Metabolic Regulation of Carbon Flux during C4 Photosynthesis I. Evidence for Parallel CO2 Fixation by Mesophyll and Bundle Sheath Cells in situ

Abstract

Summary CO2 fixation by isolated mesophyll protoplasts, bundle sheath strands and thin leaf slices of three types of C4 plants Digitaria sanguinalis (NADP-malic enzyme type), Panicum miliaceum (NAD-malic enzyme type) and Eriochloa borumensis (phosphoenolpyruvate-carboxykinase [PEP-CK] type) was investigated in the presence of appropriate inhibitors of mesophyll and bundle sheath photosynthesis. The inhibitors used were: malonate and maleate for PEP carboxylation, and D,L-glyceraldehyde for ribulose 1,5-diphosphate (RuDP) generation in all the three species; oxalate for C4 acid decarboxylation through NADP-malic enzyme in D. sanguinalis, and 3-mercaptopicolinic acid for C4 acid decarboxylation through PEP-CK in E. borumensis. In the absence of inhibitors, the C4 leaf slices fixed CO2 at very high rates at both 0.4 mM and 6 mM HCO3−, and O2 (0–100 %) had no effect. The 14C label was equally distributed between C4 acids and Calvin cycle compounds. In the absence of functional PEP carboxylation and C4 acid decarboxylation, the C4 leaf slices fixed CO2 directly into Calvin cycle compounds (the rates were 14–17 % of the control). At low HCO3− (0.4 mM) concentration, the direct fixation of CO2 by RuDP carboxylase in C4 leaf slices treated with maleate and appropriate C4 acid decarboxylase inhibitor was progressively inhibited by 21 % and 100% O2 and the label readily appeared in photorespiratory intermediates. The O2 inhibition was effectively reversed by increasing the added HCO3− concentration. Similar effects of O2 and HCO3− were observed during CO2 fixation by isolated bundle sheath stands. Pyruvate + oxaloacetate-dependent CO2 fixation (PEP carboxylation) in leaf slices treated with D,L-glyceraldehyde was increased by 2 fold at 100% O2 and the HCO3− concentration had no effect. Similar enhancement by O2 was observed with isolated mesophyll protoplasts. O2 inhibited bundle sheath photosynthesis competitively with respect to HCO3−, and reversal of O2 inhibition by CO2 was also competitive with respect to O2. The Km(CO2) for bundle sheath photosynthesis was about 11–13 µM at pH 8.2 and 37C; and increased roughly by 5-fold at 21 % O2 and 10-fold at 100 % O2. Both the observed and calculated Vmax for cell photosynthesis were similar except for D. sanguinalis in which the observed Vmax was 33 % lower than the calculated Vmax. The results indicate that 14–17 % of atmospheric CO2 entering the leaf is directly fixed by the Calvin cycle without the intervention of C4 cycle. The significance of these observations is discussed in relation to the mechanism and function of C4 photosynthesis and the C4 control of photorespiration in vivo.