The Light-Dependent and Light-Independent Reduction of Protochlorophyllide a to Chlorophyllide a

Two different pathways for protochlorophyllide a (Pchlide) reduction in photosynthetic organisms have been proved: one is strictly light-dependent whereas the second is light-independent. Both pathways occur in all photosynthetic cells except in angiosperms which form chlorophyll only through the light-dependent pathway. Most cells belonging to Eubacteria (i.e., the anoxygenic photosynthetic bacteria) synthesize bacteriochlorophyll through the light-independent pathway. This review deals with the physiological, biochemical, and molecular biological features of molecules involved in both pathways of Pchlide reduction.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Light-independent accumulation of essential chlorophyll biosynthesis- and photosynthesis-related proteins in <Emphasis Type="Italic">Pinus mugo</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings

Dark-grown seedlings of Pinus mugo Turra and Pinus sylvestris L. accumulate chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide). Pchlide reduction is a key regulatory step in Chl biosynthesis. In the dark, Pchlide is reduced by light-independent Pchlide oxidoreductase (DPOR) encoded by three plastid genes chlL, chlN, and chlB (chlLNB). To investigate the differences in chlLNB gene expressions, we compared the dark-grown and 24-h illuminated seedlings of P. mugo and P. sylvestris. Expression of these genes was found constitutive in all analyzed samples. We report light-independent accumulation of important proteins involved in Chl biosynthesis (glutamyl-tRNA reductase) and photosystem formation (D1 and LHCI). Chl and Pchlide content and plastid ultrastructure studies were also performed.     

A short overview of chlorophyll biosynthesis in algae

Chlorophylls are the most abundant classes of natural pigments and their biosynthesis is therefore a major metabolic activity in the ecosphere. Two pathways exist for chlorophyll biosynthesis, one taking place in darkness and the other requiring continuous light as a precondition. The key process for Chl synthesis is the reduction of protochlorophyllide (Pchlide). This enzymatic reaction is catalysed by two different enzymes — DPOR (dark-operative Pchlide oxidoreductase) or the structurally distinct LPOR (light-dependent Pchlide oxidoreductase). DPOR which consists of three subunits encoded by three plastid genes in eukaryotes was subject of our study. A short overview of our present knowledge of chlorophyll biosynthesis in Chlamydomonas reinhardtii in comparison with other plants is presented. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Influence of irradiance on chlorophyll synthesis in Picea abies calli cultures

Dark-grown seedlings of Picea abies (L) Karst. are able to accumulate the highest amounts of chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide) in all Pinaceae, but calli derived from 14-d-old green cotyledons of P. abies are completely white during the cultivation in the dark. Pchlide reduction is catalysed in the dark by light-independent protochlorophyllide oxidoreductase (DPOR). This enzyme complex consists of three protein subunits ChlL, ChlN and ChlB, encoded by three plastid genes chlL, chlN and chlB. Using semiquantitative RT-PCR, we observed very low expression of chlLNB genes in dark-grown calli. It seems, that chlLNB expression and thus Chl accumulation could be modulated by light in P. abies calli cultures. This hypothesis is supported by the fact, that we observed low contents of glutamyl-tRNA reductase and Flu-like protein, which probably affected Chl biosynthetic pathway at the step of 5-aminolevulinic acid formation. ChlB subunit was not detected in dark-grown P. abies calli cultures. Our results indicated limited ability to synthesize Chl in callus during cultivation in the dark.     

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 31 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, -aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.Abbreviations ALA -aminolevulinic acid - Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - POR NADPH-Protochlorophyllide oxidoreductase - xan xantha This study was initiated while one of the authors (K.A.) was on sabbatical leave in the laboratory of Dr. C. Somerville (MSU, East Lansing, Mich., USA). We are extremely grateful to Dr. Somerville and his coworkers for advice and support during this time. This research was supported by the Deutsche Forschungsgemeinschaft and the Schweizerischer Nationalfonds.     

Protochlorophyllide photoconversion mutants of Chlamydomonas reinhardtii

Summary We have developed a procedure for the isolation of Chlamydomonas reinhardtii mutants defective in light-dependent protochlorophyllide reduction (photoconversion), a key step in the biosynthesis of chlorophyll. Mutants were isolated by mutagenizing y-1-4, a temperature-sensitive yellow mutant blocked in the alternative light-independent protochlorophyllide reduction pathway, and screening for colonies which failed to green in the light at the restrictive temperature. Seven mutants were isolated which fail to photoconvert protochlorophyllide in photoconversion tests. All seven mutants have a single mutation at the pc-1 locus responsible for the defect in photoconversion. pc-1 maps close to y-5 on nuclear linkage group I. The pc-1 mutation is not itself temperature-sensitive because it blocks photoconversion at the permissive temperature when combined with the non-conditional yellow mutations y-5 and y-7. Cells containing the pc-1 mutation alone synthesize about 52% and 36% of the wildtype chlorophyll level in the dark and light, respectively, demonstrating that the light-independent protochlorophyllide reduction pathway in C. reinhardtii operates in the light.     

Protochlorophyllide Reduction: a Key Step in the Greening of Plants

The reduction of Protochlorophyllide (Pchlide) is a major regulatorystep in the biosynthesis of chlorophyll (Chl) in oxygenic phototrophs.Two different enzymes catalyze this reduction: a light-dependentenzyme (LPOR), which is unique as a consequence of its directutilization of light for catalysis; and a light-independentPchlide reductase (DPOR). Since the reduction of Pchlide inangiosperms is catalyzed exclusively by LPOR, they become etiolatedin the absence of light. LPOR, a major protein in etioplastmembranes, consists of a single polypeptide and it exists asa ternary complex with its substrates, Pchlide and NADPH. Bycontrast to the copious information about LPOR, limited informationabout DPOR has been reported. Recent molecular genetic analysesin a cyanobacterium and a green alga have revealed that at leastthe three genes, namely, chlL, chlN and chlB, encode proteinsessential for the activity of DPOR. These genes are widely distributedamong phototrophic organisms with the exception of angiospermsand Euglenophyta. This distribution seems to be well correlatedwith light-independent greening ability. These genes might havebeen lost during the evolution of gymnosperms to angiosperms.The similarities among the deduced amino acid sequences of thethree gene products and the subunits of nitrogenase suggestan evolutionary relationship between DPOR and nitrogenase. Theidentification of genes for the reduction of Pchlide providesthe groundwork for investigations of the mechanism that regulatesthe synthesis of Chl, which is closely coordinated with greeningin plants. ¹Recipient of the Plant and Cell Physiology Award for the Paperof Excellence (PCP Award), 1995.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

The nifH-Like (frxC) Gene Is Involved in the Biosynthesis of Chlorophyll in the Filamentous Cyanobacterium Plectonema boryanum

The frxC gene found in the DNA of the liverwort chloroplastencodes a protein of unknown function. The deduced amino acidsequence shows significant homology to that of the nitrogenaseFe-protein encoded by the nifH gene. We previously identifiedthe frxC and nifH genes in the filamentous cyanobacterium Plectonemaboryanum. We describe here the isolation of targeted mutantsof frxC (YFC1004) and nifH (YFH201) which were generated byinsertion of a kanamycin-resistance gene into the structuralportion of the respective genes. As expected, YFH201 cannotgrow under nitrogen-fixing conditions. However, YFC1004 growsas well as the wild type does under nitrogen-fixing, photoautotrophicand chemoheterotrophic conditions, indicating that the FrxCprotein is essential neither for nitrogen fixation nor for majorenergytransduction systems, such as photosynthesis and respiration.YFC1004 synthesizes chlorophyll (Chi) normally in the lightbut not in the dark, and it accumulates a precursor to Chi,protochlorophyllide (Pchlide) in the dark. These phenotypiccharacteristics of YFC1004 suggest that the cyanobacterium hastwo pathways for the reduction of Pchlide: a light-dependentand a lightindependent system. The FrxC protein appears to beinvolved in the light-independent reduction of Pchlide. (Received September 24, 1991; Accepted November 11, 1991)     

Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.

Summary The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3 end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 by segment from 399 to 441 by of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.     

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL ^- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL ^- strain, greening occurred at the same rate at two different light intensities (5 and 50 E m^-2s^-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding chelator protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

POR hits the road: import and assembly of a plastid protein

The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import – which is in contrast to earlier observations – and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane–associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.     

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum

Cyanobacteria have two protochlorophyllide (Pchlide) reductasescatalyzing the conversion of Pchlide to chloro-phyllide, a keystep in the biosynthetic pathway of chlorophylls (Chls); a light-dependent(LPOR) and a light-independent (DPOR) reductase. We found anopen reading frame (ORF322) in a 2,131-bp EcoRI fragment fromthe genomic DNA of the cyanobacterium Plectonema boryanum. Becausethe deduced amino acid sequence showed a high similarity tothose of various plant LPORs and the LPOR activity was detectedin the soluble fraction of Esche-richia coli cells over-expressingthe ORF322 protein, ORF322 was defined as the por gene encodingLPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolatedby targeted mutagenesiss to investigate the physiological importanceof LPOR. YFP12 grew as well as wild type under low light conditions(10-25 µE m^–2 S^–1). However, its growth wassignificantly retarded as a result of a significant decreasein its Chl content under higher light conditions (85-130 µEm^–2 s^–1). Furthermore, YFP12 stopped growing andsuffered from photobleaching under the highest light intensity(170 µE m^–2 s^–1). In contrast, a chlL-dis-rupted(DPOR-less) mutant YFC2 grew as well as wild type irrespectiveof light intensity. From these phenotypic characteristics, weconcluded that, although both LPOR and DPOR contribute to Chlsynthesis in the cells growing in the light, the extent of thecontribution by LPOR increases with increasing light intensity;without it, the cells are unable to grow under light intensitiesof more than 130 µ Em^–2s-^–. (Received September 26, 1997; Accepted November 21, 1997)     

Chlorophyll biosynthesis and chloroplast development in etiolated seedlings of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgo biloba L. is a large tree native in China with evolutionary affinities to the conifers and cycads. However unlike conifers, the gymnosperm G. biloba is not able to synthesize chlorophyll (Chl) in the dark, in spite of the presence of genes encoding subunits of light-independent protochlorophyllide oxidoreductase (DPOR) in the plastid genome. The principal aims of the present study were to investigate the presence of DPOR protein subunits (ChlL, ChlN, ChlB) as well as the key regulatory step in Chl formation: aminolevulinic acid (ALA) synthesis and abundance of the key regulatory enzyme in its synthesis: glutamyl-tRNA reductase (GluTR). In addition, functional stage of photosynthetic apparatus and assembly of pigment-protein complexes were investigated. Dark-grown, illuminated and circadian-grown G. biloba seedlings were used in our experiments. Our results clearly showed that no protein subunits of DPOR were detected irrespective of light conditions, what is consistent with the absence of Chl and Chl-binding proteins (D1, LHCI, LHCIIb) in the dark. This correlates with low ALA-synthesizing capacity and low amount of GluTR. The concentration of protochlorophyllide (Pchlide) in the dark is low and non-photoactive form (Pchlide₆₃₃) was predominant. Plastids were developed as typical etioplasts with prollamelar body and few prothylakoid membranes. Continual illumination (24 h) only slightly stimulated ALA and Chl synthesis, although Pchlide content was reduced. Prollamelar bodies disappeared, but no grana were formed, what was consistent with the absence of D1, LHCI, LHCIIb proteins. Lightinduced development of photosynthetic apparatus is extremely slow, as indicated by Chl fluorescence and gas exchange measurements. Even after 72 h of continuous illumination, the values of maximum (F_v/F_m) and effective quantum yield (Φ_PSII) and rate of net photosynthesis (P _N) did not reach the values comparable with circadian-grown plants.     

Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC).

The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.     

ATP Photosynthetic vesicles for light-driven bioprocesses

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H⁺ transporter) and TF₀F₁-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.     

The Accumulation of Protochlorophyllide in Cells of Synechocystis PCC 6714 with a Low PSI/PSII Stoichiometry

Changes in intracellular levels of Chl a precursors were examinedin relation to changes in the PSI/PSII stoichiometry in thecyanophyte Synechocystis PCC 6714. Protochlorophyllide (Pchlide)accumulated markedly in cells with a low PSI/PSII stoichiometrygrown under light that is absorbed by Chl a (PSI light) whereasno accumulation occurred in cells with a high PSI/PSII stoichiometrygrown under light absorbed by phycobilisomes (PSII light). Levelsof Pchlide in cells grown under PSI light decreased rapidlyupon a shift to PSII light. The rapid decrease in Pchlide accompanieda transient increase in chlorophyllide a, indicating that reductionof Pchlide was enhanced by shift to PSII light. The action spectrumindicated that the Pchlide decrease upon the shift to PSII lightdepended on excitation of Pchlide, suggesting that the accumulationof Pchllide was due to limited excitation of Pchlide, so thatPchlide photoreduction, under PSI light. However, comparisonof levels of Pchlide and the photosystem complexes in wild-typePlectonema boryanum with those in a mutant that lacked the darkPchlide reductase (YFC 1004) indicated that dark reduction compensatedfor the limited photoreduction under PSI light. Similar compensationby dark reduction was confirmed with Synechocystis PCC 6714.In cultures of Synechocystis under conditions where Pchlidecould not be photoreduced, accumulation of Pchlide and low PSI/PSIIstoichiometry occurred only when cells were illuminated withlight that preferentially excited PSI. The results indicatethat the low PSI/PSII stoichiometry in cells grown under PSIlight is not a result of inefficient synthesis of Chl a witha reduced rate of Pchlide photoreduction. They suggest furtherthat accumulation of Pchlide under PSI light results from retardationof the Chl a synthesis due to suppression of PSI synthesis. ¹Present address: Tsurukawa 5-15-11, Machida, Tokyo, 195 Japan.