Carbon isotope composition of respired CO2 in woody stems and leafy shoots of three tree species along the growing season: physiological drivers for respiratory fractionation.

The carbon isotope composition of respired CO2 (δ13CR) and bulk organic matter (δ13CB) of various plant compartments informs about the isotopic fractionation and substrate of respiratory processes, which are crucial to advance the understanding of carbon allocation in plants. Nevertheless, the variation across organs, species and seasons remains poorly understood. Cavity Ring-Down Laser Spectroscopy was applied to measure δ13CR in leafy shoots and woody stems of maple (Acer platanoides L.), oak (Quercus robur L.) and cedar (Thuja occidentalis L.) trees during spring and late summer. Photosynthesis, respiration, growth and non-structural carbohydrates were measured in parallel to evaluate potential drivers for respiratory fractionation. The CO2 respired by maple and oak shoots was 13C-enriched relative to δ13CB during spring, but not late summer or in the stem. In cedar, δ13CR did not vary significantly throughout organs and seasons, with respired CO2 being 13C-depleted relative to δ13CB. Shoot δ13CR was positively related to leaf starch concentration in maple, while stem δ13CR was inversely related to stem growth. These relations were not significant for oak or cedar. The variability in δ13CR suggests (i) different contributions of respiratory pathways between organs and (ii) seasonality in the respiratory substrate and constitutive compounds for wood formation in deciduous species, less apparent in evergreen cedar, whose respiratory metabolism might be less variable.

Mechanistic drivers of stem respiration: A modelling exercise across species and seasons.

Stem respiration (RS ) plays a crucial role in plant carbon budgets. However, its poor understanding limits our ability to model woody tissue and whole-tree respiration. A biophysical model of stem water and carbon fluxes (TReSpire) was calibrated on cedar, maple and oak trees during spring and late summer. For this, stem sap flow, water potential, diameter variation, temperature, CO2 efflux, allometry and biochemistry were monitored. Shoot photosynthesis (PN ) and nonstructural carbohydrates (NSC) were additionally measured to evaluate source-sink relations. The highest RS and stem growth was found in maple and oak during spring, both being seasonally decoupled from PN and [NSC]. Temperature largely affected maintenance respiration (RM ) in the short term, but temperature-normalized RM was highly variable on a seasonal timescale. Overall, most of the respired CO2 radially diffused to the atmosphere (>87%) while the remainder was transported upward with the transpiration stream. The modelling exercise highlights the sink-driven behaviour of RS and the significance of overall metabolic activity on nitrogen (N) allocation patterns and N-normalized respiratory costs to capture RS variability over the long term. These insights should be considered when modelling plant respiration, whose representation is currently biased towards a better understanding of leaf metabolism.

Limited plasticity of anatomical and hydraulic traits in aspen trees under elevated CO2 and seasonal drought.

The timing of abiotic stress elicitors on wood formation largely affects xylem traits that determine xylem efficiency and vulnerability. Nonetheless, seasonal variability of elevated CO2 (eCO2) effects on tree functioning under drought remains largely unknown. To address this knowledge gap, 1-year-old aspen (Populus tremula L.) trees were grown under ambient (±445 ppm) and elevated (±700 ppm) CO2 and exposed to an early (spring/summer 2019) or late (summer/autumn 2018) season drought event. Stomatal conductance and stem shrinkage were monitored in vivo as xylem water potential decreased. Additional trees were harvested for characterization of wood anatomical traits and to determine vulnerability and desorption curves via bench dehydration. The abundance of narrow vessels decreased under eCO2 only during the early season. At this time, xylem vulnerability to embolism formation and hydraulic capacitance during severe drought increased under eCO2. Contrastingly, stomatal closure was delayed during the late season, while hydraulic vulnerability and capacitance remained unaffected under eCO2. Independently of the CO2 treatment, elastic, and inelastic water pools depleted simultaneously after 50% of complete stomatal closure. Our results suggest that the effect of eCO2 on drought physiology and wood traits are small and variable during the growing season and question a sequential capacitive water release from elastic and inelastic pools as drought proceeds.

Efflux and assimilation of xylem-transported CO2 in stems and leaves of tree species with different wood anatomy.

Determining the fate of CO2 respired in woody tissues is necessary to understand plant respiratory physiology and to evaluate CO2 recycling mechanisms. An aqueous 13 C-enriched CO2 solution was infused into the stem of 3-4 m tall trees to estimate efflux and assimilation of xylem-transported CO2 via cavity ring-down laser spectroscopy and isotope ratio mass spectrometry, respectively. Different tree locations (lower stem, upper stem and leafy shoots) and tissues (xylem, bark and leaves) were monitored in species with tracheid, diffuse- and ring-porous wood anatomy (cedar, maple and oak, respectively). Radial xylem CO2 diffusivity and xylem [CO2 ] were lower in cedar relative to maple and oak trees, thereby limiting label diffusion. Part of the labeled 13 CO2 was assimilated in cedar (8.7%) and oak (20.6%) trees, mostly in xylem and bark tissues of the stem, while limited solution uptake in maple trees hindered the detection of label assimilation. Little label reached foliar tissues, suggesting substantial label loss along the stem-branch transition following reductions in the radial diffusive pathway. Differences in respiration rates and radial xylem CO2 diffusivity (lower in conifer relative to angiosperm species) might reconcile discrepancies in efflux and assimilation of xylem-transported CO2 so far observed between taxonomic clades.

Leaf and tree responses of young European aspen trees to elevated atmospheric CO2 concentration vary over the season.

Elevated atmospheric CO2 concentration (eCO2) commonly stimulates net leaf assimilation, decreases stomatal conductance and has no clear effect on leaf respiration. However, effects of eCO2 on whole-tree functioning and its seasonal dynamics remain far more uncertain. To evaluate temporal and spatial variability in eCO2 effects, 1-year-old European aspen trees were grown in two treatment chambers under ambient (aCO2, 400 p.p.m.) and elevated (eCO2, 700 p.p.m.) CO2 concentrations during an early (spring 2019) and late (autumn 2018) seasonal experiment. Leaf (net carbon assimilation, stomatal conductance and leaf respiration) and whole-tree (stem growth, sap flow and stem CO2 efflux) responses to eCO2 were measured. Under eCO2, carbon assimilation was stimulated during the early (1.63-fold) and late (1.26-fold) seasonal experiments. Stimulation of carbon assimilation changed over time with largest increases observed in spring when stem volumetric growth was highest, followed by late season down-regulation, when stem volumetric growth ceased. The neutral eCO2 effect on stomatal conductance and leaf respiration measured at leaf level paralleled the unresponsive canopy conductance (derived from sap flow measurements) and stem CO2 efflux measured at tree level. Our results highlight that seasonality in carbon demand for tree growth substantially affects the magnitude of the response to eCO2 at both leaf and whole-tree level.

Woody tissue photosynthesis increases radial stem growth of young poplar trees under ambient atmospheric CO2 but its contribution ceases under elevated CO2.

Woody tissue photosynthesis (Pwt) contributes to the tree carbon (C) budget and generally stimulates radial stem growth under ambient atmospheric CO2 concentration (aCO2). Moreover, Pwt has potential to enhance tree survival under changing climates by delaying negative effects of drought stress on tree hydraulic functioning. However, the relevance of Pwt on tree performance under elevated atmospheric CO2 concentration (eCO2) remains unexplored. To fill this knowledge gap, 1-year-old Populus tremula L. seedlings were grown in two treatment chambers at aCO2 and eCO2 (400 and 660 ppm, respectively), and woody tissues of half of the seedlings in each treatment chamber were light-excluded to prevent Pwt. Radial stem growth, sap flow, leaf photosynthesis and stomatal and canopy conductance were measured throughout the growing season, and the concentration of non-structural carbohydrates (NSC) in stem tissues was determined at the end of the experiment. Fuelled by eCO2, an increase in stem growth of 18 and 50% was observed in control and light-excluded trees, respectively. Woody tissue photosynthesis increased radial stem growth by 39% under aCO2, while, surprisingly, no impact of Pwt on stem growth was observed under eCO2. By the end of the growing season, eCO2 and Pwt had little effect on stem growth, leaf photosynthesis acclimated to eCO2, but stomatal conductance did not, and homeostatic stem NSC pools were observed among combined treatments. Our results highlight that eCO2 potentially fulfils plant C requirements, limiting the contribution of Pwt to stem growth as atmospheric [CO2] rises, and that radial stem growth in young developing trees was C (source) limited during early phenological stages but transitioned towards sink-driven control at the end of the growing season.

Woody tissue photosynthesis delays drought stress in Populus tremula trees and maintains starch reserves in branch xylem tissues.

Photosynthesis in woody tissues (Pwt ) is less sensitive to water shortage than in leaves, hence, Pwt might be a crucial carbon source to alleviate drought stress. To evaluate the impact of Pwt on tree drought tolerance, woody tissues of 4-m-tall drought-stressed Populus tremula trees were subjected to a light-exclusion treatment across the entire plant to inhibit Pwt . Xylem water potential (Ψxylem ), sap flow ( FH2O ), leaf net photosynthesis (Pn,l ), stem diameter variations (ΔD), in vivo acoustic emissions in stems (AEs) and nonstructural carbohydrate concentrations ([NSC]) were monitored to comprehensively assess water and carbon relations at whole-tree level. Under well-watered conditions, Pwt kept Ψxylem at a higher level, lowered FH2O and had no effect on [NSC]. Under drought, Ψxylem , FH2O and Pn,l in light-excluded trees rapidly decreased in concert with reductions in branch xylem starch concentration. Moreover, sub-daily patterns of ΔD, FH2O and AEs were strongly related, suggesting that in vivo AEs may inform not only about embolism events, but also about capacitive release and replenishment of stem water pools. Results highlight the importance of Pwt in maintaining xylem hydraulic integrity under drought conditions and in sustaining NSC pools to potentially limit increases in xylem tension.

TReSpire - a biophysical TRee Stem respiration model.

Mechanistic models of plant respiration remain poorly developed, especially in stems and woody tissues where measurements of CO2 efflux do not necessarily reflect local respiratory activity. We built a process-based model of stem respiration that couples water and carbon fluxes at the organ level (TReSpire). To this end, sap flow, stem diameter variations, xylem and soil water potential, stem temperature, stem CO2 efflux and nonstructural carbohydrates were measured in a maple tree, while xylem CO2 concentration and additional stem and xylem diameter variations were monitored in an ancillary tree for model validation. TReSpire realistically described: (1) turgor pressure to differentiate growing from nongrowing metabolism; (2) maintenance expenditures in xylem and outer tissues based on Arrhenius kinetics and nitrogen content; and (3) radial CO2 diffusivity and CO2 solubility and transport in the sap solution. Collinearity issues with phloem unloading rates and sugar-starch interconversion rates suggest parallel submodelling to close the stem carbon balance. TReSpire brings a breakthrough in the modelling of stem water and carbon fluxes at a detailed (hourly) temporal resolution. TReSpire is calibrated from a sink-driven perspective, and has potential to advance our understanding on stem growth dynamics, CO2 fluxes and underlying respiratory physiology across different species and phenological stages.

Woody tissue photosynthesis reduces stem CO2 efflux by half and remains unaffected by drought stress in young Populus tremula trees.

A substantial portion of locally respired CO2 in stems can be assimilated by chloroplast-containing tissues. Woody tissue photosynthesis (Pwt ) therefore plays a major role in the stem carbon balance. To study the impact of Pwt on stem carbon cycling along a gradient of water availability, stem CO2 efflux (EA ), xylem CO2 concentration ([CO2 ]), and xylem water potential (Ψxylem ) were measured in 4-year-old Populus tremula L. trees exposed to drought stress and different regimes of light exclusion of woody tissues. Under well-watered conditions, local Pwt decreased EA up to 30%. Axial CO2 diffusion (Dax ) induced by distant Pwt caused an additional decrease in EA of up to 25% and limited xylem [CO2 ] build-up. Under drought stress, absolute decreases in EA driven by Pwt remained stable, denoting that Pwt was not affected by drought. At the end of the dry period, when transpiration was low, local Pwt and Dax offset 20% and 10% of stem respiration on a daily basis, respectively. These results highlight (a) the importance of Pwt for an adequate interpretation of EA measurements and (b) homeostatic Pwt along a drought stress gradient, which might play a crucial role to fuel stem metabolism when leaf carbon uptake and phloem transport are limited.

Isotope ratio laser spectroscopy to disentangle xylem-transported from locally respired CO2 in stem CO2 efflux.

Respired CO2 in woody tissues radially diffuses to the atmosphere or it is transported upward with the transpiration stream, making the origin of CO2 in stem CO2 efflux (EA) uncertain, which may confound stem respiration (RS) estimates. An aqueous 13C-enriched solution was infused into stems of Populus tremula L. trees, and real-time measurements of 13C-CO2 and 12C-CO2 in EA were performed via Cavity Ring Down Laser Spectroscopy (CRDS). The contribution of locally respired CO2 (LCO2) and xylem-transported CO2 (TCO2) to EA was estimated from their different isotopic composition. Mean daily values of TCO2/EA ranged from 13% to 38%, evidencing the notable role that xylem CO2 transport plays in the assessment of stem respiration. Mean daily TCO2/EA did not differ between treatments of drought stress and light exclusion of woody tissues, but they showed different TCO2/EA dynamics on a sub-daily time scale. Sub-daily CO2 diffusion patterns were explained by a light-induced axial CO2 gradient ascribed to woody tissue photosynthesis, and the resistance to radial CO2 diffusion determined by bark water content. Here, we demonstrate the outstanding potential of CRDS paired with 13C-CO2 labelling to advance in the understanding of CO2 movement at the plant-atmosphere interface and the respiratory physiology in woody tissues.

Sugars from woody tissue photosynthesis reduce xylem vulnerability to cavitation.

Reassimilation of internal CO2 via woody tissue photosynthesis has a substantial effect on tree carbon income and wood production. However, little is known about its role in xylem vulnerability to cavitation and its implications in drought-driven tree mortality. Young trees of Populus nigra were subjected to light exclusion at the branch and stem levels. After 40 d, measurements of xylem water potential, diameter variation and acoustic emission (AE) were performed in detached branches to obtain acoustic vulnerability curves to cavitation following bench-top dehydration. Acoustic vulnerability curves and derived AE50 values (i.e. water potential at which 50% of cavitation-related acoustic emissions occur) differed significantly between light-excluded and control branches (AE50,light-excluded  = -1.00 ± 0.13 MPa; AE50,control  = -1.45 ± 0.09 MPa; P = 0.007) denoting higher vulnerability to cavitation in light-excluded trees. Woody tissue photosynthesis represents an alternative and immediate source of nonstructural carbohydrates (NSC) that confers lower xylem vulnerability to cavitation via sugar-mediated mechanisms. Embolism repair and xylem structural changes could not explain this observation as the amount of cumulative AE and basic wood density did not differ between treatments. We suggest that woody tissue assimilates might play a role in the synthesis of xylem surfactants for nanobubble stabilization under tension.

Clustering reveals cavitation-related acoustic emission signals from dehydrating branches.

The formation of air emboli in the xylem during drought is one of the key processes leading to plant mortality due to loss in hydraulic conductivity, and strongly fuels the interest in quantifying vulnerability to cavitation. The acoustic emission (AE) technique can be used to measure hydraulic conductivity losses and construct vulnerability curves. For years, it has been believed that all the AE signals are produced by the formation of gas emboli in the xylem sap under tension. More recent experiments, however, demonstrate that gas emboli formation cannot explain all the signals detected during drought, suggesting that different sources of AE exist. This complicates the use of the AE technique to measure emboli formation in plants. We therefore analysed AE waveforms measured on branches of grapevine (Vitis vinifera L. 'Chardonnay') during bench dehydration with broadband sensors, and applied an automated clustering algorithm in order to find natural clusters of AE signals. We used AE features and AE activity patterns during consecutive dehydration phases to identify the different AE sources. Based on the frequency spectrum of the signals, we distinguished three different types of AE signals, of which the frequency cluster with high 100-200 kHz frequency content was strongly correlated with cavitation. Our results indicate that cavitation-related AE signals can be filtered from other AE sources, which presents a promising avenue into quantifying xylem embolism in plants in laboratory and field conditions.