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.     

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.     

Formation of the photosynthetic apparatus during greening of cadmium-poisoned barley leaves

The effect of cadmium on the formation of the photosynthetic apparatus of greening barley (Hordeum vulgare L. cv. Triangel) leaves has been investigated. Cadmium treatment of dark-grown leaves strongly reduced the extent of chlorophyll accumulation during greening. Low-temperature fluorescence emission showed, however, that neither the synthesis nor photoconversion of protochlorophyllide was inhibited, although a blue shift of the main fluorescence emission from 685 to 668 mm was found. Chlorophyll fluorescence lifetime was followed by measuring the phase-shift angle of modulated emission. Whereas this parameter normally decreases rapidly during greening, this change proceeded noticeably slower with increasing severity according to cadmium concentration. Cadmium also decreased the variable part of fluorescence induction. These results suggest that the cadmium in greening leaves, rather than interfering with chlorophyll biosynthesis, acts mainly by disturbing the integration of chlorophyll molecules into the stable complexes required for normal functional photoysnthetic activity.     

Regulation of the carotenoid content and chloroplast development by levulinic acid

Chloroplast development and chlorophyll biosynthesis are co-regulated. To understand the mechanism of regulation of chloroplast biogenesis by chlorophyll, development of the photosynthetic apparatus was monitored during greening of etiolated barley leaf discs in the presence of levulinic acid, an inhibitor of chlorophyll biosynthesis. Although not a direct inhibitor of carotenoid biosynthesis, treatment by levulinic acid resulted in a linear reduction in both chlorophyll and carotenoid contents. Chlorophyll biosynthesis appeared to control that of carotenes. In the presence of levulinic acid, photosystem II (PSII) activity decreased while photosystem I (PSI) activity increased when expressed on a chlorophyll basis. However, the activities of both photosystem I and II decreased when expressed on a per plastid basis. As expected, in the presence of low amounts of chlorophyll, the light-harvesting chlorophyll-protein complex II (LHCPII) was not visible in Coomassie-stained gels in 20 m M levulinic acidtreated tissues, but was detected as a faint band by immunoblotting. This small amount of the LHCPII induced significant amounts of grana stacking, which was monitored as an increase in the ratio of variable to maximum fluorescence. When levulinic acid was washed from the leaf discs and the latter allowed to green in its absence, the chlorophyll and carotenoid contents and the photosynthetic activities approached the control values. Levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and removed by washing the leaves to restore the developmental process without any apparent toxic effect. Results demonstrate that biosynthesis of carotenes is regulated by that of chlorophylls and extremely low amounts of the LHCPII can induce grana stacking.     

Formation of photosynthetic pigments and quinones and development of photosynthetic activity in barley etioplasts during greening in intermittent and continuous white light

The appearance and development of photosynthetic activity, and the accumulation of chlorophylls, carotenoids and quinones, was investigated in etiolated barley shoots (Hordeum vulgare L. cv. Villa) during greening in flash light, periodic light-dark cycles, and continuous white light. Greening and the development of photosynthetic activity was delayed in flash and periodic light compared to continuous white light. Photosystem II activity occurred after 6 light-dark cycles and increased continuously during greening. After 3 h greening in continuous white light, photosystem II activity appeared with a very high rate and decreased to that of a green leaf after 50 h greening. Parallel to the development of photosynthetic activity, light stimulated the biosynthesis of prenyllipids. Moreover, chlorophylls and those carotenoids and quinones that are contained in etioplasts in relatively small amounts, were particularly enhanced in their biosynthesis. Chlorophyll a was synthesized without a lag phase during greening in flash light, whereas a 2 h lag phase occurred in continuous white light. In all three modes of illumination the formation of chlorophyll a exceeded that of chlorophyll b. After 4 flashes and 2 light-dark cycles, chlorophyll b could be detected with a very high initial a/b ratio. Higher chlorophyll a/b ratios were reached after 200 flashes (a/b=10.9) and 50 light-dark cycles (a/b=6.6) than after 50 h continuous white light (a/b=3.3). The formation of carotenes, lutein, violaxanthin and neoxanthin was also enhanced by light. This was also confirmed for plast-ouinone-9. ?-tocopherol,α-tocoquinone and phylloquinone. A comparison of the carotenoid and quinone composition of the differentiating thylakoid membrane before and after onset of photosynthesis, reveals that the photosynthetic membrane is already equipped with photosynthetic pigments and quinones before the appearance of photosystem II activity. It is concluded that during development of the photo-synthetic apparatus the thylakoid membrane with its structural and functional constituents is formed first. In a second and slower process the water splitting enzyme system and enzymes of the Calvin cycle are activated.     

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.     

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.     

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.     

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.     

Effect of clinorotation on growth and pigment biosynthesis in etiolated barley plants

Altered gravity conditions (hyper- or hypogravity) leads to changes in metabolic processes in living organisms (Kordyum, 1997). One important subject of plant space biology is the investigation of the effects of microgravity conditions on the development of the photosynthetic apparatus. The impact of microgravity on the photosynthetic apparatus of plants has been studied in a number of space-flight experiments. Particularly, the Chl (a+b) content and Chl a/b ratio were determined in several experiments, however the results did not allow understanding how microgravity affects pigment apparatus of the plants since presented contradictory results [Laurinavichius et al. 1984; Volovik et al, 1999]. To elucidate how clinorotation affects pigment apparatus formation in etiolated plants, we studied morphological characteristics and pigment (chlorophyll and carotenoids) biosynthesis in the first period of greening of barley plants.     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

Excitation kinetics of chlorophyll fluorescence during light-induced greening and establishment of photosynthetic activity of barley seedlings

Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and lightadapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material.     

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.     

The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

The light-induced development of nitrate reductase in etiolated barley shoots: An inhibitory effect of laevulinic acid

Induction of nitrate reductase EC 1.6.6.1 in etiolated barley (Hordeum vulgare L., var. Proctor) required continuous illumination and showed a lag period of about three hours. During the first 16 h of illumination the ratio NADH/NAD and NADPH/NADP, taken as a measure of internal oxidation reduction potential, declined. The inhibitor DCMU applied to whole leaves at concentrations shown to inhibit the reduction of cytochrome f by Photosystem 2 light did not inhibit the induction of nitrate reductase nor did it diminish the ratio of reduced to oxidised puridine nucleotides in the early hours of greening. It was concluded that light driven electron flow was not necessary for nitrate reductase induction. Chloramphenicol gave a slight inhibition of nitrate reductase induction. Laevulinic acid was added to greening barley leaves to inhibit tetrapyrrole pigment biosynthesis and plastid development. It strongly inhibited chlorophyll synthesis and nitrate reductase induction, with relatively little effect upon Photosystem 1 and 2 activities in isolated plastids. The activities of other inducible enzymes and control enzymes were little affected by laevulinic acid. Laevulinic acid also inhibited nitrate reductase induction by added nitrate in fully-greened illuminated plants grown in nitrate-free medium and so is unlikely to be acting through inhibition of plastid development. This inhibitor lowered the level of protohaem in whole leaves and plastids of greening barley and it is postulated that it may diminish the protohaem available for the assembly of a cytochrome b component of nitrate reductase.Abbreviations DCMU 3-(3:4-Dichlorophenyl)-1:1-dimethylurea - LA laevulinic acid     

Development of chlorophyll and hill activity

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding δ-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with δ-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.     

Effects of UV-B radiation on Chl fluorescence of greening barley (Hordeum vulgare L.) seedling

Effects of UV-B radiation on the developing chloroplast of barley (Hordeum vulgare L.) seedling during greening were determined by Chl contents, Fo, Fv and fluorescence quenching coefficients. In greening of etiolated barley seedling, the value of Fo was greatly increased after the initiation of greening. However Fv and Fv/Fo were gradually increased. In greening with the additional irradiation of UV-B radiation, the value of Fo was strikingly decreased than that of the control after the initiation of greening, but Fv was gradually decreased from than that of the control during the greening period. These results suggest that the function of light-harvesting Chl are more sensitive than photosynthetic electron transport system by UV-B. Chl contents, Fv/Fo, qP and qNP, were decreased from than that of the control during the 72 h greening, especially, qR was strikingly decreased, but qE was slightly decreased by UV-B. These suggest that the sites of inhibition by UV-B are PSII and all sites of photosynthetic electron transport system. But PQ pool seems to be slightly inhibited by UV-B.