Metabolite pools and carbon flow during C 4 photosynthesis in maize: 13 CO 2 labeling kinetics and cell type fractionation

1The CO2-concentrating shuttle (CCS) in C4 photosynthesis requires close co-operation between mesophyll cells (MCs) and bundle sheath cells (BSCs). CO2 is initially incorporated into oxaloacetate (OAA) in the MCs by phosphenolpyruvate carboxylase (PEPC), whose high affinity allows it to operate at low CO2 concentrations. OAA is converted to other four-carbon metabolites that move to the BSCs and are decarboxylated, generating a high CO2 concentration that allows efficient operation of the Calvin–Benson cycle (CBC), and three-carbon metabolites return to the MCs. C4 plants have historically been classified into three subtypes based on the major decarboxylation enzyme (;; Supplementary Fig. S1 at JXB online): the NADP-malic enzyme (ME) subtype where malate moves to the BSCs and pyruvate returns to the MCs; the NAD-ME subtype where aspartate moves to the BSCs and alanine returns to the BSCs; and the phosphoenolpyruvate carboxykinase (PEPCK) subtype where NAD-ME also contributes to decarboxylation. and aspartate and malate move to the BSCs, and PEP and alanine move back to the MCs. In some cases the shuttles also transfer energy to the BSCs, for example NADPH in the NADP-ME subtype. In many NADP-ME species, including maize, PSII in the BSCs is strongly decreased and ATP and NADPH are supplied to the BSCs via another intercellular shuttle; 3-phosphoglycerate (3PGA) moves from the BSCs to the MCs where it is reduced by phosphoglycerate kinase and NADP-glyceraldehyde-3-phosphate dehydrogenase to triose-phosphate (triose-P) which move back to the BSCs. The topology of C4 photosynthesis was established in the 1970–1980s but fundamental questions remain concerning its operation.

2One issue relates to how metabolites move between the MCs and BSCs. It was proposed in the 1970s that this occurs by diffusion. Diffusion will be facilitated by the high density of plasmodesmata and close vein spacing which means that each MC is adjacent to a BSC. Rapid diffusion requires concentration gradients between the MCs and BSCs, estimated in the range of 5–10 mM. In the 1980s, two methods were developed to enrich MCs and BSCs from maize leaves while preventing changes in metabolite levels. Both reported gradients to support malate and triose-P movement from the MCs to the BSCs, and 3PGA movement from the BSCs to the MCs, but not for pyruvate. However, it remained unclear what proportion of the overall pools were actually involved in photosynthesis, as opposed to being located in, for example, the vacuole or other cell types.

3Secondly, it may be overly simplistic to classify C4 plants into three subtypes. There is evidence for parallel operation of shuttles, including co-occurrence of enzymes and high contents and rapid labeling of diagnostic metabolites. However, part or most of the metabolite content may not be involved in C4 photosynthesis and labeling might be due to label exchange rather than net flux through the metabolite.

4Thirdly, other metabolic pathways must be modified to make them compatible with the high concentrations of metabolites that are involved in intercellular shuttles. One example is sucrose synthesis, which occurs in the MC cytosol. The affinity of maize cytosolic fructose bisphosphatase is an order weaker than that of the C3 enzyme, allowing high concentrations of triose-P to be maintained in the MCs to drive diffusion to the BSCs. It is not known how the high concentrations of organic and amino acids are shielded from respiration.

5A fourth question concerns interactions between the CCS and the 3PGA/triose-P shuttle. Interconversion of PEP and 3PGA will allow carbon to move between these shuttles, and ultimately into or out of the CBC. It has been proposed that large pools of organic acids and amino acids buffer CBC metabolite levels. However, it is not known how rapidly 3PGA and PEP are interconverted, and how movement of carbon from one shuttle to the other is regulated. Shuttle operation depends on high metabolite concentrations to drive diffusion, and efficient C4 photosynthesis depends on a correct balance between the rate at which carbon is shuttled into the BSC and its utilization by the CBC.

6It is also unclear how far photorespiration is decreased in C4 plants. Compared with C3 plants, C4 plants have lower but still substantial activities of enzymes for photorespiration. Evidence for the occurrence of photorespiration has been provided by investigating O2 dependency and 18O incorporation, and a comparative study in the Flaveria genus revealed 2- to 5-fold lower photorespiration in C4 than in C3 species. Further, photosynthesis is impaired in maize mutants with decreased glycolate oxidase activity.

7Labeling studies with 14CO2 played a key role in elucidating C4 photosynthesis. In recent years, analysis of labeling kinetics in individual metabolites has been greatly facilitated by the use of stable isotopes in combination with GC-MS or tandem liquid chromatography (LC-MS/MS). We recently established a protocol for dynamic 13CO2 labeling of whole Arabidopsis rosettes, analyzed the temporal labeling kinetics of ~40 metabolites using GC-MS and LC-MS/MS, estimated fluxes, and benchmarked them against literature values. 13CO2 labeling also provides information about the sizes of pools that are involved in photosynthesis; in many cases, only part and sometimes only a minor part of the total pool is directly involved. We now adapt this method to analyze C4 photosynthesis in maize to address the five questions discussed above. In addition, we analyze 13C labeling patterns in fractions enriched in MCs and BSCs, to determine the intercellular distributions and concentration gradients of the metabolic pools that are involved in C4 metabolism, and provide independent evidence for operation of more than one CCS.

8Carbon dioxide (13CO2, isotopic purity 99 atom%) was from Campro Scientific, and chemicals from Sigma-Aldrich, Roche, or Merck.

9Maize (Zea mays L. cv. B73) seeds were germinated in darkness in Petri dishes on moistened filter paper (3 d, 28 °C), transferred to soil in 10 cm diameter pots, grown for 5 d under 16/8 h day/night cycles (irradiance 105 µmol photons m−2 s−1, 22/18 °C, 70% relative humidity) and then under 14/10 h day/night cycles (irradiance 480 µmol photons m−2 s−1, 25/22 °C, 65% relative humidity) and used at 3 weeks for 13CO2 labeling. Arabidopsis thaliana Col-0 was grown as in. CO2 concentration in our growth facilities was ~420 ppm.

10Gas was mixed to a final concentration of 78% N2, 21% O2, and 420 ppm 12CO2/13CO2, humidified, pumped into a custom-designed labeling chamber, and exited via a PVC tube over soda lime to capture 12CO2/13CO2 [Supplementary Fig. S2A, B; see ]. The chamber (volume 320 ml) had a gas half-life of 2.6 s and 1.3 s at flow rates of 5 l min−1 and 10 l min−1, respectively. Flow at 10 l min−1 was used for pulses of up to 1 min, and 5 l min−1 for longer pulses. The chamber was made of copper with a hollow body and a transparent Plexiglas lid with a hollow vertical tube (internal diameter ~2 cm) sealed with transparent film (Toppits©). The lid was fastened using clasps, with a soft rubber O-ring between the lid and chamber, ensuring air-tight closure. An additional lamp (FL-460 Lighting Unit, Walz, Effeltrich, Germany) was placed beside the labeling chamber to ensure an irradiance in the chamber of 480 µmol photons m−2 s−1. To obtain a constant leaf temperature of 35 °C (measured in the growth chamber with a thermocouple; VWR and Testo), gas was passed through a humidifier in a water bath (gas relative humidity of 65%), and water was pumped through the hollow body of the labeling chamber. Plant material was quenched by dropping a pre-cooled (in liquid N2) copper rod with a sharp machined edge down the hollow tube in the lid into the labeling chamber, freeze-clamping a leaf disc (1.9 cm diameter, ~65 mg FW) between this rod and another copper rod fixed in the chamber, protruding into the base of the chamber and extending well below the outside of the chamber to allow pre-cooling with liquid N2 (Supplementary Fig. S2C, D). Leaf four was used after it reached full expansion, when plants were ~3 weeks old. Labeling started 2 h after the start of the light period to ensure metabolic steady state. The leaf was placed in the labeling chamber with a continuous flow of 78% N2, 21% O2, and 420 ppm 12CO2 (Supplementary Fig. S2A, B), and after 1 min 12CO2 was replaced by 13CO2. Samples were collected 10, 15, 20, 30, and 50 s, and 1, 3, 5, 10, 20, 40, or 60 min after starting labeling, in a random manner. Unlabeled samples (t=0) were collected after 1 min in an unlabeled gas mixture. Leaves were also labeled for 60 min and chased with unlabeled gas for 5 min or 20 min. Leaf discs from two plants were pooled per time point.

11To obtain material for cellular separation, 13CO2 air was applied in a transparent plastic oven bag (Pely-plastic, Wahlstedt, Germany) sealed at both ends with bag ties/clasps (Supplementary Fig. S2E). Gas entered and exited via PVC tubes inserted at the leaf base and the other end of the bag, respectively. Gas mixing, humidification, and irradiance were as described above. Labeling started at least 2 h after the start of the light period. The leaf was subjected for 1 min to a continuous flow (5 l min−1) of 78% N2, 21% O2, and 420 ppm 12CO2, and the flow then diverted for 1 min from the labeling chamber into the CO2 trap (Supplementary Fig. S2E), 12CO2 replaced by 13CO2 in the gas mixture, unlabeled gas removed from the bag by vacuum (Supplementary Fig. S2F), and the labeled gas mix then switched to the bag. Three minutes later the central leaf part (~500 mg FW) was quenched by clamping the leaf and bag between two aluminum blocks pre-cooled in liquid N2 (Supplementary Fig. S2G, H). Six leaves were harvested, and pooled in pairs to provide three biological samples.

12Arabidopsis rosettes were labeled and quenched as in, except that they were subjected to 78% N2, 21% O2, and 420 ppm 13CO2 (ambient O2) or 97% N2, 2% O2, and 420 ppm 13CO2 (low O2). Plants were placed in a transparent box and provided (5 l min−1) with 420 ppm 12CO2 and either 78% N2/21% O2 or 97% N2/2% O2 for at least 30 min prior to switching to 420 ppm 13CO2, and sampled after 5, 10, 20, 30, and 45 s, and 1, 1.5, 2, 3, 5, 10, 20, 40, and 60 min, with 3–13 replicates per time point. Unlabeled samples (t=0) were collected after at least 30 min in ambient or low O2.

13Maize or Arabidopsis material was ground to a fine powder by hand in a mortar pre-cooled with liquid N2 or in a cryo-robot, and stored at –80 °C. Samples were analyzed by LC-MS/MS and GC-MS, with authentic standards for accurate metabolite quantification, as in. We additionally analyzed aspartate, PEP, 2-phosphoglycolate (2PG), ribose-5-phosphate (R5P), and ribulose-5-phosphate+xylulose-5-phosphate (Ru5P+Xu5P) [see Supplementary Tables S1, S2 for the isotopomer-dependent MS parameters used for selected reaction monitoring (SRM) and the corconfig.cfg file used to correct for natural abundance;; ]. Amounts of the unlabeled form and each 13C isotopomers in maize samples are provided in Supplementary Table S3 and total contents in Supplementary Table S4. Total amounts of 3PGA and PEP were determined enzymatically using a Sigma-22 dual-wavelength photometer using freshly prepared extracts for PEP. 13C Enrichment and isotopomer distribution were calculated as in (Supplementary Tables S5, S6). Active and inactive pools were calculated as in Supplementary Table S3, positional 13C enrichment (C4 and C1–C3 positions) of aspartate and malate as in Supplementary Tables S7, S8, and 13C amounts in metabolites as in Supplementary Tables S7, S8.

14Maize leaves were fractionated as in, ). Four fractions were obtained by homogenizing ~1 g FW of material at low temperature, resuspending in liquid N2, and filtering sequentially through 200, 80, and 40 µm nylon meshes (Sefar, Switzerland). Activities of the MC markers NADP-malate dehydrogenase (NADP-MDH) and PEPC were measured as in with 2000- and 20 000-fold dilution (FW/extract volume), respectively. The BSC marker ribulose-1-5-bisphosphate (RuBP) was quantified by LC-MS/MS. Cell separation was performed on three biological replicates, and these were combined to calculate distribution. The intercellular distribution of a metabolite X was estimated by extrapolation. Enzyme activities and metabolite amounts in each fraction were expressed as a percentage of that in the summed fractions. For each fraction, the ratio of (average of NADP-MDH and PEPc)/RuBP; x-axis) and the ratio (metabolite X/RuBP; y-axis) were plotted against each other, and regression calculated. The intercept on the y-axis represented the proportion of metabolite X in the BSCs. Distribution was calculated for each isotopomer, the sum of all isotopomers of a metabolite and, for malate, the sum of labeled isotopomers.

15Net CO2 assimilation (An) was measured on the fouth fully expanded leaf in a gas exchange system (LiCor) attached to a Leaf Chamber Fluorometer under similar conditions to the labeling experiment (480 µmol m−2 s−1 irradiance, 35 °C, 420 ppm CO2). An was measured first at 21% O2 and then at 2% O2 in six plants with eight technical replicates per plant.

16Leaves were pulsed with 13CO2 for 0, 10, 15, 20, 30, and 50 s, and 1, 3, 5, 10, 20, 40, and 60 min, or were pulsed for 60 min followed by a 5 min or 20 min chase with 12CO2. The labeling chamber was designed to have a small volume to minimize the time delay between introduction and full equilibration of 13CO2 and to allow leaf material to be quenched instantaneously under the prevailing light regime, and without allowing access of unlabeled CO2 for even a fraction of a second. The latter is important because many metabolites turn over very rapidly during photosynthesis. A total of 35 metabolites were detected with GC-MS and LC-MS/MS including all C4 photosynthetic intermediates, almost all CBC intermediates, intermediates in starch and sucrose biosynthesis and photorespiration, and organic acids and amino acids. The amount of the unlabeled form and each 13C isotopomer (Supplementary Table S3) were used to estimate the total amount (Supplementary Table S4), 13C enrichment (% of C atoms that are labeled with 13C; Supplementary Table S5), and relative abundance of each isotopomer (Supplementary Table S6) for each metabolite and time point.

17Total metabolite content showed some variation (Supplementary Table S4). This was not related to the time of sampling (Supplementary Fig. S3). Variation in metabolite contents was seen previously between maize leaves and along the leaf developmental gradient. The pulse and chase experiments were performed on different days. Metabolite contents were in the same range in both experiments except that glucose and fructose were higher, and pyruvate and fumarate were lower in the chase experiment (Supplementary Table S4). Total content was estimated as the average of all samples from the pulse and chase, unless stated otherwise.

18We subjected the 13C enrichment kinetics of all 35 metabolites to k-means clustering based on Euclidian distance (Supplementary Fig. S4, note log10 time scale). Based on the ‘rule of thumb’, we generated four clusters. Cluster I contained metabolites whose enrichment was high (average 25%) within 10–30 s and were almost fully labeled (average 93%) after 40–60 min. It included most CBC intermediates, the starch biosynthesis intermediate ADPG, and the immediate product of RuBP oxygenation, 2PG. Cluster II contained metabolites that were labeled in the first 10–30 s although less strongly (average 11%) than cluster I, and whose enrichment rose to an average of 82% after 40–60 min. It included sucrose synthesis intermediates (G6P, G1P, and UDPG) and some metabolites involved in the CCS (PEP, aspartate). Cluster III showed very low enrichment (<1%) in the first 30 s, and rose gradually to an average value of 76% after 40–60 min. It included metabolites from the CCS (pyruvate, alanine) and photorespiration (glycine, serine). Cluster IV showed negligible labeling in the first 30 s and rose slowly to an average of 16% after 40–60 min. It included glycerate, which is the last metabolite in the photorespiration pathway, sugars, tricarboxylic acid cycle (TCA) intermediates [fumarate, succinate, 2-oxoglutarate (2OG)], many amino acids, and, unexpectedly, malate and sedoheptulose-1,7-bisphosphate (SBP).

19The 13C enrichment kinetics for most metabolites in and downstream of the CBC resembled those of Arabidopsis, showing that many features of C3 pathway topology are conserved in maize. However, there were some anomalies. First, metabolites involved in CCS such as malate, aspartate, pyruvate, alanine, and PEP were spread across three clusters (II, III, IV) and labeled more slowly than CBC intermediates. Indeed, malate was assigned to cluster IV. Secondly, the CBC intermediate SBP was assigned to cluster IV. Photorespiratory intermediates were also distributed across three clusters (I, III, IV). We considered two possible reasons for these anomalies. One is the presence of compartmented pools that are not involved in photosynthesis. The second is related to the topology of C4 photosynthesis (see below).

20When most or all of the total content of a metabolite is involved in photosynthesis, the unlabeled isotopomer will decrease to very low values and the fully labeled isotopomer will rise to become the predominant form. This pattern was seen for all CBC metabolites (Supplementary Fig. S5; Supplementary Table S6) except SBP, where >80% was present as the unlabeled form even at 60 min while the remainder was present as a mix of isotopomers with heavily labeled forms predominating. Similarly, whereas at 60 min the unlabeled form of PEP, aspartate, pyruvate, and alanine decreased to negligible levels and the fully labeled form predominated, ~60% of the total malate pool was unlabeled while most of the rest was the quadruple-labeled (i.e. fully labeled) isotopomer (Fig. 1). This pattern points to there being kinetically separated pools; an ‘active’ pool that is involved in photosynthetic flux and an ‘inactive’ pool that does not participate in photosynthesis.

21To estimate the active pool size of malate (malatea), we first summed the labeled isotopomers of malate [termed Malate(L)]. Compared with the 4-fold variation in total malate content (1.8–7 µmol g−1 FW; Supplementary Table S4), Malate(L) was fairly constant from 1 min onwards (1.1–1.7 µmol g−1 FW; Supplementary Table S3). Malate(L) at 60 min (1.3 µmol g−1 FW) was similar to the average of Malate(L) between 10 min and 60 min (1.29 µmol g−1 FW). The inactive pool of malate (Malatei) at each time point was estimated by subtracting 1.3 µmol g−1 FW from the total malate amount at that time (Supplementary Table S3). Malatei was then subtracted from Malate-0 to provide an estimate of the amount of 12C malate in the active pool at each time (Malate-0a, Supplementary Table S3). Malate-0a and the values for the labeled isotopomers were used to calculate 13C enrichment (Supplementary Tables S5) and relative isotopomer abundance (Fig. 1; Supplementary Table S6) for the active malate pool. The corrected isotopomer abundance profile (Malatea, Fig. 1) resembled that of aspartate although with noise, probably due to assumptions in estimating the active pool. A similar correction was performed for SBP (Supplementary Fig. S5; Supplementary Tables S3, S5, S6).

22PEPC incorporates bicarbonate into the C4 position of metabolites such as malate and aspartate, and carbon at the C4 position is released as CO2 in the BSCs (Supplementary Fig. S6A;;; ). Thus, early in the labeling kinetics, single-labeled isotopomers of malate and aspartate should dominate and there should be little label in decarboxylation products such as pyruvate and alanine. The appearance of multiple labeled isotopomers of malate and aspartate and labeling of pyruvate and alanine will be delayed until 13C moves from the CBC into PEP and other CCS metabolites (Supplementary Fig. S6B).

23The isotopomer patterns for malate, aspartate, pyruvate, and alanine qualitatively matched these expectations. Single-labeled isotopomers of malatea and aspartate were dominant at early time points (Fig. 1). For malatea, 50–74% was present as a single-labeled isotopomer at 10–30 s, and 4–9%, 0.6–1.2%, and 0.25–0.38% as double-, triple-, and fully labeled forms, respectively. For aspartate, 66–68%, 5–8%, 0.5–1.1%, and 0.22–0.37% was present as single-, double-, triple-, and fully labeled isotopomers, respectively. Labeling in PEP was low for the first 60 s and then rose (Fig. 1). Subsequently, intermediate-labeled forms of malatea and aspartate increased and declined, with the fully labeled form being dominant from 10 min onwards. This was accompanied by appearance of label in pyruvate and alanine (Fig. 1).

24To estimate position-dependent enrichment in the C4 position and the C1–C3 positions of malate and aspartate, we assumed all labeled molecules contained 13C at the C4 position (see Supplementary Table S7 for the calculation). This assumption is justified by the very low PEP labeling at early time points (Fig. 1; Supplementary Table S6). Estimated enrichment in the C4 position averaged 71% in the first 10–30 s, and approached 100% at 40–60 min. Enrichment in C1–C3 was negligible at early times, and from 30 s on rose gradually to >80% after 60 min (Supplementary Table S7).

25In Fig. 2A, adjusted values for 13C enrichment in malate, aspartate, and SBP, after correction for inactive pools for malate and SBP and separation of the C4 and C1–C3 positions of malate and aspartate, are superimposed on the k-means clusters in Supplementary Fig. S4. The C4 positions of malate and aspartate are shown as cluster 0 whose enrichment rises more rapidly than the CBC intermediates in cluster I. PEP labels more slowly than CBC intermediates and is in cluster II. Enrichment in the C1–C3 positions of malate and aspartate rises even more slowly, resembling pyruvate and alanine, which are in cluster III.

26The similarity of its enrichment kinetics to those of malatea indicates that aspartate contributes to the CCS. Further evidence was provided by the chase experiment in which leaves were labeled for 60 min and chased with 12CO2 for 5 min and 20 min. Label depleted more rapidly in aspartate than malate, even after correction for the inactive malate pool (Supplementary Fig. S7).

27The rapid labeling of CBC metabolites, with multiple labeled isotopomers appearing within 10 s (Supplementary Fig. S5), resembles the pattern in tobacco and Arabidopsis. After 40–60 min, 67–81% of CBC intermediates were present as fully labeled isotopomers. Incomplete labeling at the end of the kinetics was also reported in tobacco and Arabidopsis.

28Starch is synthesized from F6P via G6P, G1P, and ADPG in BSC chloroplasts, and sucrose is synthesized from triose-P via FBP, F6P, G6P, G1P, and UDPG in the MC cytosol. ADPG had enrichment kinetics similar to the CBC intermediates (Fig. 2A). This is explained because the small pool (0.7 nmol g−1 FW) will turn over rapidly, as in Arabidopsis. G6P, G1P, and UDPG were labeled more slowly than the CBC intermediates (Fig. 2A; Supplementary Fig. S5). F6P was labeled more rapidly than G6P, reflecting its additional role in BSCs as a CBC intermediate. Compared with G6P, labeling of UDPG and G1P was slightly and markedly slower. The proportion of unlabeled form at the end of the experiment was negligible for F6P (4.2%) and G6P (6.8%), and substantial for UDPG (17%) and G1P (30%), while the rest was predominantly fully labeled isotopomers. This resembles the pattern in Arabidopsis and tobacco and indicates that some of the UDPG and much of the G1P are not involved in photosynthesis.

29We combined information about metabolite content, isotopomer distribution, and the number of 13C atoms in an isotopomer to calculate the amount of 13C in each metabolite at a given time (Fig. 2B). In view of the variation in pool size, we compared two approaches: multiplying isotopomer distribution at a given time point by the average total pool size (or active pool size determined at 60 min), or using the sum of the measured isotopomer amounts at that time point (see Supplementary Table S8 for further explanation). The relatively good agreement between the approaches provided support for their reliability (shown for malate and aspartate in Supplementary Fig. S8). All 13C amounts presented in this study were calculated using the first approach.

30The estimated 13C pool in the C4 position of malate and aspartate, respectively, rose from ~700 nmol and 80 nmol 13C equivalents g−1 FW at 10 s to 1300 nmol and 100 nmol 13C equivalents g−1 FW from 60 s onwards (Fig. 2B; Supplementary Tables S7, S8). The higher value for malate reflects the 10-fold larger pool size of active malate compared with aspartate (Supplementary Table S4). Comparison of the amount of 13C in the C4 position of malate and aspartate in the initial 20 s of the labeling kinetics (Table 1) indicates that ~10-fold more carbon is shuttled into the BSCs via malate than via aspartate.

31The amount of 13C in the C4 position of malate and aspartate is large compared with the total pool of CBC intermediates (Fig. 2B). From 1 min on, it is 30- to 50-fold larger than the pool of RuBP (32 ± 11 nmol g−1 FW) and only ~3-fold smaller than the total carbon in CBC intermediates (~4100 nmol C g−1 FW). The amount of 13C in alanine is nearly 10-fold higher than that in pyruvate. It is also noteworthy that from 180 s onwards, the vast majority of the detected 13C is in sucrose (note insert in Fig. 2B). Negligible amounts of 13C were found in fumarate, succinate, 2OG, and glutamate up to 20 min and, even after 60 min, they contained only 0.01, 0.18, 0.52, and 2.2%, respectively, of the total detected label compared with 83.7% in sucrose. Although the 13C enrichment kinetics of sucrose and these organic acids and amino acids are similar (Fig. 2A), there is much more 13C in sucrose because its pool is much larger (Supplementary Table S4).

32At early time points. enrichment is higher in PEP than in pyruvate (Supplementary Table S5; Fig. 1) indicating that PEP is probably labeled from the CBC. Carbon exchange between the CBC and CCS will occur via interconversion of PEPC, 3PGA, 2PGA, and PEP via the reversible reactions catalyzed by phosphoglycerate mutase and enolase. Overall enrichment in 3PGA and 2PGA was almost 10-fold higher than in PEP at early time points (10–30 s) and was still 3-fold higher after 3 min (Fig. 1; Supplementary Table S5), implying some kinetic restriction on flow of newly assimilated 13C.

33To estimate flow of 13C from the CBC into the metabolites of the CCS, we summed the amount of 13C in PEP, in C1–C3 of malate and aspartate, and in pyruvate and alanine (i.e. 13C atoms in CCS intermediates excluding the C4 position of malate and aspartate; Table 2). It represented 8.7–13.1% of total detected 13C at the initial time points (10, 15, and 20 s).

34We found substantial label in photorespiratory metabolites. At 10–30 s, average enrichment in 2PG was 14% (Supplementary Table S5), compared with 29% and 30% in RuBP and 3PGA. Sequential increase of enrichment in 2PG, glycine, serine, and glycerate (Fig. 2A; Supplementary Fig. S9) is explained by these metabolites being in a linear pathway, with the latter three having large active pools (1.7, 84, 166, and 425 nmol g−1 FW for 2PG, glycine, serine, and glycerate, respectively). Movement through glycolate and glyoxylate, which was not measured, may lead to a further delay. Unlabeled isotopomer forms for glycine and serine plateaued between 40 min and 60 min, suggesting the presence of inactive pools (Supplementary Fig. S9; ~22% and 20%, respectively). This was observed for glycine but not serine in Arabidopsis.

35We did not estimate C flow through photorespiration from the early time points of the labeling kinetics because the 2PG pool is very small and label will have moved more into glycolate and glyoxalate, which were not detected. Also GC-MS detects only incomplete fragments of glycine, serine, and glycerate. Gas exchange measurements in ambient CO2 revealed a 5.3 ± 1.7% stimulation of net photosynthesis after decreasing O2 from 21% to 2% (Supplementary Table S9).

36We next compared labeling kinetics in maize and Arabidopsis. To distinguish between differences that are related to and independent of photorespiration, we labeled Arabidopsis at 420 ppm CO2 with either 21% or 2% O2. Enrichment of glycine, serine, and glycerate in Arabidopsis in 21% O2 (Fig. 3, red filled circles) resembled that in. The slightly lower enrichment of glycine compared with serine and glycerate is explained by an inactive pool of glycine (Supplementary Fig. S11; Supplementary Table S10; see also ). In 2% O2 (Fig. 3, red open circles), enrichment in photorespiratory intermediates was lower and rose more slowly. There were also significantly lower contents of serine and glycerate in 2% than in 21% O2 (Supplementary Fig. S10). Enrichment of CBC intermediates increased more rapidly in 2% than 21% O2, especially between 45 s and 10 min. This is probably due to faster 13C assimilation and decreased recycling of 12C from photorespiratory metabolites into the CBC.

37In maize (Fig. 3, blue filled squares), the enrichment patterns for serine and glycerate resembled those in Arabidopsis in 2% O2. Enrichment of glycine was higher than in Arabidopsis, but interpretation is complicated by the inactive pool. As expected from the higher rate of photosynthesis, enrichment in CBC metabolites in the first 3 min was higher in maize (Fig. 3, blue filled squares) than in Arabidopsis, even in 2% O2. However, between 5 min and 20 min, enrichment of CBC metabolites was similar or slightly lower in maize than in Arabidopsis in 21% O2, and was always lower than in Arabidopsis in 2% O2. By 40–60 min, enrichment in maize was similar to that in Arabidopsis. This labeling pattern indicates that in maize between 5 min and 20 min, unlabeled C flows into the CBC that probably does not derive from photorespiration.

38Maize leaf material was fractionated to enrich MCs and BSCs partially in order to estimate intercellular concentration gradients for CCS metabolites, 3PGA and triose-P, and to provide independent evidence for parallel operation of different CCSs. It is difficult from labeling kinetics, on their own, to discriminate if labeling of aspartate and alanine is due to their synthesis in one cell type and consumption in the other cell type (i.e. involvement in the CCS) or to reversible formation by aminotransferase reactions in one or both cell types without them being involved in net flux. The presence of a concentration gradient between BSCs and MCs would provide independent evidence for their involvement.

39Fractions enriched in MCs and BSCs were obtained by sequentially filtering leaf homogenates in liquid N2 through three nylon meshes with different pore sizes. BSCs are mechanically stronger and are enriched in larger particles that are retained on meshes with a larger pore size, whereas MC-enriched material is preferentially retained on smaller meshes or passes through to the final filtrate. The leaf discs obtained with the labeling chamber did not provide enough tissue for this procedure. Instead, leaves were incubated in transparent plastic bags (see Supplementary Fig. S2E, G, H and the Materials and Methods for details). A 3 min pulse was used, when all isotopomers of malate, aspartate, pyruvate, alanine, and PEP are at high enough levels to be reliably quantified.

40We first compared metabolite amounts and labeling patterns in non-filtered homogenates with those at 3 min in the kinetic experiment. Metabolite levels were similar in both experiments (Supplementary Table S4), with a few exceptions; in material for cell separation, aspartate was 3- to 4-fold higher, R5P, Ru5P+Xu5P, and RuBP were slightly higher, glucose 2-fold lower, and sucrose 7-fold lower. This may be partly because the tissue collected for cell separation included a larger part of the leaf than the leaf disc harvested with the labeling chamber. As previously mentioned, there are substantial metabolic gradients along the maize leaf blade.

4113C Enrichment in the various metabolites was higher at 3 min in the tissue fractionation experiment than at 3 min in the kinetic experiment (Supplementary Table S5). Correlation analysis revealed that 13C enrichment values in the tissue fractionation experiment resembled those after 5 min in the time kinetic experiment (Supplementary Fig. S12). This indicates that there were higher rates of photosynthesis in the tissue fractionation experiment. All key features of the time kinetic experiment were confirmed in the triplicated samples for tissue fractionation (Supplementary Table S5). All CBC metabolites were highly labeled (53 ± 5% to 69 ± 3% 13C enrichment) with the exception of SBP (9 ± 6% enrichment when not corrected for the inactive pool), ADPG was strongly labeled (63 ± 2%), malate and aspartate were present mainly in the single-labeled form (45 ± 10% for malate after correction, 52 ± 3.5% for aspartate), PEP was less enriched than 2PGA and 3PGA (30 ± 2% compared with 69 ± 3 and 67 ± 0.67%, respectively), pyruvate and alanine were relatively weakly labeled (18 ± 1% and 14 ± 2% enrichment, respectively, there was substantial labeling of glycine and progressively less of serine and glycerate (36 ± 2, 11 ± 11, and 1.9 ± 0.58%, respectively; 2PG was not analyzed in this experiment), and there was negligible labeling of succinate, 2OG, and glutamate (0.09 ± 0.16, 0.66 ± 0.27, and 0.60 ± 0.2% enrichment, respectively).

42We used PEPC and NADP-MDH activities as markers for the MCs and total RuBP as a marker for the BSCs. They were compared with isotopomer amounts in the four fractions from the net filtration (Supplementary Table S11) to estimate the distribution of each isotopomer of the other metabolites between the BSCs and MCs (Fig. 4; Supplementary Table S12). As the signals for 3PGA and 2PGA could not be differentiated in all fractions, they were combined and termed ‘PGAs’.

43As shown in Fig. 4, malate and aspartate were preferentially located in the MCs, with aspartate showing an especially marked asymmetric distribution. Alanine showed a slight preferential distribution towards the BSCs, pyruvate was preferentially located in the BSCs, PEP showed a strong preferential distribution towards the BSCs, PGAs was preferentially located in the BSCs, and triose-P in the MCs. In all cases, a similar distribution was found for the unlabeled form and each isotopomer.

44The amount in the BSCs and MCs (Table 3) was estimated by multiplying the fraction of the summed isotopomers found in the BSCs and the MCs, respectively, by the average active pool size from the time kinetic/chase experiment (Supplementary Table S4). For malate, the fraction of summed 13C isotopomers was multiplied by the active malate pool size at 60 min (Supplementary Table S3). We then related these amounts to the cytoplasmic volume since diffusion will occur between the cytoplasm of the BSCs and the MCs. For this, we assumed that BSCs occupy 19% of the leaf, that MCs have an ~3.8-fold larger volume than the BSCs, and that the cytoplasm occupies ~50% of BSC volume and 10% of MC volume. These published data allow us to take into account that the BSCs and MCs occupy a different proportion of the maize leaf, and have a different internal architecture.

45We estimated a malate concentration of 11.2 mM in the MCs and 5.2 mM in the BSCs, resulting in a large (6 mM) gradient between MCs and BSCs (Table 3). Aspartate was even more asymmetrically distributed, with estimated concentrations of 1.17 mM in the MCs and 0.21 mM in the BSCs, but the smaller pool size means that the concentration gradient (0.96 mM) was about a sixth of that for malate. These concentration gradients are in the direction needed to drive diffusion of malate and aspartate in the BSCs. Pyruvate showed a very asymmetric distribution with estimated concentrations of 1.53 mM in the MCs and 0.26 mM in the BSCs, resulting in a concentration gradient of 1.27 mM in the opposite direction to that required to drive diffusion of pyruvate from the BSCs to the MCs. Alanine had an estimated concentration of 11.5 mM in the BSCs and 10.8 mM in the MCs, leading to a small concentration gradient (0.68 mM) from the BSCs to the MCs. Alanine showed only a weakly asymmetric distribution between the MCs and BSCs, making the calculation sensitive to errors in the assumed volumes. 3PGA had a higher estimated concentration in the BSCs (4.73 mM) than in the MCs (1.87 mM), and triose-P had a higher estimated concentration in the MCs (2.73 mM) than in the BSCs (1.29 mM), providing a concentration gradient of 2.87 mM to drive diffusion of 3PGA from the BSCs to the MCs, and 1.43 mM to drive diffusion of triose-P from the MCs to the BSCs. PEP concentrations of 0.79 mM and 0.30 mM were estimated for the BSCs and MCs, respectively, providing a small (0.4 mM) concentration gradient from the BSCs to the MCs. Although the absolute magnitude is small, it is based on a highly asymmetric distribution of PEP. The PEP:3PGA ratio in the MCs (0.166) and BSCs (0.2) resemble those expected if 3PGA and PEP are close to thermodynamic equilibrium.

46Some of our results recapitulate the studies with 14CO2 that led to the elucidation of C4 photosynthesis, in particular the rapid labeling of C4 acids and slower labeling of pyruvate. A recent study of the role of the DCT2 transporter reported rapid labeling of malate and aspartate, and labeling of photorespiration intermediates. However, we have analyzed a larger set of metabolites and included longer labeling kinetics to allow separation of active and inactive pools. Further, we have determined metabolite concentrations and labeling patterns in the MCs and BSCs, and estimated concentration gradients between the MCs and BSCs. Our analyses provide information about which metabolites are involved in the CCS and how rapidly carbon is exchanged between the CBC and CCS, and raise questions about how metabolism is regulated to allow efficient operation of C4 photosynthesis.

47Our approach has some limitations. First, it is not possible to resolve CBC labeling kinetics (see also; ). However, rapid randomization aids interpretation of labeling kinetics in metabolites downstream of the CBC. Secondly, overall metabolite levels show leaf-to-leaf and within-leaf variation in maize. The labeled pools showed less variation, indicating that the variation is mainly due to pools not directly involved in photosynthesis. Thirdly, some metabolites exhibit complex labeling kinetics, with part remaining unlabeled while the remainder is present as multiple or fully labeled isotopomers. We interpret this as evidence for the presence of multiple pools, with only one being active in photosynthesis. Separation of active and inactive pools is essential to estimate concentrations and fluxes, and can be achieved by labeling for long enough to label the active pool fully. Fourthly, as MS does not provide position-specific information, we distinguished between label in the C4 and the C1–C3 positions of malate and aspartate by assuming all labeled forms have a 13C at the C4 position. This approach is supported by the similarity between the labeling kinetics we estimated for positions C1–C3 of malate and aspartate, and measured for pyruvate and alanine. Fifthly, we only obtain partial enrichment of MCs and BSCs, and errors are introduced in corrections for cross-contamination. In addition, our calculations of concentrations use published values for cell and cytoplasmic volume. For these reasons, the estimated MC and BSC concentrations are only approximations.

48An ultimate goal is to use global enrichment kinetics to model fluxes. However, this is complicated by redundancy in C4 photosynthesis. Based on labeling kinetics alone, it is difficult to decide whether 13C is detected in aspartate and alanine because they are directly involved in C4 photosynthesis or because they exchange 13C with OAA and pyruvate. A similar problem arises for formation of PEP in the BSCs, which may be labeled by decarboxylation of labeled four-carbon metabolites or 13C exchange with 3PGA. For this initial study, we restricted our calculations to early points in the time kinetics, and tried to support these calculations by estimating intercellular concentration gradients to provide independent evidence that flux is carried by a particular metabolite.

49Earlier studies reported gradients for 3PGA movement from the BSCs to the MCs, and for malate and triose-P movement from the MCs to the BSCs, but no gradient for pyruvate. These studies assumed the whole pool was involved in C4 photosynthesis, as opposed to being partly located in, for example, the vacuole or other cell types. The observation that large amounts of malate remain in darkness or at CO2 subcompensation point indicated that this might be the case. Similarly, it remained possible that compartmentation masked a gradient for pyruvate. Dynamic 13CO2 labeling allows us to measure the size and distribution of the pools that are actually involved in photosynthesis, which we term the active pool. For malate, this represents ~40% of the total pool. Labeling of the other metabolites involved in the intercellular shuttles was high by 40–60 min, showing that most of their pool is involved in C4 photosynthesis.

50The estimated concentration of the active malate pool is ~2-fold higher in the MCs than in the BSCs, resulting in an estimated gradient of ~6 mM from the MCs to the BSCs. 3PGA is preferentially located in the BSCs and triose-P in the MCs, providing estimated gradients of ~2.9 mM for 3PGA from the BSCs to the MCs, and 1.4 mM for triose-P from the MCs to the BSCs. There were also concentration gradients for further metabolites including a gradient for PEP from the BSCs to the MCs (0.4 mM), for aspartate from the MCs to the BSCs (0.96 mM), and for alanine from the BSCs to the MCs (0.68 mM) (see below for more discussion).

51The labeled pyruvate pool showed strong preferential distribution towards the MCs. It therefore remains unclear how pyruvate moves from the BSCs to the MCs. One possibility is that after its formation in the BSC chloroplasts, pyruvate is actively exported to the cytosol, or that pyruvate is actively taken up into MC chloroplasts. Active pyruvate uptake has been measured in MC chloroplasts. While a sodium-dependent transporter has been identified in Flaveria species, pyruvate transporters have not yet been identified in species such as maize where uptake is proton dependent. The transporter that exports pyruvate from BSC chloroplasts is also unknown.

52The estimated gradients for malate, 3PGA, and DHAP in our study are smaller than those previously reported for overall pools (;, b). This is probably because we used a lower irradiance (480 µmol photons m−2 s−1 compared with 1300–2500 µmol photons m−2 s−1). It should be noted that diffusion will be driven by the concentration gradient between the BSC and MC cytosols. Our calculations assume an equal distribution of metabolites between the cytoplasm and chloroplast, and the gradients will be different if the metabolite is preferentially located in one of these compartments. This may explain why the estimated concentration gradient is larger for 3PGA than for triose-P. Due to the pH gradient between the plastid stroma and the cytosol and their charge properties, 3PGA is preferentially located in the stroma and triose-P in the cytosol.

53It has been proposed that NADP-ME-type plants such as maize operate parallel CCSs via PEPCK or NAD-ME. Aspartate aminotransferase (AspAT) activity is high in maize leaves, alanine aminotransferase (AlaAT) activity is only 3- to 10-fold lower, and PEPC, AspAT, and AlaAT show very similar developmental profiles (;; data replotted in their Supplementary Fig. S3). PEPCK is expressed in the BSCs and supports high rates of aspartate-dependent photosynthesis in isolated maize BSCs. Many studies reported the presence of substantial pools of aspartate and rapid labeling of aspartate in NADP-ME species such as maize. However, these studies remained inconclusive because it was unclear how strongly aspartate was labeled and whether there was a concentration gradient to drive its movement from the MCs to BSCs.

54Our study reveals that there is a rapid and near complete labeling of aspartate, that the enrichment kinetics and isotopomer distribution of aspartate are almost identical to those of the active malate pool, and that label in aspartate is rapidly lost in a chase. They also reveal a strongly asymmetric distribution of aspartate, resulting in a distinct concentration gradient between the MCs and BSCs. The gradient (0.96 mM) is ~6-fold smaller than the estimated gradient for malate, consistent with a shuttle involving aspartate carrying ~4% of the CO2 into the BSCs. This is in quite good agreement with the initial labeling kinetics estimated for the C4 positions of malate and aspartate, which indicate that aspartate carries ~10% of the carbon into the BSCs. Our estimates are lower than the 34% estimated by (see below for further discussion).

55In the BSCs, aspartate might be deaminated and decarboxylated by PEPCK, or further reduced to malate and decarboxylated by NAD-ME or NADP-ME. In the former case, PEP is formed and would have to return to the MCs. Both the overall and the labeled pool of PEP are ~4-fold higher in the BSCs than in the MCs, providing a concentration gradient of ~0.4 mM to drive movement from the BSCs to the MCs. If aspartate moves from the MCs to the BSCs, amino groups will have to return to the MCs. postulated that they might return as alanine. Our study reveals that the labeling kinetics of alanine are almost identical to those of pyruvate, and that alanine is strongly enriched by 40–60 min, implying that most of the large alanine pool is involved in C4 photosynthesis. Further, and in contrast to pyruvate, we estimate a small concentration gradient for alanine (0.69 mM) between the BSCs and MCs. It should, however, be noted that the alanine pool showed only a slight asymmetric distribution, so this estimate will be sensitive to noise in our data and our assumptions.

56proposed that the NADP-ME species maize operates a parallel CCS via PEPCK. This proposal was based on PEPCK showing a similar developmental gradient to other C4 enzymes and having a high expression and activity in the BSCs. Our study supports this idea. The estimated concentration gradient for movement of aspartate to the BSCs was 0.96 mM, compared with gradients of 0.4 mM and 0.68 mM for movement of PEP and alanine back to the MCs. Making the reasonable assumption that fluxes are proportional to the intercellular concentration gradients, ~40% of the aspartate may be decarboxylated by PEPCK and the remainder by NAD- or NADP-ME. However, it is important to stress that these estimates are approximate. It is also possible that some aspartate is converted to malate in the BSCs and decarboxylated by NADP-ME. This pathway would not transfer reducing equivalents from the MCs to the BSCs. It operates in some NADP-ME species such as Flavaria bidentis, which have high PSII activity in the BSCs. In maize, it is presumably restricted by low BSC PSII, but a minor contribution might be possible especially if, as postulated by, PSII activity were to co-vary with aspartate decarboxylation via NADP-ME. There is some evidence that the distribution of PSII between the MCs and BSCs in maize can vary.

57It has been proposed that operation of multiple decarboxylation pathways is advantageous because it allows several metabolites to be involved in the CCS, permitting a given rate of photosynthesis to be achieved with lower concentrations of each individual metabolite. It may also confer robustness in a fluctuating environment. There may be considerable flexibility in the extent to which different decarboxylation pathways are used, even in a given species. While there was a marked asymmetric distribution of aspartate between the MCs and BSCs, and a gradient of PEP between the BSCs and MCs in the present study, gradients for aspartate and alanine were not detected previously, and gradients for PEP were either not detected or were in the opposite direction. The overall aspartate:malate ratio varies between studies, with reported values ≤0.1, as high as 0.2, or even 0.5, and <0.1 in our kinetic experiment and >0.2 in the material used to analyze intercellular distributions. While alanine levels are usually about half the overall malate content, this can vary, and the relative levels of aspartate, alanine, and malate vary between individual leaves and within a single leaf. Further experiments are needed to understand the reason for this variation, which may reflect differing contributions of NADP-ME and other decarboxylation enzymes, and its consequences for C4 photosynthesis under different environmental conditions.

58Operation of the CCS requires that the high concentrations of organic acids and amino acids are insulated from wasteful respiration, including insulation. Our labeling kinetics show that whereas the active malate pool, aspartate, pyruvate, and alanine approach full enrichment by ~20 min, there was negligible movement of 13C into TCA cycle intermediates such as succinate or 2OG, and amino acids derived from these organic acids.

59In maize, C4 photosynthesis requires two types of intercellular shuttle: the CCS that deliver CO2 to the BSCs and the 3PGA/triose-P shuttle that supplies NADPH and ATP to the BSCs. This raises the question of how they are co-ordinated. Excess CCS would result in CO2 overaccumulation in the BSCs and increased wasteful back leakage of CO2 to the MC. Up to 20–30% of the carbon incorporated by PEPC may leak back, which will decrease quantum yield because energy is required to drive the CCS. On the other hand, a low CCS would lead to a decrease in the BSC CO2 concentration and a wasteful increase in photorespiration. Flux in the shuttles will depend on the rate of diffusion, which depends on the magnitude of the concentration gradients and will therefore be constrained by total pool size. This makes it important to ask if carbon can be exchanged between the CCS and the 3PGA/triose-P shuttle and, if so, how quickly.

60Earlier studies showed that there is a large increase in the levels of pyruvate, 3PGA, and DHAP but no marked change in the level of malate during induction of photosynthesis in maize, and that higher light intensities lead to a progressive increase in the steady-state levels of pyruvate, 3PGA, and, especially, DHAP, but not of malate or PEP. It was proposed that carbon moves between the CCS and CBC, including the large pools in the 3PGA/triose-P shuttle. However, these conclusions were qualitative, and depended on comparisons at different light intensities or different times during the induction of photosynthesis.

6113CO2 labeling allows carbon flow to be investigated during steady-state photosynthesis. Our study reveals considerable movement of carbon from the CBC into the CCS, with 9–13% of the 13C fixed by RuBisCO in the first 30 s moving into CCS metabolites. Further evidence for carbon exchange between the CBC and the CCS is provided by comparing the labeling kinetics of CBC intermediates in maize with the C3 plant Arabidopsis in 2% O2 where photorespiration is decreased to mimic the situation in maize. The initial increase of enrichment in CBC intermediates is faster in maize, as expected from its higher rate of photosynthesis. However, between 5 min and 20 min CBC metabolite enrichment increases more slowly in maize than in Arabidopsis, indicating that there is an influx of unlabeled carbon into the CBC in maize. The most likely source is the C1–C3 positions of malate and aspartate, and pyruvate and alanine. It requires >20 min for their enrichment to approach that of the CBC intermediates. This 13C is derived from the CBC (see above) and, as the experiments were carried out at metabolic steady state, there must be an equivalent flux of unlabeled carbon from malate, aspartate, pyruvate, and alanine into the CBC.

62Taken together, our results reveal substantial movement of carbon between the CCS and the 3PGA/triose-P shuttle. During steady-state photosynthesis, this involves carbon exchange, but in non-steady-state conditions it will facilitate net carbon transfer. Total carbon in the CBC, including 3PGA and triose-P, was 4144 nmol g FW−1, total carbon in the CCS (positions C1–C3 of the active malate pool and aspartate, plus pyruvate and alanine) was ~11 000 nmol g FW−1, and the rate of photosynthesis was ~55 nmol CO2 g FW−1 s−1. Exchange between the CCS and CBC equivalent to 9–14% of the rate of photosynthesis could generate a 50% change in the total CBC pool including 3PGA and triose-P in ~10 min, and in the total pool of CCS metabolites in 30 min.

63Our results highlight PEPC as important for regulating allocation between the CBC and CCS. The estimated concentration of PEP in the MCs (0.38 mM) is below the measured Km of maize PEPC (0.6–1.5 mM;; ) and especially the likely Km in vivo in the presence of inhibitory metabolites and at low Mg2+ representative of that in the cytosol. Changes in PEPC activity will alter the level of PEP and, via equilibration, 3PGA, as well as allocation between the CCS and CBC.

64Our study and that of reveal a low but significant rate of photorespiration. We show that 2PG is labeled within 10 s, although less strongly than CBC intermediates. Label in 2PG represented <0.5% of fixed 13C at early time points, but the 2PG pool is very small and will turn over rapidly. The appearance of label in glycine, serine, and glycerate provides a better qualitative picture of the rate of photorespiration, and resembled the C3 plant Arabidopsis in 2% O2. Based on the response of photosynthesis in maize to O2, we estimate the rate of photorespiration in 420 ppm CO2 to be ~5% of that of carboxylation, which is ~5-fold lower than in a C3 plant in air. However, this may depend on the conditions. For example, if PEPC activity were low, metabolite concentrations in the CCS would decrease (see above) with the risk that the supply of CO2 to the BSCs is decreased and photorespiration increased. This scenario might develop, for example, when stomatal closure leads to very low internal CO2 concentrations in the MCs. It might be speculated that this is one reason for the relatively high levels of photorespiratory enzymes in maize as in such conditions photorespiration might serve to increase energy dissipation.

65Supplementary data are available at JXB online.

66Fig. S1. Three major biochemical subtypes for C4 photosynthesis.

67Fig. S2. 13C labeling systems and quenching procedures.

68Fig. S3. Metabolic content of 13CO2-labeled maize leaf during the day.

69Fig. S4. Overview of 13C labeling kinetics from primary carbon metabolism by k-means clustering.

70Fig. S5. Time-course of mass distribution of metabolites from CBC, starch, and sucrose pathways.

71Fig. S6. Scheme of positional carbon incorporation in compounds from the CO2 shuttle.

72Fig. S7. Time-course of mass distribution of metabolites in malate and aspartate during a chase.

73Fig. S8. Regression plots of 13C amounts calculated with two approaches.

74Fig. S9. Time-course of mass distribution of photorespiration cycle intermediates, amino acids, organic acids, and sugars.

75Fig. S10. Amounts of photorespiratory intermediates in Arabidopsis thaliana labeled with 13CO2 under 21% and 2% O2.

76Fig. S11. Time-course of mass distribution of glycine in Arabidopsis thaliana labeled with 13CO2 under 21% and 2% O2.

77Fig. S12. Regression plots of 13C enrichments from the kinetic experiment and material for cell separation.

78Table S1. Specific isotopomer-dependent MS parameters used in selected reaction monitoring (SRM).

79Table S2. Corconfig.txt file used with the CORRECTOR program for the correction of aspartate, PEP, 2PG, and pentose-phosphate data.

80Table S3. Amounts of unlabeled form and [13C]isotopomers for each metabolite in the time kinetic pulse–chase experiments and the cell separation experiment.

81Table S4. Metabolic content in the time kinetic pulse–chase experiments and the cell separation experiment.

82Table S5. 13C enrichment (%) of metabolites in the time kinetic pulse–chase experiments and the cell separation experiment.

83Table S6. Relative isotopomer abundance (%) in the time kinetic pulse–chase experiments and the cell separation experiment.

84Table S7. Calculation of carbon-dependent 13C enrichment in the C4 position and in C1–C3 positions of malate and aspartate.

85Table S8. Estimation of 13C amounts in the CO2 shuttle, CBC, and first intermediates of starch and sugar intermediates, photorespiratory intermediates, and additional metabolites

86Table S9. Estimation of photorespiration in maize from gas exchange.

87Table S10. Arabidopsis thaliana at ambient (21%) and low (2%) O2: 13C enrichment (%) of photorespiratory and CBC intermediates, relative isotopomer abundance (%) of glycine and serine, and amounts of photorespiratory intermediates.

88Table S11. Metabolic content and activities of enzyme markers of the fraction obtained by cell separation of maize leaves.

89Table S12. Distribution of isotopomers in BSCs and MCs of maize leaves.