Measuring carbon fixation under low temperatures

This article was originally posted on the WinterTurf project blog.

By Dominic Petrella

The amount of carbon that is fixed (i.e. photosynthesized) during winter acclimation may have a direct impact on winter survival due to increasing the amount of carbohydrates. The concentrations of carbohydrates such as sucrose, glucose, raffinose, and fructans have been shown to be important for winter survival in different turfgrasses. This has been shown in previous research on perennial ryegrass (Hoffman et al., 2010), buffalograss (Ball et al., 2002), creeping bentgrass, and Poa annua (Hoffman et al., 2014). However, we do not have a good grasp on what temperatures carbon fixation becomes limited in turfgrasses, and if some species are better at low temperature carbon fixation than others. Plants that fix more carbon at low temperatures may be more fit to survive winter. 

We have been working to develop methods to better measure carbon fixation in turfgrasses. Typical methods that measure photosynthesis utilize infrared gas analyzers (IRGA) to quantify rates of carbon fixation. While these instruments have produced plenty of valuable data on turfgrasses, they are not the best for measuring photosynthesis on turfgrasses maintained at low heights of cut and have drawbacks for low temperature experiments. We have been adapting methods based on previous research that uses carbon isotope ratios to quantify carbon fixation (Bergman et al., 2021). 

Carbon can be found in 3 isotopes: ¹²C, ¹³C, and ¹⁴C. Both ¹²C and ¹³C are stable, and ¹⁴C is radioactive. Most of the carbon dioxide in our atmosphere is made of ¹²C, but approximately 1.1% is ¹³C. Plants fix both ¹²CO₂ and ¹³CO₂, but fix a greater amount of ¹²CO₂ due to enzymatic and diffusional discrimination of ¹³CO₂ in the leaf (Cesmusak et al., 2013; van Caemmmerer et al., 2014). This discrimination can be measured and reported as a ratio of how much ¹³C is in the plant relative to ¹²C (δ¹³C, per mille, a percentage in parts per thousand rather than the typical parts per hundred, ‰). For measuring carbon fixation, we can measure a baseline δ¹³C and see how it changes when we introduce atmosphere that only contains ¹³CO₂ (i.e. pulse labeling). This gives the plant no choice on what isotope of carbon can be fixed and allows us to measure how much more ¹³C is incorporated over time. 

Graduate student Jillian Turbeville has been working to adapt simple versions of this type of method to quantify carbon fixation under shade stress and see how light quality influences photosynthesis when light intensity is low (Figure 1). Stable carbon isotope labeling involves sealing plants in an enclosed chamber, flooding them with atmosphere containing 100% ¹³CO₂ for a pre-determined amount of time, harvesting leaves, and then measuring δ¹³C using elemental analysis – isotope ratio mass spectrometry (EA-IRMS). Our next steps for this method are to see how well it works at low temperatures, and determine if low temperature tolerant perennial ryegrasses also fix more carbon at low temperatures.

References

Ball, S., Qian, Y., & Stushnoff, C. (2001). Soluble carbohydrates in two buffalograss cultivars with contrasting freezing tolerance. Journal of the American Society for Horticultural Science, 127(1), 45–49.

Bergman, M.E., González-Cabanelas, D., Wright, L.P. et al. Isotope ratio-based quantification of carbon assimilation highlights the role of plastidial isoprenoid precursor availability in photosynthesis. Plant Methods 17, 32 (2021).

Ebdon, J.S., Petrovic, A.M. and Dawson, T.E. (1998), Relationship between carbon isotope discrimination, water use efficiency, and evapotranspiration in Kentucky Bluegrass. Crop Science, 38: 157-162.

Hoffman, L., DaCosta, M., Ebdon, J.S. and Watkins, E. (2010). Physiological changes during cold acclimation of perennial ryegrass accessions differing in freeze tolerance. Crop Science, 50: 1037-1047.

Hoffman, L., DaCosta, M., Bertrand, A., Castonguay, Y., and Ebdon, J.S. (2014). Comparative assessment of metabolic responses to cold acclimation and deacclimation in annual bluegrass and creeping bentgrass. Environmental and Experimental Botany, 106: 197-206.