Calorespirometry as a phenotyping tool for selection of genotypes with high plasticity upon temperature stress – a focus on the analysis of pea seeds

Abstract

Increasing human population and climatic and edaphic changes impose needs to develop new cultivars with seeds that have grea-ter plasticity for environmental constraints and higher germinati-on rates. Improving seed vigor remains a primary objective of the agricultural industry and the seed/breeding companies that sup-port it. Phenotyping tools to assist breeding programs for pheno-typing seeds for resilience and selecting high-quality crop seeds are of interest. Because temperature is the major environmental constraint controlling growth, with a strong impact on crop yield, we have investigated the temperature dependence of respiratory parameters with calorespirometry as an alternative to conventio-nal seed phenotyping strategies. Calorespirometry has been used to assess metabolic and respiratory changes associated with cell reprogramming events, and has been proposed as a screening tool for predicting growth phenotype for temperature stress. Calore-spirometry measures metabolic heat rates (Rq) and CO2 emission rates (RCO2) of biological samples as a function of temperature allowing prediction of growth rates (Rstruct biomass) without growing plants under different environmental temperatures. Calorespiro-metry also allows the determination of the fraction of carbon sub-strate incorporated into structural biomass, i.e. carbon use effi-ciency (ε), as a function of temperature. Three pea (Pisum sativum L.) cultivars (‘Rondo’, ‘Torta de Quebrar’ and ‘Maravilha D´América’) commonly used in conventional agri-culture were selected for calorespirometry measurements and germination trials. The effect of temperature on seed germination was evaluated after an imbibition period of 16 h under dark condi-tions. Calorespirometric measurements were conducted in iso-thermal mode in a Multi-Cell Differential Scanning Calorimeter (TA Instruments, New Castle, DE) at 15, 20, 25 and 28°C. A minimum of three measurements were made on each cultivar. The heat rates, Rq, were measured directly and CO2 rates, RCO2, were deter-mined from the increase in the heat rate in the presence of a vial of NaOH, which reacts with CO2 in an exothermic reaction (-108.5 kJ/mol CO2). For data validation, a seed germination trial with the same cultivars was conducted under the same temperatures tested in the calorimeter. Substrate carbon conversion efficiencies and growth rates could not be calculated from the data collected in this study because the calorespirometric ratios, Rq/RCO2, showed that a significant portion of the CO2 was produced by anaerobic respiration.