Chloroplast biogenesis. Demonstration of the monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants

It is shown that barley (Hordeum vulgare), a dark monovinyl/light divinyl plant species, and cucumber (Cucumis sativus L.) a dark divinyl/light divinyl plant species synthesize monovinyl and divinyl protochlorophyllide in darkness from monovinyl and divinyl protoporphyrin IX via two distinct monovinyl and divinyl monocarboxylic chlorophyll biosynthetic routes. Evidence for the operation of monovinyl monocarboxylic biosynthetic routes consisted (a) in demonstrating the conversion of delta-aminolevulinic acid to monovinyl protoporphyrin and to monovinyl Mg-protoporphyrins, and (b) in demonstrating the conversion of these tetrapyrroles to monovinyl protochlorophyllide by both isolated barley and cucumber etiochloroplasts. Likewise, evidence for the operation of divinyl monocarboxylic chlorophyll biosynthetic routes consisted (a) in demonstrating the biosynthesis of divinyl protoporphyrin and divinyl Mg-protoporphyrins from delta-aminolevulinic acid, and (b) in demonstrating the conversion of the latter tetrapyrroles to divinyl protochlorophyllide. Finally, it was shown that the divinyl tetrapyrrole substrates were metabolized differently by barley and cucumber. For example, divinyl protoporphyrin, divinyl Mg-protoporphyrin, and divinyl Mg-protoporphyrin monoester were converted predominantly to monovinyl protochlorophyllide and to smaller amounts of divinyl protochlorophyllide by barley etiochloroplasts. In contrast, cucumber etiochloroplasts converted the above substrates predominantly to divinyl protochlorophyllide, although smaller amounts of monovinyl protochlorophyllide were also formed. Furthermore, it was shown that monovinyl protochlorophyllide was not formed from divinyl protochlorophyllide either in barley or in cucumber etiochloroplasts. These metabolic differences are explained by the presence of strong biosynthetic interconnections between the divinyl and monovinyl monocarboxylic routes, prior to divinyl protochlorophyllide formation, in barley but not in cucumber.     

Chloroplast Biogenesis 49 : Differences among Angiosperms in the Biosynthesis and Accumulation of Monovinyl and Divinyl Protochlorophyllide during Photoperiodic Greening

Various angiosperms differed in their monovinyl and divinyl protochlorophyllide biosynthetic capabilities during the dark and light phases of photoperiodic growth. Some plant species such as Cucumis sativus L., Brassica juncea (L.) Coss., Brassica kaber (DC.) Wheeler, and Portulaca oleracea L. accumulated mainly divinyl protochlorophyllide at night. Monocotyledonous species such as Avena sativa L., Hordeum vulgare L., Triticum secale L., Zea mays L., and some dicotyledonous species such as Phaseolus vulgaris L., Glycine max (L.) Merr., Chenopodium album L., and Lycopersicon esculentum L. accumulated mainly monovinyl protochlorophyllide at night.

Under low light intensities meant to simulate the first 60 to 80 minutes following daybreak divinyl protochlorophyllide appeared to contribute much more to chlorophyll formation than monovinyl protochlorophyllide in species such as Cucumis sativus L. Under the same light conditions, species which accumulated mainly monovinyl protochlorophyllide at night appeared to form chlorophyll preferably via monovinyl protochlorophyllide.

These results were interpreted in terms of: (a) a differential contribution of monovinyl and divinyl protochlorophyllide to chlorophyll formation at daybreak in various plant species; and (b) a differential regulation of the monovinyl and divinyl protochlorophyllide biosynthetic routes by light and darkness.

     

Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro

It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.     

Separation of monovinyl and divinyl protochlorophyllides and chlorophyllides from etiolated and phototransformed cucumber cotyledons

A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0°C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark.     

Divinyl Chlorophyll(ide) a Can Be Converted to Monovinyl Chlorophyll(ide) a by a Divinyl Reductase in Rice

3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a.Chlorophyll (Chl) is the main component of the photosynthetic pigments. Chl molecules universally exist in photosynthetic organisms and perform essential processes of harvesting light energy in the antenna systems and by driving electron transfer in the reaction centers (Fromme et al., 2003). In higher plants, there are two Chl species, Chl a and Chl b. The photosynthetic reaction centers contain only Chl a, and the peripheral light-harvesting antenna complexes contain Chl a and Chl b (Grossman et al., 1995). Chl a is synthesized from glutamyl-tRNA, and Chl b is synthesized from Chl a at the last step of Chl biosynthesis (Beale, 1999). So far, genes for all 15 steps in the Chl biosynthetic pathway have been identified in higher plants, at least in angiosperms represented by Arabidopsis (Arabidopsis thaliana; Beale, 2005; Nagata et al., 2005). Analysis of the complete genome of Arabidopsis showed that it has 15 enzymes encoded by 27 genes for Chl biosynthesis from glutamyl-tRNA to Chl b (Nagata et al., 2005). However, only six genes encoding three enzymes involved in Chl biosynthesis have been identified in rice (Oryza sativa). Magnesium chelatase comprises three subunits (ChlH, ChlD, and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both Chl and heme biosyntheses. Jung et al. (2003) characterized OsCHLH gene for the OsChlH subunit of magnesium chelatase, and Zhang et al. (2006) cloned Chl1 and Chl9 genes encoding the OsChlD and OsChlI subunits of magnesium chelatase. Chl synthase catalyzes esterification of chlorophyllide (Chlide), resulting in the formation of Chl a. Wu et al. (2007) identified the YGL1 gene encoding the Chl synthase. Chl b is synthesized from Chl a by Chl a oxygenase; Lee et al. (2005) identified OsCAO1 and OsCAO2 genes for Chl a oxygenase.According to the number of vinyl side chains, Chls of oxygenic photosynthetic organisms are classified into two groups: 3,8-divinyl Chl (DV-Chl) and 3-vinyl Chl (monovinyl Chl [MV-Chl]). Almost all of the oxygenic photosynthetic organisms contain MV-Chls, regardless of the variation in their indigenous environments (Porra, 1997). The exceptions are species of Prochlorococcus marinus, marine picophytoplanktons that contain DV-Chls as their photosynthetic pigments (Chisholm et al., 1992).Chl biosynthetic heterogeneity is assumed to originate mainly in parallel DV- and MV-Chl biosynthetic routes interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). Most of Chls carry an ethyl group or, less frequently, a vinyl group. For example, Chl a and b occur as the MV-derivatives in green plants, but Chl precursors sometimes accumulate as DV-intermediates, and the ratio between the two forms can vary depending on the species, tissue, and growth conditions (Shioi and Takamiya, 1992; Kim and Rebeiz, 1996). So far, five 8-vinyl reductase activities have been detected at the levels of DV Mg-protoporphyrin IX (Kim and Rebeiz, 1996), Mg-protomonomethyl ester (Kolossov et al., 2006), protochlorophyllide (Pchlide) a (Tripathy and Rebeiz, 1988), Chlide a (Kolossov and Rebeiz, 2001; Nagata et al., 2005), and Chl a (Adra and Rebeiz, 1998). What is not clear at this stage is whether the various 8-vinyl reductase activities are catalyzed by one enzyme of broad specificity or by a family of enzymes of narrow specificity encoded by one gene or multiple genes, as is the case for NADPH Pchlide oxidoreductases (Rebeiz et al., 2003). The issue could be settled by purification of the various putative reductases and comparison of their properties.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis as a divinyl reductase (DVR) that has sequence similarity to isoflavone reductase. Chew and Bryant (2007) demonstrated that BciA (CT1063), which is an ortholog of the Arabidopsis gene, encodes a DVR of the green sulfur bacterium Chlorobium tepidum TLS. They also considered that BchJ, which had been reported to be a vinyl reductase (Suzuki and Bauer, 1995), is not the enzyme, but it may play an important role in substrate channeling and/or regulation of bacteriochlorophyll biosynthesis. Islam et al. (2008) and Ito et al. (2008) independently identified a novel 8-vinyl reductase gene (Slr1923) in DVR-less cyanobacterium Synechocystis sp. PCC6803. However, no DVR gene has yet been identified in monocotyledonous plants.In this study, we isolated a spontaneous mutant, 824ys, from indica rice cv 824B. The mutant exhibited a yellow-green leaf phenotype throughout the growth stage, reduced level of Chls, arrested development of chloroplasts, and retarded growth rate. Map-based cloning of the mutant resulted in the identification of the OsDVR gene, showing sequence similarity to the DVR gene of Arabidopsis. In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame (ORF), resulting in three amino acid deletion in the encoded protein. HPLC analysis of Chls indicated the mutant accumulates only DV-Chls. Enzymatic analysis demonstrated that the recombinant protein expressed in Escherichia coli is able to catalyze the conversion of DV-Chl(ide) a to MV-Chl(ide) a. Therefore, this study has confirmed that the OsDVR gene encodes a functional DVR in rice.     

Monovinyl and divinyl protochlorophyllide pools in etiolated tissues of higher plants

The composition of chlorophyll-precursor pigments, particularly the contents of monovinyl (MV) and divinyl (DV) protochlorophyllides (Pchlides), in etiolated tissues of higher plants were determined by polyethylene-column HPLC (Y. Shioi, S. I. Beale [1987] Anal Biochem 162: 493-499), which enables the complete separation of these pigments. DV-Pchlide was ubiquitous in etiolated tissue of higher plants. From the analyses of 24 plant species belonging to 17 different families, it was shown that the concentration of DV-Pchlide was strongly dependent on the plant species and the age of the plants. The ratio of DV-Pchlide to MV-Pchlide in high DV-Pchlide plants such as cucumber and leaf mustard decreased sharply with increasing age. Levels of DV-Pchlide in Gramineae plants were considerably lower at all ages compared with those of other plants. Etiolated tissues of higher plants such as barley and corn were, therefore, good sources of MV-Pchlide. Absorption spectra of the purified MV- and DV-Pchlides in ether are presented and compared.     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.     

Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species

Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.     

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS

Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with δ-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.     

Chloroplast Development in Etiolated Peas: Reformation of Prolamellar Bodies in Red Light without Accumulation of Protochlorophyllide

Chloroplast development and accumulation of chlorophylls werestudied in etiolated peas (Pisum sativum L., cv. Green feast)exposed for 24 h to one of three intensities of red light orto a corresponding intensity of white light producing a similarhigh medium or low terminal chlorophyll content. Chloroplastdevelopment was assessed by counts of grana and partitions pergranum in electron micrographs. Chlorophylls were partitionedand protochlorophyllide measured in the unphytylated fraction.More sensitive estimates of protochlorophyllide were made byfluorescence. In the high- and medium-intensity red-light treatmentsconsiderable reformation of prolamellar bodies took place duringdevelopment. This occurred at a stage when no protochlorophyllidewould be detected. In the low-intensity treatments reformationwas accompanied by an accumulation of protochlorophyllide ata higher level than that obtaining in the dark.     

Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.     

Characterization and Properties of a Protochlorophyllide Ester in Leaves of Dark Grown Barley with Geranylgeraniol as Esterifying Alcohol

The protochlorophyllide ester isolated from dark grown barley leaves was shown to contain geranylgeraniol as esterifying alcohol. No phytylester was found. The qualitative analyses were performed with combined gas chromatography-mass spec-trometry. Chromatographic separation and spectrofluorometric determination of the protochlorophyll and chlorophyll pigments before and after irradiation of the dark grown leaves with light flashes at 2°C showed that part of the protochlorophyllide ester was photoconverted to chlorophyll a.     

Protochlorophyllide b does not occur in barley etioplasts

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection < 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH :protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80-84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

The Influence of Dark Adaptation Temperature on the Reappearance of Variable Fluorescence following Illumination

The effect of chilling temperatures (5°C) on chlorophyll fluorescence transients was used to study chilling-induced inhibition of photosynthesis in plant species with differing chilling sensitivities. A Brancker SF-20 fluorometer was used to measure induced fluorescence transients from both attached and detached leaves of chilling-sensitive cucumber (Cucumis sativus L. cv Ashley) and chilling-resistant pea (Pisum sativum L. cv Alaska). The rate of reappearance of the variable component of fluorescence (F_v), following a period of illumination at 25°C, was dependent on the temperature at which the leaf was allowed to dark adapt in chilling-sensitive cucumber, but not in chilling-resistant pea. In cucumber, dark adaptation at 25°C following illumination resulted in a much faster return of F_v than dark adaptation at 5°C following illumination. However, F_v reappearance during the dark adaptation period in chilling-resistant pea was temperature independent. The difference in the temperature response of F_v following illumination correlated with temperature sensitivity of these two species. The process responsible for the difference in F_v may represent a site of chilling sensitivity in the photosynthetic apparatus.     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.