Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potato plants

Journal of Experimental Botany, Vol. 66, No. 19 pp. 5769–5781, 2015 doi:10.1093/jxb/erv279 Advance Access publication 2 July 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potatoplants Marco M.Lehmann1,2,*, Katja T.Rinne1, CarolaBlessing2, Rolf T. W.Siegwolf1, NinaBuchmann2 and Roland A.Werner2 1 Laboratory of Atmospheric Chemistry, Paul Scherrer Institute (PSI), CH-5232 Villigen, Switzerland 2 Institute of Agricultural Sciences, ETH Zurich, Universitaetsstr. 2, CH-8092 Zurich, Switzerland * To whom correspondence should be addressed. E-mail: mar[email protected] Received 28 January 2015; Revised 30 April 2015; Accepted 6 May 2015 Editor: Howard Griffiths Abstract Dissimilation of carbon sources during plant respiration in support of metabolic processes results in the continuous release of CO2. The carbon isotopic composition of leaf dark-respired CO2 (i.e. δ13CR) shows daily enrichments up to 14.8‰ under different environmental conditions. However, the reasons for this 13C enrichment in leaf darkrespired CO2 are not fully understood, since daily changes in δ13C of putative leaf respiratory carbon sources (δ13CRS) are not yet clear. Thus, we exposed potato plants (Solanum tuberosum) to different temperature and soil moisture treatments. We determined δ13CR with an in-tube incubation technique and δ13CRS with compound-specific isotope analysis during a daily cycle. The highest δ13CRS values were found in the organic acid malate under different environmental conditions, showing less negative values compared to δ13CR (up to 5.2‰) and compared to δ13CRS of soluble carbohydrates, citrate and starch (up to 8.8‰). Moreover, linear relationships between δ13CR and δ13CRS among different putative carbon sources were strongest for malate during daytime (r2=0.69, P≤0.001) and nighttime (r2=0.36, P≤0.001) under all environmental conditions. Amultiple linear regression analysis revealed δ13CRS of malate as the most important carbon source influencing δ13CR. Thus, our results strongly indicate malate as a key carbon source of 13C enriched dark-respired CO2 in potato plants, probably driven by an anapleurotic flux replenishing intermediates of the Krebs cycle. Key words: Compound-specific isotope analysis (CSIA), drought, organic acids, plant respiration, stable carbon isotopes, sugars, temperature, tricarboxylic acid (TCA) cycle. Introduction The investigation of plant respiration as a major process in plant biochemistry has expanded our understanding of carbon cycling in autotrophic organisms. Plants dissimilate carbon sources for the production of intermediates and reducing equivalents in support of metabolic processes, thereby continuously releasing CO2 via plant respiration (Hopkins, 2006). Leaf-respired CO2 is mainly derived from oxidative decarboxylation reactions catalysed by enzymes from the Krebs cycle This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. Abbreviations: δ13CR, carbon isotopic composition of leaf dark-respired CO2; δ13CRS, carbon isotopic composition of putative leaf respiratory carbon sources; An, net assimilation rate; Ci, intercellular CO2 concentration; CSIA, compound-specific isotope analysis; gs, stomatal conductance; HPLC, high performance liquid chromatography; KC, Krebs cycle; LEDR, light-enhanced dark respiration; ME, malic enzyme; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; SPS, sucrose phosphate synthase; SWC, volumetric soil water content. at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from 5770 | Lehmann etal. (KC) and from interacting anabolic and catabolic reactions (Voet and Voet, 2011). Using stable isotopes, the pathway of carbon can be traced from photosynthetic carbon fixation to respiratory carbon loss. On the one hand, C3 plants discriminate heavily against 13C due to photosynthetic isotope fractionation, leading to general 13C depletion in plant biomass of about 20‰ in comparison to atmospheric CO2 (Farquhar etal., 1989). The exact magnitude of photosynthetic carbon isotope discrimination depends on the intercellular CO2 concentration (Ci) in the substomatal cavity, which is regulated by other physiological parameters such as net assimilation rate (An) and stomatal conductance (gs). Environmental conditions such as light, temperature, soil moisture, and air humidity will influence these parameters and with them the photosynthetic carbon isotope discrimination. On the other hand, the carbon isotopic composition of leaf dark-respired CO2 (i.e. δ13CR) has clearly been shown to be less negative than leaf metabolites in several plant species (Ghashghaie etal., 2003; Bowling etal., 2008; Werner and Gessler, 2011; Ghashghaie and Badeck, 2014). In a daily cycle, leaf dark-respired CO2 follows a progressive 13C enrichment during the day and a gradual 13C depletion during the course of the night (Hymus etal., 2005; Prater etal., 2006), resulting in a strong temporal variability of up to 14.8‰ (Barbour et al., 2007; Werner et al., 2009; Wegener etal., 2010), which differs among functional groups (Priault etal., 2009; Werner etal., 2009). δ13CR is thereby linked to the carbon isotopic composition of putative leaf respiratory carbon sources (i.e. δ13CRS) such as carbohydrates (soluble monoand di-saccharides, and starch) and organic acids. Previous studies showed that environmental drivers such as temperature and soil moisture influence δ13CR and δ13CRS. More negative δ13CR values with increasing temperature have been observed with short-term changes in leaf temperature during darkness in Phaseolus vulgaris (Tcherkez et al., 2003), while long-term effects of higher temperatures on δ13CR and δ13CRS have not yet been investigated under controlled conditions. Other studies have demonstrated less negative δ13CR and δ13CRS values under dry conditions compared to those under wet conditions (Duranceau et al., 1999; Ghashghaie et al., 2001). Similar observations were made in field experiments (Sun etal., 2009; Dubbert etal., 2012). Conversely, more negative δ13CR values have been found under dry conditions for Mediterranean trees and herbs such as Quercus ilex and Tuberaria guttata compared to those under wet conditions (Unger etal., 2010), which have been explained with accompanied increases in temperatures and vapour pressure deficit. Nevertheless, the combined effects of temperature and soil moisture on δ13CR and δ13CRS under controlled conditions have yet to be tested. Moreover, δ13CR is determined by various post-photosynthetic carbon isotope fractionation processes at pivotal branching points in respiratory pathways, carbon isotope effects on enzymatic reactions, and changes in respiratory substrates (for a detailed review see Werner and Gessler, 2011). The 13C enrichment in leaf dark-respired CO2 itself is thought to be a result of fragmentation fractionation processes based on heterogeneous intramolecular carbon isotope distribution in respiratory carbon sources (Tcherkez et al., 2004). For instance, C-3 and C-4 positions of glucose are known to be enriched in 13C compared to the other molecule positions due to an isotope effect of the aldolase reaction (Rossmann et al., 1991; Gleixner and Schmidt, 1997). Breakdown of glucose during glycolysis produces pyruvate with a 13C enriched C-1 position (former C-3 and C-4 positions of glucose). Thereafter, the pyruvate dehydrogenase reaction (PDH) releases the C-1 position as 13C enriched CO2, whereas the more 13C depleted acetyl-CoA residue is used in the KC (Priault et al., 2009; Werner and Gessler, 2011). Thus, a PDH dominated respiratory pathway may lead to 13C enrichment in leaf dark-respired CO2. However, the knowledge about δ13CR is often based on light-acclimated leaves, which have been transferred into darkness to allow respiratory measurements. This approach holds an unpreventable bias known as ‘light-enhanced dark respiration’ (LEDR), which needs to be taken into account when interpreting daytime δ13CR values. LEDR is a shortterm light-dark transition period, describing an increase in the amount of leaf dark-respired CO2 shortly upon darkening for about 20 min, which depends on light intensity (Atkin etal., 1998). On the one hand, LEDR may be influenced by reassembly of the KC, which is thought to be only partially active under light conditions (Tcherkez etal., 2005; Sweetlove et al., 2010; Werner and Gessler, 2011; Werner etal., 2011). On the other hand, LEDR may be driven by a breakdown of a light-accumulated malate pool, causing 13C-enriched leaf dark-respired CO2 (Barbour etal., 2007; Gessler etal., 2009; Werner etal., 2009; Barbour etal., 2011; Werner and Gessler, 2011). Malate itself is also known to be 13C enriched compared to other carbon sources (Gleixner etal., 1998; Ghashghaie et al., 2001). The 13C enrichment in malate was attributed to an anapleurotic flux via the phosphoenolpyruvate carboxylase reaction (PEPC), which fixes 13C-enriched hydrogen carbonate and replenishes KC intermediates (Melzer and O’Leary, 1987; Savidge and Blair, 2004). Thus, a possible breakdown of malate by the mitochondrial malic enzyme reaction, or within the KC, may influence δ13CR (Barbour etal., 2007; Werner etal., 2011). In addition, plants may also use to a certain extent more complex carbon sources such as lipids and proteins under severe environmental conditions or under prolonged darkness (Tcherkez et al., 2003; Usadel et al., 2008). However, the driving processes, the respiratory carbon sources, and the mechanisms causing changes in δ13CR during day and night are not fully resolved thusfar. Hence, with this study we intend to assess two major research questions. What causes the high daily variations in δ13CR? How are δ13CR and δ13CRS influenced by temperature and soil moisture conditions? Our main objectives were (i) to analyse the relationship between δ13CR and δ13CRS values and (ii) to determine changes in δ13CR and δ13CRS values, as well as in concentrations of the putative carbon sources under different environmental conditions. Therefore, we exposed potato plants to different controlled temperature and soil moisture conditions and measured δ13CR with an in-tube incubation technique, as well as δ13CRS and concentrations of soluble at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from Malate as a key carbon source of leaf dark-respired CO2 in potato | 5771 carbohydrates, organic acids and starch from leaves with compound specific isotope analysis (CSIA) on a daily basis. Materials and methods Plant material Potato plants (Solanum tuberosum L. cv. Annabell) were grown from tubers of the same size in 5 l pots filled with bark humus soil (Ökohum, Herrenhof, Switzerland) in a greenhouse, with average temperatures of 20/16°C and vapour pressure deficits (VPD) of about 0.9/0.4 kPa (day/night). The plants were exposed to a 16 h daylight period supplemented by 400 W sodium-lamps (Powertone Son-T Plus, Philips, Amsterdam, Netherlands). Forty days after planting, plants were transferred into walk-in climate chambers for acclimatization for 2 weeks. The 16 h daylight in the climate chambers had an averaged photosynthetic photon flux density of ~400µmol m-2 s-1 at leaf level, thus plants were not fully light-saturated. Before the treatment period, soil water status was optimal for at least 3 d after watering, while an individual plant consumed about 300 ml water per day. 50 ml of a 0.4% fertilizer solution (v/v, Gesal, Zürich, Switzerland) was applied twice to all plants during the whole experiment of 70d. Treatments were applied during the last 15 d of the experiment. Plants were exposed to high temperature (Thigh) of 28/23°C (day/ night) and low temperature conditions (Tlow) of 22/17°C, at a VPD of about 0.9/0.35 kPa for both temperature treatments. Three climate chambers were used for replication of each temperature treatment. Within each climate chamber there were two soil-moisture treatments with nine plants each. Dry soil moisture conditions were kept constantly at 50–60% of the daily water consumption of each individual plant, determined by weighing the entire pots. Plants under wet conditions were kept at100%. The final sampling period lasted 32 h during the last 2 d of the experiment, when dry soil conditions were established for both temperature treatments. Sampling was done on a daily basis every 2 h (nighttime) or 4 h (daytime). During sampling, individual plants had 3–6 ranks, with about four fully developed leaves per rank. Always the third-last fully developed leaf per rank was sampled at all points in time, but within 24 h only one sample was taken from each individual plant to avoid any stress response induced by sampling. Sampled leaf material was immediately frozen in liquid nitrogen and stored at −80°C. Subsequently, the leaf material was freeze-dried and milled to powder by a steel ball mill (MM200, Retsch, Haan, Germany) for all further isotopic and biochemical analyses. In addition to leaf sampling, air CO2 samples from all six climate chambers were collected at the same points in time during the sampling period, showing a mean δ13C value of −12.2‰ and typical daily variations of SD ≤1.4‰; no differences between temperature treatments (P≥0.05) and points in time (P≥0.05; linear mixed effects model) were observed during the daily cycle. Physiological measurements and biomass determination Several leaf physiological parameters were determined with an infrared gas analyser (LI-6400, LI-COR, Lincoln, Nebraska, USA), including net assimilation rate (An), intercellular CO2 concentration (Ci), and stomatal conductance (gs). All measurements were taken in the last 4 h of the daylight phase. To monitor volumetric soil water content (SWC), up to three soil moisture sensors (EC-5 and logger Em5b, Decagon Devices, Pullman, USA) were installed for each treatment. Shortly after the sampling period, total plant biomass was harvested, oven-dried (at 60°C), and weighed. The fresh tuber weight and tuber count (number of potatoes) were determined. Carbon isotope and concentration analyses δ13C values are expressed as described by Craig (1957) and modified by Coplen (2011): δ 13 sample standard C RR 1 ‰/ () =− where Rsample is the 13C/12C ratio of the sample material and Rstandard is that of the international standard VPDB (Vienna Pee Dee Belemnite). Determination of δ13CR The in-tube incubation technique was used for the collection of leaf dark-respired CO2 during daytime and nighttime (Werner et al., 2007). A leaf was placed in a 12 ml gas-tight exetainer (Labco, Lampeter, UK), which was immediately darkened with a lightproof casing to trigger leaf dark respiration. The tube was then flushed for 1 min with synthetic air until a CO2-free atmosphere was established, which was monitored with an infrared gas analyser (LI-6262, LI-COR, Lincoln, Nebraska, USA). After an incubation time of 3 min in darkness, an aliquot of dark-respired CO2 was transferred with a gas-tight syringe into a new exetainer filled with dry N2. δ13CR values were determined with an IRMS, using a modified Gasbench II (Thermo Fisher, Bremen, Germany) connected to a DeltaplusXPIRMS, similar to Zeeman etal. (2008). The transfer of the CO2 sample into a new exetainer, as well as the IRMS measuring procedure, were both tested with air of known δ13C of CO2 to ensure no isotope fractionation had occurred. Measurement precision of a quality control standard (three standards per 24 samples) was SD≤0.1‰. Determination of δ13C in bulk leaves and leafstarch Extraction of leaf starch was performed as described in previous studies (Wanek etal., 2001; Goettlicher etal., 2006; Richter etal., 2009). Leaf starch was isolated from 50 mg leaf material with methanol/ chloroform/water (MCW, 12:5:3, v/v/v) at 70°C for 30 min. Samples were centrifuged (10 000 ×g, 2 min) and supernatants removed, while the leaf-starch-containing pellets were washed with MCW and deionized water and dried at room temperature (RT). Pellets were then re-suspended in water and boiled at 99°C for 15 min to facilitate starch gelatinization. Subsequently, leaf starch was enzymatically digested with α-amylase (EC 3.2.1.1, Sigma-Aldrich, Buchs, Switzerland) at 85°C for 2 h, and cleaned with centrifugation filters to remove enzymes (Vivaspin, Sartorius, Göttingen, Germany). To determine δ13C of bulk leaves (δ13Cleaf) and starch, an elemental analyser (Flash EA 1112 Series) coupled to a DeltaplusXP-IRMS was used (both Thermo Fisher, Bremen, Germany; Werner etal., 1999). Measurements of samples, blanks, and reference material followed the identical treatment principle described by Werner and Brand (2001). The long-term precision of a quality control standard for all sequences was SD≤0.12‰. Isotopic and concentration analysis of soluble carbohydrates and organicacids Water-soluble compounds were extracted from 100 mg leaf material with water at 85°C for 30 min, similar to Streit et al. (2013). Subsequently, soluble carbohydrates and organic acids were separated by ion-exchange chromatography (Wanek et al., 2001; Goettlicher etal., 2006; Richter etal., 2009), using Dowex 50WX8 in H+-form and Dowex 1X8 in NaCOO--form (both 100–200 mesh, Sigma-Aldrich, Buchs, Switzerland). To avoid clogging of the HPLC column by polyphenols, all samples designated for carbohydrate analyses were filtered with 100 mg Sep-Pak C18 Vac RC Cartridges (Waters AG, Milford, Massachusetts, USA). Finally, all carbohydrate and organic acid samples were cleaned with 0.45µm PTFE syringe filter (Infochroma AG, Zug, Switzerland) prior to HPLC measurements. To determine δ13CRS values and the concentrations of soluble carbohydrates and organic acids, a HPLC-IRMS system consisting of a high performance liquid chromatograph coupled to a Delta V Advantage IRMS by a LC IsoLink (all Thermo Fisher, Bremen, Germany) at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from 5772 | Lehmann etal. was used according to Krummen etal. (2004). Carbohydrates were separated on a 3 × 150 mm anion-exchange column CarboPac PA20 (Dionex, Olten, Switzerland) using 2 mM NaOH as the mobile phase and a flow speed of 250µl min-1 (Boschker etal., 2008; Rinne etal., 2012). Low column temperature of 20°C was used to prevent isomerization of hexoses (Rinne etal., 2012). This enabled chromatographic separation for sucrose and glucose, but fructose δ13CRS and concentration measurements were affected by partial co-elution of fructose with other compounds. To correct δ13CRS values and to calculate concentrations from the peak areas, interspersed standard solutions in a concentration range of 20–180 ng C µl-1 were measured within each sequence. The measurement precision of δ13CRS values in all carbohydrate standards was SD<0.5‰. Below a concentration of 60 ng C µl-1, the precision of fructose standards was lower for certain batches, and therefore these results were excluded. Organic acids were separated on a 4.6 × 300 mm Allure Organic Acids column (Restek, Bellefonte, USA) at 5–10°C. The mobile phase was a 100 mM monopotassium phosphate buffer (pH 3)with a flow speed of 500μl min-1 (Hettmann etal., 2005). The measurement precision of δ13C in organic acid standards was SD<0.4‰. Low citrate concentrations from Tlow samples (<45 ng C µl-1) impeded the analytical accuracy of the δ13CRS values, therefore these samples were not taken into account. All purification steps were verified for each batch of 24 samples using 2.5 mg standard solutions of known δ13C (by EA-IRMS) for all carbohydrates and organic acids measured in this study. Differences between δ13C values before and after purification were generally ≤0.2‰, indicating no significant isotope fractionation for any standard. Mean recovery was 101 ± 6% for fructose, 96 ± 6% for glucose, 89 ± 3% for sucrose, 91 ± 3% for malate, and 86 ± 3% for citrate. Determination of starch concentration For the extraction of leaf starch for concentration analyses we used a modified method of Critchley etal. (2001). Leaf starch was isolated with 1.12 M perchloric acid from 50 mg leaf material at RT for 15 min and centrifuged (10 min, 3000×g, 4°C). The supernatant was removed and the leaf-starch-containing pellet was washed free from pigments with deionized water and ethanol. Pellets were then dried at RT, resuspended in water, and gelatinized. Subsequently, starch samples were enzymatically hydrolysed to glucose for 2 h at 37°C with a solution mix of α-amylase (EC 3.2.1.1, Sigma-Aldrich, Buchs, Switzerland) and α-amyloglucosidase (EC 3.2.1.3, Roche, Rotkreuz, Switzerland) in 220 mM sodium acetate buffer (pH 4.8). The glucose concentration was determined at 340 nm with a 96-well microplate reader (EL×800, BioTek, Luzern, Switzerland) using a coupled enzymatic reaction (Hoch etal., 2002). Potato starch was used as a standard. Glucose concentrations are expressed in molarity of starch monomers. Data analysis R version 3.0.2 (R Core Team, 2013) was used for (multiple) linear regression analyses and linear mixed effects models (R package nlme). Models included fixed effects (temperature, soil moisture, sampling time) and random effects (climate chambers, individual plants). If applicable, δ13C values and concentrations were logarithmically transformed to ensure normal distribution. For the best-fit combination of the multiple linear regression analysis, variables were excluded if P≥0.05. Results Physiological parameters and biomass Physiological parameters (An, Ci, gs, and SWC) of potato plants exposed to four different treatments were monitored during the treatment period of 15 d (Fig.1). The net assimilation rate declined during the treatment period under all four treatments (Fig.1A). During the sampling period (Fig.1A, day 15), An was significantly influenced by soil moisture (P=0.02, Table1), with lowest values (1.9µmol m-2 s-1) under Thigh and dry conditions, and highest values (5.4µmol m-2 s-1) under Tlow and wet conditions, whereas the temperature influence on An was not significant (P=0.07, Table1) but tended to cause lower An values under Thigh than under Tlow under both soil moisture conditions. The intercellular CO2 concentration increased during the treatment period for all four treatments (Fig.1B). During the sampling period (Fig.1B, day 15), Ci was independently influenced by temperature (P=0.012, Table1) and soil moisture (P=0.01, Table1), with lowest Ci (247.5µmol mol-1) under Tlow and dry conditions and highest Ci (332.8µmol mol-1) under Thigh and wet conditions. Stomatal conductance during the treatment period was lower under dry treatments compared to those under wet treatments (Fig.1C). During the sampling period (Fig.1C, day 15), gs was significantly influenced by soil moisture (P≤0.001, Table1), with lowest gs (about 0.06 mol m-2 s-1) in plants of both dry treatments and highest gs (0.22 mol m-2 s-1) in plants under Thigh and wet conditions, whereas the temperature influence under wet conditions tended to cause higher gs values under Thigh than under Tlow. The volumetric soil water content was lower under dry conditions (~7–14%) compared to wet conditions (23–27.5%) for the last 9 d of the treatment period (Fig.1D), including the sampling period (Fig.1D, day 15), where SWC was significantly affected only by soil moisture treatments (P=0.002, Table1). Generally, no significant interactions between temperature and soil moisture were observed for any parameter (Table1). In addition, only soil moisture treatments affected plant biomass (P=0.008, Table1) and tuber weight (P=0.023, Table 1) taken shortly after the sampling period, independent of temperature treatments. Highest values tended to be under Tlow and wet conditions and lowest values under Thigh and dry conditions (Tables 1, 2), indicating different stress levels created by the four treatments. Carbon isotopes in potatoleaves Daily cycles of δ13CR and δ13Cleaf δ13C values of leaf dark-respired CO2 (δ13CR) varied significantly over time (P≤0.001, Table3) with values in the range of −21.9‰ and −32‰, declining strongly during nighttime and increasing again during the daytime for all four treatments (Fig.2A). An interaction between temperature and time showed that the influence of temperature differed with time (P=0.014, Table3). Daytime δ13CR values under Thigh were up to 4.7‰ more negative compared to those under Tlow, independent of soil moisture conditions, whereas nighttime δ13CR values of both temperature treatments were very similar, particularly in the second night. Dry soil moisture conditions caused less negative δ13CR values compared to those under wet conditions during the daily cycle (P=0.013, Table3), with a maximum difference of 2.7‰, independent of temperature treatments. On average, the difference between daytime and nighttime δ13CR values was highest under Tlow and wet conditions, at 5.7‰, and lowest under Thigh and dry conditions, at 2.5‰. at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from Malate as a key carbon source of leaf dark-respired CO2 in potato | 5773 The bulk leaf material reflects all environmental conditions experienced during the whole growth period. δ13Cleaf of all treatments showed no changes during the sampling period and no interactions between treatments and time (Fig.2B; Table3). Under Thigh, δ13Cleaf values were up to 2.2‰ more negative compared to those under Tlow, resulting in a significant temperature effect independent of soil moisture conditions (P=0.022, Table3). Similarly, soil moisture showed a significant effect on δ13Cleaf (P=0.005, Table 3), independent of temperature treatments, with values up to 1.1‰ less negative under dry than under wet conditions mainly during nighttime. δ13CRS of soluble carbohydrates, organic acids, andstarch Highest δ13C values in putative leaf respiratory carbon sources (δ13CRS) were found in the organic acid malate, while ● ● ● ● ● ● ● ● ● ● ● ● 0 2 4 6 8 10 (A) An (µmol m − 2 s − 1) ●● ● ● ●● ● ● ● ●● ● 150 200 250 300 350 (B) Ci (µmol mol − 1) ●●● ● ●●● ● ● ● ● ● 0.00 0.05 0.10 0.15 0.20 0.25 (C) gs (mol m − 2 s − 1) ● ●● ●● ● ● ● ●● ●●●● ● ● ● ●● ●● ● ● ● ●● ●●●● ● ● ● ● ●● ● ● ●●● ● ●● ● ● ●● 036912 15 0.0 0.1 0.2 0.3 ●Tlow Wet Tlow Dry Thigh Wet Thigh Dry (D) SWC (m3 m−3) Treatment period (d) Fig.1. Physiological parameters under different environmental conditions during the treatment period: (A) net assimilation rate (An, µmol m-2 s-1), (B) intercellular CO2 concentration (Ci, µmol mol-1), (C) stomatal conductance (gs, mol m-2 s-1), (D) volumetric soil water content (SWC, m3/m-3). Potato plants were treated with a combination of Tlow (low temperature; closed symbols), Thigh (high temperature; open symbols), and wet (circles) or dry (triangles) conditions. Boxed areas indicate the sampling period. Means ±SE are given (n=3). Table1. Environmental influences on physiological parameters Results of linear mixed effects models testing the effects of temperature (low, high) and soil moisture (wet, dry) on physiological parameters (An, net assimilation rate; Ci, intercellular CO2 concentration; gs, stomatal conductance; SWC, volumetric soil water content),total plant biomass, tuber weight, and tuber count during the sampling period. P-values are given for treatments and their interaction. Significant differences are given in bold (P≤0.05). Parameter AnCigsSWC Plant biomass Tuber weight Tuber count Temperature 0.070 0.012 0.127 0.863 0.978 0.359 0.400 Soil moisture 0.020 0.010 0.001 0.002 0.008 0.023 0.233 Temp.:moisture 0.522 0.110 0.174 0.845 0.565 0.892 0.486 ● ● ● ● ● ●● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ●●● ● ● ●● ● ● ●● ● ●● ● ● −34 −32 −30 −28 −26 −24 −22 −20 (A) δ13CR (‰) ●● ●● ●●● ● ●●● ● ● ●● ●● ●●● ● ●●● ● ● ● ● ●● ●● ● ● ● ●●●● 048121620242 83 2 −34 −32 −30 −28 −26 −24 −22 −20 (B) Sampling period (h) δ13Cleaf (‰) ●Tlow Wet Tlow Dry Thigh Wet Thigh Dry Fig.2. Daily cycles of the carbon isotopic composition of (A) leaf dark-respired CO2 (δ13CR) and (B) bulk leaves (δ13Cleaf) under different environmental conditions during the sampling period. Potato plants were treated with a combination of Tlow (low temperature; closed symbols), Thigh (high temperature; open symbols), and wet (circles) or dry (triangles) conditions. Grey areas indicate nighttime. Means ±SE are given (n=3). at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from 5774 | Lehmann etal. soluble carbohydrates (fructose, glucose and sucrose) exhibited generally lowest δ13CRS values (Fig.3). δ13CRS of soluble carbohydrates of all treatments were in the range of −27.2‰ and −36.6‰. More negative δ13CRS values of glucose and sucrose under Thigh compared to those under Tlow were found, independent of soil moisture conditions, while less negative δ13CRS values under dry conditions compared to those under wet conditions were observed, independent of temperature treatments (Fig.3B, C; Table3). Significant interactions between temperature and time for δ13CRS of glucose (P=0.008, Table3) and sucrose (P=0.003, Table3) showed that daily cycles differed between temperatures. Additionally, soil moisture conditions caused significant temporal variations during the daily cycle in δ13CRS of sucrose (P=0.002, Table3). We observed significant linear relationships between fructose and glucose for δ13CRS (r2=0.74, P≤0.001) and concentration values (r2=0.8, P≤0.001), while relationships between the other δ13CRS values and concentrations of different carbon sources were weaker (data not shown). However, the deviant results for δ13CRS of fructose in comparison to the other sugars are assumed to reflect peak overlap issues of this sugar (Tables 3, 4). This is clearly reflected also in the concentration results (Fig.4A). Consequently, the fructose results will not be discussed further in detail. δ13CRS of malate (Fig. 3D) in the range of −24‰ and −29.3‰ and δ13CRS of citrate (Fig.3E) in the range of −29.6‰ and −32.1‰ showed no temporal variations (P=0.198 and P=0.052 for malate and citrate, respectively, Table 3). Significant interactions between temperature and soil moisture treatments were observed for δ13CRS of malate (P=0.017; Table3), resulting in larger differences between δ13CRS values of soil moisture conditions under Thigh than under Tlow (Fig.3D). Citrate showed less negative δ13CRS values under dry conditions than under wet conditions (P=0.009; Table3). δ13CRS of starch of all treatments (Fig.3F), ranging from −25.2‰ and −32.1‰, was influenced by soil moisture conditions (P=0.046, Table 3), independent of temperature treatments, while temperature showed no significant effect (P=0.107, Table 3). In addition, soil moisture conditions caused significant temporal variations during the daily cycle in δ13CRS of starch (P=0.032, Table3). Concentrations of soluble carbohydrates, organic acids, andstarch Concentrations of glucose of all treatments (Fig.4B), ranging from 27 to 95µmol g DW-1, showed no temporal variations (P=0.927, Table3). In contrast, concentrations of sucrose Table2. Biomass and tuber analyses after sampling period Total plant biomass (dry weight), tuber weight (fresh weight), and tuber count (number of potatoes) after the sampling period. Potato plants were treated with a combination of Tlow (low temperature), Thigh (high temperature), and wet or dry conditions. Means ±SE are given (n=3). Refer to Table 1 for statistical analysis. Treatments Parameter Tlow wet Tlow dry Thigh wet Thigh dry Total biomass (g) 10.6 ± 1.4 7.8 ± 1.2 10.3 ± 1 8.1 ± 0.5 Tuber weight (g) 513.9 ± 18.4 458.3 ± 15 481.4 ± 15.9 430 ± 24.2 Tuber count (no.) 21.3 ± 2.7 20.2 ± 1.7 19.3 ± 1.2 19 ± 2.3 Table3. Environmental influences on leaf dark-respired CO2 and respiratory carbon sources Results of linear mixed effects models testing the effects of temperature (low, high) and soil moisture (wet, dry) on δ13C values in different putative leaf respiratory carbon sources, bulk leaves (δ13Cleaf), and in leaf dark-respired CO2 (δ13CR), as well as on concentrations of different carbon sources during the sampling period. Results for fructose are affected by co-elution with other compounds. P-values are given for treatments, time, and their interactions. Significant differences are given in bold (P≤0.05). δ13C Parameter Fructose Glucose Sucrose Malate Citrate Starch δ13CLeaf δ13CR Temperature 0.019 0.004 0.028 0.015 n.a. 0.107 0.022 0.044 Soil moisture 0.001 0.001 0.001 0.049 0.009 0.046 0.005 0.013 Time 0.001 0.195 0.081 0.198 0.052 0.001 0.066 0.001 Temp.:moisture 0.035 0.063 0.543 0.017 n.a. 0.270 0.165 0.875 Temp.:time 0.256 0.008 0.003 0.807 n.a. 0.113 0.812 0.014 Moisture:time 0.061 0.291 0.002 0.060 0.411 0.032 0.596 0.883 Concentration Parameter Fructose Glucose Sucrose Malate Citrate Starch Temperature 0.663 0.352 0.142 0.011 n.a. 0.002 Soil moisture 0.001 0.001 0.031 0.999 0.052 0.001 Time 0.016 0.927 0.001 0.035 0.110 0.001 Temp:moisture 0.475 0.705 0.462 0.796 n.a. 0.001 Temp.:time 0.901 0.847 0.113 0.387 n.a. 0.324 Moisture:time 0.831 0.629 0.063 0.889 0.895 0.071 n.a., not available at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from Malate as a key carbon source of leaf dark-respired CO2 in potato | 5775 (Fig.4C) in the range of 23 to 159µmol g DW-1 showed clear daily variations (P≤0.001, Table3), with highest concentrations for all treatments by the end of the day, except for Thigh and dry conditions. Glucose concentrations were significantly higher under dry than under wet conditions (P≤0.001, Table3), while converse results were observed for sucrose (P=0.031, Table3). Generally, no effect of temperature on the concentration of any soluble carbohydrate was observed. Malate concentrations of all treatments (Fig.4D), ranging from 23 to 163µmol g DW-1, showed a daily pattern with declining concentrations in the beginning of the night and an increase after 2–4 h in the dark (P=0.035, Table3). In contrast to soluble carbohydrates, malate concentrations were significantly higher under Thigh than under Tlow (P=0.011, Table3), but were not affected by soil moisture treatments (P=0.999, Table3). Citrate concentrations under Thigh of ~15µmol g DW-1 were the lowest of all measured putative carbon sources available for leaf dark respiration and showed no changes due to soil moisture treatments and time (Fig.4E; Table3). Starch concentrations (Fig. 4F), ranging from 67 to 282 µmol g DW-1, showed significant temporal variations (P≤0.001, Table3), independent of any treatment. The average starch concentration of 243µmol g DW-1 under Tlow and wet conditions was clearly higher (~2.5 times) compared to those under other treatments. In addition, interactions between temperature and soil moisture treatments led to smaller differences between the values of wet and dry conditions under Thigh compared to those under Tlow (P≤ 0.001, Table3). Linear relationships between δ13CR and δ13CRS Linear regression analyses were performed to understand the biochemical link between δ13CR and δ13CRS across all treatments (Table 4; Supplementary Fig. S1). δ13CRS of malate explained most of the daily variation of δ13CR (r2=0.26, P≤ 0.001), while the explanatory power of fructose, glucose, and citrate was lower. The lowest linear relationships during the daily cycle were found between δ13CR and δ13CRS of sucrose and starch. Due to the high daily variations in δ13CR we carried out the same analysis separately for daytime and nighttime. Daytime linear relationships were generally stronger than during nighttime, with δ13CR strongly related to δ13CRS of malate, citrate, and δ13Cleaf (r2>0.6, P≤0.001), but lower related to δ13CRS of soluble carbohydrates and starch. During nighttime, δ13CRS of malate explained 36% of the variation in δ13CR, but δ13CRS ● ● ●● ●●●● ● ● ●● ●●●● ● ● ● ● ● ● ●● −37 −34 −31 −28 −25 −22 (A) Fructose ● ● ● ● ●●●● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● (B) Glucose ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ● ● ● (C) Sucrose ●● ● ●● ●● ● ●● ● ●● ●● ● ● ● ●●● ●●● 0816 24 32 −37 −34 −31 −28 −25 −22 (D) Malate ●● ● ●●●● ● 0816 24 32 (E) Citrate ●Tlow Wet Tlow Dry Thigh Wet Thigh Dry ●●●●● ●●● ●●●●● ●●● ● ●● ● ● ● ● ● 0816 24 32 (F) Starch δ 13 CRS (‰) Sampling period (h) Fig.3. Daily cycles of the carbon isotopic composition of different leaf respiratory carbon sources (δ13CRS) under different environmental conditions during the sampling period: (A) fructose, (B) glucose, (C) sucrose, (D) malate, (E) citrate, and (F) starch. Potato plants were treated with a combination of Tlow (low temperature; closed symbols), Thigh (high temperature; open symbols), and wet (circles) or dry (triangles) conditions. Results for fructose are affected by co-elution with other compounds. Grey areas indicate nighttime. Means ± SE are given (n=2–3). Table4. Relationships between δ13C of leaf dark-respired CO2 and δ13C of respiratory carbon sources Linear regression analyses relating δ13C of leaf dark-respired CO2 to δ13C of putative respiratory carbon sources and to δ13C of bulk leaves (δ13Cleaf) across all environmental conditions for daytime (0 h, 16 h, 24 h), for nighttime (2 h, 4 h, 8 h, 26 h, 28 h, 32 h), and for the total daily cycle (sampling period over 32 h). Results for fructose are affected by co-elution with other compounds. Generic regression equation y=mx+b was used. r2 values are given, stars indicate P-values. All correlation coefficients were positive. r2 Putative carbon sources Daytime Nighttime Daily Fructose 0.35*** 0.34*** 0.12*** Glucose 0.54*** 0.34*** 0.13*** Sucrose 0.59*** 0.20*** 0.04* Malate 0.69*** 0.36*** 0.26*** Citrate 0.67*** 0.28** 0.17** Starch 0.48*** 0.16** 0.06* δ13CLeaf 0.63*** 0.33*** 0.20*** *, P≤0.05; **, P≤0.01; ***, P≤0.001 at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from 5776 | Lehmann etal. of fructose and glucose, as well as δ13Cleaf, showed similarly high explanatorypower. Influence of environmental drivers and carbon sources on δ13CR Furthermore, a stepwise (backward) multiple linear regression analysis was performed to identify environmental drivers and carbon sources influencing δ13CR (Table5). Daytime/nighttime showed the strongest positive effect on δ13CR (β=0.73, P≤0.001), while δ13CRS of malate was the carbon source that affected δ13CR most (β=0.4, P≤0.001). By comparison, the influence of δ13CRS of starch and soil moisture conditions on δ13CR values wasminor. Discussion This study clearly demonstrates that different temperature and soil moisture conditions influence δ13C of leaf dark-respired CO2 (δ13CR), δ13C of different putative leaf respiratory carbon sources (δ13CRS), and concentrations of carbon sources during a daily cycle in potato leaves. Furthermore, our findings strongly indicate malate as a key carbon source of daytime and nighttime δ13CR across different environmental conditions. Influence of temperature and soil moisture on isotopic compositions After 2 weeks of treatment, we already found a clear temperature effect on δ13Cleaf, with up to 2.2‰ more negative δ13Cleaf values under Thigh conditions compared to those under Tlow conditions (Fig.2B). This is in agreement with a study showing more negative δ13C values with increasing temperature for bulk leaves of Xanthium species (Smith etal., 1976). Similar to Tcherkez etal. (2003) under short-term temperature treatments, we observed more negative δ13CR value with increasing temperature (Fig.2A), but due to our long-term treatment we found also more negative δ13CRS values (Fig.3). On the other hand, dry conditions in both of the temperature treatments caused less negative δ13Cleaf, δ13CR, and δ13CRS values compared to those under wet conditions, which is consistent with previous studies under controlled conditions (Duranceau etal., 1999; Ghashghaie etal., 2001). The isotopic results under the different environmental conditions can be directly linked to the leaf gas exchange observed during the 32 h sampling period (day 15 of the treatment period). Increasing temperature caused lower An values under both soil moisture conditions (Fig.1A; Table1), indicating that plants under Thigh were beyond the photosynthetic optimum. This result is in agreement with earlier studies, showing that cold-adapted potato plants have reduced rates of photosynthesis with temperatures above 20°C (Levy and Veilleux, 2007). Additionally, An might be also influenced by leaf ageing, since Table5. Environmental drivers and carbon sources influencing δ13C of leaf dark-respired CO2 Result of stepwise (backward) multiple linear regression analysis showing the best-fit combination of independent environmental drivers (temperature, soil moisture, daytime/nighttime), time, and δ13C of glucose, sucrose, malate, and starch as variables influencing δ13C of leaf dark-respired CO2 (δ13CR) during the sampling period in potato leaves. Standardized β-coefficients and P-values are given. Drivers and carbon sources influencing δ13CR Standardized β-coefficient P-value Daytime/nighttime 0.73 <0.001 Malate 0.40 <0.001 Starch 0.11 0.019 Soil moisture 0.14 0.013 ● ● ●● ● ●● ● ● ● ●● ● ●● ● ●●● ● ● ● ●● 0 50 100 150 200 (A) Fructose ● ●●● ● ● ● ● ● ●●● ● ● ● ● ●●●● ● ● ●● (B) Glucose ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● 0 50 100 150 200 (C) Sucrose ● ● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● 0816 24 32 0 50 100 150 200 (D) Malate ● ● ●●●●●● 0816 24 32 (E) Citrate ●Tlow Wet Tlow Dry Thigh Wet Thigh Dry ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●●● ●●●● ● 0816 24 32 0 100 200 300 400 (F) Starch Sampling period (h) Concentration (µmol g DW − 1) Fig.4. Daily cycles of the concentration of different leaf respiratory carbon sources under different environmental conditions during the sampling period: (A) fructose, (B) glucose, (C) sucrose, (D) malate, (E) citrate, and (F) starch. Potato plants were treated with a combination of Tlow (low temperature; closed symbols), Thigh (high temperature; open symbols), and wet (circles) or dry (triangles) conditions. Grey areas indicate nighttime. Results for fructose are affected by co-elution with other compounds. To facilitate comparison with other metabolites, sucrose concentrations were multiplied by 2 to count for hexose units, while starch concentrations are given in molarity of starch monomers. Note different y-axis scale in (F). Means ±SE are given (n=2–3). at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from Malate as a key carbon source of leaf dark-respired CO2 in potato | 5777 An decreased under all treatments during the treatment period. On the other hand, gs tended to higher values with increasing temperature, but only under wet conditions (Fig.1C; Table1). An increase of gs under Thigh might be triggered by increasing transpiration rates, which could be a physiological response to compensate reduced rates of An by cooling the leaf temperature under Thigh conditions. However, this was only observed in plants under Thigh and well-watered conditions, when SWC was high. Subsequently, lower carbon fixation and higher CO2 diffusion into the stomatal cavities under Thigh, in comparison to Tlow, caused an increase of Ci (Fig.1B) and more negative δ13CR and δ13CRS values (Table6). Furthermore, dry soil moisture conditions caused reduced rates of An and gs compared to those under wet conditions (Fig.1A, C; Table1), independent of temperature treatments. This can be explained with the severe drought stress, reflecting low SWC values (Fig. 1D). Consequently, plants under dry conditions experienced reduced CO2 diffusion into the stomatal cavities, leading to lower Ci and less negative δ13C values (Table6). Plants under Thigh and dry conditions showed the lowest performance during the sampling period compared to plants under other treatments, which is reflected in low An values (Fig.1A), plant biomass, tuber weight and tuber count (Table2). δ13CR and δ13CRS in these plants were expected to be the most positive compared to other treatments due to a severe drought caused by the double effect of high temperature and dry soil moisture. Instead, δ13CR and δ13CRS of the plants under the highest stress level (Thigh and dry conditions) were rather similar to those under lowest stress level (Tlow and wet conditions). This was particularly observed for δ13CRS of soluble carbohydrates and starch (Fig.3). Again, this is an indicator of low An under Thigh and dry conditions, resulting in a moderate reduction of Ci, while at the same time gs strongly reduces CO2 diffusion into the stomatal cavities, causing an increase of Ci. Consequently, this led to intermediate δ13CR and δ13CRS values under Thigh and dry conditions (Table6). In summary our findings indicate that combined effects of temperature and soil moisture conditions on δ13CR and δ13CRS could cancel out the individual effect of each driver. Environmental influences on concentrations of putative carbon sources Soil moisture and temperature affected concentrations of putative leaf respiratory carbon sources differently. Sucrose concentration decreased under dry conditions (Fig. 4C; Table3), which is in contrast to the recent study by Lemoine et al. (2013). This may be explained by reduced rates of sucrose synthesis due to lowering of the sucrose phosphate synthase reaction (SPS) (Vu et al., 1998). The decrease in the enzyme activity is probably triggered by limited rates of phloem sugar transport observed under drought (Ruehr etal., 2009). This in turn could be an explanation for lower plant biomass and tuber weight/count in response to higher temperatures and dry conditions (Tables 1, 2). Subsequently, the increase of fructose and glucose concentrations under drought may also be a consequence of lower SPS activity (Fig. 4A, B; Table 3), since the demand for both hexoses for sucrose synthesis was reduced. Additionally, increasing fructose and glucose concentrations under drought might have osmotic functionality, maintaining metabolic activity (Lemoine etal., 2013). On the other hand, malate concentrations increased with temperature (Fig.4D; Table3), which is most likely a consequence of higher PEPC activity (Chinthapalli etal., 2003). Higher malate concentrations may also support respiratory processes in the KC or regulation of stomatal opening (Finkemeier and Sweetlove, 2009). Moreover, decreased starch concentrations in leaves under treatments with higher environmental stress than Tlow and wet conditions (Fig.4F) were similar to previous findings (Lemoine etal., 2013). The result also supports the assumption that reduced amounts of assimilated carbon due to lower An under Thigh or dry conditions were used for maintenance of biochemical processes rather than for carbon storage. Additionally, this indicates that plants under Thigh or dry conditions were under severe environmental stress. Malate as a key respiratory carbon source of daytime and nighttime δ13CR The daily cycle of δ13CR was highly variable, showing less negative daytime and more negative nighttime values, while δ13CRS values generally showed lower changes during the same period (Figs 2A, 3; Table3). δ13CRS values of all treatments compared to δ13CR values were more negative for soluble carbohydrates (up to 9.3‰) and citrate (up to 4.1‰), but also less negative for starch (up to 4‰) and malate (up to 5.2‰) during the daily cycle (Figs 2A, 3). In particular, malate was strongly enriched in 13C, by up to 8.8‰, compared to all other putative carbon sources (Fig.3). This was similar to a previous study investigating metabolites in potato leaves (Gleixner etal., 1998) and indicates a possible biochemical link between 13C enriched leaf dark-respired CO2 and 13C enriched malate. For a better understanding of the overall biochemical connections between δ13CR and different putative carbon sources, we carried out linear regression analyses, independent of environmental conditions (Table4; Supplementary Fig. S1). Table6. Coherence between leaf physiological parameters and δ13C values. Leaf physiological parameters and δ13C values during the sampling period in potato plants under different treatments compared to those in potato plants growing under Tlow and wet conditions The following variables were considered: An, net assimilation rate; Ci, intercellular CO2 concentration; gs, stomatal conductance; δ13CR, δ13C of leaf dark-respired CO2; δ13CRS, δ13C of different putative respiratory carbon sources (fructose, glucose, sucrose, starch, and malate). Arrows indicate strong (↑, ↓), intermediate (↗, ↘), or no changes (→) due to the influence of treatment combinations (Tlow, low temperature; Thigh, high temperature; and wet or dry conditions). Treatments AngsCiδ13CRδ13CRS Tlow dry ↘ ↓ ↓ ↑ ↑ Thigh wet ↘ ↑ ↑ ↓ ↓ Thigh dry ↓ ↓ ↗ → → at Natural Resources Institute Finland (Luke) on November 29, 2016http://jxb.oxfordjournals.org/Downloaded from