Contribution of carbon fixed by Rubisco and PEPC to phloem export in the Crassulacean acid metabolism plant Kalanchoe daigremontiana

Abstract

Crassulacean acid metabolism (CAM) plants exhibit a complex interplay between CO(2) fixation by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), and carbon demand for CAM maintenance and growth. This study investigated the flux of carbon from PEPC and direct Rubisco fixation to different leaf carbon pools and to phloem sap over the diurnal cycle. Concentrations and carbon isotope compositions of starch, soluble sugars, and organic acids were determined in leaves and phloem exudates of Kalanchoë daigremontiana Hamet et Perr., and related to CO(2) fixation by PEPC and Rubisco. Three types of leaf carbon pools could be distinguished. (i) Starch and malate pools were dominant and showed a pattern of reciprocal mobilization and accumulation (85/54 and 13/48 mg C g(-1) DW, respective, at the beginning/end of phase I). The carbon isotope composition of these pools was compatible with predominant PEPC fixation (delta(13)C values of -13 and -11 per thousand for starch and malate compared to -11 per thousand of PEPC fixed carbon). (ii) Isotopic composition (-17 per thousand and -14 per thousand) and concentration of glucose and fructose (2 and 3 mg C g(-1) DW, respectively) were not affected by diurnal metabolism, suggesting a low turnover. (iii) Sucrose (1-3 mg C g(-1) DW), in contrast, exhibited large diurnal changes in delta(13)C values (from -17 per thousand in the evening to -12 per thousand in the morning), which were not matched by net changes in sucrose concentration. This suggests a high sucrose turnover, fed by nocturnal starch degradation and direct Rubisco fixation during the day. A detailed dissection of the carbon fixation and mobilization pattern in K. daigremontiana revealed that direct fixation of Rubisco during the light accounted for 30% of phloem sucrose, but only 15% of fixed carbon, indicating that carbon from direct Rubisco fixation was preferentially used for leaf export.

Fig. 1.

Proposed scheme of cellular carbon flow in K. daigremontiana, modified after Dodd et al. (2002), focusing on the sucrose pool. PEPC fixation, a; malate decarboxylation, b; starch accumulation, c; starch degradation, d; respiration, e; Rubisco fixation, f; export, g.

Fig. 2.

Diurnal pattern of net CO₂ uptake (solid line) and instantaneous discrimination Δ¹³C (squares) of an exemplary K. daigremontiana plant. CAM phases are denoted by dashed lines, the solid bar on the x-axis indicates the dark period. Δ¹³C is shown as the mean ±SE of three consecutive measurements.

Fig. 3.

Diurnal changes in the concentration (A) and δ¹³C (B) of starch (open symbols), malate (closed symbols), and citrate/isocitrate (shaded symbols) of K. daigremontiana. CAM phases are denoted by dashed lines, the solid bar on the x-axis indicates the dark period. Data shown are the means ±SE of five replicate plants.

Fig. 4.

Diurnal changes in the concentration (A) and δ¹³C (B) of sucrose (open symbols), fructose (closed symbols), and glucose (shaded symbols) of K. daigremontiana. CAM phases are denoted by dashed lines, the solid bar on the x-axis indicates the dark period. Data shown are the means ±SE of five replicate plants. At the end of the phases I, II, III, and IV, carbon from direct Rubisco fixation accounted for 7, 21, 19, and 54% of sucrose, respectively.

Fig. 5.

Diurnal changes in δ¹³C of sucrose in the leaf (squares) and in phloem exudates (shaded bars) in K. daigremontiana. Exudation lasted 5 h. The phases are denoted by dashed lines, the solid bar on the x-axis indicates the dark period. Data shown are the means ±SE of five replicate plants.