Transgenic perturbation of the decarboxylation phase of Crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth.
Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO(2) fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. 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 CAM CO(2) fixation. In well-watered conditions, these lines fixed all of their CO(2) in the light; they thus performed C(3) photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO(2) 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 CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.
Department of Plant Sciences, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
Louisa V
LV
Dever
Department of Plant Sciences, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
Susanna F
SF
Boxall
Department of Plant Sciences, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom.
Jana
J
Kneřová
Department of Plant Sciences, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom hartwell@liv.ac.uk.
James
http://orcid.org/0000-0001-5000-223X
J
Hartwell
D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology; D003653:Decarboxylation / Q000235:genetics* / Q000502:physiology*; D003681:Dehydration / Q000378:metabolism; D055786:Gene Knockout Techniques; D031962:Kalanchoe / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D010788:Photosynthesis / Q000235:genetics / Q000502:physiology; D030821:Plants, Genetically Modified; D011772:Pyruvate, Orthophosphate Dikinase / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism
D008293:Malates; C030298:malic acid; D013213:Starch; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D011772:Pyruvate, Orthophosphate Dikinase