Thomas Canam

Over-expression of an arabidopsis family A sucrose phosphate synthase (SPS) gene alters plant growth and fibre development

The objective of this study was to manipulate the intracellular pools of sucrose by differentially expressing exogenous sucrose phosphate synthase (SPS) and investigating its role in regulating plant growth and fibre development. Tobacco (Nicotiana tabacum cv. Xanthi) plants were transformed with an arabidopsis SPS gene under the regulation of the ubiquitously expressed tandem repeat of the 35S cauliflower mosaic virus promoter, and subject to growth trials and fibre characterization. It was apparent that over-expression of SPS resulted in substantially elevated concentrations of sink sucrose pools compared to wild-type plants, while source tissue sucrose pools remained the same. All transformed plants had significantly increased stem height, which was ascribed to internode elongation, and greater stem diameters, longer fibers and increased total dry biomass relative to the control plants. Difference in the chemical composition of either the storage or structural carbohydrates of the wild-type and SPS transgenic lines were only minor. The correlation between increased stem sucrose content and plant phenotypes with elevated SPS gene expression confirm a role for sucrose availability in controlling plant growth and fibre elongation.

Over-expression of UDP-glucose pyrophosphorylase in hybrid poplar affects carbon allocation

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2x35S) on plant metabolism and growth were investigated in hybrid poplar (Populus albaxgrandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2x35S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2x35S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis revealed significant increases in soluble sugar, starch, and cellulose contents, and concurrent decreases in lignin content. Lignin monomer composition was altered in favour of syringyl moieties. Detailed soluble metabolite analysis revealed that 2x35S::UGPase trees had as much as a 270-fold increase in the salicylic acid 2-O-beta-D-glucoside (SAG), a compound typically associated with the stress response. These data suggest that while it is possible to alter the allocation of carbon in favour of cellulose biosynthesis, whole plant changes result in unexpected decreases in growth and an increase in defence metabolites.

Sucrose phosphate synthase expression influences poplar phenology

The objective of this study was to manipulate the intracellular pools of sucrose, and investigate its role in regulating plant growth, phenology (leaf senescence and bud break) and fibre development. This objective was achieved by differentially expressing an Arabidopsis (Arabidopsis thaliana L. Heynh.) sucrose phosphate synthase (SPS) gene in hybrid poplar (Populus alba L.xPopulus grandidentata Michx.), a model system for tree biology with substantial industrial relevance in the context of short rotation forestry and a target bioenergy crop. Phenotypic differences were evident in the transgenic trees, as both the timing of bud flush and leaf senescence were altered compared to wild-type (WT) trees. Tree height and stem diameter were similar in WT and in the AtSPS transgenic trees, however, there were differences in the length of xylem fibres. Elevated concentrations of intracellular sucrose in both leaf and stem tissue of the transgenic trees are associated with a prolonged onset of senescence and an advancement in bud flush in the following spring. The association among sucrose content, tree phenology and elevated SPS gene expression implicates both enzyme and product in regulating poplar developmental processes.

Isolation and characterization of galactinol synthases from hybrid poplar

The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 2.4.1.123), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba×grandidentata). Phylogenetic analyses of the Populus GolS isoforms with other known GolS proteins suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. The predicted protein sequences of both isoforms (Pa×gGolSI and Pa×gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII resulted in Km values for UPD-galactose of 0.80 and 0.65 mM and Vmax values of 657.5 and 1245 nM min−1, respectively. Pa×gGolSI inherently possessed a broader pH and temperature range when compared with Pa×gGolSII. Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

Varied growth, biomass and cellulose content in tobacco expressing yeast-derived invertases

The effects of the expression of yeast-derived apoplastic (AI) and cytosolic (CI) invertases (EC 3.2.1.26) on biomass and structural carbohydrate accumulation in tobacco (Nicotiana tabacum L. cv. Xanthi) were evaluated. Transgenic tobacco plants expressing AI or CI under the control of either a tandem repeat of the Cauliflower Mosaic Virus 35S promoter (2X35S), or a promoter that drives xylem-localized expression (Petroselinum crispum 4-coumarate:CoA ligase promoter; 4CL) were generated. Yeast-derived invertase transcript levels, invertase protein, enzyme activity, growth parameters as well as both structural and soluble carbohydrates of stem tissue of all transformed lines were quantified. Transgenic tobacco lines expressing invertase under the control of 4CL displayed severe growth retardation with both yeast-derived isogenes. Similarly, several transformed lines expressing either AI or CI regulated by the 2X35S promoter were also shorter than wild-type (WT) plants. Despite the decreases in height, some transformed lines had significant increases in biomass. One line (2X35S::AI-1) had a biomass/height increase of 88% and an increase in stem diameter of over 40%, while a second line (2X35S::CI-5) had a biomass/height increase of 21%. A separate line (2X35S::AI-2) had a 36% increase in cellulose content, while two others (4CL::AI-2 and 4CL::AI-3) displayed significant decreases in cellulose content. The observed phenotypes can be in part explained by the levels of foreign invertase present, subcellular localization and the carbohydrate status of the tissues.

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white-rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col-0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl-4-O-methyl-d-glucopyranuronate and could target ester linkages that contribute to lignin-carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf-yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT-IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross-linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross-linking within plant cell walls.

Pretreatment of Lignocellulosic Biomass Using Microorganisms: Approaches, Advantages, and Limitations

Much of Earth’s recent geologic history is dominated by periods of extensive glaciation, with relatively low global mean temperatures and correspondingly low atmospheric CO2 concen‐ trations [1]. The current interglacial period stands out as an anomaly because the atmospheric CO2 concentration has risen sharply above the range of approximately 180-280 parts per million by volume that has defined the past 420,000 years to reach levels that are nearly 40% higher than the biosphere has experienced over this time frame [2]. This rapid increase in CO2 concentration is primarily due to the release of ancient fixed atmospheric CO2 into the modern atmosphere through the combustion of fossil fuel resources over the past 200 years. Since it is clear from ice core records that atmospheric CO2 concentration has a strong positive correlation to global temperature, it is expected that changes to global climate are forthcoming [3]. There are substantial uncertainties regarding the ability of terrestrial and oceanic carbon sinks to absorb this anthropogenic CO2 on time scales that are relevant to human society [2], so the continued release of ancient CO2 into the modern atmosphere at current rates carries with it an important risk of inducing climate changes of unknown amplitude along with a host of ancillary changes that are difficult to predict with certainty. This has led to the search for alternatives to fossil fuels to meet a rising global energy demand, and one such option is the use of extant organic matter to produce energy. This resource contains carbon that was fixed from the modern atmosphere, which means it does not result in a net increase in atmospheric CO2 upon combustion.

Differential metabolite profiles and salinity tolerance between two genetically related brown-seeded and yellow-seeded Brassica carinata lines

Brassica carinata (Ethiopian mustard) has previously been identified as a potential crop species suitable for marginal land in the North American prairies due to its relatively high salt tolerance. Two genetically related B. carinata lines with brown-seeded (BS) and yellow-seeded (YS) phenotypes were assessed for their tolerance to sodium sulfate. Specifically, each line was greenhouse-grown under 0, 50 and 100mM of salt, and analyzed after four weeks and eight weeks of treatment. Generally, the height of the BS line was greater than the YS line under both salt treatments, indicating enhanced salt tolerance of the BS line. NMR-based metabolite profiling and PCA analyses indicated a more pronounced shift in key stem metabolites after four weeks of treatment with the YS line compared to the BS line. For example, tryptophan and formate levels increased in the YS line after four weeks of 100mM salt treatment, while proline and threonine levels varied uniquely compared to other metabolites of the lines. Together, the data indicate that the brown-seeded line has greater sodium tolerance than the yellow-seed line, provide clues to the biochemical underpinnings for the phenotypic variation, and highlight the utility of B. carinata as a biorefinery crop for saline land.

Soil microbial communities alter leaf chemistry and influence allelopathic potential among coexisting plant species

While both plant–soil feedbacks and allelochemical interactions are key drivers of plant community dynamics, the potential for these two drivers to interact with each other remains largely unexplored. If soil microbes influence allelochemical production, this would represent a novel dimension of heterogeneity in plant–soil feedbacks. To explore the linkage between soil microbial communities and plant chemistry, we experimentally generated soil microbial communities and evaluated their impact on leaf chemical composition and allelopathic potential. Four native perennial old-field species (two each of Aster and Solidago) were grown in pairwise combination with each species’ soil microbial community as well as a sterilized inoculum. We demonstrated unequivocally that variation in soil microbial communities altered leaf chemical fingerprints for all focal plant species and also changed their allelopathic potential. Soil microbes reduced allelopathic potential in bioassays by increasing germination 25–54% relative to sterile control soils in all four species. Plants grown with their own microbial communities had the lowest allelopathic potential, suggesting that allelochemical production may be lessened when growing with microbes from conspecifics. The allelopathic potential of plants grown in congener and confamilial soils was indistinguishable from each other, indicating an equivalent response to all non-conspecific microbial communities within these closely related genera. Our results clearly demonstrated that soil microbial communities cause changes in leaf tissue chemistry that altered their allelopathic properties. These findings represent a new mechanism of plant–soil feedbacks that may structure perennial plant communities over very small spatial scales that must be explored in much more detail.

Genes and nitrogen fuel wood formation

Daily newspapers abound with stories of the latest discoveries of the interplay between human genetics and nutrition and its role in human health and development. For example, recent evidence has provided an incredibly strong link between genetic variation in human populations, vitamin D concentrations and the occurrence of multiple sclerosis, where genetic predisposition to the disorder is strongly influenced by diet and environment (Ramagopalan et al., 2009). Similar examples in human genetics are plentiful, with some recent reviews focusing on the combinatorial effects of genotype and diet on human health and development from a systems biology perspective (Daniel et al., 2008; van Ommen et al., 2008). Like their human counterparts, plant phenotypes are also shaped by the interaction between genotype and the availability of essential nutrients, as recent reviews focusing on a variety of crop species illustrate (e.g. Fageria et al., 2008; Rengel & Damon, 2008). Given the increasing global demand for resources derived from plants, such as food and fuel, there is a palpable urgency to establish sustainable and highly productive crops. To achieve this, it is critical that efforts are continually focused on deepening our understanding of the combined effects of genotype and nutrient availability on the growth and productivity of plant species – especially those species earmarked as having the potential to mitigate future resource crises. Identification of genotype-specific and genotype-independent responses to nutritional cues will inform future crop improvement efforts and production regimes.