Ca(2+)-dependent oxidation of cytosolic NADPH is mediated by NDB1, which is an external type II NADPH dehydrogenase in the plant mitochondrial electron transport chain. Using RNA interference, the NDB1 transcript was suppressed by 80% in Arabidopsis thaliana plants, and external Ca(2+)-dependent NADPH dehydrogenase activity became undetectable in isolated mitochondria. This was linked to a decreased level of NADP(+) in rosettes of the transgenic lines. Sterile-grown transgenic seedlings displayed decreased growth specifically on glucose, and respiratory metabolism of (14)C-glucose was increased. On soil, NDB1-suppressing plants had a decreased vegetative biomass, but leaf maximum quantum efficiency of photosystem II and CO2 assimilation rates, as well as total respiration, were similar to the wild-type. The in vivo alternative oxidase activity and capacity were also similar in all genotypes. Metabolic profiling revealed decreased levels of sugars, citric acid cycle intermediates, and amino acids in the transgenic lines. The NDB1-suppression induced transcriptomic changes associated with protein synthesis and glucosinolate and jasmonate metabolism. The transcriptomic changes also overlapped with changes observed in a mutant lacking ABAINSENSITIVE4 and in A. thaliana overexpressing stress tolerance genes from rice. The results thus indicate that A. thaliana NDB1 modulates NADP(H) reduction levels, which in turn affect central metabolism and growth, and interact with defense signaling.

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP + ratio was unaffected, the stem NADPH/NADP + ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP + ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

Summary• The effect of previous light conditions on metabolite and transcript levels was investigated in leaves of Arabidopsis thaliana during illumination and after light-enhanced dark respiration (LEDR), when dark respiration was measured.• Primary carbon metabolites and the expression of light-responsive respiratory genes were determined in A. thaliana leaves before and after 30 min of darkness following different light conditions. In addition, metabolite levels were determined in the middle of the night and the in vivo activities of cytochrome and alternative respiratory pathways were determined by oxygen isotope fractionation.• A large number of metabolites were increased in leaves of plants growing in or transiently exposed to higher light intensities. Transcript levels of respiratory genes were also increased after high light treatment. For the majority of the light-induced metabolites and transcripts, the levels were maintained after 30 min of darkness, where higher and persistent respiratory activities were also observed. The levels of many metabolites were lower at night than after 30 min of darkness imposed in the day, but respiratory activities remained similar.• The results obtained suggest that 'dark' respiration measurements, as usually performed, are probably made under conditions in which the overall status of metabolites is strongly influenced by the previous light conditions.

NADPH and NADH mediate reductant flow between cellular processes, linking central carbon and energy metabolism with intermediary metabolism, stress defence and development. Recent investigations have revealed paths of functional interactions, and have suggested that mitochondrial NADPH oxidation, especially together with the oxidative pentose phosphate pathway, is an important regulator of the cytosolic NADPH reduction level. Furthermore, stress-dependent metabolic pathways substantially affect the NADPH reduction level in particular physiological situations. The mitochondrial impact on the NADPH reduction level provides a model example of the physiological significance of the mitochondrial NAD(P)H dehydrogenase set-up, which is more complex in plants than in other organisms.