Anne Louise Maddison

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the β-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Characterization of chilling-shock responses in four genotypes of Miscanthus reveals the superior tolerance of M. × giganteus compared with M. sinensis and M. sacchariflorus

Background and Aims The bioenergy grass Miscanthus is native to eastern Asia. As Miscanthus uses C4 photosynthesis, the cooler temperatures experienced in much of northern Europe are expected to limit productivity. Identification of genetic diversity in chilling tolerance will enable breeders to generate more productive varieties for these cooler regions. Characterizing the temporal relationships between photosynthesis, carbohydrate and molecular expression of relevant genes is key to understanding genotypic differences in tolerance or sensitivity. Methods To characterize chilling responses in four Miscanthus genotypes, plants were exposed to a sudden reduction in temperature. The genotypes studied comprised of two M. sinensis, one M. sacchariflorus and one inter-species hybrid, M. × giganteus. Changes in photosynthesis (Asat), carbohydrate composition and the expression of target transcripts were observed following chilling-shock. After 4 d the decline in leaf elongation rate (LER) in the different genotypes was measured. Results Following chilling-shock the greatest decline in Asat was observed in M. sacchariflorus and one M. sinensis genotype. Carbohydrate concentrations increased in all genotypes following chilling but to a lesser extent in M. sacchariflorus. Two stress inducible genes were most highly expressed in the genotypes that experienced the greatest declines in Asat and LER. Miscanthus × giganteus retained the highest Asat and was unique in exhibiting no decline in LER following transfer to 12 °C. Conclusions Miscanthus × giganteus exhibits a superior tolerance to chilling shock than other genotypes of Miscanthus. The absence of sucrose accumulation in M. sacchariflorus during chilling-shock suggests an impairment in enzyme function. A candidate transcription factor, MsCBF3, is most highly expressed in the most sensitive genotypes and may be a suitable molecular marker for predicting chilling sensitivity.

Cell-specific expression of salicylate hydroxylase in an attempt to separate localized HR and systemic signalling establishing SAR in tobacco.

Abstract There is conflicting evidence concerning the nature of the long-distance signal responsible for establishing the systemic acquired resistance (SAR) state following a local response to an incompatible plant/pathogen interaction. We outline standard inoculation procedures and terminology for experiments used to characterize SAR in Nicotiana tabacum and show that leaf development (age) has dramatic affects on TMV lesion size which needs to be taken into account in experimental design. TMV infection was more efficient at inducing SAR than primary infection with avirulent bacteria. We have examined the effect on SAR induction of altering the accumulation of salicylic acid (SA), through the expression of a salicylate hydroxylase gene (SH-L), in different phases of lesion development using the hydrogen peroxide-responsive AoPR1 promoter and the salicylate-responsive PR1a promoter. Suppression of SA accumulation during the early phases of lesion development in AoPR1-SH-L transgenic tobacco resulted in an attenuated form of SAR compared to wild-type plants, whereas SAR was not exhibited in PR1a-SH-L plants. However, interpretation of data from these experiments was complicated by virus escape from inoculated leaves. Using a GUS reporter it was discovered that the CaMV35S promoter was not expressed constitutively in all cell types of petioles and stems, particularly phloem tissue, whereas the PR1a promoter demonstrated induced expression in the phloem following TMV infection. We suggest two hypotheses for why PR1a-SH-L transgenics do not display SAR: either the systemic expression of PR1a-SH-L is sufficient to suppress SAR, or SA synthesis or translocation in the phloem is essential for SAR.

Radiation capture and conversion efficiencies of Miscanthus sacchariflorus , M. sinensis and their naturally occurring hybrid M . × giganteus

Miscanthus is a rhizomatous C4 grass of great interest as a biofuel crop because it has the potential to produce high yields over a wide geographical area with low agricultural inputs on marginal land less suitable for food production. At the moment, a clonal interspecific hybrid Miscanthus × giganteus is the most widely cultivated and studied in Europe and the United States, but breeding programmes are developing newer more productive varieties. Here, we quantified the physiological processes relating to whole season yield in a replicated plot trial in Wales, UK. Light capture and conversion efficiency were parameterized for four carefully selected genotypes (M. sinensis, M. sacchariflorus and Miscanthus × giganteus). Differences in the canopy architecture in mature stands as measured by the extinction coefficient (k) were small (0.55–0.65). Sensitivity analysis on a mathematical model of Miscanthus was performed to quantify the accumulative intercepted photosynthetically active radiation (iPAR) in the growing season using (i) k, (ii) variation in the thermal responses of leaf expansion rate, (iii) base temperature for degree days and (iv) date start of canopy expansion. A 10% increase in k or leaf area per degree day both had a minimal effect on iPAR (3%). Decreasing base temperature from 10 to 9 °C gave an 8% increase in iPAR. If the starting date for canopy expansion was the same as shoot emergence date, then the iPAR increases by 12.5%. In M. × giganteus, the whole season above ground and total (including below ground) radiation‐use efficiency (RUE) ranged from 45% to 37% higher than the noninterspecific hybrid genotypes. The greater yields in the interspecific hybrid M. × giganteus are explained by the higher RUE and not by differences in iPAR or partitioning effects. Studying the mechanisms underlying this complex trait could have wide benefits for both fuel and food production.

Non-structural carbohydrate profiles and ratios between soluble sugars and starch serve as indicators of productivity for a bioenergy grass

Miscanthus is a perennial bioenergy crop that offers a sustainable alternative to fossil fuels. We sought to identify candidate metabolic biomarkers of productivity that may be used as a method of screening for superior individuals in breeding programmes. Our experiments were carried out over two years and two sites in four genotypes. The concentration of fructose positively correlated whereas starch and the ratio of soluble sugars to starch negatively correlated with three biomass traits: yield, stem height and growth rate. Our results show the potential of the carbohydrate metabolic profile as a biomarker of productivity in a perennial energy crop.

Predicting future biomass yield in Miscanthus using the carbohydrate metabolic profile as a biomarker

In perennial energy crop breeding programmes, it can take several years before a mature yield is reached when potential new varieties can be scored. Modern plant breeding technologies have focussed on molecular markers, but for many crop species, this technology is unavailable. Therefore, prematurity predictors of harvestable yield would accelerate the release of new varieties. Metabolic biomarkers are routinely used in medicine, but they have been largely overlooked as predictive tools in plant science. We aimed to identify biomarkers of productivity in the bioenergy crop, Miscanthus, that could be used prognostically to predict future yields. This study identified a metabolic profile reflecting productivity in Miscanthus by correlating the summer carbohydrate composition of multiple genotypes with final yield 6 months later. Consistent and strong, significant correlations were observed between carbohydrate metrics and biomass traits at two separate field sites over 2 years. Machine‐learning feature selection was used to optimize carbohydrate metrics for support vector regression models, which were able to predict interyear biomass traits with a correlation (R) of >0.67 between predicted and actual values. To identify a causal basis for the relationships between the glycome profile and biomass, a 13C‐labelling experiment compared carbohydrate partitioning between high‐ and low‐yielding genotypes. A lower yielding and slower growing genotype partitioned a greater percentage of the 13C pulse into starch compared to a faster growing genotype where a greater percentage was located in the structural biomass. These results supported a link between plant performance and carbon flow through two rival pathways (starch vs. sucrose), with higher yielding plants exhibiting greater partitioning into structural biomass, via sucrose metabolism, rather than starch. Our results demonstrate that the plant metabolome can be used prognostically to anticipate future yields and this is a method that could be used to accelerate selection in perennial energy crop breeding programmes.

Could Miscanthus replace maize as the preferred substrate for anaerobic digestion in the United Kingdom? : Future breeding strategies

Fodder maize is the most commonly used crop for biogas production owing to its high yields, high concentrations of starch and good digestibility. However, environmental concerns and possible future conflict with land for food production may limit its long‐term use. The bioenergy grass, Miscanthus, is a high‐yielding perennial that can grow on marginal land and, with ‘greener’ environmental credentials, may offer an alternative. To compete with maize, the concentration of non‐structural carbohydrates (NSC) and digestibility may need to be improved. Non‐structural carbohydrates were quantified in 38 diverse genotypes of Miscanthus in green‐cut biomass in July and October. The aim was to determine whether NSC abundance could be a target for breeding programmes or whether genotypes already exist that could rival maize for use in anaerobic digestion systems. The saccharification potential and measures of N P and K were also studied. The highest concentrations of NSC were in July, reaching a maximum of 20% DW. However, the maximum yield was in October with 300–400 g NSC plant−1 owing to higher biomass. The digestibility of the cell wall was higher in July than in October, but the increase in biomass meant yields of digestible sugars were still higher in October. Nutrient concentrations were at least twofold higher in July compared to November, and the abundance of potassium showed the greatest degree of variation between genotypes. The projected maximum yield of NSC was 1.3 t ha−1 with significant variation to target for breeding. Starch accumulated in the highest concentrations and continued to increase into autumn in some genotypes. Therefore, starch, rather than sugars, would be a better target for breeding improvement. If harvest date was brought forward to autumn, nutrient losses in non‐flowering genotypes would be comparable to an early spring harvest.