The Effect of Phytohormones on Chlorophyll(ide), Protochlorophyll(ide) and Carotenoid Formation in Greening Dark Grown Wheat Leaves

The influence of phytohormones on chlorophyll and carotenoid formation during the greening of irradiated dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull) was studied. Leaves were floated on solutions of abscisic acid, gibberellic acid and kinetin for 24 h. The chlorophyll and carotenoid contents were determined during a subsequent period of 48 h of continuous irradiation. Leaves treated with abscisic acid showed a longer lag phase and a lower rate of accumulation of chlorophyll as compared to the control than did leaves treated with gibberellic acid and kinetin. The carotenoid content was low both in leaves treated with abscisic acid and in those treated with gibberellic acid. Treatment with abscisic acid lowered the protochlorophyllide regeneration after a saturating light flash while gibberellic acid as well as kinetin had no effect. The influence of ABA was partly dependent on an increase of the wounded part of the cut leaf segments. The accumulation of protochlorophyllide in leaves treated with δ-aminolevulinic acid was not affected by the different hormonal treatments. These results suggest that the main effect of abscisic acid is probably outside the chloroplast, i.e. on the formation or transport of δ-aminolevulinic acid.     

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.     

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.     

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.     

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.     

The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark-grown seedlings of barley ( Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5-aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5-aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5-aminolaevulinic acid.     

Correlation between chlorophyllide esterification, Shibata shift and regeneration of protochlorophyllide650 in flash-irradiated etiolated barley leaves

The pigments of etiolated leaves of barley ( Hordeum vulgare L.) were analysed during dark periods after flash illumination, and the results were compared with in vivo spectroscopy of the leaves. Pretreatment of the leaves with kinetin slightly stimulated and pretreatment with NaF and anaerobiosis inhibited the esterification of chlorophyllide a (Chlide) at 10–40 min after the flash, whereas the rapid esterification within 30 s after the flash remained unchanged. Irrespective of pretreatment, the amount of esterified pigment was, at any time, identical with the amount of pigment that had shifted its absorption from 684 to 672 nm (Shibata shift). Cycloheximide (CHI) had only a small inhibitory effect on esterification, but drastically inhibited the hydrogenation of geranylgeraniol to phytol, bound to Chlide. The regeneration of long-wavelength protochlorophyllide a (Pchlide650) was stimulated by kinetin and inhibited by CHI and NaF. During the rapid phase (0–30 s after the flash), the esterification was faster than the regeneration of Pchlide650, and this, in turn, was faster than the formation of photoactive Pchlide. The kinetics changed after pretreatment with 5-aminolaevulinic acid: regeneration of Pchlide650 was the fastest reaction and the Shibata shift preceded the esterification of Chlide. The results are discussed as pigment exchange reactions at NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99.1).     

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.     

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.     

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 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.     

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.     

δ-Aminolaevulinic acid formation in greening Avena laminae

[1,5-¹⁴C]-citrate was employed to indicate complete five carbon incorporation into the chlorophylls from the labelling pattern to be found in degraded chlorophylls and the maleimides. This provides additional evidence that there may be an alternative pathway to that involving δ-aminolaevulinic acid (δ-ALA) synthetase in higher plants.     

Methylglyoxal with glycine or succinate enhances differentiation and shoot morphogenesis in Nicotiana tabacum callus

The aim of this study was to evaluate the influence of methylglyoxal (MG) on organogenesis and regeneration of tobacco (Nicotiana tabacum L.) plants from callus in media containing glycine or succinate. The best improvement in shoot proliferation and shoot length was obtained in the medium supplemented with 0.1 mM MG and 0.5 mM glycine or 0.25 mM succinate. The histological studies showed vigorous development of corm like structures and shoot organogenesis from callus tissues cultured in MG supplemented media. Biochemical studies also revealed higher content of δ-aminolaevulinic acid (a precursor of chlorophyll) and of chlorophyll.     

In vitro characterization of the inhibitor resistance of glutamate-1-semialdehyde aminotransferase from the cyanobacterium Synechococcus PCC630l GR6

Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met²⁴⁸ → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser¹⁶³ → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met²⁴⁸ → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA.     

The Conversion of Protochlorophyllide636 to Protochlorophyllide630 in Leaves Treated with δ-Aminolevulinic Acid

Absorbancy changes in dark-grown, excised wheal leaves fed with δ-aminolevulinic acid are measured in vivo. The treatment with σ-aminolevulinic acid caused accumulation of protochlorophyllide, absorbing at 636 nm. After flashlight this form is found to convert in darkness to protochlorophyllide, absorbing at 650 nm. The conversion starts instantly after the leaves have been exposed to the flashlight, and the pre-existent pool of protocholorophyllidc absorbing at 650 nm will become emptied. The conversion is completed after 15–20 minutes, when a new pool of protochlorophyllide has been filled up. This new pool is transformed to chlorophyllide by a second flash and the sequence is repeated. The conversion may be composed of two reactions, a conclusion which can be drawn from the behaviour at different temperatures. One of these reactions is fairly temperature independent while the other is temperature dependent. The action of the protochlorophyllide holochrome is discussed.     

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 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.     

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).     

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.