The bench scientist\u27s guide to statistical analysis of RNA-Seq data

RNA sequencing (RNA-Seq) is emerging as a highly accurate method to quantify transcript abundance. However, analyses of the large data sets obtained by sequencing the entire transcriptome of organisms have generally been performed by bioinformatics specialists. Here we provide a step-by-step guide and outline a strategy using currently available statistical tools that results in a conservative list of differentially expressed genes. We also discuss potential sources of error in RNA-Seq analysis that could alter interpretation of global changes in gene expression

Variation in acclimation of photosynthesis in Trifolium repens after eight years of exposure to Free Air CO2 Enrichment (FACE)

The initial stimulation of photosynthesis observed on elevation of [CO2] in grasslands has been predicted to be a transient phenomenon constrained by the loss of photosynthetic capacity due to other limitations, notably nutrients and sinks for carbohydrates. Legumes might be expected partially to escape these feedbacks through symbiotic N2 fixation. The Free‐Air Carbon dioxide Enrichment (FACE) experiment at Eschikon, Switzerland, has been the longest running investigation of the effects of open‐air elevation of [CO2] on vegetation. The prediction of a long‐term loss of photosynthetic capacity was tested by analysing photosynthesis in Trifolium repens L. (cv. Milkanova) in the spring and autumn of the eighth, ninth and tenth years of treatment. A high and low N treatment also allowed a test of the significance of exogenous N‐supply in maintaining a stimulation of photosynthetic capacity in the long‐term. Prior work in this Free Air CO2 Enrichment (FACE) experiment has revealed that elevated [CO2] increased both vegetative and reproductive growth of T. repens independent of N treatment. It is shown here that the photosynthetic response of T. repens was also independent of N fertilization under both current ambient and elevated (600 µmol mol-1) [CO2]. There was a strong effect of season on photosynthesis, with light‐saturated rates (Asat) 37% higher in spring than in autumn. Higher Asat in the spring was supported by higher maximum Rubisco carboxylation rates (Vc,max) and maximum rates of electron transport (Jmax) contributing to RuBP regeneration. Elevated [CO2] increased Asat by 37% when averaged across all measurement periods and both N fertilization levels, and decreased stomatal conductance by 25%. In spring, there was no effect of elevated [CO2] on photosynthetic capacity of leaves, but in autumn both Vc,max and Jmax were reduced by approximately 20% in elevated [CO2]. The results show that acclimation of photosynthetic capacity can occur in a nitrogen‐fixing species, in the field where there are no artificial restrictions on sink capacity. However, even with acclimation there was a highly significant increase in photosynthesis at elevated [CO2

Genetic strategies for improving crop yields.

The current trajectory for crop yields is insufficient to nourish the world's population by 20501. Greater and more consistent crop production must be achieved against a backdrop of climatic stress that limits yields, owing to shifts in pests and pathogens, precipitation, heat-waves and other weather extremes. Here we consider the potential of plant sciences to address post-Green Revolution challenges in agriculture and explore emerging strategies for enhancing sustainable crop production and resilience in a changing climate. Accelerated crop improvement must leverage naturally evolved traits and transformative engineering driven by mechanistic understanding, to yield the resilient production systems that are needed to ensure future harvests

Shifts in microbial communities in soil, rhizosphere and roots of two major crop systems under elevated CO2 and O3

Rising atmospheric concentrations of CO2 and O3 are key features of global environmental change. To investigate changes in the belowground bacterial community composition in response to elevated CO2 and O3 (eCO2 and eO3) the endosphere, rhizosphere and soil were sampled from soybeans under eCO2 and maize under eO3. The maize rhizosphere and endosphere α-diversity was higher than soybean, which may be due to a high relative abundance of Rhizobiales. Only the rhizosphere microbiome composition of the soybeans changed in response to eCO2, associated with an increased abundance of nitrogen fixing microbes. In maize, the microbiome composition was altered by the genotype and linked to differences in root exudate profiles. The eO3 treatment did not change the microbial communities in the rhizosphere, but altered the soil communities where hybrid maize was grown. In contrast to previous studies that focused exclusively on the soil, this study provides new insights into the effects of plant root exudates on the composition of the belowground microbiome in response to changing atmospheric conditions. Our results demonstrate that plant species and plant genotype were key factors driving the changes in the belowground bacterial community composition in agroecosystems that experience rising levels of atmospheric CO2 and O3

Ozone tolerant maize hybrids maintain Rubisco content and activity during long-term exposure in the field.

Ozone pollution is a damaging air pollutant that reduces maize yields equivalently to nutrient deficiency, heat, and aridity stress. Therefore, understanding the physiological and biochemical responses of maize to ozone pollution and identifying traits predictive of ozone tolerance is important. In this study, we examined the physiological, biochemical and yield responses of six maize hybrids to elevated ozone in the field using Free Air Ozone Enrichment. Elevated ozone stress reduced photosynthetic capacity, in vivo and in vitro, decreasing Rubisco content, but not activation state. Contrary to our hypotheses, variation in maize hybrid responses to ozone was not associated with stomatal limitation or antioxidant pools in maize. Rather, tolerance to ozone stress in the hybrid B73 × Mo17 was correlated with maintenance of leaf N content. Sensitive lines showed greater ozone-induced senescence and loss of photosynthetic capacity compared to the tolerant line

Advances in field-based high-throughput photosynthetic phenotyping

Gas exchange techniques revolutionized plant research and advanced understanding, including associated fluxes and efficiencies, of photosynthesis, photorespiration, and respiration of plants from cellular to ecosystem scales. These techniques remain the gold standard for inferring photosynthetic rates and underlying physiology/biochemistry, although their utility for high-throughput phenotyping (HTP) of photosynthesis is limited both by the number of gas exchange systems available and the number of personnel available to operate the equipment. Remote sensing techniques have long been used to assess ecosystem productivity at coarse spatial and temporal resolutions, and advances in sensor technology coupled with advanced statistical techniques are expanding remote sensing tools to finer spatial scales and increasing the number and complexity of phenotypes that can be extracted. In this review, we outline the photosynthetic phenotypes of interest to the plant science community and describe the advances in high-throughput techniques to characterize photosynthesis at spatial scales useful to infer treatment or genotypic variation in field-based experiments or breeding trials. We will accomplish this objective by presenting six lessons learned thus far through the development and application of proximal/remote sensing-based measurements and the accompanying statistical analyses. We will conclude by outlining what we perceive as the current limitations, bottlenecks, and opportunities facing HTP of photosynthesis

Predicting biochemical acclimation of leaf photosynthesis in soybean under in-field canopy warming using hyperspectral reflectance

Traditional gas exchange measurements are cumbersome, which makes it difficult to capture variation in biochemical parameters, namely the maximum rate of carboxylation measured at a reference temperature (Vcmax25) and the maximum electron transport at a reference temperature (Jmax25), in response to growth temperature over time from days to weeks. Hyperspectral reflectance provides reliable measures of Vcmax25 and Jmax25; however, the capability of this method to capture biochemical acclimations of the two parameters to high growth temperature over time has not been demonstrated. In this study, Vcmax25 and Jmax25 were measured over multiple growth stages during two growing seasons for field-grown soybeans using both gas exchange techniques and leaf spectral reflectance under ambient and four elevated canopy temperature treatments (ambient+1.5, +3, +4.5, and +6°C). Spectral vegetation indices and machine learning methods were used to build predictive models for Vcmax25 and Jmax25, based on the leaf reflectance. Results showed that these models yielded an R2 of 0.57–0.65 and 0.48–0.58 for Vcmax25 and Jmax25, respectively. Hyperspectral reflectance captured biochemical acclimation of leaf photosynthesis to high temperature in the field, improving spatial and temporal resolution in the ability to assess the impact of future warming on crop productivity

Uncovering hidden genetic variation in photosynthesis of field‐grown maize under ozone pollution

Ozone is the most damaging air pollutant to crops, currently reducing Midwest US maize production by up to 10%, yet there has been very little effort to adapt germ‐ plasm for ozone tolerance. Ozone enters plants through stomata, reacts to form reactive oxygen species in the apoplast and ultimately decreases photosynthetic C gain. In this study, 10 diverse inbred parents were crossed in a half‐diallel design to create 45 F1 hybrids, which were tested for ozone response in the field using free air concentration enrichment (FACE). Ozone stress increased the heritability of pho‐ tosynthetic traits and altered genetic correlations among traits. Hybrids from par‐ ents Hp301 and NC338 showed greater sensitivity to ozone stress, and disrupted relationships among photosynthetic traits. The physiological responses underlying sensitivity to ozone differed in hybrids from the two parents, suggesting multiple mechanisms of response to oxidative stress. FACE technology was essential to this evaluation because genetic variation in photosynthesis under elevated ozone was not predictable based on performance at ambient ozone. These findings suggest that selection under elevated ozone is needed to identify deleterious alleles in the world's largest commodity crop