Singlet Oxygen and Superoxide Anion Radical Detection by EPR Spin Trapping in Thylakoid Preparations.

Reactive oxygen species (ROS) are produced by energy transfer and electron transport in plant chloroplast thylakoids at non-toxic levels under normal growth conditions, but at threatening levels under adverse or fluctuating environmental conditions. Among chloroplast ROS, singlet oxygen and superoxide anion radical, respectively, produced by photosystem II (PSII) and PSI, are known to be the major ROS under several stress conditions. Both are very unlikely to diffuse out of chloroplasts, but they are instead capable of triggering ROS-mediated chloroplast operational retrograde signalling to activate defence gene expression in concert with hormones and other molecular compounds. Therefore, their detection, identification and localization in vivo or in biological preparations is a priority for a deeper understanding of their role in (concurrent) regulation of plant growth and defence responses. Here, we present two EPR spin traps, abbreviated as TEMPD-HCl and DEPMPO, to detect and identify ROS in complex systems, such as isolated thylakoids, together with some hints and cautions to perform reliable spin trapping experiments.

Physiological and Antioxidant Response to Different Water Deficit Regimes of Flag Leaves and Ears of Wheat Grown under Combined Elevated CO2 and High Temperature.

Triticum aestivum L. cv. Gazul is a spring wheat widely cultivated in Castilla y León (Spain). Potted plants were grown in a scenario emulating the climate change environmental conditions expected by the end of this century, i.e., with elevated CO2 and high temperature under two water deficit regimes: long (LWD) and terminal (TWD). Changes in biomass and morphology, the content of proline (Pro), ascorbate (AsA) and glutathione (GSH), and enzymatic antioxidant activities were analyzed in flag leaves and ears. Additionally, leaf gas exchange was measured. LWD caused a decrease in biomass and AsA content but an increase in Pro content and catalase and GSH reductase activities in flag leaves, whereas TWD produced no significant changes. Photosynthesis was enhanced under both water deficit regimes. Increase in superoxide dismutase activity and Pro content was only observed in ears under TWD. The lack of a more acute effect of LWD and TWD on both organs was attributed to the ROS relieving effect of elevated CO2. Gazul acted as a drought tolerant variety with anisohydric behavior. A multifactorial analysis showed better adaptation of ears to water deficit than flag leaves, underlining the importance of this finding for breeding programs to improve grain yield under future climate change.

Screening for Higher Grain Yield and Biomass among Sixty Bread Wheat Genotypes Grown under Elevated CO2 and High-Temperature Conditions.

Global warming will inevitably affect crop development and productivity, increasing uncertainty regarding food production. The exploitation of genotypic variability can be a promising approach for selecting improved crop varieties that can counteract the adverse effects of future climate change. We investigated the natural variation in yield performance under combined elevated CO2 and high-temperature conditions in a set of 60 bread wheat genotypes (59 of the 8TH HTWSN CIMMYT collection and Gazul). Plant height, biomass production, yield components and phenological traits were assessed. Large variations in the selected traits were observed across genotypes. The CIMMYT genotypes showed higher biomass and grain yield when compared to Gazul, indicating that the former performed better than the latter under the studied environmental conditions. Principal component and hierarchical clustering analyses revealed that the 60 wheat genotypes employed different strategies to achieve final grain yield, highlighting that the genotypes that can preferentially increase grain and ear numbers per plant will display better yield responses under combined elevated levels of CO2 and temperature. This study demonstrates the success of the breeding programs under warmer temperatures and the plants' capacity to respond to the concurrence of certain environmental factors, opening new opportunities for the selection of widely adapted climate-resilient wheat genotypes.

Genotypic Variability on Grain Yield and Grain Nutritional Quality Characteristics of Wheat Grown under Elevated CO2 and High Temperature.

The progressive rise in atmospheric CO2 concentrations and temperature associated with climate change is predicted to have a major impact on the productivity and quality of food crops. Therefore, food security is highly dependent on climate change. Following a survey with 60 bread wheat genotypes, here we investigated the genetic variation in grain yield and nutritional quality among 10 of these genotypes grown under elevated CO2 and temperature. With this purpose, the biomass production, grain yield-related traits, the grain concentration of starch, total protein, phenolic compounds, and mineral nutrients, together with the total antioxidant capacity, were determined. Variation among genotypes was found for almost all the studied traits. Higher grain and ear numbers were associated with increased grain yield but decreased grain total protein concentration and minerals such as Cu, Fe, Mg, Na, P, and Zn. Mineral nutrients were mainly associated with wheat biomass, whereas protein concentration was affected by plant biomass and yield-related traits. Associations among different nutrients and promising nutrient concentrations in some wheat genotypes were also found. This study demonstrates that the exploration of genetic diversity is a powerful approach, not only for selecting genotypes with improved quality, but also for dissecting the effect of the environment on grain yield and nutritional composition.

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro.

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis-Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min-1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

Surfing the Hyperbola Equations of the Steady-State Farquhar-von Caemmerer-Berry C3 Leaf Photosynthesis Model: What Can a Theoretical Analysis of Their Oblique Asymptotes and Transition Points Tell Us?

The asymptotes and transition points of the net CO2 assimilation (A/Ci) rate curves of the steady-state Farquhar-von Caemmerer-Berry (FvCB) model for leaf photosynthesis of C3 plants are examined in a theoretical study, which begins from the exploration of the standard equations of hyperbolae after rotating the coordinate system. The analysis of the A/Ci quadratic equations of the three limitation states of the FvCB model-abbreviated as Ac, Aj and Ap-allows us to conclude that their oblique asymptotes have a common slope that depends only on the mesophyll conductance to CO2 diffusion (gm). The limiting values for the transition points between any two states of the three limitation states c, j and p do not depend on gm, and the results are therefore valid for rectangular and non-rectangular hyperbola equations of the FvCB model. The analysis of the variation of the slopes of the asymptotes with gm casts doubts about the fulfilment of the steady-state conditions, particularly, when the net CO2 assimilation rate is inhibited at high CO2 concentrations. The application of the theoretical analysis to extended steady-state FvCB models, where the hyperbola equations of Ac, Aj and Ap are modified to accommodate nitrogen assimilation and amino acids export via the photorespiratory pathway, is also discussed.