Studies on chlorophyll formation in Chlorella protothecoides II. Enhancing effects of light and suppressive effects of glucose on the development of porphyrin-synthesizing activity during light-induced greening of etiolated algal cells

Frozen and thawed cells, as well as sonicated cell preparationsof Chlorella protothecoides, were assayed for activity to synthesizeporphyrins from added ALA or PBG. Activity was very low in etiolatedcells, and markedly developed during the process of light-inducedgreening. The development of activity was strongly suppressedby glucose. Activity for the synthesis of URO(gen) from ALAwas initially developed, then the formation of COPRO(gen)-synthesizingactivity ensued. In glucose-suppressed cells as in cells incubatedin continuous darkness, URO was the main porphyrin producedand COPRO was virtually missing in the reaction products withadded ALA, indicating that development of activity for the conversionof URO(gen) to COPRO(gen) is greatly enhanced by light and isrepressed by added glucose. Suppressive effects of CP and CHon the development of porphyrin-synthesizing activity were alsostudied. From these and other results, a tentative scheme ispresented for the enhancing effects of light and the suppressiveeffects of glucose on the development of porphyrin-synthesizingactivity in etiolated algal cells, in correlation with the effectson other processes of chlorophyll biosynthesis. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. (Received April 6, 1972; )     

EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

Studies on chloroplast development in Chlamydomonas reinhardtii I. Effect of brief illumination on chlorophyll synthesis

When dark grown cells of Chlamydomonas reinhardtii y-1 mutantwere exposed to continuous light, an immediate transformationof small amounts of protochlorophyll(ide), which had been presentin the dark grown cells, to chlorophyll was observed. Afterthis, there was a slow accumulation of chlorophyll lasting for2.5-3 hr before the start of exponential synthesis. Initialaccumulation of chlorophyll was distinctly slower at a highlight intensity (13,000 lux) than it was at moderate intensitiesof light (2,000–5,000 lux). However, the exponential synthesisof chlorophyll started after the same 2.5–3 hr of illumination. A brief pre-illumination of cells followed by incubation indarkness was effective in promoting chlorophyll synthesis undersubsequent continuous illumination at high, as well as moderatelight intensities. Pretreatment alleviated retardation of theinitial chlorophyll accumulation by light of high intensity.The promoting effect of preillumination on chlorophyll synthesiswas sufficient, even when a light impulse as short as 10 secwas given. However, the effect was dependent on length of thedark period after the short pre-illumination. The full extentof this effect was observed when the dark period was about 2.5–3hr long. Further dark incubation gradually decreased the effect. On the basis of these findings, it is assumed that a factor(s)responsible for promotion of chlorophyll (or chloroplast) synthesisin the process of greening of dark grown cells is produced duringthe dark period after a brief pre-illumination, and that thefactor is turned over at a relatively fast rate. The possiblenature of the presumed factor is discussed in relation to chloroplastdevelopment. ¹Present address: Department of Biology, Faculty of Science,Kobe University, Nada-ku, Kobe, Japan. (Received August 18, 1970; )     

Photoinhibition of 5-aminolevulinic acid formation under GO2-free condition

5-Aminolevulinate accumulation in the presence of levulinatewas followed in greening Chlorella protothecoides cells. Underthe CO₂-free condition, ALA formation was severely inhibitedby 20 W/m² white light. The inhibition was removed by CMU. Combinedaddition of CMU with N, N'-tetramethyl phenylenediamine plusascorbate again caused photoinhibition of ALA formation, whilethe addition of CMU with dithiothreitol caused severe inhibitionof ALA formation in both light and darkness. Exogenous glucose enhanced ALA formation in darkened algal celb,but not in photo- and DTT-inhibited cells. In either case, glucoseseemed to be metabolized mainly by the algal cells through theglycolysis-citric acid system. It was inferred that ALA formationwas suppressed at the site of, or related to, an enzyme reactionforming ALA. (Received June 27, 1979; )     

A two-fold action of benzyladenine on chlorophyll formation in etiolated cucumber cotyledons

Treatment of 3-day-old excised etiolated cotyledons of cucumber ( Cucumis sativus L. cv. Aonagajibai) with benzyladenine (BA) in the dark stimulates chlorophyll (Chl) formation during the lag phase (designated as 'lag elimination') and accelerates the steady-state rate of Chl formation under subsequent continuous illumination with white light. The separation of this two-fold effect is possible using two different methods of BA treatment in darkness: a brief BA treatment followed by various periods of water treatment in darkness, or various periods of continuous dark BA treatment. In either treatment, BA rapidly eliminates the lag phase (the fast-appearing effect) and after a longer time period accelerates the steady-state rate (the late-appearing effect). With a brief BA treatment, both effects decay rapidly. In contrast, with continuous BA treatment, none of the effects decay after reaching their maxima, particularly in cotyledons excised 2 days after sowing and aged for a long period before the onset of BA treatment. These facts indicate that BA acts as a trigger in stimulating Chl formation. The relationship between the actions of BA and light is discussed.     

Glucose and δ-Aminolevulinic Acid Stimulate the Dark Chlorophyll Synthesis of Rice Seedlings

This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ(Proto), Mg-protoporphyrin Ⅸ(Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark. Key words: angiosperm; rice; dark chlorophyll synthesis; glucose; δ-aminolevulinic acid; protoporphyrin Ⅸ; Mg-protoporphyrin Ⅸ; protochlorophyllide     

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.     

Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation

Effects of chloramphenicol, cycloheximide, puromycin and ethionineon the light-independent and subsequent light-dependent processesof chlorophyll formation in "glucose-bleached" cells of Chlorellaprotothecoides were studied. These substances, except puromycin,strongly suppressed different phases of chlorophyll formation.Ethionine most strongly suppressed the light-independent phaseand chloramphenicol an early, relatively short process in thelight-dependent phase of chlorophyll formation. Cycloheximideseverely suppressed all phases of chlorophyll formation. Possibleimplications of these results for the biosynthesis of chlorophyllin algal cells are discussed. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. ² Laboratory of Entomology, Faculty of Agriculture, TamagawaUniversity, Machida-shi, Tokyo, Japan (Received October 5, 1972; )     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells II. Blue Light Effect on 5-Aminolevulinic Acid Formation

5-Aminolevulinic acid (ALA) accumulation in dark-grown tobaccocallus cells in the presence of levulinic acid (LA) was followedunder blue or red light or in continuous darkness. Significantformation of ALA continued in the dark. The protochlorophyll-(ide) (Pchl) content of dark-incubated cells remained low becauseof its turnover. We inferred that the feedback inhibition ofALA synthesis by Pchl would not occur in darkincubated calluscells. ALA formation was enhanced by blue light, and this effectreached saturation at an intensity of about 800 mW.m^–2.Neither weak nor strong red light affected ALA formation. Fullenhancement of ALA formation by blue light was attained afterfairly long continuous illumination of the callus cells. Thisblue lightenhanced activity of ALA synthesis declined very slowlyduring the subsequent dark incubation. The blue light enhancement of ALA formation was observed incallus cells supplied with sucrose over a wide range of concentrations.Pchl regeneration in carbon-starved callus cells, supplied withglutamate at various concentrations, was also markedly enhancedby blue light. Respiration of the callus cells was not enhancedby blue light. A possible role of blue light in regulating ALAformation in callus cells is discussed. ¹Dedicated to the late Professor Joji Ashida. (Received September 3, 1982; Accepted April 5, 1983)     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Biosynthetic events required for lag elimination in chlorophyll synthesis in Euglena

Summary Levulinic acid, a competitive inhibitor of aminolevulinic acid dehydratase, cycloheximide, an inhibitor of translation on 89s cytoplasmic ribosomes, and chloramphenicol, an inhibitor of translation on 68s chloroplast ribosomes, are reversible inhibitors of light induced chlorophyll synthesis in resting Euglena gracilis Klebs. When dark grown resting cells are preilluminated for 2 h followed by darkness for 12 h prior to exposure to continuous light, the usual lag period in chlorophyll formation is eliminated. If cycloheximide, chloramphenicol, or levulinic acid are present during either the preillumination period or the subsequent dark period, the lag is reestablished. Only the very beginning of the dark period is sensitive to cycloheximide but the dark period is less sensitive to levulinic acid than is the light period. Exposure of preilluminated cells to cycloheximide or levulinic acid at the time of exposure to continuous illumination completely inhibits chlorophyll synthesis indicating that the potential for rapid chlorophyll synthesis generated by preillumination and a dark period does not result simply from the accumulation of porphyrin precursors. Preillumination has little effect on the development of the capacity to fix CO₂ photosynthetically. These results indicate that the control of chlorophyll formation is more complex than in higher plants and a model based on the formation of certain crucial enzymes in the porphyrin pathway, rather than simply upon the accumulation of aminolevulinic acid is presented to explain the experimental findings.Abbreviations ALA delta amino levulinic acid - CAM chloramphenicol - CEX cycloheximide - chl (ide) chlorophyll (ide) - LEV levulinic acid - pchl (ide) protochlorophyll (ide) Supported by GM14595 from the National Institutes of Health. This paper is No. 9 in the series, Events Surrounding the Early Development of Euglena ChloroplastsMicrobiology Trainee of the National Institutes of Health, Grant No. GM1586. The material in this paper is part of a dissertation submitted by S.D.S. to the Graduate Faculty of Brandeis University in partial fulfillment of the requirements for the Ph.D. Degree.     

Chlorophyll turnover in etiolated greening barley transferred to darkness: Isotopic (1-14C glutamic acid) evidence of dark chlorophyll synthesis in the absence of chlorophyll accumulation

Synthesis of chlorophyll was initiated in 5- to 6-day-old dark-grown barley (Hordeum vulgare L. cv. Clipper)seedlings by exposing them to light in the presence of 1-¹⁴ C glutamic acid supplied via the roots.The plants were then returned to darkness. At the end of light treatment (_T) and after 7 or 18 h dark treatment chlorophylls a and b were extracted, quantified (μgleaf¹). purified by HPLC to their magnesium-free derivatives (pheophytin a and b) and their molar radioactivities determined. After 2 h exposure to light followed by 6 h illumination in the presence of 1-¹⁴ C glutamic acid, seedlings had accumulated 4-7 nmol chlorophyll leaf¹ and had incorporated between 900-1 350 Bq (g fresh weight)¹ of radioactive label into the chlorophyll pool. When seedlings were transferred to darkness, label continued to be incorporated and after 18 h the radioactivity of the chlorophyll pool had increased by 300-700 Bq (g fresh weight)¹. Net chlorophyll content, however, remained constant during dark treatment. The increase in radioactivity of the chlorophyll pool in darkness represented the difference between a net increase of label incorporated into chlorophyll a and a small loss of label from chlorophyll b. The absence of measurable radioactivity in the phytol moiety of labelled chlorophyll a, extracted at the endof dark treatment, demonstrated thatincorporation of label was into the tetrapyrrole moiely of chlorophyll and not into the phytol chain. Light-independent incorporation of 1-¹⁴ C glutamic acid into chlorophyll of greening barley seedlings transferred to darkness indicates that chlorophyll synthesis continues when light is withheld. We interpret the net gain in radioactivity of chlorophyll in darkness, in the absence of a net gain in chlorophyll content, to chlorophyll turnover i.e. to simultaneous synthesis and breakdown of chlorophyll when etiolated greening barley seedlings are transferred to darkness.     

Formation of chlorophyll B, and the fluorescence properties and photochemical activities of isolated plastids from greening pea seedlings

Chlorophyll b was first detectable after 10 minutes of illumination of etiolated pea seedlings (Pisum sativum L. var Greenfeast) with continuous white light. The chlorophyll a/b ratio decreased from 300 at 10 minutes to 15 after 1 hour. There was little change in the chlorophyll a/b ratio between 1 and 2 hours, and it declined to 3 between 2 and 5 hours of illumination. In red light, the time courses of total chlorophyll synthesis and chlorophyll a/b ratio were similar to those in white light for the first 5 hours of illumination. But with increasing time of illumination with red light, there was an increase in the chlorophyll a/b ratio to 7 after 30 hours. Illumination with white light of very low intensity also gave high chlorophyll a/b ratios. Seedlings which had been illuminated for varying periods and then returned to darkness always showed an increase in chlorophyll a/b ratio during the dark period. It is concluded that the synthesis of chlorophyll b is controlled by light.     

Catabolism of [1-C]levulinic Acid by etiolated and greening barley leaves

Levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), has been used extensively in the study of ALA formation during greening. When [1-¹⁴C]LA is administered to etiolated barley (Hordeum vulgare L. var. Larker) shoots in darkness, ¹⁴CO₂ is evolved. This process is accelerated when such tissues are incubated with 2 millimolar ALA or placed under continuous illumination. Label from the C-1 of LA becomes incorporated into organic acids, amino acids, sugars, lipids, and proteins during a 4-hour incubation in darkness or in the light. This metabolism is discussed in relation to the use of LA as a tool in the study of chlorophyll synthesis in higher plants.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: I. SYNTHESES OF NUCLEIC ACIDS AND PROTEIN IN RELATION TO THE PROCESS OF REGENERATION OF CHLOROPLAST

When Chlorella protothecoides is grown mixotrophically in thelight in a medium rich in glucose and poor in nitrogen source(urea), one obtains the cells that are entirely devoid of chlorophylland containing only little RNA and protein. When these cells—referredto as "glucose-bleached" cells—are further grown in thelight with provision of nitrogen source, but without glucose,sequential syntheses of RNA, protein and chlorophyll take place.If the glucose-bleached cells are incubated in the dark underthe same nutritional condition, RNA, protein and chlorophyllare also successively formed in relatively small amounts. Thecells obtained under such a condition are, in many respects,similar to the cells that are obtained when the alga is grownin the dark in a medium poor in glucose and rich in the nitrogensource. These cells, which are called the "etiolated cells",are faintly green in color and contain larger amounts of RNAand protein compared with the chlorophyll-less glucose-bleachedcells. The glucose-bleached cells and the etiolated cells showapproximately the same content of DNA per cell. When the etiolatedcells are incubated in the light with provision of nitrogensource, but without glucose, they become green with active synthesisof chlorophyll and additional syntheses of RNA and protein. Based on these results and those to be reported later, it wasconcluded that the greening of the glucose-bleached cells involvesa light-independent phase followed by a light-requiring phasewhich entails the greening of cells and full organization ofchloroplasts, and that the latter process is essentially thesame as that taking place when the etiolated cells are incubatedin the light with provision of nitrogen source in the absenceof glucose. (Received September 5, 1964; )     

Spectral effectiveness in chlorophyll and 5-aminolevulinic acid formation during regreening of glucose-bleached cells of Chlorella protothecoides

Regreening of glucose-bleached cells of Chlorella protothecoidesis stimulated by light. Spectral effectiveness in the processshowed maxima around 370, 440 and 480 nm, suggesting a flavoproteinas primary photoreceptor. Action spectra of ALA synthesis provedto be similar to those of chlorophyll formation, indicatingthat light stimulation of greening in this alga is regulatedat the first step of chlorophyll biosynthesis. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 27, 1978; )     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

Incorporation of 5-aminolevulinic acid into chlorophyll in darkness in barley

The incorporation of radioactive aminolevulinic acid (ALA) into chlorophyll (Chl) a and b , as well as protochlorophyllide (Pchlide) in light-grown barley seedlings ( Hordeum vulgare L. cv. Clipper) transferred to darkness is demonstrated.
In the experiments described, 6-day-old, glasshouse-grown seedlings were transferred to darkness and incubated in [¹⁴C]- or [³H]- ALA for 18 h.
Chl a and b were extracted and purified to constant specific radioactivity by HPLC and TLC of their magnesium-free derivatives, pheophytin a and b . The presence of label in the tetrapyrrole ring of the Chls was established by removal of the phytol chain by alkaline hydrolysis and determination of the specific radioactivity of the chlorin e ₆ and rhodin g ₇ derivatives.
Barley seedlings that had been grown in darkness for 5 days, transferred to light for 20 h, and then returned to darkness in the presence of radioactive ALA also incorporated label into Chl. However, this was only apparent in intact seedlings. Excised leaves from greened etiolated plants did not incorporate ALA into Chl in darkness. This was consistent with the finding of Apel et al. (K. Apel, M. Motzkus and K. Dehesh, 1984. Planta 161: 550–554) and may account for their failure to obtain evidence for a light-independent protochlorophyllide reductase in greening barley.
Although the incorporation of ALA into Chl compared to Pchlide was slight (5%), the presence of label in the tetrapyrrole nucleus of Chl a and b is unequivocal evidence of a light-independent pathway of Chl biosynthesis in barley that has been exposed to light during development. Limited entry of exogenous labelled ALA into the precursor pools leading to the dark reduction of Pchlide is postulated.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

The effect of haem on chlorophyll synthesis in barley leaves

Exogenously supplied bovine haemin, fed to etiolated barley leaves, inhibited chlorophyll synthesis in leaves exposed to light. Haemin inhibited the regeneration of protochlorophyllide (P650) and the conversion of exogenously supplied δ-aminolaevulinate (ALA) to protochlorophyll (P630). The effect of haemin on chlorophyll production was overcome by incubating the leaves in water in the dark before light treatment, suggesting the operation of a rapid haem destruction mechanism in leaves. Protohaem turnover in dark-grown leaves was between 8 and 9 hr, based on the rate of degradation of erogenous haemin and the rate of protohaem breakdown in laevulinic acid (LA) treated leaves. The rate constant for haem destruction was 85 pmol/nmol/hr in the dark and 45 pmol/nmol/hr after 4 hr light. There was no evidence that light affects the synthesis of protohaem. It appears that the regulation of endogenous levels of protohaem is by breakdown and it is this mechanism which is under light control. Haem considerably decreased the incorporation of radioactivity from glycollate-[¹⁴C], glycine-[¹⁴C] and glutamate-[¹⁴C] into accumulated ALA in the presence of LA.