Transformation of plastids in soil‐shaded lowermost hypocotyl segments of bean (Phaseolus vulgaris) during a 60‐day cultivation period

The maintenance but substantial transformation of plastids was found in lowermost hypocotyl segments of soil‐grown bean plants (Phaseolus vulgaris cv. Magnum) during a 60‐day cultivation period. Although the plants were grown under natural light–dark cycles, this hypocotyl segment was under full coverage of the soil in 5–7 cm depth, thus it was never exposed to light. The 4‐day‐old plants were fully etiolated: amyloplasts, occasionally prolamellar bodies, protochlorophyllide (Pchlide) and protochlorophyll (Pchl) were found in the hypocotyls of these young seedlings. The 633 and 654 nm bands in the 77 K fluorescence emission spectra indicated the presence of Pchlide and Pchl pigments. During aging, both the Pchlide and Pchl contents increased, however, the Pchl to Pchlide ratio gradually increased. In parallel, the contribution of the 654 nm form decreased and in the spectra of the 60‐day‐old samples, the main band shifted to 631 nm, and a new form appeared with an emission maximum at 641 nm. The photoactivity had been lost; bleaching took place at continuous illumination. The inner membranes of the plastids disappeared, the amount of starch storing amyloplasts decreased. These data may indicate the general importance of plastids for plant cell metabolism, which can be the reason for their maintenance. Also the general heterogeneity of plastid forms can be concluded: in tissues not exposed to light, Pchl accumulating plastids develop and are maintained even for a long period.     

Nitrogen deficiency hinders etioplast development in stems of dark‐grown pea (Pisum sativum) shoot cultures

The effects of nitrogen (N) deprivation were studied in etiolated pea plants (Pisum sativum cv. Zsuzsi) grown in shoot cultures. The average shoot lengths decreased and the stems significantly altered considering their pigment contents, 77 K fluorescence spectra and ultrastructural properties. The protochlorophyllide (Pchlide) content and the relative contribution of the 654–655 nm emitting flash‐photoactive Pchlide form significantly decreased. The etioplast inner membrane structure characteristically changed: N deprivation correlated with a decrease in the size and number of prolamellar bodies (PLBs). These results show that N deficiency directly hinders the pigment production, as well as the synthesis of other etioplast inner membrane components in etiolated pea stems.     

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.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

Light and temperature regulation of greening in dark‐grown ginkgo (Ginkgo biloba)

The last steps of chlorophyll (Chl) biosynthesis were studied at different light intensities and temperatures in dark‐germinated ginkgo (Ginkgo biloba L.) seedlings. Pigment contents and 77 K fluorescence emission spectra were measured and the plastid ultrastructure was analysed. All dark‐grown organs contained protochlorophyllide (Pchlide) forms with similar spectral properties to those of dark‐grown angiosperm seedlings, but the ratios of these forms to each other were different. The short‐wavelength, monomeric Pchlide forms were always dominating. Etioplasts with small prolamellar bodies (PLBs) and few prothylakoids (PTs) differentiated in the dark‐grown stems. Upon illumination with high light intensities (800 μmol m^?2 s^?1 photon flux density, PFD), photo‐oxidation and bleaching occurred in the stems and the presence of ¹O₂ was detected. When Chl accumulated in plants illuminated with 15 μmol m^?2 s^?1 PFD it was significantly slower at 10°C than at 20°C. At room temperature, the transformation of etioplasts into young chloroplasts was observed at low light, while it was delayed at 10°C. Grana did not appear in the plastids even after 48 h of greening at 20°C. Reaccumulation of Pchlide forms and re‐formation of PLBs occurred when etiolated samples were illuminated with 200 μmol m^?2 s^?1 PFD at room temperature for 24 h and were then re‐etiolated for 5 days. The Pchlide forms appeared during re‐etiolation had similar spectral properties to those of etiolated seedlings. These results show that ginkgo seedlings are very sensitive to temperature and light conditions during their greening, a fact that should be considered for ginkgo cultivation.     

Plastid development in germinating wheat (Triticum aestivum) is enhanced by gibberellic acid and delayed by gabaculine

Etioplast development and protochlorophyllide (Pchlide) accumulation was studied in wheat seedlings ( Triticum aestivum L. cv. Walde, Weibull) grown in darkness on gibberellic acid (GA₃), gabaculine (3-amino-2,3-dihydrobenzoic acid), or on a combination of the two. The results were compared with the features of seedlings grown on water only. GA₃ enhanced shoot growth and promoted etioplast development. A correlation was observed between the appearance of prolamellar bodies (PLBs) and of phototransformable Pchlide. Gabaculine, a known tetrapyrrole biosynthesis inhibitor, delayed growth, slowed down the rate of PLB formation and caused structural alterations of the etioplasts up to 48 h of germination. Gabaculine also delayed the formation of phototransformable Pchlide as well as overall Pchlide biosynthesis, as determined by low-temperature fluorescence emission in vivo. The spectral blue-shift of newly formed chlorophyllide (Chlide) was delayed in irradiated dark-grown gabaculine-grown seedlings, indicating an inhibited dissociation of Chlide and NADPH-Pchlide oxidoreductase (Pchlide reductase: EC 1.3.1.33). Thus there is a close correlation between accumulation of Pchlide and etioplast development, also under conditions when development is enhanced or delayed.     

Delayed chlorophyll accumulation and pigment photodestruction in the epicotyls of dark-grown pea (Pisum sativum)

A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid chromatography analysis after continuous or flash-irradiation showed that the biosynthetic pathway from protochlorophyllide (Pchlide) to chlorophyll (Chl) a was markedly slowed down at the step of the reduction of geranylgeranyl(gg)-Chl to dihydrogeranylgeranyl (dhgg)-Chl in epicotyls, whereas phytyl-Chl was synthesized in leaves subjected to the same light treatments. Quantitative pigment analysis during continuous irradiations of different intensities also showed that significant Pchlide photodestruction occurred in epicotyls even under weak light. When both Pchlide and chlorophyllide and/or chlorophylls were present in epicotyls, Pchlide photodestruction was faster under 630-nm light than under 670-nm light, which indicates that this process is most efficiently promoted by Pchlide excitation. Pre-incubation of epicotyl segments with 10 m M ascorbate partly alleviated pigment photodestruction in white light. It is concluded that formation of photoactive Pchlide–Pchlide oxidoreductase complexes is important to prevent fast pigment photooxidation after Pchlide accumulation in the dark.     

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.     

Formation and Degradation of Protochlorophylls in Etiolated and Greening Cotyledons of Cucumber

The formation, degradation and phototransformation of protochlorophylls(Pchls) in the etiolated and greening cotyledons of cucumber(Cucumis sativus L.) werestudied using high-performance liquidchromatography. The pigment analysis of etiolated cotyledonsshowed the presence of four Pchls esterified with phytol, tetrahydrogeranylgeraniol(THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG).The content of Pchl THGG rapidly increased during dark developmentof seedlings and reached a maximal level at 4th day, then decreasedgradually. Unlike Pchl THGG, Pchl DHGG and Pchl GG showed asmall peak at 3rd day followed by a one-day lag, then accumulationbegan. The content of Pchl DHGG reached a maximal level at 12thday, then decreased rapidly, while Pchl GG continued to increaseand its maximal stage was not attained at 15th day. The contentof Pchl phytol remained very low during dark growth. These resultsmay indicate that with increasing age, the inactivation of hydrogenationof the alcohol moiety of Pchl proceeds stepwise at the sitesof Pchl THGG, Pchl DHGG and Pchl GG, in that order, withoutaffecting the esterification of Pchlide. The content of four Pchls remained unchanged before and after30-s illumination, indicating that none of the four Pchls istransformed to chlorophyll by light. Under continuous illumination,Pchls decreased exponentially or linearly at a rather slow rate.Thus, the four Pchls are not direct precursors for chlorophylland are metabolized slowly under greening. (Received December 6, 1982; Accepted April 13, 1983)     

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.     

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.     

Changing ratios of phototransformable protochlorophyll and protochlorophyllide of bean seedlings developing in the dark

Protochlorophyll (Pchl) and protochlorophyllide (Pchlide) are at comparable levels in 2-day-old (young) etiolated bean leaves (Phaseolus vulgaris L. var. Red Kidney). During subsequent development in the dark, both pigments increase, but the rate of Pchlide increase is greater than that of Pchl, leading to the commonly observed predominance of Pchlide beyond 7 days (old leaves). Both protopigments are phototransformable to their respective chlorophyll(ide) photoproducts throughout dark development. The rate of protopigment regeneration in young leaves after illumination is rapid and displays no lag, whereas this process in old leaves begins slowly and achieves only about one-fifth the rate of younger leaves. The rate of chlorophyllide esterification is also faster in the younger tissue. Since the proplastid-related properties of young bean leaves are quite similar to those of Euglena, young leaves and Euglena may represent an evolutionarily primitive case compared with older bean leaves which contain etioplasts. Since Euglena and young beans green perfectly well when exposed to light, the extensive modifications associated with prolonged dark growth do not seem to be obligatory for plastid development. The properties of older beans are viewed as being the consequence of prolonged etiolation which may provide a faster rate of plastid development and appearance of photosynthesis as the plant nears the limits of its stored reserves.     

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

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     

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.     

Early and late plastid development in response to chill stress and heat stress in wheat seedlings

Five-day-old etiolated wheat (Triticum aestivum L.) seedlings were transferred to 7°C (chill stress), 25°C (control), and 42°C (heat stress) and were kept in the dark or light for different time periods. Plastids were isolated from the control and stressed seedlings, and their low-temperature (77 K) fluorescence emission spectra were monitored. Most of the Protochlorophyllide (Pchlide) present in heat-stressed etiolated seedlings were in nonphototransformable form. The phototransformable Pchlide (F657) rapidly decreased when 5-day-old etiolated seedlings were transferred to 42°C in the dark for 24 h. A flash illumination of 0.2 s given to etiolated heat-stressed seedlings resulted in substantial arrest of Shibata shift, while in chill-stress conditions, it was only partially affected. In high temperature, due to disaggregation of polymeric Pchlide–Pchlide oxidoreductase (POR)–nicotinamide adenine dinucleotide phosphate (NADPH) molecules, the conversion of nonphototransformable Pchlide to its phototransformable form is substantially delayed resulting in impaired Shibata shift and belated development of the core antenna CP47 Photosystem II (PSII). Chill stress, however, did not disaggregate the polymeric Pchlide–POR–NADPH molecule-suppressed Pchlide and Chl synthesis and impaired of the assembly of PSII core antenna CP47 that emits F695 and PSI that emits F735. The decreased gene/protein expression and reduced posttranslational import of plastidic proteins, importantly POR in temperature-stressed plants, may be responsible for the delay in conversion of nonphototransformable to phototransformable form of Pchlide and plastid biogenesis.     

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea ( Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m⁻² s⁻¹photon flux density (PFD) white light and subsequently incubated in total darkness for 4–24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form—dominating in epicotyls of dark-grown seedling—required 18–24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4–7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.     

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.     

A new pathway of chlorophyll biosynthesis from long-wavelength protochlorophyllide Pchlide 686/676 in juvenile etiolated plants

By spectral methods, the final stages of chlorophyll formation from protochlorophyllide were studied using etiolated pea, bean, barley, wheat and maize plants in early stages (4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7–9-day-old) plants, a new reaction chain was found, which started with phototransformation of the long-wavelength form Pchlide 686/676(440) into Pchlide 653/648(440). (Pchlide 653/648(440) differs from the main known precursor form Pchlide 655/650(448)). The subsequent photoreduction of Pchlide 653/648(440) leads to the formation of Chlide 684/676(440), which is transformed into Chl 688/680(440) in the course of a dark reaction. After completion of this reaction, fast (20–30 s) quenching of the low-temperature fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II core. This revised version was published online in June 2006 with corrections to the Cover Date.