The effects of phenolic acids on the photosynthetic characteristics and growth of <Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">euramericana</Emphasis> cv. ‘Neva’ seedlings

Populus × euramericana cv. ‘Neva’ is an important tree species in northern China. In the study, we used its potted oneyear- old seedlings as experimental material and established three treatments (CK, 0.5X, and 1.0X) according to the concentrations of phenolic acids in order to examine the effects of different concentrations on the photosynthetic characteristics and growth of poplar. With increasing concentrations of phenolic acids, the net photosynthetic rate, stomatal limitation, transpiration rate, apparent quantum yield, photochemical quenching coefficient, electron transport rate, chlorophyll content, and total biomass decreased significantly. The intercellular CO₂ concentration, light-compensation point, nonphotochemical quenching, malondialdehyde content, and root/shoot ratio increased significantly. Peroxidase and superoxide dismutase activities initially decreased and then increased. We concluded that phenolic acids significantly inhibited poplar’s photosynthesis and the higher phenolic acid concentration, the greater inhibition of photosynthesis occurred. This inhibition effect was mainly caused by nonstomatal factors. Phenolic acids induced noticeable photoinhibition, resulted in the irreversible damage of membrane structure, and then changed intracellular metabolic processes. To cope with phenolic acid stress, poplar seedlings increased dissipation of excess light energy and distributed relatively more biomass to underground parts within carbon allocation.     

The recovering of breeding achievements of <Emphasis Type="Italic">Populus × leningradensis</Emphasis> bogd. and <Emphasis Type="Italic">Populus × newensis</Emphasis> bogd. Based on microsatellite analysis

The genotyping of 75 trees from poplar plantations in St. Petersburg and Leningrad oblast was conducted with microsatellite markers to identify the elite clonal varieties developed by P.L. Bogdanov in the period of 1938–1965. The information about the varieties was lost. The authentic herbarium specimens of poplar clonal varieties preserved at the St. Petersburg State Forest Technical University were used as reference genotypes. According to the results of DNA fingerprinting, we identified the clonal plantations of Populus × newesis Bogd. and Populus × leningradensis Bogd. from the Kartashevskii forest district and the arboretum of the St. Petersburg State Forest Technical University. The identified elite poplar hybrids have a higher frost resistant and a higher growth rate. They are recommended for plantation cultivation in the northwest of Russia.     

The effects of <Emphasis Type="Italic">β</Emphasis>-amino-butyric acid on resistance of cucumber against root-knot nematode, <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

In this study, the effects of β-amino-butyric acid (BABA) on root-knot nematode(Meloidogyne javanica) infection of cucumber and accumulation of total phenolic compounds, hydrogen peroxide and activity of some enzymes related to plant defense mechanisms, i.e., guaiacol peroxidase (GPOX), polyphenol oxidase (PPO), catalase (CAT) in cucumber roots infected with nematode were investigated. Results of this study show that treating the cucumber seedlings with the above elicitor significantly reduces the nematode infection level (the nematode galls, number of egg masses per plant and number of eggs per individual egg mass) compared to control. Additionally, treatment of cucumber roots by BABA and BABA + nematode, significantly increased peroxidase, polyphenol oxidase and catalase activities in root tissues, 1 day after nematode inoculation in comparison to nematode inoculated plants as control and sterile water-treated plants. Enzyme activities reached to a maximum level at 4, 4 and 3 days after nematode inoculation, respectively. Additionally, the amount of H₂O₂, a product of oxidative stress, was significantly increased in the BABA and BABA + nematode treatments in comparison to control. Such increases have occurred in two phases and maximum levels of it were observed at 5 days after inoculation. Inoculation of cucumber plants by BABA also significantly increased accumulation of total phenol in comparison to control and maximum level of it was observed at 7 days after nematode inoculation. The results suggest that the inhibitory effect of BABA on the root-knot nematode (M. javanica) may be related to its ability to enhance defense responses in the cucumber roots.     

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in <Emphasis Type="Italic">Mentha</Emphasis> × <Emphasis Type="Italic">piperita</Emphasis> cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g⁻¹ DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g⁻¹ DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.     

<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.     

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.     

Proteins responding to drought and high-temperature stress in <Emphasis Type="Italic">Populus </Emphasis>× <Emphasis Type="Italic">euramericana</Emphasis> cv. ‘74/76’

Proteomic analysis provides a powerful method of studying plant responses to stress at the protein level. In order to study stress-responsive molecular mechanisms for Populus × euramericana cv. ‘74/76’, one of the most important forest plantation tree species in subtropical and temperate regions, we analyzed the response of 2-year-old cuttings of P. × euramericana cv. ‘74/76’ to drought and high temperature using two-dimensional gel electrophoresis. More than 1,000 reproducible leaf proteins were detected in the controls and treatments, and 26 proteins were found to change notably in abundance. We identified 13 proteins affected by drought stress and 11 proteins affected by high temperature. These proteins are mainly involved in photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and putative photosystem I reaction center subunit II precursor, and detoxification (manganese superoxide dismutase and methionine sulfoxide reductase A). Furthermore, the level of the photosynthesis proteins affected greatly by the imposed stress conditions was consistent with the observed noticeable decrease in net photosynthesis rate. These studies provides a fundamental data for future research on responses to drought and high temperature, two major factors limiting the growth of forest trees during summer under recent climatic warming.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Genetic mapping in (<Emphasis Type="Italic">Populus tomentosa</Emphasis> ×<Emphasis Type="Italic"> Populus bolleana</Emphasis>) and<Emphasis Type="Italic"> P. tomentosa</Emphasis> Carr. using AFLP markers

The AFLP genetic linkage maps for two poplar cultivars were constructed with the pseudo-test-cross mapping strategy. The hybrids were derived from an interspecific backcross between the female hybrid clone TB01 (Populus tomentosa × Populus bolleana) and the male clone LM50 (P. tomentosa). A total of 782 polymorphic fragments were obtained with a PCR-based strategy using 49 enzyme-nested (EcoRI/MseI) primer combinations. Six hundred and thirty two of these fragments segregated in a 1:1 ratio (P<0.01), indicating that these DNA polymorphisms are heterozygous in one parent and null in the other. The linkage analysis was performed using Mapmaker version 3.0 with LOD 5.0 and a maximum recombination fraction () of 0.3. Map distances were estimated using the Kosambi mapping function. In the framework map for LM50 (P. tomentosa), 218 markers were aligned in 19 major linkage groups. The linked loci spanned approximately 2,683 cM of the poplar genome, with an average distance of 12.3 cM between adjacent markers. For TB01 (P. tomentosa × P. bolleana), the analysis revealed 144 loci, which were mapped to 19 major linkage groups and covered about 1,956 cM, with an average distance of 13.6 cM between adjacent markers. These maps covered about 87% and 77% of the estimated genome size of parents LM50 and TB01, respectively. The maps developed in this study lay an important foundation for future genomics research in poplar, providing a means for localizing genes controlling economically important traits in P. tomentosa.Communicated by O. Savolainen     

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.     

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.     

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.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.