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TROL integrates chloroplast redox signalling

Introduction Thylakoid membranes of chloroplasts catalyze the fundamental process of photosynthetic energy conversion. The dual genetic origin and the enormous physiological versatility, in particular its ability to manage short- and long-term changes in the light environment, are outstanding characteristics of this specialized biomembrane. Various mechanisms can regulate the distribution of excitation energy between the two photosystems, and other can convert excess excitation energy into thermal energy. A significant number of auxiliary enzymes are believed to be involved in these physiological processes. Diverse protein kinases and phosphatases, chaperones, a substantial number of proteases that catalyze regulated protein degradation, and other protein components are required during biogenesis of the photosynthetic multisubunit complexes. In this work, we used reverse genetics approach to identify novel auxiliary thylakoid component, entitled TROL, which is necessary for sustaining efficient photosynthetic electron transport under conditions of elevated light intensity. Materials and Methods Full length At4g01050 cDNA was obtained from Arabidopsis Biological Resource Center, USA and fully sequenced. Gene silencing cassette was constructed by a 3’ -5’ insertion of the cDNA into pGPTV binary vector. Construct was introduced into Arabidopsis thaliana plants by Agrobacterium-mediated floral infiltration. Selection of transformants was performed using glufosinate ammonium resistance. Arabidopsis T-DNA insertional inactivation lines were obtained from Nottingham Arabidopsis Stock Centre, UK. Position of the insertion was verified by sequencing. Homozygous plants were identified via PCR. Plants were grown under 16 hour light regime in a growth chamber. Overexpression of a 23 kDa portion of the protein was done using pRSET system. Recombinant protein was purified by using Ni-NTA affinity matrix (Qiagen). Production of polyclonal antiserum was performed by Pineda (Germany). Yeast two-hybrid screen was performed according to manufacturer’ s procedure (Stratagene). In vitro chloroplast protein import assays were performed as described in (Fulgosi et al., 1998). Localization studies using YFP fusion were performed on isolated protoplasts using Leica confocal microscope TCS SP2 AOBS. Standard co-immunoprecipitation and Western analyses were performed on various sub-chloroplast fractions. In vivo chlorophyll a fluorescence kinetics was measured by using mini-PAM (Walz). Results and discussion Molecular characterization of TROL We have used proteomics approach to obtain N-terminal amino acid sequence of a hitherto uncharacterized chloroplast protein. The sequence was used to search Arabidopsis genome database and an ORF, which encodes a hypothetical polypeptide of 466 aa residues with a predicted thylakoid targeting presequence, was identified. Extensive bioinformatics analysis revealed the existence of putative rhodanese (thiosulfate sulfurtransferase)-like domain and a repetitive module possibly involved in binding of ferredoxin:NADP+ oxidoreductase (FNR). FNR catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P) (Carrillo and Caccareli, 2003). Accordingly, the protein was named TROL (thylakoid rhodanese-like). Localization studies using C-terminal YFP fusion revealed chloroplast localization of TROL. In vitro chloroplast import assays confirmed its thylakoid localization and have demonstrated that TROL possesses characteristics of an integral membrane component. Furthermore, TROL is most likely exclusively located in non-apressed thylakoid regions. To confirm the assumption that TROL is involved in anchoring of FNR, the sequence module at the C-terminus of TROL was used as bait in two-hybrid screen using FNR gene as a target. Following yeast transformation, the expression of both protein products resulted in a number of colonies showing β -galactosidase activity. Furthermore, thylakoid membranes were solubilised with 1% Triton X-100, and TROL antibodies were used for immunoprecipitation. FNR was significantly enriched in the immunoprecipitated fraction, whereas the preimmune serum of TROL did not co-precipitate detectable amounts of FNR. TROL is necessary for photosynthesis To gain insight into the function of TROL, we have used transgenic Arabidopsis lines in which At4g01050 gene was inactivated by an insertion of a T-DNA element into the last intron at position 2278. Insertion leads to a complete depletion of TROL protein, as tested by Western analyses of cellular extracts from homozygous plants. To be able to verify results obtained from analyses of TROL knock-out plants, we have generated antisense lines in which TROL protein accumulation was lowered. In low-light conditions, both knock-out and antisense lines did not show any visible phenotype. We have investigated photosynthetic performance of wild-type leaves and both transgenic lines by using Pulse-Amplitude fluorometry and saturation pulse method. Knock-out and antisense lines had severely lowered relative electron transport rate (ETR) at light intensities exceeding 250 µ ; molPHOTONS m-2s-1. Simultaneously, the amount of nonphotochemical quenching (NPQ) increases, indicating enhanced dissipation of absorbed light energy as heat. Thus, TROL deficient thylakoids direct only a small fraction of absorbed light to photosystems. In summary, we conclude that TROL represents a thylakoid membrane docking site for the assembly of a ternary complex between FNR, ferredoxin and NAD(P). Deletion of TROL leads to overreduction of thylakoid membranes, indicating that formation of such an anchored complex is required for oriented interaction between electron donor and acceptor in order to accommodate their respective redox centres in optimal orientation for efficient electron transfer. It is tempting to speculate that TROL is a key element in regulation of thylakoid redox poise, thereby influencing a wide range of redox signals originating from photosynthetic membranes. References Fulgosi H., Vener A., Altschmied L., Herrmann R. G. and Andersson B. (1998). EMBO Journal. 17 (6), 1577-1587. Carrillo N., Caccareli E. A. (2003) Eur. J. Biochem. 270, 1900-1915.

redox regulation; thylakoid membranes; ferredoxin NAD(P) oxidoreductase

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