Jukuri, open repository of the Natural Resources Institute Finland (Luke) All material supplied via Jukuri is protected by copyright and other intellectual property rights. Duplication or sale, in electronic or print form, of any part of the repository collections is prohibited. Making electronic or print copies of the material is permitted only for your own personal use or for educational purposes. For other purposes, this article may be used in accordance with the publisher’s terms. There may be differences between this version and the publisher’s version. You are advised to cite the publisher’s version. This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Katja T. Rinne‐Garmston, Yu Tang, Elina Sahlstedt, Bartosz Adamczyk, Matthias Saurer, Yann Salmon, María del Rosario Domínguez Carrasco, Teemu Hölttä, Marco M. Lehmann, Lan Mo, Giles H. F. Young Title: Drivers of intra‐seasonal δ13C signal in tree‐rings of Pinus sylvestris as indicated by compound‐specific and laser ablation isotope analysis Year: 2023 Version: Publisher’s version Copyright: The author(s) 2023 Rights: CC BY 4.0 Rights url: https://creativecommons.org/licenses/by/4.0/ Please cite the original version: Rinne‐Garmston, K.T., Tang, Y., Sahlstedt, E., Adamczyk, B., Saurer, M., Salmon, Y. et al. (2023) Drivers of intra‐seasonal δ13C signal in tree‐rings of Pinus sylvestris as indicated by compound‐specific and laser ablation isotope analysis. Plant, Cell & Environment, 1–18 Received: 14 September 2022 | Revised: 17 May 2023 | Accepted: 27 May 2023 DOI: 10.1111/pce.14636 OR I G I NA L A R T I C L E Drivers of intra‐seasonal δ13C signal in tree‐rings of Pinus sylvestris as indicated by compound‐specific and laser ablation isotope analysis Katja T. Rinne‐Garmston1 | Yu Tang1,2 | Elina Sahlstedt1 | Bartosz Adamczyk1 | Matthias Saurer3 | Yann Salmon2,4 | María del Rosario Domínguez Carrasco2 | Teemu Hölttä2 | Marco M. Lehmann3 | Lan Mo1,2 | Giles H. F. Young1 1Stable Isotope Laboratory of Luke (SILL), Natural Resources Institute Finland (Luke), Helsinki, Finland 2Institute for Atmospheric and Earth System Research (INAR)/Forest Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland 3Forest Dynamics, Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland 4Institute for Atmospheric and Earth System Research (INAR)/Physics, Faculty of Science, University of Helsinki, Helsinki, Finland Correspondence Katja T. Rinne‐Garmston, Stable Isotope Laboratory of Luke (SILL), Natural Resources Institute Finland (Luke), Helsinki, Finland. Email: katja.rinne-garmston@luke.fi Funding information European Research Council; Academy of Finland; Swiss National Science Foundation Abstract Carbon isotope composition of tree‐ring (δ13CRing) is a commonly used proxy for environmental change and ecophysiology. δ13CRing reconstructions are based on a solid knowledge of isotope fractionations during formation of primary photosynthates (δ13CP), such as sucrose. However, δ 13CRing is not merely a record of δ13CP. Isotope fractionation processes, which are not yet fully understood, modify δ13CP during sucrose transport. We traced, how the environmental intra‐seasonal δ13CP signal changes from leaves to phloem, tree‐ring and roots, for 7 year old Pinus sylvestris, using δ13C analysis of individual carbohydrates, δ13CRing laser ablation, leaf gas exchange and enzyme activity measurements. The intra‐seasonal δ13CP dynamics was clearly reflected by δ13CRing, suggesting negligible impact of reserve use on δ 13CRing. However, δ13CP became increasingly 13C‐enriched during down‐stem transport, probably due to post‐photosynthetic fractionations such as sink organ catabolism. In contrast, δ13C of water‐soluble carbohydrates, analysed for the same extracts, did not reflect the same isotope dynamics and fractionations as δ13CP, but recorded intra‐seasonal δ13CP variability. The impact of environmental signals on δ13CRing, 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 δ13CRing. K E YWORD S CO2, phloem transport, photosynthesis: carbon reactions, stable carbon isotope (d13C), sugars Plant Cell Environ. 2023;1–18. wileyonlinelibrary.com/journal/pce | 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd. 1 | INTRODUCTION Tree‐ring stable carbon isotope record (δ13CRing) has become a commonly utilized proxy for environmental change and its impact on tree physiological processes (Helama et al., 2018; Kress et al., 2010; Rinne et al., 2010). At the same time, we do not yet fully understand the photosynthetic and post‐photosynthetic metabolic processes and associated isotope fractionations leading to a specific δ13C value of a xylem cell (Gessler et al., 2014; Schiestl‐Aalto et al., 2021), or how the role of these individual processes may depend on the time of the growing season or changing environmental conditions. These un- knowns affect the accuracy and spatial comparability of reconstruc- tions that are based on the absolute δ13CRing values, such as intrinsic water‐use efficiency [iWUE, the ratio of photosynthetic rate (A) to stomatal conductance (gs)] (Adams et al., 2020; Mathias & Thomas, 2021). They also exert a level of uncertainty for reconstructions that utilize the average δ13CRing without the knowl- edge of how and why δ13C varies within the analysed ring at a finer scale. Better knowledge of the processes that modify the δ13C of leaf photosynthates (δ13CP), such as sucrose and glucose, would also benefit studies interested in δ13C of source and sink organ respiration (Barbour et al., 2005; Werner & Gessler, 2011; Wingate et al., 2010), carbon allocation dynamics in trees (Brüggemann et al., 2011; Tang, Schiestl‐Aalto, Saurer, et al., 2022) or translocation of δ13CP signal belowground via ectomycorrhizal fungi (Hobbie et al., 2012; Högberg et al., 2010). This is because these processes are closely linked with δ13C of sucrose (δ13CSuc), which is the sugar transported from leaves to phloem and roots (Dennis & Blakeley, 2000). A large uncertainty in using tree‐ring carbon isotopes for environmental reconstructions is the potential reliance on carbohy- drate reserves, which would uncouple δ13CRing from its contemporary ambient environmental conditions. For broadleaved species, which can gain up to 30% of the total stem increment before budburst (Hinckley & Lassoie, 1981), the typical requirement of reserves for earlywood (EW) growth has been well demonstrated (Helle & Schleser, 2004) and, hence, their latewood (LW) section is normally used for environmental studies (Etien et al., 2009). Conifers, on the other hand, have several existing needle generations or, in the case of deciduous larch, bud burst typically occurs before the start of stem growth, providing fresh assimilates for cell formation (Rinne, Saurer, Kirdyanov, Loader, et al., 2015). Congruently, the studies that have followed δ13C signal from bulk water‐soluble carbohydrates (WSCs, δ13CWSC) (Pinus sylvestris, Gessler, Brandes, et al., 2009) or individual sugars (Larix gmelinii, Rinne, Saurer, Kirdyanov, Loader, et al., 2015) (P. sylvestris, Tang et al., 2023) to δ13CRing have found a close correspondence in intra‐seasonal δ13C trends between the two carbon pools. However, there is currently no consensus on the suitability of EW of boreal conifers for environmental studies (Fonti et al., 2018; Kagawa et al., 2006; Kress et al., 2009). Better knowledge of reserve use dynamics of conifers within a growing season could help to decipher whether there is the need to discard EW from environmental and tree physiological studies, such as reconstructions of iWUE (Michelot et al., 2011). Additionally, the average δ13C value of an annual ring and how well it represents the environmental conditions of that growing season are also dependent on other metabolic processes. These include isotope fractionation associated with activity of sucrose degrading invertase enzyme in leaves (Mauve et al., 2009; Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015), potential isotope fractionation during phloem loading (Bögelein et al., 2019), mixing of sugar pools of different ages during phloem transport (Brandes et al., 2006) and isotope fractionation due to respiration (Gleixner et al., 1998) or during xylem formation (Panek & Waring, 1997). Although some of these processes have been examined relatively extensively in the literature (Gessler et al., 2014 and references therein), there are not many studies that have taken the analytical approach to compound‐specific and high spatial and temporal resolution level (Rinne‐Garmston et al., 2022; and refer- ences therein). The potential of identifying and quantifying the above listed processes is much better, if examining changes in δ13C values of individual compounds, particularly of sugars, as opposed to δ13C of a bulk matter, where the impact of a metabolic process may be obscured by the different metabolic history and biochemical role of the constituent compounds. This issue was demonstrated in Bögelein et al. (2019), where the absence of diel variation in δ13C of leaf and twig phloem water‐soluble organic matter of Douglas fir (Pseudotsuga menziesii) was assigned to the high content of isotopically invariable sugar alcohols in the bulk matter. At leaf level, compound‐specific isotope analysis (CSIA) has been applied in a few studies on WSCs of conifers, to examine how environmental signals are recorded and post‐photosynthetically modified in δ13C values of individual assimilates during a growing season (Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015; Sidorova et al., 2018, 2019; Tang, Schiestl‐Aalto, Lehmann, et al., 2022). In Rinne, Saurer, Kirdyanov, Loader, et al. (2015) the intra‐seasonal records of needle δ13CSuc (δ 13CSuc_Needle) were further compared to high spatial resolution δ13CRing profiles, to examine the reasons behind the widely reported 13C‐enrichment of sink organs relative to leaves (Gessler, Brandes, et al., 2009; Gleixner et al., 1993) and evaluate the quality of seasonal climate information stored in larch (L. gmelinii) trees. However, to our knowledge, there is no high spatial resolution CSIA data available for phloem, done together with intra‐ annual δ13CRing profiling, so that it could be possible to separate the impact of individual metabolic processes on δ13CSuc along its transport route from leaves to stem xylem. The preexisting CSIA studies have focused either on examining carbon allocation patterns with the 13CO2‐labelling technique (Galiano et al., 2017; Streit et al., 2013), which has the drawback of losing the environmental and physiological information recorded in δ13C variability at natural abundance, or on studying biosynthetic pathways and isotope fractionations within a very narrow timeframe (Bögelein et al., 2019; Smith et al., 2016). Several studies have, though, examined down‐ stem isotope fractionations using phloem sap (Gessler et al., 2004), but reached contradictory outcomes on, for example, the occurrence of a 13C‐fractionation process during xylem formation (Cernusak et al., 2005; Gessler, Brandes, et al., 2009). Although sucrose is the 2 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License main constituent, phloem sap contains also a variable amount of other sugars, such as glucose (Devaux et al., 2009), and other compounds (Merchant et al., 2010) with distinctive δ13C values (Bögelein et al., 2019). Hence, there is a factor of uncertainty connected with the utilization of phloem sap for isotope fractionation studies. In this paper, we examine the environmental signal recorded in leaf δ13CSuc and how it is kept along sucrose transport pathway to phloem, tree‐rings and roots. It aims to resolve some of the controversy concerning the significance of the individual metabolic processes in the post‐photosynthetic modification of isotopic signal and to quantify their impact on δ13C. With the aid of CSIA data, it also studies the quality of environmental and tree physiological signal in δ13CWSC, and potential issues when using WSCs for isotope measurements. The study was conducted at controlled, regularly watered conditions for clones of 7‐year‐old P. sylvestris L. saplings, in which δ13CRing profiles were time‐scaled by sampling the ring during growth, concurrently with sampling of leaves, phloem and roots for carbohydrate δ13C analysis. Specifically, we seek answers to the following questions: (1) What is the impact of the postulated isotope fractionation and mixing processes occurring during phloem loading on δ13CSuc? (2) What processes modify phloem δ 13CSuc before its use to produce tree‐ring material and to what extent? (3) What is the isotopic offset between leaf sugars and tree‐rings? Further, this unique data set enables us to estimate the sensitivity of δ13CWSC to environmental changes and its suitability for determining post‐ photosynthetic fractionation processes. This detailed data set also allows us to evaluate the impact of reserve use on EW δ13C. These questions are tackled with an exceptionally diverse data set that combines for the first time intra‐seasonal analysis of δ13CSuc, glucose δ13C (δ13CGlc) and δ 13CWSC in leaves, phloem and roots with intra‐ ring δ13C profiling from laser ablation isotope ratio mass spectrome- try (LA‐IRMS). The data interpretations were supported by leaf gas exchange measurements and leaf starch analysis, and analysis of enzymes involved in sucrose synthesis and degradation in leaves, to better understand how the invertase‐related fractionation is mani- fested in δ13C values of leaf sugars (Gilbert et al., 2011). 2 | MATERIALS AND METHODS 2.1 | Plant material and experimental setup The experiment was conducted during summer 2018 in a greenhouse of University of Helsinki (60°14'N, 25°01'E). The 7‐year‐old saplings of P. sylvestris were obtained in May 17, at the beginning of the growing season (May 22 ± 13 days in Jyske et al., 2014), in 2018 from outdoor nursery garden of Haapastensyrjä (60°37′N, 24°27′E). Four different clones (clones A, B, C and D in Supporting Information: Figure S1) of P. sylvestris were available for the study (for more details, see Ryhti et al., 2022). Nine specimens of each four clones (i.e., 36 saplings in total) were dug‐out with their roots and the attached soil (below‐ground volume was ~3.5 L) for immediate transport. The average temperature (T) of May was 14.6°C in Haapastensyrjä, where the saplings had grown in open air, and 21.6°C in the greenhouse. Clones were used to minimize the non‐ environmentally driven temporal variability in the analytical results (Schuster et al., 1992), a beneficial approach for studies using destructive sampling (see Section 2.2). Four clones, instead of a single clone, were used and their measurements (e.g., δ13C) averaged for each sampling day, to obtain results that better represent the studied species rather than responses of an individual (Leavitt & Long, 1984). At the greenhouse in Helsinki, the saplings were repotted in May 17 into 7.5 L pots and filled with additional peat‐based soil. At the start of the experiment, stem diameter (near soil surface) was 21 ± 5, 21 ± 4, 21 ± 4 and 18 ± 3mm, and the height of the saplings (from the soil surface) was 120 ± 17, 113 ± 15, 104 ± 13 and 96 ± 10 cm, for clones A, B, C and D, respectively. The saplings of each clone were distributed randomly on a table with automated watering system to maintain the plants at well‐watered conditions. The watering was used each Tuesday, Thursday, Saturday and Sunday for a period of 15min. To assure equal light conditions for each sapling during the experiment, the position of each tree on the table was changed every second day. 2.2 | Sampling and instrumental measurements Destructive sampling was done between 1300 and 1700 h once a week from June 5 to July 30. On each sampling day, one sapling from each four group of clones (A, B, C and D) was randomly selected for analysis (Supporting Information: Figure S1). Immediately before the sampling, leaf gas‐exchange measurements were conducted on 1‐year‐old needles (1 N). A, gs and leaf internal concentration of CO2 (ci) were measured using a GFS‐3000 (Heinz Walz GmbH). The conditions inside the measurement cuvette (T, photosynthetically active radiation, vapour pressure deficit (VPD) and CO2 concentra- tion) were set to track ambient conditions in the greenhouse. Sampling of 1 N, current‐year needles (0 N), stem phloem and roots was conducted for analysis of WSCs. Needles were collected from several branches around the canopy to ensure representative results. Phloem, including cambium and periderm, was obtained by removing the outer bark and by separating the xylem on the basis of differences in hardness and colour (Lintunen et al., 2016). The samples were immediately placed in a cool box with ice blocks, and treated in a microwave within 2 h of collection to stop metabolic activities (Wanek et al., 2001). The samples were subsequently dried for 24 h at 60°C, and ground to fine powder. On sampling days between June 18 and July 23, 0 and 1 N were additionally collected for determining activity of the enzymes involved in sucrose synthesis and degradation. These samples were immediately snap‐freezed on dry ice and stored in −80°C before analyses. Finally, the stem of each sampled tree was micro‐cored with a Trephor instrument (Rossi et al., 2006) for xylogenesis observations, and the obtained sample was placed in ethanol‐water solution (1:1). The remaining stem was dried and stored in room T for δ13C analysis of resin extracted DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 3 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License tree‐ring (δ13CRing_RE). T and relative humidity (RH) were automati- cally recorded at 15min intervals with thermo‐ and psychrometer (Priva Hortimation). They averaged 23.3°C and 56%, respectively, for the study period. 2.3 | Intra‐annual tree‐ring δ13C analysis A disk was cut from all stems, approximately 10 cm above soil surface, collected on June 5 (the first sampling day), July 2 and July 30 (the last sampling day), to establish a time‐axis for δ13CRing_RE profiles. The disks were treated in a Soxhlet apparatus with ethanol for 48 h to remove resins and other soluble materials. Subsequently, ethanol was removed by boiling the disks for 6 h in water, which was replaced every hour, and the disks were dried at 40°C in an oven for 2 day. The samples were then sanded for a smooth, even surface with a series of finer sandpapers until all cells were clearly visible under magnification before LA‐IRMS analysis of year 2018. Any remaining wood powder from sanding was removed in an ultrasonic bath in water, and the samples were dried once more. One approximately 5mm wide segment was cut from the resin extracted disks of July 30 for determining the difference in δ13C values between resin extracted wood and tree‐ring α‐cellulose (δ13CRing_Cellulose). From these segments, the EW and LW of 2018 were separated with a scalpel into small slivers. A representative subsample was taken from each EW and LW sample for EA‐IRMS δ13C analysis. The remaining slivers were extracted to α‐cellulose, a procedure which involves removal of lignins by treatment with acidic sodium chlorite solution, and treatment with first 10% and then 17% sodium hydroxide solution to leach hemicelluloses (Loader et al., 1997; Rinne et al., 2005). High spatial resolution δ13C profiles were determined for resin extracted wood (δ13CRing) by LA‐IRMS analysis at Stable Isotope Laboratory of Luke (SILL), Finland. The operational principle was as described in Tang et al. (2023), based on Schulze et al. (2004) and Loader et al. (2017). In brief, the LA‐IRMS analysis consists of sampling a tree‐ring by laser ablation (LSX‐213 G+; Teledyne Photon Machines), combustion of the formed wood powder to CO2 in a glass reactor tube (OD = 6mm) filled with Cr2O3 and held at 700°C, collection of the CO2 in a liquid nitrogen trap, and finally heating of the trap to room temperature, which releases the CO2 to IRMS (Sercon 20‐22; Sercon Ltd.) for δ13C analysis. Water produced in the combustion reaction is separated from the sample stream using a NafionTM drying tube downstream of the reactor. The laser parameters are operated and automated sampling sequences are created via a separate software (Chromium; Teledyne Photon Machines). In this study, we analysed tree‐rings at 40 µm (40 µm wide tracks placed in a zig‐zag pattern, ‘third LA series’ in Figure 1) or 80 µm (40 µm wide tracks with 40 µm spacing, ‘first LA series’ in Figure 1) resolution, starting from the beginning of the annual ring and ending to the bark edge. ‘Raw’ δ13C values, which were calculated by the IRMS software (Callisto; Sercon Ltd.) by comparison to reference CO2 gas pulses with a known δ 13C, were calibrated against a USGS‐55 (Mexican ziricote wood, −27.13‰) and an in‐ house (yucca tree powder, −15.46‰) reference materials (powders compressed to solid pellets by manual hydraulic press). The calibration standards were placed with the samples into the laser chamber and analysed concurrently with LA‐IRMS. IAEA‐C3 cellulose paper was also measured concurrently with the samples and similarly calibrated against the USGS‐55 and in‐house references, thus giving an average δ13C value of −24.81 ± 0.16 (n = 113), which agrees well F IGURE 1 Disk sections of Pinus sylvestris examined for clone B of July 30 using LA‐IRMS. The first and second δ13C series contained compression wood (CW) whereas the third series was on normal density wood. The δ13C profiles are shown in Figure 2. LA‐IRMS, laser ablation isotope ratio mass spectrometry. 4 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License with the values reported for this material: −24.91 ± 0.49‰ (IAEA‐C3, cellulose paper) and −24.72 ± 0.04‰ (IAEA‐CH3, powder prepared from IAEA‐C3). Signal size effects on δ13C were monitored and, when necessary, corrected for by running a set of reference sample laser lines at different lengths and spot sizes to produce variable signal sizes. 2.4 | Alignment and time‐scaling of tree‐ring δ13C profiles Intra‐annual low frequency δ13C variability was similar for the analysed tree‐ring sections, enabling the alignment of the δ13CRin- g_RE series and, hence, time‐scaling of the profiles. The June 5 series were used to identify, which section of the July 30 δ13C profiles were formed before the start of the experiment, and July 2 tree‐ring group was used to determine which part of July 30 series had formed by July 2. Thereafter, phloem sugar δ13C series were incorporated together with δ13CRing_RE series into Figure 2 using the common sampling days (June 5, July 2 and July 30), and used to finalize the time‐scaling of the x‐axis. The detailed description of the alignment procedure is given in the Supporting Information: Method S1. 2.5 | Extraction and purification of sugars and starch Water soluble compounds in 0, 1 N, phloem and roots were extracted using a modified method after Wanek et al. (2001). Two microlitres reaction vials were filled with 60mg of homogenized plant powder and 1.5 mL of Milli‐Q water, stirred with vortex, and placed in a water bath at 85°C for 30min. Once the samples had cooled down, they were centrifuged at 10 000 g for 2 min. WSCs were isolated from the supernatant using three types of sample treatment cartridges, as described in Rinne et al. (2012). The WSC‐samples were then freeze‐ dried, dissolved in 1mL of Milli‐Q water, pressed through a 0.45 µm syringe filter. For 1 N, starch was extracted from the pellet of the WSC extraction by enzymatic hydrolysis (Lehmann et al., 2019; Wanek et al., 2001). Shortly, pellet in each reaction vial was washed repeatedly with 1.2mL methanol‐chloroform‐water (12:5:3, v/v/v) solution for lipid removal, followed by washes with deionized water. Subsequently, starch in each pellet was gelatinized at 99°C for 15min and hydrolysed with purified (with Vivaspin 15R; Sartorius) α‐amylase (EC 3.2.1.1; Sigma‐Aldrich) solution at 85°C for 2 h. Hydrolysed starch in supernatant was cleaned with centrifugation filters (Vivaspin 500; Sartorius). Identical treatment principle (Werner & Brand, 2001) was applied to two maize starch standards (Fluka), two wheat starch standards (Fluka) and four blanks with every batch of 40 samples. The WSC and starch samples were stored in a −20°C freezer until further use. 2.6 | CSIA of sugars with HPLC‐IRMS The WSC extracts were analysed online using a Delta V Advantage IRMS coupled with HPLC with a Finnigan LC Isolink interface (Krummen et al., 2004; Rinne et al., 2012) at WSL, Switzerland. A column T of 20°C (CarboPac PA20; Thermo Fisher Scientific) was used to prevent isomerization of hexoses with 1% NaOH as the HPLC eluent (Rinne et al., 2012). Excellent chromatographic peak separation and peak shape were obtained for sucrose and glucose of the analysed extracts (Supporting Information: Figure S2). Fructose was excluded from further analysis, because its δ13C value was affected by peak tailing and occasional co‐elution with another compound under these analytical settings (Rinne et al., 2012). Furthermore, glucose and fructose are often isotopically similar and, hence, there may not be an added benefit of obtaining fructose δ13C values for studying post‐photosynthetic fractionations (Rinne‐ Garmston et al., 2022). All extracts also contained a significant amount of sugar alcohols pinitol/myo‐inositol (‘pinitol’ from now on, Supporting Information: Figure S2), which co‐elute from the column. A dilution series (20, 40, 60, 90, 120 and 180 ng CμL.1) of external compound‐matched standard solutions (mixture of sucrose, glucose, fructose and pinitol) were analysed between every 10 samples to correct sample δ13C values (Rinne et al., 2012). The standard deviation of the repeated analytical measurements was 0.31‰ for sucrose (−25.37‰) and 0.51‰ (−10.24‰) for glucose standards. 2.7 | δ13C and concentration analysis with EA‐IRMS The 1N starch extracts in liquid were pipetted into tin capsules and freeze‐dried. The dry resin‐extracted and cellulose‐extracted samples of tree‐rings, and WSCs were weighted into tin capsules (IVA Analysentechnik). The samples were analysed for δ13C using an elemental analyser (Europa EA‐GSL; Sercon Limited) coupled to an IRMS (Sercon Limited) at SILL, Finland. The δ13C values were calibrated against IAEA‐CH3 (cellulose, –24.72‰), IAEA‐CH7 (poly- ethylene, –32.15‰) and an in‐house (sucrose, –12.22‰ and lactose, −24.66‰) reference materials. Measurement precision was 0.1‰ (SD), determined from repeated measurements of a quality control material. Starch concentrations were determined using the weight of starch in tin capsules and the weights of plant materials used for extraction, and by considering a conversion factor for the efficiency of the enzymatic conversion of starch to hydrolysed glucose (Tang, Schiestl‐Aalto, Lehmann, et al., 2022). The blanks used in starch extraction contained residuals of α‐amylase, having δ13C value of −27.62 ± 0.63‰. Blank concentration was on average 0.14 ± 3mg/mL in the final extraction solution. Sample to the average blank amount ratio was often 2 or less, causing significant uncertainty on the 1N starch δ13C results. The impact of blank correction on δ13C values is illustrated in Figure 3. DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 5 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F IGURE 2 Intra‐annual δ13C composition of tree rings and seasonal changes in δ13C of phloem sugars of Pinus sylvestris saplings. The dates on the y‐axis divide the series into three groups based on the sampling day of the saplings for tree ring δ13C analysis. June 5 tree ring δ13C series represent the xylem wood formed before the start of the experiment. July 2 tree ring δ13C series represent the wood formed by July 2, and July 30 series the wood formed by the end of the experiment. The coloured squares are the three maximum or one minimum values of wood δ13C series, and were used to align the wood δ13C series of June 5, July 2 and July 30 (see Supporting Information: Method S1). Earlywood (EW) and latewood (LW) areas and indicated. The darker LW bands indicate δ13C values between −29 and −31‰, to enhance visual comparisons. Some of the wood δ13C series were analysed on compression wood (CW) to obtain more δ13C datapoints for LW. Each phloem δ13C value represents the average of four clones (A−D) and the error bars its standard deviations. 6 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2.8 | Determination of enzyme activities for needles Enzymes were extracted as described in Nguyen et al. (2016) and Rende et al. (2017). Briefly, 250mg of needles were ground in liquid nitrogen. Enzymes were extracted with 50mM HEPES buffer, pH 8, with 1mM EDTA, 10mM MgCl2, 1 mM EGTA, 5mM dithiothreitol, 1 mM PMSF, 1% TritonX‐100, 2% glycerol and 3% PVPP. Following centrifugation (5min, 14 000 g), supernatants were filtered using VWR centrifugal filters with 10 K MWCO. Filtered extract was used to measure activity of sucrose forming and degrading enzymes. Invertase activity was measured as described in Jammer et al. (2015). Briefly, filtered extracts (20 µL) were incubated at 37°C for 30min with 5 µL of 100mM sucrose and 5 µL 0.4M reaction buffer of pH 4.5 or pH 6.8 for acid invertase (INVacid) and neutral invertase (INVneutral), respectively. For control reactions, sucrose was omitted. The amount of liberated glucose from sucrose was determined by measurement of absorbance at 405 nm in a plate reader (CLAR- IOstar; BMG Labtech) after 30min incubation at roomT with 200 µL of glucose oxidase‐peroxidase reagent, and 0.8 mgmL−1 ABTS in 0.1M potassium phosphate buffer pH 7.0. Standard curve was made on the basis of known concentration of glucose. Enzymatic activities are given as nmol of glucose per hour per dry weight of needles. Activity of sucrose synthase was measured as described in Nguyen et al. (2016). Briefly, sucrose‐degrading SuSy (SuSydegradation) activity was measured by adding 25 µL filtered extract to 200 µL of solution with 10mM sucrose and 10mM UDP (uridine 5'‐ diphosphate), pH 5.0 followed by 1 h incubation at 37°C. The same reaction, but with no UDP, was conducted to estimate sucrose‐ degrading activity caused by other enzymes existing in filtered extract. Sucrose‐synthetizing SuSy (SuSysynthesis) activity was mea- sured by adding 25 µL filtered extract to 200 µL solution with 10mM UDP‐glucose and 10mM fructose, pH 5.0 followed by 1 h incubation at 37°C. Controls did not include extract. Fructose reacted with 0.25% triphenyltetrazoliumchloride (TTC) in 1M NaOH producing red colour measured at 495mm at microplate reader (CLARIOs- tar; BMG Labtech). Standard curve was based on known concentra- tion of fructose. Enzymatic activities are given as nmol of fructose per hour per dry weight of needles. However, the use of plant extract with no in‐depth enzyme purification, like in our study, does not enable to clearly differentiate between sucrose phosphate synthase and SuSy (sucrose forming) activities and, hence, SuSysynth- esis represents the activity of both sucrose‐forming enzymes. 2.9 | Data analysis CorelDRAW Graphics Suite X7 was used for aligning the LA‐IRMS δ13C series. IBM SPSS Statistics software package was used for correlation analysis, where the average values of the four clones F IGURE 3 Measured δ13C and concentration of starch, and δ13C of glucose of 1‐year‐old needles of Pinus sylvestris saplings. For starch, each data point represents the average of four trees and the error bars its standard deviations (clones A, B, C and D). Glucose content was too low in some samples for accurate δ13C measurements. A number in the figure indicates the replication when n < 4 for the calculated δ13C average. For starch, the impact of blank correction on δ13C (13C‐depletion of values) is indicated by the red shaded area. DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 7 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License were used, and for two‐sample t‐tests. iWUE was calculated from the gas exchange data as the ratio between A and gs. δ 13C of primary photosynthates was calculated (δ13CP_calc) by the classic photo- synthetic discrimination model (Supporting Information: Method S2; Farquhar et al., 1982, 1989) using the gas exchange data of 1 N as input. 3 | RESULTS 3.1 | Water status and gas exchange VPD measured at 1400 h varied from 9.05 to 42.5 hPa between the sampling days (Figure 4), and T from 21.6°C to 35.7°C. Although the studied trees received regular watering, they were affected by water limitations, when VPD was high. This was indicated by an inverse relationship between VPD and A or gs of trees, when VPD was at maximum (June 11, June 18 and July 16, Figure 4). Due to this inconsistent relationship between these parameters, VPD did not correlate with A or gs for the entire study period. iWUE, from the gas exchange data, first declined until July 2, followed by a sharp increase, and a subsequent general decline until the end of the experiment (Figure 4). 3.2 | δ13CSuc, δ13CGlc and δ13CWSC in studied organs The temporal variation of δ13CSuc was similar for 0, 1 N, phloem and roots (Table 1), but there were significant isotopic offsets between the organs (Figure 5 and Table 2). The organ specific 13C‐enrichment of sucrose was as following: 0N < 1N = phloem< roots, where the δ13C offsets were 1.2 ± 0.8‰ [0 N vs. phloem, t(62) = 2.743, p < 0.008] and 1.0 ± 0.5‰ [phloem vs. roots, t(68) = 2.809, p < 0.006] (Table 2). When considering that 0 N contained on average 1.5 times more sucrose than 1N, the 13C‐enrichment of phloem over the two needle generations was 0.7 ± 1.0‰ according to mass balance calculation (Table 2, see also Section 3.3. for needle enzyme activity). F IGURE 4 Changes in assimilation rate (A), stomatal conductance (gs) and intrinsic water‐use efficiency (iWUE) of Pinus sylvestris with vapour pressure deficit (VPD) during the study period. Each gas exchange measurement value represents the average of four clones (A−D) and the error bars its standard deviations. 8 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License δ13CGlc series correlated with δ 13CSuc for each organ and had similar organ‐specific 13C‐enrichment as δ13CSuc, with lowest δ 13CGlc in needles and highest in roots (Table 1 and Figure 5). In roots, δ13CGlc > δ 13CSuc [1.2 ± 0.6‰, t(52) = 3.526, p < 0.001]. In phloem, δ13CGlc > δ 13CSuc, when considering the standard deviations (0.8 ± 0.6‰, n = 13). However, it should be noted that the replication was relatively low for phloem δ13CGlc series because the abundance of glucose was often low in phloem (Table 2 and Figure 5). In needles, δ13CGlc ≈ δ 13CSuc (0.4 ± 0.8‰) (Table 2). The δ13CWSC series correlated well with δ 13CSuc and/or δ 13CGlc series for all organs (Table 1) but had relatively low day‐to‐day variability. The latter was due to the presence of pinitol, whose TABLE 1 The results of correlation analysis (r‐values) for sucrose, glucose and water‐soluble carbohydrate (WSC) δ13C in different organs of Pinus sylvestris, photosynthetic parameters, climate parameters and calculated δ13C of primary photosynthates (δ13CP_calc) from leaf gas exchange measurements. δ13CSuc Photosynthetic parameters Climate parameters 0 N 1N Phloem Roots A gs δ13CP_calc iWUE VPD RH T δ13CSuc 0 N (n = 9) 0.79 a 0.79a 0.78a −0.67a −0.76a 0.60 (0.75a) 0.44 (0.66) 0.45 −0.70a 0.35 1 N (n = 9) 0.50 0.48 −0.51 −0.56 0.52 (0.54) 0.34 (0.38) 0.46 −0.42 0.50 Phloem (n = 9) 0.91b −0.49 −0.83b 0.77a (0.81a) 0.67a (0.76a) 0.11 −0.77a −0.17 Roots (n = 9) −0.32 −0.71a 0.80b (0.86b) 0.65 (0.77a) 0.22 −0.82b 0.05 δ13CGlu 0 N (n = 9) 0.91 b −0.52 −0.67a 0.59 (0.74a) 0.47 (0.71) 0.24 −0.73 0.10 1 N (n = 9) 0.75a −0.62 −0.86b 0.72a (0.86b) 0.59 (0.82b) 0.38 −0.73a 0.27 Phloem (n = 6) 0.89b −0.39 −0.85a 0.95b (1.00b) 0.82a (0.93a) 0.15 −0.78 0.02 Roots (n = 8) 0.81a −0.54 −0.71a 0.70 (0.72) 0.50 (0.54) 0.55 −0.71a 0.27 δ13CWSC 0 N (n = 9) 0.52 −0.26 −0.70 a 0.65 (0.77a) 0.61 (−0.82a) 0.00 −0.76a −0.30 1 N (n = 9) 0.24 −0.31 −0.86b 0.84b (0.90b) 0.76a (−0.88b) 0.15 −0.84b −0.14 Phloem (n = 9) 0.82b −0.35 −0.80b 0.77a (0.84b) 0.59 (0.71a) 0.30 −0.67 0.12 Roots (n = 9) 0.82b −0.52 −0.69a 0.69a (0.74a) 0.54 (0.63) 0.29 −0.66 0.22 Note: The studied organs included current‐year needles (0 N), one‐year‐old needles (1 N), phloem and roots. The determined photosynthetic and climate parameters were assimilation rate (A), stomatal conductance (gs), gas‐exchange derived δ13C value of primary photosynthates (δ13CP_calc) gas‐exchange derived intrinsic water‐use efficiency (iWUE), vapour pressure deficit (VPD), relative humidity (RH) and temperature (T). The r‐values and significance levels (a95% significance level, b99% significance level) are shown. The r‐values in brackets were obtained, when the data of July 16 (VPD maxima) was excluded. The correlation between δ13CP_calc and leaf‐internal concentration of CO2 (ci) with iWUE was −0.98 (p < 0.01). Abbreviation: WSC, water‐soluble carbohydrate. F IGURE 5 Comparison of sucrose, glucose, pinitol and water‐soluble carbohydrate (WSC) δ13C values in different tree organs with leaf internal concentration of CO2 (ci, reversed y‐axis scale) and calculated δ 13C of primary photosynthates (δ13CP_calc) from gas exchange measurements of Pinus sylvestris. For the gas exchange, 1‐year‐old needles (1 N) were used. 0 N is current‐year needles. In general, each sucrose and WSC δ13C value represent the average of four clones (A−D) and the error bars its standard deviations. Glucose content was too low in several samples for accurate CSIA. A number in the figure indicates the replication when n < 4 for the calculated δ13C average. CSIA, compound‐ specific isotope analysis. DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 9 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License carbon isotope composition (δ13CPinitol) did not show much temporal variability (Table 2 and Figure 5), and did not correlate with δ13CSuc or δ13CGlc for any organ (p > 0.05). Pinitol was 13C‐depleted relative to sugars, and its abundance and δ13C value were organ specific (Table 2 and Figure 5). There was no offset between δ13CWSC_0N and δ13CWSC_1N or between δ 13CWSC_Neelde and δ 13CWSC_Phloem, only roots showed 13C‐enrichment (Figure 5). δ13CP_calc data (−29.6 ± 1.5‰) did not significantly differ from δ13CSuc_1N (−28.4 ± 2.0‰, p > 0.05), but the daily average δ 13CP_calc values had 2.5 ± 2.3 times higher standard deviations. δ13CP_calc had significant correlations with δ13CSuc, δ 13CGlc and δ 13CWSC in the studied organs (Table 1). Statistically significant environmental (RH) and physiological signals (gs, A, ci and iWUE) were observed for δ 13CSuc_0N, δ13CSuc_Phloem and δ 13CSuc_Root, but not of δ 13CSuc_1N, despite the significant correlation between δ13CSuc_1N and δ 13CSuc_0N (Table 1). For δ13CGlc and δ 13CWSC, also the 1N series correlated. When July 16, the sampling day with the VPD maxima (Figure 4), was excluded from correlation analysis, correlations with δ13CP_calc (Figure 5a) and iWUE were generally improved (Table 1). On this day, the typical negative correlation between ci and the measured δ 13C series was inversed (Figure 5). 3.3 | Starch δ13C and concentration in 1 N Needle starch δ13C (δ13CStarch_1N) was invariable during the experi- ment, unlike needle sugars, and on average more 13C‐depleted (no blank correction: −28.9 ± 0.4‰, corrected: −30.3 ± 0.5‰) than δ13CSuc_1N (−28.2 ± 2.0‰) and δ 13CGlc_1N (−28.0 ± 1.4‰) but similar to δ13CWSC_1N (−29.1 ± 0.7‰) (Figures 3 and 5). δ 13CStarch_1N was significantly affected by blank correction (p < 0.001; δ13CBlank: −27.6 ± 0.6, n = 26), caused by low starch content in analysed samples. The correlation of starch concentration with blank corrected δ13CStarch_1N (r = 0.73, p < 0.05), but not with uncorrected δ13CStarch_1N, can be associated with the correction procedure and suggests overcorrection of the δ13CStarch_1N data. Starch concentra- tion was stable during the first 5 weeks of experiment, but then significantly declined (p < 0.01) on July 16, coinciding with the VPD maxima, and significantly increased (p < 0.01) week later. 3.4 | Enzymatic activity of needles The activity of the enzymes synthesizing sucrose or degrading it to glucose and fructose in needles was on average 3.1 times higher in 0 N than 1N (Figure 6). The enzyme activities did not correlate with δ13C or concentration series of the sugars or with the leaf gas exchange variables. Similarly, there were no significant correlations between the relative activities of the sucrose degrading enzymes, calculated as the difference between the activity of INVacid and SuSydegradation, and the offset between δ 13CSuc and δ 13CGlc. 3.5 | Intra‐annual tree‐ring δ13C variability In some of the specimens, the studied annual ring of 2018 contained compression wood, a type of reaction wood that is found in leaning conifer stems for tree support (Timell, 1986). These sections were used together with normal density wood for δ13C analysis, because their width enabled relatively high spatial resolution δ13C profiling, and since no 13C‐fractionation was observed in connection to the formation of compression wood (Supporting Information: Figure S3 and Method S3), in accordance with Walia et al. (2010) and Janecka et al. (2020). δ13CRing_RE profiles had a general increasing trend until June 5, followed by a decline that reached a distinct minimum by July 2 (Figure 2). This time period included the transition from EW to LW in tree‐ring composition. For the remaining period of the experiment, δ13C values had a general increasing trend. The overall low frequency trend in δ13CRing_RE profiles was similar to that observed for phloem δ13CSuc and δ 13CGlc series, which were fitted in Figure 2 using the common sampling days for phloem and tree‐rings (Supporting Information: Method S1) The average δ13C value of the July 2 and July 30 tree‐ring series was then calculated from Figure 2 for each phloem sampling day (Table 3). The average of all δ13C datapoints on TABLE 2 The abundance (%) of different carbohydrate compounds in the total water‐soluble carbohydrate (WSC) fraction in different organs of the Pinus sylvestris saplings, together with their average δ13C values. Abundance of WSCs (%) δ13C (‰) 0 N 1N Phloem Roots 0 N 1N Needles (0 N + 1N) Phloem Roots Sucrose 26 ± 11 17 ± 9 86 ± 27 31 ± 11 −29.5 ± 1.3 −28.3 ± 1.5 −29.0 ± 1.3 −28.3 ± 1.2 −27.3 ± 1.0 Glucose 24 ± 8 29 ± 7 16 ± 7 28 ± 8 −28.8 ± 1.2 −28.0 ± 0.9 −27.3 ± 1.1 −26.2 ± 1.1 Pinitol 62 ± 6 50 ± 10 20 ± 5 29 ± 5 −30.3 ± 0.5 −32.0 ± 0.2 −31.1 ± 0.4 −31.2 ± 0.3 WSC −29.3 ± 0.8 −29.1 ± 0.7 −29.2 ± 0.7 −29.1 ± 0.7 −27.8 ± 0.5 Note: The studied organs included current‐year needles (0 N), one‐year‐old needles (1 N), phloem and roots. The needle (0 N + 1N) δ13C value for sucrose and WSCs represents a mass balance calculation, based on the 1.5 times higher content of sucrose in 0 N than 1 N (see also Figure 6 for the correspondingly lower enzyme activity in 1 N). For glucose and pinitol, the needle (0 N + 1N) δ13C value was not calculated, because these compounds are not transported down‐stem from leaves and the values are hence irrelevant for the study. 10 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License EW was −27.2 ± 0.6‰, when calculated for each clone of July 2 and July 30 (Figure 2). The average of LW was −29.5 ± 0.45‰ for July 30 samples. When more than one segment had been measured for a clone, the average δ13C of a clone was first calculated before the calculation of the EW and LW average. The δ13C value of resin extracted EW and LW dissections of July 30 samples from EA‐IRMS analysis were −27.9 ± 0.9‰ and −29.9 ± 0.7‰, respectively, agreeing well with the calculated average δ13C values from LA‐IRMS analysis. Cellulose extracted from the same sections had δ13C values 1.2 ± 0.4‰ higher in comparison to resin extracted wood. 4 | DISCUSSION 4.1 | Environmental and physiological signal in δ13CSuc, δ13CGlc and δ13CWSC 4.1.1 | Leaves The observation that the temporal changes in leaf δ13CSuc and δ13CGlc recorded RH and gs, and were overall predicted by δ 13CP_calc (Table 1 and Figure 5) suggests a generally negligible role of old reserve use for these carbon pools. This is supported by the F IGURE 6 Activity of enzymes involved in sucrose formation and breakdown in current‐year (0 N) and 1‐year‐old (1 N) needles of Pinus sylvestris saplings. Each data point represents the average of four trees and the error bars its standard deviations (clones A, B, C and D). The temporal changes had clear similarities for the two needle generations, although the correlations were not statistically significant for these short datasets. The activities were higher in 0 N than 1 N: on average 3.3 times for neutral invertase, 5.4 times for acid invertase, 2.0 for sucrose‐ degrading sucrose synthase (SuSy) and 1.6 times for sucrose‐synthetizing SuSy. TABLE 3 The average δ13C value of the resin extracted tree‐rings of Pinus sylvestris saplings for each phloem sampling day, as calculated from Figure 2. June 5 June 11 June 18 June 25 July 2 July 9 July 16 July 23 July 30 −26.6 ± 0.7‰ −27.4 ± 1.0‰ −28.7 ± 1.1‰ −30.0 ± 0.8‰ −29.8 ± 0.6‰ −29.2 ± 0.6‰ −28.7 ± 1.0‰ −28.7 ± 0.9‰ −28.5 ± 0.8‰ DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 11 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License invariable and low starch δ13C in 1N (Figure 3). The environmentally driven δ13C of leaf sugars is in accordance with studies on mature L. gmelinii (Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015), Larix decidua (Sidorova et al., 2019) and P. sylvestris (Tang, Schiestl‐ Aalto, Lehmann, et al., 2022), where high resolution sampling during a growing season showed a significant climatic signal in δ13CSuc and δ13CGlc values with little or no sign of reserve use. In contrast, the climatic and physiological signal of δ13C from bulk leaf matter may be reduced or distorted, due to the high proportion of other compounds, such as pinitol (Table 2) or starch (Figure 3), with slow turnover rate and different metabolic history (Leppä et al., 2022; Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015; Streit et al., 2013). Similarly, Bögelein et al. (2019) reported absence of diel variation in leaf and twig phloem δ13CWSC of Douglas fir, which they assigned to large abundance of pinitol. However, the present study, which for the first time compared temporal changes in δ13CWSC with those in δ 13CSuc and δ13CGlc, suggests that δ 13CWSC can be an equally strong proxy record of intra‐seasonal environmental variability and tree physiology as δ13CP (Table 1), although with isotopic offset and smaller amplitude of variation due to the presence of pinitol (Table 2 and Figure 5). This finding is significant for future studies, considering the rarity of HPLC‐IRMS instruments for CSIA. Yet, the synchrony between δ13CWSC and δ 13CSuc may not be present for trees exposed to more significant environmental stress than encountered in this study, considering that an increase in synthesis of pinitol, for example due to drought (Ford, 1984; Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015), would cause a decline in δ13CWSC. The unconventional increase (vs. decrease) in leaf δ13C series with increase in ci observed in the July 16 sampling (Figure 5, ci with reversed y‐axis scale), the sampling day with theVPD maxima and the A minima (Figure 4), may be explained by utilization of carbohydrate reserves (Jäggi et al., 2002), as suggested by the significant decline in 1N starch concentration on July 16 (p > 0.01, Figure 3). However, the increase in δ13CGlc on July 16 is inconsistent with δ 13CStarch values, which may indicate transport of sucrose from down‐stem, such as twigs, to 1N at this time. Alternative, or complimentary mechanism, may be isotopic discrimination caused by mesophyll conductance, the diffusion of CO2 from the substomatal cavity to the carboxylation sites (Flexas et al., 2008). Mesophyll conductance has been shown to decline in response to water stress, leading to reduced 13C‐ discrimination during assimilation (Schiestl‐Aalto et al., 2021). 4.1.2 | Modification of δ13CSuc, δ 13CGlc and δ 13CWSC signal from needles to sink organs This first simultaneous comparison of δ13C data for individual sugars, together with WSCs, in leaves, phloem and roots at intra‐seasonal resolution allows us to explain some of the controversy in the literature concerning post‐photosynthetic fractionation processes. In the following, we will scrutinize the main possible mechanisms reported in the literature in light of this data set. Although results from sapling studies can be an oversimplification for mature trees growing in the field (Johnson & Ball, 1996), for example due to their smaller reserve pool size, it is this simplified experimental design that improves our ability to identify the main individual isotope fractionation processes. The temporal δ13C changes in needles were well preserved in sink organ δ13CSuc, δ 13CGlu and δ 13CWSC, However, sucrose became progressively 13C‐enriched from needles to phloem and roots, consistently throughout the experiment (Figures 5 and 7). The 13C‐ enrichment of sink organs relative to leaves has been commonly reported and the associated mechanisms widely debated (Cernusak et al., 2009; Gessler, Brandes, et al., 2009). Conventionally a bulk matter, such as WSCs or total organic matter, has been used for the δ13C analysis, which may complicate the interpretations on post‐ photosynthetic isotope fractionations, considering that each com- pound within the mixture can differ in its δ13C value due to their metabolic formation pathways having different C isotope effects (Tcherkez et al., 2011). Indeed, this was seen for needle δ13CWSC (Figure 5), which did not record the ~1.2‰ difference between sucrose in 1 N and sucrose in 0 N, because the offset was balanced by the ~1.7‰ lower δ13CPinitol_1N compared to δ 13CPinitol_0N (Table 2). Similarly, if only δ13CWSC had been analysed in the present study, it would not have been possible to observe the 13C‐enrichment of needle sugars along their transport route in phloem (Figure 7). This is because δ13C value and abundance of pinitol, constituent of WSCs, varied greatly between the organs (Table 2). CSIA studies on larch in Siberia (Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015; Rinne, Saurer, Kirdyanov, Loader, et al., 2015) proposed that the generally reported 13C‐ enrichment of sink organs relative to leaves is in large parts caused by the high abundance of 13C‐depleted pinitol in leaves and the INVacid induced 13C‐enrichment of leaf sucrose relative to hexoses (Gilbert et al., 2011; Mauve et al., 2009). However, the role of INVacid associated fractionation is inconsistent between the published studies: the level of 13C‐enrichment of sucrose over hexose is F IGURE 7 Average δ13C values and isotopic offsets (vertical numbers) between sucrose (S), glucose (G), water‐soluble carbohydrates (WSC) and tree rings of Pinus sylvestris saplings. δ13C of S, G and WSC were measured for needles, phloem and roots (Figure 5). S and G were measured with HPLC‐IRMS, WSC and wood α‐cellulose with EA‐IRMS, and resin extracted wood using laser ablation (Figure 2). 12 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License variable (e.g., Sidorova et al., 2018), even within a growing season (Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015), or not detectable (the present study). This inconsistency may indicate species‐specific differences (Dominguez & Niittylä, 2021), tree health status (Sidorova et al., 2018) or differences in the activity level of INVacid (Rinne, Saurer, Kirdyanov, Loader, et al. 2015). The present study provided the first opportunity to examine, if changes in the relative activity of INVacid and SuSydegradation in sucrose breakdown to hexoses can be used to explain temporal changes in the isotopic offset between sucrose and hexoses in leaves (Rinne, Saurer, Kirdyanov, Loader, et al., 2015), considering that no isotope fractionation is involved with activity of SuSydegradation (Gilbert et al., 2012). However, the lack of a significant correlation between the enzyme activities and δ13C values of the sugars indicates that the processes leading to a given isotopic offset is more complex and will be discussed in detail below. The 13C‐enrichment of leaf sucrose relative to leaf WSCs was not the only factor contributing to the 13C‐enrichment of sink organs relative to leaf WSCs in this study. The comparison of δ13CSuc of the studied organs demonstrates that during down‐stem transport of sucrose from leaves to roots, its carbon isotope composition has been progressively altered by one or several fractionating processes (Figure 5). Since needle δ13CSuc recorded environmental change (Section 4.1.1) and the temporal variability in this series was not dampened but instead preserved in phloem and root δ13CSuc series (Figure 5), it seems unlikely that reserve use, such as starch pools of phloem parenchyma, or mixing of sugar pools of different age (Brandes et al., 2006) could explain the isotopic offsets between the three organs. Further, 13C‐enrichment of phloem δ13CSuc relative to needle δ13CSuc cannot be ascribed to remobilization of transitory starch during the night (Gessler & Ferrio, 2022), because needle starch reserves (uncorrected: −28.9 ± 0.1‰, blank corrected: −30.3 ± 0.5‰) were overall not 13C‐enriched compared to needle sucrose (−28.3 ± 1.5‰) (Figure 3). The direction of the observed isotopic offset between starch and sugars is not in accordance with the general assumption about the relative 13C‐enrichment of starch over sucrose in leaves due to the aldolase reaction (Gleixner & Schmidt, 1997), but is in agreement with Lehmann et al. (2019), who did not detect an impact of the aldolase reaction on WSC δ13C when utilizing mutants of four nontree C3 species that lacked the enzyme phosphoglucomutase needed for starch production. These are cautionary findings for studies that link increases in δ13C of, for example, photosynthates or tree‐rings with reserve use without measuring starch δ13C. Furthermore, the 13C‐enrichment of phloem δ13CSuc relative to leaves should not be due to a higher proportion of sucrose from sunlit needles than that from sampled needles (Bögelein et al., 2019). This is because the trees were 1.4 m tall and their canopy evenly exposed to light, hence the sampled needles can be considered to be representative of the entire canopy (see Section 2.2). Also invertase activity in phloem, which was hypothe- sized in Mauve et al. (2009) to explain 13C‐enrichment of sucrose in sink organs relative to leaves, cannot fully explain our observations (Figure 5). This is because there is no primary isotope effect on glucose during invertase catalysis (Mauve et al., 2009) and, hence, the enzyme activity cannot explain the observed 13C‐enrichment of glucose relative to sucrose in phloem and roots. In contrast, the progressive 13C‐enrichment of sucrose along the pathway from leaves to phloem and roots, and the 13C‐enrichment of glucose relative to sucrose in sink organs, could be explained by fractionations in primary carbon metabolism [respiratory decarbox- ylations, pentose phosphate pathway (Bathellier et al., 2008) and phosphoenolpyruvatecarboxylase (PEPC) (Gessler, Tcherkez, et al., 2009)], leading to 13C‐enrichment of the remaining substrate (Cernusak et al., 2009; Ghashghaie et al., 2003; Gleixner et al., 1998). The impact of sink organ catabolism on δ13CSuc has been previously reported also for potato, where the 2‰ increase in tuber δ13CSuc over sprout or leaf sucrose was explained by this process (Gleixner et al., 1998; Maunoury‐Danger et al., 2009). In the present study, the increase in δ13CSuc was 0.7‰ from leaves to phloem (Figure 5) and a further 1.0‰ increase from phloem to roots (Figures 5 and 7). Although our results are consistent with fractionation reactions associated with CO2 production being the main cause of observed 13C‐enrichment in sink organ sucrose and glucose, other underlying processes may have contributed to the δ13CSuc offsets. The exclusion of 2‐year‐old needles from the present study probably overestimated the impact of phloem CO2 efflux on δ 13CSuc, considering that this needle generation likely had needle δ13CSuc similar to that in 1 N, in contrast with the metabolically active 0 N, which had relatively lower δ13CSuc values (Figures 5 and 6). However, this impact was likely small, because 0 and 1N are the dominant needle generations in P. sylvestris (80% of all needles in Kurkela et al., 2009) and because there is a major decline in physiological activity of conifer needles with age (Drenkhan et al., 2006; Robakowski & Bielinis, 2017). In the present study, the more significant role of 0 N relative to 1 N in sugar dynamics was suggested by the 1.5 times higher sucrose content in 0 N and by the 3.1 times higher activity of the studied enzymes responsible for sucrose synthesis and breakdown in 0 N (Figure 6). The fact that δ13CGlc was higher than δ 13CSuc in sink organs (Figure 5), which was statistically significant for roots and within standard deviations for phloem, may also be explained by respiration, considering that glucose is the substrate for glycolysis. The relative 13C‐enrichment in glucose (compared to sucrose) in sink organs could come from glucose 6‐phosphate consumption by catabolism, follow- ing glucose production by enzymatic sucrose cleavage. The impact of carbon loss by CO2 formation on δ 13CGlc can also explain why glucose was generally more 13C‐enriched than sucrose in sink organs but not in needles: whereas in sink organs the above discussed pentose phosphate pathway causes dark respired CO2 to be 13C‐ depleted relative to its substrate, in leaves the produced CO2 is assumed to be 13C‐enriched (Bathellier et al., 2017; Ghashghaie et al., 2003). Since glucose can be used by respiration, and that stem and root δ13CGlc recorded the environmentally driven (Table 1) changes in leaf δ13CSuc (Figure 5), it is possible that the δ 13C pattern in respired CO2 also followed fluctuations in photosynthetic 13C‐ discrimination (Gessler et al., 2007). DRIVERS OF INTRA‐SEASONAL δ13C SIGNAL IN TREE‐RINGS | 13 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Summarizing, our findings provide the missing evidence for earlier studies reporting sink organ 13C‐enrichment (Wingate et al., 2010) and could explain the observed correlations between sink organ respiration and environmental drivers (Barbour et al., 2005; Ekblad & Högberg, 2001). Future studies should, however, ascertain the role respiration on δ13C of sink organ substrates with simulta- neous measurements of δ13C of respired CO2 (Ghashghaie et al., 2003; Tcherkez et al., 2003). 4.2 | Intra‐annual tree‐ring δ13C signal 4.2.1 | Isotopic offset between leaf sugars and tree‐rings Considering that only low‐frequency trends (from 2 to 3 weeks) in climate and δ13CSuc can be preserved in tree‐rings due to the development period of a xylem cell (Gessler, Brandes, et al., 2009; Pérez‐de‐Lis et al., 2022), the modification of δ13C signal from needles to tree‐rings appears to have been relatively simple. Both temporal changes and absolute values in δ13CRing_RE series can be explained by δ13C of phloem sugars, which recorded ambient environmental conditions from leaf photosynthesis but with a systematic shift in isotope composition by fractionating reactions contributing to phloem CO2 efflux formation (Section 4.1.2). Our results indicate that the δ13CGlc of the pine saplings closely matched the δ 13C of cellulose (formed of glucose units), because the offset between resin extracted wood and glucose (1.0 ± 1.0‰, Figure 7) was very similar to the offset between resin extracted wood and cellulose (1.2 ± 0.4‰, Figure 7). Therefore, there was no evidence of the postulated isotope fractiona- tion occurring during xylem cell formation (δ13CGlc≈ δ 13CRing_Cellulose) due to, for example, activity of the fractionating INVacid (e.g., Panek & Waring, 1997; Rinne, Saurer, Kirdyanov, Loader, et al., 2015; Terwilliger et al., 2001). This finding suggests that SuSydegradation, the non‐fractionating enzyme, is responsible for providing substrate for the cellulose synthase complex, in line with Stein and Granot (2019). Evidence of 13C‐fractionating process during xylem formation are contradictory: none were found in Eucalyptus globulus between phloem sap and newly developing xylem tissue (Cernusak et al., 2005), but wholewood of Pinus halepensis was 1.4‰ 13C‐enriched compared to phloem sap (Gessler, Brandes, et al., 2009). We propose that such contradictive outcomes can be expected, when conducting δ13C analysis on a bulk matter of phloem, because: (i) phloem sap contains sugar alcohols and/or raffinose with a distinctive δ13C value (Bögelein et al., 2019; Merchant et al., 2010; Rinne, Saurer, Kirdyanov, Bryukhanova, et al., 2015), and the relative proportion of sucrose in phloem sap vary between sites and species, with for example, 80% for E. globulus in Australia (Merchant et al., 2010), 62% for Pinus pinaster in France (Devaux et al., 2009) and 50% for Citrus sinensis in another greenhouse experiment (Hijaz & Killiny, 2014); (ii) sucrose is degraded to hexoses for sink organ formation, with a systematic offset between sucrose and hexoses (Figure 5), probably due to fractionations in hexose metabolism (Section 4.1.2). This results in an apparent fractionation between phloem sucrose and wood cellulose even if enzymatic processes responsible for cellulose formation are non‐ fractionating. Thus, the analysis of phloem sap comes with uncertainties that do not allow exact estimation of isotope fractionation processes. This is clearly illustrated in Figure 7, where the isotopic offsets between δ13CRing and δ 13CWSC in the analysed organs are very deviant from those between δ13CRing and δ 13CSuc or δ 13CGlc. Consequently, without CSIA, it would not have been possible to, for example, detect the role of SuSydegradation in cellulose formation or the postulated impact of carbon loss in sink organs by CO2 formation on δ 13CRing. The observed 0.5‰ 13C‐enrichment of δ13CRing_RE relative to δ13CSuc_Needle is relevant for environmental and tree physiological reconstructions that are based on the absolute tree‐ring δ13C values, such as iWUE (Figure 7). Further, it is important to determine how this isotopic offset may vary between sites and species. The two other publications that have combined CSIA of leaf photosynthates with δ13C profiling of tree‐rings reported 1.0 and 0.9‰ higher values for δ13CRing_RE of mature L. gmelinii in central Siberia (Rinne, Saurer, Kirdyanov, Loader, et al., 2015) and mature P. sylvestris in southern Finland (Tang et al., 2023), respectively. There are several factors to consider, when evaluating such offsets in δ13C between studies. (i) There may be species‐ and site‐specific differences in INVacid‐induced fractionation, as discussed in Section 4.1. By removing the impact of INVacid on δ 13CSuc_Needle in Rinne, Saurer, Kirdyanov, Loader, et al. (2015), the difference between δ13CRing_RE and δ 13CSuc_Needle is reduced to 0.4‰, which is similar to our finding (Figure 7). (ii) The magnitude of CO2 efflux associated isotope fractionation along the pathway from leaves to stem xylem could increase with tree height, potentially leading to a bigger isotopic offset between leaf sugars and tree‐rings for mature trees, for example inTang et al. (2023). (iii) When using mature trees for studying post‐photosynthetic 13C‐fractionation, it can be challenging to obtain canopy‐representative CSIA results, considering that δ13CP is dependent on the incident solar energy received by the leaves (Bögelein et al., 2019; Schleser, 1989). Whereas this could affect the results of Tang et al. (2023), where needles were collected from the upper part of a dense canopy, it probably did not impact the finding of Rinne, Saurer, Kirdyanov, Loader, et al. (2015), where the canopies were small and tree density low, providing more or less equal light levels for needles around the canopy. Our study thus shows that the estimation of absolute values of iWUE derived from δ13CRing are still hampered by difficulties in our understanding of isotope fractionation processes between plant assimilate and ring material. The measured 13C‐enrichment of resin extracted wood relative to cellulose by 1.2‰ (Figure 7) is smaller than the 1.8 and 2‰ reported for conifers L. gmelinii and Pseudotsuga menziexii, respec- tively (Livingston & Spittlehouse, 1996; Sidorova et al., 2010). However, our results may not be directly comparable to the previous findings due to differences in chemical extraction procedures of the analysed wood. The possible reasons are (i) the duration of resin extraction, which was shorter or not reported in the previous studies, and (ii) removal of hemicelluloses during α‐cellulose extraction, considering that this step was not done in Livingston and Spittlehouse (1996) and Sidorova et al. (2010). Resins are up to 14 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6‰ depleted relative to cellulose (Schmidt & Gleixner, 1998) and hemicelluloses isotopically differ from α‐cellulose (Boettger et al., 2007), and, therefore, the presence of these substrates in Livingston and Spittlehouse (1996) and Sidorova et al. (2010) could explain the differences between our results. Another potential explanator is a difference in lignin to cellulose content ratio in the analysed samples, considering that lignin is relatively 13C‐depleted (Bowling et al., 2008) and its abundance is not constant between gymnosperms (Ana & Helena, 2017). 4.2.2 | Environmental signal in EW δ13C There was no observable evidence for use of reserves for tree‐ring growth under the studied conditions, characterized by relatively high ambient T and VPD levels, which sometimes limited gas exchange (Figure 4) and which are the likely reason for the narrow LW bands formed in 2018 (Zweifel et al., 2021). The low frequency trends and absolute values of the intra‐annual δ13CRing_RE series can be explained by the phloem sugar δ13C values (Figure 2, section 4.3.1.1), which recorded changes in ci and the subsequent post‐photosynthetic isotope fractionations caused by CO2 production (Section 4.1). For the EW section formed before June 5 (Figure 2), there is no sugar data available for a similar evaluation, however, the initial increase in EW δ13C can be explained by the acclimation of the saplings to the warmer conditions in greenhouse after their transport from the field on May 17 (Tdiff = 7°C). Hence, we propose/confirm, as hypothesized inMonson et al. (2018) and indicated inTang et al. (2023), that it may not be necessary to discard the EW section of δ13C chronologies for tree physiological and paleoenvironmental studies, when studying conifers, such as Pinus (this study) and Larix (Rinne, Saurer, Kirdyanov, Loader, et al., 2015), in boreal forests under non‐stressed to mildly stressed conditions. Apart from providing valuable information about the processes occurring during the early part of the growing season, this outcome would also give further confidence for the interpretation of δ13C chronologies constructed using the whole annual ring, for example, because of the presence of too narrow LW sections for manual dissection (Vitali et al., 2022). 5 | CONCLUSIONS Our δ13C data set, which combines compound‐specific sugar data obtained from leaves, phloem and roots at weekly resolution with high spatial resolution tree‐ring series of P. sylvestris, provided a close insight into the metabolic processes that occur and modify a δ13CP before the isotopic record is deposited in tree‐rings. The observations indicated a relatively simple pattern, where the progressive 13C‐ enrichment of δ13CP from needles via phloem to roots is perhaps explained by sink organ catabolic CO2 loss (question 2 in Introduction). There was no evidence of phloem loading distorting the δ13CP signal (question 1). The observation of lower δ13CP relative to δ 13CRing_RE and δ13CRing_Cellulose are important for studies using δ 13CRing for, for example, iWUE reconstructions (question 3). The significant environmental signal recorded in δ13CP was preserved in intra‐ annual δ13CRing at low frequency scale, demonstrating negligible role of reserve use for EW or LW formation for these saplings (question 2). This is promising also for studies aiming for high‐resolution, seasonally resolved climate reconstructions, at least when using pine species in boreal regions. The comparison of intra‐seasonal δ13CWSC and δ 13CP signals in different organs demonstrated that a bulk organic matter is unreliable proxy record for studies of post‐photosynthetic isotope fractionation, but that δ13CWSC may perform equally well as δ 13C of individual sugars for reconstructions of intra‐seasonal environmental variability and tree physiology. The outcomes of this study have relevance for the utilization of tree‐rings as a proxy of environmental change and for studies interested in δ13C of sink organ respiration and its environmental drivers (Barbour et al., 2005; Ekblad & Högberg, 2001). ACKNOWLEDGEMENTS We would like to thank Juhani Pyykkö, Iman Karmoun, Aino Seppänen, Kira Ryhti and Kaarina Pynnönen for their valuable fieldwork assistance, and Aino Seppänen, Fana Gizaw, Marine Manche for efforts in sample preparation. We are grateful to Manuela Oettli for the HPLC‐IRMS analysis. We would like to thank the Associate Editor and three anonymous reviewers for constructive comments. This study was financially supported by the European Research Council (755865), Academy of Finland (295319, 323843) and Swiss National Science Foundation (179978, 207360). DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Katja T. 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SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: Rinne‐Garmston, K.T., Tang, Y., Sahlstedt, E., Adamczyk, B., Saurer, M., Salmon, Y. et al. (2023) Drivers of intra‐seasonal δ13C signal in tree‐rings of Pinus sylvestris as indicated by compound‐specific and laser ablation isotope analysis. Plant, Cell & Environment, 1–18. https://doi.org/10.1111/pce.14636 18 | RINNE‐GARMSTON ET AL. 13653040, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/pce.14636 by D uodecim M edical Publications Ltd, W iley O nline Library on [18/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License