Investigation On The Physiological Function Of Chloroplastic NAD(P)H Dehydrogenase

The recent research indicated that, besides the linear electron transport via the two photosystems (PSII and PSI), the cyclic electron transport pathways around PSI are also essential for the operation of efficient photosynthesis, and as one of which, the NAD(P)H dehydrogenase complex (NDH)-mediated cyclic electron transport has been well focused. In the following studies, using tobacco NDH deficient mutant, we clarified for the first time that the chloroplastic NDH complex-mediated PSI cyclic electron transport is responsible for the formation of trans-thylakoid proton gradient (?pH) and ATP is generated by its coupled photophosphorylation. On the basis of this, the function of NDH pathway during light fluctuation, temperature stress, and chloroplast development, when CO2 assimilation is affected, were further investigated: (1) During illumination of the dark-adapted plants, NDH pathway provides extra ?pH and ATP for the activation and stabilization of Rubisco activase, thus contributing to the light induction of photosynthesis; (2) Under heat stress conditions, NDH-mediated cyclic and chlororespiratory electron transport are accelerated, act as a safety valve for excess electrons in addition to providing extra ?pH and ATP, so that the photooxidative damage and inhibition of CO2 assimilation caused by high temperature are mitigated; (3) At the early stage of chloroplast development when linear electron transport was less effective, NDH was abundant and the cyclic electron transport was active, thus might compensate for the energy deficiency during formation of the photosynthetic apparatus, and also functioned in dissipation of the excess light energy.1.The Role of Chloroplastic NAD(P)H Dehydrogenase in Light Induction of Photosynthesis and Its Effect on Rubisco ActivaseWhen dark-adapted plants were transferred into light, compared with the wild type, the initiation of photosynthetic O2 evolution was more delayed and the rising phase of non-photochemical chlorophyll fluorescence quenching induction curve was slower in the mutant of tobacco (Nicotiana tabacum) defective in ndhC-ndhK-ndhJ (?ndhCKJ). The building-up of trans-thylakoid proton gradient (?pH) and the generation of ATP induced by light were significantly suppressed in the mutant. Meanwhile, the rate of light-induced H2O2 production was higher in ?ndhCKJ chloroplasts. In accordance with this, the kinetics of NADPH fluorescence indicated that the photo-reduction level of NADP+ was lower while the oxidation of NADPH via reactive oxygen species (ROS) increased in ?ndhCKJ during dark-light transition. Furthermore, although there was not differences in the transcriptional level of Rubisco activase, the amount of the stromal soluble active form of Rubisco activase, as well as its ATPase activity, increased more evidently in wild type than in ?ndhCKJ after light induction, while the inactive form bound to the thylakoid exhibited higher level in ?ndhCKJ. In support of this, in vitro analysis revealed that ATP and H2O2 are responsible for the stabilization and decomposition of Rubisco activase respectively. The results suggest that PSI cyclic electron flow mediated by chloroplastic NAD(P)H dehydrogenase (NDH) is involved in light induction of photosynthesis, especially through providing ATP for the stabilization and activation of Rubisco activase.2.Chloroplastic NAD(P)H Dehydrogenase in Tobacco Leaves Functions in Alleviation of Oxidative Damage Caused by Temperature StressIn this study, we investigated the function of NDH-dependent pathway in suppressing the generation of reactive oxygen species in chloroplasts. After incubation at 42°C for more than 48 h, oxidative damages occurred in the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (?ndhCKJ), followed by wilt of the leaves, while less phenotype was found in its wild type. At 4°C, both wilted quickly. By DAB (3, 3-diaminobenzidine) staining, hydrogen peroxide (H2O2) was found to be accumulated in the leaves of ?ndhCKJ at 42°C and 4°C, and in wild-type leaves at 4°C.The maximum photochemical efficiency of PSII (Fv/Fm) decreased to a similar extent in both strains at 42°C, while it decreased more evidently in ?ndhCKJ at 4°C. The parameters linked to CO2 assimilation such as the effective photochemical efficiency of PSII (ΦPSII), the decrease of qN following the initial rise and the photosynthetic O2 evolution were inhibited more significantly in ?ndhCKJ than in wild type at 42°C, and were seriously inhibited in both strains at 4°C. The cyclic electron transport around PSI mediated by NDH was remarkably enhanced at 42°C but suppressed at 4°C. Accordingly, the proton gradient across the thylakoid membranes and light-dependent ATP synthesis were higher in wild type than in ?ndhCKJ at either 25°C or 42°C, but were barely formed at 4°C.On the other hand, analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the chlororespiration mediated by NDH was also significantly enhanced in wild type under high temperature. After leaf disks were treated with methyl viologen (MV), the photosynthetic apparatus of ?ndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH-mediated cyclic and chlororespiratory electron flow probably functioned as an electron competitor with Mehler reaction, thus to reduce the accumulation of ROS. The amount of Rubisco activase was observed to be more evidently decreased in ?ndhCKJ than that in wild type under heat stressed conditions, which was probably attributable to the different levels of both ATP and H2O2.The results indicated that the cyclic electron flow mediated by NDH could be stimulated by heat stress, to divert excess electrons via chlororespiration. The NDH pathway-coupled cyclic photophosphorylation might play important role in the regulation of CO2 assimilation under heat stress condition but was less important under chilling stress condition, thus to optimize the photosynthetic electron transport and reduce the generation of ROS.3.Possible Involvement of NAD(P)H Dehydrogenase in Dissipation and Conversion of Light Energy during Chloroplast DevelopmentUpon exposure of the etiolated pumpkin cotyledon to light, large amount of fluorescence were emitted, and the rate of protochlorophyllide (Pchlide) re-accumulation decreased rapidly within the first hour, followed by a recovery to a submaximum level after about 2-h illumination, and then decreased slowly. Accordingly, the post-illumination fluorescence rise reflecting the electron transport via NDH also exhibited a highest level after 2-h illumination, and then gradually weakened. At the same time, the accumulation of chlorophyll and appearance of P700 oxidation activity were observed, as well as the formation of photosynthetic lamellar structure from the prolamellar body, along which NDH complexes were specifically located as revealed by immunogold-labelling. Also after about 1～2-h illumination, ATP content of the cotyledon dropped to the lowest level, which might be the result of extra ATP consumption in this constructing period. The highest activity of NDH-mediated electron transport as shown by the post-illumination fluorescence rise probably operated to compensate this energy deficiency. On the other hand, the amount of NDH and PTOX (plastid terminal oxidase) decreased, especially after 8 h of greening, since when H2O2 amount in the cotyledon was also evidently reduced. Based on these results, we suppose that the light driven transformation of Pchlide and NDH-dependent electron transport might be related to the dissipation of excess light energy and energy conversion at the early stage of the development of the photosynthetic apparatus.