Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

Adsorption of lead(II) from aqueous solution onto Hydrilla verticillata

The adsorption of Pb(II) onto Hydrilla verticillata was examined in aqueous solution with parameters of pH, adsorbent dosage, contact time and temperature. The linear Langmuir and Freundlich models were applied to describe equilibrium isotherms, and both models fitted well. The monolayer adsorption capacity of Pb(II) was found as 104.2 mg/g at pH 4 and 25°C. Dubinin–Radushkevich (D–R) isotherm model was also applied to the equilibrium data. The mean free energy of adsorption (15.81 kJ/mol) indicated that the adsorption of Pb(II) onto H. verticillata may be carried out via chemical ion-exchange mechanism. Thermodynamic parameters, free energy (ΔG ⁰), enthalpy (ΔH ⁰) and entropy (ΔS ⁰) of adsorption were also calculated. These parameters showed that the adsorption of Pb(II) onto H. verticillata was a feasible, spontaneous and exothermic process in nature. The influence of Cd²⁺, Cu²⁺ and Ni²⁺ on adsorption of Pb²⁺ onto H. verticillata was studied, too. In the investigated range of operating conditions, it was found that the existence of Cd ²⁺, Cu ²⁺ and Ni ²⁺ had no impact on the adsorption of Pb²⁺.     

Effect of 24-Epibrassinolide on Chlorophyll Fluorescence and Photosynthetic CO<Subscript>2</Subscript> Assimilation in <Emphasis Type="Italic">Vicia faba</Emphasis> Plants Treated with the Photosynthesis-Inhibiting Herbicide Terbutryn

Simultaneous measurements of chlorophyll (Chl) fluorescence and CO₂ assimilation (A) in Vicia faba leaves were taken during the first weeks of growth to evaluate the protective effect of 24-epibrassinolide (EBR) against damage caused by the application of the herbicide terbutryn (Terb) at pre-emergence. V. faba seeds were incubated for 24 h in EBR solutions (2 × 10⁻⁶ or 2 × 10⁻⁵ mM) and immediately sown. Terb was applied at recommended doses (1.47 or 1.96 kg ha⁻¹) at pre-emergence. The highest dose of Terb strongly decreased CO₂ assimilation, the maximum quantum yield of PSII photochemistry in the dark-adapted state (F _V/F _M), the nonphotochemical quenching (NPQ), and the effective quantum yield (ΔF/F′_M) during the first 3–4 weeks after plant emergence. Moreover, Terb increased the basal quantum yield of nonphotochemical processes (F ₀/F _M)_, the degree of reaction center closure (1 − q _p), and the fraction of light absorbed in PSII antennae that was dissipated via thermal energy dissipation in the antennae (1 − F′_V/F′_M). The herbicide also significantly reduced plant growth at the end of the experiment as well as plant length, dry weight, and number of leaves. The application of EBR to V. faba seeds before sowing strongly diminished the effect of Terb on fluorescence parameters and CO₂ assimilation, which recovered 13 days after plant emergence and showed values similar to those of control plants. The protective effect of EBR on CO₂ assimilation was detected at a photosynthetic photon flux density (PFD) of 650 μmol m⁻² s⁻¹ and the effect on ΔF/F′_M and photosynthetic electron transport (J) was detected under actinic lightings up to 1750 μmol m⁻² s⁻¹. The highest dose of EBR also counteracted the decrease in plant growth caused by Terb, and plants registered the same growth values as controls.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

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.     

A preliminary comparison of the mariculture potential of Porphyra purpurea and Porphyra umbilicalis

Due to their rapid growth and nutrient assimilation,Porphyra spp. are good candidates for bioremediation and polyculture. The production potential of two strains of P. purpurea and P. umbilicalis from north-east USA was evaluated by measuring rates of photosynthesis (as O₂evolution) of material grown at 20 ^°C. Photosynthetic rates of P. umbilicalis were 80%higher than P. purpurea over the temperature range 5–20 ^°C, at both sub-saturating andsaturating irradiances (37 and 289 μmol photonm^-2 s^-1). Porphyra umbilicalis was more efficient at low irradiances (higher α) and had a higher P_max (23.0 vs 15.6 μmolO₂ g^-1 DW min^-1) than P.purpurea, suggesting that P. umbilicalis is a better choice for mass culture, where self-shading maybe severe. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> Swartz)

Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical, and subtropical regions. A callus induction and plant regeneration protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg l⁻¹ 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.1 or 1.0 mg l⁻¹ benzylaminopurine (BA). A multifactorial experiment demonstrated the combination of 3 mg l⁻¹ dicamba and 1.0 mg l⁻¹ BA for induction of callus resulted in 12 times higher plant regeneration frequency compared to 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone or ten times higher plant regeneration frequency than the combination of 3 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ BA. These results are expected to support the development of a genetic transformation protocol for seashore paspalum.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Does carbon isotope discrimination correlate with biological nitrogen fixation?

It has recently been reported that N₂ fixation and carbon isotope discrimination (Δ) are negatively correlated. To further test this hypothesis, a greenhouse experiment was conducted to investigate if Δ is correlated with the efficiency of lentil (Lens culinaris cv Laird) in fixing atmospheric nitrogen. Lentil seed was inoculated with one of 10 Rhizobium leguminosarum strains that varied in their effectiveness in symbiotic N₂ fixation. Carbon-13 discrimination was positively correlated with N₂ fixation (r²=0.60^*). Although the amount of N₂ fixed ranged from 1.5 mg N to 13.5 mg N shoot⁻¹, the range of Δ values was only 25.8 to 26.6%.. It is unlikely that variability of such small magnitude could be of any practical use in selecting for N₂-fixing efficiency.     

Effects of the invasive plant Mikania micrantha H.B.K. on soil nitrogen availability through allelopathy in South China

Allelopathy has been regarded as a mechanism for successful exotic plant invasion. However, it is not clear if and what effects of allelopathic substances may exert on soil nutrient. The exotic plant Mikania micrantha H.B.K. (M. micrantha) has invaded many forests in south China, and recent studies have suggested it has allelopathic potential for other plants and soil microbial community. Thus, we hypothesized that M. micrantha could influence soil nutrients and N transformation through allelopathy. We measured total C and N, NO₃ ⁻, NH₄ ⁺ and pH of the soil beneath M. micrantha and the adjacent open soil, and then measured the above soil properties after treating soil with 3 concentrations of aqueous extracts of M. micrantha (T₁: 0.005 g ml⁻¹; T₂: 0.025 g ml⁻¹; T₃: 0.100 g ml⁻¹). In addition, a bioassay was conducted to determine the allelopathic potential of the soil beneath M. micrantha. The results showed that M. micrantha significantly affected soil nutrients and N transformation. Soil beneath M. micrantha had inhibitory effects on seed germination and seedling growth of test plant, and had significantly higher C, N, ammonia, net nitrification rate than those of open soil. The plant extracts decreased soil pH, and T₁ decreased it the most, and it increased soil C and N, and T₁ represented the greatest increase in both C and N. The extracts also increased both NO₃ ⁻ and NH₄ ⁺ in soil, whereas no significant difference existed among the 3 extract treatments. Compared to the water control, the soil net mineralization rate was higher under T₁, while lower under T₂ and T₃. However, the extracts increased the soil nitrification rates under all the treatments (T₁, T₂ and T₃). Our results suggest that the water soluble allelochemicals of M. micrantha improve soil nutrient availability, through which the invasive plant M. micrantha may successfully invade and establish in new habitats.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Purification and characterization of xylanases from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> and its mutant derivative possessing novel kinetic and thermodynamic properties

Xylanases produced from a locally isolated strain of Thermomyces lanuginosus and its mutant derivative were purified to a yield of 39.1 and 42.83% with specific activities of 15,501 and 17,778 IU mg⁻¹ protein, respectively. The purification consisted of two steps i.e., ammonium sulphate precipitation, and gel filtration chromatography. The mutant enzyme showed high affinity for substrate, with a K _m of 0.098 mg ml⁻¹ as compared to wild type enzyme showing K _m of not less than 0.112 mg ml⁻¹. It was found that pH values of 8.1 and 7.3 were best for activity of the mutant and wild-type-derived enzymes, respectively. The values of pK _a of the acidic limbs of both enzymes were the same (5.0 and 4.9, respectively) but the pK _a value of the basic limb was slightly increased, indicating the participation of a carboxyl group present in a non-polar environment. Temperatures of 70 and 65°C were found optimal for mutant and wild-derived xylanase, respectively. Enzymes displayed a high thermostability showing a half life of 31.79 and 6.0 min (5.3-fold improvement), enthalpy of denaturation (ΔH*) of 146.06 and 166.95 kJ mol⁻¹, entropy of denaturation (ΔS*) of 101.44 and 174.67, and free energy of denaturation (ΔG*) of 110.25 and 105.29 kJ mol⁻¹ for mutant- and wild-organism derived enzyme, respectively at 80°C. Studies on the folding and stability of cellulase-less xylanases are important, since their biotechnological employments require them to function under extreme conditions of pH and temperature. The kinetic and thermodynamic properties suggested that the xylanase from the mutant organism is better as compared to xylanase produced from the wild type and previously reported strains of same species, and may have a potential usage in various industrial fields.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Carbon addition interacts with water availability to reduce invasive forb establishment in a semi-arid grassland

Increases in nitrogen (N) availability can favor fast-growing invasive species over slow-growing native species. One way to reduce N availability is to add labile carbon (C) to the soil, which can lead to microbial immobilization of plant available N. This method has been used, with widely varying degrees of success, to both study and control plant invasions. One reason that C addition might not work as expected is that N is not always the limiting resource for plant growth. For example, if plant growth is limited by water, changes in N availability might have little effect on invasion. Here I ask whether effects of C addition on N availability, resident plant biomass, and invasion depend on water availability in semi-arid mixedgrass prairie. Six invasive species were seeded into plots treated with a factorial combination of water (ambient or added) and N (+C, control or +N). Carbon addition reduced capture of mineral N by resin probes (by an average of 73%), and reduced biomass of resident species (from 336 g m⁻² to 203 g m⁻²), both with and without added water. In contrast, because there was little invasion in ambient-water plots, C addition reduced invasion only in added-water plots. Given added water, C addition reduced biomass of Centaurea diffusa by 95%, and prevented invasion by Gypsophila paniculata and Linaria dalmatica. Mechanisms by which C addition reduced invasion varied by species, with added C reducing the growth of individual C. diffusa plants, but reducing numbers of G. paniculata and L. dalmatica individuals.     

Croatian barberry (<Emphasis Type="Italic">Berberis croatica</Emphasis> Horvat): a new source of berberine—analysis and antimicrobial activity

By using HPLC/UV–VIS, Croatian barberry (Berberis croatica Horvat) was found to be a new source of the bioactive alkaloid berberine. Comparison of berberine content in roots, leaves, and twigs between wild specimens of B. croatica and B. vulgaris collected in Croatia showed that the roots of both species contained the highest berberine content (B. croatica 1.120–1.217%; B. vulgaris 0.805–1.424%), followed by twigs (B. croatica 0.049–0.216%; B. vulgaris 0.077–0.112%). While the berberine content in the leaves of both species was very low (between 0.002% and 0.044%), they were found to be rich in phenols and flavonols. The Student’s t-test showed a significant difference at P < 0.05 for phenol and flavonol content in the plant organs, both between species and within species. Leaf samples were most variable, while root samples were the least. Extracts from the roots of both barberry species expressed antimicrobial activity against Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10535, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231. Antimicrobial activity of leaf extracts was species-dependent. Root extracts of both species also showed lower MIC values than other extracts (MIC ≤ 87.5 mg/ml).