The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.     

Circular dichroism studies on the structure and the photochemistry of protochlorophyllide and chlorophyllide holochrome

On the basis of absorption and circular dichroism (CD) spectral measurements, we conclude that the photoreduction of protochlorophyllide to chlorophyllide in homogenates of etiolated bean seedlings (Phaseolus vulgaris L.) involves two light steps in series. Before illumination, the active protochlorophyllide occurs in a dimeric form in the holochrome protein. The initial light reaction converts one of the protochlorophyllide molecules and forms a chlorophyllide-protochlorophyllide holochrome intermediate with a weak, characteristic CD spectrum. The second light reaction subsequently converts the second protochlorophyllide in a less efficient reaction that is temperature dependent. This produces a chlorophyllide holochrome which exhibits a strong double CD characteristic of dimers and which is stable below 1°C. At higher temperatures this dimeric chlorophyllide transforms in the dark to a monomeric form with low CD amplitude. Sucrose at high concentrations (2 M) alters the chlorophyllide holochrome CD spectrum and prevents the final dark dissociation step. Analysis of the photochemical kinetics confirms the occurrence of the two-step photoreduction and supports the stoichiometry of two (proto)chlorophyllides per holochrome protein.     

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     

Chlorophyll a and b biosynthesis in the dark in etiolated leaves infiltrated by exogenous chlorophyllide a]

Exogenous chlorophyllide a was introduced into etiolated rye leaves by the vacuum-infiltration technique. Appearance and accumulation of chlorophylls a and b within the leaves are observed during continued darkening, protochlorophyllide photoreduction being avoided. The pigments are identified by the solubility in petroleum ether, paper chromatograms, the fluorescence maxima, the peculiarities of exciting light 430 and 460 nm effects on fluorescence intensity, the specific interaction with hydrochloric hydroxylamine. The conclusion is made that before illumination etioplasts already contain enzyme systems and substrates which provide esterification of chlorophyllide a to chlorophyll a and conversion of chlorophyll a into chlorophyll b.     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

Light-induced reduction of protochlorophyllide in angiosperms and chloroplast development

One of the final reactions of chlorophyll (Chl) biosynthesis, e.g: photoreduction of protochlorophyllide (Pchlid) to chlorophyllide (Chlid) is a light-induced process in Angiosperm plants and it is catalyzed by light-dependent NADPH-Pchlid oxidoreductase (1.3.1.33; LPOR). In darkness, Chl biosynthesis is stopped at the stage of Pchlid formation. Seedlings and plastids develop according to a different pattern than that observed in the light. Moreover, synthesis of some proteins of the photosynthetic apparatus is inhibited. Light triggers the Pchlid photoreduction to Chlid, which induces the cascade of biochemical reactions and structural changes leading to the assembly of thylakoid membranes. In the present paper, the current knowledge on LPOR protein, mechanism of Pchlid to Chlid photoreduction, the role of lipid structure in etioplasts as well as spectral properties of Pchlid in etiolated seedlings and model systems is summarized.     

Formation of long-wavelength chlorophyllide (Chlide695) is required for the assembly of Photosystem II in etiolated barley leaves

Chlorophyll(ide) spectroscopic properties and Photosystem II assembly, monitored by 77 K variable fluorescence, were studied in etiolated barley leaves as a function of the extent of protochlorophyllide photoreduction by a single millisecond light flash of different intensities. Variable fluorescence, measured 2 hours after the flash, was only detected when the extent of phototransformation was higher than a threshold value of 0.4. Its development paralleled the formation of a chlorophyll emission component at 685 nm, which itself derived from long-wavelength chlorophyllide with an emission maximum at 695 nm. At low flash intensities, short-wavelength chlorophyllide forms preferentially accumulated and no Photosystem II fluorescence was detected after 2 hours. Chlorophyllide esterification was independent of the extent of phototransformation. These results suggested that the formation of long-wavelength chlorophyllide was essential for further assembly of Photosystem II. This interpretation was strengthened by the observed inhibition of both long-wavelength chlorophyllide formation and of variable fluorescence development in leaves treated with -aminolevulinic acid or in untreated leaves subjected to repeated flashes of low intensity.     

Chlorophyll synthesis in green leaves and isolated chloroplasts of barley (Hordeum vulgare)

The photoreduction of protochlorophyllide was studied in leaves and isolated chloroplasts of barley. Leaves of plants which had been preilluminated for varying lengths of time were incubated with [¹⁴C]-δ- aminolevulinic acid for 2 h in the dark. The subsequent photoreduction of [¹⁴C]-protochlorophyllide was analyzed by high performance liquid chromatography of pigments extracted from illuminated leaves and plastids. The plastids used in this study were isolated in the dark from leaves at the end of the 2 h labelling period. Three major results were obtained:

• 1

  The extent of protochlorophyllide reduction in vivo was rapidly reduced as a function of the preillumination period. In 24 h preilluminated plants only a small fraction of the radioactively labelled protochlorophyllide was reduced during the subsequent light period.
• 2

  The amount of NADPH-protochlorophyllide oxidoreductase (EC 1.6.99.-) present in plastids of fully-green plants was drastically reduced relative to levels in plastids of dark-grown plants as estimated by the methods of immunoblotting of plastid proteins and immunogold labelling of ultrathin sections of the leaf tissue.
• 3

  In etiolated plants light seemed to affect the reduction of protochlorophyllide directly through the excitation of protochlorophyllide. In fully green plants, however, light also affected chlorophyll formation indirectly by the supply of NADPH via photosynthetic electron transport.

     

Participation of Free Radicals in Photoreduction of Protochlorophyllide to Chlorophyllide in an Artificial Pigment–Protein Complex

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.     

On the nature of the two pathways in chlorophyll formation from protochlorophyllide

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14–24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 °C. The product of fast esterification can be correlated with ‘short-wavelength’ chlorophyll, characterized by a fluorescence emission peak at 673–675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Properties of reformed prolamellar bodies from illuminated and redarkened etiolated wheat plants

Prolamellar bodies were isolated from etiolated leaves of wheat ( Triticum aestivum L. cv. Walde, Weibull), which were illuminated for 4 h and then grown in darkness for 16 h. The inner etiochloroplast membranes were isolated by differential centrifugation, and prolamellar bodies and thylakoids were separated on a 10–50% continuous sucrose density gradient. The reformed prolamellar bodies contained phototransformable protochlorophyllide as the main pigment as shown by low temperature fluorescence spectra and high performance liquid chromatography. After illumination with 3 flashes of white light almost all of the protochlorophyllide was transformed to chlorophyllide. In the thylakoids, however, most of the protochlorophyllide was not phototransformed. The reformed prolamellar bodies and the thylakoids showed a fluorescence emission ratio 657/633 nm of 5.6 and 0.5, respectively. Both membrane systems contained also chlorophyllide and chlorophyll synthesized during the illumination. Polyacrylamide gel electrophoresis showed the main chlorophyllide oxidoreductasse.
Teransmission and scanning electron micrographs indicated that the reformed prolamellar bodies are mainly of the "narrow" type and that the prolamellar body fraction had only a minor contamination with thylakoid membranes.
The results obtained showed that reformed prolamellar bodies isolated from illuminated redarkened etiolated wheat leaves had features very similar to the prolamellar bodies isolated from etiolated leaves. This provides support for the idea that prolamellar bodies are an important natural membrane system which plays a dynamic role in the development of the etio-chloroplasts in light.     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Soret absorption of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction trapped at low temperature

Absorption spectra at ca 100 K from 400 to 750 nm and fluorescence emission spectra at 77 K from 600 to 750 nm were obtained from: 1) etiolated leaves of the H-ordeum vulgare L. (barley) mutant albozonata 2 and SAN 9789-treated Avena sativa L. (oat) with low levels of carotenoids, and 2) preparations of protochlorophyllide holo-chrome from Phaseolus vulgaris L. cv. Commodore (bean).
This allowed clear resolution for the first time of the Soret bands of the green pigments before and after light-induced accumulation of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction and after conversion of the intermediate(s) to chlorophyllide by warming the samples to 233 K in darkness. Although the intermediate(s) differ(s) in absorption and fluorescence in the red wavelength region from both protochlorophyllide and chlorophyllide, the extinction in the Soret band is not distinguishable from that of chlorophyllide. These observations indicate that the C7-C8 double bond in ring IV of protochlorophyllide has been altered in intermediate(s) accumulated at low temperature in intense light, such that the transition state exhibits the character of a π complex.     

Influence of Anaerobiosis on Chlorophyll Biosynthesis in Greening Oat Seedlings (Avena sativa L.)

The influence of anaerobiosis for 0.5 to 15 hours on the last steps of chlorophyll biosynthesis of etiolated oat seedlings was investigated. Phototransformation of protochlorophyllide to chlorophyllide is only slightly reduced and esterification of chlorophyllide is slightly increased by pretreatment under anaerobic conditions. Pretreated plants accumulate the geranylgeraniol ester of chlorophyllide rather than the phytol ester. Enzymic hydrogenation of the esterifying alcohol geranylgeraniol to phytol is presumably inhibited by anaerobiosis.     

Protochlorophyllide oxidoreductase: "dark" reactions of a light-driven enzyme

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in the chlorophyll biosynthesis pathway. As POR is light activated, it allows intermediates in the reaction pathway to be observed by initiating catalysis with illumination at low temperatures, a technique that has recently been used to study the initial photochemistry. Here, we use low-temperature spectroscopy to show that the catalytic mechanism of POR involves two additional steps, which do not require light and have been termed the "dark" reactions. The first of these involves the conversion of the product of the initial light-driven reaction, a nonfluorescent radical species, into a new intermediate that has an absorbance maximum at 681 nm and a fluorescence peak at 684 nm. During the second dark step this species gradually blue shifts to yield the product, Chlide. The temperature dependence for each of these two processes was measured; the data revealed that these steps could only occur close to or above the "glass transition" temperature of proteins, suggesting that domain movements and/or reorganization of the protein are required for these stages of the catalytic mechanism.     

The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably δ-amino-levulinic acid synthetase) is not longer than 10 minutes.