Arabidopsis NAD-dependent isocitrate dehydrogenase mutants

NAD-dependent isocitrate dehydrogenase (IDH) is a TCA cycle enzyme that produces 2oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant is lacking the corresponding gene products. Leaf IDH activity is reduced by 92%, 60% and 43% for idhv, idhi and idhii respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in day-time sucrose, glucose, fructose, citrate, ammonium and total soluble amino acid levels. However, GC-MS metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild-type. These data suggest that IDH activity is not limiting for TCA cycle functioning and nitrogen assimilation. On the other hand, liquid-culture grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35fold), hexose-phosphate and sugar contents, while ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these non-physiological growth conditions, pyridine nucleotide levels remained relatively constant between the WT and the idhv line although some small but significant alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH contents. www.plantphysiol.org on August 29, 2017 Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.