Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and other metalloporphyrins by isolated etioplasts and developing chloroplasts

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, δ-aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in vitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H₂O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and δ-aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own δ-aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in vitro. The metalloporphyrins synthesized in vitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.     

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.     

Steady state kinetic mechanism of the Escherichia coli coenzyme A transferase.

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins when incubated in the dark in the presence of protoporphyrin and cofactors. These results constituted the first unambiguous demonstration of the insertion of magnesium into exogenous protoporphyrin in a cell-free system from higher plants. The metalloporphyrin synthetic activity did not occur in the absence of the plastids or when the plastids were heated in a 100 °C water bath for 2 min. It is thus suggested that, in higher plants, the in vitro insertion of magnesium into protoporphyrin is an enzymatic reaction.     

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.     

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins by greening cotyledons

Etiolated excised cucumber cotyledons (Cucumis sativus L. cv. Alpha Green), while greening in distilled water, synthesized and accumulated several metalloporphyrins in the absence of added substrates or inhibitors. The metalloporphyrins, undetectable by conventional spectrophotometry, exhibited distinct fluorescence emission and excitation properties in situ and in organic solvents. The metalloporphyrins were partially segregated on thin layers of silica gel H into three Chromatographic bands and the bands were eluted in methyl alcohol:acetone (4:1 $\text{v}\text{v}$). The metalloporphyrins in the eluted bands were characterized by their soret excitation and short-wavelength emission maxima. One of the metalloporphyrins of band 3 (R_f, 0.4?0.56) was identified as Mg-protoporphyrin monoester. It was accompanied by traces of two other metalloporphyrins. Band 2 (R_f, 0.32?0.48) was made up of three metalloporphyrins and had the Chromatographic mobility of endogenous protochlorophyllide. Band 1 (R_f, 0.22?0.43) was made up of two metalloporphyrins; it moved with endogenous chlorophyllide. The metalloporphyrins of band 2 and 1 exhibited fluorescence emission and excitation maxima similar to Mg-protoporphyrin monoester but slightly shifted to longer wavelengths. The Chromatographic and spectral properties of these compounds suggested that they represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The analytical techniques described in this work may prove useful in the elucidation of the enzymology between protoporphyrin IX and protochlorophyllide.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

Intraplastidic Localization of the Enzymes That Convert delta-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

The intraplastidic localization of the enzymes that catalyze the conversion of δ-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.     

Acclimation of chlorophyll biosynthetic reactions to temperature stress in cucumber (Cucumis sativus L.)

The adaptive responses of the greening process of plants to temperature stress were studied in cucumber (Cucumis sativus L. cv. Poinsette) seedlings grown at ambient (25 °C), low (7 °C) and high (42 °C) temperatures. Plastids isolated from these seedlings were incubated at different temperatures and the net syntheses of various tetrapyrroles were monitored. In plastids isolated from control seedlings grown at 25 °C, the optimum temperature for synthesis of Mg-protoporphyrin IX monoester or protochlorophyllide was 35 °C. Temperature maxima for Mg-protoporphyrin IX monoester and protochlorophyllide syntheses were shifted to 30 °C in chill-stressed seedlings. The net synthesis of total tetrapyrroles was severely reduced in heat-stressed seedlings and the optimum temperature for Mg-protoporphyrin IX monoester or protochlorophyllide synthesis shifted slightly towards higher temperatures, i.e. a broader peak was observed. To further study the temperature acclimation of seedlings with respect to the greening process, tetrapyrrole biosynthesis was monitored at 25 °C after pre-heating the plastids (28–70 °C) isolated from control, chill- and heat-stressed seedlings. In comparison to 28 °C-pre-heated plastids the percent inhibition of protochlorophyllide synthesis in 40 °C-pre-heated plastids was higher than for the control (25 °C-grown) in chill-stressed seedlings and lower than for the control in heat-stressed seedlings. Maximum synthesis of total tetrapyrroles and protoporphyrin IX was observed when chloroplasts were heated at 50 °C, which was probably due to heat-induced activation of the enzymes involved in protoporphyrin IX synthesis. Prominent shoulders towards lower or higher temperatures were seen in chill-stressed or heat-stressed seedlings, respectively. The shift in optimum temperature for tetrapyrrole biosynthesis in chill- and heat-stressed seedlings was probably due to acclimation of membranes possibly undergoing desaturation or saturation of membrane lipids. Proteins synthesized in response to temperature-stress may also play an important role in conferring stress-tolerance in plants. Received: 8 October 1998 / Accepted: 19 November 1998     

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.     

Differential distribution of chlorophyll biosynthetic intermediates in stroma, envelope and thylakoid membranes in Beta vulgaris

Stroma, envelope and thylakoid membranes were prepared from chloroplasts isolated from leaves of Beta vulgaris. Out of total plastidic protochlorophyllide, envelope membranes contained 1.5%, thylakoids had the maximum 98.48% and stroma had a trace fraction of 0.02%. Distribution of the Mg-protoporphyrin IX and its monoester was 89.0% in thylakoids, 10.0% in stroma and 1.0% in envelope. A substantial fraction (33.77%) of plastidic protoporphyrin IX was partitioned into stroma. Envelope contained 0.66% and thylakoids had 65.57% of the total plastidic protoporphyrin IX pool. The proportion of monovinyl and divinyl forms of protochlorophyllide was almost similar in intact plastid, thylakoids, and outer and inner envelope membranes suggesting a tight regulation of vinyl reductase enzyme. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma is discussed. This work was supported by a grant from the Council of Scientific and Industrial Research (38/1079/03/EMRII) to BCT.     

Chloroplast Biogenesis 65 : Enzymic Conversion of Protoporphyrin IX to Mg-Protoporphyrin IX in a Subplastidic Membrane Fraction of Cucumber Etiochloroplasts

The preparation from Percoll-purified cucumber (Cucumis sativus)etiochloroplasts of a subplastidic membrane fraction that is capable of high rates of Mg insertion into protoporphyrin IX is described. The plastid stroma was inactive when used either alone or in combination with the membrane fraction. Successful preparation of the subplastidic membrane fraction required that Mg-protoporphyrin chelatase was first stabilized by its substrate. This was achieved by lysing Percoll-purified plastids in a fortified hypotonic medium containing protoporphyrin IX prior to ultracentrifugation and separation of the stroma from the plastid membranes. Protoporphyrin IX became membrane bound. Other additives needed for enzyme activity fell into two groups: (a) those needed for enzyme stabilization during membrane preparation and (b) those involved in the primary mechanism of Mg insertion into protoporphyrin IX. Ethylenediaminetetraacetate belonged to the first group, magnesium belonged to the second group, and ATP belonged to both groups.     

Chloroplast culture VIII a new effect of kinetin in enhancing the synthesis and accumulation of protochlorophyllide invitro

By pretreating etiolated cucumber cotyledons with kinetin in the dark, it was observed that the plastids isolated from such tissues were 400% more active in the conversion of δ-aminolevulinic acid into protochlorophyllide, than plastids prepared from water-treated controls. The experimental evidence is consistent with the hypothesis that (a) the kinetin dark-pretreatment of the etiolated tissue, uncouples the joint biosynthesis of prothylakoids and protochlorophyll and results in the accumulation of excess prothylakoid membranes poorly supplied with protochlorophyllide (b) upon isolation of the plastids and incubation with δ-aminolevulinic acid, the latter is very rapidly converted into membrane-bound protochlorophyllide.     

Chloroplast biogenesis. Demonstration of the monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants

It is shown that barley (Hordeum vulgare), a dark monovinyl/light divinyl plant species, and cucumber (Cucumis sativus L.) a dark divinyl/light divinyl plant species synthesize monovinyl and divinyl protochlorophyllide in darkness from monovinyl and divinyl protoporphyrin IX via two distinct monovinyl and divinyl monocarboxylic chlorophyll biosynthetic routes. Evidence for the operation of monovinyl monocarboxylic biosynthetic routes consisted (a) in demonstrating the conversion of delta-aminolevulinic acid to monovinyl protoporphyrin and to monovinyl Mg-protoporphyrins, and (b) in demonstrating the conversion of these tetrapyrroles to monovinyl protochlorophyllide by both isolated barley and cucumber etiochloroplasts. Likewise, evidence for the operation of divinyl monocarboxylic chlorophyll biosynthetic routes consisted (a) in demonstrating the biosynthesis of divinyl protoporphyrin and divinyl Mg-protoporphyrins from delta-aminolevulinic acid, and (b) in demonstrating the conversion of the latter tetrapyrroles to divinyl protochlorophyllide. Finally, it was shown that the divinyl tetrapyrrole substrates were metabolized differently by barley and cucumber. For example, divinyl protoporphyrin, divinyl Mg-protoporphyrin, and divinyl Mg-protoporphyrin monoester were converted predominantly to monovinyl protochlorophyllide and to smaller amounts of divinyl protochlorophyllide by barley etiochloroplasts. In contrast, cucumber etiochloroplasts converted the above substrates predominantly to divinyl protochlorophyllide, although smaller amounts of monovinyl protochlorophyllide were also formed. Furthermore, it was shown that monovinyl protochlorophyllide was not formed from divinyl protochlorophyllide either in barley or in cucumber etiochloroplasts. These metabolic differences are explained by the presence of strong biosynthetic interconnections between the divinyl and monovinyl monocarboxylic routes, prior to divinyl protochlorophyllide formation, in barley but not in cucumber.     

Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide.

A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fluorescence emission spectra is developed. At room temperature (293 degrees K) protoporphyrin IX is monitored from its emission maximum, 633 nm, when excited at 400 nm (E400/F633). Protochlorophyllide is estimated at 638 nm, while being excited at 440 nm (E440/F638). Mg-protoporphyrin+Mg-protoporphyrin monoester pool has emission around 589-592 nm. Therefore the integration value of the emission band that extends from 580 to 610 nm is taken to calibrate its concentration. This spectrofluorometric method designed for the determination of protoporphyrin IX, esterified and nonesterified Mg-protoporphyrin pool, and protochlorophyllide is far superior to available spectrophotometric methods and estimates as low as 1 nM concentration of plant pigments. As minute quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates analytical uncertainties due to recovery losses caused by chromatography. However, only dilute samples can be estimated by this spectrofluorometric method as the quantitative relation between fluorescence and concentration deviates from linearity at high, i.e., above 150 nM, concentrations of pigment to be quantified.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS

Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with δ-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.     

Chloroplast Biogenesis 31: DETECTION OF AN INHIBITOR OF PROTOCHLOROPHYLL BIOSYNTHESIS IN CUCUMBER COTYLEDONS

An extract from cucumber cotyledons was shown to cause an inhibition of protochlorophyll biosynthesis and accumulation. The extract inhibited the net synthesis of protochlorophyll as well as the incorporation of δ-amino[¹⁴C]levulinic acid into protochlorophyllide and protochlorophyllide ester by excised cotyledons. The inhibition of δ-amino[¹⁴C]levulinic acid incorporation into the two protochlorophyll species was also observed in isolated etiochloroplasts before and after lysis of the plastids. The inhibition did not appear to involve the oxidation of the δ-aminolevulinic substrate or its translocation across the plastid membrane. Kinetic analysis of the rate of protochlorophyllide and protochlorophyllide ester biosynthesis in the presence and absence of the inhibitor suggested that the mode of inhibition of the two protochlorophyll species was different.     

Mechanisms of regulation and interplastid localization of chlorophyll biosynthesis.

The current concepts of chlorophyll biosynthesis, its interplastid localization, biosynthetic and biochemical heterogeneity, mechanisms of regulation of the key reactions, formation of 5-aminolevulinic acid and incorporation of magnesium into protoporphyrin IX, are reviewed. The literature and author's data demonstrate the existence of in vivo multienzyme systems synthesizing chlorophyll and its precursors as monovinyl and divinyl chemical species. Both types of the multienzyme systems synthesize 5-aminolevulinic acid and regulate this process independently. A hypothesis is considered that the function of the magnesium branch of chlorophyll biosynthesis in vivo is controlled by a mechanism through inhibition of the enzymes by their products because of the limitation of the binding sites for them in the membrane. An additional influence of light on the Mg-chelatase activity not only via the photosynthetic supply with ATP but also through the light-induced synthesis of the enzyme molecules de novo is described. Efficient energy migration from protoporphyrin IX and Mg-protoporphyrin IX (monomethyl ester) molecules to the protochlorophyllide active form detected by the author is discussed considering a close location of these pigments in plastid membranes and the enzymes participating in their formation.