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.     

Fast phototransformation of the 636 nm-emitting protochlorophyllide form in epicotyls of dark-grown pea (Pisum sativum)

The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m⁻² s⁻¹) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m⁻² s⁻¹) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.     

Tissue specific protochlorophyll(ide) forms in dark-forced shoots of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>L.)

Cuttings of grapevine (Vitis vinifera L. cv. Chardonnay) were dark-forced at least three weeks. Pigment contents, 77 K fluorescence emission, excitation spectra of the leaves, petioles, stems, transmission electron micrographs of the etioplasts from leaves, the chlorenchyma tissues of the stems were analysed. The dark-grown leaves, stems contained 8 to 10, 3 to 5 μg/g fresh weight protochlorophyllide, its esters, respectively. HPLC analysis showed that the molar ratio of the unesterified, esterified pigments was 7:3 in the shoot developed in darkness. The dark-forced leaves contained carotenoids identified as: neoxanthin, violaxanthin, antheraxanthin, lutein, β-carotene. Detailed analyses of the fluorescence spectra proved that all tissues of the dark-forced shoots had protochlorophyllide or protochlorophyll forms with emission maxima at 628, 636, 644, 655, 669 nm. The 628, 636 nm emitting forms were present in all parts of the dark-forced shoot, but dominated in the stems, which may indicate an organ specificity of the etioplast development. Variations in the distribution of the pigment forms were even found in the different tissues of the stem. The subepidermal layers were more abundant in the 655 nm form than the parenchyma cells of the inner part of the cortex, the pith. In the latter cells, the plastid differentiation stopped in intermediary stages between proplastids, etioplasts. The plastids in the subepidermal layers had developed prolamellar body structures, which were similar to those of etiolated leaves. The results highlight the importance of organ-, tissue specificity of plastid differentiation for chlorophyll biosynthesis, greening of different plant organs. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH:protochlorophyllide oxidoreductase from Avena sativa L.

Dark-grown angiosperm seedlings lack chlorophylls, but accumulate protochlorophyllide a complexed with the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase. Previous investigators correlated spectral heterogeneity of in vivo protochlorophyllide forms and a shift of chlorophyllide forms from 680 to 672 nm (Shibata shift) occurring after irradiation, with intact membrane structures which are destroyed by solubilization. We demonstrate here that the various protochlorophyllide forms and the Shibata shift which disappear upon solubilization are restored if the reconstituted complex is treated with plastid lipids and 80% (w/v) glycerol. We hypothesize that the lipids can form a cubic phase and that this is the precondition in vitro and in vivo for the observed spectral properties before and after irradiation.     

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH: protochlorophyllide oxidoreductase from Avena sativa L

Dark-grown angiosperm seedlings lack chlorophylls, but accumulate protochlorophyllide a complexed with the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase. Previous investigators correlated spectral heterogeneity of in vivo protochlorophyllide forms and a shift of chlorophyllide forms from 680 to 672 nm (Shibata shift) occurring after irradiation, with intact membrane structures which are destroyed by solubilization. We demonstrate here that the various protochlorophyllide forms and the Shibata shift which disappear upon solubilization are restored if the reconstituted complex is treated with plastid lipids and 80% (w/v) glycerol. We hypothesize that the lipids can form a cubic phase and that this is the precondition in vitro and in vivo for the observed spectral properties before and after irradiation.     

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.     

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.     

Plastid differentiation and chlorophyll biosynthesis in different leaf layers of white cabbage (Brassica oleracea cv. capitata)

The contents of protochlorophyllide, protochlorophyll and chlorophyll together with the native arrangements of the pigments and the plastid ultrastructure were studied in different leaf layers of white cabbage (Brassica oleracea cv. capitata) using absorption, 77 K fluorescence spectroscopy and transmission electron microscopy. The developmental stage of the leaves was determined using the differentiation of the stoma complexes as seen by scanning electron microscopy and light microscopy. The pigment content showed a gradual decrease from the outer leaf layer towards the central leaves. The innermost leaves were in a primordial stage in many aspects; they were large but had typical proplastids with few simple inner membranes, and contained protochlorophyllide and its esters in a 2 : 1 ratio and no chlorophyll. Short‐wavelength, not flash‐photoactive protochlorophyllide and/or protochlorophyll forms emitting at 629 and 636 nm were dominant in the innermost leaves. These leaves also had small amounts of the 644 and 654 nm emitting, flash‐photoactive protochlorophyllide forms. Rarely prolamellar bodies were observed in this layer. The outermost leaves had the usual characteristics of fully developed green leaves. The intermediary layers contained chlorophyll a and chlorophyll b besides the protochlorophyll(ide) pigments and had various intermediary developmental stages. Spectroscopically two types of intermediary leaves could be distinguished: one with only a 680 nm emitting chlorophyll a form and a second with bands at 685, 695 and 730 nm, corresponding to chlorophyll–protein complexes of green leaves. In these leaves, a large variety of chloroplasts were found. The data of this work show that etioplasts, etio‐chloroplasts or chloro‐etioplasts as well as etiolated leaves do exist in the nature and not only under laboratory conditions. The specificity of cabbage leaves compared with those of dark‐grown seedlings is the retained primordial or intermediary developmental stage of leaves in the inner layers for very long (even for a few month) period. This opens new developmental routes leading to formation of specially developed plastids in the various cabbage leaf layers. The study of these plastids provided new information for a better understanding of the plastid differentiation and the greening process .     

Abnormal etioplast development in barley seedlings infected with BSMV by seed transmission

The effect of barley stripe mosaic hordeivirus (BSMV) was studied on the ultrastructure of etioplasts, protochlorophyllide forms and the greening process of barley ( Hordeum vulgare cv. Pannónia) plants infected by seed transmission. The leaves of 7- to 11-day-old etiolated seedlings were examined by transmission electron microscopy, fluorescence and absorption spectroscopy. The etioplasts of infected seedlings contained smaller prolamellar bodies with less regular membrane structure, while prothylakoid content was higher than in the control. The protochlorophyllide content of virus-infected seedlings was reduced to 74% of the control. In the 77 K fluorescence spectra the relative amount of 655 nm emitting photoactive protochlorophyllide form decreased, and the amount of the 645 and 633 nm emitting forms increased in the infected leaves. A characteristic effect was observed in the process of the Shibata-shift: 40 min delay was observed in the infected leaves. The results of this work proved that BSMV infection delays or inhibits plastid development and the formation of photosynthetic apparatus.     

Mercury Inhibits the Activity of the NADPH:Protochlorophyllide Oxidoreductase (POR)

The effect of Hg⁺⁺ was studied on the arrangement and photoactivity of NADPH:protochlorophyllide oxidoreductase (POR) in homogenates of dark-grown wheat (Triticum aestivum L.) leaves. 77 K fluorescence emission spectra of the homogenates were recorded before and after the irradiation of the homogenates and the spectra were deconvoluted into Gaussian components. The mercury treatment caused a precipitation of the membrane particles, which was followed by a remarkable decrease of the fluorescence yield. 10^-3 M Hg⁺⁺ decreased the ratio of the 655 nm-emitting protochlorophyllide (Pchlide) form to the 633 nm-emitting form. 10^-2 M Hg⁺⁺ shifted the short wavelength band to 629–630 nm and a 655 nm form was observed which was inactive on irradiation. This inhibition may be caused by serious alteration of the enzyme structure resulting in the trans-localisation of NADPH within the active site of POR.     

Etioplasts with protochlorophyll and protochlorophyllide forms in the under‐soil epicotyl segments of pea (Pisum sativum) seedlings grown under natural light conditions

To study if etiolation symptoms exist in plants grown under natural illumination conditions, under‐soil epicotyl segments of light‐grown pea (Pisum sativum) plants were examined and compared to those of hydroponically dark‐grown plants. Light‐, fluorescence‐ and electron microscopy, 77 K fluorescence spectroscopy, pigment extraction and pigment content determination methods were used. Etioplasts with prolamellar bodies and/or prothylakoids, protochlorophyll (Pchl) and protochlorophyllide (Pchlide) forms (including the flash‐photoactive 655 nm emitting form) were found in the (pro)chlorenchyma of epicotyl segments under 3 cm soil depth; their spectral properties were similar to those of hydroponically grown seedlings. However, differences were found in etioplast sizes and Pchlide:Pchl molar ratios, which indicate differences in the developmental rates of the under‐soil and of hydroponically developed cells. Tissue regions closer to the soil surface showed gradual accumulation of chlorophyll, and in parallel, decrease of Pchl and Pchlide. These results proved that etioplasts and Pchlide exist in soil‐covered parts of seedlings even if they have a 3–4‐cm long photosynthetically active shoot above the soil surface. This underlines that etiolation symptoms do develop under natural growing conditions, so they are not merely artificial, laboratory phenomena. Consequently, dark‐grown laboratory plants are good models to study the early stages of etioplast differentiation and the Pchlide–chlorophyllide phototransformation.     

Dominance of a 675?nm chlorophyll(ide) form upon selective 632.8 or 654?nm laser illumination after partial protochlorophyllide phototransformation

The phototransformation pathways of protochlorophyllide forms were studied in 8?C14-day-old leaves of dark-germinated wheat (Triticum aestivum L.) using white, 632.8?nm He?CNe laser and 654?nm laser diode light. The photon flux density (PFD) values (0.75?C360???mol photons?m^?2?s^?1), the illumination periods (20?ms?C10?s) and the temperature of the leaves (between ?60?°C and room temperature) were varied. The 77?K fluorescence spectra of partially phototransformed leaves showed gradual accumulation or even the dominance of the 675?nm emitting chlorophyllide or chlorophyll form at room temperature with 632.8?nm of PFD less than 200???mol photons?m^?2?s^?1 or with 654?nm of low PFD (7.5???mol photons?m^?2?s^?1) up to 1?s. Longer wavelength (685 or 690?nm) emitting chlorophyllide forms appeared at illuminations under ?25?°C with both laser lights or at room temperature when the PFD values were higher or the illumination period was longer than above. We concluded that the formation of the 675?nm emitting chlorophyllide form does not indicate the direct photoactivity of the 633?nm emitting protochlorophyllide form; it can derive from 644 and 657?nm forms via instantaneous disaggregation of the newly-produced chlorophyllide complexes. The disaggregation is strongly influenced by the molecular environment and the localization of the complex.     

Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene

Background and Aims

Etiolation symptoms and the greening process are usually studied on dark-germinated seedlings and this raises the question – can these results be generalized for plants growing under field conditions? This work examines various aspects of the plastid differentiation under the covering of the achene wall, which often remains attached to the cotyledons of sunflower (Helianthus annuus) seedlings grown under light.

Methods

Cotyledons of 7- to 10-d-old sunflower seedlings grown in the dark and on light were examined. The partially covered cotyledons were sectioned into light-exposed, covered and transition zones. Pigment contents, 77 K fluorescence spectroscopy, electron microscopy and fluorescence imaging, along with fluorescence kinetic methods, were used.

Key Results

The light-exposed zone of the partially covered cotyledons was similar to cotyledons developed without achene covering. However, some of the plastids had prolamellar bodies among the granal thylakoid membranes; despite this no protochlorophyllide was detected. The fully covered, yellowish sections contained protochlorophyllide forms emitting at 633 and 655 nm and well-developed prolamellar bodies, similar to those of etiolated cotyledons. In addition, reduced amounts of chlorophyll a, chlorophyll b and stacked thylakoid membrane pairs were found in this region. The transitional sections showed a mixture of the characteristics of the covered and exposed sections. Various, but significantly different values of the photosynthetic activity parameters were found in each sector of the partially covered cotyledons.

Conclusions

The partial covering of the achene wall shades the cotyledon tissues effectively, enough to provoke the appearance of etiolation phenomena, i.e. the permanent presence of flash-photoactive protochlorophyllide complexes and prolamellar bodies (with or without protochlorophyllide), which proves that these phenomena may appear under natural illumination conditions.Key words: Cotyledon, etio-chloroplast, etioplast, etiolation, Helianthus annuus, photosynthetic activity, protochlorophyllide, prolamellar body, sunflower     

Photobiomodulation of human adipose-derived stem cells using 810 nm and 980 nm lasers operates via different mechanisms of action

Photobiomodulation (PBM) using red or near-infrared (NIR) light has been used to stimulate the proliferation and differentiation of adipose-derived stem cells. The use of NIR wavelengths such as 810 nm is reasonably well accepted to stimulate mitochondrial activity and ATP production via absorption of photons by cytochrome c oxidase. However, the mechanism of action of 980 nm is less well understood. Here we study the effects of both wavelengths (810 nm and 980 nm) on adipose-derived stem cells in vitro. Both wavelengths showed a biphasic dose response, but 810 nm had a peak dose response at 3 J/cm² for stimulation of proliferation at 24 h, while the peak dose for 980 nm was 10–100 times lower at 0.03 or 0.3 J/cm². Moreover, 980 nm (but not 810 nm) increased cytosolic calcium while decreasing mitochondrial calcium. The effects of 980 nm could be blocked by calcium channel blockers (capsazepine for TRPV1 and SKF96365 for TRPC channels), which had no effect on 810 nm. To test the hypothesis that the chromophore for 980 nm was intracellular water, which could possibly form a microscopic temperature gradient upon laser irradiation, we added cold medium (4 °C) during the light exposure, or pre-incubated the cells at 42 °C, both of which abrogated the effect of 980 nm but not 810 nm. We conclude that 980 nm affects temperature-gated calcium ion channels, while 810 nm largely affects mitochondrial cytochrome c oxidase.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

Blue organic light‐emitting diodes based on fluorene‐bridged quinazoline and quinoxaline derivatives

Two blue emitters based on fluorene‐bridged quinazoline and quinoxaline derivatives were prepared via the Suzuki reaction. Their photoluminescent properties were investigated. Furthermore, theoretical studies on these materials using the density functional theory calculation were conducted. To explore their electroluminescent properties, multilayered organic light‐emitting diodes were fabricated with the following device structure: indium–tin–oxide (180 nm)/4,4′‐bis(N‐(1‐naphthyl)‐N‐phenylamino)biphenyl (50 nm)/blue emitting materials ( 1 and 2 ) (30 nm)/bathophenanthroline (35 nm)/8‐hydroxy‐quinolinato lithium (2 nm)/Al (100 nm). Two devices showed efficient blue emission with the external quantum efficiencies of 1.58% and 1.30%, respectively, at 20 mA/cm², and Commission Internationale dÉclairage coordinates of (0.18, 0.24) and (0.19, 0.27) at 6.0 V. These results suggest that the self‐aggregation properties of emitters would have considerable effects on their photoluminescent and electroluminescent properties.     

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.     

Synthesis and photoluminescence characteristics of Sm3+‐doped Bi4Si3O12 red‐emitting phosphor

A series of novel red‐emitting Sm³⁺‐doped bismuth silicate phosphors, Bi₄Si₃O₁₂:xSm³⁺ (0.01 ≤ x ≤ 0.06), were prepared via the sol–gel route. The phase of the synthesized samples calcinated at 800 °C is isostructural with Bi₄Si₃O₁₂ according to X‐ray diffraction results. Under excitation with 405 nm light, some typical peaks of Sm³⁺ ions centered at 566, 609, 655 and 715 nm are found in the emission spectra of the Sm³⁺‐doped Bi₄Si₃O₁₂ phosphors. The strongest peak located at 609 nm is due to ⁴G_5/2–⁶H_7/2 transition of Sm³⁺. The luminescence intensity reaches its maximum value when the Sm³⁺ ion content is 4 mol%. The results suggest that Bi₄Si₃O₁₂:Sm³⁺ may be a potential red phosphor for white light‐emitting diodes. Copyright © 2016 John Wiley & Sons, Ltd.     

Protochlorophyllide and POR development in dark‐grown plants with different proportions of short‐wavelength and long‐wavelength protochlorophyllide spectral forms†

The effect of leaf developmental age on the protochlorophyllide (Pchlide) spectral forms and the expression of messenger RNA (mRNA) encoding NADPH‐protochlorophyllide oxidoreductase (POR) were investigated. Four plant species, maize, wheat, pea and the lip1 mutant of pea, known to have different composition of the spectral forms of Pchlide, were used. In very young plants short‐wavelength Pchlide with a fluorescence emission at 631 nm was dominating. Long‐wavelength Pchlide fluorescing mainly around 655 nm increased during development, which led to a relative decrease of the short‐wavelength forms. During ageing of the leaves, the short‐wavelength forms slightly increased again. The different proportions of short‐ and long‐wavelength Pchlide spectral forms were, however, found to vary with the developmental stage in a species specific pattern. The steady‐state level of POR mRNA and the amount of the POR protein were similar in species dominated by short‐wavelength forms and in species dominated with long‐wavelength forms. Even if POR is necessary for the formation of the long‐wavelength Pchlide form it is not the only limiting factor for formation of long‐wavelength Pchlide forms in mature plants.     

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