Expression of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis> α-glucosidase in<Emphasis Type="Italic"> Lactococcus lactis</Emphasis>

The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable -glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic -glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 °C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of -glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.     

Screening interspecific hybrids of<Emphasis Type="Italic"> Populus</Emphasis> (<Emphasis Type="Italic">P. ciliata</Emphasis> ×<Emphasis Type="Italic"> maximowiczii</Emphasis>) using AFLP markers

Hybrids of Populus ciliata × maximowiczii are very vigorous and outperform both the parents in growth performance and yield. Genetic evaluation of 24 of these interspecific hybrids along with the two mother trees (Populus ciliata), and five male-parent (Populus maximowiczii) genotypes was carried out using the AFLP marker assay. Eight AFLP primer combinations detected 428 markers, of which 280 (66%) were polymorphic. Genetic relationships within the samples were evaluated by generating the similarity matrix based on Jaccards coefficient. The phenetic dendrograms, as well as the PCO plots, separated the hybrids and the two parent species into three distinct clusters. The hybrids grouped closer to the P. ciliata (female parent) cluster as compared to the P. maximowiczii (male parent) cluster. The hybrid cluster contained internal groupings, which correlated to some extent with growth performance. The four best performing hybrids (42m1, 65m1, 23m2, Cm2-5-20/91) formed a distinct sub-cluster. Data from a single primer combination was sufficient for distinguishing the hybrids from the parents and assigning paternity. The hybrids showed 22 markers that were absent in P. ciliata but were monomorphically present in all the hybrids, suggesting outcrossing and common paternity. Further, these 22 markers were found in all the P. maximowiczii genotypes confirming it as the male parent. These male-specific markers can be converted to SCAR markers and used for rapid screening of the P.ciliata × maximowiczii hybrids. The primer combination E-AAC × M-CAA was identified as most suitable for ascertaining true hybridity. AFLP proves to be a useful tool for screening of P. ciliata × maximowiczii hybrids at the early stages of development.Communicated by H.F. Liskens     

<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.     

Micropropagation of<Emphasis Type="Italic"> Thapsia garganica</Emphasis>—a medicinal plant

The micropropagation of T. garganica, a medicinally important plant, was investigated as an option for conservation purposes as wild populations are becoming sparse. A Murashige and Skoog agar medium supplemented with 0.5 mg l(-1) alpha-naphthaleneacetic acid and 1.5 mg l(-1) benzyl-6-adenine significantly improved the production of multiple shoots directly from petiole and leaflet explants compared to other plant growth regulator (PGR) combinations. Medium free of PGRs promoted rooting at a low incidence. Acclimatisation was low--52% plantlet survival--as micropropagated plantlets were highly susceptible to fungal rot once removed from culture. Application of antifungal agents to in vitro-derived plantlets as pre- and post-acclimatisation treatments during transplanting significantly reduced ex vitro mortality.     

Molecular population genetics of the<Emphasis Type="Italic">β-esterase</Emphasis> gene cluster of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We have investigated nucleotide polymorphism at theβ-esterase gene cluster including theEst-6 gene andψEst-6 putative pseudogene in four samples ofDrosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in bothEst-6 andψEst-6. Total nucleotide diversity is twice inψEst-6 as inEst-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within theβ-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected withinEst-6 and, to a much greater extent, withinyEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for theβ-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in theb-esterase gene cluster. However there are some ’footprints’ of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection betweenEst-6 andψEst-6 may play an important role in the evolution of theβ-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene.Est-6 andyEst-6 may represent an indivisible intergenic complex (‘intergene’) in which each single component (Est-6 orψEst-6) cannot separately carry out the full functional role.     

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.     

<Emphasis Type="Italic">Marcetia candolleana</Emphasis> (<Emphasis Type="Italic">Melastomeae</Emphasis> – <Emphasis Type="Italic">Melastomataceae</Emphasis>), a new species from Bahia (Brazil)

Summary   Marcetia candolleana A. K. A. Santos & A. B. Martins, is apparently restricted to Mucugê, Bahia (Brazil), where it occurs in areas of campo rupestre vegetation. This new species is closely related to the sympatric M. mucugensis Wurdack, but can be easily recognised by its semi-prostate to procumbent habit, reddish glandular-hirsute indument, loose and flexuous branches, leaves with inconspicuous reticulation on the abaxial surface, connectives very shortly prolonged below the thecae, style curved towards the apex, not exceeding the anthers, and pendulous fruit.

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Resumo   Marcetia candolleana A. K. A. Santos & A. B. Martins, é aparentemente restrita a Mucugê, Bahia (Brasil), onde ocorre em áreas de campo rupestre. Esta nova espécie é proximamente relacionada à M. mucugensis Wurdack, mas pode ser facilmente reconhecida por seu hábito semi-prostado a procumbente, indumento glandular-hirsuto, vináceo, ramos flexuosos, folhas inconspicuamente reticuladas na face abaxial, conectivos muito curtamente prolongados abaixo das tecas, estilete curvo no ápice, n?o ultrapassando o comprimento das anteras, e fruto pêndulo.

     

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     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Endophytic fungi associated with the Antarctic grass <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> Desv. (<Emphasis Type="Italic">Poaceae</Emphasis>)

Deschampsia antarctica Desv. (Poaceae) represents one of the two vascular plants that have colonized the Antarctic continent, which is usually exposed to extreme environmental conditions. In this work, we have characterized the endophytic fungi associated with the leaves of D. antarctica. Endophytic fungi were recovered from 91 individual plants from different points of Admiralty Bay at King George Island, Antarctica. A total of 26 fungal isolates were obtained from 273 leaf fragments. All isolates were identified by analysis of the sequences of the internal transcribed spacer region (ITS) of the rDNA. Alternaria and Phaeosphaeria were the most frequent genera associated with the plant. Other fungal isolates were identified as Entrophospora sp. and several undescribed Ascomycete species. An interesting result was obtained for the isolates UFMGCB 215 and UFMGCB 262, which were related to fungi associated with bryophytes present in boreal ecosystems. Some isolates showed low identity in the ITS sequences to sequences of fungal species deposited in GenBank, suggesting that these fungi could be new species. This work is the first report on fungal endophytes associated with leaves of the Antarctic grass D. antarctica.     

Diversity of<Emphasis Type="Italic"> Mhc</Emphasis> class I and IIB genes in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

In order to understand the expression and evolution of host resistance to pathogens, we need to examine the links between genetic variability at the major histocompatibility complex (Mhc), phenotypic expression of the immune response and parasite resistance in natural populations. To do so, we characterized the Mhc class I and IIB genes of house sparrows with the goal of designing a PCR-based genotyping method for the Mhc genes using denaturing gradient gel electrophoresis (DGGE). The incredible success of house sparrows in colonizing habitats worldwide allows us to assess the importance of the variability of Mhc genes in the face of various pathogenic pressures. Isolation and sequencing of Mhc class I and IIB alleles revealed that house sparrows have fewer loci and fewer alleles than great reed warblers. In addition, the Mhc class I genes divided in two distinct lineages with different levels of polymorphism, possibly indicating different functional roles for each gene family. This organization is reminiscent of the chicken B complex and Rfp-Y system. The house sparrow Mhc hence appears to be intermediate between the great reed warbler and the chicken Mhc, both in terms of numbers of alleles and existence of within-class lineages. We specifically amplified one Mhc class I gene family and ran the PCR products on DGGE gels. The individuals screened displayed between one and ten DGGE bands, indicating that this method can be used in future studies to explore the ecological impacts of Mhc diversity.     

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.     

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

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

Molecular understandings on ‘the never thirsty’ and apomictic <Emphasis Type="Italic">Cenchrus</Emphasis> grass

The genus Cenchrus comprises around 25 species of ‘bristle clade’ grasses. Cenchrus ciliaris (buffel grass) is a hardy, perennial range grass that survives in poor sandy soils and limiting soil moisture conditions and, due to the very same reasons, this grass is one of the most prevalent fodder grasses of the arid and semi-arid regions. Most of the germplasms of Cenchrus produce seeds asexually through the process of apomeiosis. Therefore, the lack of sufficient sexual lines has hindered the crop improvement efforts in Cenchrus being confined to simple selection methods. Many attempts have been initiated in buffel grass to investigate the various molecular aspects such as genomic signatures of different species and genotypes, molecular basis of abiotic stress tolerance and reproductive performance. Even though it is an important fodder crop, molecular investigations in Cenchrus lack focus and the molecular information available on this grass is scanty. Cenchrus is a very good gene source for abiotic stress tolerance and apomixis studies. Biotechnological interventions in Cenchrus can help in crop improvement in Cenchrus as well as other crops through transgenic technology or marker assisted selection. To date no consolidated review on biotechnological interventions in Cenchrus grass has been published. Therefore we provide a thorough and in depth review on molecular research in Cenchrus focusing on molecular signatures of evolution, tolerance to abiotic stress and apomictic reproductive mechanism.     

<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.     

The slow wound-response of<Emphasis Type="Italic"> γVPE</Emphasis> is regulated by endogenous salicylic acid in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Vacuolar processing enzyme (VPE) is a cysteine protease responsible for the maturation of various vacuolar proteins in higher plants. The Arabidopsis thaliana (L.) Heynh. VPE gene, encoding a VPE homologue, is slowly up-regulated in both local and systemic leaves in response to wounding. To clarify the activation mechanism of VPE, we examined the accumulation of VPE mRNA after hormone treatments or after wounding in wild-type and various mutant plants of Arabidopsis. Both ethylene and jasmonic acid (JA) are known as signal molecules that activate the wound-responsive genes. However, treatment with exogenous JA had little effect on the VPE response, although JA activated the vegetative storage protein (VSP) gene, a typical wound-responsive gene. Wounding activated VPE even in two ethylene-insensitive plants (etr1-1 and ein2-1). Thus, the wound-induced expression of VPE was independent of ethylene and JA. We found that the wound-induced expression of VPE was reduced in two SA-deficient plants (pad4-1 and NahG), while the wound-induced expression of VSP increased in these mutants. Appreciable accumulation of SA was not observed in either the local or systemic leaves after wounding. These results suggest that endogenous SA enhances the wound-induced expression of VPE and attenuates the wound-induced expression of VSP, although SA is not a wound-signal that directly activates these genes.Abbreviations ABA abscisic acid - GST glutathione S-transferase - INA 2,6-dichloroisonicotinic acid - JA jasmonic acid - MeJA methyl jasmonate - PR pathogenesis-related - RBCS Rubisco small subunit - SA salicylic acid - VPE vacuolar processing enzyme - VSP vegetative storage protein