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

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

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

A physiological signal derived from sun-induced chlorophyll fluorescence quantifies crop physiological response to environmental stresses in the U.S. Corn Belt

Sun-induced chlorophyll fluorescence (SIF) measurements have shown unique potential for quantifying plant physiological stress. However, recent investigations found canopy structure and radiation largely control SIF, and physiological relevance of SIF remains yet to be fully understood. This study aims to evaluate whether the SIF-derived physiological signal improves quantification of crop responses to environmental stresses, by analyzing data at three different spatial scales within the U.S. Corn Belt, i.e. experiment plot, field, and regional scales, where ground-based portable, stationary and space-borne hyperspectral sensing systems are used, respectively. We found that, when controlling for variations in incoming radiation and canopy structure, crop SIF signals can be decomposed into non-physiological (i.e. canopy structure and radiation, 60% ∼ 82%) and physiological information (i.e. physiological SIF yield, ΦF, 17% ∼ 31%), which confirms the contribution of physiological variation to SIF. We further evaluated whether ΦF indicated plant responses under high-temperature and high vapor pressure deficit (VPD) stresses. The plot-scale data showed that ΦF responded to the proxy for physiological stress (partial correlation coefficient, r p= 0.40, p\u3c 0.001) while non-physiological signals of SIF did not respond (p\u3e 0.1). The field-scale ΦF data showed water deficit stress from the comparison between irrigated and rainfed fields, and ΦF was positively correlated with canopy-scale stomatal conductance, a reliable indicator of plant physiological condition (correlation coefficient r= 0.60 and 0.56 for an irrigated and rainfed sites, respectively). The regional-scale data showed ΦF was more strongly correlated spatially with air temperature and VPD (r= 0.23 and 0.39) than SIF (r= 0.11 and 0.34) for the U.S. Corn Belt. The lines of evidence suggested that ΦF reflects crop physiological responses to environmental stresses with greater sensitivity to stress factors than SIF, and the stress quantification capability of ΦF is spatially scalable. Utilizing ΦF for physiological investigations will contribute to improve our understanding of vegetation responses to high-temperature and high-VPD stresses

Translation and preliminary validation of a Korean version of the parental reflective functioning questionnaire

This study aimed to explore the factor structure, reliability, and validity of a Korean translation of the Parental Reflective Functioning Questionnaire (PRFQ). The PRFQ consists of three subscales: prementalizing modes , certainty about mental states , and interest and curiosity in mental states . A convenience sample of 163 Korean parents completed the K‐PRFQ. Exploratory factor analysis showed three factors mapped on to the original PRFQ factors, but items from the original prementalizing modes subscale clustered into two additional factors. Data from a subsample (n = 67) showed that the certainty about mental states and interest and curiosity in mental states subscales correlated positively with more optimal self‐reported parenting. We discuss the validity of using the PRFQ in collectivistic culture