Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane

Abstract.

Chloroplasts or chromoplasts were purified from sweet-pepper (Capsicum annuum L. cv. Yolo Wonder) fruits and analysed with respect to their enzymic equipment, the transport properties across the envelope membrane, and for the presence of a functional oxidative pentose-phosphate pathway (OPPP). It was demonstrated that both types of plastid contain enzyme activities that allow glycolysis and OPPP. During the developmental conversion from chloroplasts to chromoplasts the activities of enzymes catalysing potentially rate-limiting reactions in glycolysis increased considerably. Most enzyme activities involved in the plastidic OPPP stayed constant or decreased during ripening, but transaldolase activity increased by more than 500%. To analyse whether pepper fruit chromoplasts are able to use exogenously supplied carbohydrates for the OPPP we measured the rate of ¹⁴CO₂ release after application of radioactively labelled precursors. Isolated pepper fruit chromoplasts used exogenously supplied [U¹⁴C]glucose- 6-phosphate (Glc6P) as a precursor for the OPPP. The metabolic flux through this pathway was stimulated by the presence of additional compounds which require reducing equivalents for further conversion, e.g. nitrite, or 2-oxoglutarate plus glutamine. The [¹⁴C]Glc6P-driven OPPP in isolated chromoplasts exhibited saturation with rising concentrations of Glc6P, reaching highest rates at an external concentration of about 2 mM. Exogenously given [U¹⁴C]glucose 1-phosphate (Glc1P)′ did not lead to a release of ¹⁴CO₂, indicating that this hexose phosphate is not taken up into the intact plastid. Using a proteoliposome system in which the envelope membrane proteins from sweet-pepper chromoplasts were functionally reconstituted we demonstrated that Glc6P is transported in counter-exchange with inorganic phosphate (P_i) or other phosphorylated intermediates. The Glc6P was taken up into proteoliposomes with an apparent K _m of 0.34 mM. Surprisingly, in contrast to tomato fruit plastids, isolated chromoplasts from sweet-pepper fruits do not possess a phosphate translocator allowing the uptake of Glc1P. Rising exogenous concentrations of dihydroxyacetone phosphate strongly inhibited the metabolic flux through the OPPP. This observation is discussed with respect to the presence of two phosphate translocator proteins in the envelope of sweet-pepper chromoplasts and with respect to possible metabolic changes occurring in heterotrophic tissues during development.