The localization of magnesium-protoporphyrin and protochlorophyllide in separated prolamellar bodies and prothylakoids of wheat treated with 8-hydroxyquinoline and δ-aminolevulinic acid

Dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were treated in darkness with 8-hydroxyquinoline and δ-aminolevulinic acid in order to accumulate magnesium-protoporphyrin and/or magnesium-protoporphyrin monomethylester. Prolamellar bodies and prothylakoids were separated from the treated leaves by sucrose density gradient centrifugation. About 90% of the recovered magnesium-protoporphyrin/magnesium-protoporphyrin monomethylester and about 75% of the recovered protochlorophyllide was found in the prothlakoid fraction. The significance of the distribution pattern of the chlorophyll precursors between the prolamellar bodies and the prothylakoids is discussed. The results indicate that the prothylakoids are the site for synthesis of membrane-bound chlorophyll precursors and that phototransformable protochlorophyllide is a constituent of prolamellar bodies as well as of prothylakoids.     

Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha'-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha'-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins.     

Native state,energetic interaction of chlorophyll precursors and intraplastid location of S-adenosyl-L-methionine: Mg-protoporphyrin IX methyltransferase in etiolated leaves

Low temperature fluorescence spectra (FS) and fluorescence excitation spectra (FES) of protoporphyrin IX (Proto), Mg-protoporphyrin IX and its monomethyl ester (MgProto-ME) and protochlorophyllide (Pchlide) in etiolated barley leaves treated with 5-aminolevulinic acid and/or 2,2'-dipyridyl were studied. The spectra of Proto and MgProto-ME showed a little dependence on temperature of registration and exhibited similarity to low temperature spectra in diluted organic and buffer solutions. However, a red wavelength shift for Soret bands of Proto and MgProto-ME was observed due to porphyrin interaction with bovine serum albumin in 0.05 M, Na2HPO4 solution at room temperature. Disaggregating treatments had no effect on Proto and MgProto-ME spectra in plants. These results suggested that in etiolated leaves Proto and MgProto-ME molecules were in a monomer state. The spectral properties of these molecules were determined by interaction of porphyrins with proteins and other plastid membrane components. The spectral analyses indicated an efficient energy migration from Proto and MgProto-ME molecules to active form of Pchlide which emitted at 656nm, and no energy transfer from carotenoids to porphyrins in vivo. These findings suggested that Proto and MgProto-ME from carotenoids, and close location of these porphyrins and photoactive Pchlide in etioplast membranes. The latter conclusion was strongly supported by an observation that in etiolated leaves, S-adenosyl-L-methionin:Mg-protoporphyrin IX methyltransferase, which converts MgProto into MgProtoME, were located not only in prothylakoids but also in prolamellar bodies containing photoactive Pchlide.     

The Influence of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, on Chlorophyll Synthesis in Leaves during Flash Illumination

In the presence of large accumulations of protochlorophyllide, derived from exogenous δ-aminolevulinic acid, chlorophyll synthesis in excised leaves of two varieties of barley was less than in untreated leaves. In oat leaves the accumulated protochlorophyllide, from exogenous δ-laminolevulinic acid, stimulated chlorophyll synthesis to above the control level. — These relationships could only be demonstrated when phtodestruction of pigments was minimised by the use of flash illumination (2 milliseconds every 3 minutes). — These was no evidence from in vivo absorption spectra that the pigments in the barley leaves were different to those in leaves studied by other workers. However, the presence of the accumulated protochlorophyllide appeared to prevent the shift of the chlorophyll absorption maximum from 673 nm to 677 nm. — Possible mechanisms of inhibition are discussed.     

Porphyrin Biosynthesis in Cell-free Homogenates from Higher Plants

The porphyrin and phorbin biosynthetic activity of etiolated cucumber (Cucumis sativus, L.) cotyledons was compared to that of cotyledonary homogenates. Etiolated cotyledons incubated with δ-aminolevulinic acid accumulate protoporphyrin, coproporphyrin, small amounts of Mg protoporphyrin monoester, and trace amounts of uroporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into free porphyrins, protochlorophyllide, protochlorophyllide phytyl ester, and Mg protoporphyrin monoester. Homogenates incubated with δ-aminolevulinic acid likewise accumulate coproporphyrin, uroporphyrin, Mg coproporphyrin, and trace amounts of protoporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into Mg protoporphyrin monoester, Mg coproporphyrin, and free porphyrins. However, the capacity to synthesize protochlorophyllide and protochlorophyllide phytyl ester is lost and the endogenous protochlorophylls gradually disappear. Mg protoporphyrin monoester represents the terminal biosynthetic step in this cell-free system.     

THE PORPHYRIN REQUIREMENTS OF HAEMOPHILUS INFLUENZAE AND SOME FUNCTIONS OF THE VINYL AND PROPIONIC ACID SIDE CHAINS OF HEME

1. Iron protoporphyrin IX was required for the growth of H. influenzae. It could be replaced by protoporphyrin IX. When grown on protoporphyrin evidence was obtained for the presence of Fe porphyrin in the organism. It was concluded that the organism could insert iron into the protoporphyrin ring. 2. In the smooth strains, other porphyrins containing no iron such as deutero-, hemato-, meso-, and coproporphyrins could not replace protoporphyrin for growth. Since protoporphyrin has two vinyl groups which other porphyrins lack, it was concluded that the two vinyl groups were essential for growth. 3. When porphyrins lacking vinyl groups were converted chemically into iron porphyrins and then supplied to the organisms it was found that these iron porphyrins supported growth. It was concluded that the "smooth" organisms were able to insert iron only into the porphyrin containing the vinyl groups; i.e., protoporphyrin. One function of the vinyl groups then was to permit iron to be inserted biologically into the porphyrin ring. 4. An anomalous behavior in the rough Turner strain was observed and discussed. This organism was able to insert iron into mesoporphyrin at low concentrations but was inhibited by this compound at higher concentrations. In all other reactions with the porphyrins this rough strain behaved in the same was as did the smooth strains. 5. All strains which were grown on iron porphyrins lacking vinyl groups could not reduce nitrate to nitrite. When grown on protoporphyrin or Fe protoporphyrin reduction of nitrate occurred. It was concluded that the nitrate-reducing mechanism required the presence of the vinyl groups either for its formation or function. 6. The porphyrins lacking iron and lacking vinyl groups inhibited the growth of H. influenzae on Fe protoporphyrin. The inhibition between a porphyrin and Fe protoporphyrin was a competitive one. It was suggested that the porphyrin inhibited the growth-promoting properties of Fe protoporphyrin by attaching on to a particular apoprotein, thus preventing the formation of a heme catalyst. Likewise, competition between two growth-promoting Fe porphyrins for apoenzymes could be shown to occur. 7. Protoporphyrin and Fe protoporphyrin supported growth. When their propionic acid side chains were esterified they no longer supported growth. It was suggested that the esterified carboxyl groups could not attach to the specific apoproteins to form the heme enzymes and so could not act to support growth. For the same reason the inhibitory action of porphyrins lacking vinyl groups could be prevented by esterifying their propionic acid groups.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

Reversal of alpha,alpha'-Dipyridyl-induced Porphyrin Synthesis in Etiolated and Greening Red Kidney Bean Leaves

The chemical induction of porphyrin synthesis has been investigated in etiolated and greening leaves of Phaseolus vulgaris L. var. Red Kidney. When these leaves are incubated in darkness with solutions of transition metal ion chelators such as α,α′-dipyridyl, 1,10-phenanthroline, pyridine-2-aldoxime, or other related aromatic heterocyclic nitrogenous bases, they synthesize large amounts of protochlorophyllide and Mg protoporphyrins. Greening leaves produce more porphyrin than do etiolated leaves under such conditions. If the leaves are then transferred to 1 millimolar solutions of various transition metal salts such as Fe²⁺, Zn²⁺, or Co²⁺ (but not Mn²⁺ or Mg²⁺), Mg protoporphyrin (monomethyl ester) synthesis immediately ceases and the pigment(s) rapidly disappear(s); protochlorophyllide synthesis gradually diminishes during 4 to 8 hours of treatment. The loss in Mg protoporphyrin(s) can be accounted for by a simultaneous increase in protochlorophyllide in partially greened leaves but not in etiolated leaves. In the latter, the decline in Mg protoporphyrin(s) initiated by the application of Zn²⁺ is retarded by low temperature and anaerobiosis but not by respiratory inhibitors. Cycloheximide inhibits the loss of Mg protoporphyrin(s) but does not affect their conversion to protochlorophyllide.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

Oxygen Consumption Stimulated by δ-Aminolevulinic Acid in Darkness and during Irradiation of Dark Grown Wheat Leaves

Dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull), treated with δ-aminolevulinic acid in darkness, showed an increased oxygen uptake as measured by a Warburg method. The production of CO₂ was also increased in darkness, giving an RQ ? 1. The increased respiration was dependent on the treatment time as well as on the concentration of the δ-aminolevulinic acid. Potassium cyanide suppressed both the normal and the increased respiration. The treatment with δ-aminolevulinic acid caused accumulation of high amounts of protochlorophyllide. Levulinic acid suppressed the increased oxygen uptake as well as the protochlorophyllide accumulation in δ-aminolevulinic acid treated leaves. Irradiation rapidly decreased the protochlorophyllide content with a simultaneous increase in oxygen uptake over the dark value. The peak value of the increase in oxygen uptake was reached after about 5 min. The light induced oxygen uptake was dependent on the amount of PChlide present at the onset of irradiation. Also the CO₂ production was increased during the first minutes of irradiation but soon fell under the buffer control value. Neither potassium cyanide nor heat denaturation affected the oxygen uptake in light in contrast to the effect on the CO₂ production, which was blocked by heat denaturation. The increased oxygen uptake in light initially seems to be a purely photochemical process leading to a release of CO₂, which release is probably an enzymatic process induced by the photo-oxidative decomposition of pigment.     

Chloroplast biogenesis. Biosynthesis and accumulation of protochlorophyll by isolated etioplasts and developing chloroplasts.

Etioplasts and developing chloroplasts were isolated from etiolated Cucumis cotyledons that were irradiated with white fluorescent light for various periods of time. The endogenous porphyrins and phorbins of the isolated plastids were partitioned between hexane, hexane-extracted aqueous acetone and a lipoprotein precipitate. Spectrofluorometric determinations were performed on these fractions without further fractionation. For quantitative determinations, the fluorescence amplitudes of the various fluorescent components were corrected for fluorescence emission overlap by sets of simultaneous equations. Developing chloroplasts contained endogenous amounts of the following metabolites: Protochlorophyllide, protochlorophyllide ester, Mg-protoporphyrin monoester + longer-wavelength metalloporphyrins and protoporphyrin. The protochlorophyll pool consisted mainly of protochlorophyllide. The latter was heterogeneous and consisted of at least two chemically related protochlorophyllides. In contrast to developing chloroplasts, irradiated etioplasts contained mostly protochlorophyllide ester and smaller amounts of protochlorophyllide. Upon incubation of developing chloroplasts and irradiated etioplasts with δ-aminolevulinic acid and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium and phosphate), a net synthesis and accumulation of protochlorophyllide, Mg-protoporphyrin monoester + longer-wavelength metalloporphyrins, protoporphyrin, coproporphyrin and uroporphyrin were observed. Small amounts of zinc-coproporphyrin and zinc-uroporphyrin were also formed. In some experiments a net synthesis of protochlorophyllide ester was also observed. This report represents the first account of the unambiguous net synthesis of protochlorophyll in vitro.     

Photochlorophyllide (P650) turnover in dark-grown barley leaves

Laevulinate (LA) induced an increase in protochlorophyllide (P650) in dark-grown ageing barley leaves. The increase was due to a suppression of a P650 breakdown mechanism. The LA inhibition of P650 destruction allowed an estimate to be made of turnover of P650 in ageing etiolated leaves. The rate constant for P650 destruction in 8-day-old dark-grown leaves was 139 pmol/nmol/hr with a half life of 5 hr.     

Effect of 1,10-phenanthroline on ultrastructure of pea leaves

Summary We have examined ultrastructural changes of mesophyll cells in pea leaves induced by the photodynamic herbicide 1,10-phenanthroline (Phe). Dark incubation of pea plants did not cause any damage in plants or changes in the ultrastructure of mesophyll cells. Two hours of illumination after pretreatment with Phe caused photooxidative damage in plant but was not sufficient to markedly change the ultrastructure, although dilation of endoplasmic reticulum (ER) cisternae occurred. Illumination for 12 h caused inhibition of grana formation in pretreated plants. These ultrastructural changes and the inhibition of chlorophyll (Chl) accumulation may be due to the inhibition of transport of certain proteins to the plastids, diminished accumulation of chlorophyll proteins (e.g., LHCP) and a decrease in activity of the chlorophyll synthetase.Abbreviations ALA 5-aminolevulinate - 2,2 2,2-bipyridyl - Chl chlorophyll - ER endoplasmic reticulum - 8H 8-hydroxyquinoline - LHCP light-harvesting chlorophyll a/b-binding proteins - PBs prolamellar bodies - Mg-Proto Mg-protoporphyrin - Mg-Proto-Me Mg-protoporphyrin monomethyl ester - Pchlide protochlorophyllide - Phe 1,10-phenanthroline - Proto protoporphyrin IX     

On the Relationship between Ribulose Diphosphate Carboxylase and Protochlorophyllide Holochrome of Phaseolus vulgaris Leaves

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100.Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.     

The Pool Size of Protochlorophyllide during Different Stages of Greening of Dark Grown Wheat Leaves

The pool size of protochlorophyllide in wheat leaves irradiated for 5 minutes to 6 hours was studied. Protochlorophyllide then accumulated in the dark, but the pool size of regenerated protochlorophyllide was considerably smaller in leaves irradiated for six hours than in leaves irradiated for 5 minutes. The decrease in pool size of regenerated protochlorophyllide was found to take place at the time when the chlorophyll formation had accelerated and reached the linear phase. The protochlorophyllide accumulated is the form with absorption maximum at 650 nm, which is phototransformed to chlorophyllide with maximum absorption at 684 nm. This species goes through the Shibata shift when formed even after 6 hours of irradiation. If leaves, irradiated for 1 or 6 hours, were fed with δ-amino-levulinic acid the protochlorophyllide synthesis was only 1.2 times faster in the leaves irradiated for 6 hours than in those irradiated for 1 hour. In the case of leaves fed with δ-amino-levulinic acid the absorption maximum of protochlorophyllide is at 636 nm and the absorption maximum of the chlorophyllide formed is at 672 nm.     

Purification of protochlorophyllide holochrome

Phototransformable protochlorophyllide holochrome was prepared from etiolated bean leaves. The detergent Triton X-100 in the presence of glycerol and tricine-KOH buffer (pH 8) enhanced the extractability, specific activity, and phototransformability of the holochrome. Purification was achieved by polyethylene glycol-6000 precipitation and hydroxyl-apatite, DEAE-cellulose, and agarose chromatography. The presence of Triton X-100 permitted removal of the carotenoid contamination from the holochrome. The 678-nm absorption maximum of newly formed chlorophyllide a holochrome shifts to 672 nm in a temperature-dependent manner. The purified holochrome contains 0.24 g of protein per μmole of protochlorophyllide. Estimation of the molecular weight of the holochrome by gel filtration on agarose revealed the presence of aggregates of approximately 550,000 and 300,000. There are at least 2 chromophores per 550,000 molecular weight.     

Breakdown of photosynthetic pigments and lipids in spinach leaves with ozone fumigation: Role of active oxygens

Dark-grown wheat leaves ( Triticum L. cv. Starke II Weibull) were illuminated repeatedly with light flashes giving partial phototransformation of protochlorophyllide to chlorophyllide. After short flashes (e.g. 15 ms red light, 250 W m⁻²), transforming only a minor part of the protochlorophyllide present, the first more stable chlorophyll(ide) measured ca 15 s after the phototransformation had its absorption maximum in the red around 672 nm. It stayed there during the following 30 min in darkness. After longer flashes (e.g. 125 ms), transforming a larger portion of the protochlorophyllide, the chlorophyll(ide) formed had its maximum absorption more towards 684 nm and shifted to 672 nm during a subsequent period in darkness. Thus, in this case a Shibata shift took place.
The conditions which produce the "stable" 672 nm form, without a Shibata shift, are discussed. The presence of large amounts of non-transformed protochlorophyllide remaining after the phototransformation seems to be important. Under such conditions it is possible that the Shibata shift is completed within a very short time.
Also the possible existence of two kinds of phototransformable protochlorophyllide is discussed. According to this idea one of the two protochlorophyllide forms produces a chlorophyllide absorbing at 672 nm shortly after phototransformation without having passed a Shibata shift. The other protochlorophyllide form photo-transforms to a chlorophyllide which proceeds through the Shibata shift.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.