Carbohydrate Storage Research Articles

Carbohydrate storage in cells: a laboratory activity for the assessment of glycogen stores in biological tissues.

Carbohydrates and fats constitute our primary energy sources. The importance of each of these energy substrates varies across cell types and physiological conditions. For example, the brain normally relies almost exclusively on glucose oxidation, whereas skeletal muscle shifts from lipids toward higher carbohydrate oxidation rates as exercise intensity increases. Understanding how carbohydrates are stored in our cells and which tissues contain significant carbohydrate stores is crucial for health professionals, especially given the role of carbohydrate metabolism in various pathophysiological conditions. This laboratory activity uses a simple and low-cost iodine binding method to quantify glycogen in mouse skeletal muscle and liver samples. By integrating the results of this activity with literature data, students can determine overall glycogen storage in the human body. The primary goal of the activity is to enhance students' understanding of the importance and limitations of glycogen stores in energy metabolism.NEW & NOTEWORTHY Carbohydrates are one of the primary energy sources utilized by our cells. Liver and skeletal muscle glycogen, which are the main carbohydrate reserves in the body, play a central role in energy metabolism, especially during periods of fasting and exercise. In this laboratory activity, students measure glycogen levels in tissues to gain insights into how carbohydrates are stored in our cells and understand the role and limitations of liver and muscle carbohydrate stores.

Forward modelling of white spruce radial growth at trailing edge demonstrates high plasticity

Climate conditions throughout the 21st century across much of western Canada's boreal forest have been drier than normal leading to significant impacts on forest productivity and tree growth. Determining the limiting factors of radial growth in common boreal tree species under current and future conditions is crucial to reconcile how they will continue to respond to climate change. In this study, we used a network of 26 white spruce tree-ring chronologies south of its natural range as an artificially constructed trailing edge to assess climate-growth relationships and limiting factors by identifying seasonal climate relationships and using the Vaganov-Shashkin Lite (VS-Lite) model, respectively. Analysis revealed that white spruce in the study region is positively correlated to precipitation and negatively associated with temperature from the current and previous growing seasons. VS-Lite showed that moisture was the primary limiting factor in growth across the entire study area. The modelled radial growth from the VS-Lite model showed moderate success (14 of 26 site models were significant) and was more variable than measured tree-ring index values. The model systematically overestimated drought-related impacts on radial growth, especially during the middle portion of the growth season (June–August) indicating white spruce likely employs a conservative approach to carbohydrate storage as the region is much more susceptible to moisture deficits. The outcome of our research illustrates that white spruce may be resilient to hotter, drier conditions through environmental plasticity in the Boreal Plains.

Cropping and Pruning Systems of Primocane Raspberries in the Subtropical Climate

Raspberry production is limited to cold temperate areas of high latitude due to the requirement of low temperatures for flowering and fruiting from most cultivars. However, primocane cultivars, as they are less demanding in cold conditions, represent a possible alternative that suits regions with a subtropical climate. The cultivar Heritage primocane raspberry was investigated in the Cwa climate, in three production systems (PS), during two crop cycles. In PS1, canes were hard pruned at ground level after primocane fruiting. In PS2, canes were tipped to promote subapical bud break for a second harvest. In PS3, canes were tipped again after the second harvest to induce a third harvest. PS1 had the lowest yield, however, after two cycles; in plants of this system it was observed the highest root weight, and starch content. Raspberries subjected to subapical pruning show lower carbohydrate storage in the root system. The production systems had little influence on fruit qualities, in both cycles. The cultivation of cv. Heritage raspberry primocane, in the subtropical Cwa climate can be carried out with sequential pruning, allowing for the production of commercial fruits with harvests distributed over the months, without any reduction in the postharvest quality of the fruits produced.

Impact of climate change on the kelp Laminaria digitata – simulated Arctic winter warming

The Arctic is seasonally exposed to long periods of low temperatures and complete darkness. Consequently, perennial primary producers have to apply strategies to maximize energy efficiency. Global warming is occurring in the Arctic faster than the rest of the globe. The highest amplitude of temperature rise occurs during Polar Night. To determine the stress resistance of the ecosystem-engineering kelp Laminaria digitata against Arctic winter warming, non-meristematic discs of adult sporophytes from Porsangerfjorden (Finnmark, Norway) were kept in total darkness at 0°C and 5°C over a period of three months. Physiological variables, namely maximum quantum yield of photosynthesis (Fv/Fm) and dry weight, as well as underlying biochemical variables including pigments, storage carbohydrates, total carbon and total nitrogen were monitored throughout the experiment. Although all samples remained in generally good condition with Fv/Fm values above 0.6, L. digitata performed better at 0°C than at 5°C. Depletion of metabolic products resulted in a constant decrease of dry weight over time. A strong decrease in mannitol and laminarin was observed, with greater reductions at 5°C than at 0°C. However, the total carbon content did not change, indicating that the sporophytes were not suffering from “starvation stress” during the long period of darkness. A decline was also observed in the accessory pigments and the pool of xanthophyll cycle pigments, particularly at 5°C. Our results indicate that L. digitata has a more active metabolism, but a lower physiological and biochemical performance at higher temperatures in the Arctic winter. Obviously, L. digitata is well adapted to Arctic Polar Night conditions, regardless of having its distributional center at lower latitudes. Despite a reduced vitality at higher temperatures, a serious decline in Arctic populations of L. digitata due to winter warming is not expected for the near future.

Wood nutrients: Underexplored traits with functional and biogeochemical consequences.

Resource storage is a critical component of plant life history. While the storage of nonstructural carbohydrates in wood has been studied extensively, the multiple functions of mineral nutrient storage have received much less attention. Here, we highlight the size of wood nutrient pools, a primary determinant of whole-plant nutrient use efficiency, and a substantial fraction of ecosystem nutrient budgets, particularly tropical forests. Wood nutrient concentrations also show exceptional interspecific variation, even among co-occurring plant species, yet how they align with other plant functional traits and fit into existing trait economic spectra is unclear. We review the chemical forms and location of nutrient pools in bark and sapwood, and the evidence that nutrient remobilization from sapwood is associated with mast reproduction, seasonal leaf flush, and the capacity to resprout following damage. We also emphasize the role wood nutrients are likely to play in determining decomposition rates. Given the magnitude of wood nutrient stocks, and the importance of tissue stoichiometry to forest productivity, a key unresolved question is whether investment in wood nutrients is a relatively fixed trait, or conversely whether under global change plants will adjust nutrient allocation to wood depending on carbon gain and nutrient supply.

Megafire smoke exposure jeopardizes tree carbohydrate reserves and yield.

The global incidence of megafires is on the rise, leading to extensive areas being shrouded in dense smoke for prolonged periods, spanning days or weeks1. Here, by integrating long-term regional observations of non-structural carbohydrate content in trees across California's Central Valley with spatiotemporal satellite data, we present compelling evidence that dense smoke plumes negatively impact carbohydrate stores in three tree species: Prunus dulcis, Pistacia vera and Juglans regia. Our findings show that the presence of smoke causes a significant decrease in total non-structural carbohydrates, with reductions in the accumulation of both soluble sugar and starch reserves. This decline in carbohydrate levels persists through the trees' dormancy period into the next season's bloom, culminating in a reduced yield. Our results highlight a previously unrecognized wildfire threat that could affect plant health and ecosystem stability in both agricultural and natural environments.

Integrative LC-MS and GC-MS metabolic profiling unveils dynamic changes during barley malting

Malting involves complex biochemical transformations affecting sensory and quality attributes. Despite extensive research on storage carbohydrates and proteins in malting, the lack of a detailed metabolic understanding of this process limits our ability to assess and enhance malt quality. This study employed untargeted GC-MS and LC-MS metabolite profiling across six malting timepoints to identify 4980 known metabolites, 82 % of which exhibited significant changes during the malting process. Here we identified stage-dependent metabolic shifts and dynamic chemical classes and pathways between each studied stage. These results can guide the fine-tuning of malting conditions to improve malt quality for beer production and other malt-based applications. Additionally, metabolites with antimicrobial properties were identified, underscoring the interplay between barley and microbial metabolic processes during malting. Further research into these microbial metabolites and cognate microbes may lead to novel malting assessment traits for high-quality and safe malted barley.

Photoperiod and temperature interactions drive the latitudinal distribution of Laminaria hyperborea (Laminariales, Phaeophyceae) under climate change.

Due to global rises in temperature, recent studies predict marine species shifting toward higher latitudes. We investigated the impact of interacting abiotic drivers on the distribution potential of the temperate kelp Laminaria hyperborea. The ecosystem engineering species is widespread along European coasts but has not yet been observed in the High Arctic, although it can survive several months of low temperatures and darkness. To investigate its ability to extend northward in future, we conducted a long-term multifactorial experiment with sporophytes from Porsangerfjorden, Norway-close to the species' documented northernmost distribution margin. The samples were exposed to three different photoperiods (PolarDay, LongDay, and PolarNight) at 0°C, 5°C, and 10°C for 3 months. Optimum quantum yield of photosynthesis (Fv/Fm), dry weight, pigments, phlorotannins, and storage carbohydrates were monitored. Both physiological and biochemical parameters revealed that L. hyperborea was strongly influenced by the different photoperiods and their interaction with temperature, while temperature alone exerted only minor effects. The Fv/Fm data were integrated into a species distribution model to project a possible northward expansion of L. hyperborea. The combination of extended day lengths and low temperatures appeared to be the limiting reason for northward spread of L. hyperborea until recently. However, with water temperatures reaching 10°C in summer, this kelp will be able to thrive also in the High Arctic. Moreover, no evidence of stress to Arctic winter warming was observed. Consequently, L. hyperborea has a high potential for spreading northward with further warming which may significantly affect the structure and function of Arctic ecosystems.

Evaluating cellular roles and phenotypes associated with trehalose degradation genes in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.

Glut1 Functions in Insulin-Producing Neurons to Regulate Lipid and Carbohydrate Storage in Drosophila.

Obesity remains one of the largest health problems in the world, arising from the excess storage of triglycerides (TAGs). However, the full complement of genes that are important for regulating TAG storage is not known. The Glut1 gene encodes a Drosophila glucose transporter that has been identified as a potential obesity gene through genetic screening. Yet, the tissue-specific metabolic functions of Glut1 are not fully understood. Here, we characterized the role of Glut1 in the fly brain by decreasing neuronal Glut1 levels with RNAi and measuring glycogen and TAGs. Glut1RNAi flies had decreased TAG and glycogen levels, suggesting a nonautonomous role of Glut1 in the fly brain to regulate nutrient storage. A group of hormones that regulate metabolism and are expressed in the fly brain are Drosophila insulin-like peptides (Ilps) 2, 3, and 5. Interestingly, we observed blunted Ilp3 and Ilp5 expression in neuronal Glut1RNAi flies, suggesting Glut1 functions in insulin-producing neurons (IPCs) to regulate whole-organism TAG and glycogen storage. Consistent with this hypothesis, we also saw fewer TAGs and glycogens and decreased expression of Ilp3 and Ilp5 in flies with IPC-specific Glut1RNAi. Together, these data suggest Glut1 functions as a nutrient sensor in IPCs, controlling TAG and glycogen storage and regulating systemic energy homeostasis.

The role of melatonin in delaying senescence and maintaining quality in postharvest horticultural products.

The postharvest lifespan of horticultural products is closely related to loss of nutritional quality, accompanied by a rapid decline in shelf life, commercial value, and marketability. Melatonin (MT) application not only maintains quality but also delays senescence in horticultural products. This paper reviews biosynthesis and metabolism of endogenous MT, summarizes significant effects of exogenous MT application on postharvest horticultural products, examines regulatory mechanisms of MT-mediated effects, and provides an integrated review for understanding the positive role of MT in senescence delay and quality maintenance. As a multifunctional molecule, MT coordinates other signal molecules, such as ABA, ETH, JA, SA, NO, and Ca2+, to regulate postharvest ripening and senescence. Several metabolic pathways are involved in regulation of MT during postharvest senescence, including synthesis and signal transduction of plant hormones, redox homeostasis, energy metabolism, carbohydrate metabolism, and degradation of pigment and cell wall components. Moreover, MT regulates expression of genes related to plant hormones, antioxidant systems, energy generation, fruit firmness and colour, membrane integrity, and carbohydrate storage. Consequently, MT could become an emerging and eco-friendly preservative to extend shelf life and maintain postharvest quality of horticultural products.

Shortage of storage carbohydrates mainly determines seed abscission in Torreya grandis ‘Merrillii’

Torreya grandis cv. Merrillii is a well-known nut in South China with high nutritional value. Severe premature seed abscission limits the industrial development of T. grandis by causing serious economic losses. However, the physiological mechanisms of seed abscission in T. grandis remain poorly understood. To gain insight into the relationships between carbohydrate status and seed abscission, three-year-old seed-bearing branches were taken as representative materials for the entire tree. Furthermore, the time course of changes in the photosynthetic rate and the non-structural carbohydrate (NSC) dynamics were monitored in the main sources (the one-year-old and two-year-old shoots), and the dry weight and NSC levels of sinks (the seeds, current female cone cluster, and current vegetative cluster) across all seed development stages were recorded. The cumulative seed abscission rate significantly increased, reaching 91.5% from 0 to 72 days after seed protrusion time (SPT). NSC levels in the main sources significantly decreased by 56%–79%, accompanied by a significantly increased photosynthesis rate of 17.1%–49.1% during that period and increased NSC levels in the three sinks. The gene expression level of cell wall invertase (TgCWIN) was significantly correlated with sucrose, fructose, and glucose levels. The carbon storage capacity of the main sources significantly decreased from 6.03 to 3.14 mmol C · d−1, with a stable photosynthetic capacity, from 0 to 72 days after SPT, whereas the carbon demand of the three sinks showed a continuously increasing trend from 3.14 to 7.71 mmol C · d−1. In addition, sucrose supplementation significantly decreased the cumulative seed abscission rate. These results suggest that storage carbohydrates play a major role in the regulatory mechanism of seed abscission in T. grandis. Our study provides a theoretical basis for improving T. grandis yield through establishing a better carbon balance between sources and sinks using timely fertilization or proper pruning procedures.

Neutral lipid and chrysolaminarin metabolic pathways during nitrogen starvation and recovery in the diatom Phaeodactylum tricornutum

Oleaginous diatoms, including the marine model species Phaeodactylum tricornutum, have been identified as promising sources of compounds with a biotechnological interest including triacylglycerol and the soluble polysaccharide chrysolaminarin. These two molecules are accumulated in cells under nutrient starvation conditions, however, their consumption after nutrient replenishment lack a precise characterization. In this study, two ecotypes of P. tricornutum (Pt1 and Pt4) have been subjected to a nitrate starvation followed by a resupply to monitor their response through biochemical assays and gene expression quantification. We highlighted that both ecotypes experienced a two-step response to nitrogen resupply, with a rapid initial consumption of stored soluble carbohydrates probably allowing the restart of photosynthesis and protein synthesis, followed by a consumption of neutral lipids fueling cell division. Some genes were particularly upregulated after resupply, especially those encoding the lipases Phatr3_EG02408 and Phatr3_EG00720 and the exoglucosidase Phatr3_J43302, suggesting their role in degradation processes. Additionally, ecotype Pt4 recovered faster from stress than ecotype Pt1, possibly due to differences in specific gene regulations. Overall, these findings provide potential targets for understanding lipid and carbohydrate degradation, and for creating high-lipid producing strains.

Yield development and nutrient offtake in contrasting miscanthus hybrids under green and brown harvest regimes

AbstractHarvest time is an important variable that determines the yield of miscanthus biomass, its possible end uses, and the nutrient offtake from the field. Green harvests result in a higher yield and greater nutrient removal from the field. Brown miscanthus harvests, carried out in late winter or early spring, result in lower yields and a lower nutrient offtake, whereby the harvested biomass is better suited to use in combustion. To look at the long‐term impact of green harvests on miscanthus, this experiment followed the yield development of two miscanthus hybrids subjected to green and brown harvests over a period of seven years at one site in Southern Germany. The standard commercial hybrid Miscanthus × giganteus (Mxg) was compared with a novel late‐ripening Miscanthus sinensis hybrid: Syn55. Average yields of Mxg were 19.9 t ha−1 for green harvests and 13.2 t ha−1 for brown harvests compared to 13.9 and 12.9 t ha−1 for green and brown harvested Syn55, respectively. Yields of Mxg were very different for green and brown harvests; green harvested Mxg had very high nutrient offtake, while brown harvested Mxg had the lowest nutrient offtakes of all treatments. Syn55 showed a less marked difference between green and brown harvests likely due to its tendency to retain its leaves over winter. Syn55 was however not tolerant of a green harvest, with yields of brown harvested stands surpassing the yield of green harvested stands in several years. Although Mxg demonstrated consistently high yields when harvested in October, some signs of yield decline were detected in both hybrids when harvested green, which was due to insufficient carbohydrate relocation. Alternating green and brown harvests are recommended to allow stands to replenish carbohydrate stores and to form a litter layer.

Effects of artificial management on culm properties of Dendrocalamus brandisii.

The artificial cultivation and management were extensively carried out in Dendrocalamus brandisii stands. However, the influences of artificial management on the anatomical and chemical characteristics of the bamboo culms were unknown. In this study, the fiber morphology, chemical composition and sugar accumulation of the D. brandisii culms with management and without management were compared in order to determine the influences of artificial management on bamboo culms. The results indicated that artificial management had a significant influence on the fiber morphology, resulting in shorter fiber length, larger L/T ratio, and smaller W/Lu value. However, the management not only increased the contents of moisture, ash, SiO2, and extractive, but also increased the holocellulose contents and decreased the lignin contents, as compared to those without management. Moreover, the management significantly increased the endogenous carbohydrates storage in the culms so as to improve the shoot production. The bamboos under management conditions could still be utilized as a raw material for papermaking. This provided a theoretical basis for the artificial management of D. brandisii stands.

Cytology, metabolomics, and proteomics reveal the grain filling process and quality difference of wheat

Comparative proteomics and non-target metabolomics, together with physiological and microstructural analyses of wheat grains (at 15, 20, 25, and 30 days after anthesis) from two different quality wheat varieties (Gaoyou 5766 (strong-gluten) and Zhoumai 18) were performed to illustrate the grain filling material dynamics and to search for quality control genes. The differential expressions of 1541 proteins and 406 metabolites were found. They were mostly engaged in protein metabolism, stress/defense, energy metabolism, and amino acid metabolism, and the metabolism of stored proteins and carbohydrates was the major focus of the latter stages. The core proteins and metabolites in the growth process were identified, and the candidate genes for quality differences were screened. In conclusion, this study offers a molecular explanation for the establishment of wheat quality, and it aids in our understanding of the intricate metabolic network between different qualities of wheat at the filling stage.

Central transcriptional regulator controls photosynthetic growth and carbon storage in response to high light

Carbon capture and biochemical storage are some of the primary drivers of photosynthetic yield and productivity. To elucidate the mechanisms governing carbon allocation, we designed a photosynthetic light response test system for genetic and metabolic carbon assimilation tracking, using microalgae as simplified plant models. The systems biology mapping of high light-responsive photophysiology and carbon utilization dynamics between two variants of the same Picochlorum celeri species, TG1 and TG2 elucidated metabolic bottlenecks and transport rates of intermediates using instationary 13C-fluxomics. Simultaneous global gene expression dynamics showed 73% of the annotated genes responding within one hour, elucidating a singular, diel-responsive transcription factor, closely related to the CCA1/LHY clock genes in plants, with significantly altered expression in TG2. Transgenic P. celeri TG1 cells expressing the TG2 CCA1/LHY gene, showed 15% increase in growth rates and 25% increase in storage carbohydrate content, supporting a coordinating regulatory function for a single transcription factor.

MeGT2.6 increases cellulose synthesis and active gibberellin content to promote cell enlargement in cassava.

Cassava, a pivotal tropical crop, exhibits rapid growth and possesses a substantial biomass. Its stem is rich in cellulose and serves as a crucial carbohydrate storage organ. The height and strength of stems restrict the mechanised operation and propagation of cassava. In this study, the triple helix transcription factor MeGT2.6 was identified through yeast one-hybrid assay using MeCesA1pro as bait, which is critical for cellulose synthesis. Over-expression and loss-of-function lines were generated, and results revealed that MeGT2.6 could promote a significant increase in the plant height, stem diameter, cell size and thickness of SCW of cassava plant. Specifically, MeGT2.6 upregulated the transcription activity of MeGA20ox1 and downregulated the expression level of MeGA2ox1, thereby enhancing the content of active GA3, resulting in a large cell size, high plant height and long stem diameter in cassava. Moreover, MeGT2.6 upregulated the transcription activity of MeCesA1, which promoted the synthesis of cellulose and hemicellulose and produced a thick secondary cell wall. Finally, MeGT2.6 could help supply additional substrates for the synthesis of cellulose and hemicellulose by upregulating the invertase genes (MeNINV1/6). Thus, MeGT2.6 was found to be a multiple regulator; it was involved in GA metabolism and sucrose decomposition and the synthesis of cellulose and hemicellulose.

Intensive Microalgal Cultivation and Tertiary Phosphorus Recovery from Wastewaters via the EcoRecover Process.

Mixed community microalgal wastewater treatment technologies have the potential to advance the limits of technology for biological nutrient recovery while producing a renewable carbon feedstock, but a deeper understanding of their performance is required for system optimization and control. In this study, we characterized the performance of a 568 m3·day-1 Clearas EcoRecover system for tertiary phosphorus removal (and recovery as biomass) at an operating water resource recovery facility (WRRF). The process consists of a (dark) mix tank, photobioreactors (PBRs), and a membrane tank with ultrafiltration membranes for the separation of hydraulic and solids residence times. Through continuous online monitoring, long-term on-site monitoring, and on-site batch experiments, we demonstrate (i) the importance of carbohydrate storage in PBRs to support phosphorus uptake under dark conditions in the mix tank and (ii) the potential for polyphosphate accumulation in the mixed algal communities. Over a 3-month winter period with limited outside influences (e.g., no major upstream process changes), the effluent total phosphorus (TP) concentration was 0.03 ± 0.03 mg-P·L-1 (0.01 ± 0.02 mg-P·L-1 orthophosphate). Core microbial community taxa included Chlorella spp., Scenedesmus spp., and Monoraphidium spp., and key indicators of stable performance included near-neutral pH, sufficient alkalinity, and a diel rhythm in dissolved oxygen.

Legacy effects of early cotyledon removal on the growth, carbon and nitrogen storage, and drought response of Quercus variabilis seedlings

Predation of cotyledons can reduce short-term survival and growth in emerging oak seedlings, potentially affecting species recruitment. However, the physiological basis of reduced seedling performance after early cotyledon predation and the mid-term consequences of cotyledon predation on seedling drought tolerance remain poorly understood. We performed an experiment where we simulated predation by systematically removing cotyledons from Quercus variabilis seedlings at seven growth stages during emergence in the first growing season. We assessed survival, growth, and nitrogen (N) and non-structural carbohydrate (NSC) storage. Subsequently, in the second growing season, we assessed drought tolerance in seedlings with cotyledons removed at four growth stages in the previous season, and subjected seedlings to two drought levels to assess overall performance. During the first growing season, seedling survival showed less sensitivity to cotyledon removal compared to growth, N and NSC storage, which were significantly reduced when cotyledons were removed within the first 4 days after emergence. Early cotyledon removal (4 days after emergence) in the first growing season enhanced drought tolerance in the second growing season by reducing leaf embolism vulnerability, but it did not affect leaf drought tolerance traits such as the osmotic potential or the modulus of elasticity. Early cotyledon removal had a lasting effect on seedling growth and N and NSC storage building in the second growing season. Additionally, it exacerbated the negative consequences of drought on seedlings growth and N storage building, but it did not influence survival or NSC storage. Our findings evidence that early cotyledon removal in emerging oak seedlings has significant short- and medium-term repercussions on growth and capacity to store N and NSC, and it hinders the capacity of seedlings to respond to drought despite early cotyledon removal increases leaf drought tolerance. These functional legacies can influence oak seedling recruitment.