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Continuous Chlorophyll Degradation Accompanied by Chlorophyllide and Phytol Reutilization for Chlorophyll Synthesis in Synechocystis Sp

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Continuous Chlorophyll Degradation Accompanied by Chlorophyllide and Phytol Reutilization for Chlorophyll Synthesis in Synechocystis Sp View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1767 (2007) 920–929 www.elsevier.com/locate/bbabio Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803 ⁎ Dmitrii Vavilin, Wim Vermaas School of Life Sciences and Center for the Study of Early Events in Photosynthesis, Arizona State University, Box 874501, Tempe, AZ 85287, USA Received 3 January 2007; received in revised form 23 March 2007; accepted 27 March 2007 Available online 3 April 2007 Abstract Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of 13C-labeled glucose or 15N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of 13C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of 13C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (12Por12Phy), (ii) 13C-labeled chlorophyll (13Por13Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was 13C-labeled, whereas the other constituent of the molecule remained unlabeled (13Por12Phy and 12Por13Phy). The kinetics of 12Por12Phy disappearance, presumably due to chlorophyll de- esterification, and of 13Por12Phy, 12Por13Phy, and 13Por13Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of 12Por12Phy was three-fold faster in a photosystem I-less strain than in a photosystem II- less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de- esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of 13Por12Phy and 12Por13Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process. © 2007 Elsevier B.V. All rights reserved. Keywords: Chlorophyll biosynthesis; Chlorophyll degradation; Mass spectrometry; Stable isotope labeling; Cyanobacteria 1. Introduction condensation of isopentenyl and dimethylallyl units form gera- nylgeranyl diphosphate, which can be converted to phytyl∼PP Chlorophyll a is the prevailing form of chlorophyll in plants by a NADPH-dependent enzyme (ChlP) that sequentially and green algae and the sole form of chlorophyll in many reduces three double bonds of geranylgeranyl. Chlorophyll cyanobacteria. As most other chlorophylls, chlorophyll a synthase (ChlG) catalyzes the last step of chlorophyll biosynth- consists of two moieties, the porphyrin macrocycle chlorophyl- esis by esterifying chlorophyllide with phytyl∼PP or with lide a and a branched-carbon phytyl tail. The chlorophyllide and geranylgeranyl∼PP [4]. In the latter case, ChlP reduces three phytyl moieties are formed by the tetrapyrrole and isoprenoid double bonds of the geranylgeranyl moiety of chlorophyll and biosynthesis pathways, respectively (Fig. 1; for reviews on converts it to phytylated chlorophyll [5], the dominant form of chlorophyll and isoprenoid biosynthesis see Ref. [1–3]). Eight chlorophyll in photosynthetic species. Phytyl∼PP also serves as 5-aminolevulinate molecules are required to make chlorophyl- a precursor for the synthesis of phylloquinone (vitamin K1) and lide, a cyclic tetrapyrrole with four pyrrole nitrogen atoms tocopherols (including vitamin E), in which phytol constitutes coordinating a central Mg2+ ion. In the biosynthesis of phytol, the lipophilic tail moiety, as in chlorophyll. Degradation of chlorophyll catalyzed by chlorophyllase starts by hydrolytic cleavage of the ester bond yielding chloro- Abbreviations: OD730, optical density at 730 nm; PS, photosystem; phytyl∼P, phytyl monophosphate; phytyl∼PP, phytyl pyrophosphate phyllide and free phytol. Chlorophyllide can be further degraded ⁎ Corresponding author. Tel.: +1 480 965 6250; fax: +1 480 965 6899. through the removal of Mg followed by oxidative opening of the E-mail address: [email protected] (W. Vermaas). tetrapyrrole macrocycle (reviewed in [1]). In plants, massive 0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2007.03.010 D. Vavilin, W. Vermaas / Biochimica et Biophysica Acta 1767 (2007) 920–929 921 13 13 μ chlorophyll breakdown initiated by chlorophyllase occurs dur- mixture of 1 mM C-glucose and 2.4 mM NaH CO3.20 M atrazine was also added to the wild-type and Δslr1652 cells. Every 24 h, the cultures were ing leaf senescence and fruit ripening. Interestingly, active chlo- 13 13 supplemented with additional portions of C-glucose and NaH CO3 (1 mM rophyllases have been found in vegetative plant tissues of all and 2.4 mM, respectively). ages [6,7], indicating that chlorophyll hydrolysis is not neces- 15N-labeling experiments were performed essentially as described earlier sarily accompanied by a net decrease in chlorophyll content. [14]. In brief, cell cultures grown to OD730 of 0.9 in BG-11 medium containing The fate of phytol released upon chlorophyll degradation is regular NaNO3 as nitrogen source were diluted with fresh BG-11 medium α without NaNO3, to a final OD730 of 0.3. The cultures were then supplemented unclear. Some bacteria are known to catabolize phytol via - 15 15 β with a mixture of Na NO3 and ( NH4)2SO4 to a final concentration of 2 mM and -oxidation pathways [8]. There are several reports of and 9 mM, respectively. 13 13 13 phytol that is esterified to fatty acids in leaves of higher plants C-glucose and NaH CO3 with C enrichment of at least 99%, and 15 15 15 [9,10], mosses [11], and dinoflagellates [12]. Also, it has been Na NO3 and ( NH4)2SO4 with N enrichment of at least 98% were purchased suggested that phytol can reenter central metabolism serving as from Cambridge Isotope Laboratories Inc. (Andover, MA). a precursor for tocopherol and phylloquinone synthesis ([10,13] and references therein). 2.3. Pigment extraction and purification We have studied chlorophyll stability in Synechocystis sp. 13 15 To determine the incorporation of C into chlorophyll, 15 to 60 mL portions PCC 6803 by incubating cells in the presence of N-labeled of Synechocystis cell culture labeled with 13C were harvested by centrifugation, 15 salts [14]. Using this N-chlorophyll labeling approach, under and chlorophyll was extracted from the pellet with 0.5 mL of methanol and normal growth conditions the overall degradation of the immediately subjected to HPLC separation as described earlier [14]. Fractions porphyrin macrocycle moiety of chlorophyll in Synechocystis containing chlorophyll were collected, dried under vacuum, and stored at −80 °C until further use. In order to perform quantitative mass-spectrometry was found to be very slow (half-time of many days). In the 13 13 analysis of C-labeled pigments, HPLC-purified chlorophyll was converted to present paper, C labeling was used to analyze separately the pheophytin by dissolving the pigment in 0.4 mL of methanol/water mixture (9:1 turnover of both the chlorophyllide a and phytyl moieties of the v/v) containing 20 μL of 0.1% HCl. After a 10- to 20-min incubation, pigments chlorophyll a molecule. We demonstrate that in actively were subjected to a second round of HPLC. As the Rt of pheophytin is growing Synechocystis cells chlorophyll dephytylation occurs significantly longer as compared to that of chlorophyll (21.3 and 17.6 min, faster than the irreversible degradation of the chlorophyll respectively), this procedure allowed obtaining pigment free of contaminants that co-elute with chlorophyll. macrocycle. Substantial amounts of chlorophyllide and phytol To measure the incorporation of 15N into chlorophyll and chlorophyllide, formed upon chlorophyll dephytylation are reused to make new 300 mL portions of PS I-less cell culture were harvested by centrifugation, the chlorophyll molecules, indicating that a chlorophyll de- pigments were extracted from the pellet with 2.5 mL of methanol, and the extract esterification/chlorophyllide re-esterification cycle operates was adjusted to 10 mL with a mixture of acetone:water:0.1 N NH4OH (56:11:8 continuously in cyanobacterial cells. v/v/v). Chlorophyll was extracted from the solvent mixture first with 10 mL of n-hexane and then with 3 mL of n-hexane. The hexane fractions containing chlorophyll and the remaining hexane-extracted acetone fraction containing 2. Materials and methods chlorophyllide were dried under vacuum and the dried pigments were subsequently subjected to the HPLC purification. 2.1. Strains and growth conditions 2.4. Mass spectroscopy A photosystem (PS) II-less strain of Synechocystis sp. PCC 6803 was constructed by sequentially transforming wild type cells with genomic DNA Positive ion mass spectra of pheophytin were obtained by matrix-assisted isolated from mutants with truncated psbB [15] and deleted psbDIC genes. The laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry deletion of psbDIC was originally introduced in Synechocystis sp. PCC 6803 by replacing a 2818-bp XmnI/SfiI fragment, which covered the entire psbDIC coding region together with 212 bp upstream
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