Influence of starch deficiency on photosynthetic and post-photosynthetic carbon isotope fractionations

Carbon isotope composition of starch-deficient mutants is primarily explained by photosynthetic ¹³C fractionations, and thus plant physiological responses to changes in carbon storage, rather than by post-photosynthetic ¹³C fractionations. 

Abstract

Carbon isotope (¹³C) fractionations occurring during and after photosynthetic CO₂ fixation shape the carbon isotope composition (δ¹³C) of plant material and respired CO₂. However, responses of ¹³C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ¹³C of organic matter (δ¹³C_OM), concentrations and δ¹³C of potential respiratory substrates, δ¹³C of dark-respired CO₂ (δ¹³C_R), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ¹³C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ¹³C_OM, δ¹³C_R, and δ¹³C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ¹³C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic ¹³C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ¹³C variations in starch-deficient mutants are primarily explained by photosynthetic ¹³C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism.

Introduction

Short-term variation in the carbon isotope composition (δ¹³C) in plant-respired CO₂ and in the related respiratory substrates is of wide interest for plant ecophysiological studies investigating carbon allocation (Brueggemann et al., 2011; Hagedorn et al., 2016; Galiano Pérez et al., 2017), for reconstructions of plant functional responses to climatic conditions (Ehleringer et al., 1997; Gessler et al., 2014; Ehlers et al., 2015), and for the plant biochemical community measuring and modeling metabolic carbon fluxes (Werner et al., 2011; Szecowka et al., 2013; Sweetlove et al., 2014). The enzymatic and diffusional carbon isotope fractionations during photosynthesis are well known and can be mathematically modeled (Farquhar et al., 1989), with the ratio of leaf internal (c_i or, more precisely, chloroplastic c_c) to atmospheric (c_a) CO₂ concentrations as a key parameter for the ¹³C depletion of organic matter relative to atmospheric CO₂. The c_i/c_a ratio is regulated by plant physiological parameters such as the photosynthetic assimilation rate (A_n) and stomatal conductance (g_s) in response to environmental conditions (Brugnoli et al., 1988; Scheidegger et al., 2000). Subsequently, the photosynthetic assimilates, such as leaf (transitory) starch and sugars, and downstream metabolites undergo additional, so-called post-photosynthetic (synonym: post-carboxylation) ¹³C fractionation processes during the maintenance of plant metabolism, respiratory processes, carbon allocation, and/or biosynthesis of various primary or secondary metabolites (Werner and Gessler, 2011; Werner et al., 2011). However, given that post-photosynthetic ¹³C fractionation processes can overlap with each other or with the c_i/c_a-driven photosynthetic ¹³C fractionations (Brandes et al., 2006), their magnitude and influence on short-term δ¹³C variation in leaf dark-respired CO₂ (δ¹³C_R) and respiratory substrates is still under debate (Werner and Gessler, 2011; Gessler et al., 2014).

One of the most important post-photosynthetic ¹³C fractionations is probably related to an equilibrium isotope effect on the plastidic fructose-1,6-bisphosphate aldolase (pFBA) reaction (Brugnoli et al., 1988; Gleixner et al., 1993; Gleixner and Schmidt, 1997). This isotope effect potentially explains the heterogeneous ¹³C distribution in carbohydrates that has been observed in glucose derived from C₃ beet sucrose and C₄ corn starch (Rossmann et al., 1991) or from transitory starch in potato and beet leaves (Gleixner et al., 1993; Gilbert et al., 2012). The pFBA reaction reflects a metabolic branching point favoring ¹³C in fructose-1,6-bisphosphate under equilibrium conditions (Fig. 1). This ¹³C enrichment is passed to transitory starch (or ribulose-1,5-bisphosphate), whereas the ¹³C depleted triose phosphates are exported into the cytosol for sugar biosynthesis (e.g. sucrose) and glycolysis. Such a mechanism probably explains why transitory starch has been found to be more ¹³C enriched than sugars in beet leaves (Gleixner et al., 1993). The pFBA reaction has been suggested to cause diel δ¹³C variation in sugars and starch (Brugnoli et al., 1988; Gleixner et al., 1993; Goettlicher et al., 2006; Gessler et al., 2007; Lehmann et al., 2015), thereby also influencing δ¹³C_R and the apparent respiratory ¹³C fractionation (e; Ghashghaie et al., 2001, 2003; Werner et al., 2009). In fact, diel δ¹³C variations in starch and in the organic acid malate have been identified as main drivers of diel changes in δ¹³C_R across different soil moisture and temperature conditions in potato plants (Lehmann et al., 2015). Diel δ¹³C variation in leaf starch and sugars related to the pFBA reaction has also been found to partially influence the δ¹³C of phloem-exported sugars and source to sink ¹³C fractionations (Goettlicher et al., 2006; Gessler et al., 2008; Barthel et al., 2011), and thus ultimately the δ¹³C of tree-rings (Jäggi et al., 2002; Gessler et al., 2014; Rinne et al., 2015).

Fig. 1.

Reaction scheme for potential post-photosynthetic ¹³C fractionations via pFBA in pgm mutant plants. Photosynthetic triose phosphates from the CBB cycle are linked via the pFBA reaction to starch biosynthesis in the chloroplast of wild-type plants. An equilibrium isotope effect (EIE) on the pFBA causes the triose phosphates used for starch biosynthesis to be more ¹³C enriched than those exported to the cytosol for sugar (e.g. sucrose) biosynthesis (Gleixner and Schmidt, 1997). In contrast, the main route for starch biosynthesis is blocked in pgm mutants. The surplus of ¹³C enriched triose phosphates in the chloroplast might be transported to the cytosol, potentially causing a ¹³C enrichment in sugars. Additional bypass reactions for the starch residue in pgm mutants are indicated. CBB cycle, Calvin–Benson–Bassham cycle; pFBA and cFBA, plastidic and cytosolic fructose-1,6-bisphosphate aldolase; PGM, phosphoglucomutase; TPT, triosephosphate translocator; Glc-6-PT, Glc-6-phosphate translocator; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate.

Both photosynthetic and post-photosynthetic ¹³C fractionations (e.g. via pFBA or respiration) are likely to be closely connected to leaf starch metabolism. Biosynthesis and storage of leaf starch occur in the chloroplast and are under strong control by the circadian clock, leading to significant diel variation in starch concentrations (Zeeman et al., 2007; Stitt and Zeeman, 2012; Kolling et al., 2015). Starch functions as a highly flexible buffer molecule, balancing carbon supply and demand, and was identified as an important factor maintaining plant performance and growth (Huber and Hanson, 1992; Sulpice et al., 2009). The leaf starch content also shows strong seasonal (Jäggi et al., 2002) and species-specific variation (Ivanova et al., 2008), and is additionally influenced by abiotic stresses such as drought (Lehmann et al., 2015; Galiano Pérez et al., 2017; Thalmann and Santelia, 2017). The regulation and pathways of starch biosynthesis are still under debate (Streb et al., 2009; Geigenberger, 2011).

To study starch biosynthesis and degradation, knockout mutants have proven to be invaluable (Stitt and Zeeman, 2012). However, mutant plants have only rarely been combined with stable isotope analysis. Pulses of ¹³CO₂ have been applied to mutant plants to study biosynthetic pathways (Figueroa et al., 2016; Baslam et al., 2017), but very few studies have included carbon isotope analysis at natural isotope abundances to investigate δ¹³C differences among starch fractions or explore respiratory ¹³C fractionations (Scott et al., 1999; Duranceau et al., 2001). Thus, it is not yet clear whether plant mutants are helpful for elucidating ¹³C fractionations and the resulting ¹³C isotope signature of organic compounds.

Mutant plants lacking the expression of a functional plastidial phosphoglucomutase (PGM) have negligible starch concentrations. The enzyme catalyzes the reversible interconversion of glucose-6-phosphate to glucose-1-phosphate (EC 2.7.5.1) and is the main route responsible for starch production (Periappuram et al., 2000; Streb et al., 2009). The remaining starch residue in the pgm mutant has been suggested to be produced via cytosolic bypass reactions (Geigenberger, 2011). A number of pgm mutants have been isolated from species such as Arabidopsis thaliana (Caspar et al., 1985), Nicotiana sylvestris (Hanson and McHale, 1988), Pisum sativum (Harrison et al., 2000), and Mesembryanthemum crystallinum (Cushman et al., 2008). The mutants feature a phenotype with a reduced growth rate (Caspar et al., 1985; Huber and Hanson, 1992; Geiger et al., 1995) but increased sugar concentrations during the day, higher leaf to phloem export, and enhanced root respiration (Brauner et al., 2014).

Findings from some studies have indicated a reduction of assimilation rates in pgm mutants compared with wild-type plants (Caspar et al., 1985; Huber and Hanson, 1992; Geiger et al., 1995; Sun et al., 1999). This may cause differences in photosynthetic ¹³C fractionations and thus changes in δ¹³C values of plant material, but the magnitude of such an effect has yet to be determined. On the other hand, the starch deficiency in pgm mutants probably influences post-photosynthetic ¹³C fractionations via pFBA. The reduced need for hexose-phosphates in the chloroplasts of pgm mutants causes an increased flux, in the form of triose-phosphates, to the cytosol, leading to soluble sugar biosynthesis. We therefore expect that the equilibrium isotope effect on the pFBA reaction is expressed to a lesser extent or not at all in the direction of starch, causing a ¹³C enrichment of cytosolic sugars and all downstream metabolites in pgm mutants compared with wild-type plants. Moreover, the PGM-knockout might also affect apparent respiratory ¹³C fractionations, given that the absence of starch metabolism may lead to changes in the δ¹³C of potential respiratory substrates (i.e. sugar, starch, and organic acids) and their pool sizes, thereby causing variation in δ¹³C_R. The study of pgm mutants may therefore help improve our mechanistic understanding of ¹³C fractionations in plants.

Here we performed several experiments with pgm mutants and wild-type plants of different species. We hypothesized that the starch deficiency induced by the PGM-knockout leads to changes in (i) photosynthetic (via leaf gas exchange) and (ii) post-photosynthetic ¹³C fractionations (via pFBA and respiration) and thus to δ¹³C variation in organic matter, dark-respired CO₂, and potential respiratory substrates. To test our hypothesis, we first screened pgm mutants and wild-type plants of four species for average differences in assimilate concentrations and δ¹³C values. We then investigated short-term ¹³C fractionation mechanisms in N. sylvestris pgm mutants and wild-type plants by analyzing the δ¹³C_OM, δ¹³C_R, and δ¹³C of individual compounds (e.g. sugars, starch, malate, and citrate) and their concentrations during a diel cycle. Finally, we measured the leaf gas exchange in both N. sylvestris genotypes to determine if potential δ¹³C differences caused by the PGM-knockout are influenced more by photosynthetic or post-photosynthetic ¹³C fractionations.

Materials and methods

Plant material

Experiment 1

We first screened wild-type and pgm genotypes of the four species A. thaliana, M. crystallinum, N. sylvestris, and P. sativum. All four species, including the facultative Crassulacean acid metabolism (CAM) plant M. crystallinum, use the C₃ photosynthetic pathway (δ¹³C values less than –30‰, Fig. 2C). Plants were grown from seeds in a climate-controlled growth chamber. Light was provided during a 12 h photoperiod with a photosynthetic photon flux density (PPFD) of ~160 µmol m^–2 s^–1. Relative humidity was constantly at 70% during the day/night cycle, while temperature was 22 °C during the day and 18 °C during the night. Two weeks after germination, plantlets were transplanted into 160 ml pots filled with potting soil. Timing of sampling was adapted to the different growth habits of the four species but occurred during the exponential growth phase of each species, prior to flowering or pot limitation. A.thaliana and M. crystallinum plants were sampled 28 d after germination, while P. sativum and N. sylvestris were sampled after 17 d and 42 d, respectively. At the end of each sampling day, leaf discs (20 mm²) of three individuals were punched and transferred to reaction vials, frozen in liquid nitrogen to inactivate metabolism, and immediately stored at –80 °C.

Fig. 2.

Screening of pgm mutants (experiment 1). Concentrations of starch (A) and soluble sugars (B), as well as δ¹³C values in organic matter (C) and sugars (D), measured in leaf discs at the end of the day in pgm mutants and wild-type plants of four species. Mean values ±1 SD are given (n=3).

Experiment 2

N.sylvestris wild-type and pgm mutant plants were grown from seeds under identical growth conditions to those in experiment 1. Two weeks after germination, plantlets were transplanted into 1 liter pots filled with potting soil. After 8 weeks, samples were taken over a 24 h period: fully expanded leaves from individual plants (n=3) were sampled after 0, 4, 8, 12, 14, 20, and 24 h, transferred to paper bags, and frozen in liquid nitrogen. In addition, aliquots of leaf dark-respired CO₂ and climate chamber air were taken at each point in time (see methods below).

Experiment 3

For leaf gas exchange measurements, an additional batch of N. sylvestris wild-type and pgm mutant plants were grown under conditions identical to those described above. Measurements were performed at three points in time on individual pgm mutants and wild-type plants (n=5). Leaf material was sampled on the next day after 4 h of light, as described above.

Isotope ratio analyses of leaf dark-respired CO₂

Leaf dark-respired CO₂ was sampled at the above-listed time points using the in-tube incubation method originally described by Werner et al. (2007) and modified by Lehmann et al. (2015). In short, leaf material was placed in 12 ml gas-tight glass vials (‘Exetainer’, Labco Ltd, Lampeter, Ceredigion, UK) and flushed with CO₂-free synthetic air (Pangas, Dagmersellen, Switzerland). The exetainer was immediately darkened for 4 min, and subsequently an aliquot of the sample air (now with respired CO₂) was transferred with a gas-tight syringe into a second exetainer pre-filled with dry N₂ (N₂ 5.0, Pangas). In addition, climate chamber air samples were taken and transferred to N₂-filled exetainers. The gaseous samples in the exetainers were then analyzed using a modified Gasbench II (Zeeman et al., 2008) connected to a Delta^PlusXP isotope ratio mass spectrometer (Thermo-Fisher, Bremen, Germany) with a precision of ~0.1‰ (SD) for a quality control standard (400 ppm CO₂ in artificial air).

Isotope ratio analysis of organic compounds

Extraction and purification of organic compounds

Leaf samples from all experiments were freeze-dried (Beta 2-8 LD plus, Martin Christ, Osterode am Harz, Germany) and subsequently milled to a fine powder with a steel ball-mill (MM 200, Retsch, Haan, Germany) for further chemical analyses. Owing to biomass limitations, some of the leaf material from experiment 1 was pooled for δ¹³C analysis of sugars (Table 1).

Table 1.

Comparison of modeled and observed δ¹³C values in Nicotiana sylvestris pgm mutants and wild-type (WT) plants

Nicotiana sylvestris	Experiment 3			Experiment 2
genotype	c i	δ13CM	δ13COM	δ13COM	Sugars	Starch	Malate	Citrate	δ13CR
WT	164.9±29.4	–27.4±1.7	–27.6±0.2	–32.6±0.4	–32.2±0.7	–31.8±0.6	–24.4±2.1	–25.3±2.0	–28.9±2.5
pgm	204.3±28.9	–29.7±1.7	–28.7±0.6	–33.4±0.4	–34.3±0.7	–33.1±1.1	–26.3±1.6	–27.9±2.7	–30.3±2.3
pgm − WT	39.4	–2.3	–1.1	–0.8	–2.1	–1.3	–1.9	–2.6	–1.4

Modeled δ¹³C values of assimilates (δ¹³C_M, ‰, Equation 1) derived from diel average c_i values (µmol mol^–1) and observed δ¹³C values of organic matter (δ¹³C_OM) are shown (all from experiment 3). The diel averages for δ¹³C_OM, δ¹³C of dark-respired CO₂ (δ¹³C_R), and δ¹³C of different substrates are also given (all from experiment 2). For all parameters, the genotype difference (pgm−WT) is indicated. Mean values ±1 SD are shown.

For isotope analysis, 100 mg of dry leaf material was transferred into a 2 ml reaction vial and the water-soluble compounds (WSCs) were extracted in 1.5 ml of deionized water at 85 °C for 30 min in a water bath according to Lehmann et al. (2015). After centrifugation (10 000 g, 2 min), the supernatant holding the WSC fraction was transferred into a new reaction vial and stored at –20 °C for further purification, while the pellet was used for isolation of starch (see below). Soluble carbohydrates (‘sugars’) and the bulk organic acid fractions were isolated from the WSC fraction by ion-exchange chromatography, as described by Lehmann et al. (2015). In brief, Dowex 50WX8 in hydrogen form and Dowex 1X8 in formate form (both 100–200 mesh, Sigma-Aldrich, Buchs, Switzerland) were packed in 5 ml syringes (B. Braun, Melsungen, Germany) arranged in a custom-built rack so that the outlet of the cation exchanger (Dowex 50WX8) syringe was connected to the inlet of the anion exchanger (Dowex 1X8) syringe. After extensive flushing of the Dowex material with deionized water, the WSC fraction (~1 ml) was added to the upper ion exchanger and the neutral sugar fraction was eluted with 30 ml of deionized water. Subsequently, the organic acid fraction was eluted from the anion exchanger with 35 ml of 1 M HCl. All aqueous and acidic samples were frozen at –20 °C and freeze-dried (Hetosicc CD 52-1, Heto, Birkerød, Denmark). The remaining pellet was dissolved in 1 ml of deionized water. All samples were stored at –20 °C until isotope analysis.

Leaf starch was isolated from the remainder of the hot water extraction by enzymatic hydrolysis (Richter et al., 2009; Lehmann et al., 2015). In short, the pellet was washed several times with 1.5 ml of a methanol/chloroform/water (MCW) solution and subsequently with deionized water, and then bench-dried overnight. On the next day, the pellet was additionally oven-dried at 60 °C for 1 h to fully remove chloroform residues, suspended in deionized water, and boiled at 100 °C for 15 min in a water bath to gelatinize starch. The starch was hydrolyzed with a heat-stable α-amylase (EC 3.2.1.1, Sigma-Aldrich) at 85 °C for 2 h and then centrifuged (10 000 g, 2 min). The supernatant was freed from enzymatic residues by using centrifugation filters (Vivaspin 500, Sartorius, Göttingen, Germany) and stored in 2 ml reaction vials at –20 °C until isotope analysis.

δ¹³C values of bulk leaf organic matter, sugars, and starch

δ¹³C analysis of bulk organic matter, sugars, and starch was performed using an elemental analyzer (Flash, ThermoFisher) coupled to a Delta^PlusXP isotope ratio mass spectrometer (Werner et al., 1999; Brooks et al., 2003). Leaf material was weighed into Sn capsules (5×9 mm, Säntis, Teufen, Switzerland), and aliquots of solubilized sugars and starch were pipetted into the Sn capsules and oven-dried at 60 °C. Positioning of the samples, blanks, and laboratory standards, as well as the referencing of the measurement, was done as suggested by Werner and Brand (2001). Measurement precision of a long-term quality control standard was typically better than 0.2‰ (SD). The applied chemical isolation methods were shown to be free of isotope fractionation by analysis of commercial standard materials with respect to the measurement precision.

δ¹³C values and concentrations of individual organic acids

The analysis of δ¹³C values and concentrations of individual organic acids (citrate and malate) was performed by coupling an Isolink HPLC device to a DeltaV isotope ratio mass spectrometer (all Thermo-Fisher; Lehmann et al., 2015, 2016b). Before analysis, the aqueous bulk organic acid fractions have to be passed through a 0.45 µm syringe filter (Infochroma, Zug, Switzerland). Organic acids were separated on a 4.6×300 mm Allure Organic Acids column (Restek, Bellefonte, PA, USA) at 8 °C using 100 mM KH₂PO₄ (pH 3) as a mobile phase with a flow rate of 500 µl min^–1. All organic compounds were oxidized to CO₂ at 99 °C using Na₂S₂O₈ under acidic conditions. The CO₂ was subsequently separated from the mobile phase and measured for its isotope ratio and concentration in the IRMS. Every 10 samples, a set of malate and citrate laboratory standards of different concentrations (10–180 ng C μl^–1) was analyzed. Offset corrections to EA-IRMS δ¹³C values and determination of concentrations were performed according to Rinne et al. (2012). The applied method was shown to be free of isotope fractionation by analysis of standard materials with respect to the measurement precision, which was typically better than 0.4‰ (SD).

Sugar and starch concentration measurements

Soluble sugars were directly extracted from leaf discs (experiment 1) or 10 mg of leaf powder (experiment 2) by two sequential additions of 80% ethanol and one addition of 50% ethanol, where the supernatant was recovered and pooled. During each extraction, the sample was heated to 80 °C for 30 min. Sugars were photometrically measured according to Mueller-Roeber et al. (1992). Starch in the remaining pellet was extracted and photometrically measured as described in Hostettler et al. (2011). Sugar and starch concentrations are presented in μmol glucose equivalents per mg dry mass calculated using a pre-determined conversion factor.

Leaf gas exchange measurements

A cuvette with a measurement area of 6 cm² coupled to a Licor 6400 was used for leaf gas exchange measurements (all supplied by LI-COR, Lincoln, NE, USA). Temperature and relative humidity conditions in the cuvette were 21.9±0.5 °C and 63.9±7.3%, respectively, throughout all diurnal measurements. A flow rate of 250 µmol s^–1, CO₂ concentration of 400 µmol mol^–1 CO₂, PPFD of 160 µmol m^–2 s^–1, and leaf temperature of 22.0 °C were constantly maintained. Climatic and light conditions in the cuvette were thus nearly identical to growth conditions.

Data analysis and calculations

Leaf gas exchange data were used to model δ¹³C values of recent assimilates (δ¹³C_M). The calculation was based on standard ¹³C discrimination models (Farquhar et al., 1982):

$${\displaystyle \begin{array}{l}{\displaystyle {\delta }^{13}{\mathrm{C}}_{\mathrm{M}}={\delta }^{13}{\mathrm{C}}_{\mathrm{A}\mathrm{i}\mathrm{r}}-(\mathrm{a}+(\mathrm{b}-\mathrm{a})\ast {\mathrm{c}}_{\mathrm{i}}/{\mathrm{c}}_{\mathrm{a}})}\end{array}}$$ (1)

where δ¹³C_Air reflects the diel average of climate chamber air (–13.7±1.2‰, mean ±1 SD), a stands for the diffusional isotope fractionation (4.4‰), b for the enzymatic isotope fractionation (27‰, mainly due to Rubisco-catalyzed carboxylation reactions), and c_i/c_a is the ratio of leaf internal and atmospheric CO₂ concentrations (c_a was held constant at 400 µmol mol^-1).

The diel apparent respiratory ¹³C fractionation was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle e={\delta }^{13}{\mathrm{C}}_{\mathrm{S}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}}-{\delta }^{13}{\mathrm{C}}_{\mathrm{R}}}\end{array}}$$ (2)

where δ¹³C_Substrate is the average δ¹³C value of a respiratory substrate, and δ¹³C_R is the average δ¹³C value of leaf dark-respired CO₂ during a 24 h diel cycle.

Linear mixed effects models were used to test the individual effects of species and genotype (experiment 1) or genotype and time (experiment 2) on gas exchange, δ¹³C values, and concentrations, as well as interactions between these effects. If no significant interaction between individual effects was found, the model was repeated without the interaction term. The individual plant ID was included as a random effect. Effects with P-values <0.05 were considered to be statistically significant. Where not indicated otherwise, all given errors denote standard errors. All statistical analyses were performed in R version 3.4.4. (R Core Team, 2018).

Results

Experiment 1: screening of pgm mutants and wild-type plants

Functioning of the PGM-knockout in all screened species was demonstrated by clearly lower starch concentrations in pgm mutants than in wild-type plants (Fig. 2A). Sugar concentrations were generally higher in pgm mutants than in wild-type plants, with the greatest difference in N. sylvestris plants, while only M. crystallinum plants showed little difference in sugar concentrations (Fig. 2B). Isotope analysis of leaf bulk organic matter showed a clear δ¹³C difference of 1.6‰ between pgm and the wild type for N. sylvestris plants (Fig. 2C) but not for other species. A similar tendency was found for δ¹³C values of sugars (Fig. 2D), with the largest differences being up to 2.0‰ for sugars of A. thaliana and M. crystallinum. Whether the results were statistically significant for each species could not be determined because all three replicates were pooled for the isotope analysis of sugars. In summary, the effect of the PGM-knockout was greatest for δ¹³C_OM and δ¹³C values of sugars in N. sylvestris plants, which also showed the largest difference in sugar and starch concentrations between the two genotypes.

Experiment 2: diel cycle of N. sylvestris pgm mutants versus wild-type plants

Given the above-mentioned results, we further investigated the mechanisms of ¹³C fractionation in N. sylvestris pgm mutants and wild-type plants by measuring δ¹³C values of leaf bulk organic matter (δ¹³C_OM), assimilates, organic acids, and dark-respired CO₂ (δ¹³C_R) during a diel cycle (Fig. 3; Table 1). In general, we observed clear effects of time and genotype for δ¹³C_OM and δ¹³C_R, and for δ¹³C values of all substrates (P<0.001 for both time and genotype). δ¹³C_OM values, ranging from –32.2 to –33.8 for both genotypes and all points in time, were on a diel average 0.8‰ more negative in pgm mutants than in wild-type plants. δ¹³C values of sugars and starch, ranging from –31.3‰ to –35.2‰ for both genotypes and all points in time, were on a diel average 2.1‰ and 1.3‰ more negative in pgm mutants than in wild-type plants. On a diel average, starch was 0.4‰ and 1.2‰ ¹³C enriched compared with sugars for wild-type plants and pgm mutants, respectively.

Fig. 3.

Diel δ¹³C variation (‰) in dark-respired CO₂ and substrates in leaves of Nicotiana sylvestris wild-type plants (WT; filled circles) and pgm mutants (experiment 2; open circles). The gray shaded area indicates night-time. Error bars can be smaller than data point symbols. Mean values ±1 SE are given (n=2–3).

In comparison with the δ¹³C values of plant assimilates, the δ¹³C_R and δ¹³C values of malate and citrate were less negative (except for some δ¹³C values at 0, 20, and 24 h) and showed wider variation during the diel cycle for both genotypes (Fig. 3; Table 1). δ¹³C_R values ranging from –25.7‰ to –33.6‰ increased by 4.3‰ during the day and decreased by 5.9‰ during the night for both genotypes. δ¹³C_R values were on a diel average ~1.4‰ more negative in pgm mutants than in wild-type plants, with the exception that after 8 h of illumination δ¹³C_R values were 3.6‰ more negative. δ¹³C values of malate and citrate, ranging from –22.5‰ to –31.9‰ for both genotypes and all points in time, were on a diel average 1.9‰ and 2.6‰ more negative for pgm mutants than for wild-type plants. δ¹³C values of malate showed an increase of 1‰ for pgm mutants and 2‰ for wild-type plants during the day, while a similar decrease of 4.1‰ during the night was observed for both genotypes. Also, δ¹³C values of citrate for pgm mutants showed an increase of 1.9‰ during the day and a decrease of 5.9‰ during the night, while the values for wild-type plants steadily decreased by 4.1‰ during the diel cycle. Thus, δ¹³C_R and δ¹³C values of all substrates were clearly more negative for pgm mutants than for wild-type plants.

We also measured assimilate and organic acid concentrations during a diel cycle in both pgm mutants and wild-type N. sylvestris plants (Fig. 4). As expected, the pgm mutant showed only very low diel starch concentrations, while wild-type plants showed a clear diel cycle, with the lowest and highest concentrations occurring at the beginning and end of the day (P<0.001 for the interaction between time and genotype). In contrast, sugar concentrations showed a clear diel cycle in pgm mutants, with values up to 76% higher during the day and up to 53% lower during the night compared with values in wild-type plants, which showed no clear diel cycle (P<0.05 for the interaction between time and genotype). Moreover, malate and citrate concentrations were both on average 28.5% lower in pgm mutants than in wild-type plants during the diel cycle (P<0.01). Malate concentrations were highest at the end of the day (nearly twice higher at 8 h in wild-type plants compared with concentrations in pgm mutants) and lowest at the end of the night in both genotypes (P<0.001), while citrate concentrations showed no distinct diel cycle in either genotype (P>0.05). Thus, both assimilate and organic acid metabolism were strongly influenced by the PGM-knockout.

Fig. 4.

Diel variation in substrate concentrations (µmol g DW^–1) in leaves of Nicotiana sylvestris wild-type plants (WT; filled circles) and pgm mutants (experiment 2; open circles). Sugar and starch concentrations are normalized to hexose units to facilitate comparison. The gray shaded area indicates night-time. Please note the y-axis scale differences between the upper and lower panels. Error bars can be smaller than data point symbols. Mean values ±1 SE are given (n=2–3).

Experiment 3: leaf gas exchange measurements and modeling of δ¹³C_M

The PGM-knockout affected the assimilation rate (A_n; P<0.001) and stomatal conductance to water vapor (g_s; P<0.05), causing up to 2.0 μmol m^–2 s^–1 and 14.5 mmol m^–2 s^–1 lower values, respectively, in pgm mutants than in wild-type plants of N. sylvestris (Fig. 5). Although the genotype differences in A_n and g_s tended to increase during the diurnal cycle, no clear significant temporal variations during the light period were observed (P>0.05). The changes in A_n and g_s caused up to 54.0 µmol mol^–1 higher intercellular CO₂ concentrations (c_i) in pgm mutants than in wild-type plants (P<0.001), but again without clear temporal variation (P>0.05).

Fig. 5.

Leaf gas exchange measurements in Nicotiana sylvestris wild-type plants (WT; filled circles) and pgm mutants (open circles) during a 12 h light period (experiment 3). A_n, net assimilation rate (µmol m^–2 s^–1), g_s, stomatal conductance (mmol m^–2 s^–1), and C_i, intercellular CO₂ concentration (µmol mol^–1). Mean values ±1 SE are given (n=5).

The c_i values derived from leaf gas exchange measurements were used to model the δ¹³C of assimilates (δ¹³C_M; Table 1, Equation 1). δ¹³C_M values of both genotypes were on a diel average consistent with the measured δ¹³C_OM values of wild-type plants from the same experiment. δ¹³C_OM differences between experiment 2 and 3 were probably caused by temporal δ¹³C differences in the climate chamber air. In particular, fossil fuel CO₂ emissions in winter have most probably caused lower δ¹³C_Air and thus lower δ¹³C_OM for plants of experiment 2 than for those of experiment 3, which was conducted in summer. The genotype difference in δ¹³C_M of 2.3‰ was similar to that for sugars (2.1‰), malate (1.9‰), and citrate (2.6‰), but tended to be greater than the difference observed for δ¹³C_OM (0.8–1.1‰) and δ¹³C_R (1.4‰). Thus, the genotype difference in both modeled and observed δ¹³C values showed a consistent ¹³C depletion of pgm mutants compared with wild-type plants across all experiments.

Apparent respiratory ¹³C fractionation and correlations with δ¹³C of substrates

We calculated the diel average apparent respiratory ¹³C fractionation using Equation 2. The e values differed among the potential respiratory substrates for both genotypes (Table 2), with the highest values observed for malate and citrate (up to 4.5‰) and the lowest values observed for sugars, starch, and bulk organic matter (up to –3.9‰). No clear significant differences were observed between the genotypes for any substrate (P>0.05, t-test). In addition, we investigated the correlation between individual δ¹³C_R values and the δ¹³C values of different substrates for the two genotypes and all points in time (Fig. 6). We found a strong correlation for malate (R=0.81, P<0.001) and citrate (R=0.63, P<0.001) but a weaker correlation for sugars (R=0.31, P<0.05) and no correlation for starch (R=0.09, P>0.05). An analysis of covariance (ANCOVA) showed that the relationship between δ¹³C_R and each δ¹³C_Substrate was not influenced by the PGM-knockout (P>0.05). In addition, genotype differences across the four species in the δ¹³C of sugars were found to be negatively related to a genotype difference in sugar concentrations (r²=0.61, P=0.118) and positively related to those in starch concentrations (r²=0.86, P=0.024; Fig. 7).

Table 2.

Average diel apparent respiratory ¹³C fractionation (e, Equation 2) for different potential respiratory substrates in leaves of Nicotiana sylvestris wild-type and pgm mutant plants (all from experiment 2)

Substrate	e (‰)
	Wild type	pgm
Organic matter	–3.7±0.5	–3.1±0.5
Sugars	–3.4±0.6	–3.9±0.5
Starch	–3.1±0.5	–2.8±0.6
Malate	4.5±0.3	4.0±0.4
Citrate	3.6±0.3	2.4±0.4

No significant differences between genotypes for any substrate were observed (t-test, P>0.05). Mean values ±1 SE are given (n=19–21).

Fig. 6.

Correlations between δ¹³C values of dark-respired CO₂ (δ¹³C_R) and different respiratory substrates (δ¹³C_Substrate) in leaves of Nicotiana sylvestris wild-type plants (WT; filled circles) and pgm mutants (open circles) for both genotypes and all points in time. Pearson correlation coefficients (R) and P-values (***P <0.001, *P <0.05) are given.

Fig. 7.

Relationships between genotype differences (pgm−WT) in δ¹³C values of sugars (δ¹³C_Substrate, ‰) and sugar (A) and starch (B) concentrations in leaves of Nicotiana sylvestris at the end of the day. The coefficient of determination (r²) and P-value are given in each case, based on linear regression (solid line).

Discussion

Our screening (experiment 1) confirmed that pgm mutants had lower starch concentrations but higher sugar concentrations at the end of the day compared with wild-type plants for most species (Fig. 2A, B), clearly demonstrating that the primary assimilate metabolism was affected by the PGM-knockout across all species. As shown for N. sylvestris plants (experiment 2), the sugar pool in pgm mutants was used up during the night, similar to the starch pool in wild-type plants for various biosynthetic and respiratory processes (Fig. 4). This demonstrates that the PGM-knockout leads to a re-routing of freshly assimilated triose phosphates towards cytosolic sugar biosynthesis, as plastidic starch biosynthesis is prevented in mutant plants (Streb and Zeeman, 2012). Thus, the lack of starch as an important temporary carbon sink and chemical energy buffer in pgm mutants is at least partially counterbalanced by higher sugar biosynthesis. The pgm mutant therefore has great potential as a tool to study ¹³C fractionation responses related to changes in the diel starch metabolism.

On the post-photosynthetic ¹³C fractionation via pFBA in response to pgm-induced starch deficiency

We hypothesized that post-photosynthetic ¹³C fractionation via pFBA plays an important role in the δ¹³C variation in plant material. We expected that, owing to the blocked starch biosynthesis in pgm mutants, the equilibrium isotope effect on the pFBA reaction would be only slightly or not at all expressed (Fig. 1). Given that more triose phosphates are exported to the cytosol under such conditions, more ¹³C enriched sugars and, by extension, more ¹³C enriched organic matter should be produced in pgm mutants than in wild-type plants. However, carbon isotope analysis of leaf organic matter and sugars of the four species revealed, in contrast to our hypothesis, either no clear δ¹³C differences or a ¹³C depletion in pgm mutants compared with wild-type plants of N. sylvestris (Fig. 2C, D). This shows that the pFBA-related post-photosynthetic ¹³C fractionation is probably not the main reason for the observed δ¹³C differences between the genotypes.

Theoretically, only one out of six triose phosphate molecules after CO₂ fixation is used for sugar/starch synthesis, while the other five molecules enter the regeneration part of the Calvin–Benson–Bassham cycle. The PGM-knockout might therefore affect the kinetic operation conditions of the pFBA reaction much less than expected. For example, the ¹³C enrichment of starch compared with sugars was 0.8‰ greater on a diel average in pgm mutants than in wild-type plants (Fig. 3). This is a surprising result given that the main route of starch biosynthesis is blocked by the PGM-knockout in the mutant plant. The ¹³C enrichment of the starch residue compared with that of sugars in pgm mutants must therefore be caused by a different biosynthetic pathway such as a cytosolic bypass reaction. Higher glucose-6-phosphate concentrations in chloroplasts of pgm mutants than in those of wild-type plants have been observed (Kofler et al., 2000). Assuming that pFBA is operating normally in pgm mutants, glucose-6-phosphate molecules could still show the typical ¹³C enrichment induced by pFBA. Transport of glucose-6-phosphate to the cytosol via the glucose-6-phosphate translocator in connection with the glucose-1-phosphate transport from the cytosol to the chloroplast (Fettke et al., 2011) would mean that the plastidic PGM could be bypassed by the cytosolic isoenzyme. Such a mechanism might explain why starch is more ¹³C enriched compared with sugars in the pgm mutant compared with wild-type plants.

The low temporal variation in the δ¹³C of starch and sugars is in line with observations that there is no clear circadian rhythm in δ¹³C of leaf bulk sugars and starch of other plant species (Sun et al., 2009; Lehmann et al., 2016b). However, this result contradicts previous findings that starch-related post-photosynthetic ¹³C fractionations influence (e.g. via pFBA) short-term δ¹³C variation in leaf and phloem assimilates (Gessler et al., 2008; Kodama et al., 2011; Lehmann et al., 2015). These opposing observations stress that species-specific differences need to be considered when modeling temporal δ¹³C variation in assimilates, including the extent of isotope effects on enzymatic reactions, compartmentalization of sugar/starch pools, and their pool sizes and turnover rates. In summary, potential post-photosynthetic ¹³C fractionation via pFBA cannot explain the observed δ¹³C difference between pgm mutants and wild-type plants.

pgm-induced starch deficiency affects daytime organic acid metabolism but not apparent respiratory ¹³C fractionations

The organic acid metabolism (i.e. δ¹³C values and concentrations of malate and citrate) and δ¹³C_R were clearly affected by the PGM-knockout-induced starch deficiency (Figs 3, 4). Both organic acids were generally ¹³C enriched compared with starch and sugars of both genotypes. This pattern can be explained by the activity of the phosphoenolpyruvate carboxylase (PEPC) reaction that catalyzes the conversion of phosphoenolpyruvate and hydrogen carbonate to oxaloacetate with a net isotope discrimination against ¹³C of –5.7‰ relative to CO₂ in equilibrium with hydrogen carbonate (Farquhar et al., 1989). The ¹³C enriched oxaloacetate functions as a precursor for malate and citrate. The PEPC reaction is therefore assumed to reflect an anaplerotic flux in C₃ plants that replenishes withdrawn carbon skeletons from the tricarboxylic acid (TCA) cycle (Werner et al., 2011; Lehmann et al., 2015). However, it is generally accepted that the TCA cycle is not fully functional during the day due to light inhibition of key enzymes (Hanning and Heldt, 1993; Tcherkez et al., 2005; Sweetlove et al., 2010). Given the light-induced limitation of the TCA cycle, the light-activated PEPC reaction (together with the non-inhibited malate dehydrogenase reaction) must be responsible for the often-observed accumulation of malate (via oxaloacetate) during the day (Scheible et al., 2000; Gessler et al., 2009; Igamberdiev and Bykova, 2018). In fact, we observed a simultaneous increase in δ¹³C values and concentration of malate for both N. sylvestris genotypes, although these increases were lower in pgm mutants than in wild-type plants (Figs 3, 4). This strongly suggests that the anaplerotic PEPC flux is down-regulated in pgm mutants during the day, potentially owing to the increase in sugar concentration which supplies glycolysis and the TCA cycle with additional carbon skeletons (Figs 2, 4).

Moreover, we observed that diel δ¹³C_R values were strongly related to δ¹³C values of malate and citrate for both genotypes but were weakly or not at all correlated with δ¹³C values of sugars and starch, respectively (Fig. 6). This result is in line with findings from previous studies that malate is a key substrate for leaf dark-respired CO₂ (Gessler et al., 2009; Lehmann et al., 2015, 2016b), particularly shortly after darkening, as shown in an experiment with position-specific ¹³C-labeled malate (Lehmann et al., 2016a). However, despite the lower daytime δ¹³C values and concentrations of malate in N. sylvestris pgm compared with wild-type plants (Figs 3, 4), we generally observed no genotype differences in the average diel apparent respiratory ¹³C fractionation for various potential substrates (Table 2) or in the δ¹³C relationships between dark-respired CO₂ and substrates (Fig. 6). Thus, starch deficiency induced by the PGM-knockout had no clear influence on apparent respiratory ¹³C fractionations.

pgm-induced starch deficiency causes photosynthetic ¹³C fractionations

Interestingly, the differences in sugar and starch concentrations between genotypes were related to the δ¹³C difference in sugars across all species (Fig. 7), with the largest δ¹³C difference corresponding to the largest differences in sugar and starch concentrations between N. sylvestris genotypes. This suggests that the changes in the assimilate pool cause ¹³C fractionations. It has been demonstrated in several studies that an increase in sugar concentrations (as observed in pgm mutants) decreases the photosynthetic activity of a plant (Krapp and Stitt, 1995; Paul and Driscoll, 1997; Burkle et al., 1998). This hypothesis is supported by our gas exchange measurements, which showed lower A_n and g_s values and higher c_i values in pgm mutants than in wild-type N. sylvestris plants during the day (Fig. 5). In particular, the lower assimilation rates in pgm mutants are widely supported by findings in A. thaliana and N. sylvestris plants (Caspar et al., 1985; Huber and Hanson, 1992; Geiger et al., 1995; Sun et al., 1999). Thus, the changes in starch and sugar pool sizes in response to the PGM-knockout (Fig. 7) have influenced leaf gas exchange and thus caused differences in photosynthetic ¹³C fractionations, explaining the observed ¹³C depletion in the organic matter, sugars, organic acids, and dark-respired CO₂ in pgm mutants (Figs 2, 3).

To determine whether photosynthetic ¹³C fractionations are actually the main driver of the δ¹³C differences observed between the N. sylvestris genotypes, we modeled δ¹³C values of assimilates from leaf gas exchange measurements and found that δ¹³C_M and δ¹³C_OM values from the same experiment were in good agreement (Table 1). Most importantly, the δ¹³C_M difference between the two genotypes of 2.3‰ was similar to the differences observed for sugars and organic acids. This again indicates that δ¹³C differences in fresh assimilates caused by the pgm-induced starch deficiency are primarily driven by photosynthetic ¹³C fractionations. Further, the smaller genotype difference for δ¹³C_OM of 0.8–1.1‰ compared with the difference for δ¹³C_M might be explained by structural components in organic matter. For example, cellulose reflects and integrates all leaf gas exchange variation that occurs during the period of growth or leaf expansion. Given that pgm mutants grow differently from wild-type plants (Huber and Hanson, 1992), the leaf gas exchange and thus the photosynthetic ¹³C fractionations that have shaped the δ¹³C of cellulose and organic matter might differ from those that have shaped the δ¹³C of assimilates measured at the end of the growth period. In addition, potential isotope fractionations related to sugar and starch biosynthesis may cancel each other out during the diel buildup of organic matter, reducing the genotype differences in δ¹³C_OM compared with δ¹³C_M. Overall, we conclude that the majority of δ¹³C genotype differences can be primarily explained by leaf gas exchange adaptations and thus by c_i/c_a-driven photosynthetic ¹³C fractionations, while potential post-photosynthetic ¹³C fractionations in response to the PGM-knockout are of minor importance.

Conclusions and implications

Through this investigation, we showed that the δ¹³C values of organic matter, substrates, and dark-respired CO₂ are strongly influenced by differences in leaf gas exchange and corresponding photosynthetic ¹³C fractionations. Changes in assimilate pool sizes due to the PGM-knockout may have induced the photosynthetic ¹³C fractionations by suppressing the photosynthetic activity of the mutant plants. δ¹³C variability in plant material might therefore be indicative of increases in sugar concentrations or changes in the sugar/starch ratio. It is likely that the observed shift in δ¹³C due to starch deficiency might also occur in non-genetically modified plants in response to changes in environmental conditions (e.g. drought) or nutrient availability; this topic should be investigated in future studies.

Moreover, we demonstrated that post-photosynthetic ¹³C fractionation (e.g. via pFBA or respiration) is not or only slightly affected by pgm-induced starch deficiency. Instead the isotopic signature of both plant mutants and wild-type plants is dominated by the photosynthetic ¹³C fractionation processes. This might have important implications for the reconstruction of leaf gas exchange responses to climatic conditions of the past using δ¹³C values of plant compounds or other biomarkers (Ehleringer et al., 1997; Gessler et al., 2014).

Finally, we conclude that δ¹³C changes in plant material may not only contain information on physiological and biochemical processes but might also be helpful for inferring and reconstructing genetic responses. Our findings might therefore be interesting for retrospective studies on tree decline and mortality using tree-ring growth patterns in combination with stable isotope analysis of carbon, oxygen, and hydrogen (Scheidegger et al., 2000; Cormier et al., 2018; Gessler et al., 2018) and with genome analysis (Heer et al., 2018). Our mutant approach thus paves the way for future studies exploring the biochemical and genetic background of isotope fractionations in plants.