My research develops and applies stable isotope analysis as a tool to address fundamental questions at the interface between plants and their environment. I have focused on taking a highly mechanistic approach to tackle critical research challenges in environmental plant physiology. A central theme of my research involves in-depth exploration of physiological, biophysical and biochemical mechanisms behind oxygen, hydrogen and carbon isotopic variability during key plant physiological processes including transpiration, photosynthesis and post-photosynthetic metabolisms. My key contributions to the field are as the following:
(1) Experimental demonstration of the mechanisms underlying leaf water isotope enrichment.
As water is transpired to the atmosphere, the lighter isotopes of water (H, 16O) evaporate preferentially over the heavy isotopes (D, 18O), resulting in an enrichment of leaf water. The evaporatively enriched signal of leaf water (Delta_L) provides a cornerstone of several isotope-based ecological applications including reconstruction of plant ecophysiological performance and assessment of terrestrial water and carbon cycle. The promising applications underscore the need for a solid understanding of the mechanistic controls over leaf water enrichment. Towards this goal I carried out a common garden experiment to show, for the first time, that pathlength for water movement inside the leaf, -- a critical component of the Delta_L model, -- exhibits a universal scaling relationship with transpiration rate (Song et al. 2013 PC&E). In a subsequent experiment specifically focusing on cotton plants, I set up a measurement system coupling laser isotope spectrometry with a gas exchange system to allow for determination of delta_18O and delta_2H of evaporative site water within the leaf with high degree of certainty under both steady and non-steady state conditions. With the collected data I re-examined the evidence related to within-leaf water isotope heterogeneity predicted by theoretical models and further demonstrated that a simpler, two-pool model was sufficient to predict enrichment in cotton leaves (Song et al. 2015 New Phyto). These results improved our understanding of the influence of leaf physiology on fractionation processes underpinning Delta_L, and thus have significant ramifications for the various ecological applications that stem from Delta_L-related signals.
(2) Improving understanding of the “non-climatic” aspect of plant cellulose delta_18O signal
There is currently wide-spread interest among paleo-environmental scientists to employ the delta_18O analysis of tree ring cellulose as a tool to reconstruct past environmental/climatic conditions. This is because climatic factors such as temperature, precipitation and relative humidity are known to exert significant controls on delta_18O of tree-ring cellulose (delta_18O_cell). Within the context of d18Ocell – climate relationships, it is important to recognize that some non-climatic factors such as species characteristics can also affect d18Ocell. The non-climatic factors are often considered as “noises” in delta_18O_cell based paleo-climate studies, and therefore have attracted much less attention in the scientific community than climatic factors that are often the target “signals” of reconstructive interest. However, it can be argued that without sufficient knowledge of the “noises”, one’s ability to effectively extract climatic signals from delta_18O_cell is unavoidably compromised. Furthermore, the isotopic “noises”, if carefully analyzed, can also be used to reveal novel, retrospective insight into plant ecophysiological performance in the context of a changing environment (Song et al. 2011 New Phyto; Helliker et al. Oecologia 2018; Liancourt et al. 2020 Global Change Biol).
In this context, we designed a field experiment to examine non-climatic aspects of tree-ring delta_18O_cell variation. Our contribution was demonstrating that a biochemical fractionation factor that controls the degree (p_ex) of organic oxygen exchange with the unenriched xylem water during post-photosynthetic processes plays a pivotal role in driving delta_18O_cell difference between evergreen coniferous (i.e. pines) and broad-leaved deciduous (i.e. oaks) tree species (Song et al. 2014a PC&E). In another study performed under controlled environmental conditions, we showed that variation in p_ex is significantly associated with variation in how fast the pool of non-structural carbohydrates, such as sucrose, is turning over during cellulose synthesis (Song et al. 2014b PC&E). Both of these studies have contributed to bringing to the current realization by the scientific community that the “pex being constant” assumption should not be taken for granted, and thus represent a step forward in improving mechanistic understanding of isotopic effect during plant cellulose synthesis.
(3) Innovative use of laser spectrometry in plant water isotope studies.
I have demonstrated through years of research that innovative use of laser spectrometry has the potential to revolutionize the research field of plant water isotopes. Aside from the afore-mentioned application for determining water isotope signals at the evaporative sites of the leaf, the laser-gas exchange coupled system could also be explored to trace the exponential trajectory of the variations in isotope signatures of transpiration during its approach towards the steady state. By taking advantage of this unique measurement power we were able to make the first rigorous experimental testing of the nonsteady state model of leaf water enrichment (Song et al. 2015 PC&E). Moreover, we developed a novel, direct-equilibration based method for measuring leaf water isotope ratios in a wide range of species, bypassing the need for water extraction as in the conventional methods. CO2 laser spectrometry remains a critical component of this method as it can provide rapid and low-cost solution for determining delta_18O of the CO2 molecules after direct equilibration with water in leaf samples (Song & Barbour 2016 New Phyto).
In the most recent example showcasing the “magic power” of laser spectrometry, we purposedly designed a whole-plant gas exchange system to couple with LGR water isotope analyser, so to make possible unbiased determination of stem xylem water delta_2H through steady-state monitoring of whole-plant transpired vapor delta_2H. Using this measurement system, we show that significant deuterium depletion in cryogenically-extracted stem water from xylem water is universally present in various plant species investigated, and further that the “deuterium depletion” phenomenon is not caused by isotope fractionation during root water uptake as traditionally thought, but rather is rooted in a cryogenic extraction-associated methodological artifact. Using the unraveled magnitude of “deuterium depletion” as a correction factor to adjust extraction artifact in an earlier study of world-wide variation in deuterium offsets of xylem water, we show that the extraction error-corrected result tends to nullify support for ecohydrological separation as a globally widespread phenomenon. This surprising result signals a clear need of re-assessing the paradigm-shifting concept of “two water worlds” through appropriately incorporating a measurement artifact (Chen et al. 2020 PNAS, Song et al. 2021a, 2021b PNAS).
(1) Experimental demonstration of the mechanisms underlying leaf water isotope enrichment.
As water is transpired to the atmosphere, the lighter isotopes of water (H, 16O) evaporate preferentially over the heavy isotopes (D, 18O), resulting in an enrichment of leaf water. The evaporatively enriched signal of leaf water (Delta_L) provides a cornerstone of several isotope-based ecological applications including reconstruction of plant ecophysiological performance and assessment of terrestrial water and carbon cycle. The promising applications underscore the need for a solid understanding of the mechanistic controls over leaf water enrichment. Towards this goal I carried out a common garden experiment to show, for the first time, that pathlength for water movement inside the leaf, -- a critical component of the Delta_L model, -- exhibits a universal scaling relationship with transpiration rate (Song et al. 2013 PC&E). In a subsequent experiment specifically focusing on cotton plants, I set up a measurement system coupling laser isotope spectrometry with a gas exchange system to allow for determination of delta_18O and delta_2H of evaporative site water within the leaf with high degree of certainty under both steady and non-steady state conditions. With the collected data I re-examined the evidence related to within-leaf water isotope heterogeneity predicted by theoretical models and further demonstrated that a simpler, two-pool model was sufficient to predict enrichment in cotton leaves (Song et al. 2015 New Phyto). These results improved our understanding of the influence of leaf physiology on fractionation processes underpinning Delta_L, and thus have significant ramifications for the various ecological applications that stem from Delta_L-related signals.
(2) Improving understanding of the “non-climatic” aspect of plant cellulose delta_18O signal
There is currently wide-spread interest among paleo-environmental scientists to employ the delta_18O analysis of tree ring cellulose as a tool to reconstruct past environmental/climatic conditions. This is because climatic factors such as temperature, precipitation and relative humidity are known to exert significant controls on delta_18O of tree-ring cellulose (delta_18O_cell). Within the context of d18Ocell – climate relationships, it is important to recognize that some non-climatic factors such as species characteristics can also affect d18Ocell. The non-climatic factors are often considered as “noises” in delta_18O_cell based paleo-climate studies, and therefore have attracted much less attention in the scientific community than climatic factors that are often the target “signals” of reconstructive interest. However, it can be argued that without sufficient knowledge of the “noises”, one’s ability to effectively extract climatic signals from delta_18O_cell is unavoidably compromised. Furthermore, the isotopic “noises”, if carefully analyzed, can also be used to reveal novel, retrospective insight into plant ecophysiological performance in the context of a changing environment (Song et al. 2011 New Phyto; Helliker et al. Oecologia 2018; Liancourt et al. 2020 Global Change Biol).
In this context, we designed a field experiment to examine non-climatic aspects of tree-ring delta_18O_cell variation. Our contribution was demonstrating that a biochemical fractionation factor that controls the degree (p_ex) of organic oxygen exchange with the unenriched xylem water during post-photosynthetic processes plays a pivotal role in driving delta_18O_cell difference between evergreen coniferous (i.e. pines) and broad-leaved deciduous (i.e. oaks) tree species (Song et al. 2014a PC&E). In another study performed under controlled environmental conditions, we showed that variation in p_ex is significantly associated with variation in how fast the pool of non-structural carbohydrates, such as sucrose, is turning over during cellulose synthesis (Song et al. 2014b PC&E). Both of these studies have contributed to bringing to the current realization by the scientific community that the “pex being constant” assumption should not be taken for granted, and thus represent a step forward in improving mechanistic understanding of isotopic effect during plant cellulose synthesis.
(3) Innovative use of laser spectrometry in plant water isotope studies.
I have demonstrated through years of research that innovative use of laser spectrometry has the potential to revolutionize the research field of plant water isotopes. Aside from the afore-mentioned application for determining water isotope signals at the evaporative sites of the leaf, the laser-gas exchange coupled system could also be explored to trace the exponential trajectory of the variations in isotope signatures of transpiration during its approach towards the steady state. By taking advantage of this unique measurement power we were able to make the first rigorous experimental testing of the nonsteady state model of leaf water enrichment (Song et al. 2015 PC&E). Moreover, we developed a novel, direct-equilibration based method for measuring leaf water isotope ratios in a wide range of species, bypassing the need for water extraction as in the conventional methods. CO2 laser spectrometry remains a critical component of this method as it can provide rapid and low-cost solution for determining delta_18O of the CO2 molecules after direct equilibration with water in leaf samples (Song & Barbour 2016 New Phyto).
In the most recent example showcasing the “magic power” of laser spectrometry, we purposedly designed a whole-plant gas exchange system to couple with LGR water isotope analyser, so to make possible unbiased determination of stem xylem water delta_2H through steady-state monitoring of whole-plant transpired vapor delta_2H. Using this measurement system, we show that significant deuterium depletion in cryogenically-extracted stem water from xylem water is universally present in various plant species investigated, and further that the “deuterium depletion” phenomenon is not caused by isotope fractionation during root water uptake as traditionally thought, but rather is rooted in a cryogenic extraction-associated methodological artifact. Using the unraveled magnitude of “deuterium depletion” as a correction factor to adjust extraction artifact in an earlier study of world-wide variation in deuterium offsets of xylem water, we show that the extraction error-corrected result tends to nullify support for ecohydrological separation as a globally widespread phenomenon. This surprising result signals a clear need of re-assessing the paradigm-shifting concept of “two water worlds” through appropriately incorporating a measurement artifact (Chen et al. 2020 PNAS, Song et al. 2021a, 2021b PNAS).