The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress.

After incubation at 42 degrees C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (deltandhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of deltandhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42 degrees C for 6 h, the photochemical activity declined more markedly in deltandhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (deltapH), while the consumption of deltapH was highly inhibited in deltandhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO2 assimilation by providing extra deltapH, thus reducing the photooxidative damage.

Chloroplastic NAD(P)H dehydrogenase in tobacco leaves functions in alleviation of oxidative damage caused by temperature stress.

In this study, the function of the NAD(P)H dehydrogenase (NDH)-dependent pathway in suppressing the accumulation of reactive oxygen species in chloroplasts was investigated. Hydrogen peroxide accumulated in the leaves of tobacco (Nicotiana tabacum) defective in ndhC-ndhK-ndhJ (DeltandhCKJ) at 42 degrees C and 4 degrees C, and in that of wild-type leaves at 4 degrees C. The maximum quantum efficiency of PSII decreased to a similar extent in both strains at 42 degrees C, while it decreased more evidently in DeltandhCKJ at 4 degrees C. The parameters linked to CO(2) assimilation, such as the photochemical efficiency of PSII, the decrease of nonphotochemical quenching following the initial rise, and the photosynthetic O(2) evolution, were inhibited more significantly in DeltandhCKJ than in wild type at 42 degrees C and were seriously inhibited in both strains at 4 degrees C. While cyclic electron flow around PSI mediated by NDH was remarkably enhanced at 42 degrees C and suppressed at 4 degrees C. The proton gradient across the thylakoid membranes and light-dependent ATP synthesis were higher in wild type than in DeltandhCKJ at either 25 degrees C or 42 degrees C, but were barely formed at 4 degrees C. Based on these results, we suggest that cyclic photophosphorylation via the NDH pathway might play an important role in regulation of CO(2) assimilation under heat-stressed condition but is less important under chilling-stressed condition, thus optimizing the photosynthetic electron transport and reducing the generation of reactive oxygen species.

Response of NAD(P)H dehydrogenase complex to the alteration of CO2 concentration in the cyanobacterium Synechocystis PCC6803.

An NADPH-specific NDH-1 sub-complex was separated by native-polyacrylamide gel electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis PCC6803. Low CO2 caused an increase in the activity of this sub-complex quickly, accompanied by an evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that can reflect the activity of NDH-1-mediated cyclic electron transport. During incubation with high CO2, the activities of NDH-1 sub-complex and PSI-driven NADPH oxidation as well as the protein level of NdhK slightly increased at the beginning, but decreased evidently in various degrees along with incubation time. These results suggest that CO2 concentration in vitro as a signal can control the activity of NDH-1 complex, and NDH-1 complex may in turn function in the regulation of CO2 uptake.

[Separation of hydrophobic NAD(P)H dehydrogenase subcomplexes from cyanobacterium Synechocystis PCC6803].

Many efforts have been paid to the separation of an integrated NA(D)PH dehydrogenase (NDH) complex. Several hydrophilic subcomplexes of NDH have been purified from the cyanobacterium Synechocystis PCC6803. However, no hydrophobic NDH subcomplex has ever been separated from cyanobacteria yet. In this paper, two NDH subcomplexes were separated from n-dodecyl beta-D-maltoside(DM)-treated whole cell extracts of Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Both subcomplexes contained the hydrophobic subunit NdhA, suggesting that they were hydrophobic NDH subcomplexes. Of the two subcomplexes, only one subcomplex contained NdhH. These subcomplexes showed NADPH-nitroblue tetrazolium (NBT) oxidoreductase activity and could specifically oxidize NADPH when several quinone analogues were used as electron acceptors, such as ferricyanide, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichlorophenol indophenol (DCPIP), duroquinone, ubiquinone-0 (UQ-0), etc.

Effects of low CO2 on NAD(P)H dehydrogenase, a mediator of cyclic electron transport around photosystem I in the cyanobacterium synechocystis PCC6803.

The expression and activity of type-1 NAD(P)H dehydrogenase (NDH-1) was compared between cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO(2) conditions. Western analysis indicated that L-cells contain higher amounts of the NDH-1 subunits, NdhH, NdhI and NdhK. An NADPH-specific subcomplex of NDH-1 showed higher NADPH-nitroblue tetrazolium oxidoreductase activity in L-cells. The activities of both NADPH-menadione oxidoreductase and light-dependent NADPH oxidation driven by photosystem I were much higher in L-cells than in H-cells. The initial rate of re-reduction of P700(+) following actinic light illumination in the presence of DCMU under background far-red light was enhanced in L-cells. In addition, rotenone, a specific inhibitor of NDH-1, suppressed the relative rate of post-illumination increase in Chl fluorescence of L-cells more than that of H-cells, suggesting that the involvement of NDH-1 in cyclic electron flow around photosystem I was enhanced by low CO(2). Taken together, these results suggest that NDH-1 complex and NDH-1-mediated cyclic electron transport are stimulated by low CO(2) and function in the acclimation of cyanobacteria to low CO(2).

Low concentrations of NaHSO(3) increase cyclic photophosphorylation and photosynthesis in cyanobacterium Synechocystis PCC6803.

Application of NaHSO(3) solution at low concentrations (20-200 muM) to the culture medium enhanced photosynthetic oxygen evolution in cyanobacterium Synechocystis PCC6803 by more than 10%. The slow phase of ms-DLE was strengthened, showing that the transmembrane proton motive force related to photophosphorylation was enhanced. It was also observed that dry weight as well as ATP content under illuminated conditions were both increased after the treatment, indicating that low concentrations of NaHSO(3) could enhance the supply of ATP and thus increase biomass accumulation. In accord with the promotion in the photosynthetic oxygen evolution and ATP content, the transient increase in chlorophyll fluorescence after the termination of actinic light was increased; and meanwhile, the half-time of re-reduction of P700(+) in the presence of DCMU after a pulse light under background far-red light was shortened by approximately 30%, indicating that cyclic electron flow around PS I was accelerated by the treatment. Based on these results it is suggested that the increase in photosynthesis in Synechocystis PCC6803 caused by low concentrations of NaHSO(3) solution might be due to the stimulation of the cyclic electron flow around PS I and thus the increase in photophosphorylation.

The Involvement of Plastoquinone in the Pyocyanine-mediated Cyclic Electron Transport around Photosystem I.

2,5 dibromo-3-methyl-5-isopropyl-p-benzoquinone (DBMIB), an inhibitor of plastoquinone, inhibited photosystem I cyclic electron transport mediated by pyocyanine of low concentration, but had no effect on that mediated by phenazine methosulphate (PMS). In the presence of pyocyanine, the thylakoids displayed a transient post-illumination increase in chlorophyll fluorescence which resembled that displayed in leaves. The above results indicate the involvement of plastoquinone in the pyocyanine-mediated cyclic electron transport around photosystem I.

The Involvement of Ferredoxin-NADP Reductase in the Photosynthetic Cyclic Electron Transport.

As an inhibitor of ferredoxin-NADP reductase (FNR), heparin strongly inhibited the Fd-mediated photoreduction of NADP in spinach thylakoids at very low concentration (micromole), and also inhibited the delayed light emission induced by Fd/NADP. Under anaerobic conditions, when DCMU was present to block electron transfer from photosystem II, the Fd-mediated cyclic electron transport around photosystem I as indicated by the quenching of 9-aminoacridine can be measured. Heparin could strongly inhibit this cycle, but had no significant effect on the PMS-mediated cyclic electron transport around photosystem I. Heparin did not inhibit the electron transport from phototsystem I to ferredoxin, which was demonstrated by measuring the photoreduction of ferredoxin. The above results indicate that heparin inhibits ferredoxin-mediated cyclic electron transport by inhibiting the activity of FNR, and support the hypothesis of the involvement of FNR in cyclic electron transport.

Pyocyanine Promoted the Photoreduction of Cytochrome b-559 in Chloroplasts.

A light-induced absorbance increase at 559 nm was observed in spinach chloroplasts in the presence of pyocyanine. The reaction was induced by red light (650 nm) but not by far-red light (720 nm). The light-dark difference spectra indicated that the reduced component was cytochrome b-559. The effect of pyocyanine was nearly saturated at a concentration of 1 &mgr;M and was independent of the transmembrane proton gradient. The pyocyanine-promoted photoreduction of cytochrome b-559 was inhibited by DCMU and DBMIB. In cyanide-poisoned chloroplasts pyocyanine still promoted the photoreduction of cytochrome b-559. In chloroplasts treated with CCCP the oxidation of cytochrome b-559 can be induced by red light of low intensity. Pyocyanine reversed the effects of CCCP so as to promote the reduction of cytochrome b-559 by red light of both high and low intensities. We assumed that pyocyanine affected the light-induced redox reaction of cytochrome b-559 by affecting the protonation of the cytochrome.