Ndh Mutant Research Articles

Deficiency in NDH-cyclic electron transport retards heat acclimation of photosynthesis in tobacco over day and night shift.

In order to cope with the impact of global warming and frequent extreme weather, thermal acclimation ability is particularly important for plant development and growth, but the mechanism behind is still not fully understood. To investigate the role of NADH dehydrogenase-like complex (NDH) mediated cyclic electron flow (CEF) contributing to heat acclimation, wild type (WT) tobacco (Nicotiana tabacum) and its NDH-B or NDH-C, J, K subunits deficient mutants (ΔB or ΔCJK) were grown at 25/20°C before being shifted to a moderate heat stress environment (35/30°C). The photosynthetic performance of WT and ndh mutants could all eventually acclimate to the increased temperature, but the acclimation process of ndh mutants took longer. Transcriptome profiles revealed that ΔB mutant exhibited distinct photosynthetic-response patterns and stress-response genes compared to WT. Metabolite analysis suggested over-accumulated reducing power and production of more reactive oxygen species in ΔB mutant, which were likely associated with the non-parallel recovery of CO2 assimilation and light reactions shown in ΔB mutant during heat acclimation. Notably, in the warm night periods that could happen in the field, NDH pathway may link to the re-balance of excess reducing power accumulated during daytime. Thus, understanding the diurnal cycle contribution of NDH-mediated CEF for thermal acclimation is expected to facilitate efforts toward enhanced crop fitness and survival under future climates.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

Improved Hydrogen Peroxide Stress Resistance of Zymomonas mobilis NADH Dehydrogenase (ndh) and Alcohol Dehydrogenase (adhB) Mutants

Unintended shifts in stress resistance of microbial strains with engineered central metabolism may impact their growth and production performance under oxidative, lignocellulosic, solvent, and other stress conditions, and as such, must be taken into account in bioprocess design. In the present work, we studied oxidative stress resistance in mutant strains of the facultatively anaerobic, ethanologenic bacterium Zymomonas mobilis with modified respiratory (inactivated NADH dehydrogenase Ndh, by disruption of ndh) and ethanologenic (inactivated iron-containing alcohol dehydrogenase isoenzyme ADH II, by disruption of adhB) catabolism, using exogenously added H2O2 in the concentration range of 2–6 mM as the oxidative stressor. Both mutations improved H2O2 resistance and enhanced catalase activity by a factor of 2–5, while the overexpression of Ndh had an opposite effect. Strains with a catalase-negative background were unable to grow already at 1 mM hydrogen peroxide, and their H2O2 resistance did not depend on AdhB or Ndh expression levels. Hence, the improved resistance of the ndh and adhB mutants to H2O2 resulted from their elevated catalase activity. The interrelation between these mutations, the catabolic redox balance, catalase activity, and oxidative stress defense in Z. mobilis is discussed.

Computational study to understand mechanism of isoniazid drug resistance caused by mutation (R268H) in NADH dehydrogenase of Mycobacterium tuberculosis

NADH dehydrogenase (Ndh) of Mycobacterium tuberculosis is essential for conversion of NADH to NAD+ in the presence of FMN. An increased NADH/NAD+ ratio was reported due to mutation (R268H) in Ndh, causing INH resistance. To study the effect of this mutation on Ndh, molecular dynamics (MD) simulation analysis was performed for both wild and mutant models independently as well as for docked complexes (Ndh-NADH and Ndh-FMN). Simulation study showed that mutation (R268H) affected the secondary structure of the enzyme giving extra stability to the mutant model R268H as observed in the root mean square deviation (RMSD) plot. Furthermore, it was observed that both wild-type and mutant models of Ndh were quite stable in complex with NADH but in case of FMN, the Ndh mutant appears to be more unstable and might be the reason for decreasing NAD+ concentrations thus hindering INH-NAD adduct formation resulting in isoniazid resistance.

Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis.

Cyclic electron flux (CEF) around Photosystem I (PS I) is difficult to quantify. We obtained the linear electron flux (LEFO2) through both photosystems and the total electron flux through PS I (ETR1) in Arabidopsis in CO2-enriched air. ΔFlux = ETR1 – LEFO2 is an upper estimate of CEF, which consists of two components, an antimycin A-sensitive, PGR5 (proton gradient regulation 5 protein)-dependent component and an insensitive component facilitated by a chloroplastic nicotinamide adenine dinucleotide dehydrogenase-like complex (NDH). Using wild type as well as pgr5 and ndh mutants, we observed that (1) 40% of the absorbed light was partitioned to PS I; (2) at high irradiance a substantial antimycin A-sensitive CEF occurred in the wild type and the ndh mutant; (3) at low irradiance a sizable antimycin A-sensitive CEF occurred in the wild type but not in the ndh mutant, suggesting an enhancing effect of NDH in low light; and (4) in the pgr5 mutant, and the wild type and ndh mutant treated with antimycin A, a residual ΔFlux existed at high irradiance, attributable to charge recombination and/or pseudo-cyclic electron flow. Therefore, in low-light-acclimated plants exposed to high light, ΔFlux has contributions from various paths of electron flow through PS I.

Reduction of dNTP levels enhances DNA replication fidelity in vivo

ATP is the most important energy source for the maintenance and growth of living cells. Here we report that the impairment of the aerobic respiratory chain by inactivation of the ndh gene, or the inhibition of glycolysis with arsenate, both of which reduce intracellular ATP, result in a significant decrease in spontaneous mutagenesis in Escherichia coli. The genetic analyses and mutation spectra in the ndh strain revealed that the decrease in spontaneous mutagenesis resulted from an enhanced accuracy of the replicative DNA polymerase. Quantification of the dNTP content in the ndh mutant cells and in the arsenate-treated cells showed reduction of the dNTP pool, which could explain the observed broad antimutator effects. In conclusion, our work indicates that the cellular energy supply could affect spontaneous mutation rates and that a reduction of the dNTP levels can be antimutagenic.

Anaerobiosis Induced State Transition: A Non Photochemical Reduction of PQ Pool Mediated by NDH in Arabidopsis thaliana

BackgroundNon photochemical reduction of PQ pool and mobilization of LHCII between PSII and PSI are found to be linked under abiotic stress conditions. The interaction of non photochemical reduction of PQ pool and state transitions associated physiological changes are critically important under anaerobic condition in higher plants.Methodology/FindingsThe present study focused on the effect of anaerobiosis on non-photochemical reduction of PQ pool which trigger state II transition in Arabidopsis thaliana. Upon exposure to dark-anaerobic condition the shape of the OJIP transient rise is completely altered where as in aerobic treated leaves the rise is unaltered. Rise in F o and F J was due to the loss of oxidized PQ pool as the PQ pool becomes more reduced. The increase in Fo′ was due to the non photochemical reduction of PQ pool which activated STN7 kinase and induced LHCII phosphorylation under anaerobic condition. Further, it was observed that the phosphorylated LHCII is migrated and associated with PSI supercomplex increasing its absorption cross-section. Furthermore, evidences from crr2-2 (NDH mutant) and pgr5 mutants (deficient in non NDH pathway of cyclic electron transport) have indicated that NDH is responsible for non photochemical reduction of the PQ pool. We propose that dark anaerobic condition accelerates production of reducing equivalents (such as NADPH by various metabolic pathways) which reduce PQ pool and is mediated by NDH leading to state II transition.Conclusions/SignificanceAnaerobic condition triggers non photochemical reduction of PQ pool mediated by NDH complex. The reduced PQ pool activates STN7 kinase leading to state II transition in A. thaliana.

Electron transport and oxidative stress in Zymomonas mobilis respiratory mutants

The ethanol-producing bacterium Zymomonas mobilis is of great interest from a bioenergetic perspective because, although it has a very high respiratory capacity, the respiratory system does not appear to be primarily required for energy conservation. To investigate the regulation of respiratory genes and function of electron transport branches in Z. mobilis, several mutants of the common wild-type strain Zm6 (ATCC 29191) were constructed and analyzed. Mutant strains with a chloramphenicol-resistance determinant inserted in the genes encoding the cytochrome b subunit of the bc (1) complex (Zm6-cytB), subunit II of the cytochrome bd terminal oxidase (Zm6-cydB), and in the catalase gene (Zm6-kat) were constructed. The cytB and cydB mutants had low respiration capacity when cultivated anaerobically. Zm6-cydB lacked the cytochrome d absorbance at 630 nm, while Zm6-cytB had very low spectral signals of all cytochromes and low catalase activity. However, under aerobic growth conditions, the respiration capacity of the mutant cells was comparable to that of the parent strain. The catalase mutation did not affect aerobic growth, but rendered cells sensitive to hydrogen peroxide. Cytochrome c peroxidase activity could not be detected. An upregulation of several thiol-dependent oxidative stress-protective systems was observed in an aerobically growing ndh mutant deficient in type II NADH dehydrogenase (Zm6-ndh). It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.

Two Proteins Homologous to PsbQ are Novel Subunits of the Chloroplast NAD(P)H Dehydrogenase

The PsbQ-like (PQL) proteins 1 and 2, previously shown to be located in the thylakoid lumen of Arabidopsis thaliana, are homologous to PSII oxygen-evolving complex protein PsbQ. Nevertheless, pql mutants showed no defects in PSII but instead the activity of the chloroplast NAD(P)H dehydrogenease (NDH) complex was severely impaired. In line with this observation, the NDH subunits were low in abundance in pql mutants, and, conversely, ndh mutants strongly down-regulated the accumulation of the PQL proteins. In addition, the PQL2 protein was up-regulated in mutant plants deficient in the PSI complex or the thylakoid membrane-bound ferredoxin-NADP(+) oxidoreductase, whereas in pql mutants the PSI complex was slightly up-regulated. Taken together, the two PQL proteins are shown to be novel subunits of the lumenal protuberance of the NDH complex.

AtCYP20-2 is an auxiliary protein of the chloroplast NAD(P)H dehydrogenase complex

AtCYP20-2 is one of 16 immunophilins in thylakoid lumen. The presence of the isomerase domain in AtCYP20-2, an enrichment of AtCYP20-2 in the stroma membranes and it’s co-migration with NAD(P)H dehydrogenase (NDH) in native gels provide evidence that AtCYP20-2 is an auxiliary protein of NDH. When different NDH mutants were studied, AtCYP20-2 was found to be strongly reduced especially in mutants deficient in the membrane domain of NDH, thus suggesting a role in the assembly of NDH hydrophobic domain. Lack of AtCYP20-2, however, did not lead to severe malfunction of NDH, indicating redundancy in the function of lumenal immunophilins.

Novel Nuclear-encoded Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.

Electron transport activities of Arabidopsis thaliana mutants with impaired chloroplastic NAD(P)H dehydrogenase

The activities of electron transport are compared between wild-type Arabidopsis and two Arabidopsis mutants deficient for the chloroplastic NAD(P)H dehydrogenase (NDH) which catalyzes cyclic electron transport around photosystem I. The quantum yield of photosystem II and the degree of non-photochemical quenching of chlorophyll fluorescence were of similar levels in the two NDH-deficient mutants and the wild type under non-stressed standard growth conditions. Stromal over-reduction was induced in Arabidopsis NDH mutants with high light treatment, as is the case in tobacco NDH mutants. However, unlike tobacco mutants, photoinhibition was not observed in the Arabidopsis NDH mutants.

Characterization of NADH dehydrogenases of Pseudomonas fluorescens WCS365 and their role in competitive root colonization.

The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization. Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization. In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201. The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P. fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN. It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity. In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas. Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions. The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth. By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere. In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization.

Reply from H-U. Koop, W. Kofer and K. Steinmüller

Reply from H-U. Koop, W. Kofer and K. Steinmüller

In vitro synthesis of the respiratory NADH dehydrogenase of Escherichia coli. Role of UUG as initiation codon.

The respiratory NADH dehydrogenase of Escherichia coli has been synthesized in vitro in a coupled transcription--translation system with cloned deoxyribonucleic acid (DNA) as template. The identity of the protein produced was confirmed by paper chromatography and electrophoresis of tryptic peptides. [35S]Methionine-labeled tryptic peptides from the in vitro product were shown to comigrate with authentic methionine-containing tryptic peptides from the purified enzyme. Using a transcription-translation system derived from an ndh mutant, it was shown that the enzyme produced in vitro was incorporated into membrane vesicles of the mutant to give functional, cyanide-sensitive NADH oxidase activity. Radiochemical N-terminal sequencing of the synthesized NADH dehydrogenase showed that the product was a mixture of three different species, with N-formylmethionine, methionine, or threonine at the N terminus. The results indicated that only partial N-terminal processing was occurring in vitro and that the first residue of the unprocessed NADH dehydrogenase is N-formylmethionine. Since DNA sequencing has shown that this residue is encoded by UUG [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. (in press)], this work verifies the role of UUG as a normal initiation codon.

Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles.

Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli.

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.

Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Ap r and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8–10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50–60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.