Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803.

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.

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803.

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE(-) mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with--presumably--allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.

15N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems.

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with ((15)NH(4))(2)SO(4) and Na(15)NO(3). Pigments extracted from cells were separated by HPLC and incorporation of the (15)N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time (tau) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 micromol photons m(-2) s(-1) was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation (tau approximately 200-400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4-5 fold (tau approximately 50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics.

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range. Assessment of isotope ratio measurement variability suggests substantial contributions from natural measurement variability. A better understanding of this variable will allow assessment of the contribution of sequence dependence. Both variables must be understood before meaningful mixing experiments for relative expression proteomics are performed. Subtle modification of isotope ratio ( approximately 1-2% increase in (13)C) had no effect upon either the ability of data-dependent acquisition software or database searching software to trigger tandem mass spectrometry or match MSMS data to peptide sequences. More severe modification of isotope ratio caused a significant drop in performance of both functionalities. Development of software for deconvolution of isotope ratio concomitant with protein identification using LC-MSMS, or any other proteomics strategy, is underway (Isosolv). The identified peptide sequence is then be used to provide elemental composition for accurate isotope ratio decoding and the potential to control for specific amino acid biases should these prove significant. It is suggested that subtle modification of isotope ratio proteomics (SMIRP) offers a convenient approach to in vivo isotope coding of plants and might ultimately be extended to mammals including humans.

Isolation of functional photosystem II core particles from the Cyanobacterium synechocystis sp. PCC 6803.

This chapter contains the description of several methods used for the isolation of functional photosystem (PS)II core particles from wild-type (wt), PSI-less, and CP47 histidine-tagged cells of the cyanobacterium Synechocystis sp. PCC 6803. These protocols discuss the cultivation of PSI-containing and PSI-less cells, isolation of thylakoid membranes, purification of PSII core particles using a weak cation exchange or metal affinity column chromatography, and characterization of the final preparation. The described isolation procedures, which normally yield PSII particles highly active in oxygen evolution, can be easily adapted for obtaining preparations from different types of Synechocystis mutants with modified PSII.

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria.

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.

Methods for the isolation of functional photosystem II core particles from the cyanobacterium Synechocystis sp. PCC 6803.

This chapter contains the description of several methods used for the isolation of functional photosystem II (PS II) core particles from wild type, photosystem I-less, and CP47 histidine-tagged cells of the cyanobacterium Synechocystis sp. PCC 6803. The presented protocols cover cultivation of photosystem I-containing and photosystem I-less cells, isolation of thylakoid membranes, purification of PS II core particles using a weak cation exchange or metal affinity column chromatography, and characterization of the final preparation. These isolation procedures yield highly active oxygen-evolving PS II particles and can be easily adapted for obtaining preparations from Synechocystis mutants with genetically modified photosystem II.

Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C.

Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated beta-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.

The presence of chlorophyll b in Synechocystis sp. PCC 6803 disturbs tetrapyrrole biosynthesis and enhances chlorophyll degradation.

Both chlorophyll (Chl) a and b accumulate in the light in a Synechocystis sp. PCC 6803 strain that expresses higher plant genes coding for a light-harvesting complex II protein and Chl a oxygenase. This cyanobacterial strain also lacks photosystem (PS) I and cannot synthesize Chl in darkness because of the lack of chlL. When this PS I-less/chlL(-)/lhcb(+)/cao(+) strain was grown in darkness, small amounts of two unusual tetrapyrroles, protochlorophyllide (PChlide) b and pheophorbide (pheide) b, were identified. Accumulation of PChlide b trailed that of PChlide a by several days, suggesting that PChlide a is an inefficient substrate of Chl a oxygenase. The presence of pheide b in this organism suggests a breakdown of Chl b via a pathway that does not involve conversion to a-type pigments. When the PS I-less/chlL(-) control strain was grown in darkness, Chl degradation was much slower than in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain, suggesting that the presence of Chl b leads to more rapid turnover of Chl-binding proteins and/or a more active Chl degradation pathway. Levels and biosynthesis kinetics of Chl and of its biosynthetic intermediates are very different in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain versus in the control. Moreover, when grown in darkness for 14 days, upon the addition of delta-aminolevulinic acid, the level of magnesium-protoporphyrin IX increased 60-fold in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain (only approximately 2-fold in the PS I-less/chlL(-) control strain), whereas the PChlide and protoheme levels remained fairly constant. We propose that a b-type PChlide, Chl, or pheide in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain may bind to tetrapyrrole biosynthesis regulatory protein(s) (for example, the small Cab-like proteins) and thus affect the regulation of this pathway.

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant.

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.

Multiple deletions of small Cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: consequences for pigment biosynthesis and accumulation.

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA-scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of delta-aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/ch/L-/scpE- strain, whereas PChlide accumulated in the PS I-less/chlL-/scpB- strain. In the PS I-less/chlL- control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.