Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I

Abstract

To evaluate the physiological significance of cyclic electron f low around photosystem (PS) I, we used a reverse genetic approach to focus on 11 chloroplast genes that encode homologs of mitochondrial complex I subunits (ndhA-K). Since their discovery, the exact function of the respiratory components in plant chloroplasts has been a matter of discussion. We disrupted one of these genes (ndhB) in tobacco by chloroplast transformation. Analysis of the transient increase in chlorophyll f luorescence after actinic light illumination and the redox kinetics of P700 (reaction center chlorophylls of PS I) suggest that the cyclic electron f low around PS I is impaired in the ndhB-deficient transformants. Transformants grew normally in a greenhouse, suggesting that the cyclic electron f low around PS I mediated by ndh gene products is dispensable in tobacco under mild environmental conditions.Photosynthetic electron flow provides the first stable products of photosynthesis: NADPH and ATP. Despite the importance of this electron flow, a fundamental problem remains unsolved; that is, how an appropriate balance between the production of NADPH and ATP is maintained. To answer this question, the contributions of the Q cycle, cyclic electron flow around photosystem (PS) I, and pseudocyclic electron flow (water-water cycle) in chloroplast energetics must be evaluated quantitatively (1). There is little doubt that cyclic electron flow around PS I provides extra ATP in some cellular processes, such as N 2 fixation in cyanobacterial heterocysts (2) and CO 2 concentration in cyanobacterial and C4 photosynthesis (3-8). However, it is unclear whether this cyclic electron flow contributes to the supply of ATP during steady-state photosynthesis in nonspecialized photosynthetic cells of higher plants (1, 9, 10).Although molecular biological dissection using a reverse genetic approach is an effective means to evaluate the physiological significance of cyclic electron flow around PS I, it has not been attempted because of a lack of information about the genes responsible for the electron flow. However, the discovery of an ndhB-deficient mutant of Synechocystis PCC6803 that lacked cyclic electron flow around PS I led to the idea that electron f low is mediated by the respiratory complex, NAD(P)H dehydrogenase, in cyanobacteria (4-8).Eleven ndh genes encoding homologs of mitochondrial complex I subunits are also present in the chloroplast genome of higher plants (11,12). Although respiratory function is limited to the mitochondria, a respiratory complex, NAD(P)H dehydrogenase, may catalyze cyclic electron flow around PS I in chloroplasts, as in cyanobacteria. However, the existence of NAD(P)H dehydrogenase-mediated electron flow in higher plants is still a matter of controversy (1, 13), because genes for the crucial flavoprotein subunits have not yet been identified (14). Moreover, physiological evidence alone has been insufficient to show an NAD(P)H dehydrogenase-mediated pathway for cyclic electron flow around PS I in higher plants...

Increased Sensitivity of Photosynthesis to Antimycin A Induced by Inactivation of the Chloroplast ndhB Gene. Evidence for a Participation of the NADH-Dehydrogenase Complex to Cyclic Electron Flow around Photosystem I

“…In the WT, a transient increase in the fluorescence level was observed before reaching the F 0 level, this effect being absent in ndhB Ϫ . This confirms recent work reporting that the postillumination fluorescence rise is absent in ndh inactivated mutants (Burrows et al, 1998;Cournac et al, 1998;Kofer et al, 1998;Shikanai et al, 1998). Figure 2A shows that qP rapidly increased during the first 2 min of illumination in both WT and ndhB Ϫ before reaching progressively a plateau.…”

“…More recently, based on P700 ϩ rereduction measurements performed in barley thylakoids, Scheller (1996) proposed the existence of an antimycin-insensitive cyclic electron transport around PSI. The involvement of the NDH complex in cyclic electron flow in association with other pathways was shown in cyanobacteria (Mi et al, 1992;Yu et al, 1993) and recently suggested in higher plants from in vitro experiments performed on broken chloroplasts (Endo et al, 1998). Based on a differential sensitivity to antimycin A of PQ reduction in the WT and in a ndhB Ϫ mutant, these authors concluded to the existence of two pathways, one of them involving the NDH complex.…”

“…Previous studies, based on the disappearance of the transient postillumination rereduction of PQ in ndhinactivated mutants (Burrows et al, 1998;Cournac et al, 1998;Kofer et al, 1998;Shikanai et al, 1998) or on a decrease of the P700 ϩ reduction rate in the dark (Burrows et al, 1998) already concluded to an involvement of the NDH complex in intersystem chain reduction and therefore to its potential implication during cyclic electron transport. It appears from our experiments that the NDH activity is involved in cyclic electron transport together with the antimycinsensitive pathway.…”

“…To elucidate the function of the plastidial NDH complex in C 3 plants, ndh genes were inactivated by chloroplast transformation of tobacco (Nicotiana tabacum var Petit Havana) in different laboratories. Inactivation of ndhB, ndhC, ndhK, and ndhJ genes revealed that the NDH complex is dispensable for plant growth under standard conditions (Burrows et al, 1998;Shikanai et al, 1998;Horvath et al, 2000). The absence of a transient postillumination increase in chlorophyll fluorescence in all NDH-inactivated plastid transformants led to conclude that the NDH complex is involved in the dark reduction of the plastoquinone (PQ) pool, this phenomenon being considered as an after effect of cyclic electron flow around PSI (Burrows et al, 1998;Cournac et al, 1998;Kofer et al, 1998;Shikanai et al, 1998).…”

Structural and functional characterization of ferredoxin‐NADP+‐oxidoreductase using knock‐out mutants of Arabidopsis

“…In cyclic electron transfer, electrons can be recycled from reduced ferredoxin or NADPH either directly to plastoquinone (Clark et al, 1984;Zhang et al, 2001), or alternatively via the type I NAD(P)H dehydrogenase (NDH-1) complex to plastoquinone and subsequently to the cytochrome (cyt)-b 6 f complex. The latter route is known to be involved in dark reduction of the plastoquinone pool (Burrows et al, 1998;Joet et al, 2001;Kofer et al, 1998;Shikanai et al, 1998). The change in pH (DpH) generated by cyclic electron transfer is important for production of ATP, but also for the induction of thermal dissipation of absorbed energy from PSII antennae (Johnson, 2005).…”