Integrated phenotyping reveals coordinated physiological responses to cold, freezing and recovery in Ramonda resurrection plants

Abstract

Resurrection plants provide insights into the physiological responses that enable survival under extreme dehydration and low-temperature stress. This study presents an integrated phenotyping analysis of cold and freezing responses and subsequent recovery in Ramonda serbica and R. nathaliae. Using chlorophyll fluorescence, multispectral reflectance and gas-exchange analysis, combined with regression modelling, we characterized changes in photosynthetic and water-related traits during sequential cooling, freezing-induced dehydration and rehydration. Both species underwent pronounced dehydration during freezing and showed recovery of water status and photosynthetic function following rehydration. Cooling and freezing were accompanied by declines in PSII efficiency and non-photochemical quenching (NPQ), while gas-exchange measurements during cooling showed reductions in CO2 assimilation and stomatal conductance. During recovery, PSII efficiency increased towards pre-stress levels, whereas NPQ was rapidly reactivated, consistent with the restoration of regulated photoprotective energy dissipation. The two species showed similar responses in relative water content, PSII efficiency and multispectral traits, while species-dependent differences were most evident in NPQ and in the temporal dynamics of several gas-exchange traits. NDVI recovered to near pre-stress values, whereas specific green reflectance remained below initial levels at the end of the recovery period, indicating asynchronous recovery among spectral traits. Overall, the results indicate that R. serbica and R. nathaliae share reversible physiological responses to freezing-induced dehydration, with differences between species primarily in the timing and magnitude of particular recovery processes rather than fundamentally different tolerance strategies. Integrating phenotyping with temporal modelling provides a framework for identifying coordinated and asynchronous components of stress and recovery responses in resurrection plants.