The Effect of Diuron and 5-Aminolevulinic Acid on the Maintenance of Stable Chlorophyll Content in Greening Barley Leaves

Chlorophyll (Chl) accumulation and delayed luminescence of PSII were compared in greening barley leaves pretreated and untreated with diuron (DCMU) in the etiolated state, and reactions of two photosystems were studied in the plastids isolated from the pretreated and untreated leaves. The effect of treatment in light of post-etiolated leaves after 40-h illumination with 5-aminolevulinic acid (ALA), on the content of Chl and its precursor, protochlorophyllide (PChld) was also studied. The pretreatment of etiolated leaves with DCMU did not affect the rate of greening and the stable level of Chl content in barley. ALA, when introduced to leaves after the termination of Chl accumulation, increased PChld, but not Chl level. We suppose that the primary cause of greening cessation in etiolated leaves is the inhibition and cessation of the synthesis of apoproteins of pigment–protein complexes. The exhaustion of binding sites for newly synthesized Chl molecules leads to their retention in the so-called retroinhibitory pool of Chl, thus resulting in the inhibition of ALA synthesis by a negative feedback mechanism.     

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.     

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.     

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 effect of simulated microgravity on formation of the pigment apparatus in etiolated barley seedlings

The effect of horizontal clinorotation on the dynamics of the accumulation of the main photosynthetic pigments in the greening of 6-day-old etiolated barley seedlings has been studied. The content of protochlorophillide, the direct precursor of chlorophyll a, in clinorotated seedlings in the dark was 9–20% lower than in the control group. After exposure of barley seedlings to light for 12 h under clinorotation, chlorophyll accumulation lagged behind the control by 45% and reached the control value after 48–72 h. The total content of carotenoids increased many fold during greening; at the first stage the carotenoid level in clinorotated seedlings was less than in the control. The synthesis rates of δ-aminolevulinic acid and δ-aminolevulinate dehydratase activity in clinorotated seedlings were slower than in the control after 24 h of greening and after 72 h of greening reaching the control values. The activity of Mg-protoporphyrin IX chelatase catalyzing the incorporation of Mg ions in the structure of chlorophyll a, did not change when exposed to clinorotation. The results we obtained show inhibition of the initial stages of chlorophyll biosynthesis in the conditions of simulated microgravity. The light, to a certain extent, decreases the negative effect of microgravity on the formation of the photosynthetic apparatus in plants.     

Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.     

Chloroplast protection in greening leaves

Changes in photosynthetic activity, leaf pigments and the activities of enzymes that scavenge damaging oxygen species in chloroplasts were followed during the greening of 8-day-old etiolated pea (Pisum sativum L. cv. Meteor) seedlings. Accumulation of chlorophyll and carotenoids was accompanied by development of photosynthetic activity. Carotenoids present in etiolated leaves, and the high ratio of carotenoid to chlorophyll detected during the early hours of greening are suggested to provide important protection against singlet oxygen. Superoxide dismutase, ascor-bate peroxidase and glutathione reductase, which scavenge superoxide and hydrogen peroxide in chloroplasts, are present at high activities in etiolated leaves and throughout greening. The mechanisms by which developing chloroplasts may generate damaging oxygen species, and the role of these scavengers during greening is discussed.     

Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably δ-amino-levulinic acid synthetase) is not longer than 10 minutes.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

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

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

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.     

Characterization of protoheme levels in etiolated and greening plant tissues

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).     

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

Chlorophyll turnover in etiolated greening barley transferred to darkness: Isotopic (1-14C glutamic acid) evidence of dark chlorophyll synthesis in the absence of chlorophyll accumulation

Synthesis of chlorophyll was initiated in 5- to 6-day-old dark-grown barley (Hordeum vulgare L. cv. Clipper)seedlings by exposing them to light in the presence of 1-¹⁴ C glutamic acid supplied via the roots.The plants were then returned to darkness. At the end of light treatment (_T) and after 7 or 18 h dark treatment chlorophylls a and b were extracted, quantified (μgleaf¹). purified by HPLC to their magnesium-free derivatives (pheophytin a and b) and their molar radioactivities determined. After 2 h exposure to light followed by 6 h illumination in the presence of 1-¹⁴ C glutamic acid, seedlings had accumulated 4-7 nmol chlorophyll leaf¹ and had incorporated between 900-1 350 Bq (g fresh weight)¹ of radioactive label into the chlorophyll pool. When seedlings were transferred to darkness, label continued to be incorporated and after 18 h the radioactivity of the chlorophyll pool had increased by 300-700 Bq (g fresh weight)¹. Net chlorophyll content, however, remained constant during dark treatment. The increase in radioactivity of the chlorophyll pool in darkness represented the difference between a net increase of label incorporated into chlorophyll a and a small loss of label from chlorophyll b. The absence of measurable radioactivity in the phytol moiety of labelled chlorophyll a, extracted at the endof dark treatment, demonstrated thatincorporation of label was into the tetrapyrrole moiely of chlorophyll and not into the phytol chain. Light-independent incorporation of 1-¹⁴ C glutamic acid into chlorophyll of greening barley seedlings transferred to darkness indicates that chlorophyll synthesis continues when light is withheld. We interpret the net gain in radioactivity of chlorophyll in darkness, in the absence of a net gain in chlorophyll content, to chlorophyll turnover i.e. to simultaneous synthesis and breakdown of chlorophyll when etiolated greening barley seedlings are transferred to darkness.     

Some effects of enhanced UV-B irradiation on the growth and composition of plants

Barley (Hordeum vulgare), corn (Zea mays), bean (Phaseolus vulgaris), and radish (Raphanus sativus) seedlings were continuously irradiated under a lighting device for 5–10 d at an increased ultraviolet (UV)-B fluence rate. In their growth parameters, composition, and leaf surface, these four species responded differently to the increased UV-B exposure. Bean seedlings suffered the most serious effects, radish and barley less, and corn was hardly influenced at all. In all plant species, the fresh weight, the leaf area, the amounts of chlorophylls, carotenoids and the galactolipids of the chloroplasts were reduced. The lipid content of the corn and bean seedlings also diminished. But all the irradiated plants showed a rise in their protein content compared to the control plants. The content of flavonoids increased in barley and radish seedlings by about 50%. The effects on growth parameters and composition were more extensive with increasing UV-B fluence rates, at least as shown in the case of barley seedlings. The fresh weights fell proportionally with the chlorophylls and carotenoids. In contrast, the flavonoid content of barley leaves rose parallel to the increasing UV-B fluence rates and reached 180% of the value in the control plants with the highest UV-B fluence rate. Scorching appeared regularly in the form of bronze leaf discoloration at the highest UV-B fluence rates. Scanning electron micrographs of the leaf surface of UV-B irradiated plants showed deformed epidermal structures.Abbreviations MGDG monogalactosyldiglyceride - DGDG digalactosyldiglyceride - SL sulfoquinovosyldiglyceride - PG phosphatidylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - LA leaf are - FW fresh weight - DW dry weight - SEM scanning electron microscopy - C total carotenoids - Chl total chlorophyll     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.     

Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b₆/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b₆, and Rieske Fe-S center protein in cytochrome b₆/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b₆/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b₆, and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.