Russian Journal of Plant Physiology, Vol. 48, No. 2, 2001, pp. 148–154. Translated from Fiziologiya Rastenii, Vol. 48, No. 2, 2001, pp. 177–184. Original Russian Text Copyright © 2001 by Rassadina, Lezhneva, Yaronskaya, Taran, Averina.
The Biogenesis of the Photosynthetic Apparatus and the Activity of Chlorophyll Biosynthesis in a Plastome Mutant of Sunflower V. V. Rassadina*, L. A. Lezhneva*, E. B. Yaronskaya*, S. F. Taran**, and N. G. Averina* *Institute of Photobiology, National Academy of Sciences of Belarus, Akademicheskaya ul. 27, Minsk, 220072 Belarus; fax: (10 375 17) 284-2359; e-mail:
[email protected] ** Institute of Biology, Rostov State University, Rostov-on-Don Received August 19, 1999
Abstract—It was demonstrated that, in the phenotypically colorless leaves of a sunflower (Helianthus annuus L.) plastome mutant with a heavily reduced level of chlorophyll, all pigment–protein complexes of the photosynthetic apparatus typical for the wild type were present. However, the ratio between them was changed. During aging of the mutant leaves, pigment–protein complexes of photosystem I were destroyed first followed by those of photosystem II. Chlorophyll a/b-containing light-harvesting complex II turned out to be the most stable. This conforms to an increased content of lutein and violaxanthin in mutant leaves. A synchrony of the decreases in the chlorophyll and 5-aminolevulinic acid (ALA) contents throughout all ontogenetic stages of the colorless mutant leaves made it possible to suggest that a decrease in the synthesis and resynthesis of chlorophyll during the formation and development of such leaves is caused by the inhibition of an initial stage of this process, namely, the biosynthesis of ALA molecules. The activity of the enzymes converting ALA into protochlorophyllide did not limit chlorophyll biosynthesis. Possible mechanisms controlling the synthesis of ALA destined for chlorophyll formation are discussed. Key words: Helianthus annuus - sunflower plastome mutant (albina type) - 5-aminolevulinic acid - S-adenosylL-methionine: Mg-protoporphyrin IX methyltransferase - carotenoids - Mg-protoporphyrin IX and its monomethyl ester - protochlorophyllide - protoporphyrin IX - chlorophyll
INTRODUCTION The development of chloroplast structure and functions in photosynthesizing organisms is under the genetic control of the interrelated nuclear and chloroplast genomes, which ensures a coordinated synthesis of pigment and protein components of the photosynthetic apparatus. The chloroplast genome consists of several tens of genes encoding separate components of the photosynthetic apparatus. They include proteins of the reaction centers of both photosystems [1] and the trnE gene encoding tRNAGlu active in the synthesis of the first specific precursor of chlorophyll, viz., the molecules of 5-aminolevulinic acid [2]. Information on the enzymes of the chlorophyll biosynthetic chain and on the chlorophyll a/b-binding polypeptides of the photosynthetic apparatus is localized in the nuclear genome [3, 4]. These proteins are synthesized by cytoplasmic ribosomes and transported into plastids, where they are processed and associated with definite structural elements of organelles. Evidence for the mechanisms controlling the expression of nuclear genes encoding chloroplast proteins is scarce [5, 6]. A hypothesis concernAbbreviations: ALA—5-aminolevulinic acid; FS—fluorescence spectra; LHCII—light-harvesting complex II; MT—S-adenosylL-methionine: Mg-protoporphyrin IX methyltransferase; MgPP(E)—Mg-protoporphyrin IX (its monomethyl ether); Pchlide—protochlorophyllide; PP—protoporphyrin IX; PSI— photosystem I; PSII—photosystem II.
ing the existence of a specific plastidal factor transmitting information on the state and the developmental stage of chloroplasts to the nuclear genome is widely discussed at present [7, 8]. The cell responds to this plastidal signal by providing an optimal transcription of nuclear genes that encode a number of chloroplast proteins. However, there is no information concerning the nature of this plastidal factor and very little evidence concerning its effect on the expression of genes that encode the enzymes of the chlorophyll biosynthetic chain [9, 10]. The use of chlorophyll-deficient nuclear and plastome mutants of higher plants with a disrupted structural organization of photosynthetic membranes as well as the analysis of enzymes involved in the chlorophyll biosynthesis in these mutants might contribute to the elucidation of this problem. Only a few pigment mutants contain lesions directly related to chlorophyll synthesis [11]. In other cases, pigments are lost due to pleiotropic effects induced by a breakdown of the metabolism or structure of a cell [12]. Phenotypically colorless leaves of the sunflower (Helianthus annuus L.) plastome mutant (2-24 line) demonstrate drastic changes in the chloroplast ultrastructure characterized by a virtually complete absence of thylakoid membranes, a decrease in the number of plastidal and cytoplasmic ribosomes, and the lack of some LHCII polypeptides [13, 14]. All these facts sug-
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gest that, in this mutant, the plastidal factor controlling the activity of a number of nuclear genes, which encode chloroplast proteins, is disrupted [14]. The objective of this work was to determine whether the disruption of chlorophyll biosynthesis is directly related to the pigment deficiency in mutant leaves. We also investigated special features of the biogenesis of the photosynthetic apparatus in the mutant. MATERIALS AND METHODS Plant cultivation. Sunflower (Helianthus annuus L.) plants were grown in a 14/10-h light/dark regime using LB-40 luminescence tubes (an irradiance of 11 W/m2) at 22 or 26°C to a 38-day age. Phenotypically colorless leaves of a mutant (2-24 line, albina type) were investigated. Green leaves of parent 3629 line were used as a control. ALA accumulation. Detached leaves were incubated on the 0.05 M solution of levulinic acid in a 0.1 M Tris– HCl buffer, pH 7.5, either under illumination or in darkness for 3 h. The ALA determination was carried out according to [15]. A coefficient of molar extinction equal to 6.8 × 104/(M cm) was used for calculating the ALA content [16]. The pellet left after ALA extraction was used for determining the protein content according to Lowry et al. [17]. Determination of the activity of S-adenosyl-Lmethionine: Mg-protoporphyrin IX methyltransferase. Enzyme activity was determined as the amount of Mgprotoporphyrin IX monomethyl ester formed in leaf homogenates in the presence of exogenous S-adenosylL-methionine and Mg-protoporphyrin IX [18]. Resynthesis of protochlorophyllide and accumulation of porphyrins from exogenous ALA. To study Pchlide resynthesis, plants were kept in darkness for 17 h. To accumulate chlorophyll precursors formed from exogenous ALA, detached leaves were incubated in darkness on a solution of 5 mM ALA, pH 3.2, for 17 h. Recording of fluorescence and absorption spectra in vivo. Uncorrected FS of leaves in vivo were recorded at –196°C at the split width of 2 nm for both exciting and recording monochromators. The latter was equipped with an OC-13 light filter [19]. Absorption spectra were recorded at –196°C using a UVIKON 931 spectrophotometer (Kontron Instruments, Germany). Determination of the pigment content. The contents of chlorophylls a and b were determined from the absorption spectra of leaf extracts prepared in 85% acetone [20] or from the FS of leaves of low chlorophyll content. The total carotenoid content was estimated according to [20]. Yellow pigments were extracted with diethyl ether and then separated by paper chromatography. Chromatograms were developed in a 3 : 1 (v/v) mixture of benzene and light petroleum (70–100°C fraction). Carotenoid zones were eluted by light petroleum (carotene) or ethanol (lutein and violaxanthin), RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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and their absorption spectra were recorded using a UVIKON 931 spectrophotometer. The carotenoid content was calculated according to [21]. PP, MgPP(E), Pchlide, and chlorophyllide were extracted with the acetone : 0.1 N NH4OH (9 : 1, v/v) mixture, and the extracts obtained were treated by hexane. The content of the above compounds was estimated using the FS of the extracts according to [22, 23]. Reagents. ALA, S-adenosyl-L-methionine, PP, and Tris–HCl were obtained from Sigma (United States). The table presents arithmetic means from five independent experiments and their standard errors. RESULTS Mutant plants were grown in a light/dark regime, under an irradiance of 11 W/m2, and at 22°C. According to appearance, the plants could be divided into three groups: 12–15% of them had completely colorless leaves, 3–5% of plants had variegated leaves, and the rest had green leaves. Only completely colorless leaves were used as experimental objects. Cotyledons of both mutant and wild-type plants were identical in their phenotype. The plastome mutation demonstrated its clearcut dependence on light and temperature [24]. Thus, cultivation of plants at 26°C and above resulted in the development of pigment deficiency not only in the leaves but also in the cotyledons of mutants. The plants with colorless leaves perished. Stored reserve substances and cotyledon photosynthesis maintained the growth and development of these plants for 30– 45 days, and then cotyledons and the whole plants died. High light intensity stimulated the manifestation of the mutation. Even at the early developmental stage, at the age of 10–12 days, mutant plants differed from the wild-type ones in the phenotype of the first true leaves. The former were characterized by a near absence of color and by a low chlorophyll content comprising about 2% of that in the control. Plant aging was accompanied by a decrease in the level of chlorophyll to 0.2% by the 20th day. Thereafter, it remained virtually constant (Fig. 1). Chlorophyll stability in plants depends on its association with the apoproteins of pigment–protein complexes of the photosynthetic apparatus and on the presence of carotenoids in the photosynthetic membrane. The analysis of low-temperature FS recorded in vivo demonstrated that, in young 10-day-old mutant leaves, chlorophyll was present in all parts of the leaf and its spectral maxima were similar to those in wild-type leaves (733, 695, and 686 nm) (Fig. 2). However, in the mutant, the ratio between various chlorophyll forms was changed: chlorophyll bands typical of the PSI (733 nm) and PSII (695 nm) were significantly reduced as compared to the 686 nm band belonging to the chlorophyll a/b-containing antenna complex of the PSII [25, 26]. With a decrease in the chlorophyll content in No. 2
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Fig. 1. Relative content of (1) chlorophylls a + b, (2) carotenoids, and (3) ALA formed during the incubation of mutant sunflower leaves in 0.05 M levulinic acid for 3 h in the light. The content of pigments and ALA in wild-type green leaves is taken as 100%.
mutant leaves, the bands of PSI complexes (luminescence at 733 nm and absorption at 688, 697, and 708 nm) were reduced and subsequently disappeared, and then PSII complexes (luminescence at 695 and absorption at 682 nm) were destroyed (Figs. 2, 3). Chlorophyll a/bcontaining LHCII was more stable. The 647-nm band (chlorophyll b) proved to be more reduced than that at 677 nm caused by the main form of chlorophyll a (Fig. 3). Chlorophyll a to b ratios in mutant and wildtype leaves were 4.5 and 2.9, respectively. With aging, chlorophyll distribution in mutant leaves became heterogeneous. In the more aged, distal regions of the 14-day-old leaves, there was only one spectral form of chlorophyll with a fluorescence maximum at 680– 682 nm, while in the older, 21-day-old plants the luminescence maximum shifted to 676 nm. In the oldest, 35-day-old plants, chlorophyll was present only in the young leaf tissue. The relative content of carotenoids in the mutant leaves was higher than that of chlorophyll and amounted to 20–30% and 8–10% of that in wild-type leaves in 10- to18- and 20- to 38-day-old plants, respectively (Fig. 1). The ratio of carotenoids to chlorophyll was virtually age-independent, its average value being 4.3 in the mutant and 0.12 in the wild-type leaves. The qualitative composition of yellow pigments was determined in the cotyledons of chlorophyll-deficient mutant plants grown at 26°C (Fig. 4). The total carotenoid content in mutant and wild-type cotyledons was virtually the same. However, the relative content of βcarotene, one of the components of PSI and PSII reaction centers, was strongly reduced, whereas the content of lutein and violaxanthin, the components of LHC, was higher than in the wild type. Subsequently, the activity of multienzyme systems participating in chlorophyll synthesis was assessed. Formation of ALA is the most sensitive reaction in this process [11, 27]. Incubation of colorless mutant leaves in the 0.05 M levulinic acid for 3 h in light resulted in a
Fluorescence intensity, arb. units
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Fig. 2. Noncorrected fluorescence spectra (1) of the 10-dayold wild-type sunflower leaves and (2) of the 10-day-old and (3) 18-day-old mutant sunflower leaves. FS were recorded at the 440-nm excitation light and –196°C.
lower accumulation of ALA than in the wild-type leaves (Fig. 1). There is a clear-cut difference between the percentages of ALA and chlorophyll in the mutant. Leaves with 2.0% and 0.2% of chlorophyll contained 40% and as much as 10–15% ALA, respectively. ALA synthesis was light-dependent in both types of plants. In darkness, ALA accumulation in the wild-type and mutant seedlings decreased 2.2-fold and 1.7-fold, respectively, as compared to that in the light (data not shown). The ability of plants for Pchlide resynthesis in darkness was also investigated. This allowed us to estimate the activity of the multienzyme system performing the synthesis of ALA and its transformation in Pd. Figure 5 demonstrates FS of the hexane-treated water–acetone extracts from the leaves of colorless mutants and parent plants kept in the dark for 17 h. The mutant accumulates on the average 0.20 ± 0.04 nmol Pchlide per unit fr wt, i.e., 30% of the Pd level resynthesized in the wildtype leaves. Further illumination resulted in quenching Pchlide luminescence. This might be related to photoreduction or photodestruction of the pigment. Earlier, we showed that, in young leaves, resynthesized Pchlide
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is represented by two spectral forms, photoinactive Pchlide635 and photoactive Pchlide656 associated with Pchlide oxidoreductase; older leaves contained only Pchlide635 [28]. We failed to find any other intermediate products of chlorophyll formation in either type of plant. The restriction of chlorophyll synthesis at the stage of ALA formation can be removed by introducing exogenous ALA into the leaves. The analysis of chlorophyll precursors thus accumulated made it possible to find out the presence of specific blocks in the chlorophyll-formation chain and to evaluate the potential ability of leaves for chlorophyll synthesis. Data presented in the table show that the incubation with exogenous ALA in darkness for 17 h resulted in the accumulation of chlorophyll precursors PP, MgPP(E), and Pchlide. Their amounts in the mutant were significantly lower than in the control leaves. The Pd content in the former comprised 14.3% of that in the green leaves of parent plants. However, the content of other chlorophyll precursors, PP and MgPP(E), accumulated from exogenous ALA differed little from that in the wild-type leaves. The evaluation of the specific activity of MT was also carried out. This enzyme participates in the magnesium branch of the porphyrin biosynthesis at the stage of MgPP methylation [18]. MT activity was esti-
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0 650 700 Wavelength, nm
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Fig. 5. Fluorescence spectra of water–acetone extracts from (a) wild-type and (b) mutant suflower leaves (1) before and (2) after a 17 h incubation of plants in the dark. Spectra were recorded at 435-nm excitation light and room temperature.
mated as the amount of MgPPE formed per mg protein per unit time by incubating leaf homogenate in the presence of exogenous substrates. The activity in mutant leaves was shown to be twice as high as that in parent leaves. At the same time, the enzyme activity calculated for the fresh weight was the same in the control and mutant plants (table). It cannot be excluded that the higher enzyme activity in colorless leaves calculated per mg protein is caused by a lower protein content in the mutant [14]. DISCUSSION The plastome mutation of sunflower manifests itself as a decrease in chlorophyll content already at the stage of the first true leaf formation (Fig. 1). Young mutant No. 2
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Accumulation of PP, MgPP(E), and Pchlide in the presence of 5 mM exogenous ALA and the activity of MT in mutant and wild-type sunflower leaves Index PP MgPP(E) Pchlide Pchlide/PP Pchlide/MgPP(E) MgPPE, nmol/g fr wt MgPPE, nmol/mg protein
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Porphyrin accumulation from exogenous ALA, nmol/g fr wt 23.91 ± 4.41 100% 3.07 ± 0.80 1.76 ± 0.54 100% 0.29 ± 0.02 10.58 ± 1.10 100% 1.52 ± 0.42 0.44 0.49 6.01 5.24 MT activity 55.92 ± 9.21 100% 55.02 ± 8.45 3.51 ± 1.22 100% 6.67 ± 3.35
leaves contained all the pigment–protein complexes of the photosynthetic apparatus typical for the wild-type leaves, but the ratio between them was changed (Figs. 2, 3). The considerable reduction of the 733-nm band attributed to PSI in the low-temperature FS of leaves [25, 26] is usually typical for partially differentiated chloroplasts [29]. Because the expression of the mutation was found to be light-dependent, it can be suggested that, in the mutant, one of the pathways of light signal transduction is disrupted, resulting in the blocking of PSI formation [29]. A gradual decrease in the chlorophyll and carotenoid contents observed during plant development seems to be associated with photodestructive processes. The reduction of PSI observed at the early stages of mutant leaf development was increased with leaf age. It manifested itself in the disappearance of long-wave fluorescence and of the bands at 688, 697, and 708 nm in the spectra of the leaf absorption second derivative (Fig. 3). It was also accompanied by a decrease in the fluorescence at 695 nm, thus indicating a gradual disappearance of PSII. A decrease in the content of PS complexes in the mutant was correlated with changes in the relative content of carotenoids. There is a decrease in the content of β-carotene, which is a component of both photosystems, and an increase in the content of lutein and violaxanthin, which are components of LHC. The disappearance of the luminescence bands typical for the complexes of both photosystems was accompanied by the emergence of 676- to 681-nm band in the FS of mutant leaves. This band was located close to the spectral form of chlorophyll typical for LHCII [26]. In this case, the spectrum of the absorption second derivative of such leaves clearly displayed the bands of chlorophyll a (661, 668, and 677 nm) and chlorophyll b (647 nm), which form this complex (Fig. 3). It could be suggested that the different resistances of these pigments to photodestruction cause a high chlorophyll a/chlorophyll b ratio (4.5) in the mutant. The 647-nm band of chlorophyll b can be clearly seen to be more reduced as compared to that of
12.8% 16.5% 14.3%
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the main form of chlorophyll a as a constituent of LHCII (677 nm). Thus, the mutant is characterized by a considerable decrease in the chlorophyll content. However, mechanisms affecting the assembly of pigment–protein complexes of the photosynthetic apparatus and providing protein carriers for the remainder of chlorophyll are not disrupted. Our results do not contradict published data on the very low level of cab gene transcription in the light observed in a number of chlorophyll-deficient or chlorophyll-devoid mutants of higher plants [30]. The PAGE of chloroplast proteins from the mutant demonstrated a strong reduction of LHC proteins in the 27- to 28-kD range, whereas other protein components were found in both mutant and wild-type leaves (data not shown). These data are consistent with earlier results [14]. The light-dependence of ALA synthesis in the mutant found here enables one to identify the portion of ALA that is designed for chlorophyll formation [27, 31, 32]. This dependence can indicate that, in mutant leaves of colorless phenotype, there is both a breakdown and a resynthesis of pigment components of the pigment–protein complexes of the photosynthetic apparatus. Of course, the efficiency of this process was significantly less than that in wild-type leaves. We believe that a strong deterioration of chloroplast differentiation in sunflower leaves resulted in a sharp decrease in the activity of chlorophyll biosynthesis related to the inhibition of the earliest stage of the process, namely the formation of ALA molecules. A synchrony in the decrease in ALA and chlorophyll contents was observed at all stages of mutant leaf ontogeny (Fig. 1). Earlier, we demonstrated [28] that the lack of chlorophyll in mutant tissues is caused by the termination of the Pchlide resynthesis and, probably, of the formation of endogenous ALA designed for chlorophyll synthesis [27]. Attention is drawn to the fact that ALA synthesis in colorless leaves in the light is sufficiently high in spite of a low content of chlorophyll. This may indicate that there is an intense synthesis of the nonchlorophyll tet-
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rapyrroles, hemes, thus demonstrating the stability of the multienzyme system for the synthesis of ALA designed for the hemin branch. In the sunflower mutant, a virtually unknown regulatory mechanism controlling the synthesis of ALA designed for chlorophyll synthesis seems to be active. Earlier, it was demonstrated that, in the light, a chlorophyll b-devoid barley mutant is incapable of synthesizing an ALA fraction designed for the formation of chlorophylls a + b participating in the synthesis of chlorophyll a/b-containing LHC [27]. It has been shown [33] that, in this mutant, the system of the synthesis of the enzymes converting chlorophyll a into chlorophyll b is genetically disrupted. It is possible that there is a regulatory mechanism of chlorophyll biosynthesis according to which, in the case of a lack or deterioration of the enzyme catalyzing the final stage of this process, the formation of the initial substrate of the biosynthesis, namely, ALA molecules, is not necessary. In these cases, the mechanism controlling the initial and final stages of chlorophyll biosynthesis could correct the ALA synthesis. In particular, it could correct ALA formation within the framework of those multienzyme complexes of chlorophyll synthesis in which the final stages of the process were deteriorated. Actually, in the colorless leaves of the sunflower mutant, we have found a clear correlation between the activity of the enzymes catalyzing the final stages of the chlorophyll biosynthesis, viz. esterification of chlorophyllide a with the formation of chlorophyll a and the reverse reaction of conversion of chlorophyll b into chlorophyll a, and the activity of the enzyme system of the ALA synthesis [28]. It is of interest that the existence of Pd oxidoreductase, as proved by the presence of the Pchlide656 band in FS, was not correlated with the activity of the ALA synthesis. Actually, the segments of leaf with a low chlorophyll content performed the resynthesis of Pchlide represented by only the Pchlide635 form. This fact can explain the inconsistency between the rather high activity of Pchlide synthesis in mutant leaves (30% of the control level) and the low content of the end product, chlorophyll. The activities of other enzymes participating in the chlorophyll synthesis could easily be recorded by the accumulation of a number of intermediate products, such as Pchlide, MgPP(E), and Pd, in the presence of exogenous ALA. In the mutant, the amount of Pchlide accumulated from the exogenous substrate comprised only 14% of that in wild-type leaves. This is to say that, in mutant leaves, the potential for the chlorophyll synthesis dependent on the total amount of multienzyme systems of this synthesis is considerably decreased. At the same time, the quantitative ratios between Pchlide and its precursors accumulated from exogenous ALA differed little in both types of plants (table). Therefore, it could be suggested that, in mutant-plant plastids, in spite of the decrease in the total amount of mutienzyme complexes of chlorophyll synthesis, the activity of the individual complexes and their component enzymes RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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differed little from that in wild-type leaves. The determination of the activity of MT and ALA dehydratase [34] showed that these enzymes did not limit chlorophyll formation in the mutant. Thus, the main result of this investigation consists in demonstrating that all types of pigment–protein complexes of the photosynthetic apparatus are present in the leaves of the sunflower plastome mutant with a heavily reduced content of chlorophyll. With the aging of the mutant leaves, photodestruction of pigments is accompanied by the destruction of, first, PSI complexes and, then, those of PSII. Chlorophyll a/b-containing LHCII turned out to be more stable, which is consistent with the increased content of lutein and violaxanthin in the mutant. A synchrony in the decrease in chlorophyll and ALA contents at all stages of colorless mutant leaf ontogeny makes it possible to suggest that the decreased synthesis and renewal of chlorophyll during formation and development of such leaves is caused by the inhibition of the earliest stage of the process, the biosynthesis of ALA molecules. The activity of other enzymes that convert ALA into Pchlide did not limit chlorophyll synthesis in the mutant. ACKNOWLEDGMENTS The authors are grateful to N.V. Shalygo, Senior Researcher, Institute of Photobiology, National Academy of Sciences of Belarus, for help in performing protein separation by PAGE. This work was supported by the International Science Foundation, project no. MWVOOO. REFERENCES 1. Sigura, M., The Chloroplast Genome, Plant Mol. Biol., 1992, vol. 19, pp. 149–168. 2. Schön, A., Krupp, G., Gough, S., Berry-Lowe, S., Kannangara, C.G., and Söll, D., The RNA Required for the First Step of Chlorophyll Biosynthesis Is a Chloroplast tRNA, Nature, 1986, vol. 322, pp. 281–284. 3. Bukharov, A.A. and Abdulaev, N.G., Polypeptides of Chlorophyll–Protein Complexes in the Chloroplast Thylakoid Membrane, Biol. Membr. (Moscow), 1990, vol. 7, pp. 1221–1255. 4. Kannangara, C.G., Andersen, R.V., Pontopiddan, B., Willows, R., and von Wettstein, D., Enzymic and Mechanistic Studies on the Conversion of Glutamate to 5-Aminolevulinate, The Biosynthesis of Tetrapyrrole Pigments. CIBA Foundation Symposium 180, Chadwick, D.J. and Ackrill, K., Eds., Chichester: John Wiley and Sons, 1994, pp. 3–25. 5. He, Z.-H., Li, J., Sundqvist, C., and Timko, M.P., Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.), Plant Physiol., 1994, vol. 106, pp. 537–546. 6. Papenbrock, J., Mock, H.-P., Kruse, E., and Grimm, B., Expression Studies in Tetrapyrrole Biosynthesis: Inverse Maxima of Magnesium Chelatase and Ferrochelatase No. 2
2001
154
7.
8. 9.
10.
11. 12. 13.
14.
15.
16.
17. 18.
19.
20.
21. 22.
RASSADINA et al. Activity during Cyclic Photoperiods, Planta, 1999, vol. 208, pp. 264–273. Oelmüller, R., Photooxidative Destruction of Chloroplasts and Its Effect on Nuclear Gene Expression and Extraplastidic Enzyme Levels, Photochem. Photobiol., 1989, vol. 49, pp. 229–239. Taylor, W.C., Regulatory Interactions between Nuclear and Plastid Genomes, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1989, vol. 40, pp. 211–233. Hess, W.R., Müller, A., Nagy, F., and Börner, R., Ribosome-Deficient Plastids Affect Transcription of LightInduced Nuclear Genes: Genetic Evidence for a PlastidDerived Signal, Mol. Gen. Genet., 1994, vol. 242, pp. 305–312. Bolle, C., Sopory, S., Lübberstedt, T., Klosgen, R.B., Herrmann, R.G., and Oelmüller, R., The Role of Plastids in the Expression of Nuclear Genes for Thylakoid Proteins Studied with Chimeric β-Glucuronidase Gene Fusions, Plant Physiol., 1994, vol. 105, pp. 1355–1364. Von Wettstein, D., Gough, S., and Kannangara, C.G., Chlorophyll Biosynthesis, Plant Cell, 1995, vol. 7, pp. 1039–1057. Suzuki, J.Y., Bollivar, D.W., and Bauer, C.E., Genetic Analysis of Chlorophyll Biosynthesis, Annu. Rev. Genet., 1997, vol. 31, pp. 61–89. Beletskii, Yu.D., Iskusstvennye mutatsii khloroplastov u vysshikh rastenii (Artificial Mutations in Higher Plant Chloroplasts), Rostov-na-Donu: Rostov. Gos. Univ., 1989. Taran, S.F., Some Biochemical Features of Plastid Ultrastructure in Sunflower Plastome Mutants, Cand. Sci. (Biol.) Dissertation, Rostov-na-Donu: Rostov. Gos. Univ., 1991. Miller, G.W., Denney, A., Wood, J.K., and Welkie, G.W., Light-Induced Delta-Aminolevulinic Acid in DarkGrown Barley Seedlings, Plant Cell Physiol., 1979, vol. 20, pp. 131–143. Mauzerall, D. and Granick, S., The Occurrence and Determination of 5-Aminolevulinic Acid and Porphobilinogen in Urine, J. Biol. Chem., 1956, vol. 219, pp. 435–446. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem., 1951, vol. 193, pp. 265–275. Ellsworth, R.K. and Dullagham, J.P., Activity and Properties of (–)-S-Adenosyl-L-Methionine: MagnesiumProtoporphyrin IX Methyltransferase in Crude Homogenates from Wheat Seedlings, Biochim. Biophys. Acta, 1972, vol. 268, pp. 327–333. Domanskii, V.P., Samoilenko, A.G., and Lis, P.I., Spectrofluorimeter Automatically Controlled by a MicroElectronic Computer, Vestsi Akad. Nauk BSSR, Ser. Biyal. Navuk, 1986, pp. 100–103. Shlyk, A.A., Chlorophyll and Carotenoid Determination in the Extracts from Green Leaves, Biokhimicheskie metody v fiziologii rastenii (Biochemical Methods in Plant Physiology), Pavlinova, O.A., Ed., Moscow: Nauka, 1971, pp. 154–170. Britton, G., UV/Visible Spectroscopy, Carotenoids, vol. 1B, Spectroscopy, Britton, G. et al., Eds., Basel: Birkhauser, 1995, pp. 1–62. Averina, N.G., Sidorko, V.V., and Shalygo, N.V., Spectrofluorimetric Method for Protoporphyrin IX Determi-
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26.
27.
28.
29.
30.
31.
32.
33.
34.
nation in Leaves Treated with 5-Aminolevulinic Acid, Vestsi Akad. Nauk BSSR, Ser. Biyal. Navuk, 1984, pp. 102–105. Averina, N.G., Shalygo, N.V., and Fradkin, L.I., Determination of Monomethyl Ester of Mg-Protoporphyrin IX in Barley and Wheat Leaves, Vestsi Akad. Nauk BSSR, Ser. Biyal. Navuk, 1980, pp. 25–29. Rassadina, V.V., Lezhneva, L.A., and Averina, N.G., The Effect of Light Intensity and Temperature on the Manifestation of Pigment-Deficiency in the Plastome Mutant of Sunflower (Helianthus annuus L.) Line 2-24, Tezisy Dokl. 3-go S”ezda Belarus. O–va Fotobiol. Biofiz. (Abstr. 3rd Meet. Soc. Photobiol. Biophys. Belarus), Minsk, 1998, p. 71. Franck, F. and Strzalka, K., Detection of the Photoactive Protochlorophyllide–Protein Complex in the Light during the Greening of Barley, FEBS Lett., 1992, vol. 309, pp. 73–77. Ladygin, V.G., Structural and Functional Organization of Photosystems in Chlamydomonas reinhardtii Chloroplasts, Fiziol. Rast. (Moscow), 1998, vol. 45, pp. 741–762 (Russ. J. Plant Physiol., Engl. Transl.). Averina, N.G., Rassadina, V.V., and Yaronskaya, E.B., Biosynthesis of 5-Aminolevulinic Acid in Plants and Its Regulation, Fotobiologiya i membrannaya biofizika (Photobiology and Biophysics of Membranes), Volotovskii, I.D., Ed., Minsk: Tekhnoprint, 1999, pp. 6–19. Vezitskii, A.Yu., Lezhneva, L.A., Shcherbakov, R.A., Rassadina, V.V., and Averina, N.G., Activity of Chlorophyll Synthetase and Chlorophyll b Reductase in the Chlorophyll-Deficient Plastome Mutants (albina type) of Sunflower (Helianthus annuus L.), Fiziol. Rast. (Moscow), 1999, vol. 46, pp. 580–585 (Russ. J. Plant Physiol., Engl. Transl.). Kusnetsov, V., Bolle, C., Lübberstedt, T., Sopory, S., Herrmann, R.G., and Oelmüller, R., Evidence that the Plastid Signal and Light Operate via the Same cis-Acting Elements in the Promoters of Nuclear Genes for Plastid Proteins, Mol. Gen. Genet., 1996, vol. 252, pp. 631–639. Hess, W.R., Schendel, R., Börner, T., and Rüdiger, W., Reduction of mRNA Level for Two Nuclear Encoded Light Regulated Genes in the Barley Mutant Albastreins Is Not Correlated with Phytochrome Content and Activity, J. Plant Physiol., 1991, vol. 138, pp. 292–298. Shlyk, A.A., Rudoi, A.B., and Vezitzskii, A.Y., Metabolism of Chlorophyll Pigments at Centers of Biosynthesis during the Initial Stages of Greening of Etiolated Seedlings, Proc. II Int. Congr. Photosynthesis Research, Forti, G. et al., Eds., Hague, 1972, vol. 3, pp. 2289–2297. Rudoi, A.B., Chkanikova, R.A., and Shlyk, A.A., Fast Photoregulation of Protochlorophyllide Biosynthesis during Greening of Barley Etiolated Leaves, Dokl. Akad. Nauk SSSR, 1978, vol. 242, pp. 1450–1453. Rudoi, A.B. and Shcherbakov, R.A., Analysis of the Chlorophyll Biosynthetic System in a Chlorophyll bLess Barley Mutant, Photosynth. Res., 1998, vol. 58, pp. 71–80. Lezhneva, L.A., Rassadina, V.V., Yaronskaya, E.B., Radyuk, M.S., Taran, S.F., and Averina, N.G., Activity of Fe Branch of Tetrapyrrole Biosynthesis in the Chlorophyll-Deficient Plastome Mutant of Sunflower (Heliantus annuus L.), Fiziol. Rast. (Moscow), 2000, vol. 47, pp. 69–74 (Russ. J. Plant Physiol., Engl. Transl.).
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
Vol. 48
No. 2
2001
