Protochlorophyll(ide) forms in hypocotyls of dark-grown bean (Phaseolus vulgaris)

The protochlorophyll(ide) forms and plastid ultrastructure were investigated in hypocotyls of dark-grown seedlings of kidney bean ( Phaseolus vulgaris L. cv. Brede zonder draad). By deconvolution of the fluorescence emission spectra into Gaussian components three protochlorophyll(ide) forms were found with maxima at 633, 642 and 657 nm, respectively. The ratio of protochlorophyll(ide) emitting at 657 nm to protochlorophyll(ide) emitting at 633 nm decreased downwards the hypocotyl. The gradient was established already after 4 days in dark-grown Phaseolus and was also seen in hypocotyls of 7-day-old dark-grown plants of 8 other species. Ultrastructural observations revealed a plastid developmental sequence along the hypocotyl. Plastids in the upper parts of the hypocotyl contained prolamellar bodies typical of etiolated leaves while those in the lower parts contained only stroma lamellae. Immunological detection of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) on nitrocellulose membranes after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) indicated the occurrence of the enzyme in upper, middle and lower sections of hypocotyls and in the root tips.     

Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies

The localization of NADPH-protochlorophyllide oxidoreductase (PChlide reductase, EC 1.6.99.–) in dark-grown and in irradiated dark-grown leaves of wheat ( Triticum aestivum L. cv. Walde) was investigated by subjecting thin sections of Lowicryl K4M-embedded leaf pieces to a monospecific antiserum raised against PChlide reductase followed by protein A-gold. A well-preserved antigenicity of the tissue was achieved by polymerizing the resin under UV-light at low temperature. In dark-grown leaves PChlide reductase was found in prolamellar bodies only. In leaves irradiated for 30 min with white light PChlide reductase was found not only in the transformed prolamellar bodies but also to a large extent in connection with the prothylakoids. The localization of PChlide reductase is discussed in relation to fluorescence emission spectra of the dark-grown and greening leaves. We conclude that the light-dependent transformation of protochlorophyllide to chlorophyllide initiates a translocation of PChlide reductase from the prolamellar bodies to the prothylakoids.     

Protochlorophyll forms in roots of dark-grown plants

Protochlorophyll was found in roots of dark-grown plants of seven species investigated. It was identified by absorbance and fluorescence spectra of acetone and ether extracts. Chlorophyll was also found in roots of one pea species. The concentration of protochlorophyll was usually highest in young root tips and decreased upwards along the roots. The maxima of the in vivo absorbance spectra of the species studied varied between 634 and 638 nm. Low temperature in vivo fluorescence emission spectra had two maxima, one at ca 633 and the other at ca 642 nm, when the wavelengths of the excitation light were 440 and 460 nm, respectively. In vivo fluorescence excitation spectra displayed a shift of the excitation maximum from 438 to 445 nm, when emission varied from 620 to 647.5 nm. Deconvolution of these three types of spectra into Gaussian components made it possible to identify two spectral forms of protochlorophyll: protochlorophyll_629–633 and protochlorophyll_638–642.     

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.     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea ( Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.     

Spectral forms of protochlorophyllide in prolamellar bodies and prothylakoids fractionated from wheat etioplasts

The relation between the different protochlorophyllide (PChlide) forms in isolated etioplast inner membranes was dependent on the concentration of sucrose and NADPH in the isolation media. Etioplasts were prepared from wheat ( Triticum aestivum L. cv. Starke II, Weibull) by differential centrifugation. The etioplasts were freed of envelope and stroma and the etioplast inner membranes were exposed to a concentration series of sucrose. Fluorescence emission spectra revealed a positive correlation between the emission ratio 657/633 nm and the sucrose concentration in which the membranes were suspended. Addition of NADPH prevented the degradation of 657 nm emission caused by low sucrose concentrations. PChlide already altered to PChide_628–632 could not re-form PChlide_650–657 after the addition of NADPH in darkness. Prolamellar bodies and prothylakoids were separated in a bottom-loaded sucrose density gradient in the presence of NADPH. The dominating PChlide-protein complex in the prolamellar bodies was PClide_650–657. Only minor amounts of PChlide_628–632 were found in these membranes. The prothylakoids had a higher content of PChlide_628–632, relative to PChlide_650–657, than the prolamellar bodies, as judged from absorption and fluorescence spectra. After phototransformation the fluorescence emission at 633 nm increased relative to the emission from phototransformed PChlide indicating an efficient energy transfer between PChlide_628–632 and PChlide_650–657 before irradiation.     

Formation of short-wavelength chlorophyll(ide) after brief irradiation is correlated with the occurrence of protochlorophyll(ide)636–642 in dark-grown epi- and hypocotyls of bean (Phaseolus vulgaris)

Chlorophyll formation capacity along the seedling of bean ( Phaseolus vulgaris L. cv. Brede zonder draad) was investigated. After 7 days of irradiation a gradient was formed, where the primary leaf contained ca 300 times more chlorophyll per gram fresh weight than the lower hypocotyl section and ca 20 times more than the epicotyl. Similar chlorophyll gradients but at lower levels were seen when the seedlings were first placed in darkness for 7 days and then irradiated for 1, 2 or 7 days. Ultrastructural investigation of seedlings grown for 7 days in darkness and then irradiated for 24 h revealed a more developed inner membrane system with grana stacks in plastids of cells in the uppermost hypocotyl section compared to plastids of cells in lower hypocoty] sections. The higher up on the seedling the more the ratio increased of protochlorophyll(ide) emitting at 657 nm to short-wavelength protochlorophyll(ide). After flash irradiation of the different sections, fluorescence emission spectra with maxima at 680 and 690 nm, respectively, were observed, indicating the formation of short- and long wavelength chlorophyll(ide) forms. The lower the ratio of protochlorophyll(ide) emitting at 657 nm to the short-wavelength protochlorophyll(ide), the less long-wavelength chlorophyll(ide) was formed after irradiation. However, after continuous irradiation long-wavelength chlorophyll(ide) was formed. In dark grown roots, where only short-wavelength protochlorophyll forms were present, it was not possible to transform protochlorophyll to chlorophyll by flash irradiation. Possible explanations for this phenomenon are discussed.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

The Correlated Appearance of Prolamellar Bodies, Protochlorophyll(ide) Species, and the Shibata Shift during Development of Bean Etioplasts in the Dark

The structure and physiology of the etioplast was investigated in developing primary leaves of 3- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Increase in total protochlorophyll(ide) content followed that of leaf fresh weight. In 3- to 4-day-old bean leaves more than 50% of the protochlorophyll(ide) is in the form of protochlorophyll(ide) 628, which is nontransformable by light. Most of the transformable pigment is protochlorophyll(ide) 635, with smaller amounts of protochlorophyll(ide) 650. During leaf development from the 3rd to the 7th day phototransformable protochlorophyll(ide) with an absorbance maximum at 650 nm accumulates faster than nontransformable protochlorophyll(ide) or protochlorophyll(ide) 635. This increase in protochlorophyll(ide) 650 is correlated with the formation and enlargement of prolamellar bodies.     

Developmental Physiology of Bean Leaf Plastids III. Tube Transformation and Protochlorophyll (ide) Photoconversion by a Flash Irradiation

A light flash of about 1 millisecond duration elicits tube transformation in paracrystalline prolamellar bodies as well as maximal protochlorophyll(ide) photoconversion in etiolated bean leaves (Phaseolus vulgaris L.). These findings support a more detailed hypothesis on the linkage between tube transformation and protochlorophyll(ide) photoconversion than has been offered previously.     

Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts

Prolamellar bodies and prothylakoids from etioplasts of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were separated by sucrose density gradient centrifugation. Top-loaded and bottom-loaded sucrose gradients were compared. As a consequence of avoiding long time exposure of the membranes to low sucrose concentrations, separation in bottom-loaded gradients, as compared to separation in top-loaded gradients, resulted in a sharper and more narrow band of prothylakoids, and in better preservation of phototransformable protochlorophyllide, especially in the prothylakoids. In bottom-loaded gradients, the prothylakoids were found concentrated in a band at a density of 1.20 g'ml⁻¹. The prolamellar bodies were found at a density of 1.17 g'ml⁻¹. In top-loaded gradients the prothylakoids were found at a lower density than the prolamellar bodies. The prothylakoid fraction contained about 60% of the recovered protochlorophyllide and about 85% of the recovered protein. Absorption and fluorescence emission spectra revealed a higher amount of phototransformable protochlorophyllide, in relation to non-phototransformable, in the prolamellar body fraction than in the prothylakoid fraction. Polyacrylamide gel electrophoresis indicated a high proportion of protochlorophyllide reductase in the prolamellar bodies. Chloroplast ATPase (CF₁) was found predominantly in the prothylakoid fraction. Thus, our results strongly indicate the presence of phototransformable protochlorophyllide in the prolamellar bodies proper, while the main bulk of proteins are located in the prothylakoids.     

Protochlorophyllide photo-transformation and chlorophyll(ide) formation in barley etioplast fractions

Localization of protochlorophyll(ide) (Pchlide) forms and chlorophyllide (Chlide) transformation process were studied by using comparative analyses of de-convoluted 77 K fluorescence spectra of barley etioplast stroma and different membrane fractions obtained by sucrose gradient centrifugation. Non-photoactive 633 nm Pchlide form was mainly located in the envelope-prothylakoid membrane mixture while the photoactive 657 nm Pchlide was dominant pigment in the prolamellar body membrane and in the soluble etioplast fraction (stroma). When these fractions were exposed to a saturating flash, conversion of photoactive Pchlide into 697 nm Chlide was preferential in the prolamellar body and in the stroma, while the 676 nm Chlide was dominant pigment form in the envelope-prothylakoid fraction. These spectral characteristics are considered to reflect molecular composition and organization of the pigment-protein complexes specific for each etioplast compartment.     

The polypeptide composition of highly purified prolamellar bodies and prothylakoids from wheat (Triticum aestivum) as revealed by silver staining

The inner membranes from wheat ( Triticum aestivum L. cv. Walde, Weibull) etioplasts were separated by density centrifugation. The etioplasts were broken by osmotic shock and the inner membranes were split by the sheering forces when pressed through a syringe needle. Membrane fractions representative of prolamellar bodies and prothylakoids, respectively, were achieved by separation on a 20–50% continuous sucrose density gradient followed by different purification procedures. The membrane contents of the isolated fractions were characterized by low temperature fluorescence spectra, sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron micrographs. The prolamellar body and the prothylakoid fractions had a fluorescence emission ratio 657/633 nm of 18 and 0.9, respectively. The main part of the total amount of PChlide was found in the prolamellar body fraction. The electrophoretograms stained with Coomassie Blue showed the presence of mainly two polypeptides. The NADPH-protochlorophyllide oxidoreductase was the dominating polypeptide in the prolamellar body fraction, and the α and β subunits of the coupling factor 1 of chloroplast ATP synthase the dominating polypeptides in the prothylakoid fraction. Silver staining revealed at least 4 additional prominent bands with molecular weights of 86, 66, 34 and 28 kDa. The polypeptide composition of the prolamellar body is thus more complex than earlier judged after Coomassie Blue staining. The function of these polypeptides is unknown, but the knowledge of their presence is important in understanding the formation and function of the prolamellar body.     

On the aggregational states of protochlorophyllide and its protein complexes in wheat etioplasts

The inner membranes from wheat ( Triticum aestivum L. cv. Walde) etioplasts were separated into membrane fractions representative of prolamellar bodies and prothylakoids by differential and gradient centrifugations. The isolated fractions were characterized by absorption-, low-temperature fluorescence-, and circular dichroism (CD) spectroscopy, by high performancy liquid chromatography and by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
The prolamellar body fraction was enriched in NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1), and in protochlorophyllide showing an absorption maximum at 650 nm and a fluorescence emission maximum at 657 nm. Esterified protochlorophyllide was mainly found in the prothylakoid fraction. The carotenoid content was qualitatively the same in the two fractions. On a protein basis the carotenoid content was about three times higher in the prolamellar body fraction than in the prothylakoid fraction. The CD spectra of the membrane fractions showed a CD couplet with a positive band at 655 nm, a zero crossing at 643–644 nm and a negative band at 623–636 nm. These results differ from earlier CD measurements on protochlorophyllide holochrome preparations. The results support the interpretation that protochlorophyllide is present as large aggregates in combination with NADPH and NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at −14 °C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium.These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

Developmental Physiology of Bean Leaf Plastids II. Negative Contrast Electron Microscopy of Tubular Membranes in Prolamellar Bodies

Proplastids and prolamellar bodies with tubular membranes were isolated from the dark grown primary leaves of bean seedlings (Phaseolus vulgaris L.). The combination of fluorescence microscopy and negative contrast electron microscopy provided the tentative identification of protochlorophyll holochrome as a constituent of prolamellar body membranes and new evidence for solution-filled channels within the tubular membrane systems of prolamellar bodies.     

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

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.     

Tentoxin does not cause chlorosis in greening mung bean leaves by inhibiting photophosphorylation

Effects of the fungal toxin, tentotoxin, on development and chlorophyll accumulation of plastids of primary leaves of mung bean [ Vigna radiata (L.) Wilczek cv. Berken] were studied using spectrophotometric, electrophoretic, and microscopic procedures. In etioplasts of control tissues both prolamellar bodies and prothylakoids occurred, whereas small vesicles were associated with structurally distinct prolamellar bodies in tentoxin-affected etioplasts. As determined by in vivo spectrophotometry, tentoxin-affected etioplasts had 25% less phototransformable protochlorophyll(ide) and 35% less non-phototransformable protochlorophyll(ide) than had control etioplasts after 5 days of dark seedling growth. Tentoxin had no effect on the rate of the Shibita shift. Protochlorophyll(ide) resynthesis in the dark immediately after protochlorophyll(ide) phototransformation was five to six times slower in tentoxintreated than in control tissues. Effects on chlorophyll(ide) content were observed within 30 min of the beginning of continuous white light exposure. In vivo measurement of cytochrome f redox activity revealed that this cytochrome was linked to light-driven electron flow in control tissues within 20 min of the beginning of continuous white light, whereas in the tentoxin-treated tissues there was no linkage (despite the presence of cytochrome f ) at any time. Coupling factor 1 was present and had potential ATPase activity in both control and tentoxin-affected plastids. There was about sixteen times more chlorophyll in control than in tentoxin-treated tissues in continuous as well as in intermittent (2 min light/118 min dark) light. These data are consistent with the view that tentoxin disrupts normal etioplast and chloroplast development through a mechanism unrelated to photophosphorylation.