The Calvin cycle inevitably produces sugar-derived reactive carbonyl methylglyoxal during photosynthesis: a potential cause of plant diabetes

Abstract

Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O2 evolution (R = 0.88; P < 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves.

Keywords: Advanced glycation end-products; Methylglyoxal; Photosynthesis; Triose phosphate isomerase.

Fig. 1

Chromatograms of o-phenylenediamine (OPD) derivatives of sugar-derived reactive carbonyls (RCs). 3-Phosphoglycerate (3-PGA) was absent (A) and present (B) in the reaction mixtures. Reaction mixtures (1 ml) that contained chloroplasts (40 µg of Chl) in the absence and presence of 10 mM 3-PGA were illuminated in red light (>640 nm, 400 µmol photons m⁻² s⁻¹) at 25°C. Typical chromatograms of sugar-derived RCs are shown. AU indicates relative absorbance units at 312 nm. The three lines indicate different light exposure times (black, 0 min; red, 10 min; and blue, 20 min). Arrows indicate sugar-derived RCs, the levels of which increased in the presence of 3-PGA. Glyoxal (GLO) and methyglyoxal (MG) were identified from the retention times of the commercially purchased compounds. (C) MG was quantified at the indicated time after illumination in the absence (black bars) or presence (red bars) of 3-PGA. HPLC of sugar-derived RCs is described in the Materials and Methods section. Values are expressed as means ± standard deviations of three independent experiments.

Fig. 2

Light dependency of methyglyoxal (MG) production in chloroplasts. The reaction mixtures (1 ml) containing chloroplasts (40 µg of Chl) in the presence (A) and absence (B) of 10 mM 3-phosphoglycerate (3-PGA) were illuminated at the indicated intensity of red light for 10 min. Black bar, in the dark; red bars, exposure at the indicated light intensity. Values are expressed as means ± SD of three independent experiments.

Fig. 3

Dependence of the O₂ evolution rate (A) and methyglyoxal (MG) synthesis rate (B) on the intensity of red light in chloroplasts. The reaction mixture (1 ml) containing 40 µg of chloroplasts and 10 mM 3-phosphoglycerate (3-PGA) was illuminated at the indicated light intensity. The O₂ evolution rate was calculated from the steady-state increase in [O₂] at 10 min. The MG synthesis rate was calculated from the concentration of MG at 10 min. Values are expressed as means ± SE of five independent experiments.

Fig. 4

The relationship between the O₂ evolution rate and methyglyoxal (MG) synthesis rate. The O₂ evolution rate and MG synthesis rate were obtained from Fig. 3. (A) The O₂ evolution rates were plotted against the MG synthesis rates. These rates were estimated at 25°C. The positive linear line was the fitted line with the linear regression model. (B) Both the O₂ evolution rates and MG synthesis rates were estimated at 35°C (data not shown), and the O₂ evolution rates were plotted against the MG synthesis rates. The positive linear dotted line is the line fitted with the linear regression model. The correlation coefficient (R) and P-value are shown. Values were obtained from five independent experiments.

Fig. 5

Chromatograms of o-phenylenediamine (OPD) derivatives of sugar-derived reactive carbonyls (RCs). Black line, 40 Pa CO₂; Red line, 90 Pa CO₂. Typical chromatograms of sugar-derived RCs are shown. AU indicates the relative absorbance units based on leaf area at 312 nm. Arrows indicate sugar-derived RCs, the levels of which increased at 90 Pa CO₂ (compared with 40 Pa CO₂). Glyoxal (GLO) and methyglyoxal (MG) were identified from the retention times of the commercially purchased compounds. Insets show typical MG and GLO peaks, which were normalized with the baseline. HPLC of sugar-derived RCs is described in the Materials and Method. R.T., retention time.