The sensitivity of root water uptake to cold root temperature follows species-specific upper elevational distribution limits of temperate tree species.

Physiological water stress induced by low root temperatures might contribute to species-specific climatic limits of tree distribution. We investigated the low temperature sensitivity of root water uptake and transport in seedlings of 16 European tree species which reach their natural upper elevation distribution limits at different distances to the alpine treeline. We used 2H-H2O pulse-labelling to quantify the water uptake and transport velocity from roots to leaves in seedlings exposed to constant 15°C, 7°C or 2°C root temperature, but identical aboveground temperatures between 20°C and 25°C. In all species, low root temperatures reduced the water transport rate, accompanied by reduced stem water potentials and stomatal conductance. At 7°C root temperature, the relative water uptake rates among species correlated positively with the species-specific upper elevation limits, indicating an increasingly higher sensitivity to lower root zone temperatures, the lower a species' natural elevational distribution limit. Conversely, 2°C root temperature severely inhibited water uptake in all species, irrespective of the species' thermal elevational limits. We conclude that low temperature-induced hydraulic constraints contribute to the cold distribution limits of temperate tree species and are a potential physiological cause behind the low temperature limits of plant growth in general.

Water loss after stomatal closure: quantifying leaf minimum conductance and minimal water use in nine temperate European tree species during a severe drought.

Residual canopy transpiration (Emin_canop) is a key physiological trait that determines trees' survival time under drought after stomatal closure and after trees have limited access to soil water. Emin_canop mainly depends on leaf minimum conductance (gmin) and vapor pressure deficit. Here we determined the seasonal variation of gmin and how gmin is related to interspecies variation in leaf cuticular and stomatal traits for nine European tree species in a mature forest. In addition, we determined the species-specific temperature responses of gmin. With this newly obtained insight, we calculated Emin_canop for the nine species for one day at our research site during the 2022 central European hot drought. Our results show that at ambient temperatures gmin ranged from 0.8 to 4.8 mmol m-2 s-1 across the nine species and was stable in most species throughout the growing season. The interspecies variation of gmin was associated with leaf cuticular and stomatal traits. Additionally, gmin exhibited strong temperature responses and increased, depending on species, by a factor of two to four in the range of 25-50 °C. For the studied species at the site, during a single hot drought day, Emin_canop standardized by tree size (stem basal area) ranged from 2.0 to 36.7 L m-2, and non-standardized Emin_canop for adult trees ranged from 0.3 to 5.3 L. Emin_canop also exhibited species-specific rapid increases under hotter temperatures. Our results suggest that trees, depending on species, need reasonable amounts of water during a drought, even when stomates are fully closed. Species differences in gmin and ultimately Emin_canop can, together with other traits, affect the ability of a tree to keep its tissue hydrated during a drought and is likely to contribute to species-specific differences in drought vulnerability.

Uniform carbon reserve dynamics along the vertical light gradient in mature tree crowns.

Understanding the within-tree variability of non-structural carbohydrates (NSC) is crucial for interpreting point measurements and calculating whole-tree carbon balances. Yet, little is known about how the vertical light gradient within tree crowns influences branch NSC concentrations and dynamics. We measured NSC concentrations, irradiance and key leaf traits in uppermost, sun-exposed and lowest, shaded branches in the crowns of mature, temperate trees from nine species with high temporal resolution throughout one growing season. Measurements from two additional years allowed us to test the generality of our findings among climatically contrasting years. Despite the vertical light gradient, we found very similar seasonal NSC dynamics and concentrations between sun and shade branches in most species. This can at least partially be explained by acclimations in SLA and photosynthetic leaf traits compensating the different light availability between the top and bottom canopy. Only in the ring-porous species Quercus and Fraxinus, starch refilling after budbreak was slower in lower branches. End-of-season NSC concentrations were similar between canopy positions and among observation years. Only Fagus had 40 and 29% lower starch concentrations by the end of the extremely dry year 2020, relative to the other two years. We show that NSC measured anywhere in a tree crown is often representative of the whole crown. Overall, our results suggest that carbon reserve dynamics in trees are largely insensitive to both microclimatic gradients and inter-annual climatic variation, and only deviate under severe carbon deficits, as was presumably the case with Fagus in our study.

Stem growth phenology, not canopy greening, constrains deciduous tree growth.

Canopy phenology is a widely used proxy for deciduous forest growth with various applications in terrestrial ecosystem modeling. Its use relies on common assumptions that canopy greening and stem growth are tightly coordinated processes, enabling predictions on the timing and the quantity of annual tree growth. Here, we present parallel observations of canopy and stem growth phenology and annual stem increment in around 90 deciduous forest trees with diffuse-porous (Fagus sylvatica, Acer pseudoplatanus, Carpinus betulus) or ring-porous (Quercus robur × petraea) wood anatomy. These data were collected in a mixed temperate forest at the Swiss-Canopy-Crane II site, in 4 years with strongly contrasting weather conditions. We found that stem growth resumption lagged several weeks behind spring canopy greening in diffuse-porous but not in ring-porous trees. Canopy greening and stem growth resumption showed no or only weak signs of temporal coordination across the observation years. Within the assessed species, the seasonal timing of stem growth varied strongly among individuals, as trees with high annual increments resumed growth earlier and also completed their main growth earlier. The length of main growth activity had no influence on annual increments. Our findings not only challenge tight temporal coordination of canopy and stem growth phenology but also demonstrate that longer main growth activity does not translate into higher annual increments. This may compromise approaches modeling tree growth and forest productivity with canopy phenology and growth length.

Chronic in situ tissue cooling does not reduce lignification at the Swiss treeline but enhances the risk of 'blue' frost rings.

In their 2013 paper, Lenz et al. illustrated how trees growing at the low-temperature limit respond to a chronic in situ warming or cooling by 3 K, by employing Peltier-thermostated branch collars that tracked ambient temperatures. The micro-coring-based analysis of seasonal tree ring formation included double-staining microtome cross sections for lignification, but these data had not been included in the publication. In this short communication, we complement these data, collected in 2009 at the Swiss treeline, and we show that a 3 K cooling that corresponds to a 500-600 m higher elevation, had no influence on lignification. However, when a frost event occurred during the early part of ring formation, the 3 K cooling produced a blue (non-lignified) layer of cells, followed by normally lignified cells for the rest of the season. Hence, the event did not affect the cambium, but interrupted cell wall maturation in cells that were in a critical developmental stage. We conclude, that chronic cooling does not affect lignification at treeline, but it increases the risk of frost damage in premature xylem tissue.

Networking the forest infrastructure towards near real-time monitoring - A white paper.

Forests account for nearly 90 % of the world's terrestrial biomass in the form of carbon and they support 80 % of the global biodiversity. To understand the underlying forest dynamics, we need a long-term but also relatively high-frequency, networked monitoring system, as traditionally used in meteorology or hydrology. While there are numerous existing forest monitoring sites, particularly in temperate regions, the resulting data streams are rarely connected and do not provide information promptly, which hampers real-time assessments of forest responses to extreme climate events. The technology to build a better global forest monitoring network now exists. This white paper addresses the key structural components needed to achieve a novel meta-network. We propose to complement - rather than replace or unify - the existing heterogeneous infrastructure with standardized, quality-assured linking methods and interacting data processing centers to create an integrated forest monitoring network. These automated (research topic-dependent) linking methods in atmosphere, biosphere, and pedosphere play a key role in scaling site-specific results and processing them in a timely manner. To ensure broad participation from existing monitoring sites and to establish new sites, these linking methods must be as informative, reliable, affordable, and maintainable as possible, and should be supplemented by near real-time remote sensing data. The proposed novel meta-network will enable the detection of emergent patterns that would not be visible from isolated analyses of individual sites. In addition, the near real-time availability of data will facilitate predictions of current forest conditions (nowcasts), which are urgently needed for research and decision making in the face of rapid climate change. We call for international and interdisciplinary efforts in this direction.

Lack of hydraulic recovery as a cause of post-drought foliage reduction and canopy decline in European beech.

European beech (Fagus sylvatica) was among the most affected tree species during the severe 2018 European drought. It not only suffered from instant physiological stress but also showed severe symptoms of defoliation and canopy decline in the following year. To explore the underlying mechanisms, we used the Swiss-Canopy-Crane II site and studied in branches of healthy and symptomatic trees the repair of hydraulic function and concentration of carbohydrates during the 2018 drought and in 2019. We found loss of hydraulic conductance in 2018, which did not recover in 2019 in trees that developed defoliation symptoms in the year after drought. Reduced branch foliation in symptomatic trees was associated with a gradual decline in wood starch concentration throughout summer 2019. Visualization of water transport in healthy and symptomatic branches in the year after the drought confirmed the close relationship between xylem functionality and supported branch leaf area. Our findings showed that embolized xylem does not regain function in the season following a drought and that sustained branch hydraulic dysfunction is counterbalanced by the reduction in supported leaf area. It suggests acclimation of leaf development after drought to mitigate disturbances in canopy hydraulic function.

Negative effects of low root temperatures on water and carbon relations in temperate tree seedlings assessed by dual isotopic labelling.

Low root zone temperatures restrict water and carbon (C) uptake and transport in plants and may contribute to the low temperature limits of tree growth. Here, we quantified the effects of low root temperatures on xylem conductance, photosynthetic C assimilation and phloem C transport in seedlings of four temperate tree species (two broad-leaved and two conifer species) by applying a simultaneous stable isotope labelling of 2H-enriched source water and 13C-enriched atmospheric CO2. Six days before the pulse labelling, the seedlings were transferred to hydroponic tubes and exposed to three different root temperatures (2, 7 and 15 °C), while all seedlings received the same, warm air temperatures (between 18 and 24 °C). Root cooling led to drought-like symptoms with reduced growth, leaf water potentials and stomatal conductance, indicating increasingly adverse conditions for water uptake and transport with decreasing root temperatures. Averaged across all four species, water transport to leaves was reduced by 40% at 7 °C and by 70% at 2 °C root temperature relative to the 15 °C treatment, while photosynthesis was reduced by 20 and 40% at 7 and 2 °C, respectively. The most severe effects were found on the phloem C transport to roots, which was reduced by 60% at 7 °C and almost ceased at 2 °C in comparison with the 15 °C root temperature treatment. This extreme effect on C transport was likely due to a combination of simultaneous reductions of phloem loading, phloem mass flow and root growth. Overall, the dual stable isotope labelling proved to be a useful method to quantify water and C relations in cold-stressed trees and highlighted the potentially important role of hydraulic constraints induced by low soil temperatures as a contributing factor for the climatic distribution limits of temperate tree species.

Trunk radial growth, water and carbon relations of mature apple trees on two size-controlling rootstocks during severe summer drought.

The use of size-controlling rootstocks is central to modern high-density fruit production systems. While biological mechanisms responsible for vigor control are not fully understood, differences in water relations and carbohydrate storage ability have been suggested as two potential factors. To better understand the processes that control growth vigor, we analyzed the trunk radial variation at seasonal and diurnal timescales and measured the midday leaf water potential (ΨMD), leaf gas exchange and concentrations of non-structural carbohydrates (NSC) in apple trees of variety 'Jonagold' grafted on two rootstocks of contrasting growth vigor (dwarfing J-TE-G vs invigorating J-TE-H). The measurements were conducted during an exceptionally hot and dry summer. We found that smaller annual trunk radial increments in dwarfed trees were primarily due to an earlier cessation of trunk secondary growth. The interdiurnal trunk circumference changes (ΔC) were slightly lower in dwarfed trees, and these trees also had fewer days with positive ΔC values, particularly during the driest summer months. The trunks of dwarfed trees shrank gradually during the drought, showed less pronounced diurnal variation of trunk circumference and the maximum trunk daily shrinkage was only weakly responsive to the vapor pressure deficit. These results indicated that lower turgidity in the cambial region may have limited the trunk radial expansion in dwarfed trees during the hot and dry days. Dwarfed trees also maintained lower ΨMD and leaf gas exchange rates during the summer drought. These parameters decreased in parallel for both rootstock combinations, suggesting their similar drought sensitivity. Similar concentrations and seasonal dynamics of NSC in both rootstock combinations, together with their similar spring growth rates, suggest that NSC reserves were not directly limiting for growth. Our results support the prominent role of water relations in rootstock-induced size-controlling mechanisms and highlight the complexity of this topic.

Climate Change Modulates Multitrophic Interactions Between Maize, A Root Herbivore, and Its Enemies.

How climate change will modify belowground tritrophic interactions is poorly understood, despite their importance for agricultural productivity. Here, we manipulated the three major abiotic factors associated with climate change (atmospheric CO2, temperature, and soil moisture) and investigated their individual and joint effects on the interaction between maize, the banded cucumber beetle (Diabrotica balteata), and the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora. Changes in individual abiotic parameters had a strong influence on plant biomass, leaf wilting, sugar concentrations, protein levels, and benzoxazinoid contents. Yet, when combined to simulate a predicted climate scenario (Representative Concentration Pathway 8.5, RCP 8.5), their effects mostly counter-balanced each other. Only the sharp negative impact of drought on leaf wilting was not fully compensated. In both current and predicted scenarios, root damage resulted in increased leaf wilting, reduced root biomass, and reconfigured the plant sugar metabolism. Single climatic variables modulated the herbivore performance and survival in an additive manner, although slight interactions were also observed. Increased temperature and CO2 levels both enhanced the performance of the insect, but elevated temperature also decreased its survival. Elevated temperatures and CO2 further directly impeded the EPN infectivity potential, while lower moisture levels improved it through plant- and/or herbivore-mediated changes. In the RCP 8.5 scenario, temperature and CO2 showed interactive effects on EPN infectivity, which was overall decreased by 40%. We conclude that root pest problems may worsen with climate change due to increased herbivore performance and reduced top-down control by biological control agents.

Rapid hydraulic collapse as cause of drought-induced mortality in conifers.

Understanding the vulnerability of trees to drought-induced mortality is key to predicting the fate of forests in a future climate with more frequent and intense droughts, although the underlying mechanisms are difficult to study in adult trees. Here, we explored the dynamic changes of water relations and limits of hydraulic function in dying adults of Norway spruce (Picea abies L.) during the progression of the record-breaking 2018 Central European drought. In trees on the trajectory to drought-induced mortality, we observed rapid, nonlinear declines of xylem pressure that commenced at the early onset of xylem cavitation and caused a complete loss of xylem hydraulic conductance within a very short time. We also observed severe depletions of nonstructural carbohydrates, though carbon starvation could be ruled out as the cause of the observed tree death, as both dying and surviving trees showed these metabolic limitations. Our observations provide striking field-based evidence for fast dehydration and hydraulic collapse as the cause of drought-induced mortality in adult Norway spruce. The nonlinear decline of tree water relations suggests that considering the temporal dynamics of dehydration is critical for predicting tree death. The collapse of the hydraulic system within a short time demonstrates that trees can rapidly be pushed out of the zone of hydraulic safety during the progression of a severe drought. In summary, our findings point toward a higher mortality risk for Norway spruce than previously assumed, which is in line with current reports of unprecedented levels of drought-induced mortality in this major European tree species.

Soil nutrient availability alters tree carbon allocation dynamics during drought.

Drought alters allocation patterns of carbon (C) and nutrients in trees and eventually impairs tree functioning. Elevated soil nutrient availability might alter the response of trees to drought. We hypothesize that increased soil nutrient availability stimulates root metabolism and C allocation to belowground tissues under drought stress. To test this hypothesis, we subjected 3-year-old Pinus sylvestris L. saplings in open-top chambers during two subsequent years to drought using three different water treatments (100, 20 and 0% plant available water in the soil) and two soil nutrient regimes (ambient and nitrogen-phosphorus-potassium (N-P-K) fertilization corresponding to 5 g N m-2 year-1) and released drought thereafter. We conducted a 15N and 13C labeling experiment during the peak of the first-year drought by injecting 15N labeled fertilizer in the soil and exposing the tree canopies to 13C labeled CO2. The abundance of the N and C isotopes in the roots, stem and needles was assessed during the following year. Carbon uptake was slightly lower in drought-stressed trees, and extreme drought inhibited largely the N uptake and transport. Carbon allocation to belowground tissues was decreased under drought, but not in combination with fertilization. Our results indicate a potential positive feedback loop, where fertilization improved the metabolism and functioning of the roots, stimulating C allocation to belowground tissues. This way, soil nutrients compensated for drought-induced loss of root functioning, mitigating drought stress of trees.

Reaching Natural Growth: The Significance of Light and Temperature Fluctuations in Plant Performance in Indoor Growth Facilities.

Recommendations for near-natural plant growth under indoor conditions have been described without considering environmental fluctuations, which might have important consequences for researchers and plant producers when comparing results from indoor facilities with natural ecosystems or production. Previous authors proposed that differences in temperature, light quantity, and the lack of their variation are sources of deviations between indoor and outdoor experiments. Here, we investigated the effect of fluctuating light, temperature, and humidity in an indoor environment on plant performance. Seven plant species from different functional plant types were grown outdoors during summer and spring. The same species were then grown in indoor growth chambers under different scenarios of climate complexity in terms of fluctuations of temperature, air humidity, and light: 1) fixed night and day conditions, 2) daily sinusoidal changes, and 3) variable conditions tracking the climate records from the field trials. In each scenario, the average of the environmental variables was the same as in the respective field trial. Productivity-, gas exchange-, and leaf pigment-traits were measured in all plants at the end of the experiments. The plant trait responses were highly dependent on species and treatment, but general trends were observed. The variable condition yielded lower biomass compared to the fixed and sinusoidal conditions, together with a higher specific leaf area and increased chlorophyll concentrations. A principal component analysis (PCA) across all plant traits in response to climatic conditions suggested that at least a sinusoidal fluctuation is recommended for a more natural-like plant performance in indoor growth facilities. However, prevailing significant differences for several traits between field- and indoor-grown plants even under variable climates indicate that additional factors other than those controllable in standard phytotrons (e.g., wind speed and direction, leaf and soil temperature) can still significantly bias plant performance in indoor facilities.

Reaching Natural Growth: Light Quality Effects on Plant Performance in Indoor Growth Facilities.

To transfer experimental findings in plant research to natural ecosystems it is imperative to reach near to natural-like plant performance. Previous studies propose differences in temperature and light quantity as main sources of deviations between indoor and outdoor plant growth. With increasing implementation of light emitting diodes (LED) in plant growth facilities, light quality is yet another factor that can be optimised to prevent unnatural plant performance. We investigated the effects of different wavelength combinations in phytotrons (i.e., indoor growth chambers) on plant growth and physiology in seven different plant species from different plant functional types (herbs, grasses and trees). The results from these experiments were compared against a previous field trial with the same set of species. While different proportions of blue (B) and red (R) light were applied in the phytotrons, the mean environmental conditions (photoperiod, total radiation, red to far red ratio and day/night temperature and air humidity) from the field trial were used in the phytotrons in order to assess which wavelength combinations result in the most natural-like plant performance. Different plant traits and physiological parameters, including biomass productivity, specific leaf area (SLA), leaf pigmentation, photosynthesis under a standardised light, and the respective growing light and chlorophyll fluorescence, were measured at the end of each treatment. The exposure to different B percentages induced species-specific dose response reactions for most of the analysed parameters. Compared with intermediate B light treatments (25 and/or 35% B light), extreme R or B light enriched treatments (6% and 62% of B respectively) significantly affected the height, biomass, biomass allocation, chlorophyll content, and photosynthesis parameters, differently among species. Principal component analyses (PCA) confirmed that 6% and 62% B light quality combinations induce more extreme plant performance in most cases, indicating that light quality needs to be adjusted to mitigate unnatural plant responses under indoor conditions.

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.

Plant respiration: Controlled by photosynthesis or biomass?

Two simplifying hypotheses have been proposed for whole-plant respiration. One links respiration to photosynthesis; the other to biomass. Using a first-principles carbon balance model with a prescribed live woody biomass turnover, applied at a forest research site where multidecadal measurements are available for comparison, we show that if turnover is fast the accumulation of respiring biomass is low and respiration depends primarily on photosynthesis; while if turnover is slow the accumulation of respiring biomass is high and respiration depends primarily on biomass. But the first scenario is inconsistent with evidence for substantial carry-over of fixed carbon between years, while the second implies far too great an increase in respiration during stand development-leading to depleted carbohydrate reserves and an unrealistically high mortality risk. These two mutually incompatible hypotheses are thus both incorrect. Respiration is not linearly related either to photosynthesis or to biomass, but it is more strongly controlled by recent photosynthates (and reserve availability) than by total biomass.

Revisiting the relative growth rate hypothesis for gymnosperm and angiosperm species co-occurrence.

PREMISE OF THE STUDY: It is unclear to what extent the co-occurrence of angiosperm and gymnosperm species in some marginal ecosystems is explained by reduced growth in angiosperms due to carbon (C) limitation and by high stress tolerance in gymnosperms associated with lack of vessels and resource conservation. METHODS: We examined growth patterns and traits associated with C balance in four evergreen angiosperm species (including one vesselless species, Drimys winteri) and three gymnosperm tree species of a cold-temperate rainforest in southern Chile. We measured the mean basal area increment for the first 50 (BAI50 ) and the last 10 years (BAI10 ), wood density, leaf lifespan, and nonstructural carbohydrate (NSC) concentrations in different organs. KEY RESULTS: BAI50 was 6-fold higher in angiosperms than in gymnosperms and ca. 4-fold higher in Drimys than in the fastest-growing gymnosperm. BAI10 and aboveground NSC concentrations were significantly higher and leaf lifespan lower in angiosperms than in gymnosperms; these differences, however, were largely driven by the slow growth and low NSC concentrations of the Cupressaceae species (Pilgerodendron uviferum), while the two Podocarpaceae had BAI10 and NSC concentrations similar to angiosperms. In angiosperms, NSC and starch concentrations were generally higher in species with lower BAI10 , indicating no severe C limitation. CONCLUSIONS: The co-occurrence of angiosperms and gymnosperms in cold-temperate rainforests of southern Chile is not explained by growth disadvantages and C limitation in angiosperms. Long leaf longevity, but not lack of vessels, appeared to favor resource conservation and C balance in some gymnosperms (Podocarpaceae).

High carbon storage in carbon-limited trees.

The concentrations of nonstructural carbohydrates (NSCs) in plant tissues are commonly used as an indicator of total plant carbon (C) supply; but some evidence suggests the possibility for high NSC concentrations during periods of C limitation. Despite this uncertainty, NSC dynamics have not been investigated experimentally under long-term C limitation. We exposed saplings of 10 temperate tree species differing in shade tolerance to 6% of ambient sunlight for 3 yr to induce C limitation, and also defoliated one species, Carpinus betulus, in the third season. Growth and NSC concentrations were monitored to determine C allocation. Shade strongly reduced growth, but after an initial two-fold decrease, NSC concentrations of shaded saplings recovered to the level of unshaded saplings by the third season. NSC concentrations were generally more depleted under shade after leaf flush, and following herbivore attacks. Only under shade did artificial defoliation lead to mortality and depleted NSC concentrations in surviving individuals. We conclude that, irrespective of shade tolerance, C storage is maintained under prolonged shading, and thus high NSC concentrations can occur during C limitation. Yet, our results also suggest that decreased NSC concentrations are indicative of C limitation, and that additional leaf loss can lead to lethal C shortage in deep shade.

Standardized protocols and procedures can precisely and accurately quantify non-structural carbohydrates.

Non-structural carbohydrates (NSCs), the stored products of photosynthesis, building blocks for growth and fuel for respiration, are central to plant metabolism, but their measurement is challenging. Differences in methods and procedures among laboratories can cause results to vary widely, limiting our ability to integrate and generalize patterns in plant carbon balance among studies. A recent assessment found that NSC concentrations measured for a common set of samples can vary by an order of magnitude, but sources for this variability were unclear. We measured a common set of nine plant material types, and two synthetic samples with known NSC concentrations, using a common protocol for sugar extraction and starch digestion, and three different sugar quantification methods (ion chromatography, enzyme, acid) in six laboratories. We also tested how sample handling, extraction solvent and centralizing parts of the procedure in one laboratory affected results. Non-structural carbohydrate concentrations measured for synthetic samples were within about 11.5% of known values for all three methods. However, differences among quantification methods were the largest source of variation in NSC measurements for natural plant samples because the three methods quantify different NSCs. The enzyme method quantified only glucose, fructose and sucrose, with ion chromatography we additionally quantified galactose, while the acid method quantified a large range of mono- and oligosaccharides. For some natural samples, sugars quantified with the acid method were two to five times higher than with other methods, demonstrating that trees allocate carbon to a range of sugar molecules. Sample handling had little effect on measurements, while ethanol sugar extraction improved accuracy over water extraction. Our results demonstrate that reasonable accuracy of NSC measurements can be achieved when different methods are used, as long as protocols are robust and standardized. Thus, we provide detailed protocols for the extraction, digestion and quantification of NSCs in plant samples, which should improve the comparability of NSC measurements among laboratories.