In vivo X-ray microtomography locally affects stem radial growth with no immediate physiological impact.

Micro-computed tomography (µCT) is a non-destructive X-ray imaging method used in plant physiology to visualize in-situ plant tissues that enables assessments of embolized xylem vessels. Whereas evidence for X-ray-induced cellular damage has been reported, the impact on plant physiological processes such as carbon (C) uptake, transport, and use are unknown. Yet, these damages could be particularly relevant for studies that track embolism and C fluxes over time. We examined the physiological consequences of µCT-scanning for xylem embolism over three months by monitoring net photosynthesis (Anet), diameter growth, chlorophyll concentration (Chl), and foliar non-structural carbohydrate (NSC) content in four deciduous tree species: hedge maple (Acer campestre), ash (Fraxinus excelsior), European hornbeam (Carpinus betulus), and sessile oak (Quercus petraea). C transport from the canopy to the roots was also assessed through 13C labelling. Our results show that monthly X-ray application did not impact foliar Anet, Chl, NSC content, and C transport. Although X-ray effects did not vary between species, the most pronounced impact was observed in sessile oak, marked by stopped growth and stem deformations around the irradiated area. The absence of adverse impacts on plant physiology for all the tested treatments indicates that laboratory-based µCT systems can be used with different beam energy levels and doses without threatening the integrity of plant physiology within the range of tested parameters. However, the impacts of repetitive µCT on the stem radial growth at the irradiated zone leading to deformations in sessile oak might have lasting implications for studies tracking plant embolism in the longer-term.

Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques.

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO2 and H2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (gs ) and net photosynthesis rate (An ) decoupled across all tested species, with gs increasing but An decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency.

The mobilization and transport of newly fixed carbon are driven by plant water use in an experimental rainforest under drought.

Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem 13CO2 pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought.

Finding balance: Tree-ring isotopes differentiate between acclimation and stress-induced imbalance in a long-term irrigation experiment.

Scots pine (Pinus sylvestris L.) is a common European tree species, and understanding its acclimation to the rapidly changing climate through physiological, biochemical or structural adjustments is vital for predicting future growth. We investigated a long-term irrigation experiment at a naturally dry forest in Switzerland, comparing Scots pine trees that have been continuously irrigated for 17 years (irrigated) with those for which irrigation was interrupted after 10 years (stop) and non-irrigated trees (control), using tree growth, xylogenesis, wood anatomy, and carbon, oxygen and hydrogen stable isotope measurements in the water, sugars and cellulose of plant tissues. The dendrochronological analyses highlighted three distinct acclimation phases to the treatments: irrigated trees experienced (i) a significant growth increase in the first 4 years of treatment, (ii) high growth rates but with a declining trend in the following 8 years and finally (iii) a regression to pre-irrigation growth rates, suggesting the development of a new growth limitation (i.e. acclimation). The introduction of the stop treatment resulted in further growth reductions to below-control levels during the third phase. Irrigated trees showed longer growth periods and lower tree-ring δ13 C values, reflecting lower stomatal restrictions than control trees. Their strong tree-ring δ18 O and δ2 H (O-H) relationship reflected the hydrological signature similarly to the control. On the contrary, the stop trees had lower growth rates, conservative wood anatomical traits, and a weak O-H relationship, indicating a physiological imbalance. Tree vitality (identified by crown transparency) significantly modulated growth, wood anatomical traits and tree-ring δ13 C, with low-vitality trees of all treatments performing similarly regardless of water availability. We thus provide quantitative indicators for assessing physiological imbalance and tree acclimation after environmental stresses. We also show that tree vitality is crucial in shaping such responses. These findings are fundamental for the early assessment of ecosystem imbalances and decline under climate change.

Hydrogen isotope fractionation in carbohydrates of leaves and xylem tissues follows distinct phylogenetic patterns: a common garden experiment with 73 tree and shrub species.

Recent methodological advancements in determining the nonexchangeable hydrogen isotopic composition (δ2 Hne ) of plant carbohydrates make it possible to disentangle the drivers of hydrogen isotope (2 H) fractionation processes in plants. Here, we investigated the influence of phylogeny on the δ2 Hne of twig xylem cellulose and xylem water, as well as leaf sugars and leaf water, across 73 Northern Hemisphere tree and shrub species growing in a common garden. 2 H fractionation in plant carbohydrates followed distinct phylogenetic patterns, with phylogeny reflected more in the δ2 Hne of leaf sugars than in that of twig xylem cellulose. Phylogeny had no detectable influence on the δ2 Hne of twig or leaf water, showing that biochemistry, not isotopic differences in plant water, caused the observed phylogenetic pattern in carbohydrates. Angiosperms were more 2 H-enriched than gymnosperms, but substantial δ2 Hne variations also occurred at the order, family, and species levels within both clades. Differences in the strength of the phylogenetic signals in δ2 Hne of leaf sugars and twig xylem cellulose suggest that the original phylogenetic signal of autotrophic processes was altered by subsequent species-specific metabolism. Our results will help improve 2 H fractionation models for plant carbohydrates and have important consequences for dendrochronological and ecophysiological studies.

Covariation between oxygen and hydrogen stable isotopes declines along the path from xylem water to wood cellulose across an aridity gradient.

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ18 O and δ2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ18 O and δ2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ2 H was well-correlated between leaf and branch, there was an offset in δ18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ18 O is used to reconstruct climatic scenarios. Conversely, comparing δ2 H and δ18 O patterns may reveal environmentally induced shifts in metabolism.

Unenriched xylem water contribution during cellulose synthesis influenced by atmospheric demand governs the intra-annual tree-ring δ18 O signature.

The oxygen isotope composition (δ18 O) of tree-ring cellulose is used to evaluate tree physiological responses to climate, but their interpretation is still limited due to the complexity of the isotope fractionation pathways. We assessed the relative contribution of seasonal needle and xylem water δ18 O variations to the intra-annual tree-ring cellulose δ18 O signature of larch trees at two sites with contrasting soil water availability in the Swiss Alps. We combined biweekly δ18 O measurements of soil water, needle water, and twig xylem water with intra-annual δ18 O measurements of tree-ring cellulose, xylogenesis analysis, and mechanistic and structural equation modeling. Intra-annual cellulose δ18 O values resembled source water δ18 O mean levels better than needle water δ18 O. Large parts of the rings were formed under high proportional exchange with unenriched xylem water (pex ). Maximum pex values were achieved in August and imprinted on sections at 50-75% of the ring. High pex values were associated with periods of high atmospheric evaporative demand (VPD). While VPD governed needle water δ18 O variability, we estimated a limited Péclet effect at both sites. Due to a variable pex , source water has a strong influence over large parts of the intra-annual tree-ring cellulose δ18 O variations, potentially masking signals coming from needle-level processes.

Updating the dual C and O isotope-Gas-exchange model: A concept to understand plant responses to the environment and its implications for tree rings.

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors.

Drivers of intra-seasonal δ13 C signal in tree-rings of Pinus sylvestris as indicated by compound-specific and laser ablation isotope analysis.

Carbon isotope composition of tree-ring (δ13 CRing ) is a commonly used proxy for environmental change and ecophysiology. δ13 CRing reconstructions are based on a solid knowledge of isotope fractionations during formation of primary photosynthates (δ13 CP ), such as sucrose. However, δ13 CRing is not merely a record of δ13 CP . Isotope fractionation processes, which are not yet fully understood, modify δ13 CP during sucrose transport. We traced, how the environmental intra-seasonal δ13 CP signal changes from leaves to phloem, tree-ring and roots, for 7 year old Pinus sylvestris, using δ13 C analysis of individual carbohydrates, δ13 CRing laser ablation, leaf gas exchange and enzyme activity measurements. The intra-seasonal δ13 CP dynamics was clearly reflected by δ13 CRing , suggesting negligible impact of reserve use on δ13 CRing . However, δ13 CP became increasingly 13 C-enriched during down-stem transport, probably due to post-photosynthetic fractionations such as sink organ catabolism. In contrast, δ13 C of water-soluble carbohydrates, analysed for the same extracts, did not reflect the same isotope dynamics and fractionations as δ13 CP , but recorded intra-seasonal δ13 CP variability. The impact of environmental signals on δ13 CRing , and the 0.5 and 1.7‰ depletion in photosynthates compared ring organic matter and tree-ring cellulose, respectively, are useful pieces of information for studies exploiting δ13 CRing .

Estimating intra-seasonal photosynthetic discrimination and water use efficiency using δ13C of leaf sucrose in Scots pine.

Sucrose has a unique role in recording environmental and physiological signals during photosynthesis in its carbon isotope composition (δ13C) and transport of the signal to tree rings. Yet, instead of sucrose, total organic matter (TOM) or water-soluble carbohydrates (WSC) are typically analysed in studies that follow δ13C signals within trees. To study how the choice of organic material may bias the interpretation of δ13C records, we used mature field-grown Scots pine (Pinus sylvestris) to compare for the first time δ13C of different leaf carbon pools with δ13C of assimilates estimated by a chamber-Picarro system (δ13CA_Picarro), and a photosynthetic discrimination model (δ13CA_model). Compared with sucrose, the other tested carbon pools, such as TOM and WSC, poorly recorded the seasonal trends or absolute values of δ13CA_Picarro and δ13CA_model. Consequently, in comparison with the other carbon pools, sucrose δ13C was superior for reconstructing changes in intrinsic water use efficiency (iWUE), agreeing in both absolute values and intra-seasonal variations with iWUE estimated from gas exchange. Thus, deriving iWUE and environmental signals from δ13C of bulk organic matter can lead to misinterpretation. Our findings underscore the advantage of using sucrose δ13C to understand plant physiological responses in depth.

Rhizosphere activity in an old-growth forest reacts rapidly to changes in soil moisture and shapes whole-tree carbon allocation.

Drought alters carbon (C) allocation within trees, thereby impairing tree growth. Recovery of root and leaf functioning and prioritized C supply to sink tissues after drought may compensate for drought-induced reduction of assimilation and growth. It remains unclear if C allocation to sink tissues during and following drought is controlled by altered sink metabolic activities or by the availability of new assimilates. Understanding such mechanisms is required to predict forests' resilience to a changing climate. We investigated the impact of drought and drought release on C allocation in a 100-y-old Scots pine forest. We applied 13CO2 pulse labeling to naturally dry control and long-term irrigated trees and tracked the fate of the label in above- and belowground C pools and fluxes. Allocation of new assimilates belowground was ca. 53% lower under nonirrigated conditions. A short rainfall event, which led to a temporary increase in the soil water content (SWC) in the topsoil, strongly increased the amounts of C transported belowground in the nonirrigated plots to values comparable to those in the irrigated plots. This switch in allocation patterns was congruent with a tipping point at around 15% SWC in the response of the respiratory activity of soil microbes. These results indicate that the metabolic sink activity in the rhizosphere and its modulation by soil moisture can drive C allocation within adult trees and ecosystems. Even a subtle increase in soil moisture can lead to a rapid recovery of belowground functions that in turn affects the direction of C transport in trees.

Drought impacts on tree carbon sequestration and water use - evidence from intra-annual tree-ring characteristics.

The impact of climate extremes on forest ecosystems is poorly understood but important for predicting carbon and water cycle feedbacks to climate. Some knowledge gaps still remain regarding how drought-related adjustments in intra-annual tree-ring characteristics directly impact tree carbon and water use. In this study we quantified the impact of an extreme summer drought on the water-use efficiency and carbon sequestration of four mature Norway spruce trees. We used detailed observations of wood formation (xylogenesis) and intra-annual tree-ring properties (quantitative wood anatomy and stable carbon isotopes) combined with physiological water-stress monitoring. During 41 d of tree water deficit, we observed an enrichment in 13 C but a reduction in cell enlargement and wall-thickening processes, which impacted the anatomical characteristics. These adjustments diminished carbon sequestration by 67% despite an 11% increase in water-use efficiency during drought. However, with the resumption of a positive hydric state in the stem, we observed a fast recovery of cell formation rates based on the accumulated assimilates produced during drought. Our findings enhance our understanding of carbon and water fluxes between the atmosphere and forest ecosystems, providing observational evidence on the tree intra-annual carbon sequestration and water-use efficiency dynamics to improve future generations of vegetation models.

Tree allocation dynamics beyond heat and hot drought stress reveal changes in carbon storage, belowground translocation and growth.

Heatwaves combined with drought affect tree functioning with as yet undetermined legacy effects on carbon (C) and nitrogen (N) allocation. We continuously monitored shoot and root gas exchange, δ13 CO2 of respiration and stem growth in well-watered and drought-treated Pinus sylvestris (Scots pine) seedlings exposed to increasing daytime temperatures (max. 42°C) and evaporative demand. Following stress release, we used 13 CO2 canopy pulse-labeling, supplemented by soil-applied 15 N, to determine allocation to plant compartments, respiration and soil microbial biomass (SMB) over 2.5 wk. Previously heat-treated seedlings rapidly translocated 13 C along the long-distance transport path, to root respiration (Rroot ; 7.1 h) and SMB (3 d). Furthermore, 13 C accumulated in branch cellulose, suggesting secondary growth enhancement. However, in recovering drought-heat seedlings, the mean residence time of 13 C in needles increased, whereas C translocation to Rroot was delayed (13.8 h) and 13 C incorporated into starch rather than cellulose. Concurrently, we observed stress-induced low N uptake and aboveground allocation. C and N allocation during early recovery were affected by stress type and impact. Although C uptake increased quickly in both treatments, drought-heat in combination reduced the above-belowground coupling and starch accumulated in leaves at the expense of growth. Accordingly, C allocation during recovery depends on phloem translocation capacity.

Species variation in the hydrogen isotope composition of leaf cellulose is mostly driven by isotopic variation in leaf sucrose.

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ2 H of leaf sucrose along with the δ2 H and δ18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ2 H of sucrose explained 66% of the δ2 H variation in cellulose (R2 = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( ε sucrose : -126% to -192‰) rather than by variation in leaf water δ2 H itself. ε sucrose was positively related to dark respiration (R2 = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R2 = 0.38), but only when ε sucrose was fixed to the literature accepted value of - 171 ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ2 H of sugars is needed.

A high-temperature water vapor equilibration method to determine non-exchangeable hydrogen isotope ratios of sugar, starch and cellulose.

The analysis of the non-exchangeable hydrogen isotope ratio (δ2 Hne ) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ2 Hne values of non-structural carbohydrates (NSC) such as sugar and starch do not yet exist. Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways (i.e., C3 , C4 and CAM). δ2 Hne of commercial sugars and starch from different classes and sources, ranging from -157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ2 Hne values of sugar are lowest in C3 (-92.0‰), intermediate in C4 (-32.5‰) and highest in CAM plants (6.0‰), with NSC being 2 H-depleted compared to cellulose and sugar being generally more 2 H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2 H-fractionation processes, for improving mechanistic δ2 Hne models for leaf and tree-ring cellulose and for further development of δ2 Hne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology.

Increasing temperature and vapour pressure deficit lead to hydraulic damages in the absence of soil drought.

Temperature (T) and vapour pressure deficit (VPD) are important drivers of plant hydraulic conductivity, growth, mortality, and ecosystem productivity, independently of soil water availability. Our goal was to disentangle the effects of T and VPD on plant hydraulic responses. Young trees of Fagus sylvatica L., Quercus pubescens Willd. and Quercus ilex L. were exposed to a cross-combination of a T and VPD manipulation under unlimited soil water availability. Stem hydraulic conductivity and leaf-level hydraulic traits (e.g., gas exchange and osmotic adjustment) were tracked over a full growing season. Significant loss of xylem conductive area (PLA) was found in F. sylvatica and Q. pubescens due to rising VPD and T, but not in Q. ilex. Increasing T aggravated the effects of high VPD in F. sylvatica only. PLA was driven by maximum hydraulic conductivity and minimum leaf conductance, suggesting that high transpiration and water loss after stomatal closure contributed to plant hydraulic stress. This study shows for the first time that rising VPD and T lead to losses of stem conductivity even when soil water is not limiting, highlighting their rising importance in plant mortality mechanisms in the future.

Constraining parameter uncertainty for predicting oxygen and hydrogen isotope values in fruit.

Understanding δ18O and δ2H values of agricultural products like fruit is of particular scientific interest in plant physiology, ecology, and forensic studies. Applications of mechanistic stable isotope models to predict δ18O and δ2H values of water and organic compounds in fruit, however, are hindered by a lack of empirical parameterizations and validations. We addressed this lack of data by experimentally evaluating model parameter values required to model δ18O and δ2H values of water and organic compounds in berries and leaves from strawberry and raspberry plants grown at different relative humidities. Our study revealed substantial differences between leaf and berry isotope values, consistent across the different relative humidity treatments. We demonstrated that existing isotope models can reproduce water and organic δ18O and δ2H values for leaves and berries. Yet, these simulations require organ-specific model parameterization to accurately predict δ18O and δ2H values of leaf and berry tissue and water pools. We quantified these organ-specific model parameters for both species and relative humidity conditions. Depending on the required model accuracy, species- and environment-specific model parameters may be justified. The parameter values determined in this study thus facilitate applications of stable isotope models where understanding δ18O and δ2H values of fruit is of scientific interest.

High resilience of carbon transport in long-term drought-stressed mature Norway spruce trees within 2 weeks after drought release.

Under ongoing global climate change, drought periods are predicted to increase in frequency and intensity in the future. Under these circumstances, it is crucial for tree's survival to recover their restricted functionalities quickly after drought release. To elucidate the recovery of carbon (C) transport rates in c. 70-year-old Norway spruce (Picea abies [L.] KARST.) after 5 years of recurrent summer droughts, we conducted a continuous whole-tree 13 C labeling experiment in parallel with watering. We determined the arrival time of current photoassimilates in major C sinks by tracing the 13 C label in stem and soil CO2 efflux, and tips of living fine roots. In the first week after watering, aboveground C transport rates (CTR) from crown to trunk base were still 50% lower in previously drought-stressed trees (0.16 ± 0.01 m h-1 ) compared to controls (0.30 ± 0.06 m h-1 ). Conversely, CTR below ground, that is, from the trunk base to soil CO2 efflux were already similar between treatments (c. 0.03 m h-1 ). Two weeks after watering, aboveground C transport of previously drought-stressed trees recovered to the level of the controls. Furthermore, regrowth of water-absorbing fine roots upon watering was supported by faster incorporation of 13 C label in previously drought-stressed (within 12 ± 10 h upon arrival at trunk base) compared to control trees (73 ± 10 h). Thus, the whole-tree C transport system from the crown to soil CO2 efflux fully recovered within 2 weeks after drought release, and hence showed high resilience to recurrent summer droughts in mature Norway spruce forests. This high resilience of the C transport system is an important prerequisite for the recovery of other tree functionalities and productivity.

Dynamics of initial carbon allocation after drought release in mature Norway spruce-Increased belowground allocation of current photoassimilates covers only half of the carbon used for fine-root growth.

After drought events, tree recovery depends on sufficient carbon (C) allocation to the sink organs. The present study aimed to elucidate dynamics of tree-level C sink activity and allocation of recent photoassimilates (Cnew ) and stored C in c. 70-year-old Norway spruce (Picea abies) trees during a 4-week period after drought release. We conducted a continuous, whole-tree 13 C labeling in parallel with controlled watering after 5 years of experimental summer drought. The fate of Cnew to growth and CO2 efflux was tracked along branches, stems, coarse- and fine roots, ectomycorrhizae and root exudates to soil CO2 efflux after drought release. Compared with control trees, drought recovering trees showed an overall 6% lower C sink activity and 19% less allocation of Cnew to aboveground sinks, indicating a low priority for aboveground sinks during recovery. In contrast, fine-root growth in recovering trees was seven times greater than that of controls. However, only half of the C used for new fine-root growth was comprised of Cnew while the other half was supplied by stored C. For drought recovery of mature spruce trees, in addition to Cnew , stored C appears to be critical for the regeneration of the fine-root system and the associated water uptake capacity.

Memory of environmental conditions across generations affects the acclimation potential of scots pine.

Long generation times have been suggested to hamper rapid genetic adaptation of organisms to changing environmental conditions. We examined if environmental memory of the parental Scots pines (Pinus sylvestris L.) drive offspring survival and growth. We used seeds from trees growing under naturally dry conditions (control), irrigated trees (irrigated from 2003 to 2016), and formerly irrigated trees ("irrigation stop"; irrigated from 2003-2013; control condition since 2014). We performed two experiments, one under controlled greenhouse conditions and one at the experimental field site. In the greenhouse, the offspring from control trees exposed regularly to drought were more tolerant to hot-drought conditions than the offspring from irrigated trees and showed lower mortality even though there was no genetic difference. However, under optimal conditions (high water supply and full sunlight), these offspring showed lower growth and were outperformed by the offspring of the irrigated trees. This different offspring growth, with the offspring of the "irrigation-stop" trees showing intermediate responses, points to the important role of transgenerational memory for the long-term acclimation of trees. Such memory effects, however, may be overridden by climatic extremes during germination and early growth stages such as the European 2018 mega-drought that impacted our field experiment.