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.     

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 .     

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.     

Formation of the Pigment Apparatus in Etiolated Barley Leaves under the Influence of Levulinic Acid

The effects of levulinic acid (LA) on the synthesis of pigments and the membrane system of etioplasts were studied in etiolated leaves of barley (Hordeum vulgare L.). Growing in the solution of LA during a six-day period, which started one day after the soaking of seeds, resulted in a retardation of leaf growth, more than a twofold decrease in the level of carotenoids and protochlorophyllide (Pd) in the leaf tissue, and suppression of the synthesis of long-wave form of Pd₆₅₅; these effects depended on the LA concentration. In etioplasts isolated from the seedlings treated with 50 M LA and containing predominantly a short-wave form of Pd with a peak of fluorescence at 632 nm (–196°C), there was a membrane fraction whose location in the sucrose density gradient was identical to that of prolamellar bodies (PLB) in the control plants. The content of Pd and carotenoids in this fraction calculated on a protein basis was 2.46 and 1.3 times lower than in control seedlings, while the relative content of Pd oxidoreductase (POR) essentially did not change. Thus, the suppression of Pd synthesis did not affect translocation of POR from the cytoplasm to the membranes of etioplasts. In the PLB membranes, there was no transfer of energy from the molecules of lipophilic fluorescent probe pyrene (excitation at 337, 278, and 296 nm) to Pd; however, under pigment deficiency, the production therein of pyrene excimer form at the expense of energy transfer from protein tryptophanyls (excitation at 278 and 296 nm) became more efficient, which indicated changes in protein–lipid interactions. The obtained results suggest that the short-wave form of Pd₆₃₂ accumulating in etioplasts under the suppressed synthesis of tetrapyrroles is not a free pigment.     

Biogenesis of photosynthetic apparatus under the inhibition of energy processes in de-etiolated seedlings of barley (Hordeum vulgare L.)

Transformation of protochlorophyllide forms in etiolated barley seedlings and biogenesis of photosynthetic apparatus in greening leaves of 7-day-old etiolated barley seedlings (Hordeum vulgare L.) were studied under the inhibition of energy processes during illumination. Repression of electron transport between photosystem 2 and 1 (PS2 and PS1, respectively) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) inhibited the photochemical activity of PS2 but did not affect chlorophyll biosynthesis and ATP content in leaves compared to the control. Inhibition of mitochondrial electron transport with sodium azide increased relative content of nonphotoactive protochlorophyllide in etiolated leaves, decreased the content of ATP, chlorophylls, and carotenoids and completely suppressed the functional activity of PS 2. The inhibitor of glycolysis sodium fluoride affected all the parameters even more strongly. We observed synchronism in the accumulation of chlorophylls and carotenoids during greening for all inhibitor variants other than fluoride (correlation coefficient, r, equal to 0.98, 0.97, 0.97, and 0.47 with the significance level of 0.01; 0.015; 0.015, and 0.27 for control, diuron, azide, and sodium fluoride, respectively). The change in chlorophyll content under the influence of inhibitors positively correlated with the amount of ATP in the leaf tissue (for 24 h greening, r = 0.97 with significance level of 0.015). We suggest that sources of ATP involved in the synthesis of chlorophyll during greening of etiolated barley seedlings are mostly of non-plastid origin.     

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.     

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

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

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

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

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.     

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.     

Protochlorophyllide b does not occur in barley etioplasts

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection < 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH :protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80-84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

Chloroplast biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants

It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C. (2003) J. Biol. Chem. 278, 800-806). It has also been claimed that extraction of barley etioplasts with 100% acetone containing 0.1% diethyl pyrocarbonate prevents the conversion of Pchlide b to Pchlide a and leads to the detection of large amounts of Pchlide b in the isolated etioplasts. In this work the extraction protocol of Reinbothe et al. is compared with the more conventional 80% aqueous acetone extraction method. No Pchlide b was detected either in etiolated barley leaves or isolated barley etioplasts irrespective of the extraction protocol. On the other hands, small amounts of Pchlide b were detected in green barley leaves and isolated chloroplasts, extracted either with 80% acetone or 100% acetone containing 0.1% diethyl pyrocarbonate. It is concluded that the proposed occurrence of a light-harvesting POR-Pchlide-a,b complex in etiolated plant tissues is untenable, and its ensuing consequences and implications, for the greening process, are irrelevant.     

MACROMOLECULAR PHYSIOLOGY OF PLASTIDS : VIII. Pigment and Membrane Formation in Plastids of Barley Greening under Low Light Intensity

Sequential changes occurring in the etioplasts of the primary leaf of 7-day-old dark-grown barley seedlings upon continuous illumination with 20 lux have been investigated by electron microscopy, in vivo spectrophotometry, and thin-layer chromatography. Following photoconversion of the protochlorophyllide pigment to chlorophyllide and the structural transformation of the crystalline prolamellar bodies, the tubules of the prolamellar bodies are dispersed into the primary lamellar layers. As both chlorophyll a and b accumulate, extensive formation of grana takes place. After 4 hr of greening, protochlorophyllide starts to reaccumulate, and concomitantly both large and small crystalline prolamellar bodies are formed. This protochlorophyllide is rapidly photoconverted upon exposure of the leaves to high light intensity, which also effects a rapid reorganization of the recrystallized prolamellar bodies into primary lamellar layers.     

High salt stress induces swollen prothylakoids in dark-grown wheat and alters both prolamellar body transformation and reformation after irradiation

High salinity causes ion imbalance and osmotic stress in plants. Leaf sections from 8-d-old dark-grown wheat (Triticum aestivum cv. Giza 168) were exposed to high salt stress (600 mM) and the native arrangements of plastid pigments together with the ultrastructure of the plastids were studied using low-temperature fluorescence spectroscopy and transmission electron microscopy. Although plastids from salt-treated leaves had highly swollen prothylakoids (PTs) the prolamellar bodies (PLBs) were regular. Accordingly, a slight intensity decrease of the short-wavelength protochlorophyllide (Pchlide) form was observed, but no change was found in the long-wavelength Pchlide form emitting at 656 nm. After irradiation, newly formed swollen thylakoids showed traversing stromal strands. The PLB dispersal was partly inhibited and remnants of the PLBs formed an electron-dense structure, which remained after prolonged (8 h) irradiation. The difference in fluorescence emission maximum of the main chlorophyll form in salt-stressed leaves (681 nm) and in control leaves (683 nm) indicated a restrained formation of the photosynthetic apparatus. Overall chlorophyll accumulation during prolonged irradiation was inhibited. Salt-stressed leaves returned to darkness after 3 h of irradiation had, compared with the control, a reduced amount of Pchlide and reduced re-formation of regular net-like PLBs. Instead, the size of the electron-dense structures increased. This study reports, for the first time, the salt-induced swelling of PTs and reveals traversing stromal strands in newly formed thylakoids. Although the PLBs were intact and the Pchlide fluorescence emission spectra appeared normal after salt stress in darkness, plastid development to chloroplasts was highly restricted during irradiation.     

Photoreduction of Protochlorophyllide to Chlorophyllide in 2-d-old Dark-Grown Bean (Phaseolus vulgaris cv. Commodore) Leaves. Comparison with 10-d-old Dark-Grown (Etiolated) Leaves

Two-d-old leaves which do not contain prolamellar bodies synthesizeactive protochlorophyllide in darkness. When protochlorophyllideis photoreduced by one intense white flash, a main chlorophyllidespecies emitting at 690 nm is formed. After the photoreduction,the emission maximum is shifted to 675 nm within 5s. This resultsuggests that in young leaves, chlorophyllide formed after oneflash is quickly released from the active site of NADPH: protochlorophyllideoxidoreductase. This interpretation is strenghtened by time-resolvedfluorescence measurements at room temperature, showing that675 nm emitting chlorophyllide does not transfer excitationenergy to the 696 nm emitting chlorophyllide which is formedin very low amount. In 10-d-old bean leaves, the 690 nm chlorophyllideemitting species formed after one short flash undergoes thewellknown rapid and Shibata spectral shifts. The 675 nm emittingchlorophyllide appears only as a shoulder. At both ages, thefluorescence intensity of the active protochlorophyllide stronglydecreases during and after photoreduction, suggesting rapidmodifications in the close environment of the pigment. Key words: Bean, chlorophyllide, etioplast, proplastid, protochlorophyllide     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.     

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.