Knockdown of glycine decarboxylase complex alters photorespiratory carbon isotope fractionation in Oryza sativa leaves.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration.

Introduction

In C3 plants, Rubisco operates in the leaf pan class="Chemical">mesophyll cells, where n>n class="Chemical">CO2 and O2 compete to react with ribulose-1,5-bisphosphate (RuBP). The carboxylation of RuBP results in the formation of two molecules of 3-phosphoglycerate (3-PGA) that are integrated into the Calvin–Benson cycle. Alternatively, the oxygenation of RuBP produces one molecule of 3-PGA and one 2-phosphoglycolate (2-PG). The 2-PG is primarily recycled via photorespiration through a complex and energy-consuming set of reactions, which spans the chloroplasts, cytosol, peroxisomes, and mitochondria (Bauwe ; Betti ). By scavenging 2-PG, photorespiration removes a strong inhibitor of enzymes in photosynthetic carbon metabolism (Anderson, 1971; Kelly and Latzko, 1976; Peterhansel ; Walker ) and recovers up to one molecule of 3-PGA for every two molecules of 2-PG. Nevertheless, a minimum of one out of four 2-PG carbon atoms is released as CO2 by the glycine decarboxylase complex (GDC) and can be lost by the plant (Bauwe, 2018).

The GDC is an atypical mitochondrial four-protein system, comprised of three enzymes (P-, T-, and L-protein) and the H-protein, which is a small lipoylated protein (Somerville and Ogren, 1982; Douce ; Bauwe, 2018). GDC plays a critical role in the photorespiratory cycle by catalyzing the conversion of two molecules of n class="Chemical">glycine into n>n class="Chemical">serine and one molecule of CO2 and NH3 (Somerville, 2001; Maurino and Peterhansel, 2010). However, in the absence of the H component, the GDC cannot oxidize glycine (Douce ; Parys and Jastrzębski, 2008), which can accumulate. In C3 plants, the impaired activity of the H-subunit leads to a knockdown (KD) of GDC activity and a photorespiratory phenotype (Ewald ). Plants with reduced GDC activity typically have lower rates of leaf photosynthesis, a depletion of Calvin cycle metabolites, an impairment of photorespiratory nitrogen re-assimilation, and the accumulation of photorespiratory metabolites (e.g. glycine) under current ambient CO2 and O2 partial pressures (Wingler ; Timm and Bauwe, 2013; Lin ).

This buildup of leaf photorespiratory metabolites can have a negative feedback effect on Calvin cycle activity. For example, n class="Chemical">glyoxylate produced by n>n class="Chemical">glycolate oxidation negatively impacts on the activation state of Rubisco (Wingler ; Peterhansel ). Additionally, disruption of the photorespiratory pathway may lead to alternative decarboxylation reactions of accumulated pools of photorespiratory intermediates, such as glyoxylate and hydroxypyruvate in the peroxysomes (Wingler , 2000; Tcherkez, 2006; Peterhansel ), and an increase in the ratio of moles of photorespiratory CO2 released per mole of O2 reacting with RuBP (α; Cousins , 2011; Walker and Cousins, 2013; Timm ). Furthermore, the accumulation of photorespiratory intermediates could also affect the rates of leaf CO2 evolved in the dark (Rd, μmol CO2 m−2 s−1) and the 13C composition of Rd (δ13CRd, ‰) (Ghashghaie ; Tcherkez ).

The multiple leaf metabolic reactions simultaneously consuming and releasing pan class="Chemical">CO2 in the light make it difficult to determine how changes in photorespiration affect rates of leaf net n>n class="Chemical">CO2 assimilation (A), mesophyll CO2 conductance (gm), refixation of (photo)respired CO2, and mitochondrial non-photorespiratory respiration rates (RL). However, photosynthesizing leaves discriminate against 13C during CO2 diffusion from the atmosphere to the chloroplast stroma (through both the air and liquid phases), and during carboxylation, photorespiration, and mitochondrial non-photorespiratory respiration processes, with a specific 13C fractionation for each diffusional or biochemical step (Evans ). The observed leaf net discrimination against 13C in the light (Δo, ‰) can be modeled with four 13C fractionation terms (‰): Δi, which accounts for the 13C discrimination during CO2 diffusion from the atmosphere to the intercellular air space and for the Rubisco 13C fractionation (~29‰, Ubierna and Farquhar, 2014; von Caemmerer ); Δgm, which accounts for the 13C discrimination during CO2 diffusion in the liquid phase to chloroplast stroma and depends on the magnitude of gm; and Δf and Δe which are associated with photorespiration and mitochondrial non-photorespiratory respiration activity, respectively (von Caemmerer and Evans, 1991; Flexas ; Tazoe ; Evans and von Caemmerer, 2013). Δf is primarily attributed to the glycine–serine reaction catalyzed by GDC, which releases CO2 depleted in 13C compared with substrate and tends to decrease Δo (Farquhar ; Ghashghaie ; Lanigan ). In contrast, Δe may increase or decrease Δo in relation to the difference between 13C composition (‰) of CO2 entering the leaf chamber during measurements and in the plant growth chamber (Gillon and Griffiths, 1997; Ghashghaie ).

The photorespiratory fractionation (f, ‰) estimated in vivo in multiple C3 species ranges between 8‰ and 16.2‰ relative to photosynthetic products (Ghashghaie ; Evans and von Caemmerer, 2013), with 11‰ predicted from the theory (Tcherkez, 2006). However, under photorespiratory conditions, when Rubisco pan class="Chemical">oxygenation exceeds the capan>city of the photorespiratory recycling of n>n class="Chemical">2-PG or in the presence of disruption of the photorespiratory pathway, Δf and f may vary due to changes in α associated with alternative decarboxylation of photorespiratory intermediates (Cousins , 2011; Walker and Cousins, 2013). Alternative photorespiratory bypasses may occur in the chloroplasts (e.g. glyoxylate may be enzymatically reduced back to glycolate or further oxidized to CO2, but with no RuBP regenerated; see Kebeish ), peroxysomes (non-enzymatic decarboxylation of glyoxylate to formate using H2O2 as oxidizing agent may lead to formation of serine; catalase may be also involved as reported in Wingler ), mitochondria (enzymatic oxidation of glycolate to glyoxylate with release of CO2 and synthesis of glycine; see Niessen ), and cytosol (enzymatic reduction of hydroxypiruvate to glycerate; see Timm ).

The aim of the present study was to test how changes in pan class="Chemical">carbon flux through the photorespiratory pathway influenced leaf n>n class="Chemical">CO2 and 13CO2 isotope exchange, both in the light and in the dark, in transgenic plants of Oryza sativa with the GDC H-subunit KD in mesophyll cells. Both gdch-KD and wild-type (WT) plants were grown under low photorespiratory conditions (atmospheric CO2 partial pressure of 184.2 Pa) to minimize any pleiotropic effects. In the light, measurements of leaf–atmosphere CO2 and stable carbon isotope exchange were performed under low photorespiratory and photorespiratory conditions (atmospheric O2 partial pressure of 1.84 kPa or 18.4 kPa, respectively, and CO2 partial pressure of 27.6 Pa). The disruption of the photorespiratory pathway in the gdch-KD plants was characterized by leaf photosynthetic traits, Δo, Δf, f, α, Rd, and δ13CRd, compared with the WT.

Materials and methods

Plant material

Generation of GDC-H knockdown transgenic rice lines

The generation and the characterization of three pan class="Species">Oryza sativa n>n class="Gene">gdch-KD transgenic lines, including gdch-38, was previously described by Lin . Line gdch-38 was selected for analysis in the present study since in Lin it had shown a more consistent photorespiratory-deficient phenotype under different O2:CO2 growing and measuring conditions compared with the other two gdch-KD lines. Untransformed O. sativa cv. IR64 line A009 (WT) was used as negative control for comparison with the gdch-KD line.

Plant growth conditions

Two batches of 10 transgenic n class="Gene">gdch-38 line (T4 generation) and 10 WT plants of n>n class="Species">O. sativa cv. IR64 were grown consecutively in a controlled-environment growth chamber (Gch; Bigfoot series, BioChambers Inc., Winnipeg, MB, Canada) at the School of Biological Sciences at Washington State University, Pullman, WA (USA). All plants were individually grown in 4 liter free drainage pots; soil, irrigation, and fertilization were as in Giuliani .

The daily photoperiod was 14 h, from 8.00 h to 22.00 h standard time. Light was provided by F54T5/841HO Fluorescent 4100 K and 40 W halogen incandescent bulbs (Philips) and was supplied in a bell-shaped pattern; that is, with increasing photosynthetic photon flux density (PPFD) during the first 2 h, a maximum PPFD of 600 μmol photons m−2 s−1 incident on the plant canopy for 10 h, and decreasing PPFD in the last 2 h. Air temperature (tair) was set at 22 °C in the dark period; after switching on the light, tair tracked the PPFD pattern; that is, it ramped during the first 2 h from 22 °C to 26 °C, then 26 °C for 10 h, and decreased to 22 °C in the last 2 h photoperiod. Air relative humidity was maintained at ~70%, corresponding to a maximum air vapor pressure deficit (VPD) of ~1.6 kpan class="Chemical">Pa. During the light period, the n>n class="Chemical">CO2 partial pressure (pCO2) in the Gch atmosphere was elevated to 184.2 Pa (2000 μmol mol−1). The 13C composition of the atmospheric CO2 during the light period (δ13CGch) was −41.6‰ and −30.6‰ for the first and second batch of grown plants, respectively. The δ13CGch was determined as described in Supplementary Methods S1 at JXB online, and was a proxy of the 13C composition of the CO2 in the tank used (during the second plant growing cycle a new tank was needed and no tank with 13CO2 composition comparable with the previous one was available).

Leaf biochemical analysis

Protein content

Protein immunoblot analysis was performed to determine the leaf abundance of GDC H-, P-, and T-subunits in fully expanded leaves of 4- to 5-week-old transgenic n class="Gene">gdch-KD and WT plants. For each genotype, two sepan>rate protein extractions were performed, each one using the leaf tissue collected from two plants, according to Koteyeva . Protein concentration was determined for each extract with an RC DC protein quantification kit (Bio-Rad, Hercules, CA, USA) and 20 µg of protein per extract were sepan>rated by 10% (w/v) n>n class="Chemical">SDS–PAGE for the GDC P-subunit or 15% (w/v) for GDC H- and T-subunits. Proteins were then transferred to a nitrocellulose membrane and immunoblots (n=2 for both gdch-KD and WT) were performed according to Koteyeva with primary antibodies for anti-Pisum sativum L. GDC H-, P-, and T-subunits (1:10 000) raised in rabbit (courtesy of Dr D. Oliver, Iowa State University). The L-subunit was not detected because antibodies were unavailable. The band intensities were quantified with ImageJ 1.37 software (NIH, USA).

Malate content

The leaf portions used for photosynthesis analysis in pan class="Gene">gdch-KD and WT plants (n=5) were sampled immediately after the leaf–atmosphere gas exchange measurements and frozen in liquid n>n class="Chemical">N2. Malate content per unit leaf surface area (mmol malate m−2) was then determined with a spectrophotometry-based assay as described by Hatch (1979), with modifications by Edwards .

Leaf physiological analysis

Coupled measurements of leaf–atmosphere CO2, H2O, and 13CO2 exchange

Measurements were performed in Pulpan class="Chemical">lman, WA, USA with a mean atmospheric pressure of 92.1 kn>n class="Chemical">Pa. Two LI-6400XT portable gas analyzers (LI-COR Biosciences, Lincoln, NE, USA; detecting 12CO2) operating as open systems were coupled to a tunable diode laser absorption spectroscope, which detects 12CO2 and 13CO2 isotopologs (TDLAS model TGA200A, Campbell Scientific, Inc., Logan, UT, USA; Bowling ; Barbour ; Ubierna ; Stutz ; Sun ). Additional technical information on the system setup are available in Supplementary Methods S2.

For the leaf photosynthesis measurements, each LI-COR was equipped with a 2×3 cm leaf chamber (pan class="Chemical">Lch) assembled with an LED light source (6400-02B; LI-COR Biosciences). Alternatively, leaf dark respiration measurements were performed using an 8×10 cm custom-built n>n class="Chemical">Lch having an adaxial glass window, and with a volume of ~100 cm3 (Barbour , based on Sharkey ). The chamber had a hollowed stainless steel frame sealed with a closed-cell foam gasket and was connected to a circulating water bath for temperature control in the lumen. Before dark respiration measurements, the leaf portions included in the Lch were exposed to the light, which was supplied by a LI-COR 6400-18 light source placed adjacent to the glass window.

Protocol for coupled measurements of leaf–atmosphere CO2, H2O, and 13CO2 exchange

The mid to distal portions of two fully expanded leaves from the same stem on 4- to 5-week-old plants (n=4 for n class="Gene">gdch-KD; n=5 for WT) grown under δ13CGch of −41.6‰ were used for leaf photosynthetic measurements. The leaves were positioned to cover the 6 cm2 Lch section area. Measurements were taken from 10.00 h until 16.00 h standard time under an O2 partial pressure (pO2) of 18.4 kPa (approximately the current atmospheric pO2) and 1.84 kPa, pCO2 (Ca) of 27.6 Pa, and 13C composition of CO2 (from a pressurized tank) entering the Lch (δin) of −48.0‰. PPFD was set at 1500 µmol photons m−2 s−1, leaf temperature (tleaf) at 25 °C, and leaf to air VPD was kept between 1.0 kPa and 1.5 kPa. The airflow rate through the LI-COR system was 300 µmol s−1 (~0.48 l min−1). In particular, a Ca below current ambient pCO2 (which was ~37 Pa) was chosen to amplify, under a pO2 of 18.4 kPa, the signals of the photorespiratory-deficient phenotype in the gdch-KD plants compared with the WT.

Under each experimental pan class="Chemical">O2 condition, leaf portions were acclimated for ~30 min and data were recorded for ~30–40 min. The rate of net n>n class="Chemical">CO2 assimilation per unit (one side) leaf surface area (A, µmol CO2 m−2 s−1), stomatal conductance to CO2 diffusion (gsC, μmol CO2 m−2 s−1 Pa−1), intercellular pCO2 (Ci, Pa), and the ratio Ci/Ca were determined.

For leaf dark respiration measurements, n class="Gene">gdch-KD and WT plants (n=4) grown at a δ13CGch of both −41.6‰ and −30.6‰ were used. Two plants per day (one gdch-KD and one WT) were taken out of the Gch at 9.30 h standard time and the mid to distal portions of 8–9 fully expanded leaves, similar to those used for the photosynthetic analysis, were enclosed in the custom-built Lch to cover the section area of ~76 cm2. Leaf portions were first exposed to a PPFD of 750 µmol photons m−2 s−1 for 20 min, 500 µmol photons m−2 s−1 for 15 min (at tleaf of 25 °C), and 100 µmol photons m−2 s−1 for 5 min (at tleaf of 30 °C). Measurements were taken under a pO2 of 1.84 kPa or 18.4 kPa for plants grown at a δ13CGch of −41.6‰ or −30.6‰, respectively. Ca was set at 35.0 Pa, and the airflow rate through the LI-COR was changed from 700 µmol s−1 to 500 µmol−1, and from 500 µmol s−1 to 350 µmol s−1 tracking the decreasing PPFD. A CO2 cartridge from a set of cartridges with δ13C from −6.2‰ to −4.8‰ was used, one per day, as CO2 source (the mean δin for all experimental conditions is shown in Supplementary Table S1). The different (higher) δ13CO2 composition entering the Lch with respect to the Gch (−41.6‰) was chosen to have the leaf carbon assimilates produced in the Lch with dissimilar (higher) δ13C signatures compared with those previously produced in the Gch. After 40 min of leaf light exposure, darkness was imposed in the Lch. Leaf CO2 evolution was measured at a pO2 of 18.4 kPa and tleaf of 30 °C for 195 min to determine the dynamics of the dark respiration rate per unit (one side) of leaf surface area (Rd, µmol CO2 m−2 s−1) and corresponding δ13C (δ13CRd, ‰). The tleaf was set at 30 °C to enhance the precision of the dark measurements. Additionally, three plants (n=3) of the gdch-KD line and of the WT were taken out of the growth chamber at 12.00 h standard time 3 d after their use for measurements, and darkened at 25 °C for 24 h. Subsequently, leaf dark CO2 evolution was measured at a tleaf of 30 °C and a pO2 of 18.4 kPa to determine Rd(24h) (µmol CO2 m−2 s−1) and δ13CRd(24h) (‰). The blade portions used for dark measurements on WT and gdch-KD plants were sampled and dried in a ventilated oven at 55 °C for 48 h to determine leaf dry mass per (one side) unit of leaf surface area (LMA, g m−2).

For each n class="Gene">gdch-KD and WT plant used for leaf photosynthesis measurements, the n>n class="Chemical">13C signature of leaf dry matter (δ13Cdm, ‰) and leaf total N content as a fraction (%) of dry matter were determined as described in Supplementary Methods S3, and the leaf total N content per unit leaf surface area (g m−2) was calculated. The descriptions, values, and units of abbreviations and symbols are listed in Table 1.

Table 1.

Description of the abbreviations, symbol, value (as in Evans and von Caemmerer, 2013), and unit of the environmental parameters and leaf variables used in this study

Abbreviation	Description
Gch	Growth chamber
GDC	Glycine decarboxylase complex
gdch-KD	Transgenic GDC H-subunit knockdown
Lch	Leaf chamber
LEDR	Light-enhanced dark respiration
NH3	Ammonia
NH4+	Ammonium cation
PDH	Pyruvate dehydrogenase
RuBP	Ribulose 1,5-bisphosphate
TCA	Tricarboxylic acid
2-PG	2-phosphoglycolate
3-PGA	3-phosphoglycerate
Symbol	Environmental parameters/leaf variables	Value and unit
A	Net CO2 assimilation rate per unit (one side) leaf surface area	µmol CO2 m−2 s−1
a	13C fractionation during CO2 diffusion (in air) through stomata	4.4‰
b 3	Rubisco 13C fractionation	29.0‰
C a	CO2 mole fraction or CO2 partial pressure set in the leaf chamber	µmol mol−1; Pa
C c	CO2 mole fraction or CO2 partial pressure in the chloroplast	µmol mol−1; Pa
C i	CO2 mole fraction or CO2 partial pressure in the intercellular air space	µmol mol−1; Pa
C in	CO2 mole fraction entering the leaf chamber	µmol mol−1
C out	CO2 mole fraction leaving the leaf chamber	µmol mol−1
C s	CO2 mole fraction at the leaf surface	µmol mol−1
f	Photorespiratory 13CO2 fractionation	‰
g m	Mesophyll conductance to CO2 diffusion from the substomatal cavity to the chloroplast stroma	µmol CO2 m−2 s−1 Pa−1
g sC	Stomatal conductance to CO2 diffusion	µmol CO2 m−2 s−1 Pa−1
LMA	Leaf dry mass per (one side) unit surface area	g m−2
pCO2	Partial pressure of CO2	Pa
pO2	Partial pressure of O2	kPa
PPFD	Photosynthetic photon flux density	µmol photons m−2 s−1
R d	Dark respiration rate per unit (one side) leaf surface area	µmol CO2 m−2 s-1
R d(24h)	R d after 24 h dark	µmol CO2 m−2 s−1
R d(30min)	R d after 30 min dark	µmol CO2 m−2 s−1
R d(3h)	R d after 3 h dark	µmol CO2 m−2 s−1
R d(6min)	R d after 6 min dark	µmol CO2 m−2 s−1
R L	Light mitochondrial non-photorespiratoy respiration rate per unit (one side) leaf surface area	µmol CO2 m−2 s−1
t	Correction factor for ternary effects	‰
t air	Air temperature	°C
t leaf	Leaf temperature	°C
VPD	Vapor pressure deficit	kPa
α	Moles of CO2 released in the photorespiratory pathway per mole of O2 reacting with RuBP	mol CO2 mol−1 O2
Δe	13C discrimination associated wtih mitochondrial non-photorespiratory respiration	‰
Δf	13C discrimination associated with photorespiration	‰
Δgm	13C discrimination associated with mesophyll conductance to CO2 diffusion	‰
Δi	13C discrimination due to carboxylation, boundary layer and stomatal CO2 diffusion	‰
Δo	Observed (instantaneous) leaf net discrimination against 13CO2 in the light	‰
Γ	CO2 compensation point	µmol mol−1; Pa
Γ*	CO2 compensation point in absence of mitochondrial non-photorespiratory respiration	µmol mol−1; Pa
δin	δ13C of CO2 entering the leaf chamber	‰
δout	δ13C of CO2 leaving the leaf chamber	‰
δRdGch_substr	Fractional contribution of respiratory substrates from Gch carbon assimilates to δ13C of dark-evolved CO2	‰/‰
δRdLch_substr	Fractional contribution of respiratory substrates from Lch carbon assimilates to δ13C of dark-evolved CO2	‰/‰
δ13C	13C composition of CO2	‰
δ13Cdm	13C signature of leaf dry matter	‰
δ13CGch	13C composition of atmospheric CO2 in the growth chamber during the photoperiod	‰
δ13CLch_Ph	Representative δ13C of carbon assimilates produced in the Lch	‰
δ13CRd	δ13C of CO2 evolved by leaves in the dark	‰
δ13CRd(24h)	δ13C of CO2 evolved by leaves after 24 h dark	‰
δ13CRd(30min)	δ13C of CO2 evolved by leaves after 30 min dark	‰
δ13CRd(3h)	δ13C of CO2 evolved by leaves after 3 h dark	‰
δ13CRd(6min)	δ13C of CO2 evolved by leaves after 6 min dark	‰

Leaf net 13CO2 discrimination in the light and mesophyll conductance to CO2 diffusion

The observed leaf net discrimination against pan class="Chemical">13CO2 in the light (Δo, ‰) was calculated by mass balance from the TDLAS measurements according to Evans . Under photorespiratory conditions (18.4 kn>n class="Chemical">Pa pO2), the 13CO2 fractionation for photorespiration (f, ‰) in the gdch-KD plants was calculated based on Evans and von Caemmerer (2013). Briefly, the value of f was determined by modeling the leaf net discrimination against 13CO2 (Δo) as a function of the 13C discrimination fractions associated with CO2 diffusion from the atmosphere to the intercellular air space and with carboxylation (Δi), with CO2 diffusion in liquid phase to chloroplast stroma (Δ), mitochondrial non-photorespiratory respiration (Δe), and photorespiration (Δf). The equation Δo=Δi−Δgm−Δf−Δe can be rearranged so that Δf=Δi−Δo−Δgm−Δe and f can be estimated by substituting Δf with to get . An f value of 16.2‰ was taken from Evans and von Caemmerer (2013) and assumed for WT plants. The input parameters needed to calculate f include the leaf mitochondrial respiration rate in the light (RL, µmol CO2 m−2 s−1), the CO2 compensation point in the absence of mitochondrial non-photorespiratory respiration (Γ*, μmol mol−1), and mesophyll CO2 conductance (gm, mol CO2 m−2 s−1). Values of RL at a tleaf of 25 °C were modeled for both genotypes from the corresponding Rd at 30 °C after 3 h in the dark [Rd(3h), μmol CO2 m−2 s−1] following leaf photosynthesis under atmospheric pO2 of 18.4 kPa using the temperature response function in Bernacchi . The Γ* was modeled based on von Caemmerer (2000), as described in Supplementary Methods S4, and was significantly different (P<0.05) between WT and gdch-KD plants, 45.0±1.7 SE (n=4) μmol mol−1 and 53.3±0.6 SE (n=3) μmol mol−1, respectively. Finally, gm was estimated based on leaf–atmosphere CO2 and 13CO2 exchange data, according to Evans and von Caemmerer (2013). Specifically, 13C-based gm was calculated in the gdch-KD and WT plants at 1.84 kPa pO2, but only in WT plants under 18.4 kPa pO2, using an f value of 16.2‰. The 13C-based gm cannot be calculated in gdch-KD plants at 18.4 kPa pO2 because gm and f are not independent variables in the applied procedure. Therefore, at 18.4 kPa, the gm values of gdch-KD plants were set the same as for the WT. This assumes that the 13C-based gm integrates the within-leaf resistances affecting CO2 movement across the cell wall, plasma membrane, and the chloroplast membranes, and that this cumulative resistance does not differ between gdch-KD and WT plants. This assumption is supported by the fact that the 18O-based gm, which was determined by analysis of leaf–atmosphere 18O exchange according to Ubierna , Kolbe and Cousins (2018), and Sonawane and Cousins (2019), was not significantly different between the gdch-KD and WT plants at 18.4 kPa pO2 (Supplementary Table S2). The 18O-based gm is not strictly associated with the biochemistry of photosynthesis as is the 13C-based gm and therefore cannot be used to estimate f. The values of 13C-based gm for gdch-KD and WT plants at each pO2 were used to calculate the corresponding pCO2 in the chloroplasts (Cc, Pa) by applying Fick’s first law.

The Γ* was defined in terms of Rubisco kinetic properties according to Jordan and Ogren (1984), and the estimate of n class="Chemical">CO2 released per n>n class="Chemical">O2 reacting with RuBP (α) was determined for the gdch-KD plants versus α set equal to 0.5 in the WT as described in Supplementary Methods S5. A sensitivity analysis for the dependency of f on Γ* and α is also described in Supplementary Methods S5.

13C composition of leaf dark-evolved CO2 and contributions of leaf chamber and growth chamber assimilates to substrates feeding leaf dark respiration

The pan class="Chemical">13C composition of the dark-evolved n>n class="Chemical">CO2 determining Rd (δ13CRd, ‰) was calculated according to Barbour as described by Evans .

The substrates feeding leaf dark respiration were from pan class="Chemical">carbon assimilates produced in the n>n class="Chemical">Lch and in the Gch. Given δ13CRd( as the mean values of δ13C for dark-evolved CO2 at time i from light–dark transition, the fractional contribution of Lch assimilates to δ13CRd( (δRdLch_substr(, ‰/‰) was calculated for gdch-KD and WT plant types after leaf photosynthesis under both O2 levels as

where δpan class="Chemical">13CRd( was determined by steps of 3 min over 195 min in the dark; δ13CRd(24h) is the mean δ13CRd after 24 h in the dark as shown in Supplementary Table S1; and δ13CLch_Ph (‰) is the representative δ13C of gdch-KD or WT carbon assimilates produced in the Lch at a pO2 of 1.84 kPa or 18.4 kPa before the light–dark transition (values are shown in Supplementary Table S1). The assumptions underlying Equation 1 and the calculation of δ13CLch_Ph are reported in Supplementary Methods S6.

Based on the total fractional contributions of pan class="Chemical">Lch and Gch n>n class="Chemical">carbon assimilates to δ13CRd equal to 1.0, the complementing fractional contribution of Gch assimilates to δ13CRd( [δRdGch_substr(, ‰/‰] was calculated for both plant types after leaf photosynthesis under both O2 levels as

In addition, to make a combined analysis of the data collected in the two pan class="Chemical">O2 experimental conditions possible, the δ13CRd generated from plants grown at the more depleted δ13CGch were edited to cancel out the bias in the δ13CGch effect on δ13CRd with respect to the other batch of plants. In particular, the δ13CRd following leaf photosynthesis at the lower O2 experimental level were edited through the procedure described in Supplementary Methods S7.

Leaf CO2 compensation points in the presence of RL

Leaf–atmosphere gas exchange measurements were taken with an LI-6400XT portable gas analyzer equipped with the 2×3 cm n class="Chemical">Lch on n>n class="Gene">gdch-KD and WT plants (n=4) at a PPFD of 1500 µmol photons m−2 s−1, tleaf of 25 °C, leaf to air VPD between 1.0 kPa and 1.5 kPa, Ca decreasing from 35.0 Pa to 3.7 Pa, and at a pO2 of 1.84 kPa or 18.4 kPa. For each leaf, a least square regression analysis of the response of A (µmol CO2 m−2 s−1) to Ci (Pa) was applied to the initial slope (for Ci≤9.2 Pa) to determine the CO2 compensation point in the presence of RL (Γ, Pa).

Statistical analysis

Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). A linear mixed effects model (PROC MIXED) was used with plant type (n class="Gene">gdch-KD and WT) and n>n class="Chemical">O2 level (pO2 of 1.84 kPa and 18.4 kPa) as fixed factors and leaves as random factor nested within plant type. The effects of plant type, O2 level, and plant type×O2 level interaction on A, gsC, Ci, Ci/Ca, Δo, Cc, Γ, Rd(6m), δ13CRd(6m), Rd(30m), δ13CRd(30m), Rd(3h), and δ13CRd(3h) were assessed. A PROC MIXED procedure was applied as a one-way ANOVA to determine the plant type (fixed factor) effect on the following traits: total N content, malate content, Γ*, Δo, Δi, Δi−Δo, Δgm, Δe, Δf, Rd(24h), δ13CRd(24h), LMA, and δ13Cdm. A one-sample t-test (P<0.05) was applied to test the difference of f or α modeled for the gdch-KD plants compared with a constant f or α value assumed in the WT. A two-sample t-test (P<0.05) was applied to test the difference between gdch-KD and WT gm at a pO2 of 1.84 kPa, and WT gm at the two O2 levels. For each plant type, a three-parameter non-linear model was fit to the Rd and δ13CRd responses determined over the 195 min in the dark after leaf photosynthesis at each O2 experimental level. In particular, the δ13CRd values associated with the lower O2 level had been first edited as described in Supplementary Methods S7, and then used for the analysis. The fitting model y=θ1e–θ2+θ3 was employed where x are minutes from 0 to 195 by steps of three, and y are Rd or δ13CRd values; θ1, θ2, and θ3 are the range, slope, and lower asymptote (or floor) parameters, respectively, which were determined using non-linear least squares with the iterative Gauss–Newton algorithm. Specifically, for Rd or δ13CRd responses, the range parameter corresponds to the difference between initial and lower asymptote values, and the slope is the exponential rate of change. An extra sum of squares F-test was applied to define the significance (P<0.05) of the effects of plant type and O2 level (main effects), and plant type×O2 level interactions on the three parameters of Rd or δ13CRd fitting models.

Results

Leaf GDC protein and malate content

Leaves of n class="Gene">gdch-KD plants had 21% (±2 SE) H-protein content compan>red with the WT, while the P- and T-protein content was 77% (±6 SE) and 83% (±2 SE) of that of the WT, respectively (Fig. 1; n=2). n>n class="Chemical">Malate content in leaf samples taken immediately after measurements of leaf photosynthesis were 0.49±0.08 mmol m−2 and 0.38±0.02 mmol m−2 (mean ±SE; n=5) in gdch-KD and WT plants, respectively (P=0.29).

Fig. 1.

Immunoblot analysis for GDC P-, T-, and H-subunits in mature leaves of gdch-KD compared with WT plants. The protein molecular weight of each subunit (kDa) is shown. Subunit protein abundances for gdch-KD plants are mean percentage values of the WT (n=2).

Immunoblot analysis for GDC P-, T-, and H-subunits in mature leaves of pan class="Gene">gdch-KD compan>red with WT plants. The protein molecular weight of each subunit (kDa) is shown. Subunit protein abundances for n>n class="Gene">gdch-KD plants are mean percentage values of the WT (n=2).

Leaf photosynthetic responses

There was no observable difference in growth phenotypes between the pan class="Gene">gdch-KD and WT plants when they were grown under 184.2 n>n class="Chemical">Pa pCO2 (2000 μmol mol−1). However, at approximately current ambient CO2 and O2 partial pressures, the net rate of CO2 assimilation (A) was lower in the gdch-KD compared with the WT but there was no significant difference in A between plant types under low photorespiratory conditions, when pO2 was reduced to 1.84 kPa (Table 2). There was, however, a significant effect of O2 level on gsC (negative effect) and Ci/Ca (positive effect) but not a plant type effect (Table 2). There was a significant plant type×O2 level interaction on Δo, which showed higher values for the gdch-KD compared with the WT at a pO2 of 18.4 kPa, but no difference at a pO2 of 1.84 kPa (Table 2). There was no significant plant type effect on gm at a pO2 of 1.84 kPa (P=0.586), and no O2 effect on gm in the WT (P=0.701; Table 2). There was, however, a significant effect of plant type on Cc, which showed comparable values in the gdch-KD and WT plants at 1.84 kPa pO2 and higher values in the gdch-KD plants compared with the WT at 18.4 kPa pO2 (modeled based on equal gm in both transgenic and WT plants). In addition, O2 level had a positive effect on Cc (Table 2). The Γ in the gdch-KD compared with WT plants was significantly lower under 1.84 kPa pO2 but higher under 18.4 kPa pO2 (Table 3). There was no significant difference in leaf N content between plant types, with means of 2.30±0.08 SE g m−2 and 2.31±0.14 SE g m−2 in gdch-KD and WT leaves, respectively (n=4).

Table 2.

Leaf photosynthetic traits estimated on gdch-KD and WT plants under approximately current ambient and below current ambient O2 levels (pO2 of 18.4 kPa and 1.84 kPa, respectively) at Ca of 27.6 Pa.

Plant-type	pO2	A	g sC	C i	C i /C a	g m a	C c	Δo
	(kPa)	(µmol CO2 m−2 s−1)	(µmol CO2 m−2 s−1 Pa−1)	(Pa)		(µmol CO2 m−2 s−1 Pa−1)	(Pa)	(‰)
gdch-KD	1.84	24.7±1.4	3.47±0.53	18.1±0.8	0.66±0.03	4.56±0.43	12.6±1.4	14.9±0.7
	18.4	6.3±0.3	1.37±0.11	22.1±0.2	0.80±0.01		20.6±0.2	23.7±0.5
WT	1.84	21.6±1.2	2.78±0.43	18.5±0.9	0.67±0.03	3.80±0.76	12.6±0.5	14.3±0.8
	18.4	14.3±0.8	2.45±0.31	20.5±0.5	0.74±0.02	4.07±0.14	17.0±0.6	17.8±0.3
Significance	Plant type	P=0.050	P=0.629	P=0.411	P=0.411	–	P=0.031	P=0.003
	pO2	P<0.001	P=0.018	P=0.003	P=0.003	–	P=0.000	P<0.001
	Plant type×pO2	P=0.002	P=0.057	P=0.171	P=0.171	–	P=0.033	P=0.003

Values are the mean±SE (n=4). Significance (P<0.05) of the effects of plant type, pO2, and plant type×pO2 interaction were evaluated by SAS PROC MIXED.

No significant differences were evaluated by a two sample t-test (significance for P<0.05) between the gdch-KD and WT gm values at a pO2 of 1.84 kPa (P=0.586), and the WT gm values at the two pO2 values (P=0.701).

Table 3.

CO2 compensation points (Γ) determined under low photorespiratory (1.84 kPa pO2) and photorespiratory (18.4 kPa pO2) conditions on gdch-KD and WT plants

Plant-type	pO2	Γ
	(kPa)	(Pa)
gdch-KD	1.84	0.25±0.06
	18.4	5.48±0.04
WT	1.84	0.62±0.12
	18.4	4.54±0.18
Significance	Plant type	P=0.055
	pO2	P<0.001
	Plant type×pO2	P=0.001

Values are the mean ±SE (n=3 for gdch-KD at a pO2 of 18.4 kPa; n=4 otherwise). Significance (P<0.05) for the effects of plant type, pO2, and plant type×pO2 interaction was evaluated by SAS PROC MIXED.

Leaf photosynthetic traits estimated on pan class="Gene">gdch-KD and WT plants under approximately current ambient and below current ambient n>n class="Chemical">O2 levels (pO2 of 18.4 kPa and 1.84 kPa, respectively) at Ca of 27.6 Pa.

Values are the mean±SE (n=4). Significance (P<0.05) of the effects of plant type, pan class="Chemical">pO2, and plant type×n>n class="Chemical">pO2 interaction were evaluated by SAS PROC MIXED.

No significant differences were evaluated by a two sample t-test (significance for P<0.05) between the n class="Gene">gdch-KD and WT gm values at a n>n class="Chemical">pO2 of 1.84 kPa (P=0.586), and the WT gm values at the two pO2 values (P=0.701).

pan class="Chemical">CO2 compensation points (Γ) determined under low photorespiratory (1.84 kn>n class="Chemical">Pa pO2) and photorespiratory (18.4 kPa pO2) conditions on gdch-KD and WT plants

Values are the mean ±SE (n=3 for n class="Gene">gdch-KD at a n>n class="Chemical">pO2 of 18.4 kPa; n=4 otherwise). Significance (P<0.05) for the effects of plant type, pO2, and plant type×pO2 interaction was evaluated by SAS PROC MIXED.

Δo plotted versus Ci/Ca showed a similar response in the n class="Gene">gdch-KD and WT plants at 1.84 kn>n class="Chemical">Pa pO2 but was significantly greater in the gdch-KD compared with WT plants at 18.4 kPa pO2 (Fig. 2; see Table 2 for statistical analysis). The gdch-KD plants had significantly lower Δ, Δf, and Δe compared with the WT under a pO2 of 18.4 kPa (Fig. 3A). Additionally, the gdch-KD plants had a significantly lower f with mean values of 3.4±0.5‰ SE (n=4) compared with 16.2‰ in the WT under approximately current ambient pO2 (Fig. 3B; P<0.001). A significantly higher α was determined at 18.4 kPa pO2 in gdch-KD plants, with a mean value of 0.59±0.01 SE (n=3), versus 0.5 assumed for the WT (P<0.01). There was a negative linear dependency of f on Γ* and on α (Supplementary Methods S5; Fig. S1A and B, respectively); however, there was a positive linear dependency of f on gm and RL, with a greater sensitivity to gm (Supplementary Fig. S2A ands B, respectively).

Fig. 2.

Leaf 13CO2 net discrimination in the light (Δo) versus Ci/Ca under a pO2 of 1.84 kPa and 18.4 kPa for individual gdch-KD and WT plants. The line represents the leaf 13CO2 net discrimination modeled in relation to Ci/Ca as Δ13Cmod=a+(b3−a)×Ci/Ca (Farquhar ) where a=4.4‰ and b3=29.0‰. Δ13Cmod is a proxy of Δi as described by Evans and von Caemmerer (2013). Open symbols are for gdch-KD and filled symbols for WT plants. Circles are for a pO2 of 1.84 kPa and squares for a pO2 of 18.4 kPa.

Fig. 3.

Leaf 13CO2 net discrimination and discrimination fractions in the light, and 13CO2 photorespiratory fractionation for gdch-KD versus WT plants determined based on Evans and von Caemmerer (2013). (A) Observed leaf net 13CO2 discrimination in the light (Δo), and modeled 13C discrimination fractions for gdch-KD (n=3) and the WT (n=4) at an atmospheric pO2 of 18.4 kPa. Δi is the additive 13CO2 discrimination during CO2 diffusion from atmosphere to intercellular air space and due to carboxylation; Δi−Δo is comprised of three terms: Δ, which is the 13CO2 fractionation fraction during CO2 diffusion in the liquid phase to chloroplast stroma, and Δe and Δf, which are the 13C fractionation fractions associated with light mitochondrial non-photorespiratory respiration and photorespiration, respectively. Δf was calculated as Δf=Δi−Δo−Δgm−Δe. Values are mean ±SE. (B) 13CO2 fractionation for photorespiration (f) in gdch-KD plants calculated at an atmospheric pO2 of 18.4 kPa from versus f of 16.2‰ in the WT. Values for gdch-KD plants are the mean ±SE (n=3). Significance (P<0.05) of the effect of plant type on the variables in (A) was evaluated by SAS PROC MIXED as a one-way ANOVA; * for 0.01

Leaf pan class="Chemical">13CO2 net discrimination in the light (Δo) versus Ci/Ca under a n>n class="Chemical">pO2 of 1.84 kPa and 18.4 kPa for individual gdch-KD and WT plants. The line represents the leaf 13CO2 net discrimination modeled in relation to Ci/Ca as Δ13Cmod=a+(b3−a)×Ci/Ca (Farquhar ) where a=4.4‰ and b3=29.0‰. Δ13Cmod is a proxy of Δi as described by Evans and von Caemmerer (2013). Open symbols are for gdch-KD and filled symbols for WT plants. Circles are for a pO2 of 1.84 kPa and squares for a pO2 of 18.4 kPa.

Leaf pan class="Chemical">13CO2 net discrimination and discrimination fractions in the light, and n>n class="Chemical">13CO2 photorespiratory fractionation for gdch-KD versus WT plants determined based on Evans and von Caemmerer (2013). (A) Observed leaf net 13CO2 discrimination in the light (Δo), and modeled 13C discrimination fractions for gdch-KD (n=3) and the WT (n=4) at an atmospheric pO2 of 18.4 kPa. Δi is the additive 13CO2 discrimination during CO2 diffusion from atmosphere to intercellular air space and due to carboxylation; Δi−Δo is comprised of three terms: Δ, which is the 13CO2 fractionation fraction during CO2 diffusion in the liquid phase to chloroplast stroma, and Δe and Δf, which are the 13C fractionation fractions associated with light mitochondrial non-photorespiratory respiration and photorespiration, respectively. Δf was calculated as Δf=Δi−Δo−Δgm−Δe. Values are mean ±SE. (B) 13CO2 fractionation for photorespiration (f) in gdch-KD plants calculated at an atmospheric pO2 of 18.4 kPa from versus f of 16.2‰ in the WT. Values for gdch-KD plants are the mean ±SE (n=3). Significance (P<0.05) of the effect of plant type on the variables in (A) was evaluated by SAS PROC MIXED as a one-way ANOVA; * for 0.01

Leaf dark respiration responses

In the n class="Gene">gdch-KD and WT plants, the Rd showed a hyperbolic decrease over the 3 h in the dark after leaf light exposure under different n>n class="Chemical">O2 levels, with a noticeable rapid decline in the first hour; however, Rd was higher following leaf photosynthesis at a pO2 of 18.4 kPa compared with 1.84 kPa. (Fig. 4). A significant positive O2 effect on Rd responses of gdch-KD and WT plants was inferred based on significantly higher floor (μmol CO2 m−2 s−1; P<0.0001) and range (μmol CO2 m−2 s−1; P<0.0001) parameters after leaf photosynthesis at pO2 of 18.4 kPa compared with 1.84 kPa in the non-linear model fit to the Rd responses (Supplementary Tables S3, S4). In particular, based on the spot measurements, a significant positive O2 effect was determined on Rd(6min) (together with a significant plant type effect), Rd(30min), and Rd(3h) (Table 4). A significant plant type effect on Rd responses was inferred based on a statistically larger range (P=0.023) and less steep rate of exponential change (slope, μmol CO2 m−2 s−1 min−1; P<0.0001) for the gdch-KD versus WT plants (Supplementary Tables S3, S4). After leaf photosynthesis at a pO2 of 18.4 kPa, a significant plant type effect on Rd (with higher Rd determined in the gdch-KD plants versus the WT; see Fig. 4) was driven by the significantly less steep Rd slope (P<0.001) in gdch-KD compared with WT plants. In addition, following leaf photosynthesis at a pO2 of 18.4 kPa, a change in Rd for ~75% of the Rd range occurred in WT plants within the first 30 min after light–dark transition; in contrast, this fractional variation took ~90 min in the gdch-KD plants (Fig. 4). The mean values of RL inferred from Rd(3h) were 0.59±0.03 SE μmol CO2 m−2 s−1 for gdch-KD and 0.56±0.03 SE μmol CO2 m−2 s−1 for WT plants after leaf photosynthesis under a pO2 of 1.84 kPa (n=4). In contrast, RL was 0.98±0.12 SE μmol CO2 m−2 s−1 for gdch-KD and 0.82±0.09 SE μmol CO2 m−2 s−1 for WT plants after leaf exposure to a pO2 of 18.4 kPa (n=4). For the RL values, a non-significant plant type effect and a significant effect of the O2 level can be inferred from the significance of Rd(3h) (see Table 4). In addition, no significant difference in leaf dry mass per unit surface area (LMA) was determined between gdch-KD and WT plants, with values of 43.6±2.7 SE g m−2 and 44.5±1.5 SE g m−2 (n=4), respectively.

Fig. 4.

Dynamics of leaf dark respiration rate (Rd) determined during ~3 h in the dark on gdch-KD (open symbols) and WT (filled symbols) plants after leaf photosynthesis under a pO2 of 1.84 kPa (circles) or 18.4 kPa (squares). Symbols correspond to the mean ±SE (n=4) determined every 3 min.

Table 4.

Leaf dark respiration rates (Rd) at 30 °C and 13CO2 composition of dark-evolved CO2 (δ13CRd) determined on gdch-KD versus the WT after 6 min [Rd(6min) and δ13CRd(6min); n=4], 30 min [Rd(30min) and δ13CRd(30min); n=4], 3 h [Rd(3h) and δ13CRd(3h); n=4], and 24 h [Rd(24h) and δ13CRd(24h); n=3] in the dark following leaf exposure to light under approximately current ambient and below current ambient O2 levels (pO2 of 18.4 kPa and 1.84 kPa, respectively)

Plant-type	pO2	R d(6min)	δ13CRd(6min)	R d(30min)	δ13CRd(30min)	R d(3h)	δ13CRd(3h)	R d(24h)	δ13CRd(24h)
	(kPa)	(µmol CO2 m-2 s-1)	(‰)	(µmol CO2 m−2 s−1)	(‰)	(µmol CO2 m−2 s−1)	(‰)	(µmol CO2 m−2 s−1)	(‰)
gdch-KD	1.84	1.48±0.05	−39.2±0.9*	1.04±0.06	−47.3±1.6*	0.81±0.04	−56.0±0.7*	0.79±0.02	−58.0±0.5*
WT	1.84	1.23±0.04	−40.6±1.2*	1.04±0.01	−47.2±1.7*	0.77±0.04	−54.1±0.7*	0.69±0.02	−58.6±1.0*
Significance								P=0.045	P=0.705
gdch-KD	18.4	2.59±0.29	−45.6±1.7	1.98±0.20	−54.1±0.9	1.34±0.17	−55.6±1.2	0.69±0.06	−58.1±0.1
WT	18.4	2.13±0.07	−43.2±0.9	1.47±0.08	−50.4±2.8	1.12±0.13	−52.5±1.8	0.74±0.08	−58.6±0.6
Significance	Plant type	P=0.042	P=0.723	P=0.110	P=0.349	P=0.215	P=0.132	P=0.596	P=0.410
	pO2	P=0.0001	P=0.032	P=0.0009	P=0.035	P=0.004	P=0.429	–	–
	Plant type×pO2	P=0.407	P=0.238	P=0.069	P=0.375	P=0.326	P=0.753	–

Values are the mean ±SE; the asterisks indicate means from δ13CRd values edited according to Supplementary Method S7. Significance (P<0.05) of the effects of plant type, pO2, and plant type×pO2 interaction was evaluated by SAS PROC MIXED. The effect of plant type on Rd(24h) and δ13CRd(24h) was evaluated at a pO2 of 1.84 kPa or 18.4 kPa by one-way ANOVA (significance for P<0.05).

Leaf dark respiration rates (Rd) at 30 °C and pan class="Chemical">13CO2 composition of dark-evolved n>n class="Chemical">CO2 (δ13CRd) determined on gdch-KD versus the WT after 6 min [Rd(6min) and δ13CRd(6min); n=4], 30 min [Rd(30min) and δ13CRd(30min); n=4], 3 h [Rd(3h) and δ13CRd(3h); n=4], and 24 h [Rd(24h) and δ13CRd(24h); n=3] in the dark following leaf exposure to light under approximately current ambient and below current ambient O2 levels (pO2 of 18.4 kPa and 1.84 kPa, respectively)

Values are the mean ±SE; the asterisks indicate means from δpan class="Chemical">13CRd values edited according to Supplementary Method S7. Significance (P<0.05) of the effects of plant type, n>n class="Chemical">pO2, and plant type×pO2 interaction was evaluated by SAS PROC MIXED. The effect of plant type on Rd(24h) and δ13CRd(24h) was evaluated at a pO2 of 1.84 kPa or 18.4 kPa by one-way ANOVA (significance for P<0.05).

Dynamics of leaf dark respiration rate (Rd) determined during ~3 h in the dark on pan class="Gene">gdch-KD (open symbols) and WT (filled symbols) plants after leaf photosynthesis under a n>n class="Chemical">pO2 of 1.84 kPa (circles) or 18.4 kPa (squares). Symbols correspond to the mean ±SE (n=4) determined every 3 min.

In n class="Gene">gdch-KD and WT plants, the δ13CRd estimated over the 3 h after light–dark transition showed a negative hyperbolic pattern, with most of the δ13CRd variation occurring in the first 30 min (Fig. 5A, B). A tight positive correlation between Rd and δ13CRd over the 3 h dark period was determined after leaf photosynthesis at a pO2 of 1.84 kPa for both plant types (r>0.90). After leaf photosynthesis at a pO2 of 18.4 kPa, a positive correlation between Rd and δ13CRd with r=0.75 and r=0.78 was determined for gdch-KD and the WT, respectively. Statistical analysis of a non-linear model fit to the δ13CRd responses showed a significantly lower δ13CRd range after leaf photosynthesis at a pO2 of 18.4 kPa (‰; P<0.0001) compared with 1.84 kPa pO2. In contrast, the floor parameter was non-significantly different between the O2 levels (Supplementary Tables S5, S6). These statistical results indicate a significant effect of the O2 level during previous leaf light exposure on the δ13CRd values of both plant types over 3 h in the dark (with lower δ13CRd at a pO2 of 18.4 kPa, and higher δ13CRd at a pO2 of 1.84 kPa). The spot δ13CRd measurements also showed significantly lower δ13CRd(6min) and δ13CRd(30min) after leaf photosynthesis at a pO2 of 18.4 kPa compared with 1.84 kPa (Table 4). There was no significant plant type effect on the δ13CRd over the 3 h in the dark (Table 4; Supplementary Table S6) and there was no difference for δ13Cdm between gdch-KD and the WT (Supplementary Table S1).

Fig. 5.

13CO2 composition associated with Rd (δ13CRd) determined during ~3 h in the dark in gdch-KD (open symbols) and WT (filled symbols) plants. (A) δ13CRd after leaf photosynthesis under a pO2 of 1.84 kPa edited (see Supplementary Methods S7) to remove the effect of a lower atmospheric δ13CO2 compared with (B) while growing the plants. (B) Distributions of δ13CRd after leaf photosynthesis under a pO2 of 18.4 kPa. Symbols correspond to the mean ±SE (n=4) determined every 3 min.

pan class="Chemical">13CO2 composition associated with Rd (δ13CRd) determined during ~3 h in the dark in gdch-KD (open symbols) and WT (filled symbols) plants. (A) δ13CRd after leaf photosynthesis under a pO2 of 1.84 kPa edited (see Supplementary Methods S7) to remove the effect of a lower atmospheric δ13CO2 compared with (B) while growing the plants. (B) Distributions of δ13CRd after leaf photosynthesis under a pO2 of 18.4 kPa. Symbols correspond to the mean ±SE (n=4) determined every 3 min.

Over the 3 h after the light–dark transition, the fractional contribution of n class="Chemical">Lch assimilates to δ13CRd (δRdLch_substr, ‰/‰) showed a decreasing hyperbolic pattern for both gdch-KD and WT plants (Fig. 6), with no significant differences between plant types and O2 levels.

Fig. 6.

Distributions over ~3 h in the dark of the fractional contributions (total contribution equal to 1.0) to δ13CRd of recent Lch carbon assimilates (δRdLch_substr, ‰/‰) and Gch assimilates (δRdGch_substr, ‰/‰) for gdch-KD (open symbols) and WT (filled symbols) after leaf light exposure at a pO2 of (A) 1.84 kPa and (B) 18.4 kPa. The first values are at 3 min after light–dark transition. Symbols correspond to the mean determined every 3 min (n=4). Continuous lines represent logarithmic trend lines (R2 >0.90) for gdch-KD (lower) and the WT (higher), respectively.

Distributions over ~3 h in the dark of the fractional contributions (total contribution equal to 1.0) to δpan class="Chemical">13CRd of recent n>n class="Chemical">Lch carbon assimilates (δRdLch_substr, ‰/‰) and Gch assimilates (δRdGch_substr, ‰/‰) for gdch-KD (open symbols) and WT (filled symbols) after leaf light exposure at a pO2 of (A) 1.84 kPa and (B) 18.4 kPa. The first values are at 3 min after light–dark transition. Symbols correspond to the mean determined every 3 min (n=4). Continuous lines represent logarithmic trend lines (R2 >0.90) for gdch-KD (lower) and the WT (higher), respectively.

Discussion

Altered photorespiratory metabolism and leaf photosynthetic traits

Based on leaf protein analysis, n class="Gene">gdch-KD plants had ~21, 77, and 83% of GDC H-, P-, and T-protein abundance, respectively, compan>red with the WT. Previous studies reported how GDC activity is linearly correlated with H-protein accumulation (Wingler ; Lin ). Additionally, in agreement with Lin , the n>n class="Gene">gdch-KD plants in the current study showed an expected photorespiratory-deficient phenotype. Under photorespiratory conditions, a disruption of the photorespiratory pathway negatively affects the rate of net CO2 assimilation (A) due to accumulation of metabolites that inhibit the Calvin–Benson cycle and restrict RuBP regeneration (Wingler ). Specifically, leaf glycine level is a sensitive indicator of altered photorespiratory carbon flow (Blackwell ; Timm ). For example, gdch-KD mutants of Arabidopsis, barley, and rice had substantial increases in leaf contents of glycine under ambient pO2 (Bauwe and Kolukisaoglu, 2003; Lin ). This accumulation of glycine and its precursors (P-glycolate, glycolate, and glyoxylate) in the gdch-KD plants has been suggested to alter photorespiratory carbon metabolism (Peterhansel , 2013a). These changes have important implications for understanding and modeling leaf carbon metabolism, because they may influence the stoichiometry of CO2 released per oxygenation reaction (α) and the CO2 compensation point (Γ) (see Cousins , 2011; Walker and Cousins, 2013).

In the present study, the pan class="Gene">gdch-KD plants had greater Γ compan>red with the WT under 18.4 kn>n class="Chemical">Pa pO2, as previously reported by Lin . This may be partially due to enhanced RL, which Igamberdiev and Bykova reported was needed to compensate for the lack of photorespiratory regulation of redox and energy balance (Igamberdiev ). The increase in Γ in the gdch-KD plants could also be associated with a higher α leading to an increasing Γ* compared with the WT. It has been previously suggested that α increased in Arabidopsis mutants lacking peroxysomal hydroxypuruvate reductase (Cousins ) and the peroxysomal malate dehydrogenase (Cousins ). However, these previous publications did not determine whether these disruptions to photorespiration influenced leaf CO2 isotope exchange.

Leaf net 13C discrimination in the light and photorespiratory 13C fractionation

Under leaf photorespiratory conditions, the change in leaf net discrimination against pan class="Chemical">13CO2 (Δo) in the n>n class="Gene">gdch-KD plants compared with WT plants was caused by a higher Ci/Ca, lower Δ, greater Δe, and lower Δf (Fig. 3A). However, the lower Δ in the gdch-KD plants with respect to the WT was due to a reduction in A, since WT gm was applied to both plant types (see Δ equation in Evans and von Caemmerer, 2013). In addition, a difference in Δe between gdch-KD and WT plants was related to proportional changes in RL/(A+RL) and (Ci−Γ*) (see Δe equation in Evans and von Caemmerer, 2013).

The term Δf is dependent on , but, despite the higher Γ* in the pan class="Gene">gdch-KD relative to WT plants, it was lower in the transgenic plants caused by the lower f. In WT plants, f is primarily attributed to the n>n class="Chemical">13C discrimination associated with the decarboxylation of glycine catalyzed by GDC (Tcherkez ; Tcherkez, 2006). While Rooney (1988) determined an in vitro f of 7–8‰ for Glycine max (soybean), Igamberdiev , 2004) reported f for several species between 9.8 and 13.7‰, and Ghashghaie reported an f of >9–11‰ for Senecio species. Additionally, Evans and von Caemmerer (2013) determined an in vivo f of 16.2‰ in Nicotiana tabacum. Based on Farquhar , and according to O’Leary (1988) and Tcherkez (2006),

where pan class="Chemical">glycine is assumed to have the same n>n class="Chemical">13C signature of recently fixed carbon. Therefore, an increase in δ13C of the released CO2 during photorespiration corresponds to a linear decrease in f. There is also a negative linear dependency of f on Γ* and α, as shown in Supplementary Methods S5; Fig. S1A, B.

In C3 plants, most of the pan class="Chemical">CO2 released by photorespiration tends to be through GDC (Badger, 1985; Bauwe ). However, previous reports have suggested that alternative reactions can release n>n class="Chemical">CO2 when the flux of glycolate into the photorespiratory cycle exceeds its metabolic capacity, or when the traditional photorespiratory pathway has been genetically disrupted (Cousins , 2011; Timm ; Peterhansel ). The GDC multienzyme system requires all subunits to function (Douce ); in the present study, since a low level of the H-subunit was determined in gdch-KD plants, some residual activity for GDC is expected in the transgenic plants. A change in 13C fractionation associated with the knockdown of GDC activity is therefore unlikely because the products of the glycine decarboxylation reaction (NH4+, NADH, and methylene-tetrahydrofolate) can be readily processed by downstream reactions in the glycolate pathway (Bauwe ; Maurino and Peterhansel, 2010). Thus, the higher α and lower f in the gdch-KD compared with the WT suggest an increased flow of photorespiratory carbon through alternative decarboxylation reactions, independent of the GDC, and a buildup of photorespiratory metabolites.

Leaf dark respiration and 13C isotopic composition of dark-evolved CO2

Leaves of C3 plants in the first 30 min after light–dark transition largely respire metabolites (n class="Chemical">carbohydrates and n>n class="Chemical">organic acids) recently produced in the light (Cornic, 1973; Rademacher ; Barbour ; Werner ; Werner and Gessler, 2011; Lehmann , 2016) and show high rates of CO2 evolution (named as LEDR, light-enhanced dark respiration; Atkin ). While the activity of pyruvate dehydrogenase (PDH) and metabolism in the tricarboxylic acid (TCA) cycle are the major mitochondrial decarboxylations in the dark, they are partially inhibited in the light (Ghashghaie ; Tcherkez , 2008; Barbour ). It has been suggested that LEDR mostly depends on a buildup of malate and fumarate in the light, which are then rapidly decarboxylated after the light–dark transition (Atkin ; Barbour ; Tcherkez ). However, there is evidence for species-specific differences (Lehmann ; Gessler ). Overall, Rd in the gdch-KD and WT plants showed an expected negative hyperbolic pattern during 3 h in the dark following leaf photosynthesis. The results of the present study indicate that leaf photosynthesis under photorespiring conditions, before the light–dark transition, led to an additional buildup of TCA cycle substrates in the gdch-KD plants compared with the WT. In fact, the gdch-KD plants had a significantly higher Rd over 3 h dark following leaf photosynthesis under the approximately current ambient O2 level (with leaf blades having no significantly different LMA), compared with the WT; this suggests a greater accumulation of metabolites in the light, in particular photorespiratory intermediates, as respiratory substrates to feed Rd. More precisely, a restricted photorespiratory pathway in the light may lead to an accumulation of 2-carbon metabolites in the gdch-KD plants.

The increase in LEDR has been reported to come from the decarboxylation of pan class="Chemical">13C heavier metabolites, primarily n>n class="Chemical">malate, and the decline in LEDR rates and δ13CRd over time due to a decrease in malate availability (Barbour ; Gessler ). In the gdch-KD plants, the cumulative leaf respired CO2 over 30 min after leaf photosynthesis at a pO2 of 18.4 kPa was 4.1 mmol CO2 m−2 higher with respect to the WT; theoretically, if this enhancement of Rd in the gdch-KD plants was due to malate alone this would require ~1 mmol malate m−2. The non-significant differences in the leaf malate content determined during the light period between gdch-KD and the WT suggest that metabolites other than malate, such as photorespiratory intermediates, may have contributed to the greater LEDR rates in the gdch-KD plants compared with the WT. A substantial part of the malate in leaves is also stored in vacuoles, as observed in C4 plants (Hatch, 1979; Arrivault ), and not readily available for LEDR.

In the n class="Gene">gdch-KD and WT plants presented here, the δ13CRd decreased during the 3 h dark period, tracking the decline in Rd (Fig. 5A, B). Over the 3 h of darkness, there was an increase in the contribution to δ13CRd from respiratory substrates generated during plant growth (δRdGch_substr, ‰/‰) for both plant types and O2 experimental conditions. Regardless of plant type or O2 treatment, the δRdLch_substr went from ~50% after 6 min from the light–dark transition to ~30% after 30 min in the dark, while after 3 h in the dark it represented only ~10% [see Fig. 6; data of δ13CRd(3h) approaching δ13CRd(24h) are shown in Table 4]. Tcherkez estimated on sunflower (Helianthus annuus) that recent assimilates provide 40–60% of the substrates for Rd (via a pool with a half-life of several hours) both in the light and in the dark. A similar contribution of recent assimilates to Rd was determined by Nogués on French bean (Phaseolus vulgaris) leaves during ~2 h in the dark following illumination, which indicates that leaf respiration was fed by a mixture of recent and older substrates.

The tendency for a lower leaf δpan class="Chemical">13CRd in n>n class="Gene">gdch-KD plants compared with the WT following leaf light exposure under photorespiratory conditions may partially depend on the higher Δo in the gdch-KD plants during leaf photosynthesis in the Lch at approximately current ambient pO2. A greater Δo would cause (recent) carbon assimilates synthetized in the Lch (Supplementary data Table S1) to produce more depleted respiratory substrates and a lower δ13CRd in the gdch-KD compared with the WT. It is also possible that higher Δo in the gdch-KD compared with WT plants during growth under enriched atmospheric pCO2 and current ambient pO2 may have produced Gch assimilates feeding Rd over 3 h after the light–dark transition with slightly lower δ13C compared with the WT.

The pan class="Chemical">13C fractionation during leaf dark respiration can change depending on species and environmental conditions (Ghashghaie ; Priault ; Werner ; Lehmann ). In addition, δ13CRd is influenced by the isotopic signatures of respiratory substrates, from diverse non-homogeneous isotope distributions in the substrates (positional effects) and the different relative activities of decarboxylation pathways. However, decreasing δ13CRd over time is mainly dependent on the origin of respiratory substrates, where CO2 released from pyruvate decarboxylation is 13C enriched (compared with total organic matter) but relatively 13C depleted from acetyl-CoA metabolism through the TCA cycle (Tcherkez ). Under continuous darkness and constant tair, it has been shown that δ13CRd decreases due to a switch in respiratory substrates from carbohydrates to more 13C-depleted substrates such as lipids or proteins (Ghashghaie ; Tcherkez ).

The similar δpan class="Chemical">13CRd(24h) in n>n class="Gene">gdch-KD versus WT plants implies that the long-term substrates for the TCA cycle produced in the Gch were 13C isotopically similar. This is further supported by similar leaf δ13Cdm between the gdch-KD and WT plants. Interestingly, δ13CRd(24h) was more depleted than δ13Cdm, in agreement with Tcherkez who had found that CO2 evolved in the dark by French bean leaves in a condition of carbohydrate starvation had a lower δ13C than total leaf organic matter. This denotes potential changes in dark respiration substrates, such as carbohydrate oxidation producing 13C-enriched CO2 and β-oxidation of fatty acids producing 13C-depleted CO2 when compared with total organic matter.

Conclusions

Under photorespiratory conditions, the n class="Gene">gdch-KD plants had altered n>n class="Chemical">13C discrimination fractions in the light, with a lower Δf caused by a reduced f. This change in Δf and the lower Δgm lead to a higher Δo in the gdch-KD plants in comparison with the WT. The lower f in the gdch-KD plants was attributed to a greater α compared with the WT, suggesting the occurrence of alternative photorespiratory reactions in the GDC-impaired plants. In addition, the enhanced Rd in the gdch-KD compared with WT plants after photorespiratory leaf photosynthesis indicated that the photorespiratory disruption led to an additional buildup of metabolites in the light that were decarboxylated by the TCA cycle in the dark. The tendency for a more depleted δ13CRd in the gdch-KD plants compared with the WT after photorespiratory leaf photosynthesis was mainly ascribed to a higher Δo before the light–dark transition and differences in the δ13C of the substrates feeding Rd. These results indicate that an alteration in photorespiratory carbon metabolism can have a significant effect on leaf CO2 exchange and 13CO2 discrimination, both in the light and in the dark.

Supplementary data

Supplementary data are available at JXB online.

Methods S1. Estimate of the pan class="Chemical">13CO2 composition of the growth chamber atmosphere during the light period.

Methods S2. Additional technical information on the system setup to measure online leaf atmosphere pan class="Chemical">CO2, n>n class="Chemical">H2O, and 13CO2 exchange.

Methods S3. Estimate of the pan class="Chemical">13C signature and total N content in the leaf biomass.

Methods S4. Estimate of the pan class="Chemical">CO2 compensation point in the absence of mitochondrial non-photorespiratory respiration.

Methods S5. Estimate of α, and evaluation of the sensitivity of f to Γ* and α (shown in Fig. S1).

Methods S6. Estimate of the fractional contribution of respiratory substrates from leaf chamber and growth chamber pan class="Chemical">carbon assimilates to the n>n class="Chemical">13C composition of dark-evolved CO2.

Methods S7. Editing of the pan class="Chemical">13C composition of dark-evolved n>n class="Chemical">CO2 for plants grown at an atmospheric 13C composition of −41.6‰.

Fig. S1 Sensitivity of the f parameter to Γ* and α.

Fig. S2. Sensitivity of the f parameter to gm, RL, and e'.

Table S1. Data used to calculate the fractional contributions of leaf chamber and growth chamber pan class="Chemical">carbon assimilates to n>n class="Chemical">13C composition of leaf dark-evolved CO2.

Table S2. Values of 18O-based gm.

Table S3. Statistics for the model used to fit leaf dark respiration rates.

Table S4. Significance for the model used to fit leaf dark respiration rates.

Table S5. Statistics for the model used to fit the pan class="Chemical">13C composition of leaf dark respiration rates.

Table S6. Significance for the model used to fit pan class="Chemical">13C composition of leaf dark respiration rates.

Click here for additional data file.

Author contributions

SK, SC, pan class="Disease">H-CL, RAC, WPQ, and JMH generated the transgenic plant material; SvC, RG, RTF, GEE, and ABC planned and designed the experiments; RG performed leaf gas and isotope exchange measurements and analysis; NK performed the biochemical analysis; RG, SvC, RTF, GEE, and ABC interpreted the data; and RG, ABC, and GEE developed the manuscript.