Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

Kinetics and quantum yield of photoconversion of protochlorophyll(ide) to chlorophyll(ide) a

The kinetics of photoconversion of protochlorophyll(ide) to chlorophyll(ide) a were investigated in dark-grown barley leaves and in a preparation of protochlorophyll holochrome subunits. In the subunits the conversion obeyed first-order kinetics. This indicates that the excitation of protochlorophyll(ide), energy loss through deexcitation, and the reduction of excited protochlorophyll(ide) are all reactions that follow first-order kinetics with respect to protochlorophyll(ide) in protochlorophyll holochrome subunits.In contrast, photoconversion in leaves obeyed neither first- nor second-order kinetics. This prompted the postulation of an additional route within macromolecular units of protochlorophyll holochrome, whereby energy is lost from excited protochlorophyll(ide) by a reaction that is not first order. Such a process might be energy transfer from excited protochlorophyll(ide) to newly-formed chlorophyll(ide) a.A dynamic model describing photoconversion in macromolecular units was derived. The model is consistent with the observed progress of photoconversion in barley leaves and in protochlorophyll holochrome subunits from barley.Determinations of the quantum yield of photoconversion in protochlorophyll holochrome subunits gave values of 0.4–0.5 molecules · quantum^?1. Estimates of the initial quantum yield of the photoconversion process in leaves fell into the same range. The dynamic model allows predictions on the progressively decreasing quantum yield as the photoconversion proceeds in macromolecular units.     

Protochlorophyll(ide) in a Blue-Green Alga

During growth under far-red (>650 nm) light, Anacystis nidulans accumulates protochlorophyllide to concentrations about one-tenth of the chlorophyll. From whole cell fluorescence spectra, protochlorophyll(ide) was identified also in another blue-green, and in a red, alga grown in far-red light.     

Photoactive Subunits of Protochlorophyll(ide) Holochrome

A stable, soluble, and photoactive protochlorophyll(ide) complex has been extracted from dark-grown barley (Hordeum vulgare L.) leaves with buffer containing saponin and glycerol. After ammonium sulfate precipitation, the redissolved pigment complex was partially purified by chromatography on Sephadex gels in the presence of saponin. With the assumptions that the pigment complex from barley has the same shape and density as the proteins used for calibration, its molecular weight is 63,000. Photoactive protochlorophyll(ide) complex isolated from bean (Phaseolus vulgaris L.) and chromatographed by the same procedures has an aparent molecular weight of 100,000 or greater. No chromatographic separation of photoactive and inactive protochlorophyll(ide) complexes was observed. Photoconversion of protochlorophyll(ide) to chlorophyll(ide) did not change the chromatographic behavior of the pigment complex.     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coefficient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ 1 mole^–1 cm^–1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assuming that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.Abbreviations PChl(ide) protochlorophyll(ide) - Chl(ide) chlorophyll(ide)     

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.     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coeffecient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ l mole^-1 cm^-1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assurning that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.     

Regulation of Protochlorophyll(ide) Levels in Dark-Grown Non-Dividing Euglena 3. Proplastid Structure during Loss and Regeneration of Protochlorophyll(ide)

Cells of Euglena gracilis Klebs var. bacillaris Cori growingin darkness on a complete medium have small undifferentiatedproplastids. On transfer to an incomplete (resting) medium indarkness, the cells cease division within 72 h. During thistime the proplastid expands and several prothylakoids and prolamellarbodies develop even though phototransformable protochlorophyll(ide)[PT-Pchl(ide)] is decreasing. As PT-Pchl(ide) decreases furtherand reaches a stable plateau after 4–5 more days in darkness,the proplastid structure becomes highly reduced. Forty minutesof light plus a one h dark period, or addition of glutamateor malate for 7 h does not change the proplastid structure significantlyeven though PT-Pchl(ide) returns to the level found in growingcells. Upon prolonged incubation in darkness after light treatment(72 h) an expanded proplastid containing prothylakoids, prolamellarbodies and membrane whorls with mitochondria in close associationis seen; most of the cellular paramylum is lost during thisperiod leaving cavities in the cytoplasm. Without light, prolongedincubation in darkness (72 h) with malate leads to accumulationof cellular paramylum but no change in proplastid structurewhile prolonged treatment with glutamate (72 h) allows the formationof a few prothylakoids but no prolamellar bodies. ¹Supported by Grants GM 14595 from the National Institutes ofHealth. ²Permanent address: Department of Microbiology, Tokyo MedicalCollege, 6-1-1 Shinjuku, Tokyo 160, Japan. ³Abraham and Etta Goodman Professor of Biology. (Received July 23, 1983; Accepted September 22, 1983)     

slr1923 of Synechocystis sp. PCC6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide)

The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F(420)H(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex I. In this study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than the wild type under 100 microE m(-2) s(-1) but did not grow under high light intensity (300 microE m(-2) s(-1)). The cellular content of chlorophyll was lower in the mutant, and the absorption spectrum showed a shift in the absorption peak of the Soret band to a longer wavelength by about 10 nm compared with the wild type. It was found, by high-performance liquid chromatography analysis, that the retention time of chlorophyll of the mutant is shorter than that of the wild type and that the peak wavelength of the Soret band was also shifted to a longer wavelength by 11 nm. Proton nuclear magnetic resonance analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with a vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl)chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that the Slr1923 protein is essential for the conversion from divinylchlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).     

Protochlorophyll(ide) forms in hypocotyls of dark-grown bean (Phaseolus vulgaris)

The protochlorophyll(ide) forms and plastid ultrastructure were investigated in hypocotyls of dark-grown seedlings of kidney bean ( Phaseolus vulgaris L. cv. Brede zonder draad). By deconvolution of the fluorescence emission spectra into Gaussian components three protochlorophyll(ide) forms were found with maxima at 633, 642 and 657 nm, respectively. The ratio of protochlorophyll(ide) emitting at 657 nm to protochlorophyll(ide) emitting at 633 nm decreased downwards the hypocotyl. The gradient was established already after 4 days in dark-grown Phaseolus and was also seen in hypocotyls of 7-day-old dark-grown plants of 8 other species. Ultrastructural observations revealed a plastid developmental sequence along the hypocotyl. Plastids in the upper parts of the hypocotyl contained prolamellar bodies typical of etiolated leaves while those in the lower parts contained only stroma lamellae. Immunological detection of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) on nitrocellulose membranes after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated the occurrence of the enzyme in upper, middle and lower sections of hypocotyls and in the root tips.     

The Effect of Phytohormones on Chlorophyll(ide), Protochlorophyll(ide) and Carotenoid Formation in Greening Dark Grown Wheat Leaves

The influence of phytohormones on chlorophyll and carotenoid formation during the greening of irradiated dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull) was studied. Leaves were floated on solutions of abscisic acid, gibberellic acid and kinetin for 24 h. The chlorophyll and carotenoid contents were determined during a subsequent period of 48 h of continuous irradiation. Leaves treated with abscisic acid showed a longer lag phase and a lower rate of accumulation of chlorophyll as compared to the control than did leaves treated with gibberellic acid and kinetin. The carotenoid content was low both in leaves treated with abscisic acid and in those treated with gibberellic acid. Treatment with abscisic acid lowered the protochlorophyllide regeneration after a saturating light flash while gibberellic acid as well as kinetin had no effect. The influence of ABA was partly dependent on an increase of the wounded part of the cut leaf segments. The accumulation of protochlorophyllide in leaves treated with δ-aminolevulinic acid was not affected by the different hormonal treatments. These results suggest that the main effect of abscisic acid is probably outside the chloroplast, i.e. on the formation or transport of δ-aminolevulinic acid.     

A Reversible Conversion of Phototransformable Protochlorophyll(ide)(656) to Photoinactive Protochlorophyll(ide)(656) by Hydrogen Sulfide in Etiolated Bean Leaves

The relationship of phototransformable protochlorophyll-(ide) to photoinactive protochlorophyll(ide) has been studied in the primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Subjecting the leaves to an atmosphere of H₂S causes an immediate loss of phototransformable protochlorophyll(ide)₆₅₀ and a simultaneous increase in photoinactive protochlorophyll(ide)₆₃₃. When such leaves are returned to air or N₂, the absorbance at 650 nm increases, whereas the absorbance at 633 nm decreases and photoactivity is restored. The reversion of protochlorophyll-(ide)₆₃₃ to protochlorophyll(ide)₆₅₀ is one-half complete in 3 minutes at 22 C in 8-day-old leaves. Ninety-five per cent recovery of protochlorophyll(ide)₆₅₀ is obtained when exposure to H₂S is less than 3 minutes in duration; longer periods reduce the reversion capacity proportionately. The leaves are relatively undamaged by brief exposures to H₂S, as judged by electron microscopy and by their ability to synthesize chlorophyll under continuous illumination. Hydrogen sulfide has no immediate effect upon the absorption properties of a partially purified preparation of the protochlorophyll(ide) holochrome, an etioplast suspension, or leaves subjected to freezing and thawing. Compounds such as HCN and HN₃ cause an irreversible conversion of protochlorophyll(ide)₆₅₀ to protochlorophyll(ide)₆₃₃ with total loss of photoactivity. Sulfhydryl agents, such as β-mercaptoethanol and cysteine, cause a slow, irreversible transformation of the photoactive pigment to the photoinactive form and inhibit the ability of the leaves to synthesize chlorophyll under continuous illumination. The results obtained suggest that H₂S may alter the interaction between the source of hydrogens on the protein moiety of the holochrome and the chromophore in vivo by reducing a disulfide bond in the protein, thereby causing a reversible conformational change in the complex.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Some observations on chlorophyll(ide) synthesis by isolated etioplasts.

1. A modified procedure for the isolation of etioplasts from dark-grown barley is described and the regeneration of phototransformable protchlorophyll(ide) was demonstrated in the isolated plastids. 2. On exposure of the etioplasts to a long-term flash illumination, chlorophyll(ide) synthesis from a precursor pool, which includes all the protochlorophyllide, was demonstrated. 3. Added delta-aminolaevulinic acid failed to be significantly incorporated into chlorophyll(ide) in the etioplasts despite its extensive incorporation into porphyrin precursors of chlorophyll and haem compounds. The findings are discussed in terms of the inability of etioplasts to carry out the metal-insertion step in chlorophyll synthesis. 4. An elevated chlorophyll(ide) concentration was attained in the etioplasts by increasing the size of the utilizable precursor pool by pre-feeding whole plants with delta-aminolaevulinic acid, isolating the etioplasts and subjecting them to the flash illumination.     

Biosynthesis of chlorophyll from protochlorophyll(ide) in green plant leaves

Using spectral methods, the biosynthesis of protochlorophyll(ide) and chlorophyll(ide) in green plant leaves was studied. The main chlorophyll precursors in the green leaves (as in etiolated leaves) were photoactive photocholorophyll(ide) forms Pchl(ide)655/650(448) and Pchl(ide)653/648(440). The contributions into Chl biosynthesis of the shorter-wavelength precursor forms ,which were accumulated in darkened green leaves as well, were completely absent (of Pchl(ide) 633/628(440)) or insignificant (of Pchl(ide)642/635(444)).     

Phototransformation of protochlorophyll(ide) by a proplastid fraction from Euglena

The phototransformation of protochlorophyll(ide) (Pchl(ide)) to chlorophyll(ide) (Chl(ide)) can be demonstrated in a proplastid fraction from Euglena gracilis Klebs var. bacillaris Cori if appropriate conditions are employed. Pigments were measured fluorometrically in acetone extracts of cell or organelles. Pchl(ide) and the phototransformation to Chl(ide) are at their highest levels in cells grown in darkness on normal or low vitamin B₁₂-containing medium (pH 3.5) to the late exponential phase (1.2–1.4 × 10⁶ cells ml^?1). Late exponential cells on low B¹² medium yield a proplastid fraction that contains Pchl(ide) which is phototransformed to Chl(ide) when illuminated with red light (5.6 W m^?2 for 4 min) in the presence of 10 mM Hepes, 20 mM TES, 0.5 mM potassium phosphate (pH 7.4), 70 mM sorbitol, 5 mM DTT, 5 mM ATP, 5 mM fructose-1, 6-bisphosphate, 10 mM malate and 2 mM MgCl²; intact organelles appear to be involved since deletion of osmoticum gives a lower activity, and addition of NAD(P)H is without effect. Phototransformation of Pchl(ide) to Chl(ide) in red light shows Bunsen-Roscoe reciprocity between fluence rate and duration of illumination. Although mitochondria are present, they do not appear to be involved since inhibitors of respiration and uncouplers of oxidative phosphorylation fail to block the phototransformation. The percentage phototransformation of Pchl(ide) to Chl(ide) in late exponential normal B¹² cells is 61 ± 10, and is 52 ± 3 in low B¹² cells. About 67% of the activity in low B¹² cells is recovered in the proplastid fraction incubated with the complete incubation mixture in saturating light. In both types of cells and in the proplastid fraction, the stoichiometry of conversion of Pchl(ide) to Chl(ide) is about 1:1 (mol/mol).     

Protochlorophyllide photo-transformation and chlorophyll(ide) formation in barley etioplast fractions

Localization of protochlorophyll(ide) (Pchlide) forms and chlorophyllide (Chlide) transformation process were studied by using comparative analyses of de-convoluted 77 K fluorescence spectra of barley etioplast stroma and different membrane fractions obtained by sucrose gradient centrifugation. Non-photoactive 633 nm Pchlide form was mainly located in the envelope-prothylakoid membrane mixture while the photoactive 657 nm Pchlide was dominant pigment in the prolamellar body membrane and in the soluble etioplast fraction (stroma). When these fractions were exposed to a saturating flash, conversion of photoactive Pchlide into 697 nm Chlide was preferential in the prolamellar body and in the stroma, while the 676 nm Chlide was dominant pigment form in the envelope-prothylakoid fraction. These spectral characteristics are considered to reflect molecular composition and organization of the pigment-protein complexes specific for each etioplast compartment.     

Beitrag zur Anfärbung gebundener (maskierter) Lip(o)ide mit Sudanschwarz B

Ohne ZusammenfassungMit 6 Textabbildungen     

Chlorophyll Synthesis from Protochlorophyll(ide) in Chill-Stressed Maize (Zea mays L.)

Illumination of aetiolated maize at temperatures lower than20 °C results in negligible accumulation of chlorophyll.Illumination of leaf tissue, previously incubated in 10 molm^–3 ALA in darkness, shows only a slight conversion ofprotochlorophyll(ide) to chlorophyll a and b at temperaturesless than 20 °C. A refined procedure for measuring photosynthesisby photo-acoustic spectroscopy in leaves that differ in chlorophyllcontent is presented. Studies of photosynthesis in aetiolatedseedlings illuminated at different temperatures by photo-acousticspectroscopy suggests that impairment of the chlorophyll pathwayis paralleled by an aberrant development of the thylakoid membrane. Key words: Protochlorophyll(ide), temperature, photo-acoustic spectroscopy, membrane biogenesis