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.     

The Influence of Varying Light Intensities on the Photo-transformation of Protochlorophyllide636 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

Leaves treated with δ-aminoievulinic acid accumulate protochlorophyllide₆₃₆ in large amounts. Due to a continuous conversion of protochlorophyllide₆₃₆ (nonphototransformable) into protochlorophyllide₆₅₀ (phototransformable) in weak red light, the photoreduction of protochlorophyllide to chlorophyllide can proceed for at least 20 minutes and results in a chlorophyllide content of the leaves three times higher than that in untreated leaves. The half time for this chlorophyllide accumulation is 55 seconds. A photodestruction of the pigments takes place at high light intensities or if the content of protochlorophyllide₆₃₆ is high. The conversion of protochlorophyllide₆₃₆ to chlorophyllide is dependent on the light intensity used for phototransformation of protochlorophyllide₅₅₀ The conversion of PChlide₆₄₆ was not limiting for chlorophyllide formation within the range of the light intensity used. The extrapolation of a double reciprocal plot of chlorophyllide formation, rate versus light intensity gives a maximal value of 8.7 μg chlorophyllide per g fresh weight and min. The conversion of protochlorophyllide₃₆₃ to protochlorophyllide₆₅₀ is believed to depend on the available sites of an apophotoenzyme.     

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide₆₅₀) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide₆₃₂) which is not. Dark-grown leaves placed in solutions of δ-aminolevulinic acid accumulate protochlorophyllide₆₃₂. Protochlorophyllide₆₅₀ and protochlorophyllide₆₃₂ can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide₆₅₀ in the dark, even in the presence of excess δ-aminolevulinic acid, except when a stockpile of protochlorophyllide₆₃₂ is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide₆₅₀ regeneration is accompanied by a decrease in protochlorophyllide₆₃₂. These experiments suggest that protochlorophyllide₆₃₂ may be converted to protochlorophyllide₆₅₀.     

Differential effects of gabaculin and laevulinic acid on protochlorophyllide regeneration

Abstract The effects of gabaculin (3-amino 2,3-dihydrobenzoic acid) and laevulinic acid on the regeneration of protochlorophyllide from exogenous δ-aminolaevulinic acid in leaves of dark-grown barley (Hordeum vulgare) after a brief light treatment were compared. Gabaculin, a potent inhibitor of chlorophyll biosynthesis, did not inhibit this process showing that it affects the formation of δ-aminolaevulinic acid rather than its further metabolism. Laevulinic acid, which is an inhibitor of δ-aminolaevulinic acid dehydratase, prevented regeneration of protochlorophyllide provided pools of intermediates in the biosynthetic sequence were depleted. Formation of relatively large amounts of protochlorophyllide in some experiments suggests a lack of control in the utilization of δ-aminolaevulinic acid for protochlorophyllide synthesis.     

The role of a long-wavelength pigment form in the chlorophyll biosynthesis

Photoconversion of protochlorophyllide₆₅₀ form was observed in etiolated leaves illuminated with long-wavelength—690 nm—light. This process showed Shibata shift and was found to have a strong temperature dependence between 20 and –40°C. The low rate of reaction, the strong temperature dependence and calculations on the spectral overlap integral of absorption and fluorescence bands in this spectral region indicate that the phototransformation of the 650 nm form of protochlorophyllide may be caused by a back energy migration from a long-wavelength pigment form absorbing around 690 nm; this pigment form is probably a long-wavelength form of protochlorophyll/ide.     

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[¹⁴C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[¹⁴C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.     

In Vivo Measurements of the Photo-Oxidation of Chlorophyll Pigments in Dark Grown Wheat Leaves after Treatment with δ-Aminolevulinic Acid

Dark-grown leaves of wheat fed with δ-aminolevulinic acid accumulate protochlorophyllide₆₃₆ in excess. After the leaves had been illuminated with high intensity red light (154 W × m^?2) for half a minute, a treatment which blocks the phototrans-formation protochlorophyllide chlorophyllide, the sensitivity of chlorophyllide and protochlorophyllide to light was examined. The decrease in pigment content, caused by photo-oxidation was found to be very close to a second order reaction. The second order “rate constant” for decrease in absorbance was found to be eight times greater for the formed chlorophyllide than for protochlorophyllide. The light intensity dependence of the decomposition was found to be linear within the intensity range used (E= 25 – 154 W × m^?2). In samples in which the pigments had been heat denatured, it was possible to photodecompose the chlorophyllide without affecting the protochlorophyllide. The results are discussed in connection with the theory of a photodynamic action involving oxygen in the singlet state (¹ΔO₂).     

Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus

In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as β-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed.     

Effect of light intensity on the formation of carotenoids in normal and mutant maize leaves

Carotenoid composition in leaves of normal, lycopenic and ζ-carotenic mutants of Zea mays were investigated. In lycopenic leaves, in addition to lycopene, phytoene, phytofluene, δ- and γ-carotene, trace amounts of α- and β-carotene and antheraxanthin were identified. Low light promoted accumulation of α- and β-carotene; high light brought about an increase in antheraxanthin content. In the leaves of the ζ-carotenic mutant, phytoene, phytofluene and ζ-carotene were synthesized. Illumination of low intensity stimulated carotenoid synthesis to a slight extent. Relative amounts of carotenoid components were essentially the same as in etiolated material, except for a small increase in cis-ζ-carotene. Under high intensity illumination, carotenoids were rapidly destroyed.     

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.     

Glycine metabolism and chlorophyll synthesis in barley leaves

α-Hydroxypyridine methane sulphonic acid (HPMS), isonicotinyl hydrazide (INH) and nialamide inhibit chlorophyll synthesis in etiolated barley leaves exposed to light. HPMS lowered the rate of protochlorophyllide regeneration but had little effect on the synthesis of protochlorophyll (P630) from exogenous $\text{δ}$-aminolaevulinic acid (ALA). The addition of glycine to HPMS treated leaves partially overcame the inhibition of chlorophyll synthesis. Glycine-[¹⁴C] was readily incorporated into ALA in dark-grown leaves. HPMS treatment increased the sp. act. of ALA in leaves fed glycine-[¹⁴C]. Glycollate oxidation was lower in extracts from HPMS treated leaves. Plants may therefore have two pathways for ALA production with the glutamate pathway becoming more important in conditions where photorespiration is high.     

Oak seedlings grown in different light qualities

Oak seedlings (Quercus robur L.) were germinated in darkness for 3 weeks and then given continuous long wavelength far-red light (LFR; wavelengths longer than 700 nm). A control group of seedlings was kept in darkness. After 2 additional weeks the chlorophyll formation ability in red light was examined in the different seedlings. The stability of the protochlorophyll(ide) and chlorophyll(ide) forms to high intensity red irradiation was also measured. Oak seedlings grown in darkness accumulated protochlorophyll(ide) (6 μg per g fresh matter). Absorption spectra and fluorescence spectra indicated the presence of more protochlorophyll(ide)_628–632 than protochlorophyllide_650–657. The level of protochlorophyll(ide) was higher in leaves of plants cultivated in LFR light (13 μg per g fresh matter) than in leaves of dark grown plants. 12% of the protochlorophyll(ide) was esterified in both cases. The level of protochlorophyll(ide)_628–632 in LFR grown oaks varied with the age of the leaves, being higher in the older (basal) leaves, but also in the very youngest (top-most) leaves. The ability of the leaves to form photostable chlorophyll in red light showed a similar age dependence, being low in rather young and in older leaves. A low ability to form photostable chlorophyll thus appears to be correlated with a high content of protochlorophyll(ide)_628–632. Upon irradiation only the protochlorophyllide_650–657 was transformed to chlorophyllide. After this phototransformation the chlorophyllide peak at 684 nm shifted to 671 nm within about 30 min in darkness. This shift took place without any accompanying change in photostability of the chlorophyll(ide). Upon irradiation with strong red light a similar shift took place within one minute. This indicates that the chlorophyllide after phototransformation was rather loosely bound to the photoreducing enzyme. The development towards photostable chlorophyll forms consists of three phases and is discussed.     

The After-Effect of Water Stress on Chlorophyll Formation during Greening and the Levels of Abscisic Acid and Proline in Dark Grown Wheat Seedlings

Cut seedlings of wheat plants (Triticum aestivum L. cv. Starke II Weibull) between 6 and 7 days old were water stressed in darkness by exposing them to air of 35% relative humidity 2.5 to 20 h. This treatment resulted in a water potential of -11 bars in the leaves after 20 h. The leaves were then rewatered and irradiated. The chlorophyll formation that took place in fully turgid leaves during the greening was markedly decreased in the case of the water-stress pretreatmet. and especially the lag phase was prolonged. The longer the stress pretreatment the more evident was the subsequent effect on chlorophyll formation. However, no linear relationship was found between the amount of stress and the chlorophyll content. Protochlorophyllide regeneration from endogenously formed δ-aminolevulinic acid was markedly decreased even after the shortest water-stress period. However, protochlorophyllide accumulation from exogenously supplied δ-aminolevulinic acid was only slightly decreased following the water-stress pretreatment. Further more, the ratio of protochlorophyllide₆₅₀ to protochlorophyllide₆₂₈ was slightly reduced by the same conditions. During the stress period both abscisic acid and proline were accumulated in the leaves. The content of abscisic acid increased up to six times the normal level during water stress lasting for 20 h. The increase of proline was about three-fold for similar treatment. After rewatering the leaves the levels of both abscisic acid and proline rapidly declined and reached. 10 h later, the levels found in unstressed seedlings. The increase in abscisic acid during water stress associated with impaired chlorophyll metabolism suggested that the after-effect of water stress might be linked to chlorophyll metabolism through abscisic acid or some of its metabolites. The changes in proline content open the possibility that this substance could function as a reserve substance for the formation of chlorophyll after the discon tinuation of the stress.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Chlorophyll formation and glycine metabolism in laevulinic acid treated barley leaves

Laevulinic acid (LA) inhibited chlorophyll formation and δ-aminolaevulinic acid (ALA) accumulation in dark-grown barley leaves. Mole ratios (ALA: chlorophyll × 8) indicate that LA decreased ALA production by about 30%. The turnover of glycine-[¹⁴C] in 7-day-old leaves treated with LA was 70% slower than in control tissue and this resulted in an increase in endogenous glycine. Total amino acid also increased in LA treated leaves. The data indicate that any contribution made by glycine to ALA synthesis in LA-treated barley leaves would be significantly restricted.     

Effects of Irradiance and Copper on the Activity of Ascorbate Oxidase in Detached Rice Leaves

The effects of copper on the activity of ascorbic acid oxidasc (AAO) in detached rice leaves under both light and dark conditions and in etiolated rice seedlings were investigated. CuSO₄ increased AAO activity in detached rice leaves in both light and darkness, however, the induction in darkness was higher than in the light. In the absence of CuSO₄, irradiance (40 μmol m^-2 s^-1) resulted in a higher activity of AAO in detached rice leaves than dark treatment. Both CuSO₄ and CuCl₂ increased AAO activity in detached rice leaves, indicating that AAO is activated by Cu. Sulfate salts of Mg, Mn, Zn and Fe were ineffective in activating AAO in detached leaves. CuSO₄ was also observed to increase AAO activity in the roots but not in shoots of etiolated rice seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Glycine metabolism in etiolated barley leaves on exposure to light

Of a large number of amino acids examined, changes in glycine were the only ones which were correlated with the ability of dark-grown barley leaves to synthesise protochlorophyllide, δ-aminolaevulinic acid and chlorophyll on exposure to light. A rapid depletion was found in endogenous glycine in barley leaves after day 7. Illumination of the leaves increased the rate of glycine depletion. Glycine concentrations were high throughout the young leaf. The top and middle leaf sections however, which had maximal chlorophyll synthesising potential exhibited the most pronounced decrease in glycine as the leaf aged. Using glycine-[¹⁴C] pulse techniques the half life of glycine in 7 and 14-day-old dark-grown leaves was 3.5 and 4.4 min respectively. Light treatment lengthened the half life to 6.9 and 12.1 min in 7 day and 14-day-old-leaves. Sustained illumination continued to decrease glycine turnover.     

The turnover of chlorophyll in greening wheat leaves

A method for the estimation of chlorophyll turnover in wheat leaves is presented. This is based on the inhibition of chlorophyll synthesis by treatment of the cut leaves with laevulinic acid (LA), a competitive inhibitor of δ-aminolaevulinic acid dehydratase. The turnover of chlorophyll in young, greening leaves, given short periods of light was a relatively rapid process. However, in seedlings exposed to light for longer periods the turnover became progressively slower, and was measured in days rather than hours.