Effects of calcium on chlorophyll synthesis and stability in the early phase of greening in cucumber cotyledons

The effects of calcium on chlorophyll accumulation and its stability in the early phase of greening in cucumber cotyledons were investigated. Chlorophyll accumulation was hardly affected by dark preincubation of cotyledons with 10 millimolar calcium solution, but was inhibited almost completely when 50 or 100 millimolar solution was used. On the other hand, 50 millimolar calcium inhibited δ-aminolevulinic acid formation in the light by only 75%. Calcium had little effect on the lag for initiation of protochlorophyllide₆₅₀ regeneration, but slowed down the rate of accumulation of protochlorophyllide₆₅₀. In calcium-treated cotyledons, the chlorophyll formed by primary photoconversion was quickly decomposed in the dark. The present results show that calcium inhibited chlorophyll accumulation by inhibiting δ-aminolevulinic acid formation in the light and by stimulating the decomposition of newly formed chlorophyll, both effects being completely prevented by potassium.     

The Conversion of Photoinactive Protochlorophyllide(633) to Phototransformable Protochlorophyllide(650) in Etiolated Bean Leaves Treated with delta-Aminolevulinic Acid

The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering δ-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide₆₃₃ causes a marked increase in the extent of regeneration of protochlorphyllide₆₅₀ following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide₆₅₀ regeneration in control leaves was found to be three times longer than the half-time for conversion of chlorophyllide₆₇₈ to chlorophyllide₆₈₃ at 22 C. The results are consistent with the hypothesis that protochlorophyllide₆₃₃ is a direct precursor of protochlorophyllide₆₅₀ and that the protein moiety of the protochlorophyllide holochrome acts as a “photoenzyme” in the conversion of protochlorophylide to chlorophyllide.     

Rapid regeneration of protochlorophyllide(650)

The rate of regeneration of protochlorophyllide₆₅₀ was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding δ-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides₆₃₅ but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous δ-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0°, suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides₆₅₀ holochrome.     

The Relationship between Chlorophyllide Accumulation, the Amount of Protochlorophyllide636 and Protochlorophyllide650 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

The influence of different amounts of protochlorophyl-lide₆₃₆ and protochlorophyllide₃₅₀ on light Induced chloro-phyllide formation was. studied in wheat leaves treated with δ-aminolevulinic acid. The phototransformation of proto-chlorophyllide was performed with weak red light. This transformation is unaffected by the δ-aminolevulinic acid treatment, whilst the accumulation of chlorophyllide, both the rate and the amount, is greatly stimulated by moderate amounts of protochlorophyllide₆₃₆. The presence of large amounts of protochlorophyllide₆₃₆ decreases the rate of chlorophyllide formation, but increases the final amount of chloro-phyllide formed. A decreased level of protochlorophyllide₆₅₀, obtained by treatment with N_aN₃, results in a decreased transformation rate. Inhibitors; of protein synthesis do not seem to influence the transformation of protochlorophyllide₆₃₆ to chlorophyllide, suggesting that no new synthesis of protein is required. The experimental results indicate that the final steps in chlorophyll biosynthesis are protochlorpnyllide₆₃₆→ protochlorophyllide₆₅₀→ chlorophyllide → chlorophyll.     

Controls on chlorophyll synthesis in barley

In 7- to 10-day-old leaves of etiolated barley (Hordeum vulgare), all of the enzymes that convert δ-aminolevulinic acid to chlorophyll are nonlimiting during the first 6 to 12 hours of illumination, even in the presence of inhibitors of protein synthesis. The limiting activity for chlorophyll synthesis appears to be a protein (or proteins) related to the synthesis of δ-aminolevulinic acid, presumably δ-aminolevulinic acid synthetase. Protein synthesis in both the cytosol and plastids may be required to produce nonlimiting amounts of δ-aminolevulinic acid. The half-life of a limiting protein controlling the synthesis of δ-aminolevulinic acid appears to be about 1½ hours, when determined with inhibitors of protein synthesis. Acceleration of chlorophyll synthesis by light is not inhibited by inhibitors of nucleic acid synthesis, but is inhibited by inhibitors of protein synthesis. A model for control of chlorophyll synthesis is proposed, based on a light-induced activation at the translational level of the synthesis of proteins forming δ-aminolevulinic acid, as well as the short half-life of these proteins. Evidence is presented confirming the idea that the holochrome on which protochlorophyllide is photoreduced to chlorophyllide functions enzymatically.     

Studies on the regeneration of protochlorophyllide after brief illumination of etiolated bean leaves

The effects of various inhibitors of nucleic acid and protein synthesis on protochlorophyllide synthesis in dark-grown Phaseolus vulgaris var. Red Kidney have been studied. Actinomycin D, chloramphenicol, and puromycin inhibit the regeneration of protochlorophyllide holochrome (detected as a 650 mμ absorption peak) in vivo in darkness after photoconversion of endogenous protochlorophyllide a to chlorophyllide a; this inhibition does not occur in similarly treated leaves supplied with δ-aminolevulinic acid.

These data suggest that the regeneration of protochlorophyllide results from the synthesis of RNA and enzymes required for the production of δ-aminolevulinate.

     

The effect of kinetin on chlorophyll synthesis in ageing etiolated barley leaves exposed to light

Etiolated barley seedlings lose the ability to produce chlorophyll and soluble protein on exposure to light with increasing age. Similarly, the production of δ-aminolaevulinic acid-dehydratase and succinyl-CoA synthetase is decreased in older etiolated leaves exposed to light. The rate of protochlorophyllide₆₅₂ regeneration decreased well before the rates of exogenous δ-aminolaevulinic acid conversion to protochlorophyllide₆₃₂ was affected by ageing. Application of kinetin retarded these ageing symptoms in the etiolated leaves.     

Development of chlorophyll and hill activity

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding δ-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with δ-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.     

Studies of Delta-Aminolevulinic Acid Dehydrase from Skeletonema costatum, a Marine Plankton Diatom

The accumulation of δ-aminolevulinic acid and activities of δ-aminolevulinic acid dehydrase were examined in the marine diatom, Skeletonema costatum, grown in the presence of levulinic acid. Levulinic acid concentrations greater than 10 mm affect growth and morphology, and inhibit chlorophyll synthesis. The algae recover from the effects of levulinic acid after 48 hours of exposure. The recovery is characterized by increased cellular cholorphyll content, decreased δ-aminolevulinic acid accumulation, decreased 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-enhanced in vivo fluorescence, and the induction of a levulinic acid-activated δ-aminolevulinic acid dehydrase which does not follow Michaelis-Menten kinetics. The data indicate that levulinic acid blocks may be ineffective in vivo, and that δ-aminolevulinic acid is metabolized to amino and dicarboxylic acids. δ-Aminolevulinic acid dehydrase activities are used to estimate the capacity for chlorophyll synthesis. Results suggest this diatom may be capable of rapid chlorophyll turnover, which would allow the plant to light-shade adapt on the time scales appropriate to vertical mixing rates in the sea.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues

δ-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. δ-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of δ-aminolevulinic acid dehydrase. The rate of δ-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of δ-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, δ-aminolevulinic acid accumulation ceased.     

Studies on the Biosynthesis and Metabolism of delta-Aminolevulinic Acid in Chlorella

The regulation of chlorophyll synthesis in Chlorella was examined at the level of the formation and metabolism of δ-aminolevulinic acid. δ-Aminolevulinic acid synthetase activity could not be detected in broken cell preparations, and exogenously supplied δ-aminolevulinic acid was taken up only in the presence of dimethylsulfoxide, with a corresponding production of porphobilinogen.     

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.     

Control of chlorophyll production in rapidly greening bean leaves

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.

Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.

Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of δ-amino-levulinic acid partially relieves the inhibition by chloramphenicol.

It is suggested that light regulates chlorophyll synthesis by controlling the availability of δ-aminolevulinic acid, possibly by mediating the formation of an enzyme of δ-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

     

Dependence of Betaine Stimulation of Vitamin B12 Overproduction on Protein Synthesis

The betaine-stimulated differential synthesis of vitamin B₁₂, i.e., the increase in B₁₂ per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, δ-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B₁₂ synthesis is a result of its stimulation of synthesis of δ-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B₁₂ and δ-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

(—) S-adenosyl-L-methionine-magnesium Protoporphyrin Methyltransferase, an Enzyme in the Biosynthetic Pathway of Chlorophyll in Zea mays

The enzyme (—) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (—) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays.

Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin.

The following scheme of chlorophyll synthesis in higher plants is proposed: δ-aminolevulinic acid → → → protoporphyrin → magnesium protoporphyrin → magnesium protoporphyrin monomethyl ester → → → chlorophyll a.

     

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris

An enzyme catalyzing the formation of δ-aminolevulinic acid by transamination of γ,δ-dioxovaleric acid with l-α-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in δ-aminolevulinic acid metabolism in plants is not clearly understood.     

A Mechanism of Chlorosis Caused by 1,3-Dimethyl-4-(2,4-dichlorobenzoyl)-5-hydroxypyrazole, a Herbicidal Compound

In organic solvents, 1,3-dimethyl-4-(2,4-dichlorobenzoyl)-5-hydroxypyrazole (DTP) converted chlorophyll a and b extracted from rice seedlings (Oryza sativa L. `Kinmaze') into pheophytin a and b, respectively. On comparing the chlorophyll-converting activity of DTP to those of acetic, glycolic, 2,4-dichlorobenzoic, monochloroacetic, 2,6-dichlorobenzoic, pyruvic, and dichloroacetic acids, it was demonstrated that DTP induced H<sup>+</sup> into chlorophyll specifically. 5-Hydroxypyrazoles, which seem to be dissociable, converted chlorophyll into pheophytin in vitro. These compounds also induced chlorosis in sedge seedlings (Cyperus serotinus Rottb.), when the seedlings were grown in media containing these compounds. However, 5-hydroxypyrazoles, which seem to be undissociable, and analogs having no hydroxy group caused neither the chlorophyll conversion in vitro nor chlorosis in the seedlings. Chlorosis in barnyardgrass seedlings (Echinochloa crus-galli Beauv. var. oryzicola Ohwi) induced by DTP was reversed by cultivating the seedlings in media containing DTP plus NaOH, KOH, NH<sub>4</sub>OH, Ca(OH)<sub>2</sub>, sodium acetate, sodium pyruvate, sodium succinate, or sodium fumarate. Accumulation of the vinyl pheoporphyrin fraction in 4- day-old etiolated radish cotyledons (Raphanus sativus L. `Minowase 2') was enhanced by incubating the cotyledons with δ-aminolevulinic acid in the dark. However, simultaneous treatment with δ-aminolevulinic acid and DTP reduced accumulation of the fraction and promoted formation of the uro, copro, and protoporphyrin fractions. These results suggest that DTP blocks the synthesis of protochlorophyllide in intact plants and induces consequent chlorosis, and the H⁺ -donating activity of DTP might cause the reduction of protochlorophyllide biosynthesis.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues

δ-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various ¹⁴C-labeled precursors and levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase. The radioactivity in the accumulated δ-aminolevulinic acid was measured.     

Levels of (+/-) Abscisic Acid and Xanthoxin in Spinach under Different Environmental Conditions

Kinetin-induced changes in dry weight, soluble protein content, δ-aminolevulinic acid dehydratase activity, and chlorophyll content of two clones of Nicotiana tabacum L. callus were studied. Kinetin brought about a marked increase in the δ-aminolevulinic acid dehydratase activity of nongreen tissue just before induction of greening. The experimental data suggested a possible induction of specific chloroplast protein(s) during the kinetin-induced greening of nongreen tobacco tissue. Kinetin caused a decline in the δ-aminolevulinic acid dehydratase activity and chlorophyll content of the green callus used in the present study.     

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.