Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth

摘要

Mitochondrial NAD-malic enzyme () and/or cytosolic/plastidic NADP- combined with the cytosolic/plastidic pyruvate orthophosphate dikinase () catalyze two key steps during light-period malate decarboxylation that underpin secondary CO2 fixation in some Crassulacean acid metabolism () species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate species Kalanchoë fedtschenkoi with reduced activities of NAD- or . Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CO2 fixation. In well-watered conditions, these lines fixed all of their CO2 in the light; they thus performed C3 photosynthesis. The alternative malate decarboxylase, NADP-, did not appear to compensate for the reduction in NAD-, suggesting that NAD- was the key decarboxylase for . The activity of other enzymes was reduced as a consequence of knocking out either NAD- or activity, particularly phosphoenolpyruvate carboxylase () and href="#def2" rid="def2" class=" def">PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of href="#def4" rid="def4" class=" def">PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of href="#def4" rid="def4" class=" def">PPC phosphorylation, href="#def4" rid="def4" class=" def">PPC kinase transcript levels and activity, and the classic circadian rhythm of href="#def3" rid="def3" class=" def">CAM CO2 fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing href="#def3" rid="def3" class=" def">CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.