Stable <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD₆₀₀ of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

Isolation and characterization of two T-box genes from sponges,the phylogenetically oldest metazoan taxon

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-Bra and Sd-Tbx2 sequences and their intron-exon structures demonstrate their basal position in the phylogeny of the T-box family, and allows us to hypothesize a model of the phylogenetic evolution of all T-box genes. Furthermore, we report the presence of two different products of alternative splicing of Sd-Bra, and demonstrate that they exist in different phosphorylation and glycosylation states in the sponge tissue. Sd-Bra expression in tissue and 3D-cell aggregates (primmorphs) is analyzed, suggesting that Sd-Bra might also have a role in Porifera morphogenesis.Edited by N. SatohThe sequences from Suberites domuncula reported here are deposited in the EMBL/GenBank data base: the cDNA of Brachyury (Sd-Bra; accession number AJ544242) as well as the second T-box gene Sd-Tbx2 (AJ544241).     

武汉市15种阔叶乔木滞尘能力与叶表微形态特征

以武汉市15种常见的阔叶乔木为研究对象,通过3级滤膜过滤法测定了各乔木单位叶面积滞留不同粒径颗粒物(TSP、PM_>10、PM₁₀、PM_2.5)的质量,并通过扫描电镜观察比较了15种乔木的叶表面微形态结构,分析了微形态对植物滞尘能力的影响。结果表明:15种乔木单位叶面积的滞尘量存在显著差异(P<0.05),综合滞尘能力最强的植物为二球悬铃木、桂花和石楠,除以上3者外,女贞和广玉兰分别具有较强的滞留PM₁₀和PM_2.5的能力;加杨滞留TSP和PM_>10的能力最弱,玉兰滞留PM₁₀和PM_2.5的能力最弱。各乔木单位叶面积滞留PM_2.5和PM₁₀的质量分别占总粉尘量的0.7%—8.9%和3.6%—33.9%。叶表面微结构观察表明,叶表面粗糙、褶皱较多,或被有蜡质层的植物有利于粉尘颗粒物的附着。相关性分析表明,植物单位叶面积的滞尘量与叶表面沟槽的宽度呈显著相关,...     

Molecular Identification of Fomitiporia mediterranea and Eutypa lata/Libertella blepharis in Platanus × acerifolia

The studies of woody organs‐affecting diseases of Platanus × acerifolia can be hampered by the finding of fungi whose identification is difficult with mycological techniques. In a previous study on Platanus × acerifolia affected by severe cankers, Fomitiporia mediterranea/punctata‐like fungi, not associated with fruit bodies and Libertella sp. were recovered. Due to the severity of the associated symptoms, a characterization of fungal ribosomal DNA genes was undertaken in the present study, aimed to specific identification of the pathogens. From DNA of Fomitiporia‐vegetative isolates and Fomitiporia‐fruit body isolates, included in the study for comparison (from fruit body on plane trees typical of F. mediterranea/punctata), and Libertella sp., DNA fragments were amplified in polymerase chain reaction (PCR) with the use of ITS5/ITS4 and 5.8S‐R/LR7 primer pairs. Sequence alignments showed that Fomitiporia‐vegetative/fruit body isolates had highly homologous ITS5/ITS4 sequences, with a nucleotide identity of 98–100%. All the Fomitiporia isolates from plane tree showed 97–100% and 91–94% of nucleotide identity respectively with ITS5/ITS4 sequences of known strains of F. mediterranea and F. punctata, extracted from GenBank. The strong similarity of these identity ranges with those obtained within F. mediterranea (98–100%) and between the two Fomitiporia species (90–94%) confirms the identification of the isolates from plane tree as F. mediterranea. Sequence comparison between Libertella sp. from plane tree and known strains of Eutypa lata/L. blepharis showed 94–99% of nucleotide identity. The comparison with eight additional species of Eutypa showed 90–93% of nucleotide identity. As previously reported for the different taxa within Diatrypaceae, also ITS5/ITS4 sequence of Libertella sp. from plane tree exhibited 11‐bp tandem repeats motifs. Results of sequence alignments, of phylogenetic trees, and of the putative restriction map, identify the Fomitiporia isolates of this work as F. mediterranea, and the isolates of Libertella sp. as E. lata/L. blepharis. For comparative purposes, ITS5/ITS4 sequences of Spongipellis pachyodon and Fomes fomentarius from plane tree, were also obtained in this work.     

Chalcone synthase-like gene in the liverwort, <Emphasis Type="Italic">Marchantia paleacea</Emphasis> var. <Emphasis Type="Italic">diptera</Emphasis>

A chalcone synthase (CHS)-like gene, MpCHSLK1, was isolated from liverwort, Marchantia paleacea var. diptera. Phylogenetic analysis revealed that MpCHSLK1 is closely related to stilbene synthase of the whisk fern, Psilotum nudum. Southern blot analysis using an MpCHSLK1 probe revealed that the gene belongs to a small gene family. Northern blot analysis indicated that CHS-like genes were expressed in either the mother plants or photoautotrophic cells. In photoautotrophic cells, the CHS-like genes were expressed light-dependently, and this expression was completely inhibited by the photosynthetic electron transport inhibitor, DCMU.Abbreviations CHS Chalcone synthase - DCMU 3-(3,4-Dichlorophenyl)-1-1-dimethylurea - POR Protochlorophyllide oxidoreductase - STS Stilbene synthase     

Mosaic Structure of Pathogenicity Islands in <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

A gene complex, dot/icm, located in two independent chromosomal loci of L. pneumophila, the causative agent of Legionnaires' disease, is related to virulence. To investigate the evolutionary pattern of these pathogenicity islands of L. pneumophila, portions of four genes in the dot/icm complex, namely, dotA, dotB, icmB, and icmT, were amplified, sequenced, and phylogenetically analyzed, in addition to rpoB, which encodes an RNA polymerase -subunit. The nucleotide sequences and phylogenetic analyses of these five genes of 96 L. pneumophila strains revealed that several subgroups of L. pneumophila proliferated clonally. However, incongruent gene tree topologies and the results of statistical testing (Templeton Willcoxon signed-ranked and incongruence length differences tests) indicated that the evolutionary histories of these genes within the pathogenicity islands are not uniform, and that they constitute a mosaic structure. In addition, the non-uniform grouping of some reference strains suggests that intraspecific recombination might be still occurring in nature or in the laboratory.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

Phenotypic characterization and genomic analysis of the <Emphasis Type="Italic">Shigella sonnei</Emphasis> bacteriophage SP18

A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages.     

Phylogeny and the Evolution of the <Emphasis Type="Italic">Amylase</Emphasis> Multigenes in the <Emphasis Type="Italic">Drosophila montium</Emphasis> Species Subgroup

Abstract To investigate the phylogenetic relationships and molecular evolution of α-amylase (Amy) genes in the Drosophila montium species subgroup, we constructed the phylogenetic tree of the Amy genes from 40 species from the montium subgroup. On our tree the sequences of the auraria, kikkawai, and jambulina complexes formed distinct tight clusters. However, there were a few inconsistencies between the clustering pattern of the sequences and taxonomic classification in the kikkawai and jambulina complexes. Sequences of species from other complexes (bocqueti, bakoue, nikananu, and serrata) often did not cluster with their respective taxonomic groups. This suggests that relationships among the Amy genes may be different from those among species due to their particular evolution. Alternatively, the current taxonomy of the investigated species is unreliable. Two types of divergent paralogous Amy genes, the so-called Amy1- and Amy3-type genes, previously identified in the D. kikkawai complex, were common in the montium subgroup, suggesting that the duplication event from which these genes originate is as ancient as the subgroup or it could even predate its differentiation. Thc Amy1-type genes were closer to the Amy genes of D. melanogaster and D. pseudoobscura than to the Amy3-type genes. In the Amy1-type genes, the loss of the ancestral intron occurred independently in the auraria complex and in several Afrotropical species. The GC content at synonymous third codon positions (GC3s) of the Amy1-type genes was higher than that of the Amy3-type genes. Furthermore, the Amy1-type genes had more biased codon usage than the Amy3-type genes. The correlations between GC3s and GC content in the introns (GCi) differed between these two Amy-type genes. These findings suggest that the evolutionary forces that have affected silent sites of the two Amy-type genes in the montium species subgroup may differ.     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors

To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC^c type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE “quartet model,” an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.