Transcriptional analysis of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> roots challenged with the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

Background  

Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms.     

Survival and growth of <Emphasis Type="Italic">Juniperus</Emphasis> seedlings in <Emphasis Type="Italic">Juniperus</Emphasis> woodlands

Juniperus woodlands are widely distributed in western North America. Few studies of seedling emergence, long-term survival, growth or mortality of the dominant Juniperus spp. in these woodlands have been carried out. Consequently, regeneration dynamics in these woodlands are poorly understood. Juniperus ashei is the dominant woody plant in the majority of woodland and savanna communities of the Edwards Plateau region in central Texas. We examined the emergence, mortality and growth of various spatial and temporal cohorts of J. ashei seedlings over an eight or nine-year period. Greatest emergence was found during the cool, mostly winter months and under the canopy of mature J. ashei trees. Emergence was significantly inversely related to temperature and significantly linearly related to rainfall, but only if the monthly rainfall and emergence were offset by one to four months. Greatest survival occurred below the J. ashei canopy, but greatest growth was at the canopy edge. Emerging seedlings were not from the current years seed crop, but from one or more previous years seed crops. Greatest mortality occurred mostly during the summer months and in the grassland habitat. There was a significant inverse logarithmic or exponential relationship between mean monthly temperature and mean monthly mortality. A large number of J. ashei seedlings or immature plants with reduced growth were found beneath the canopy of mature trees. These plants seem to serve as a seedling bank, providing the source of recruitment into the population should the overstory trees be removed. Survival of the two canopy cohorts with known emergence dates declined with time (negative exponential function) and was 1.0–3.4% after eight or nine years depending on the cohort. The pre-existing cohort seemed to have constant mortality (and presumably replacement), with about 8% of the population dying each year. Higher growth rates for seedlings were found at the edge of the established woodland canopy, which suggests that conditions in the edge habitat or possibly in canopy gaps are best for growth beyond the seedling stage.     

<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.     

Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.     

Cadmium stress affects seed germination and seedling growth in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench by changing the activities of hydrolyzing enzymes

Seed germination, one of the most important phases in the life cycle of a plant, is highly responsive to existing environment. Hydrolyzing enzymes play a major role in the mobilization of food reserves by hydrolyzing carbohydrates, proteins and fats. This paper reports on the effect of Cd toxicity on seed germination and the activities of hydrolyzing enzymes, like acid phosphatases (ACPs), proteases and α-amylases in Sorghum bicolor (L.) Moench. The metal uptake by embryonic axes and seeds was quantified. We found that sorghum could tolerate up to 0.5 mM Cd. At concentrations above 3.0 mM, seed germination was adversely affected with a complete cessation of seedling growth. All investigated hydrolyzing enzymes exhibited a significant decrease in activity with increasing Cd concentrations. The isozyme profiles indicated the loss of one or two isozymes of ACP, induction of a new isozyme for total protease (at 3.0 mM Cd) and a decline in the intensity of α-amylase isozymes. SEM studies revealed that Cd affected a change in root hair density. SEM investigations also confirmed the assay results of the inhibition of starch mobilization from endosperm. This suggested an inhibition of the hydrolysis of reserve carbohydrates and translocation of hydrolyzed sugars, ultimately resulting in decreased germination and disruption of seedling growth. Because sorghum is an important dryland crop, its response to the presence of Cd in agro-ecosystems and Cd-induced phytotoxicity during seed germination and seedling growth needs critical investigation.     

Rapid and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis>) employing standard binary vectors and <Emphasis Type="Italic">bar</Emphasis> gene as a selectable marker

Key message

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker.

Abstract

Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7–12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40–50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

     

Biochemical and physiological peculiarities of the interactions between <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Sorghum bicolor</Emphasis> in the presence of phenanthrene

The effect of phenanthrene, a polycyclic aromatic hydrocarbon (PAH) at concentrations of 0, 10, and 100 mg/kg and the bacterium Sinorhizobium meliloti P221 on root exudation of Sorghum bicolor L. Moench was studied in laboratory vegetative experiments. Inoculation of the bacterium promoted plant resistance to the pollutant stress and increased their acclimation rate and biomass formation. The ability of this microorganism to produce a phytohormone, indolyl-3-acetic acid, and to degrade phenanthrene, resulted in morphological changes of the plant root system and in the changed intensity of root exudation. In root exudates of sorghum, enzyme activities towards the metabolites formed during microbial degradation of PAH were revealed, which is indicative of a direct involvement of plants in PAH degradation in the rhizosphere as well as of the coupled plant-microbial metabolism in the course of xenobiotic degradation in the root zone. In phenanthrene-contaminated soil, sorghum was found to support selectively the development of the S. meliloti P221 population.     

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.     

Complete chloroplast genome sequences of <Emphasis Type="Italic">Hordeum vulgare</Emphasis>, <Emphasis Type="Italic">Sorghum bicolor</Emphasis> and <Emphasis Type="Italic">Agrostis stolonifera</Emphasis>, and comparative analyses with other grass genomes

Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5' end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19-37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16-21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C-U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Betulin inhibits cariogenic properties of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by targeting <Emphasis Type="Italic">vicRK</Emphasis> and <Emphasis Type="Italic">gtf</Emphasis> genes

Streptococcus mutans, a multivirulent pathogen is considered the primary etiological agent in dental caries. Development of antibiotic resistance in the pathogen has created a need for novel antagonistic agents which can control the virulence of the organism and reduce resistance development. The present study demonstrates the in vitro anti-virulence potential of betulin (lup-20(29)-ene-3β,28-diol), an abundantly available plant triterpenoid against S. mutans UA159. Betulin exhibited significant dose dependent antibiofilm activity without affecting bacterial viability. At 240 µg/ml (biofilm inhibitory concentration), betulin inhibited biofilm formation and adherence to smooth glass surfaces by 93 and 71 % respectively. It reduced water insoluble glucan synthesis by 89 %, in conjunction with down regulation of gtfBC genes. Microscopic analysis confirmed the disruption in biofilm architecture and decreased exopolysaccharide production. Acidogenicity and aciduricity, key virulence factors responsible for carious lesions, were also notably affected. The induced auto-aggregation of cells upon treatment could be due to the down regulation of vicK. Results of gene expression analysis demonstrated significant down-regulation of virulence genes upon betulin treatment. Furthermore, the nontoxic effect of betulin on peripheral blood mononuclear cells even after 72 h treatment makes it a strong candidate for assessing its suitability to be used as a therapeutic agent.     

<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.     

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.     

Effects of pH on NaCl tolerance of American elm (<Emphasis Type="Italic">Ulmus americana</Emphasis>) seedlings inoculated with <Emphasis Type="Italic">Hebeloma crustuliniforme</Emphasis> and <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

In the present study, we investigated the effects of pH treatments on NaCl tolerance in mycorrhizal and non-mycorrhizal American elm. American elm (Ulmus americana) seedlings were inoculated with Hebeloma crustuliniforme, Laccaria bicolor or with both mycorrhizal fungi and subsequently subjected to different pH solutions (pH 3, 6 and 9) containing 0 mM (control) and 60 mM NaCl for 4 weeks. Inoculation with the mycorrhizal fungi did not have a large effect on seedling dry weights when the pH and NaCl treatments were considered independently. However, when the inoculated seedlings were treated with 60 mM NaCl at pH 3 or 6, shoot to root ratios and root hydraulic conductivity were higher compared with non-inoculated plants, likely reflecting changes in seedling water flow properties. At pH 6, transpiration rates were about twofold lower in non-inoculated plants treated with NaCl compared with non-treated controls. For NaCl-treated H. crustuliniforme- and L. bicolor-inoculated plants, the greatest reduction of transpiration rates was at pH 9. Treatment with 60 mM NaCl reduced leaf chlorophyll concentrations more in non-inoculated compared with inoculated plants, with the greatest, twofold, decrease occurring at pH 6. At pH 3, root Na concentrations were higher in inoculated than non-inoculated seedlings; however, there was no effect of inoculation on root Na concentrations at pH 6 and 9. Contrary to the roots, the leaves of inoculated plants had lower Na concentrations at pH 6 and 9, but not at pH 3. The results point to an interaction between ECM fungi and root zone pH for salt tolerance of American elm.     

<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.     

Fine Mapping of <Emphasis Type="Italic">qDor7</Emphasis>, a Major QTL Affecting Seed Dormancy in Sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench)

Seed dormancy is a key domestication trait for major crops, which is acquired in long-term systems development processes and enables the survival of plants in adverse natural conditions. It is a complex trait under polygenic control and is affected by endogenous and environmental factors. In the present study, a major seed dormancy QTL in sorghum (Sorghum bicolor (L.) Moench), qDor7, detected previously, was fine mapped using a large, multi-generational population. The qDor7 locus was delimited to a 96-kb region which contains 16 predicted gene models. These results lay a solid foundation for cloning qDor7. In addition, the functional markers tightly linked to the seed dormancy QTL may be used in marker-assisted selection for seed dormancy in sorghum.