Endophytic <Emphasis Type="Italic">Bacillus megaterium</Emphasis> and exogenous stimuli affect the quinonemethide triterpenes production in adventitious roots of <Emphasis Type="Italic">Peritassa campestris</Emphasis> (Celastraceae)

The root bark of Peritassa campestris (Cambess.) A.C. Sm. (Celastraceae) accumulates quinonemethide triterpenes (QMTs), an important class of bioactive compounds that shows potent antitumor activity. The production of these metabolites is difficult by both chemical synthesis, because of the complex molecular structure, and extraction from plant resources, because of the low yield. Thus, the aim of this work was to evaluate the influence of some important factors on the synthesis of QMTs to increase their production in adventitious roots grown in vitro. The effects of luminosity, mechanical damage to the tissue, source and concentration of carbon, auxins, macronutrient and micronutrient concentrations and the elicitation with its endophytic microorganism, Bacillus megaterium, isolated from roots grown in vitro were evaluated. Additionally, we compared the production of QMTs of roots in vitro with that of P. campestris roots bark in natura. Our results showed that all stimulating agents affected the biosynthesis of QMTs, with the exception of luminosity. The pattern of QMTs produced was different for the in vitro and in natura roots, including the accumulation of the majority QMTs: the in vitro roots accumulated maytenin (1) and 22β-hydroxy-maytenin (2), and the in natura roots showed the accumulation of maytenin (1), 22β-hydroxy-maytenin (2), 20α-hydroxy-maytenin (3), and maytenol (4). Therefore, we concluded that the biosynthesis of QMTs by P. campestris roots is affected by biotic and abiotic factors.     

<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.     

Comparative genomics using <Emphasis Type="Italic">Fugu</Emphasis> reveals insights into regulatory subfunctionalization

Background  

A major mechanism for the preservation of gene duplicates in the genome is thought to be mediated via loss or modification of cis-regulatory subfunctions between paralogs following duplication (a process known as regulatory subfunctionalization). Despite a number of gene expression studies that support this mechanism, no comprehensive analysis of regulatory subfunctionalization has been undertaken at the level of the distal cis-regulatory modules involved. We have exploited fish-mammal genomic alignments to identify and compare more than 800 conserved non-coding elements (CNEs) that associate with genes that have undergone fish-specific duplication and retention.     

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.     

<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.     

<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.     

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.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

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.     

Nucleotide substitutions in <Emphasis Type="Italic">rolC</Emphasis> and <Emphasis Type="Italic">nptII</Emphasis> gene sequences during long-term cultivation of <Emphasis Type="Italic">Panax ginseng</Emphasis> cell cultures

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Secretome profiling reveals temperature-dependent growth of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Aspergillus fumigatus is a ubiquitous opportunistic fungus. In this study, systematic analyses were carried out to study the temperature adaptability of A. fumigatus. A total of 241 glycoside hydrolases and 69 proteases in the secretome revealed the strong capability of A. fumigatus to degrade plant biomass and protein substrates. In total, 129 pathogenesis-related proteins detected in the secretome were strongly correlated with glycoside hydrolases and proteases. The variety and abundance of proteins remained at temperatures of 34°C–45°C. The percentage of endo-1,4-xylanase increased when the temperature was lowered to 20°C, while the percentage of cellobiohydrolase increased as temperature was increased, suggesting that the strain obtains carbon mainly by degrading xylan and cellulose, and the main types of proteases in the secretome were aminopeptidases and carboxypeptidases. Only half of the proteins were retained and their abundance declined to 9.7% at 55°C. The activities of the remaining β-glycosidases and proteases were merely 35% and 24%, respectively, when the secretome was treated at 60°C for 2 h. Therefore, temperatures >60°C restrict the growth of A. fumigatus.     

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.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.