Gas exchange measurements in the unsteady state

Leaf level gas exchange is a widely used technique that provides real-time measurement of leaf physiological properties, including CO2 assimilation (A), stomatal conductance to water vapour (gsw) and intercellular CO2 (Ci). Modern open-path gas exchange systems offer greater portability than the laboratory-built systems of the past and take advantage of high-precision infrared gas analyzers and optimized system design. However, the basic measurement paradigm has long required steadystate conditions for accurate measurement. For CO2 response curves, this requirement has meant that each point on the curve needs 1–3 min and a full response curve generally requires 20–35 min to obtain a sufficient number of points to estimate parameters such as the maximum velocity of carboxylation (Vc,max) and the maximum rate of electron transport (Jmax). For survey measurements, the steady-state requirement has meant that accurate measurement of assimilation has required about 1–2 min. However, steady-state conditions are not a strict prerequisite for accurate gas exchange measurements. Here, we present a new method, termed dynamic assimilation, that is based on first principles and allows for more rapid gas exchange measurements, helping to make the technique more useful for high throughput applications

Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. ‘‘Alamo’’ with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the TDNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol- HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin

Insect resistance of a full sib family of tetraploid switchgrass \u3ci\u3ePanicum virgatum\u3c/i\u3e L. with varying lignin levels

Little information is available on insect resistance mechanisms and inheritance in biomass grasses. Although reduction of lignin in biomass grasses in order to increase the efficiency of fermentation may result in increased susceptibility to insect feeding, other resistance mechanisms may be more important. Field grown leaves of two tetraploid parent (Kanlow N1, Summer) and 14 progeny switchgrass (Panicum virgatum L.) plant clones selected for a diversity of plant form and ranges in lignin levels were tested for leaf resistance to feeding by the fall armyworm (Spodoptera frugiperda J. E. Smith), a grass feeding insect pest. Although lignin generally appeared important as a resistance mechanism only at early season stages, replicate clones of two low lignin progeny plants generally remained resistant to fall armyworm feeding. Mechanical damaging increased resistance to fall armyworm feeding in several of these plants. Degrees of resistance were sometimes associated with leaf form of progeny. These results indicate there are likely multiple insect resistance mechanisms operating at different stages in switchgrass, and that segregation of some mechanisms appears related to growth form of the plants

Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

Genetic modification of herbaceous plant cell walls to increase biofuels yields is a primary bioenergy research goal. Using two switchgrass populations developed by divergent breeding for ruminant digestibility, the contributions of several wall-related factors to ethanol yields was evaluated. Field grown low lignin plants significantly out yielded high lignin plants for conversion to ethanol by 39.1% and extraction of xylans by 12%. However, across all plants analyzed, greater than 50% of the variation in ethanol yields was attributable to changes in tissue and cell wall architecture, and responses of stem biomass to diluteacid pretreatment. Although lignin levels were lower in the most efficiently converted genotypes, no apparent correlation were seen in the lignin monomer G/S ratios. Plants with higher ethanol yields were associated with an apparent decrease in the lignification of the cortical sclerenchyma, and a marked decrease in the granularity of the cell walls following dilute-acid pretreatment

A new simple method for labeling field crops with stable isotope tracers

Numerous systems have been used to label plants with 13C, which differ in design and complexity depending upon the desired experimental goals. However, most of these systems have generally been applied to greenhouse grown plants. Here, we report on a relatively simple 13C labeling system designed to label crops such as switchgrass (Panicum virgatum L.) grown in the greenhouse or small field plots. The main goals of this study were to validate the system and establish performance benchmarks. We constructed and field tested a simple design plexiglass chamber that was sealed at the soil level with a buried rubber apron. Chamber air was circulated through an infrared gas analyzer to monitor CO2 levels within the chamber. Provisions were made to control temperatures and minimize condensation inside the chamber during labeling. Control experiments using the empty chamber both under greenhouse and field settings showed that decline in CO2 levels was only due to plant CO2 absorption and not due to leakage. Results indicated that the system had generally suitable performance characteristics in both greenhouse and field settings. Isotope enrichment data from our studies revealed that switchgrass fixed 13CO2 that was injected into the chamber within 15-20 min labeling periods. The mean and standard deviation of leaf ᵹ13C values across nine plants enclosed in the chamber was 34 ± 8.9 and 96.1 ± 23.6 for the single and double labeling experiments, respectively. Results indicate that a chamber of this construction type can be effectively used also for labeling other crop plants

Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

Genetic modification of herbaceous plant cell walls to increase biofuels yields is a primary bioenergy research goal. Using two switchgrass populations developed by divergent breeding for ruminant digestibility, the contributions of several wall-related factors to ethanol yields was evaluated. Field grown low lignin plants significantly out yielded high lignin plants for conversion to ethanol by 39.1% and extraction of xylans by 12%. However, across all plants analyzed, greater than 50% of the variation in ethanol yields was attributable to changes in tissue and cell wall architecture, and responses of stem biomass to diluteacid pretreatment. Although lignin levels were lower in the most efficiently converted genotypes, no apparent correlation were seen in the lignin monomer G/S ratios. Plants with higher ethanol yields were associated with an apparent decrease in the lignification of the cortical sclerenchyma, and a marked decrease in the granularity of the cell walls following dilute-acid pretreatment

Genetic Resources for the Improvement of Switchgrass (\u3cem\u3ePanicum virgatum\u3c/em\u3e L.) for Biomass and Forage

Switchgrass (Panicum virgatum L.) is an important forage and biomass species for many parts of the USA. Switchgrass can be of several ploidies. Octoploid cultivars are most often used in forage and conservation settings, while the tetraploid cultivars are mostly targeted for bioenergy end-uses, due to their higher biomass yields. Switchgrass populations also occur as upland and lowland ecotypes, and constitute different heterotic groups. Switchgrass is mostly an obligate outcrosser resulting in substantial genotypic and phenotypic variation within populations. In the last ~15 years, significant resources have been dedicated to both breeding and understanding the genomic makeup of this plant, with a focus on bioenergy. This investment has resulted in the development of elite lines as well as a considerable increase in available genetic, physiological, and biomass-related information. The United States Department of Agriculture-Agricultural Research Service has been a major player in these developments (Mitchell and Schmer, 2012; Vogel et al., 2011). With significant improvements in DNA-sequencing technologies (High Throughput Sequencing, HTS), it has become possible to undertake large-scale analysis of both the genomic and functional genomic components of switchgrass. One such undertaking by the United States Department of Energy-Joint Genomics Institute has provided a draft assembly and annotation of the switchgrass genome (www.phytozome.org). This remarkable resource has permitted a complete utilization of HTS to analyze gene expression using RNA-Seq and related bioinformatic pipelines. Large-scale studies that are performed using field-grown plants and populations with well-characterized phenotypic traits, it increases the likelihood of discovering molecular events that underpin phenomena of interest. Even though lowland tetraploid cultivars have higher biomass yields than upland tetraploid cultivars, they can suffer significant winter-kill in more northern locations (Central Great Plains of the USA). Winter-kill is associated with the loss of rhizomes and other perenniating structures resulting in a complete or partial loss of tillering ability in the following seasons. Partial attrition of tiller production serves to limit new rhizome growth in successive years. One or more cycles of winter kill will ultimately kill the plant. We are trying to understand the cellular metabolism associated with the onset of rhizome dormancy and to connect the links between tiller/leaf senescence and rhizome metabolism using field grown plants from diverse populations, HTS and RNA-Seq

Next-Generation Sequencing of Crown and Rhizome Transcriptome from an Upland, Tetraploid Switchgrass

The crown and rhizome transcriptome of an upland tetraploid switchgrass cultivar cv Summer well adapted to the upper Midwest was investigated using the Roche 454-FLX pyrosequencing platform. Overall, approximately one million reads consisting of 216 million bases were assembled into 27,687 contigs and 43,094 singletons. Analyses of these sequences revealed minor contamination with non-plant sequences (\u3c 0.5%), indicating that a majority were for transcripts coded by the switchgrass genome. Blast2Gos comparisons resulted in the annotation of ~65% of the contig sequences and ~40% of the singleton sequences. Contig sequences were mostly homologous to other plant sequences, dominated by matches to Sorghum bicolor genome. Singleton sequences, while displaying significant matches to S. bicolor, also contained sequences matching non-plant species. Comparisons of the 454 dataset to existing EST collections resulted in the identification of 30,177 new sequences. These new sequences coded for a number of different proteins and a selective analysis of two categories, namely, peroxidases and transcription factors, resulted in the identification of specific peroxidases and a number of low-abundance transcription factors expected to be involved in chromatin remodeling. KEGG maps for glycolysis and sugar metabolism showed high levels of transcript coding for enzymes involved in primary metabolism. The assembly provided significant insights into the status of these tissues and broadly indicated that there was active metabolism taking place in the crown and rhizomes at post-anthesis, the seed maturation stage of plant development

Hookworm Infection and Environmental Factors in Mbeya Region, Tanzania: A Cross-sectional, Population-based study.

Hookworm disease is one of the most common infections and cause of a high disease burden in the tropics and subtropics. Remotely sensed ecological data and model-based geostatistics have been used recently to identify areas in need for hookworm control. Cross-sectional interview data and stool samples from 6,375 participants from nine different sites in Mbeya region, south-western Tanzania, were collected as part of a cohort study. Hookworm infection was assessed by microscopy of duplicate Kato-Katz thick smears from one stool sample from each participant. A geographic information system was used to obtain remotely sensed environmental data such as land surface temperature (LST), vegetation cover, rainfall, and elevation, and combine them with hookworm infection data and with socio-demographic and behavioral data. Uni- and multivariable logistic regression was performed on sites separately and on the pooled dataset. Univariable analyses yielded significant associations for all ecological variables. Five ecological variables stayed significant in the final multivariable model: population density (odds ratio (OR) = 0.68; 95% confidence interval (CI) = 0.63-0.73), mean annual vegetation density (OR = 0.11; 95% CI = 0.06-0.18), mean annual LST during the day (OR = 0.81; 95% CI = 0.75-0.88), mean annual LST during the night (OR = 1.54; 95% CI = 1.44-1.64), and latrine coverage in household surroundings (OR = 1.02; 95% CI = 1.01-1.04). Interaction terms revealed substantial differences in associations of hookworm infection with population density, mean annual enhanced vegetation index, and latrine coverage between the two sites with the highest prevalence of infection. This study supports previous findings that remotely sensed data such as vegetation indices, LST, and elevation are strongly associated with hookworm prevalence. However, the results indicate that the influence of environmental conditions can differ substantially within a relatively small geographic area. The use of large-scale associations as a predictive tool on smaller scales is therefore problematic and should be handled with care

Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin