_R-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

Report on non-temperature related variations in CO<Subscript>2</Subscript> efflux rates from young tree stems in the dormant season

Respiration rates are reported to increase exponentially with temperature. Respiration rates of woody tissues are commonly measured as CO₂ efflux rates () from that tissue. However, this paper describes clear variations in stem that were not related to temperature for the case of a young beech (Fagus sylvatica L.) and oak (Quercus robur L.) tree during the dormant season. The CO₂ concentration ([CO₂]) in the xylem of the beech tree showed similar temperature-independent variations. The trees were grown in a growth chamber in which radiation patterns and temperature were kept constant. was measured with an IRGA connected to cuvettes surrounding a stem segment. Xylem [CO₂] was measured in situ using a CO₂ microelectrode. Depressions in and [CO₂] occurred during the light period, despite equal temperatures in the light and dark period. Explanations found in literature for discrepancies in the exponential relationship between temperature and are the influence of (1) sap flow or (2) decreased cell water content. However, (1) the variations were observed in the dormant season, when no sap flow was observed yet, and (2) reduced cell water content was not likely to be apparent as differences in stem transpiration rates between the dark and light period were not significant. Hence, previously formulated theories failed to explain our results. This work therefore provides a new ground for discussion on other possible causes of daytime depressions in . One might be the refixation of respired CO₂ by corticular photosynthesis in the stem parts adjacent to the stem segment enclosed by the cuvette.     

Inheritance of foliar stable carbon isotope discrimination and third-year height in Pinus taeda clones on contrasting sites in Florida and Georgia

Quantifying foliar stable carbon isotope discrimination (Δ) is a powerful approach for understanding genetic variation in gas exchange traits in large populations. The genetic architecture of Δ and third-year height is described for more than 1,000 clones of Pinus taeda tested on two contrasting sites. for Δ was 0.14 (±0.03), 0.20 (±0.07), and 0.09 (±0.04) at Florida, Georgia, and across sites, respectively. for stable carbon isotope discrimination ranged from 0.25 (±0.03) at the Florida site to 0.33 (±0.03) at the Georgia site, while the across-site estimate of was 0.19 (±0.02). For third-year height, ranged from 0.13 (±0.05) at the Georgia site to 0.20 (±0.06) at the Florida site with an across-site estimate of 0.09 (±0.05). Broad-sense heritability estimates for third-year height were 0.23 (±0.03), 0.28 (±0.03), and 0.13 (±0.02) at the Florida site, Georgia site, and across sites, respectively. Type B total genetic correlation for Δ was 0.70 ± 0.06, indicating that clonal rankings were relatively stable across sites, while for third-year height, rankings of clones were more unstable across the two trials . Third-year height and Δ were negatively correlated at the parental , full-sib family , and clonal levels, suggesting that genetic variation for Δ in P. taeda may be a result of differences in photosynthetic capacity. We conclude that Δ may be a useful selection trait to improve water-use efficiency and for guiding deployment decisions in P. taeda.     

Effects of common soil anions and pH on the uptake and accumulation of perchlorate in lettuce

A mechanistic understanding of perchlorate () entry into plants is important for establishing the human health risk associated with consumption of contaminated produce and for assessing the effectiveness of phytoremediation. To determine whether common soil anions affect uptake and accumulation in higher plants, a series of competition experiments using lettuce (Lactuca sativa L.) were conducted between (50 nM) and (4–12 mM), (1–10 mM), or Cl⁻ (5–15 mM) in hydroponic solution. The effects of (0–5 mM) and pH (5.5–7.5) on uptake were also examined. Increasing in solution significantly reduced the amount of taken up by green leaf, butter head, and crisphead lettuces. Sulfate and Cl⁻ had no significant effects on uptake in lettuce over the concentrations tested. Increasing pH significantly reduced the amount of taken up by crisphead and green leaf lettuces, whereas increasing significantly reduced uptake in butter head lettuce. The inhibition by across all lettuce genotypes suggests that may share an ion carrier with , and the decrease in uptake with increasing pH or provides macroscopic evidence for cotransport across the plasma membrane.     

Genetic structure of an insect-pollinated and bird-dispersed tropical tree in vegetation fragments and corridors: implications for conservation

In the vegetation corridors that connect small remnants of undisturbed primary forest in the Lavras landscape (Brazil), Protium spruceanum is a representative of a mass-flowering insect-pollinated and bird-dispersed tree. Allozyme variation was quantified from five forest remnants (N = 150) from secondary vegetation corridors linking them (N = 80) to generate information for genetic conservation. The species adhered to H-W equilibrium in all fragments in most of the loci. The results indicated high gene diversity in the fragments and corridors positively correlated with the plant density (r = 0.742, R ² = 0.551, d.f. = 4). We did not find evidence of inbreeding within fragments nor overall The genetic differentiation among remnants was low Evidence of recent bottlenecks by anthropogenic disturbance was detected in fragments (P < 0.05, Wilcoxon sign-rank test). The minimal viable population was estimated for conservation in situ, indicating fragments with possibilities of maintaining genetic equilibrium diversity in the short term (except F3) and in the long term (only F5). The ratios was also calculated to contribute to vegetation enrichment, area recovery or creation of new vegetation corridors. We found high levels of gene diversity in the vegetation corridors, genetic identity with the fragments and absence of inbreeding. Thus, our results suggest that landscape management strategies should therefore consider both the creation of new vegetation corridors and the protection of extant ones.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

Bifurcation Analysis of Genetically Engineered Pacemaking in Mammalian Heart

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I _K1), or insertion of the hyperpolarisation-activated funny current (I _f). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance () of I _K1 and I _f as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As _K1 is decreased by ≈74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of _f induced periodicity via a subcritical Hopf bifurcation at _f≈ 0.25 mSμF⁻¹. Stable autorhythmic action potentials occurred at _f > 0.329 mSμF⁻¹.     

Sources of variability in spotted owl population growth rate: testing predictions using long-term mark–recapture data

For long-lived iteroparous vertebrates that annually produce few young, life history theory predicts that reproductive output (R) and juvenile survival should influence temporal variation in population growth rate (λ) more than adult survival does. We examined this general prediction using 15 years of mark–recapture data from a population of California spotted owls (Strix occidentalis occidentalis). We found that survival of individuals ≥1 year old (ϕ) exhibited much less temporal variability , where CV is coefficient of variation, than R and that R was strongly influenced by environmental stochasticity. Although λ was most sensitive ( ; log-transformed sensitivity) to ϕ and much less sensitive to either R or juvenile survival (survival rate of owls from fledging to 1 year old; ), we estimated that R contributed as much as ϕ to the observed annual variability in λ. The contribution of juvenile survival to variability in λ was proportional to its These results are consistent with the hypothesis that natural selection may have favored the evolution of longevity in spotted owls as a strategy to increase the probability of experiencing favorable years for reproduction. Our finding that annual weather patterns that most affected R (temperature and precipitation during incubation) and ϕ (conditions during winter related to the Southern Oscillation Index) were equally good at explaining temporal variability in λ supports the conclusion that R and ϕ were equally responsible for variability in λ. Although currently accepted conservation measures for spotted owl populations attempt to enhance survival, our results indicated that conservation measures that target R may be as successful, as long as actions do not reduce ϕ.     

Homology Modeling of Hemagglutinin/Protease [HA/P (vibriolysin)] from <Emphasis Type="Italic">Vibrio Cholerae</Emphasis>: Sequence Comparision,Residue Interactions and Molecular Mechanism

Vibrio cholerae produces a zinc-containing and calcium-stabilized soluble hemagglutinin/protease, which has been earlier shown to have the ability to cleave several physiologically important substrates including mucin, fibronectin and lactoferin. This study presents homology modeling of hemagglutinin/protease (vibriolysin) from Vibrio cholerae in the presence of inhibitor HPI [N-(1-carboxy-3-phenylpropyl)-phenylalanyl-alpha-aspargine]. The 3D structure was predicted based on its sequence homology with Pseudomonas aeruginosa elastase (PAE). Comparison of the 3D structures of PAE and HA/P reveals a remarkable similarity having a conserved α + β domain. The inhibitor shows similar binding features as seen in other metalloproteases of M4 peptidase family. The study also highlights the key catalytic residues as well as the residues at the S₁ and binding sub-sites. The similarities between the two proteins provide support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. The fact that both enzymes are secreted as zinc-containing proteases, led us to further hypothesize that they may play similar role in pathogenesis.     

A bias correction for estimates of effective population size based on linkage disequilibrium at unlinked gene loci*

Analysis of linkage disequilibrium (=mean squared correlation of allele frequencies at different gene loci) provides a means of estimating effective population size (N _e) from a single sample, but this method has seen much less use than the temporal method (which requires at least two samples). It is shown that for realistic numbers of loci and alleles, the linkage disequilibrium method can provide precision comparable to that of the temporal method. However, computer simulations show that estimates of N _e based on for unlinked, diallelic gene loci are sharply biased downwards ( in some cases) if sample size (S) is less than true N _e. The bias is shown to arise from inaccuracies in published formula for when S and/or N _e are small. Empirically derived modifications to for two mating systems (random mating and lifetime monogamy) effectively eliminate the bias (residual bias in % in most cases). The modified method also performs well in estimating N _e in non-ideal populations with skewed sex ratio or non-random variance in reproductive success. Recent population declines are not likely to seriously affect , but if N has recently increased from a bottleneck can be biased downwards for a few generations. These results should facilitate application of the disequilibrium method for estimating contemporary N _e in natural populations. However, a comprehensive assessment of performance of with highly polymorphic markers such as microsatellites is needed.The US Governmentȁ9s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Improving heterologous polyketide production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by overexpression of an <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l⁻¹ . In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.     

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4 sm -haplotype of Japanese pear

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, ‘Osa-Nijisseiki’, which is a bud mutant of ‘Nijisseiki’ (S ₂ S ₄), has a stylar-part mutant -haplotype, which lacks the S ₄-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the -haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S ₄-homozygote, and assembled a BAC contig of 570 kb around the S ₄-RNase. Genomic PCR of DNA from S ₄- and -homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S ₄-RNase. The -haplotype lacks 34 predicted open reading frames (ORFs) including the S ₄-RNase and a pollen-specific F-box protein gene (termed as S ₄ F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the -haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the -haplotype. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-term changes in nutrient concentrations of the Changjiang River and principal tributaries

We present long-term nutrient data on the Changjiang River (Yangtze River) at six hydrological stations and eight principal tributaries during the period 1958–1985. Three patterns of temporal changes were observed in nitrate and nitrite : minimal variations in the upper catchment area, rapid increases in the middle watershed towards the end of the 1970s, and a gradual increase in the lower drainage basin. Prior to the 1970s, the level of throughout the Changjiang River system remained fairly constant. In the 1980s, however, this changed, with the lowest values in the upper Changjiang changing rapidly to the highest in the middle reaches and then declining slowly but steadily in the lower courses. Compared to and ammonium and soluble reactive phosphorus (SRP) showed smaller increases or no long-term variations, while dissolved silica (DSi) concentration generally decreased at most stations. These three patterns of and changes in the Changjiang River system were reflective of the difference in chemical fertilizer use and landscape features (e.g., slope, soil type and water body area) of the drainage basins of the primary tributaries. The decreases in DSi were most likely attributed to a reduction in suspended sediment loading due to dam constructions and increasing diatom consumption. The increase in and with a reduction in DSi concentrations in the Changjiang River could have significant effects on the stoichiometric balance of nutrients delivered to the East China Sea and the ecosystem in this dynamic region.     

Anaerobic Ammonium Oxidation (Anammox) in Chesapeake Bay Sediments

Anaerobic ammonium oxidation (anammox) has recently been recognized as a pathway for the removal of fixed N from aquatic ecosystems. However, the quantitative significance of anammox in estuarine sediments is variable, and measurements have been limited to a few estuaries. We measured anammox and conventional denitrification activities in sediments along salinity gradients in the Chesapeake Bay and two of its sub-estuaries, the Choptank River and Patuxent River. Homogenized sediments were incubated with ^14/15N amendments of , , and to determine relative activities of anammox and denitrification. The percent of N₂ production due to anammox (ra%) ranged from 0 to 22% in the Chesapeake system, with the highest ra% in the freshwater portion of the main stem of upper Chesapeake Bay, where water column concentrations are consistently high. Intermediate levels of relative anammox (10%) were detected at locations corresponding to tidal freshwater and mesohaline locations in the Choptank River, whereas anammox was not detected in the tidal freshwater location in the Patuxent River. Anammox activity was also not detected in the seaward end of Chesapeake Bay, where water column concentrations are consistently low. The ra% did not correlate with accumulation rate in anoxic sediment incubations, but ra% was related to water column concentrations and salinity. Anammox bacterial communities were also examined by amplifying DNA extracted from the upper Chesapeake Bay sediment with polymerase chain reaction (PCR) primers that are specific for 16S rRNA genes of anammox organisms. A total of 35 anammox-like sequences were detected, and phylogenetic analysis grouped the sequences in two distinct clusters belonging to the Candidatus “Scalindua” genus.     

Protective Effect of Nitric Oxide against Oxidative Damage in <Emphasis Type="Italic">Arabidopsis</Emphasis> Leaves under Ultraviolet-B Irradiation

Nitric oxide (NO) is a key molecule involved in many physiological processes. To characterize its roles in the tolerance of Arabidopsis thaliana to ultraviolet-B (UV-B), we investigated the effect of a reduced endogenous NO level on oxidative damage to wild-type and mutant (Atnoa1) plants. Under irradiation, hydrogen peroxide was accumulated more in mutant leaves than in the wild type. However, the amounts of UV-B-absorbing compounds (flavonoids and anthocyanin) and the activities of two antioxidant enzymes—catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11)—were lower in leaves of the former. Supplementing with sodium nitroprusside, an NO donor, could alleviate the oxidative damage to mutant leaves by increasing flavonoid and anthocyanin contents and enzyme activities. In comparison, , an inhibitor of nitric oxide synthase, had the opposite effects on oxidation resistance in wild-type leaves. All these results suggest that nitric oxide acts as a signal for an active oxygen-scavenging system that protects plants from oxidative stress induced by UV-B irradiation.     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.