A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Seasonal abundance and reproductive season of <Emphasis Type="Italic">Chauliops fallax</Emphasis> (Heteroptera: Malcidae) on kudzu <Emphasis Type="Italic">Pueraria lobata</Emphasis>

We investigated the seasonal abundance and seasonal dynamics of reproduction in the stalk-eyed bug Chauliops fallax Scott, which is described as a minor pest of bean plants such as soybean and a minor cause of pecky rice. We counted the number of adult males and females, mating pairs, and nymphs of C. fallax on kudzu plants, Pueraria lobata (Willd.), in the southern part of Okayama City, Japan, from 2005 to 2007 and in 2009. Two peaks in abundance for mating pairs were found in late May and August. Both male and female solitary adults appeared from late April, and two peaks of solitary adults were found in May and September. Two clearly separate peaks of nymphs were found in late June and late August. Therefore, it seems that C. fallax produces two generations a year in the southern Okayama area. These data may provide essential information for pest control, and therefore, we discuss them from an applied entomological standpoint to predict invasions by C. fallax into agriculture fields from weeds that grow around and/or in fields.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Allelopathic Potential of <Emphasis Type="Italic">Pueraria thunbergiana</Emphasis>

The allelopathic potential of Pueraria thunbergiana was investigated under laboratory conditions. The powder of freeze-dried leaves of P. thunbergiana inhibited the germination and the growth of roots and shoots of cress, lettuce, timothy and ryegrass. Significant reductions in the germination and growth of roots and shoots were observed as the powder concentration increased in all bioassays. The putative compounds causing the inhibitory effect of the powder were isolated and determined by their spectral data as cis.trans- and trans,trans-xanthoxin This revised version was published online in July 2006 with corrections to the Cover Date.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

Androgenesis in the vine cacti <Emphasis Type="Italic">Selenicereus</Emphasis> and <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

Anther culture techniques were applied to develop a methodology for producing haploid vine cactus plants. Anthers with most microspores in the middle uninucleate developmental stage from the tetraploid species Selenicereus megalanthus and the diploid species Hylocereus polyrhizus and H. undatus were cultured on basal MS medium supplemented with picloram and 6-benzyladenine (BA). H. polyrhizus and H. undatus anthers were also cultured on MS medium containing either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid (2,4-D). Pro-embryo development started after three days of culture. A direct androgenic embryo response was achieved in S. megalanthus with and without picloram/BA, while H. polyrhizus exhibited non-regenerative callus formation. Only a single direct androgenic embryo was obtained in H. polyrhizus (with 0.1 mg/l TDZ). H. undatus required a cold pre-treatment of 4°C for 24 h in 0.3 M D-mannitol to produce a response, but only calluses were obtained and they did not regenerate. S. megalanthus and H. polyrhizus embryos converted into plantlets after transfer to MS medium in the light. Rooted plants were acclimatized successfully, and most plants showed normal phenotypes. Flow cytometry and cytological studies revealed monoploid, haploid, dihaploid, and mixoploid plants. This study showed that androgenesis is strongly species- and culture-medium-dependent, thus revealing new perspectives in the genetics and breeding of vine cactus species. To the best of our knowledge, this is the first report on the production of monoploid, haploid and dihaploid plants in Cactaceae.