Pchlide Reduction Research Articles

Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

In chlorophyll biosynthesis, the light-activated enzyme protochlorophyllide oxidoreductase catalyzes trans addition of hydrogen across the C-17-C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). This unique light-driven reaction plays a key role in the assembly of the photosynthetic apparatus, but despite its biological importance, the mechanism of light-activated catalysis is unknown. In this study, we show that Pchlide reduction occurs by dynamically coupled nuclear quantum tunneling of a hydride anion followed by a proton on the microsecond time scale in the Pchlide excited and ground states, respectively. We demonstrate the need for fast dynamic searches to form degenerate "tunneling-ready" configurations within the lifetime of the Pchlide excited state from which hydride transfer occurs. Moreover, we have found a breakpoint at -27 degrees C in the temperature dependence of the hydride transfer rate, which suggests that motions/vibrations that are important for promoting light-activated hydride tunneling are quenched below -27 degrees C. We observed no such breakpoint for the proton-tunneling reaction, indicating a reliance on different promoting modes for this reaction in the enzyme-substrate complex. Our studies indicate that the overall photoreduction of Pchlide is endothermic and that rapid dynamic searches are required to form distinct tunneling-ready configurations within the lifetime of the photoexcited state. Consequently, we have established the first important link between photochemical and nuclear quantum tunneling reactions, linked to protein dynamics, in a biologically significant system.

ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Differential Operation of Dual Protochlorophyllide Reductases for Chlorophyll Biosynthesis in Response to Environmental Oxygen Levels in the Cyanobacterium Leptolyngbya boryana

Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO(2)/N(2)). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed.

Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates

The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four 'dark' steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

Protochlorophyllide reduction - what is new in 2005?

Because the transformation of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is an irradiation-dependent process, it is at the heart of the photosynthetic membrane biogenesis, turnover, and adaptation to changes of the environment. I review here the new data published during the year 2004 on Pchlide reduction to Chlide.

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.

An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrested.

A key reaction in the biosynthesis of chlorophylls (Chls) a and b from cyanobacteria through higher plants is the strictly light-dependent reduction of protochlorophyllide (Pchlide) a to chlorophyllide (Chlide) a. Angiosperms, unlike other photosynthetic organisms, rely exclusively upon this mechanism to reduce Pchlide and hence require light to green. In Arabidopsis, light-dependent Pchlide reduction is mediated by three structurally related but differentially regulated NADPH:Pchlide oxidoreductases, denoted as PORA, PORB, and PORC. The PORA and PORB genes, but not PORC, are strongly expressed early in seedling development. In contrast, expression of PORB and PORC, but not PORA, is observed in older seedlings and adult plants. We have tested the hypothesis that PORB and PORC govern light-dependent Chl biosynthesis throughout most of the plant development by identifying porB and porC mutants of Arabidopsis, the first higher plant por mutants characterized. The porB-1 and porC-1 mutants lack the respective POR transcripts and specific POR isoforms because of the interruption of the corresponding genes by a derivative of the maize Dissociation (Ds) transposable element. Single por mutants, grown photoperiodically, display no obvious phenotypes at the whole plant or chloroplast ultrastructural levels, although the porB-1 mutant has less extensive etioplast inner membranes. However, a light-grown porB-1 porC-1 double mutant develops a seedling-lethal xantha phenotype at the cotyledon stage, contains only small amounts of Chl a, and possesses chloroplasts with mostly unstacked thylakoid membranes. PORB and PORC thus seem to play redundant roles in maintaining light-dependent Chl biosynthesis in green plants, and are together essential for growth and development.

Enzymology below 200 K: the kinetics and thermodynamics of the photochemistry catalyzed by protochlorophyllide oxidoreductase.

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH. As POR is light-dependent, catalysis can be initiated by illumination of the enzyme-substrate complex at low temperatures, making it an attractive model for studying aspects of biological proton and hydride transfers. The early stages in the photoreduction, involving Pchlide binding and an initial photochemical reaction, have been studied in vitro by using low-temperature fluorescence and absorbance measurements. Formation of the ternary POR-NADPH-Pchlide complex produces red shifts in the fluorescence and absorbance maxima of Pchlide, allowing the dissociation constant for Pchlide binding to be measured. We demonstrate that the product of an initial photochemical reaction, which can occur below 200 K, is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A696) that is suggested to represent an ion radical complex. The temperature dependence of the rate of A696 formation has allowed the activation energy for the photochemical step to be calculated and has shown that POR catalysis can proceed at much lower temperatures than previously thought. Calculations of differences in free energy between various reaction intermediates have been calculated; these, together with the quantum efficiency for Pchlide conversion, suggest a quantitative model for the thermodynamics of the light-driven step of Pchlide reduction.

The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.

Protochlorophyllide‐independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear‐encoded protein that is post‐translationally imported to the plastid. In barley and Arabidopsis thaliana, the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse‐grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse‐grown plants were incubated with Δ‐aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse‐grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma‐enriched fractions from chloroplasts and etioplasts of barley indicated that no post‐import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.

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.

Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction in vitro: insight into the mechanism of chlorophyll formation in light-adapted plants.

The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllide oxidoreductase B (PORb) has been analyzed by low-temperature fluorescence spectroscopy by using purified barley PORb overexpressed heterologously in Escherichia coli as a fusion protein with the maltose-binding protein. We show that the PORb-catalyzed PChlide reduction reaction consists of two steps, one photochemical and the other nonphotochemical. The initial photochemical reaction follows a single quantum mechanism and leads to the formation of an unstable intermediate with mixed pigment electronic structure and an EPR spectrum that suggests the presence of a free electron. The second step involves the spontaneous conversion of the unstable intermediate into chlorophyllide as defined by its spectroscopic characteristics and migration on an HPLC column. Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major changes in enzyme conformation or pigment rearrangement within the active site. The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are consistent with one molecule of substrate bound per active monomer in solution. The PORb-catalyzed reaction in vitro is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reaction sequence observed operates in planta.

Chlorophyll alpha synthesis upon interruption and deletion of por coding for the light-dependent NADPH: protochlorophyllide oxidoreductase in a photosystem-I-less/chlL- strain of Synechocystis sp. PCC 6803.

The gene coding for the light-dependent NADPH:protochlorophyllide oxidoreductase (POR) was interrupted or deleted in a Synechocystis sp. PCC 6803 strain lacking photosystem I (PS I) as well as ChlL, which takes part in light-independent catalysis of protochlorophyllide reduction. Interruption of por by a kanamycin-resistance cartridge between the codons for M263 and V264 (about 83% into the coding region) did not abolish POR activity, but resulted in a decrease in the protochlorophyllide-(PChlide)-binding capacity of POR. Deletion of por in the PS I-less/chlL- strain generated a mutant [PS I-less/chlL-/por (del)] which accumulated both monovinyl-PChlide and divinyl-PChlide and excreted PChlides into the medium. This mutant also synthesized small amounts of protochlorophyllide dihydrogeranylgeraniol ester (protochlorophyll) when it was grown under light-activated heterotrophic growth conditions. However, the mutant was still able to synthesize small amounts of normal chlorophyll a under weak continuous illumination, even though the quantum yield of chlorophyll a formation was reduced. Either protochlorophyll or PChlide reduction by an unspecific reductase or by a ChlB/ChlN complex could account for chlorophyll a synthesis in the PS I-less/chlL-/por (del) strain. Functional photosystem II (PS II) was assembled in this mutant, but the PS II/chlorophyll ratio was fourfold lower than in the PS I-less strain with normal chlorophyll synthesis. The PS I-less/chlL-/por (del) mutant had a 77-K fluorescence emission maximum at 685 nm but no peak or shoulder at 695 nm when the cells were excited at 435 nm. Much of the chlorophyll in the PS I-less/chlL-/por (del) mutant therefore seems to be associated with components other than PS II.

Regulation of Synthesis of PSI in the Cyanophytes Synechocystis PCC6714 and Plectonema boryanum during the Acclimation of the Photosystem Stoichiometry to the Light Quality

The effects of light quality on the formation of the PSI complex were examined in Synechocystis PCC6714 and in Plectonema boryanum. The rate of increase in levels of core polypeptides of PSI, PsaA/B, doubled upon shift from Chi a-absorbed light (PSI light) to phycobilisome-absorbed light (PSII light). The elevated rate was decreased upon the reverse shift. Half time of the acceleration was approximately 10 min, and that of the decrease was approximately 4 min. The rate of degradation of the polypeptides was far lower than the rate of the increase under either light regime. Neither synthesis nor degradation of the PsbA and PsbC polypeptides of PSII was significantly altered by the light quality. We conclude that synthesis of the PSI complex is chromatically regulated to allow adjustments in photosystem stoichiometry. The level of mRNA for PsaA/B was not altered by the light regime. Anomalous inhibition by chloramphenicol suggested that the regulation occurs at a step(s) other than the peptide elongation step, perhaps at the initiation of the ribosome cycle or at the insertion of Chi a for the stabilization of the polypeptides. The photoreduction of protochlorophyllide (Pchlide) was compared with the synthesis of the polypeptides in a mutant of Plectonema boryanum that lacked Pchlide dark reductase (YFC1004). The results indicated that the synthesis of stable PsaA/B polypeptides was not limited by the reduction of Pchlide, although the synthesis did depend on a supply of Chi a.

Protochlorophyllide Reduction: a Key Step in the Greening of Plants

The reduction of Protochlorophyllide (Pchlide) is a major regulatory step in the biosynthesis of chlorophyll (Chl) in oxygenic phototrophs. Two different enzymes catalyze this reduction: a light-dependent enzyme (LPOR), which is unique as a consequences of its direct utilization of light for catalysis; and a light-independent Pchlide reductase (DPOR). Since the reduction of Pchlide in angiosperms is catalyzed exclusively by LPOR, they become etiolated in the absence of light. LPOR, a major protein in etioplast membranes, consists of a single polypeptide and it exists as a ternary complex with its substrates, Pchlide and NADPH. By contrast to the copious information about LPOR, limited information about DPOR has been reported. Recent molecular genetic analyses in a cyano-bacterium and a green alga have revealed that at least the three genes, namely, chlL, chlN and chlB, encode proteins essential for the activity of DPOR. These genes are widely distributed among phototrophic organisms with exception of angiosperms and Euglenophyta. This distribution seems to be well correlated with light-independent greening ability. These genes might have been lost during the evolution of gymnosperms to angiosperms. The similarities among the deduced amino acid sequences of the three gene products and the subunits of nitrogenase suggest an evolutionary relationship between DPOR and nitrogenase. The identification of genes for the reduction of Pchlide provides the groundwork for investigations of the mechanism that regulates the synthesis of Chl, which is closely coordinated with greening in plants.

Regulation of Chlorophyll Biosynthesis in Angiosperms.

Light controls plant growth and development in a pleiotropic manner. For example, light dictates a pattern of gene expression that leads to photomorphogenesis (Kendrick and Kronenberg, 1994). In the absence of light, an alternative developmental process, skotomorphogenesis, takes place. Photomorphogenesis and skotomorphogenesis are controlled by several different photoreceptors, such as Pr and Pfr and blue-light-absorbing photoreceptors. During skotomorphogenesis, the germinating seedling utilizes all of the nutrients contained in the seed to establish conditions that allow the plant to harvest even traces of light. The hypocotyl elongates dramatically just to place the cotyledons above the soil. Within the cotyledons, proplastids differentiate into etioplasts, and a large supply of the tetrapyrrole pigment precursor Pchlide is built up in the prolamellar bodies of the etioplasts. Together with NADPH, Pchlide is bound to the PORA. This ternary complex is photoactive and thus able to immediately photoreduce the Pchlide to Chlide as soon as the cotyledons are exposed to light (for review, see Ryberg and Sundqvist, 1991). During photomorphogenesis, hypocotyl elongation is suppressed, but cotyledon unfolding and expansion proceed normally. Chloroplast development as well as Pchlide reduction occur in parallel and collectively lead to the rapid greening of the plant. Pioneering experiments performed in Granick's group almost 40 years ago shed some light on the regulatory mechanisms that govern Chl biosynthesis in higher plants. When angiosperm plants were germinated in the dark, their cotyledons appeared pale yellow. If such etiolated seedlings were incubated with ALA, a common precursor -of all tetrapyrrole pigments, they turned greenish (Granick, 1959). This apparent greening was not caused by the synthesis of Chl but was due to the accumulation of excess Pchlide, the immediate precursor of Chlide. Similar to leaf tissues, isolated plastids were found in later studies to accumulate excess Pchlide when they were fed ALA in the dark (Kannangara and Gough, 1977). Taken together, these basic experiments showed that all of the enzymes necessary for the conversion of ALA to Pchlide must be present in dark-grown angiosperm plants, that the entire pathway is likely to operate in the plastid, and that the only known light-requiring reaction for the synthesis of Chl is the reduction of Pchlide to Chlide. It is the aim of this review to summarize our current knowledge on key regulatory steps of Chl biosynthesis in angiosperms.

PORA and PORB, Two Light-Dependent Protochlorophyllide-Reducing Enzymes of Angiosperm Chlorophyll Biosynthesis.

Chloroplasts are the most abundant organelles in a plant cell. Besides their familiar roles in photosynthesis, chloroplasts also perform important functions during numerous other metabolic processes, such as nitrogen assimilation, amino acid biosynthesis, and tetrapyrrole production. ln particular, a major route of chloroplast anabolism is devoted to the synthesis of chlorophyll (Chl; von Wettstein et al., 1995). As the “pigment of life,” Chl plays a fundamental role in the energy absorption and transduction activities of all photosynthetic organisms (Bogorad, 1967; von Wettstein et al., 1971). In angiosperms, Chl synthesis is dependent on light (Granick, 1950). NADPH:protochlorophyllide oxidoreductase (POR; EC 1.3.1.33) catalyzes the only known light-requiring step of tetrapyrrole biosynthesis, the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) (Griffiths, 1978; Apel et al., 1980). Both the POR(A) enzyme and its substrate, Pchlide, accumulate to high levels in dark-grown plants. Together with NADPH, they form a ternary complex that instantaneously converts Pchlide to Chlide when illuminated. The operation of PORA hence helps prevent photodynamic damage by large amounts of not immediately photoconvertible Pchlide. The function of PORA is confined to the very early stages of transition from etiolated to light growth (Apel, 1981; Batschauer and Apel, 1984; Mosinger et al., 1985; Forreiter et al., 1990). The amounts of both PORA protein and porA mRNA decrease drastically soon after the beginning of illumination (Mapleston and Griffiths, 1980; Santel and Apel, 1981; Forreiter et al., 1990), due in part to rapid proteolytic turnover of the enzyme protein (Kay and Griffiths, 1983; Hauser et al., 1984). To perform the reduction of Pchlide to Chlide during the final stages of the light-induced greening and to sustain Chl

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 3:1 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, delta-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.

Identification of a nifDK-Like Gene (ORF467) Involved in the Biosynthesis of Chlorophyll in the Cyanobacterium Plectonema boryanum

The frxC gene, which is found in chloroplast DNA (ctDNA) and in cyanobacteria, encodes a protein that is required for the light-independent reduction of protochlorophyllide (Pchlide) to chlorophyllide a (Chlide). A DNA fragment downstream of frxC in the filamentous cyanobacterium Plectonema boryanum was cloned and analyzed. Sequencing of the DNA fragment revealed an open reading frame (ORF) that encoded a protein of 467 amino acid residues (designated ORF467), which showed extensive homology to the proteins encoded by genes on ctDNAs (ORF465 in liverwort, gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469 protein of the cyanobacterium Synechocystis sp. strain PCC 6803. We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivated by the insertion of a kanamycin-resistance gene into the coding region. YFM6D-3 exhibited a phenotype similar to that of YFC1004, an frxC-disrupted mutant, which did not synthesize chlorophyll (Chl) and accumulated Pchlide, a precursor to Chl, in the dark. These phenotypic characteristics of YFM6D-3 indicate that the light-independent reduction of Pchlide requires not only the FrxC protein but also the ORF467 protein. The amino acid sequences of the homologues of ORF467 exhibit low but significant similarity to those of the alpha and beta subunits of nitrogenase MoFe-protein, suggesting a phylogenetic relationship between the light-independent Pchlide reductase and nitrogenase, as is observed between the FrxC protein and the Fe-protein of nitrogenase.

Incorporation of 5‐aminolevulinic acid into chlorophyll in darkness in barley

The incorporation of radioactive aminolevulinic acid (ALA) into chlorophyll (Chl) a and b, as well as protochlorophyllide (Pchlide) in light‐grown barley seedlings (Hordeum vulgare L. cv. Clipper) transferred to darkness is demonstrated.In the experiments described, 6‐day‐old, glasshouse‐grown seedlings were transferred to darkness and incubated in [14C]‐ or [3H]‐ ALA for 18 h.Chl a and b were extracted and purified to constant specific radioactivity by HPLC and TLC of their magnesium‐free derivatives, pheophytin a and b. The presence of label in the tetrapyrrole ring of the Chls was established by removal of the phytol chain by alkaline hydrolysis and determination of the specific radioactivity of the chlorin e6 and rhodin g7 derivatives.Barley seedlings that had been grown in darkness for 5 days, transferred to light for 20 h, and then returned to darkness in the presence of radioactive ALA also incorporated label into Chl. However, this was only apparent in intact seedlings. Excised leaves from greened etiolated plants did not incorporate ALA into Chl in darkness. This was consistent with the finding of Apel et al. (K. Apel, M. Motzkus and K. Dehesh, 1984. Planta 161: 550–554) and may account for their failure to obtain evidence for a light‐independent protochlorophyllide reductase in greening barley.Although the incorporation of ALA into Chl compared to Pchlide was slight (5%), the presence of label in the tetrapyrrole nucleus of Chl a and b is unequivocal evidence of a light‐independent pathway of Chl biosynthesis in barley that has been exposed to light during development. Limited entry of exogenous labelled ALA into the precursor pools leading to the dark reduction of Pchlide is postulated.