Purification and characterization of NADP+-linked isocitrate dehydrogenase from Scots pine. Evidence for different physiological roles of the enzyme in primary development

摘要

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase [glutamate-ammonia ligase]/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH cytosolic isoform was detected in green cotyledons of Scots pine (Pinus sylvestris) and maritime pine (P. pinaster) seedlings. The Scots pine enzyme was purified and showed molecular and kinetic properties similar to those described for NADP+-IDH from angiosperms, with a higher catalytic efficiency (105 M-1 s-1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for thesynthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.