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Abstract. Stable isotope analysis is a powerful tool for assessing plant carbon and water relations and their impact on biogeochemical processes at different scales. Our processbased understanding of stable isotope signals, as well as technological developments, has progressed significantly, opening new frontiers in ecological and interdisciplinary research. This has promoted the broad utilisation of carbon, oxygen and hydrogen isotope applications to gain insight into plant carbon and water cycling and their interaction with the atmosphere and pedosphere. Here, we highlight specific areas of recent progress and new research challenges in plant carbon and water relations, using selected examples covering scales from the leaf to the regional scale. Further, we discuss strengths and limitations of recent technological Published by Copernicus Publications on behalf of the European Geosciences Union. C. Werner et al.: Progress and challenges in using stable isotopesdevelopments and approaches and highlight new opportunities arising from unprecedented temporal and spatial resolution of stable isotope measurements.

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

Summary• Temporal dynamics in carbon isotope ratios of ecosystem respiration ( δ 13 C R ) were evaluated on hourly, daily and annual timescales in a Mediterranean woodland. Emphasis was given to the periods of transition from wet to dry season and vice versa, when the system turns from a net carbon sink to a source. The constancy of nocturnal δ 13 C R was tested.• The relationship between δ 13 C R (determined through Keeling plots) and environmental factors was evaluated through time-lag analysis.• δ 13 C R exhibited high annual variation ( > 7‰). During the transition periods, δ 13 C R correlated significantly with factors influencing photosynthetic discrimination, soil respiration, and whole-canopy conductance. Time-lags differed between belowand above-ground variables, and between seasons. A shift in regression parameters with environmental factors indicated seasonal differences in ecosystem responsiveness (e.g. temperature acclimation). δ 13 C R exhibited substantial nocturnal enrichment ( > 4‰) from dusk to dawn.• These data indicate pronounced short-term dynamics in δ 13 C R at hourly to daily timescales and a modulated response to environmental drivers. Substantial shortterm changes in nocturnal δ 13 C R may have important implications for the sampling protocols of nocturnal Keeling plots.