Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L

International audienceDuring daffodilflower development,chloroplasts differentiate into photosynthetically inactive chromoplasts, which have lostfunctional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase(PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6fcomplex, ATP synthase and several isoforms of ferredoxin-NADP+oxidoreductase (FNR) and of ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6fcomplex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors forthe NDH complex, the cytochrome b6fcomplex, FNR and redox-inactive Fd in which the iron was replaced by gallium. Our data suggest anelectron flowvia twoseparatepathways, both reducing plastoquinone and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd,and cytochrome bh of the cytochrome b6fcomplex and does not result in the pumpingofprotons across the membrane. In the second,electron transport takes place via the NDH complex using preferentially NADH but also NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH-complex is responsible for the generation of the proton gradient. We propose a new model for chromorespiration which may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6fcomplex during cyclic electron transport in chloroplasts

On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate