Metabolic engineering of the Chl d-dominated cyanobacterium Acaryochloris marina: production of a novel Chl species by the introduction of the chlorophyllide a oxygenase gene

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.     

Binding affinity of Chl b for the Chl a-binding sites in PSI core complexes

Most Chl a in PSI complexes was removed without any loss of P700 by ether treatment, yielding antenna-depleted P700-Chl a protein complexes (CP1s) with a Chl a/P700 ratio of 12. On addition of about 60 molecules of Chl b per P700 with phosphatidylglycerol, about 20 molecules of Chl b per P700 were bound to the complexes. The ratio of the bound Chl b to the added Chl b was about one-third, irrespective of the amount of Chl b added. The same partition ratio was obtained on reconstitution with Chl a, suggesting that the binding affinity of Chl b for the Chl a-binding sites is similar to that of Chl a. The relative quantum efficiency of P700 photooxidation, determined by the increase in its initial rate, increased in proportion to the increase in number of bound Chl b molecules. The degree of the increase was the same as expected if the bound Chl b had the same antenna activity as the bound Chl a. The bound Chl b emitted fluorescence with a peak at 660 nm, and its yield was as high as the Chl a remaining in the complexes. However, the excitation spectrum of the Chl a fluorescence, detected at 680 nm, was almost the same as the absorption spectrum of the Chl b-bound complexes, indicating efficient energy transfer of the bound Chl b to Chl a. These results suggest that Chl b primarily occupies the Chl a-binding sites close to the reaction center region, acting as an efficient antenna for P700.     

Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris.

Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.     

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

The oligomeric states of the photosystems and the light-harvesting complexes in the Chl b-less mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.     

P700: the primary electron donor of photosystem I.

The primary electron donor of photosystem I, P700, is a chlorophyll species that in its excited state has a potential of approximately -1.2 V. The precise chemical composition and electronic structure of P700 is still unknown. Recent evidence indicates that P700 is a dimer of one chlorophyll (Chl) a and one Chl a'. The Chl a' and Chl a are axially coordinated by His residues provided by protein subunits PsaA and PsaB, respectively. The Chl a', but not the Chl a, is also H-bonded to the protein. The H-bonding is likely responsible for selective insertion of Chl a' into the reaction center. EPR studies of P700(+*) in frozen solution and single crystals indicate a large asymmetry in the electron spin and charge distribution towards one Chl of the dimer. Molecular orbital calculations indicate that H-bonding will specifically stabilize the Chl a'-side of the dimer, suggesting that the unpaired electron would predominantly reside on the Chl a. This is supported by results of specific mutagenesis of the PsaA and PsaB axial His residues, which show that only mutations of the PsaB subunit significantly alter the hyperfine coupling constants associated with a single Chl molecule. The PsaB mutants also alter the microwave induced triplet-minus-singlet spectrum indicating that the triplet state is localized on the same Chl. Excitonic coupling between the two Chl a of P700 is weak due to the distance and overlap of the porphyrin planes. Evidence of excitonic coupling is found in PsaB mutants which show a new bleaching band at 665 nm that likely represents an increased intensity of the upper exciton band of P700. Additional properties of P700 that may give rise to its unusually low potential are discussed.     

Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to P(D1) and P(D2). These findings clearly indicate that Chl a is required for water oxidation in PS II.     

Spectrophotometric method of controlled pheophytinization for the determination of both chlorophylls and pheophytins in plant extracts

Conventional spectrophotometric methods of chlorophyll (Chl) measurement with double-wavelength readings of absorbances corresponding to the peak values of both Chl a and Chl b are usable only is pheophytins (Pheo) are absent in the pigment extract. We present here a kinetic method of controlled phenophytinization of the Chl present in acetone/water (90/10, v/v) exctracts that allows the measurement of both Chl a and b, as well as of the initial Pheo preexisting in the plant material at the moment of the extraction. This method gives better accuracy in Chl b determination than conventional methods, particularly when Chl a/Chl b ratios are greater than 5. Examples are given illustrating the usefulness of this method.     

Effect of the spectral composition of light on the mechanism of reaction of chlorophyll oxidation]

Photochemical oxidation of chlorophyll "a" on excitation of the pigment in different spectrum region (400-800 nm) was studied by flash photolysis. The absorption spectrum of chlorophyll cation-radical Chl+ was obtained and the values of extinction coefficient found. An attempt was made of photochemical generation of dication form of chlorophyll. Thermodynamical calculation supporting the possibility of the following reaction is presented: (Chl+)*+Ae -- Chl2"Ae. The absence of Chl2+ is explained by a short life time of the excited cation-radical of chlorophyll (Chl+)*. The effect of the wave length of excited light on the kinetics of the decay of chlorophyll cation-radical is studied. It is shown that on excitation of chlorophyll "a" with white light the life time of Chl+ decreases and its death is described by an equation of first order, which is explained by the formation of ion-radical of the electron acceptor resulting from the direct excitation of Ae.     

Use of the atLEAF+ chlorophyll meter for a nondestructive estimate of chlorophyll content

At present, chlorophyll meters are widely used for a quick and nondestructive estimate of chlorophyll (Chl) contents in plant leaves. Chl meters allow to estimate the Chl content in relative units - the Chl index (CI). However, using such meters, one can face a problem of converting CI into absolute values of the pigment content and comparing data acquired with different devices and for different plant species. Many Chl meters (SPAD-502, CL-01, CCM-200) demonstrated a high degree of correlation between the CI and the absolute pigment content. A number of formulas have been deduced for different plant species to convert the CI into the absolute value of the photosynthetic pigment content. However, such data have not been yet acquired for the atLEAF+ Chl meter. The purpose of the present study was to assess the applicability of the atLEAF+ Chl meter for estimating the Chl content. A significant species-specific exponential relationships between the atLEAF value (corresponding to CI) and extractable Chl a, Chl b, Chl (a+b) for Calamus dioicus and Cleistanthus sp. were shown. The correlations between the atLEAF values and the content of Chl a, Chl b, and Chl (a+b) per unit of leaf area was stronger than that per unit of dry leaf mass. The atLEAF value- Chl b correlation was weaker than that of atLEAF value-Chl a and atLEAF value-Chl (a+b) correlations. The influence of light conditions (Chl a/b ratio) on the atLEAF value has been also shown. The obtained results indicated that the atLEAF+ Chl meter is a cheap and convenient tool for a quick nondestructive estimate of the Chl content, if properly calibrated, and can be used for this purpose along with other Chl meters.     

Phosphorescence study of chlorophyll d photophysics. Determination of the energy and lifetime of the photo-excited triplet state. Evidence of singlet oxygen photosensitization

Chlorophyll d (Chl d) is the major pigment in both photosystems (PSI and II) of the cyanobacterium Acaryochloris marina, whose pigment composition represents an interesting alternative in oxygenic photosynthesis. While abundant information is available relative to photophysical properties of Chl a , the understanding of Chl d photophysics is still incomplete. In this paper, we present for the first time a characterization of Chl d phosphorescence, which accompanies radiative deactivation of the photoexcited triplet state of this pigment. Reliable information was obtained on the energy and lifetime of the Chl d triplet state in frozen solutions at 77?K using diethyl ether and aqueous dispersions of Triton X100 as solvents. It is shown that triplet Chl d is effectively populated upon photoexcitation of pigment molecules and efficiently sensitizes singlet oxygen phosphorescence in aerobic solutions under ambient conditions. The data obtained are compared with the previous results of the phosphorescence studies of Chl a and Pheo a, and their possible biological implications are discussed.     

The nature of the photosystem II reaction centre in the chlorophyll d-containing prokaryote, Acaryochloris marina.

Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.     

Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana

Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Effects of Chromatic Adaptation on the Photochemical Apparatus of Photosynthesis in Porphyridium cruentum

Cells of Porphyridium cruentum were grown in different colors of light which would be absorbed primarily by chlorophyll (Chl) (red and blue light) or by the phycobilisomes (green or two intensities of cool-white fluorescent light), and samples of these cells were frozen to −196 C for measurements of absorption and fluorescence emission spectra. Cells grown in the high intensity white light had least of all of the photosynthetic pigments, a higher ratio of carotenoid/Chl, but essentially the same ratio of phycobilin to Chl as cells grown in the low intensity white light. The ratio of photosystem II (PSII) to photosystem I (PSI) pigments was affected by light quality; the ratios of phycobilin to Chl and of short wavelength (PSII) Chl to long wavelength (PSI) Chl were both greater in the cells grown in red or blue light.     

Characterization of three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II isolated from the green alga, Dunaliella salina

Three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II (LHC II) were isolated from the thylakoid membranes of Dunaliella salina grown under different irradiance conditions. Cells grown under a low intensity light condition (80 micromol quanta m(-2) s(-1)) contained one form of LHC II, LHC-L. Two other forms of LHC II, LHC-H1 and LHC-H2, were separated from the cells grown under a high intensity light condition (1,500 micromol quanta m(-2) s(-1)). LHC-L and LHC-H1 showed an apparent particle size of 310 kDa and contained four polypeptides of 31, 30, 29 and 28 kDa. LHC-H2, with a particle size of 110 kDa, consisted of 30 and 28 kDa polypeptides. LHC-L contained 7.5 molecules of Chl a, 3.2 of Chl b and 2.1 of lutein per polypeptide, analogous to the content in higher plants. LHC-H1, with 5.6 molecules of Chl a, 2.5 of Chl b and 1.8 of lutein per polypeptide was similar to that in the green alga Bryopsis maxima. LHC-L and LHC-H1 maintained high efficiency energy transfer from Chl b and lutein to Chl a molecules. LHC-H2 showed a high Chl a/b ratio of 7.5 and contained 3.4 molecules of Chl a, 0.5 of Chl b and 1.4 of lutein per polypeptide. Chl b and lutein could not completely transfer the excitation energy to Chl a in LHC-H2.