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.     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea ( Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.     

Oak seedlings grown in different light qualities

Oak seedlings (Quercus robur L.) were germinated in darkness for 3 weeks and then given continuous long wavelength far-red light (LFR; wavelengths longer than 700 nm). A control group of seedlings was kept in darkness. After 2 additional weeks the chlorophyll formation ability in red light was examined in the different seedlings. The stability of the protochlorophyll(ide) and chlorophyll(ide) forms to high intensity red irradiation was also measured. Oak seedlings grown in darkness accumulated protochlorophyll(ide) (6 μg per g fresh matter). Absorption spectra and fluorescence spectra indicated the presence of more protochlorophyll(ide)_628–632 than protochlorophyllide_650–657. The level of protochlorophyll(ide) was higher in leaves of plants cultivated in LFR light (13 μg per g fresh matter) than in leaves of dark grown plants. 12% of the protochlorophyll(ide) was esterified in both cases. The level of protochlorophyll(ide)_628–632 in LFR grown oaks varied with the age of the leaves, being higher in the older (basal) leaves, but also in the very youngest (top-most) leaves. The ability of the leaves to form photostable chlorophyll in red light showed a similar age dependence, being low in rather young and in older leaves. A low ability to form photostable chlorophyll thus appears to be correlated with a high content of protochlorophyll(ide)_628–632. Upon irradiation only the protochlorophyllide_650–657 was transformed to chlorophyllide. After this phototransformation the chlorophyllide peak at 684 nm shifted to 671 nm within about 30 min in darkness. This shift took place without any accompanying change in photostability of the chlorophyll(ide). Upon irradiation with strong red light a similar shift took place within one minute. This indicates that the chlorophyllide after phototransformation was rather loosely bound to the photoreducing enzyme. The development towards photostable chlorophyll forms consists of three phases and is discussed.     

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     

High salt stress induces swollen prothylakoids in dark-grown wheat and alters both prolamellar body transformation and reformation after irradiation

High salinity causes ion imbalance and osmotic stress in plants. Leaf sections from 8-d-old dark-grown wheat (Triticum aestivum cv. Giza 168) were exposed to high salt stress (600 mM) and the native arrangements of plastid pigments together with the ultrastructure of the plastids were studied using low-temperature fluorescence spectroscopy and transmission electron microscopy. Although plastids from salt-treated leaves had highly swollen prothylakoids (PTs) the prolamellar bodies (PLBs) were regular. Accordingly, a slight intensity decrease of the short-wavelength protochlorophyllide (Pchlide) form was observed, but no change was found in the long-wavelength Pchlide form emitting at 656 nm. After irradiation, newly formed swollen thylakoids showed traversing stromal strands. The PLB dispersal was partly inhibited and remnants of the PLBs formed an electron-dense structure, which remained after prolonged (8 h) irradiation. The difference in fluorescence emission maximum of the main chlorophyll form in salt-stressed leaves (681 nm) and in control leaves (683 nm) indicated a restrained formation of the photosynthetic apparatus. Overall chlorophyll accumulation during prolonged irradiation was inhibited. Salt-stressed leaves returned to darkness after 3 h of irradiation had, compared with the control, a reduced amount of Pchlide and reduced re-formation of regular net-like PLBs. Instead, the size of the electron-dense structures increased. This study reports, for the first time, the salt-induced swelling of PTs and reveals traversing stromal strands in newly formed thylakoids. Although the PLBs were intact and the Pchlide fluorescence emission spectra appeared normal after salt stress in darkness, plastid development to chloroplasts was highly restricted during irradiation.     

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.     

Pigment formation in potato tubers (Solanum tuberosum) exposed to light followed by darkness

Potato tubers ( Solanum tubersoum cvs Bintje and King Edward). never exposed to light, lack chlorophyllous pigments. Continuous irradiation results in chlorophyll (Chl) formation and induces the ability for protochlorophyll (Pchl) formation when the tubers are brought back to darkness. Pigment synthesis takes place in both blue and red light, but blue light is more effective than red in starting the greening process. The pigment formation is strongest in the layers just below the periderm with a steep gradient inwards. Small amounts of Chl formed after irradiation. slowly fade away during extended darkness. However, the Chl formed after long time of irradiation is remarkably stable. Irradiated potatoes, placed in darkness, form Pchl with a fluorescence emission peak at 633 nm. A maximal level is reached after ca 7 days. Resolution of the Pchl spectrum suggests the presence of small amounts of a pigment with an emission maximum at around 642 nm. No sign of the Pchl with emission maximum at 657 nm, which dominates in etiolated leaves, is found. A faint Chl fluorescence indicates that some Pchl, probably the 642 nm form, is phototransformed into Chl in weak light. The Chl formation in the potato tuber is discussed in relation to that of roots and leaves.     

Plastid differentiation and chlorophyll biosynthesis in different leaf layers of white cabbage (Brassica oleracea cv. capitata)

The contents of protochlorophyllide, protochlorophyll and chlorophyll together with the native arrangements of the pigments and the plastid ultrastructure were studied in different leaf layers of white cabbage (Brassica oleracea cv. capitata) using absorption, 77 K fluorescence spectroscopy and transmission electron microscopy. The developmental stage of the leaves was determined using the differentiation of the stoma complexes as seen by scanning electron microscopy and light microscopy. The pigment content showed a gradual decrease from the outer leaf layer towards the central leaves. The innermost leaves were in a primordial stage in many aspects; they were large but had typical proplastids with few simple inner membranes, and contained protochlorophyllide and its esters in a 2 : 1 ratio and no chlorophyll. Short‐wavelength, not flash‐photoactive protochlorophyllide and/or protochlorophyll forms emitting at 629 and 636 nm were dominant in the innermost leaves. These leaves also had small amounts of the 644 and 654 nm emitting, flash‐photoactive protochlorophyllide forms. Rarely prolamellar bodies were observed in this layer. The outermost leaves had the usual characteristics of fully developed green leaves. The intermediary layers contained chlorophyll a and chlorophyll b besides the protochlorophyll(ide) pigments and had various intermediary developmental stages. Spectroscopically two types of intermediary leaves could be distinguished: one with only a 680 nm emitting chlorophyll a form and a second with bands at 685, 695 and 730 nm, corresponding to chlorophyll–protein complexes of green leaves. In these leaves, a large variety of chloroplasts were found. The data of this work show that etioplasts, etio‐chloroplasts or chloro‐etioplasts as well as etiolated leaves do exist in the nature and not only under laboratory conditions. The specificity of cabbage leaves compared with those of dark‐grown seedlings is the retained primordial or intermediary developmental stage of leaves in the inner layers for very long (even for a few month) period. This opens new developmental routes leading to formation of specially developed plastids in the various cabbage leaf layers. The study of these plastids provided new information for a better understanding of the plastid differentiation and the greening process .     

The Absence of Protochlorophyll(ide) Accumulation in Algae with Inhibited Chlorophyll Synthesis

Golenkinia, Chlorella protothecoides, and mutant C-2A′ of Scenedesmus were grown in darkness and on media in which chlorophyll synthesis is reduced significantly. The pigments were analyzed by spectrophotometry or by paper chromatography and compared with similar extracts from light-grown algae and dark-grown beans. No protochlorophyll(ide) was present in the dark-grown algae indicating that chlorophyll synthesis is blocked by a mechanism other than feedback regulation of aminolevulinic acid synthesis by protochlorophyll(ide) which has been proposed for flowering plants.     

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.     

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.     

Involvement of photobleaching and inhibition of protochlorophyll(ide) accumulation in tentoxin effects on greening of mung bean seedlings

Several types of evidence indicate that tentoxin-caused reduction of chlorophyll accumulation in greening primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] is due to both photobleaching and decreased protochlorophyll(ide) synthesis. Greening was greater under dim (2.5 μmol m_-2 s_-1) far-red or white light than under bright (180 to 200 μmol m_-2 s_-1) white light in tentoxin-treated tissues, whereas there was a positive correlation between fluence rate and greening in control tissues. Under continuous white light (100 μmol m_-2 s_-1) chorophyll(ide) accumulation was slower in tentoxin-treated than in control tissues. This was caused by greater photobleaching of newly formed chlorophyll(ide), as well as by decreased protochlorophyll(ide) synthesis. Photobleaching did not affect protochlorophyll(ide) synthesis in control or tentoxin-treated tissues. Chlorophyll(ide) was less stable in tentoxin-treated than in control tissues during a 24 h period of darkness. Plastids of tentoxin-treated tissues had all of the chlorophyll-proteins of control plants. Etioplasts of tentoxin-treated plants contained normal galactolipid contents, but galactolipids in these plants were greatly reduced in white light. Reduced chlorophyll accumulation caused by tentoxin is apparently the result of both photodestruction and of reduced synthesis of chlorophyll.     

Chloroplast biogenesis. Detection of monovinyl protochlorophyll(ide) b in plants

The occurrence of protochlorophyllide b and protochlorophyllide b phytyl ester in green plants is described. The chemical structure of protochlorophyllide b phytyl ester was established by proton nuclear magnetic resonance, fast atom bombardment mass spectroscopic analysis, and chemical derivatization coupled to electronic spectroscopic analysis. The macrocycles of protochlorophyll(ide) b are identical to those of conventional protochlorophyll(ide) except for the presence of a formyl group instead of a methyl group at position 3 of the macrocycles. They differ from chlorophyll(ide) b by the presence of an oxidized double bond at positions 7 and 8 of the macrocycles. The trivial name protochlorophyll(ide) b is proposed to differentiate these two tetrapyrroles from conventional protochlorophyll(ide), which in turn will be referred to as protochlorophyll(ide) a. Protochlorophyll(ide) b appears to be widely distributed in green plants. Its molar extinction coefficients in 80% acetone and diethyl ether are reported. The impact of this discovery on the heterogeneity of the chlorophyll a and b biosynthetic pathways is discussed.     

Comparison of Light-dependent Oxygen Uptake, Protochlorophyll(ide)-650 Photoconversion, and Chlorophyll Disappearance in Wheat Etioplasts

Red light exposures given to dark-grown wheat seedlings (Triticum aestivum L.) prior to etioplast isolation reduced the ability of these organelles to consume O₂. The same preharvest red light exposures also decreased protochlorophyll(ide) content of etioplasts. In addition, regeneration of both O₂ uptake rates as well as protochlorophyll(ide) levels followed a parallel time course. These similarities suggested that photoconversion of protochlorophyll(ide)-650 to chlorophyll(ide) may mediate some process with O₂ as the electron acceptor. This process appears to involve photooxidation of nonphotoconvertible protochlorophyll(ide) as well as of newly formed chlorophyll(ide). This hypothesis is further supported by the observations that: (a) the in vitro light induced O₂ uptake phenomenon was observed in solubilized protochlorophyll(ide) holochrome preparations; and (b) photoinduced O₂ uptake was reduced to zero rate by light exposure time equivalent to that required for chlorophyll(ide) and nonphotoconvertible protochlorophyll(ide) destruction.     

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.     

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.     

Protochlorophyll(ide) accumulation and degradation in the dark and photoconversion to chlorophyll in the light in pigment mutant C-2A' of Scenedesmus obliquus

Pigment mutant C-2A' of Scenedesmus obliquus accumulates only traces of chlorophyll, when grown heterotrophically in the dark. Immediately upon transfer of cells into fresh medium protochlorophyllide and protochlorophyll are formed, which accumulate to their maximum concentrations within 8 to 12 h. Subsequently, this protochlorophyll(ide) is degraded in the dark, but not transformed into chlorophyll. After 6–8 days of dark growth no protochlorophyll(ide) can be detected any more. The protochlorophyll(ide) pool of cultures, which contain reduced concentrations, can be reestablished either by addition of glucose or illumination with blue light; both increase the rate of respiration.
By low temperature spectroscopy in vivo and by absorption and fluorescence emission spectroscopy of pigment extracts it is shown that the protochlorophyllide accumulated in freshly inoculated cultures can be converted to chlorophyll in light.
From the action spectrum for chlorophyll formation after addition of glucose it can be seen that protochlorophyllide 636 and 649 are present and are photoconvertible in this mutant.     

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.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations.

1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening.