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.     

Light-regulated pigment interconversion in pheophytin/chlorophyll-containing complexes formed during plant leaves greening

Upon illumination of etiolated maize leaves the photoconversion of protochlorophyllide Pchlide 655/650 into chlorophyllide Chlide 684/676 was observed. It was shown that chlorophyllide Chlide 684/676 in the dark is transformed into pheophytin Pheo 679/675 and chlorophyll Chl 671/668 by means of two parallel reactions, occurring at room temperature: Chlide 684/676. The formed pheophytin Pheo 679/675 was unstable and in the dark was transformed into chlorophyll Chl 671/668 in a few seconds: Pheo 679/675 Chl 671/668. The last reaction is reversed by the light: Chl/668 Pheo 679/675. Thus, on the whole in the greening etiolated leaves this process occurs according to the following scheme:The observed light-regulated interconversion of Mg-containing and Mg-free chlorophyll analogs is activated by ATP and inhibited by AMP.Abbreviations Chl- chlorophyll - Chlide- chlorophyllide - Pchlide- protochlorophyllide - Pheo- pheophytin - PS II RC- Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to the maximum position of the low-temperature fluorescence band (nm), the second number to the maximum position of the longwave absorption band     

The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.     

High salt stress in wheat leaves causes retardation of chlorophyll accumulation due to a limited rate of protochlorophyllide formation

When exposed to salt stress, leaves from dark-grown wheat seedlings ( Triticum aestivum , cv. Giza 168) showed reduced accumulation of chlorophyll during irradiation. To elucidate the mechanism behind salt-influenced reduction of chlorophyll biosynthesis, we have investigated the effect of salt stress on the spectral forms of Pchlide, the phototransformation of Pchlide to Chlide, the Shibata shift, the regeneration of Pchlide and the accumulation of Pchlide from 5-aminolevulinic acid (ALA). We found that the phototransformation of Pchlide to Chlide was not affected by salt stress. The blue shift (Shibata shift) of newly formed Chlide was delayed both after flash irradiation and in continuous light. The reformation of Pchlide in darkness after a flash irradiation or after a period of 3-h irradiation was retarded in the salt-treated leaves. However, after a 20-h dark period, Pchlide was reformed even in salt-treated leaves but the formation of short-wavelength Pchlide was suppressed. Compared to controls, salt treatment also reduced the amount of Pchlide accumulated in leaves floated on ALA. The increase in the low temperature fluorescence emission spectrum at 735 nm, which occurred gradually during several hours of irradiation with continuous light in control leaves, was completely suppressed in salt-treated leaves. It is concluded that salt stress inhibits chlorophyll accumulation partly by reducing the rate of porphyrin formation but, as discussed, also by a possible reduction in the formation of chlorophyll-binding proteins.     

Prolonged salt stress alters the ratios of protochlorophyllide spectral forms in dark-grown wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and influences chlorophyll <Emphasis Type="Italic">a</Emphasis> accumulation following irradiation

To examine the effects of salt stress on dark-grown wheat (Triticum aestivum), seedlings of the salt-tolerant cultivar Sids 1 and the susceptible cultivar Giza 168 were grown in darkness for 14 days in nutrient solution with and without 200 mM of supplementary salt (100 mM of NaCl and 100 mM of KCl). During this time, we monitored their protochlorophyllide (Pchlide) contents, ratios of photoactive to non-photoactive forms of Pchlide (from 655/633-nm emission ratios in their 77 K fluorescence emission spectra) and (following flash irradiation) ratios of newly formed chlorophyllide (Chlide) to non-photoactive Pchlide. In addition, the accumulation of chlorophyll a in leaf sections was monitored during prolonged (24 h) irradiation. The results depended on the developmental state of the seedlings. However, the salt stress treatment caused marked increases in both Pchlide contents in dark-grown leaves and in Chlide contents following irradiation of leaf sections of both cultivars. The ratio of phototransformable to non-phototransformable Pchlide and the abundance of newly formed Chlide were also increased by the salt stress. Further, leaves of salt-stressed seedlings consistently accumulated more chlorophyll a than leaves of unstressed seedlings when floating on the nutrient solution (with or without supplementary salt) in continuous white light. The findings are consistent with the hypothesis that increased levels of the long-wavelength form of Pchlide contribute to protective mechanisms against salt stress.     

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.     

Induction of the Photosystem 2 Dark Formation in Etiolated Leaves with the Involvement of Exogenous Chlorophyllides

The delayed luminescence (DL) of photosystem 2 (PS2) after infiltration of 7-d-old etiolated barley leaves with chlorophyllides (Chlide) a or b followed by 2.5 h dark incubation was studied. Chlide a caused a very weak DL of PS2 just at the beginning of irradiation and the intensity of this DL was not higher when the infiltration medium contained 2 mM of NADPH. Chlide b was a somewhat more efficient inducer of PS2 formation in the dark and NADPH enhanced this efficiency 4.5 times though it did not affect the amount of esterified Chlides. The photoconversion of endogenous Pchlide led to a much higher intensity of the DL in comparison with the infiltration of Chlides, while the total amount of chlorophyll (Chl) formed was almost unchanged. The use of Chlide b together with the acetone extract from green leaves, devoid of pigments, resulted in the DL intensity comparable with that observed after Pchlide photoconversion followed by 2.5 h incubation in the dark. Dark formation of active PS2 in etiolated leaves was shown for the first time. Thus the dark formation of active PS2 may require Chl b, NADPH, and some unidentified water-soluble factor(s), synthesized in the dark after a short irradiation of etiolated leaves and inherent in green leaves. This revised version was published online in September 2006 with corrections to the Cover Date.     

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.     

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

Correlation between chlorophyllide esterification, Shibata shift and regeneration of protochlorophyllide650 in flash-irradiated etiolated barley leaves

The pigments of etiolated leaves of barley ( Hordeum vulgare L.) were analysed during dark periods after flash illumination, and the results were compared with in vivo spectroscopy of the leaves. Pretreatment of the leaves with kinetin slightly stimulated and pretreatment with NaF and anaerobiosis inhibited the esterification of chlorophyllide a (Chlide) at 10–40 min after the flash, whereas the rapid esterification within 30 s after the flash remained unchanged. Irrespective of pretreatment, the amount of esterified pigment was, at any time, identical with the amount of pigment that had shifted its absorption from 684 to 672 nm (Shibata shift). Cycloheximide (CHI) had only a small inhibitory effect on esterification, but drastically inhibited the hydrogenation of geranylgeraniol to phytol, bound to Chlide. The regeneration of long-wavelength protochlorophyllide a (Pchlide650) was stimulated by kinetin and inhibited by CHI and NaF. During the rapid phase (0–30 s after the flash), the esterification was faster than the regeneration of Pchlide650, and this, in turn, was faster than the formation of photoactive Pchlide. The kinetics changed after pretreatment with 5-aminolaevulinic acid: regeneration of Pchlide650 was the fastest reaction and the Shibata shift preceded the esterification of Chlide. The results are discussed as pigment exchange reactions at NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99.1).     

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.     

The photoenzymatic cycle of NADPH: protochlorophyllide oxidoreductase in primary bean leaves (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) during the first days of photoperiodic growth

The photoenzymatic cycle of the light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) was investigated in situ during early stages of development of bean leaves under light-dark cycles (LDC). In the experimental system used in this study, prolamellar bodies developed during night periods and disappeared during light periods. This was accompanied by changes in the photoactive to non-photoactive Pchlide ratio, which was higher at the end of the light period, and tended to increase with the number of LDC's. Flash-induced absorbance changes in the Chlide absorption region (700 nm) were used in order to monitor the formation of short- and long-wavelength forms of Chlide (C670-675 and C682-694), which correspond to free Chlide and aggregated Chlide-NADPH-LPOR complexes, respectively. The ratio of long-wavelength to short-wavelength Chlides after one flash increased with the number of LDC's, and was higher in leaves collected at the end of light periods, compared to leaves collected at the end of night periods. During light periods, photoactive Pchlide regeneration and Chlide phytylation were completed within 1 min after flash-induced formation of long-wavelength Chlide. The results show for the first time that the photoenzymatic LPOR cycle proceeds through similar steps, but at much faster rates, during photoperiodic greening than in the previously studied leaves of etiolated plants. In particular, the parallel formation of two Chlide species always occurs, but the ratio of the two species depends on the ratio of photoactive to non-photoactive Pchlide and on light or dark adaptation.     

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.     

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     

Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness

During illumination of dark-grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark-grown plants, maize ( Zea mays cv. Sundance), wheat ( Triticum aestivum cv. Kosack), pea ( Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato ( Solanum lycopersicum ), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re-accumulated. The proportions of different Pchlide forms within the pool of re-accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark-grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long-wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash-induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark-grown leaves and were specific for the plant variety. Thus Chl formation in both dark-grown and light-grown leaves can occur in a similar way through the main photoactive long-wavelength form of Pchlide.     

Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves

Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.     

Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, we describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of (1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and (2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, (3) high concentrations of ATP, MgCl(2), and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg(2+) for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.