Trees are increasingly exposed to hot droughts due to CO 2 -induced climate change. However, the direct role of [CO 2 ] in altering tree physiological responses to drought and heat stress remains ambiguous.Pinus halepensis (Aleppo pine) trees were grown from seed under ambient (421 ppm) or elevated (867 ppm) [CO 2 ]. The 1.5-yr-old trees, either well watered or drought treated for 1 month, were transferred to separate gas-exchange chambers and the temperature gradually increased from 25°C to 40°C over a 10 d period. Continuous whole-tree shoot and root gasexchange measurements were supplemented by primary metabolite analysis.Elevated [CO 2 ] reduced tree water loss, reflected in lower stomatal conductance, resulting in a higher water-use efficiency throughout amplifying heat stress. Net carbon uptake declined strongly, driven by increases in respiration peaking earlier in the well-watered (31-32°C) than drought (33-34°C) treatments unaffected by growth [CO 2 ]. Further, drought altered the primary metabolome, whereas the metabolic response to [CO 2 ] was subtle and mainly reflected in enhanced root protein stability.The impact of elevated [CO 2 ] on tree stress responses was modest and largely vanished with progressing heat and drought. We therefore conclude that increases in atmospheric [CO 2 ] cannot counterbalance the impacts of hot drought extremes in Aleppo pine.

Climate extremes are likely to occur more frequently in the future, including a combination of heat waves and drought. However, the responses of trees to combined stress and their post-stress recovery are not fully understood yet. Therefore, this study investigated the responses of semi-arid Pinus halepensis seedlings to moderate drought, heat and combined heat-drought stress, as well as post-stress recovery. The seedlings were grown under controlled conditions and exposed to two 4-days-long heat periods, reaching air temperature maxima of 42 • C and vapor pressure deficit (VPD) of 7 kPa. Day-and nighttime canopy gas exchange was measured and differences in shoot and root allocation of non-structural carbohydrate (NSC) compounds (soluble sugars, starch, cyclitols, and carboxylic acids) assessed. Fluorescence parameters, nitrate levels, proline content and shoot water potential (ψ) provided additional indicators for stress severity and recovery performance. During the heat periods, net photosynthesis and stomatal conductance decreased immediately. This decline was modest under well-watered conditions, with transpiration and dark respiration rates remaining high and despite reductions in root NSC content, trees recovered following heat release. This was not the case in the heat-drought treatment, where stress resulted in high mortality rates and the few surviving seedlings showed reduced gas exchange rates and low root NSC content, while leaf nitrate and proline remained elevated even 3 weeks after heat release. Shoot ψ indicated that hydraulic failure was not the reason for mortality in the heat-drought seedlings. Instead, we argue that low transpiration rates, which resulted in needle temperatures >47 • C during heat stress (6 • C above air temperature) have caused irreversible damage. In summary, it could be demonstrated that heat waves in combination with moderate drought can either result in increased mortality or, if the seedlings survive, in delayed recovery. This highlights the potential of an increase in heat wave temperatures to trigger forest decline in semi-arid regions.

Drought‐induced tree mortality is expected to occur more frequently under predicted climate change. However, the extent of a possibly mitigating effect of simultaneously rising atmospheric [CO2] on stress thresholds leading to tree death is not fully understood, yet. Here, we studied the drought response, the time until critical stress thresholds were reached and mortality occurrence of Pinus halepensis (Miller). In order to observe a large potential benefit from eCO2, the seedlings were grown with ample of water and nutrient supply under either highly elevated [CO2] (eCO2, c. 936 ppm) or ambient (aCO2, c. 407 ppm) during 2 years. The subsequent exposure to a fast or a slow lethal drought was monitored using whole‐tree gas exchange chambers, measured leaf water potential and non‐structural carbohydrates. Using logistic regressions to derive probabilities for physiological parameters to reach critical drought stress thresholds, indicated a longer period for halving needle starch storage under eCO2 than aCO2. Stomatal closure, turgor loss, the duration until the daily tree C balance turned negative, leaf water potential at thresholds and time‐of‐death were unaffected by eCO2. Overall, our study provides for the first‐time insights into the chronological interplay of physiological drought thresholds under long‐term acclimation to elevated [CO2].

The cause of reduced leaf-level transpiration under elevated CO2 remains largely elusive. Here, we assessed stomatal, hydraulic and morphological adjustments in a long-term experiment on Aleppo pine (Pinus halepensis) seedlings germinated and grown for 22–40 months under elevated (eCO2; c. 860 ppm) or ambient (aCO2; c. 410 ppm) CO2. We assessed if eCO2-triggered reductions in canopy conductance (gc) alter the response to soil or atmospheric drought and are reversible or lasting due to anatomical adjustments by exposing eCO2 seedlings to decreasing [CO2]. To quantify underlying mechanisms, we analyzed leaf abscisic acid (ABA) level, stomatal and leaf morphology, xylem structure, hydraulic efficiency, and hydraulic safety. Effects of eCO2 manifested in a strong reduction in leaf-level gc (-55%) not caused by ABA and not reversible under low CO2 (c. 200 ppm). Stomatal development and size were unchanged while stomatal density increased (+18%). An increased vein-to-epidermis distance (+65%) suggested a larger leaf resistance to water flow. This was supported by anatomical adjustments of branch xylem having smaller conduits (-8%) and lower conduit lumen fraction (-11%), which resulted in a lower specific conductivity (-19%) and leaf-specific conductivity (-34%). These adaptations to CO2 did not change stomatal sensitivity to soil or atmospheric drought, consistent with similar xylem safety thresholds. In summary, we found reductions of gc under elevated CO2 to be reflected in anatomical adjustments and decreases in hydraulic conductivity. As these water savings were largely annulled by increases in leaf biomass, we do not expect alleviation of drought stress in a high CO2 atmosphere.

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