Knock-out of the Plastid-encoded PetL Subunit Results in Reduced Stability and Accelerated Leaf Age-dependent Loss of the Cytochrome b6f Complex

Abstract

The cytochrome-b 6 f complex, a key component of the photosynthetic electron transport chain, contains a number of very small protein subunits whose functions are not well defined. Here we have investigated the function of the 31-amino acid PetL subunit encoded in the chloroplast genome in all higher plants. Chloroplast-transformed petL knock-out tobacco plants display no obvious phenotype, suggesting that PetL is not essential for cytochrome b 6 f complex biogenesis and function (Fiebig, A., Stegemann, S., and Bock, R. (2004) Nucleic Acids Res. 32, 3615-3622). We show here that, whereas young mutant leaves accumulate comparable amounts of cytochrome b 6 f complex and have an identical assimilation capacity as wild type leaves, both cytochrome b 6 f complex contents and assimilation capacities of mature and old leaves are strongly reduced in the mutant, indicating that the cytochrome b 6 f complex is less stable than in the wild type. Reduced complex stability was also confirmed by in vitro treatments of isolated thylakoids with chaotropic reagents. Adaptive responses observed in the knockout mutants, such as delayed down-regulation of plastocyanin contents, indicate that plants can sense the restricted electron flux to photosystem I yet cannot compensate the reduced stability of the cytochrome b 6 f complex by adaptive up-regulation of complex synthesis. We propose that efficient cytochrome b 6 f complex biogenesis occurs only in young leaves and that the capacity for de novo synthesis of the complex is very low in mature and aging leaves. Gene expression analysis indicates that the ontogenetic down-regulation of cytochrome b 6 f complex biogenesis occurs at the post-transcriptional level.The Cyt-bf 2 is the smallest of the three thylakoid membraneintrinsic multiprotein complexes of photosynthetic electron transport. Cyt-bf functions as a dimer with a molecular mass of 220 kDa and consists of nine subunits per monomer (1). It catalyzes the rate-limiting step of linear electron transport, oxidizing PQ generated by PSII and reducing PC. PC then diffuses through the thylakoid lumen toward PSI and reduces the photooxidized chlorophyll a dimer in the PSI reaction center, P 700 . The PQ-PC oxidoreductase activity of the Cyt-bf is coupled to the translocation of at least two protons per transported electron into the thylakoid lumen, thus contributing to the proton motif force required for ATP synthesis (2).The Cyt-bf is the predominant point of flux control of linear electron transport (3-6); its amount varies strongly in response to growth conditions and the developmental state of the plant, thus adjusting photosynthetic electron flux to the metabolic demand for ATP and NADPH. The mechanisms underlying these adjustments of Cyt-bf concentration are currently not understood. There are some indications that Cyt-bf is a relatively stable complex with lifetimes at least in the range of several days (7), but how Cyt-bf biogenesis is regulated during plant development is completely unknown. Cyt-bf biogenesis is a highly com...

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen

“…Evidence for this scenario comes from our characterization of a DpetL mutant suffering from accelerated leaf age-dependent loss of cyt-bf and reduced electron flux capacity due to absence of a small subunit of the cyt-bf. As the ATP synthase is not degraded in parallel with cyt-bf, but instead maintained at wild-type levels, nonphotochemical quenching in these mutants is compromised (Schö ttler et al, 2007b).…”

A Mediator of Singlet Oxygen Responses in Chlamydomonas reinhardtii and Arabidopsis Identified by a Luciferase-Based Genetic Screen in Algal Cells

“…Chlorophyll a fluorescence was recorded using a pulse-amplitude modulated fluorometer (DUAL-PAM-100; Heinz Walz) on intact plants at room temperature as described previously (Schöttler et al, 2007a(Schöttler et al, , 2007b. Plants were dark-adapted for 15 min prior to determination of the maximum photochemical efficiency of PSII (F v /F m ).…”

The Plastid Genome-Encoded Ycf4 Protein Functions as a Nonessential Assembly Factor for Photosystem I in Higher Plants

“…To test if the defect can be attributed to a specific complex, we next determined the contents of the components of the photosynthetic electron transport chain (PSII, Cyt bf, plastocyanin, and PSI; Fig. 3) by spectroscopic methods (Schöttler et al, 2007a(Schöttler et al, , 2007b. These analyses revealed that the Dycf4 mutants displayed a specific reduction in PSI contents (Fig.…”

“…The contents of PSII, Cyt bf, plastocyanin, and PSI were determined in thylakoids prepared as described previously (Schöttler et al, 2004). PSI was quantified from P 700 difference absorption signals at 830 to 870 nm in solubilized thylakoids using the Dual-PAM instrument (Schöttler et al, 2007a(Schöttler et al, , 2007b. Contents of PSII and Cyt bf were determined from difference absorption measurements of cytochrome b 559 and Cyt bf, respectively.…”