Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts

Etiolated barley (Hordeum vulgare L.) seedlings were treated with heat shock (HS). The heat treatment was conducted daily for 1 h at 40°C over 6 days and led to shortening of leaves and coleoptiles, an increase in the etioplast volume and prothylakoid length, and to a decrease in the size of paracrystalline prolamellar bodies (PLB). As a result of HS treatment, stimulation of carotenoid and protochlorophyllide (Pchlide) synthesis as well as an increase in the relative content of the Pchlide short-wavelength form (Pchlide₆₃₀) were observed in the leaf tissue of seven-day-old seedlings 12 h after the last HS treatment. HS had no effect on the overall amount of Pchlide-oxidoreductase (POR) in leaves and PLB membranes and did not suppress the Pchlide photoreduction in vivo. PLB membranes, isolated from the HS-treated seedlings, possessed a higher Pchlide and carotenoid content as calculated on total protein basis. These membranes showed more intense protein fluorescence than PLB from untreated plants, whereas hydrophobicity of the microenvironment of the fluorescent amino-acid residues remained unchanged. Studies using pyrene (lipophilic fluorescent probe emitted in Pchlide and carotenoid absorption bands) showed that HS increases the fluidity of membrane lipids in PLB membranes and that the pigments accumulated in these membranes are located in the region of lipid–protein contact site. The results are discussed in relation to the adaptive role of protein–protein and pigment–protein–lipid interactions in etioplast membranes under stress.     

Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.     

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.     

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.     

Alterations of NADPH:protochlorophyllide oxidoreductase quantity and lipid composition in etiolated barley seedlings infected by Barley stripe mosaic virus (BSMV)

To understand the phenomenon by which infection of seed-transmitted Barley stripe mosaic virus (BSMV) alters membrane structures and inhibits protochlorophyllide biosynthesis of dark-grown barley ( Hordeum vulgare L.) plants, we analysed the presence of NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) and the galactolipid content and fatty acid composition. The amount of POR in etioplasts of infected leaves, compared with non-infected leaves, was reduced, as measured by immunoelectron microscopy and Western blot. These results are in agreement with the previously described reduction of the ratio of the photoactive 650 nm to non-photoactive 630 nm absorbing protochlorophyllide forms ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The galactolipid content was lower in infected leaves. Monogalactosyl-diacylglycerol (MGDG) content was reduced to 40% and digalactosyl-diacylglycerol to 55% of control plants on a fresh weight basis. In infected plants, the proportion of linolenic acid decreased in both galactolipids. The lower amount of highly unsaturated fatty acids and the reduced abundance of MGDG correlated well with the previously detected reduction in the membrane ratio of prolamellar body (PLB) to prothylakoid ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The reduced amount of POR and the above described alterations in the lipid composition resulted in a disturbed structure of PLBs. As a consequence, pigment synthesis and the greening process were inhibited in infected cells, in turn explaining the appearance of chlorotic stripes of BSMV-infected barley leaves. Our results show that BSMV infection can be detected at a very early stage of leaf development.     

Photoactive pigment—enzyme complexes of chlorophyll precursor in plant leaves

This review summarizes contemporary data on structure and function of photoactive pigment--enzyme complexes of the chlorophyll precursor that undergoes photochemical transformation to chlorophyllide. The properties and functions of the complex and its principal components are considered including the pigment (protochlorophyllide), the hydrogen donor (NADPH), and the photoenzyme protochlorophyllide oxidoreductase (POR) that catalyzes the photochemical production of chlorophyllide. Chemical variants of the chlorophyll precursor are described (protochlorophyllide, protochlorophyll, and their mono- and divinyl forms). The nature and photochemical activity of spectrally distinct native protochlorophyllide forms are discussed. Data are presented on structural organization of the photoenzyme POR, its substrate specificity, localization in etioplasts, and heterogeneity. The significance of different POR forms (PORA, PORB, and PORC) in adaptation of chlorophyll biosynthesis to various illumination conditions is considered. Attention is paid to structural and functional interactions of three main constituents of the photoactive complex and to possible existence of additional components associated with the pigment-enzyme complex. Historical aspects of the problem and the prospects of further investigations are outlined.     

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80–84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field‐grown plants. Map‐based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non‐photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short‐day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate‐dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light‐dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

The effect of 2,4-dichlorophenoxyacetic acid and (2-chloroethyl)-trimethylammonium chloride on chlorophyll synthesis in barley leaves

Summary 2.4-dichlorophenoxyacetic acid (2.4-D) and (2-chloroethyl)-trimethylammonium chloride (CCC) inhibit chlorophyll synthesis and protochlorophyllide ₆₅₂ regeneration in 6–8 day old barley leaves whilst having little effect on the rates of protochlorophyll ₆₃₂ synthesis from exogenous -aminelevulinic acid (ALA) and ALA-dehydratase activity. Longer pretreatments with 2.4-D and CCC show it is only after 50 to 60 hr that the rates of P₆₃₂ production from exogenous ALA and ALA-dehydratase activity are affected. Similar response times were obtained for chloramphenicol (CAP). The results indicate that 2.4-D and CCC may act by directly inhibiting specific plastid-protein synthesis similar to CAP. Hence it seems that it is only those proteins (enzymes) having a rapid turnover that are affected first i.e. the enzymes necessary for ALA synthesis in the plastid.Abbreviations used ALA -aminolevulinic acid - CAP chloramphenicol - CCC (2-chloroethyl)-trimethylammonium chloride - 2.4-D 2-4-dichlorophenoxyacetic acid - P₆₅₂ prodochlorophyllide with maximum in-vivo absorption at 652 nm - P₆₈₄ chlorophyllide absorbing at 684 nm - P₆₇₀ chlorophyllide absorbing at 670 nm - P₆₃₂ pigment absorbing at 632 nm synthesised from exogenous ALA - PBG Porphobilinogen P. R. Shewry is in receipt of a Science Research Council Studentship award.     

The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature- dependent enzymatic activity, being inactive at 32°C, but partially active at 24°C. Mutations in predicted - helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted -sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.     

Disintegration of the prolamellar body structure at high concentrations of Hg2+

The effects of high concentrations of Hg (2+) (10 (-2) M and 10 (-3) M) were investigated on the ultrastructure and on the light-induced transformation of isolated prolamellar bodies (PLBs) of dark-grown wheat leaves. Our earlier work on wheat leaf homogenates ( , Plant Biology 6, 358 - 368) showed that, depending on the concentration, Hg (2+) reacts with protochlorophyllide, NADPH and the NADPH : protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme and induces disaggregation of the macrodomain structure of this latter. Spectroscopic analyses confirmed that 15 min incubation with 10 (-2) M Hg (2+) at 4 degrees Celsius completely inhibited the activity of POR also in isolated PLBs. Ultrastructural investigations revealed the loosening of the PLB structure in the Hg (2+)-treated sample, i.e., intensive vesicle formation on the surface of the PLB membranes. The hexagonal geometry of the inner lattice was not disturbed, however, the unit cell size significantly increased. The disruption of the PLB membranes upon irradiation was studied after 40 min incubation with 10 (-3) M Hg (2+) at 4 degrees Celsius and a subsequent irradiation for 40 min at 20 degrees Celsius. Equimolar concentrations (10 (-3) M) of NADPH and Hg (2+) were added to the samples 10 min prior or after the addition of Hg (2+). Our results suggest that Hg (2+) accelerates the disruption of the PLB membranes and that NADPH can only partially prevent this process. These membrane transformations were similar to those observed in the initial steps of the Shibata shift of control samples.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Visualization and characterization of prolamellar bodies with atomic force microscopy

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1–2 μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1 μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25 min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl₂ also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.     

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.     

Far-red induced changes of the protochlorophyllide components in wheat leaves

Biosynthesis of chlorophyll is partly controlled by the phytochrome system. In order to study the effects of an activated phytochrome system on the protochlorophyllide (PChlide) biosynthesis without accompanying phototransformation to chlorophyll, wheat seedlings (Triticum aestivum L. cv. Starke II Weibull) were irradiated with long wavelength far-red light of low intensity. Absorption spectra were measured in vivo after different times in the far-red light or in darkness. The relationship between the different PChlide forms, the absorbance ratio _{650nm636 nm} changed with age in darkness, and the change was more pronounced when the leaves were grown in far-red light. Absorption spectra of dark-grown leaves always showed a maximum in the red region at 650 nm. For leaves grown in far-red light the absorption at 636 nm was high, with a maximum at the 5 day stage where it exceeded the absorption at 650 nm. At the same time there was a maximum in the total amount of PChlide accumulated in the leaves, about 30% more than in leaves grown in darkness. But the amount of the directly phototransformable PChlide, mainly PChlide_650–657, was not increased. The amount of PChlide_628–632, or more probably the amount of (PChlide_628–632, + PChlide _636–657) was thus higher in young wheat leaves grown in far-red light than in those grown in darkness. After the 5 day stage the absorption at 636 nm relative to 650 nm decreased with age, and at the 8 day stage the spectra were almost the same in both types of leaves. Low temperature fluorescence spectra of the leaves also showed a change in the ratio between the different PChlide forms. The height of the fluorescence peak at 632 nm relative to the peak at 657 nm was higher in leaves grown in far-red light than in dark-grown leaves. – After exposure of the leaves to a light flash, the half time for the Shibata shift was measured. It increased with age both for leaves grown in darkness and in far-red light; but in older leaves grown in far-red light (7–8 days) the half time was slightly longer than in dark-grown leaves. – The chlorophyll accumulation in white light as well as the leaf unrolling were faster for leaves pre-irradiated with far-red light. The total length of the seedlings was equal or somewhat shorter in far-red light, but the length of the coleoptile was markedly reduced from 8.1 ± 0.1 cm for dark-grown seedlings to 5.2 ± 0.1 cm for seedlings grown in far-red light.