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.     

THE EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

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.     

Occurrence of protochlorophyll and its phototransformation to chlorophyll in mutant C-2A' of Scenedesmus obliquus

The pigment mutant C-2A' of the unicellular green alga Scenedesmus obliquus accumulates protochlorophyllide and small amounts of protochlorophyll in darkness. Protochlororphyll was isolated and characterized by thin layer chromatography and absorption and fluorescence spectroscopy. The protochlorophyll was photoreduced by light to chlorophyll both in vitro and in vivo.     

Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts

The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.     

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.     

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.     

Ricerche Sulla Morfogenesi Dei Plastidi Clorofilliani

Abstract

Studies on chloroplast morphogenesis. The effect of sucrose feeding and light intensity on the plastids of etiolated plants. — The changes in the fine structure of the plastids of etiolated Bean plants, dipped into water or in various sucrose concentrations, for 24, or more, hours, and exposed to conditions of darkness and weak light, were studied at the electron microscope, and protochlorophyll, chlorophyll a and chlorophyll b contents were determined. When the etiolated plants are dipped into water or sucrose solutions, in the dark, rows of tubules and lamellae, often stacked and resembling small grana, are formed in the plastids. These structural changes of plastids of the plants exposed to conditions of darkness, where no protochlorophyll was converted to chlorophyll a, are quite similar to those described by several Authors for plants exposed to light conditions and thought to coincide with the protochlorophyll-chlorophyll transformation. Thus, the preservation of the « Kristallgitterstruktur » of the vesicular centers, or, instead, their transformation into a lapse cluster of tubules, does not seem to be related with protochlorophyll accumulation in them; indeed, an increase of protochlorophyll contents was observed both with the preservation of the crystalline structure, and with its transformation, and low protochlorophyll contents did not always coincide with the transformation of the vesicular centers. In the plants exposed to weak light (1 ft-c) there is chlorophyll a and b accumulation, and a more pronounced tendency toward stacking of tubules and lamellae. In the plants exposed to weak light, dipped into water or sucrose solutions, at the lowest concentrations, and for the shortest periods, the vesicular centers are transformed into clusters of tubules; but with higher sucrose concentrations, or longer dipping periods, their crystalline structure is preserved, just as if their preservation would depend only by an adeguate nutrients supply.

The arrangement of normal lamellae and the formation of grana connected by intergrana lamellae occur, anyhow, only when the etiolated plants are exposed to « high » light (630 ft-c). But chlorophyll accumulation is possible under « weak » light (and a stacking of tubules and lamellae, resembling small grana, also occurs), when sucrose is supplied. The achievement of the « normal », complete structure of the chloroplast is, therefore, here interpreted in the sense that it represents only the functional aspect of its organization, determined by a light intensity favorable to its photosyntetic activity, which is not directly necessary for the synthesis of the contistuents of the lamellar system (chlorophylls, phospholipids).     

Biosynthesis and accumulation of microgram quantities of chlorophyll by developing chloroplasts in vitro

Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, δ-aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro.     

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.     

A protein–protochlorophyll complex obtained from inner seed coats of Cucurbita pepo: The resolution of its two pigment groups into true protochlorophyll and a pigment related to bacterial protochlorophyll

1. The inner seed coats of Cucurbita pepo were extracted with aqueous acetone and found to contain pigments with spectra similar to that of protochlorophyll. 2. When the fruits of C. pepo were stored the amount of protochlorophyll-like material in the inner seed coats increased and a form of protochlorophyll absorbing at longer wavelength was apparently formed. 3. The pigment was resolved into two forms of protochlorophyll by chromatography on sugar columns. One form with absorption maxima in ether at 432, 535, 571 and 623mmu was spectroscopically identical with plant protochlorophyll; the other, with absorption maxima at 438, 537, 574 and 624mmu, was spectroscopically identical with bacterial protochlorophyll isolated from the tan mutant of Rhodopseudomonas spheroides. The two phaeoporphyrins obtained from the seed-coat pigments closely resemble the corresponding phaeoporphyrin derivatives of plant protochlorophyll and bacterial protochlorophyll in spectroscopic and partition properties. 4. The pigment in the cells of inner seed coat of C. pepo is concentrated in discrete particles of about 1.7mu diameter. Extracts of the seed coats in a glycerol-glycine buffer were similar in spectroscopic properties to the crude protochlorophyll holochrome, but were not light-transformable. 5. After partial purification of the glycerol-glycine buffer extracts a pigment-protein complex was obtained with absorption maxima at considerably longer wavelengths than in organic solvents. 6. Preparations of the seed-coat protochlorophyll, in the presence of bovine serum albumin, adsorbed on filter paper or in colloidal solution, did not have absorption bands shifted so far to the red region as the natural protein complex isolated from the seed coat. 7. It is suggested that bacterial protochlorophyll (magnesium 2,4-divinylphaeoporphyrin a(5) methyl ester) is involved in the biosynthesis of chlorophyll in both plants and photosynthetic bacteria.     

The plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein

Illumination of dark-grown barley plants induces a massive insertion of the light-harvesting chlorophyll a/b protein into the developing thylakoid membrane. In addition to the onset of chlorophyll synthesis, light induces specifically the appearance of a prominent mRNA species which codes for a polypeptide of Mr 29500. This component was identified as a precursor of the apoprotein of the light-harvesting chlorophyll a/b protein. The precursor has an Mr larger than the authentic protein by approximately 4000. Studies of the chlorophyll-b-less mutant chlorina f2 of barley offer the first clue to the mechanism which controls the light-dependent mRNA formation. The induction of the mRNA coding for the aproprotein of the light-harvesting chlorophyll a/b protein does not seem to be linked directly to the assembly process of the light-harvesting structure and does not require chlorophyll b. It is proposed that light exerts its influence on the mRNA formation by a reaction which is different from the phototransformation of protochlorophyll(ide) to chlorophyll(ide).     

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.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.     

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     

Expression of genes for plastid membrane proteins in barley under intermittent light conditions

Barley plants grown under intermittent light show a plastid membrane composition intermediate between those of etioplasts and chloroplasts. In particular protochlorophyll reductase disappears from the membranes whereas the 32000 protein, coded for by chloroplast DNA, becomes integrated into the membranes. The light-harvesting chlorophyll a/b protein does not accumulate within the membranes even after 11 d of development, while the corresponding mRNA can already be observed after 4 d and is translated under in vivo conditions.Abbreviations LHCP light-harvesting chlorophyll a/b protein - IL intermittent light - LD light-dark (12-h day) - EGTA ethyleneglycol-bis(oxy-ethylenenitrile)tetraacetic acid     

Formation of short-wavelength chlorophyll(ide) after brief irradiation is correlated with the occurrence of protochlorophyll(ide)636–642 in dark-grown epi- and hypocotyls of bean (Phaseolus vulgaris)

Chlorophyll formation capacity along the seedling of bean ( Phaseolus vulgaris L. cv. Brede zonder draad) was investigated. After 7 days of irradiation a gradient was formed, where the primary leaf contained ca 300 times more chlorophyll per gram fresh weight than the lower hypocotyl section and ca 20 times more than the epicotyl. Similar chlorophyll gradients but at lower levels were seen when the seedlings were first placed in darkness for 7 days and then irradiated for 1, 2 or 7 days. Ultrastructural investigation of seedlings grown for 7 days in darkness and then irradiated for 24 h revealed a more developed inner membrane system with grana stacks in plastids of cells in the uppermost hypocotyl section compared to plastids of cells in lower hypocoty] sections. The higher up on the seedling the more the ratio increased of protochlorophyll(ide) emitting at 657 nm to short-wavelength protochlorophyll(ide). After flash irradiation of the different sections, fluorescence emission spectra with maxima at 680 and 690 nm, respectively, were observed, indicating the formation of short- and long wavelength chlorophyll(ide) forms. The lower the ratio of protochlorophyll(ide) emitting at 657 nm to the short-wavelength protochlorophyll(ide), the less long-wavelength chlorophyll(ide) was formed after irradiation. However, after continuous irradiation long-wavelength chlorophyll(ide) was formed. In dark grown roots, where only short-wavelength protochlorophyll forms were present, it was not possible to transform protochlorophyll to chlorophyll by flash irradiation. Possible explanations for this phenomenon are 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.     

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.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.