Localized temperature adaptation of cottonwoods from elevational ecoregions in the Rocky Mountains

To investigate temperature adaptation of cool-climate trees, we studied 14 cottonwood genotypes from riparian (streamside) zones in three ecoregions differing in elevation in the Rocky Mountains of Alberta, Canada. Black cottonwoods (Populus trichocarpa) were collected from the higher, cooler montane and intermediate (aspen) parkland, and narrowleaf cottonwoods (P. angustifolia) were collected from the lower, warmer fescue prairie. The genotypes were grown in growth chambers under three temperature regimes reflecting the ecoregion variation. Sapling growth (dry weight) varied significantly across temperatures and for the genotypes from within and particularly across the ecoregions. Significant temperature × genotype interactions further indicated differentiation of the temperature response. Growth of the montane clones increased by 209% between 15/10 and 20/15°C and was 37% lower at 25/20°C. In contrast, genotypes from the lower ecoregions grew more slowly at the cool and intermediate temperatures (59 and 58% of montane) and then demonstrated constant (−3% parkland) or slightly increased (+16% prairie) growth at 25/20°C. This suggests the existence of P. trichocarpa ecotypes, localized populations with different temperature responses. This differentiation may explain our previous observation of comparable growth across these ecoregions despite substantial temperature variation, and the existence of ecotypes would produce a range of responses to climate warming that should produce an upward shift of the mountain ecoregions.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Two Novel Membrane Proteins,TcpD and TcpE,Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.     

The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn 4451 , consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243–261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598–707). In these mutants, removal of residues 598–707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533–583). Analysis of mutations within the aa 243–261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.     

Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA₂₀ and GA₈, in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.Semidwarfism is a valuable trait in many crop species and agricultural environments. In cereal crops, it can result in decreased lodging, increased yield, and improved harvest index (Dalrymple, 1985; Hedden, 2003). Therefore, it was a critical foundation of the “Green Revolution” that resulted in large improvements of yield in wheat (Triticum aestivum) and rice (Oryza sativa; Hargrove and Cabanilla, 1979; Perovic et al., 2008). Semidwarfism has had substantial benefits for fruit tree production, where it enables earlier fruit bearing, higher yields, and easier harvests in orchards (Battisini and Battisini, 2005). Semidwarf woody species are also extensively used in ornamental horticulture, where they allow more compact forms to be fit into small areas around homes and on streets and reduce the need for pruning to avoid interference with structures and transmission lines (Busov et al., 2003).Although against the current orthodoxy of forest tree breeding, where height growth is emphasized, semidwarfism might also have benefits for wood and biomass production (Bradshaw and Strauss, 2001). Such trees could be useful if they were less prone to wind throw due to their shorter, stockier forms and expected greater allocation to roots. Reduced stature could also result in less bending and slanting of trunks in the face of wind and gravity on hillslopes and thus reduce the extent of reaction wood formation, which degrades the performance and value of solid wood and pulp products. Reduced height and increased allocation of growth to roots might enhance stress tolerance, soil nutrient uptake, bioremediation, and carbon sequestration.Semidwarfism can be achieved by the modification of several types of genes and physiological mechanisms, but the most prevalent and advanced forms in agriculture affect GAs or their signaling (for review, see Busov et al., 2008). GAs are endogenous plant hormones that influence several aspects of plant growth and development, including seed germination, leaf expansion, shoot growth, cell division, flower induction, and fruit development (Sun and Gubler, 2004; Fleet and Sun, 2005; Swain and Singh, 2005). With respect to shoot growth, the most obvious effect of GA is its promotion of stem elongation by stimulating both cell elongation and division (Marth et al., 1956). GA modification also has significant effects on plant biochemistry, changing the amounts and distribution of a wide variety of metabolites in shoots and roots (Rossetto et al., 2003; Chen et al., 2004; Busov et al., 2006).Little is known about how semidwarfism affects belowground growth. GA has been shown to play a controlling role in lateral root development (Gou et al., 2010), and GA and ethylene synergistically promote both the initiation and growth of adventitious roots (Osmont et al., 2007). In tomato (Solanum lycopersicum), isogenic GA-deficient mutants (gib) allocate more biomass to roots compared with shoots (Nagel et al., 2001). In poplar (Populus spp.), semidwarf transgenic plants grown in vitro had a lower shoot-to-root ratio, which was at least partly due to proliferation of lateral roots (Busov et al., 2006; Gou et al., 2010).As a domestication trait, semidwarfism has been proposed as a means for mitigating the spread of transgenic plants within and outside of crop environments (Al-Ahmad et al., 2005). The genetic dominance of most semidwarfism transgenes would cause reduced height growth in transgene-containing progeny, reducing their ability to compete for light. Moreover, because of the close linkage of the semidwarfism genes to other genes that were cointroduced on the same plasmid, they would also powerfully retard their spread or introgression, even in cases where the linked transgene would, on their own, impart a selective advantage. However, there have been very few plant species where this concept has been explicitly tested (Al-Ahmad and Gressel, 2006; Gressel and Valverde, 2009), and we know of no examples in woody or perennial plants.To study the effects of semidwarfism genes in a woody plant grown under field conditions, we inserted a number of dominant GA-modifying transgenes into hybrid poplar (Populus tremula × Populus alba), the widely recognized model woody plant for genomics and biotechnology (Herschbach and Kopriva, 2002; Brunner et al., 2004a; Tuskan et al., 2004). Most of the genes studied were overexpressed forms of GA 2-oxidase, GA-Insensitive (GAI), or Repressor of GAI-Like (RGL), all known to cause semidwarfism in other plant species. GA 2-oxidase is a major GA catabolic enzyme in plants, and GAI and RGL are negative regulators of the GA signal transduction pathway (Appleford et al., 2007; Busov et al., 2008). The transgenic trees were first analyzed in the greenhouse (Busov et al., 2006) and then assayed for their effect on height growth in a 2-year field trial (Zawaski et al., 2011), from which we selected 10 transgenic events that grew at approximately three-quarters the rate of wild-type trees. The goal was to select semidwarf trees whose phenotype was not so severe as to be irrelevant to possible crop uses but strong enough to give a clear phenotype in a field study. In this study, we analyzed changes in a number of morphological, physiological, and growth traits and investigated the prospect for semidwarfism to be used as a mitigation tool to reduce the frequency of spread of transgenic and exotic species.