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.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

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.     

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.     

Dependence of the early steps of chlorophyll(ide) synthesis on respiration in dark-grown Euglena gracilis

Protochlorophyll(ide) disappearance and chlorophyll(ide) accumulation, in dark-grown Euglena, promoted by series of actinic light flashes, have been followed by in vivo fluorescence measurements. The data show that chlorophyll(ide) accumulation is biphasic, i.e., there is an initial rapid phase followed by a slower linear phase. The linear phase is highly dependent on flash frequency and on cell respiration whereas the initial phase is much less affected by these factors. It is concluded that dark-grown cells contain a limited pool of phototransformable protochlorophyll(ide); once this pool is exhausted, its reformation and/or the synthesis of some unknown metabolite necessary for the photoreduction appears to be dependent on respiration.     

Effect of Benzyladenine Treatment Duration on delta-Aminolevulinic Acid Accumulation in the Dark, Chlorophyll Lag Phase Abolition, and Long-Term Chlorophyll Production in Excised Cotyledons of Dark-Grown Cucumber Seedlings

Cotyledons excised from dark-grown cucumber (Cucumis sativus L. cv. Aonagajibai) seedlings were incubated in the dark with the cytokinin benzyladenine for different time periods. Then, various greening parameters were examined, including protochlorophyll(ide) to chlorophyll(ide) photoconversion and δ-aminolevulinic acid accumulations in the dark, both triggered by a 5-minute red-light pulse.     

Photoconvertible protochlorophyll(ide) in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.     

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.     

The Correlated Appearance of Prolamellar Bodies, Protochlorophyll(ide) Species, and the Shibata Shift during Development of Bean Etioplasts in the Dark

The structure and physiology of the etioplast was investigated in developing primary leaves of 3- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Increase in total protochlorophyll(ide) content followed that of leaf fresh weight. In 3- to 4-day-old bean leaves more than 50% of the protochlorophyll(ide) is in the form of protochlorophyll(ide) 628, which is nontransformable by light. Most of the transformable pigment is protochlorophyll(ide) 635, with smaller amounts of protochlorophyll(ide) 650. During leaf development from the 3rd to the 7th day phototransformable protochlorophyll(ide) with an absorbance maximum at 650 nm accumulates faster than nontransformable protochlorophyll(ide) or protochlorophyll(ide) 635. This increase in protochlorophyll(ide) 650 is correlated with the formation and enlargement of prolamellar bodies.     

Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2-(alpha,alpha,alpha,-trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences

The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 μg/ml without affecting cell viability. Although the inhibition is immediate and complete, in resting cells no decrease in already synthesized carotenoids is found indicating a lack of turnover. In cells growing in the dark, carotenoids are diluted out as the cells divide. Cells dividing in the light in the presence of SAN 9789, eventually lose viability, presumably because of photooxidations usually prevented by carotenoids. During 72 hours of light-induced plastid development in dark-grown resting cells, none of the usual carotenoids increase while phytoene accumulates, indicating that SAN 9789 blocks carotenoid synthesis at this point. Chlorophyll synthesis and membrane formation are also blocked by the herbicide, but these inhibitions appear to be secondary to the inhibition of carotenoid synthesis. That carotenoid levels are strongly correlated with and may control the synthesis of chlorophyll and the formation of plastid membranes is suggested by the following data. (a) If dark-grown dividing cells are placed in the presence of the herbicide for various periods, rested and exposed to light in the presence of the drug, different amounts of carotenoids remain in the cells and the amount of chlorophyll finally synthesized is proportional to the amount of carotenoids present. (b) Photodestruction of chlorophyll is excluded, since the same amounts of chlorophyll are formed at intensities of 10 to 100 foot-candles of light. (c) Photoconversion of protochlorophyll(ide) to chlorophyll(ide) in dark-grown cells is not blocked by the herbicide. (d) Initial rates of chlorophyll synthesis are the same in treated and nontreated cells. (e) The extent of membrane formation appears to parallel the amount of carotenoids present as judged by electron microscopy.     

The State of Protochlorophyll and Chlorophyll in Corn Roots

The protochlorophyll(ide) present in primary roots of dark-grown corn (Zea mays) seedlings has an in vivo absorption maximum at 634 nm. Red light converts the pigment to chlorophyll(ide) a with an absorption maximum at 675 nm.     

Photoconvertible protochlorophyll(ide)635650 in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.     

Studies on chloroplast development in Chlamydomonas reinhardtii IV. Control of rapid chlorophyll formation in greening y-1 cells

Effects of protein synthesis inhibitors, CAP and CHI, on diegreening of Chlamydomonas reinhardtii y-1 cells, particularlyon die P-factor formation (19) in the early phase, were studied.Chlorophyll synthesis in the normal greening process, whichis divided into three phases, was strongly inhibited by bothantibiotics, although the inhibition by CAP was weaker in themiddle and late phases. The development of potential for rapidchlorophyll formation (P-factor formation) that takes placein dark-grown cells during dark incubation following brief illuminationwas completely blocked by CHI, but not by CAP. A "CHI-sensitive"period for the P-factor formation was restricted to the initial30 min during the dark incubation following brief illumination(10 min). This initial 30-min period appeared to correspondto the time of protochlorophyll(ide) formation which was inhibitedby CHI. Light-dependent conversion of protochlorophyll(ide) to chlorophylland also the subsequent protochlorophyll(ide) synthesis, whichis "CHI-sensitive" seem to be prerequisite for the inductionof P-factor synthesis. A possible control mechanism involvedin the early phase of the greening process in y-1 cells is discussed. (Received February 12, 1976; )     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Events Surrounding the Early Development of Euglena Chloroplasts: I. Induction by Preillumination 1

Preillumination, followed by a dark period prior to exposure of dark-grown nondividing cells of Euglena gracilis var. bacillaris to normal lighting conditions for chloroplast development, results in potentiation, or abolishment of the usual lag in chlorophyll accumulation. The degree of potentiation is a function of the length of the preillumination period, the intensity of preilluminating light, and the length of the dark period interposed before re-exposure to continuous light for development. The optimal conditions are found to be: 90 minutes of preillumination with white light at an intensity greater than 30 microwatts per square centimeter (14 foot candles) followed by a dark period of at least 12 hours. Reciprocity is not found between duration and intensity of preilluminating light. Preillumination with blue light and red light was found to be the most effective in promoting potentiation, and the ratio of effectiveness of blue to green to red is consistent with protochlorophyll-(ide) being the photoreceptor. Although red light is effective, there is no reversal by far red light, and these facts, taken together with the effectiveness of blue light, suggest that the phytochrome system is not involved. The amount of chlorophyll formed at the end of preillumination is proportional to the resulting potentiation, suggesting that the amount of protochlorophyll(ide) removed or chlorophyll(ide) formed regulates this phenomenon. Potentiated and nonpotentiated cells show comparable rates of protochlorophyll(ide) resynthesis, suggesting that this is not the limiting factor in nonpotentiated cells. Although light is required for protochlorophyll(ide) conversion in chlorophyll synthesis, a brief preillumination seems also to initiate the production of components in the subsequent dark period which, in nonpotentiated cells, are ordinarily synthesized during the lag period under continuous illumination. These components are necessary to sustain maximal rates of subsequent chlorophyll accumulation.     

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.     

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.     

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).     

Spectrophotometric quantitation of protochlorophyll(ide): Specific absorption and molar extinction coefficients reconsidered

To allay doubt about how to calculate molar extinction eoeffieients from the specific absorption coefficients of protoehlorophyll(ide), dark-grown leaf segments and saponin protochlorophyll(ide) holochrome subunits frotn barley ( Hordeum vulgare L.) and cotyledons of cucumber ( Cucumis sativus L.) were extracted with acetone without or following prior, brief illumination. Absorbance data and eoeffieients of chlorophyll a were used to derive extinetion eoeffieients of protoehlorophyll(ide); 626 nm (range: 30 to 35 1 mmol^-1 cm^-1) without recourse to published coefficients c protochlorophyll(ide). These results combined with evaluation of how the origin; coefficients were obtained, argue for using the molecular weight of protochlorophy rather than protochlorophyllide to calculate molar extinction eoeffieients from th specific absorption coefficients.