The photoenzymatic cycle of NADPH: protochlorophyllide oxidoreductase in primary bean leaves (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) during the first days of photoperiodic growth

The photoenzymatic cycle of the light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) was investigated in situ during early stages of development of bean leaves under light-dark cycles (LDC). In the experimental system used in this study, prolamellar bodies developed during night periods and disappeared during light periods. This was accompanied by changes in the photoactive to non-photoactive Pchlide ratio, which was higher at the end of the light period, and tended to increase with the number of LDC's. Flash-induced absorbance changes in the Chlide absorption region (700 nm) were used in order to monitor the formation of short- and long-wavelength forms of Chlide (C670-675 and C682-694), which correspond to free Chlide and aggregated Chlide-NADPH-LPOR complexes, respectively. The ratio of long-wavelength to short-wavelength Chlides after one flash increased with the number of LDC's, and was higher in leaves collected at the end of light periods, compared to leaves collected at the end of night periods. During light periods, photoactive Pchlide regeneration and Chlide phytylation were completed within 1 min after flash-induced formation of long-wavelength Chlide. The results show for the first time that the photoenzymatic LPOR cycle proceeds through similar steps, but at much faster rates, during photoperiodic greening than in the previously studied leaves of etiolated plants. In particular, the parallel formation of two Chlide species always occurs, but the ratio of the two species depends on the ratio of photoactive to non-photoactive Pchlide and on light or dark adaptation.     

Light-regulated pigment interconversion in pheophytin/chlorophyll-containing complexes formed during plant leaves greening

Upon illumination of etiolated maize leaves the photoconversion of protochlorophyllide Pchlide 655/650 into chlorophyllide Chlide 684/676 was observed. It was shown that chlorophyllide Chlide 684/676 in the dark is transformed into pheophytin Pheo 679/675 and chlorophyll Chl 671/668 by means of two parallel reactions, occurring at room temperature: Chlide 684/676. The formed pheophytin Pheo 679/675 was unstable and in the dark was transformed into chlorophyll Chl 671/668 in a few seconds: Pheo 679/675 Chl 671/668. The last reaction is reversed by the light: Chl/668 Pheo 679/675. Thus, on the whole in the greening etiolated leaves this process occurs according to the following scheme:The observed light-regulated interconversion of Mg-containing and Mg-free chlorophyll analogs is activated by ATP and inhibited by AMP.Abbreviations Chl- chlorophyll - Chlide- chlorophyllide - Pchlide- protochlorophyllide - Pheo- pheophytin - PS II RC- Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to the maximum position of the low-temperature fluorescence band (nm), the second number to the maximum position of the longwave absorption band     

The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.     

Evidence of chlorophyll synthesis pathway alteration in desiccated barley leaves

In etiolated leaves, saturating flash of 200 ms induces phototransformation of protochlorophyllide (Pchlide) F655 into chlorophyllide (Chlide), then into Chl through reactions which do not need light sensibilisation. The synthesis of Chl is known to be slowed down in etiolated leaves exposed to desiccation stress. In order to analyse the intensity and time-course of Chlide transformation into Chl, we used the fluorescence emission of etiolated leaves previously exposed to a 200 ms saturating flash. We used low-temperature fluorescence spectroscopy to reveal the inhibition site of Chl synthesis in etiolated barley leaves exposed to water stress. Shibata shift appears as the main target point of the water deficit. It was found that water deficit inhibits partially active Pchlide F655 regeneration. Also, esterification of Chlide into Chl is impaired. It appears that these inhibitory effects alter the appearance of PSII active reaction centres.     

Spectroscopic analysis of desiccation-induced alterations of the chlorophyllide transformation pathway in etiolated barley leaves

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A(668)F(676). The formation of Chl(ide) A(668)F(676), so-called "free Chlide," was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis.     

Protochlorophyllide-NADP+ and protochlorophyllide- NADPH complexes and their regeneration after flash illumination in leaves and etioplast membranes of dark-grown wheat

The fast (1 min) regeneration process of the photoactive Pchlide forms after a light flash was studied in etiolated wheat leaves, and this process was simulated in vitro by incubating etioplast inner membranes of wheat with excess NADPH or NADP+. The 77 K fluorescence spectra were recorded after flash illumination, dark incubation and a subsequent flash illumination of the samples. A non-photoactive Pchlide form with an emission maximum at 650 nm was transiently detected in leaves during regeneration of a photoactive Pchlide form with an emission maximum at 654 nm. Gaussian deconvolution of fluorescence spectra of isolated membranes showed that this 650 nm form appeared in conditions of excess NADP+, as suggested in previous studies. Additionally a Pchlide form emitting at 638.5 nm was detected in the same conditions. The analysis of the spectra of leaves at different times after a flash indicated that these two non-photoactive forms are involved as intermediates in the regeneration of photoactive Pchlide. This regeneration is in correlation with the production of the Chlide form emitting at 676 nm. The results demonstrate that, in vivo, part of the NADPH:protochlorophyllide oxidoreductase is reloading with nonphotoactive Pchlide on a fast time-scale and that the 676 nm Chlide form is the released product of the phototransformation in this process.     

High salt stress in wheat leaves causes retardation of chlorophyll accumulation due to a limited rate of protochlorophyllide formation

When exposed to salt stress, leaves from dark-grown wheat seedlings ( Triticum aestivum , cv. Giza 168) showed reduced accumulation of chlorophyll during irradiation. To elucidate the mechanism behind salt-influenced reduction of chlorophyll biosynthesis, we have investigated the effect of salt stress on the spectral forms of Pchlide, the phototransformation of Pchlide to Chlide, the Shibata shift, the regeneration of Pchlide and the accumulation of Pchlide from 5-aminolevulinic acid (ALA). We found that the phototransformation of Pchlide to Chlide was not affected by salt stress. The blue shift (Shibata shift) of newly formed Chlide was delayed both after flash irradiation and in continuous light. The reformation of Pchlide in darkness after a flash irradiation or after a period of 3-h irradiation was retarded in the salt-treated leaves. However, after a 20-h dark period, Pchlide was reformed even in salt-treated leaves but the formation of short-wavelength Pchlide was suppressed. Compared to controls, salt treatment also reduced the amount of Pchlide accumulated in leaves floated on ALA. The increase in the low temperature fluorescence emission spectrum at 735 nm, which occurred gradually during several hours of irradiation with continuous light in control leaves, was completely suppressed in salt-treated leaves. It is concluded that salt stress inhibits chlorophyll accumulation partly by reducing the rate of porphyrin formation but, as discussed, also by a possible reduction in the formation of chlorophyll-binding proteins.     

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).     

Pigment-protein complexes of illuminated etiolated leaves

Photoconversion of protochlorophyllide in etiolated leaves of Avena sativa L., var. Pennal or Peniarth and Phaseolus vulgare L., var. `The Prince' results in the sequential appearance of spectrally distinct chlorophyllide complexes (Chlide 678, 684, and 672). This paper reports on the generation of similar forms in vitro, under controlled conditions, using well characterized etioplast membranes enriched in the enzyme protochlorophyllide reductase. Excess NADP<sup>+</sup> and NADPH stabilize complexes related to Chlide 678 and Chlide 684, respectively, whereas addition of exogenous Pchlide induces formation of a species related to Chlide 672. Evidence is provided to support the suggestion that Chlide 678 and Chlide 684 represent ternary complexes of the enzyme protochlorophyllide reductase, with Chlide and either NADP<sup>+</sup> (Chlide 678) or NADPH (Chlide 684). Chlide 672 is seen as `free' pigment dissociated from the enzyme. The role of Pchlide in this dissociation, observed spectroscopically as the `Shibata shift,' is discussed.     

Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves

Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness

During illumination of dark-grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark-grown plants, maize ( Zea mays cv. Sundance), wheat ( Triticum aestivum cv. Kosack), pea ( Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato ( Solanum lycopersicum ), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re-accumulated. The proportions of different Pchlide forms within the pool of re-accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark-grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long-wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash-induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark-grown leaves and were specific for the plant variety. Thus Chl formation in both dark-grown and light-grown leaves can occur in a similar way through the main photoactive long-wavelength form of Pchlide.     

Interconversions of Chlorophylls a and b Synthesized from Exogenous Chlorophyllides a and b in Etiolated and Post-Etiolated Rye Seedlings

A comparative study of reciprocal conversions of chlorophylls a and b (Chl aand Chl b) in etiolated and post-etiolated rye seedlings (Secale cereale L.) was performed. The production of these pigments was initiated by infiltration of exogenous chlorophyllides a and b (Chlide a and b). It was shown that Chlide b, when infiltrated into etiolated rye seedlings, was esterified, producing Chl b. A major portion of Chl b (more than 80%) was transformed into Chl aduring long-term seedling dark exposure. The high rate of Chl b conversion into Chl a in the pool of pigments of exogenous origin was also observed during the lag-phase when there was no chlorophyll formation from endogenous precursors. The infiltration of Chlide a resulted in Chl a formation. The efficiency of its conversion into Chl b was low (about 1%) in the etiolated seedlings but increased during their greening. In the post-etiolated seedlings infiltrated with Chlide b, which were preliminary illuminated for 6–12 h, the Chl /Chl a ratio was almost similar in the pools of pigments synthesized from both exogenous and endogenous precursors. The rates of direct and reverse reactions responsible for the interconversion of Chl aand Chl b depended on the stage of the formation of the photosynthetic apparatus during greening of etiolated seedlings, when the particular structural components are formed in a definite sequence.     

A new pathway of chlorophyll biosynthesis from long-wavelength protochlorophyllide Pchlide 686/676 in juvenile etiolated plants

By spectral methods, the final stages of chlorophyll formation from protochlorophyllide were studied using etiolated pea, bean, barley, wheat and maize plants in early stages (4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7–9-day-old) plants, a new reaction chain was found, which started with phototransformation of the long-wavelength form Pchlide 686/676(440) into Pchlide 653/648(440). (Pchlide 653/648(440) differs from the main known precursor form Pchlide 655/650(448)). The subsequent photoreduction of Pchlide 653/648(440) leads to the formation of Chlide 684/676(440), which is transformed into Chl 688/680(440) in the course of a dark reaction. After completion of this reaction, fast (20–30 s) quenching of the low-temperature fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II core. This revised version was published online in June 2006 with corrections to the Cover Date.     

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445-448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlide a and thereby may regulate the steady-state level of light-harvesting POR-Pchlide complex.     

Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, we describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of (1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and (2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, (3) high concentrations of ATP, MgCl(2), and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg(2+) for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.     

Glucose and δ-Aminolevulinic Acid Stimulate the Dark Chlorophyll Synthesis of Rice Seedlings

This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ(Proto), Mg-protoporphyrin Ⅸ(Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark. Key words: angiosperm; rice; dark chlorophyll synthesis; glucose; δ-aminolevulinic acid; protoporphyrin Ⅸ; Mg-protoporphyrin Ⅸ; protochlorophyllide     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea ( Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m⁻² s⁻¹photon flux density (PFD) white light and subsequently incubated in total darkness for 4–24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form—dominating in epicotyls of dark-grown seedling—required 18–24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4–7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.