Background

Plants produce energy in the form of ATP from glucose oxidation through mitochondrial respiration (Glucose + oxygen = carbon dioxide + water). In order to differentiate from photorespiration inherent to photosynthesis, dark respiration measurements are made in the absence of light (Graham, 1980). Mitochondria are essential organelles involved in energy production, amino acid biosynthesis, iron-sulfur (Fe-S) cluster cofactor biosynthesis, and other metabolic pathways that affect growth and stress responses (Lill, 2009). We identified two mitochondria-localized Fe-S cluster proteins, NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), as novel players in plant defense responses (Fonseca et al., 2020). In the current manuscript, we describe the methods used in this previous research to test a possible effect of the nfs1 and fh mutants, and overexpression lines, on mitochondrial respiration using measurements of dark respiration of the entire shoot and root. This simple protocol can be performed using plants grown entirely in controlled conditions in growth chambers. It involves the flux measurement of the dark respiration by-product CO₂ from tissues in small airtight chambers, using an LI-850 CO₂/H₂O gas analyzer (LICOR) in a dark room with a supplemental photosynthetically inactive yellow LED light. The CO₂ flux for each plant is measured during a 2 min 30 s interval using the software LI-8x0 v1.02 (LICOR). Specific shoot and root respiration rates (nmol CO₂ gfw^-1 s^-1) are calculated from the raw CO₂ flux data using R (v.3.6.0; https://www.r-project.org/), where linear regression is used to calculate the CO₂ flux rate with the dead band set at 60 s, and then divided by total shoot fresh weight.

Prior descriptions of plant respiration measurements often included complex apparatus consisting of independent equipment to move air and measure CO₂ with an infrared gas analyzer, and using a mass flow controller (Martin et al., 1981; Condori-Apfata et al., 2019). More recently, photosynthesis instruments that are an order of magnitude more expensive than the gas analyzer used here have been used with custom modifications (Tomaz et al., 2010). As an alternative to measuring the production of CO₂, other protocols make use of oxygen sensors to instead measure O₂ depletion (Li et al., 2013). In comparison, we have accomplished the same goals using less expensive and relatively less complex off-the-shelf parts that can be assembled and used by any plant laboratory. In addition, we have made the R scripts for data analysis publicly available to ensure the reproducibility of the method.

This protocol will be useful for researchers who need to measure dark respiration as a proxy for changes in mitochondrial abundance and/or activity, as we have done inFonseca et al. (2020). However, respiration has also been hypothesized to be a potential target for increasing metabolic efficiency, as has been done for photosynthesis (Amthor et al., 2019). Simulation results highlight the opportunity to target respiration as an unappreciated avenue for crop improvement, where both leaf and root respiration need to be considered for the whole-plant carbon allocation analysis (Holland et al., 2020). Indeed, a similar protocol as outlined here was used to measure in-light root respiration of 1,104 wheat seedlings to conduct a genome wide association study (Guo et al., 2021). This work found substantial heritable variation for specific root respiration as well as candidate genes responsible for that variation. Bunce (2021) showed that CO₂ depletion methods for respiration closely match the total mass reduction estimates of respiration, validating the approach. Therefore, this reproducible protocol can have an impact on multiple fields of plant biology, ranging from understanding the molecular basis of respiration to study respiration as a breeding target.