Arnold, P. A.; Harris, R. J.; Aitken, S. M.; Hoek, M. M.; Cook, A. M.; Leigh, A.; Nicotra, A. B.

Evaluating the drivers of variation in plant thermal tolerance limits requires a clearer understanding of how methodological matters can lead to different tolerance estimates. Chlorophyll fluorometry - to measure the temperature-dependent change in FV/FM - is a well-established approach to derive tolerance thresholds of photosystem II (PSII) in plants, but one-off, time-specific thermal exposures do not consider the fundamental dose-dependent effect of heat. The resurgent thermal death time (TDT) approach integrates both the temperature intensity and the exposure duration to derive time-based critical temperature thresholds and sensitivity parameters. We build upon this foundation to develop a protocol for evaluating thermal load sensitivity (TLS; non-lethal heat stress) of PSII in plants. Through five experiments across four diverse species, we tested the moderating effects of light, leaf sectioning, time since collection, and the temporal dynamics of FV/FM recovery. There were dramatic changes in tolerance threshold estimates based on thermal load (i.e. dose-dependent) effects on FV/FM, and strong effects of light intensity during heat and the presence of light post-heat. We offer recommendations pertaining to method implementation and discuss future empirical avenues. Appraising cumulative heat stress will enhance the utility of thermal tolerance estimates - the TLS approach outlined here moves us toward a new standard.