Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

Light-responsive metabolite and transcript levels are maintained following a dark-adaptation period in leaves of Arabidopsis thaliana.

• 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.

Involvement of mitochondria in the control of plant cell NAD(P)H reduction levels.

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.

Suppression of the external mitochondrial NADPH dehydrogenase, NDB1, in Arabidopsis thaliana affects central metabolism and vegetative growth.

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.

An alternatively spliced domain of the NDC1 NAD(P)H dehydrogenase gene strongly influences the expression of the ACTIN2 reference gene in Arabidopsis thaliana.

In plant respiratory chains, alternative pathways for NAD(P)H oxidation are mediated by type II NAD(P)H dehydrogenases belonging to the NDA, NDB, and NDC families. For the latter type, Arabidopsis thaliana contains a single gene, NDC1, whose functional role has not previously been analyzed in the plant. We found that A. thaliana NDC1 is alternatively spliced. Four base pairs at the 3' end of intron 5 are spliced out in NDC1-1, but retained in the NDC1-2 mRNA, which therefore contains a truncated reading frame. Both variants are conserved in dicotyledonous and monocotyledonous plants and their relative abundance varies between organs and in response to light. Three analyzed NDC1 T-DNA insertion lines all displayed an early bolting phenotype. A dramatic upregulation of ACTIN2 was characteristic of two lines containing T-DNA inserts upstream of intron 5, whereas a line with an insertion downstream of the NDC1-2 reading frame had an ACTIN2 expression level identical to the wildtype. Thus, the alternatively spliced 5' domain of NDC1 strongly influences the expression of the functionally unrelated ACTIN2, which is a common reference gene for quantitative RT-PCR. Also for other reference genes, strong expressional effects were observed when comparing various mutants and wildtypes in microarray databases.

A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

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.