Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves

Abstract

Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C(3) photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO(2) fixation rate in leaves, a novel in vivo (13)C-labelling system was developed with (13)CO(2) for the kinetic determination of metabolic turnover that was the time-course of the (13)C-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO(2) fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.

Fig. 1.

The path of carbon in photosynthesis in a tobacco leaf. Abbreviations: ADP-Glc, adenosine-5′-diphosphate glucose; BPGA, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetonephosphate; E4P, erythrose-4-phosphate; FBP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; PGA, 3-phosphoglycerate; R5P, ribose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; S6P, sucrose-6-phosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose-1,7-bisphosphate; UDP-Glc, uridine-5′-diphosphate glucose; Xu5P, xylulose-5-phosphate. Numbers in circles denote enzymes as follows: (1) RuBP carboxylase/oxygenase (Rubisco); (2) GAP dehydrogenase; (3) FBP aldolase; (4) fructose-1,6-bisphosphatase (FBPase); (5) SBP aldolase; (6) sedoheptulose-1,7-bisphosphatase (SBPase); (7) phosphoribulokinase; (8) phosphoglucose isomerase (PGI); (9) phosphoglucomutase (PGM); (10) ADP-glucose pyrophosphorylase; (11) sucrose phosphate synthase.

Fig. 2.

Photosynthesis chamber for feeding ¹³CO₂ into a tobacco leaf (A) and flow path of mixed gasses (B). Air is 20% O₂ and 80% N₂. 1, light guide; 2, PAM probe; 3, gas-exchange chamber; 4, leaf cutter; 5, liquid nitrogen reservoir. (This figure is available in colour at JXB online.)

Fig. 3.

Mass distribution of RuBP at 0 min (A), 1 min (B), and 10 min (C) after initiation of ¹³C-labelling from ¹³CO₂. Q1, m/z of deprotonated precursor ion; Q3, m/z of product ion.

Fig. 4.

Time-course of mass distribution of sugar phosphates in tobacco leaves under 1000 ppm ¹³CO₂ conditions. The mass distribution of ADP-glucose and UDP-glucose was calculated for the glucose moieties. Ru5P was not separated from Xu5P by capillary electrophoresis. Values are the averages of measurements of three different tobacco plants, ±SEM. Closed diamonds (black), m₀; open circles (blue), m₁; closed triangles (light blue), m₂; open squares (green), m₃; open diamonds (orange), m₄; closed circles (pink), m₅; open triangles (purple), m₆; closed squares (grey), m₇. m_i represents the relative isotopomer abundance for each metabolites in which i¹³C atoms are incorporated. 100% means the isotopomer abundance corresponds to the pool size of the metabolite. (This figure is available in colour at JXB online.)

Fig. 5.

Time-course for fraction of C as ¹³C fraction in tobacco leaves under 200 ppm (circles) and 1000 ppm (filled diamonds) ¹³CO₂ conditions.

Fig. 6.

Metabolic turnover rate of metabolites in tobacco leaves under 200 ppm (grey bars) and 1000 ppm (white bars) ¹³CO₂ condition. Values are the averages of measurements of three different tobacco plants, ±SEM.