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Ultrastructural Organization of Leaves of Transgenic Tobacco Overexpressing Histone H1 from Arabidopsis thaliana
Annals of Botany 84 : 329–335, 1999 Article No. anbo.1999.0925, available online at http:\\www.idealibrary.com on
Ultrastructural Organization of Leaves of Transgenic Tobacco Overexpressing Histone H1 from Arabidopsis thaliana J O A N N A S! L U S A R C Z Y K*, M A R T A P R Y M A K O W S KA-B O S AK†, M A R C I N P R Z E W Ł O K A†, A N D R Z E J J E R Z M A N O W S KI† ‡ and M I E C Z Y S Ł A W K U R A S! §R * Department of Ecology and Enironmental Protection, Pedagogical School, ul.Konopnickiej 15, Kielce, Poland, † Laboratory of Plant Molecular Biology, Warsaw Uniersity, ul. PawinT skiego 5a, Warsaw, Poland, ‡ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul.PawinT skiego 5a, 02-106 Warsaw, Poland and § Department of Plant Morphogenesis, Warsaw Uniersity, ul. Banacha 2, Warsaw, Poland Received : 8 January 1999
Returned for revision : 19 March 1999
Accepted : 13 May 1999
We investigated the anatomical and ultrastructural features of transgenic tobacco plants that overexpressed a gene of histone H1 from Arabidopsis thaliana. The overexpression of the heterologous gene resulted in more than a 2n5fold increase over the physiological level of the histone H1 : DNA ratio in chromatin. H1-overexpressing plants had a distinct mutant phenotype characterized by dwarf appearance and severely hampered flowering. These changes were accompanied by extensive and unusual heterochromatinization of nuclei occurring in all leaf parenchymal cells but not in leaf epidermal cells. The observed anomalies in the growth rate and size of the cells and in nuclei\chloroplast proportions in histone H1-overexpressing plants suggest that the H1 : DNA ratio can influence some specialized cellular functions involving the cytoskeleton, and nuclear\organellar interactions which are of importance for the normal development of a plant. # 1999 Annals of Botany Company Key words : Transgenic tobacco plant, histone H1 overexpression, heterochromatinization.
INTRODUCTION Eukaryotic DNA is assembled into chromatin in which it is packed by histone proteins into nucleosomes (van Holde, 1989 ; Wolffe, 1994). The inner (core) particle of the nucleosome is formed by an octamer of core histones : H2A, H2B, H3 and H4, around which is wrapped 146 base pairs of DNA. The application, in 1989, of the mutational analysis of core histone genes in yeast revealed for the first time that apart from a purely architectural role in packing DNA, the core histones are involved in many regulatory functions, predominantly concerning transcription. In particular, it was shown that the flexible N terminal tails of histone H3 and H4 participate in activation and repression of transcription of the defined genes (reviewed in Paranjape, Kamakaka and Kadanoga, 1994). In addition to core histones, a complete nucleosome contains a molecule of another type of protein, classified as linker or H1-class histone, which is bound to a variable length linker DNA extending between neighbouring core particles. There are usually several sequence variants of histone H1 in a single multicellular eukaryotic organism. In some organisms, characteristic changes in the profile of H1 variants were found to accompany development. However, in contrast to the abundance of data on the function of core histones, the role in chromatin of histone H1 is far less clear. Typical metazoan linker histone has a three-dimensional structure with a central globular domain flanked by mostly unstructured N- and C-terminal tails. For a long time the presence of linker histones in chromatin was thought 0305-7364\99\090329j07 $30.00\0
essential for folding of the chromatin fibre and histone H1 was considered a general repressor of transcription. The current view, based on analysis of the condensation properties of H1-depleted chromatin, is that linker histones while able to facilitate and guide the proper folding of a nucleosomal fibre, are not the cause of the folding per se (van Holde and Zlatanowa, 1996). As regards the functional role of H1, a key finding was the demonstration that the ciliated protozoan Tetrahymena in which linker histones have been knocked-out by genetic manipulation, grew normally (Shen et al., 1995). This suggested that linker histones are not essential for critical functions such as replication, transcription and protein synthesis. It was later shown that in a histone H1 knock-out strain of Tetrahymena the number of mature RNAs produced by genes transcribed by polymerases I and III and most genes transcribed by polymerase II remained unchanged (Shen and Gorovsky, 1996). These data were supplemented by the demonstration that Xenopus egg extracts depleted of linker histones assemble normal nuclei that are capable of initiating replication and condensing their chromosomes (Ohsumi, Katagiri and Kishimoto, 1993 ; Dasso, Dimitrov and Wolffe, 1994). The inhibition by H1-targeted ribozyme of the accumulation of somatic H1 in the developing embryo of Xenopus did not lead to activation of the vast majority of genes whose expression is required for viability of the embryonic cells (Kandolff, 1994 ; Bouvet, Dimitrov and Wolffe, 1994). In laboratory experiments with transgenic plants, it was shown that overexpression of a somatic-type of histone H1 in tobacco that led to an over two-fold # 1999 Annals of Botany Company
330
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elevation of the physiological H1 : DNA ratio in chromatin and a marked increase of the heterochromatinization of nuclei, had little effect on basal cellular functions (Prymakowska et al., 1996). This indicated that the saturation of chromatin sites with H1 does not lead to marked changes in expression of constitutive genes. Thus, the data from different organisms strongly support the view that linker histones are not general repressors of transcription in io. However, there is evidence that H1 can be involved in highly selective regulation of specific classes of genes. In the H1 knock-out strain of Tetrahymena, two genes, ngoA, a gene of unknown function, and CyP, which encodes a cysteine protease, were shown to be regulated by histone H1 in opposite directions (Shen and Gorovsky, 1996). In an analysis of the early stages of tissue differentiation in Xenopus, it was shown that the accumulation of somatic H1 in chromatin is linked to a selective transcriptional silencing of regulatory genes required for mesodermal differentiation (Steinbach, Wolffe and Rupp, 1997). In our work on tobacco we also noticed that the overexpression of histone H1 in plants, while not hampering the ability of transgenic plants to grow and differentiate, exerted a rather selective effect at the stage of flowering. In the most severe phenotypes, flowers were not formed or were discarded at the early flower bud stage. In order to investigate further the possible ultrastructural background of these specific effects, we subjected transgenic tobacco plants characterized by a high level of transgenic H1 protein to detailed cytological analysis. We hoped to define the anomalies that could become the target of more focused molecular investigation.
MATERIALS AND METHODS Leaves of transgenic tobacco (Nicotiana tabacum sp.) containing the gene for Arabidopsis thaliana L. histone H1 were used. Methods of obtaining and cultivating the plants have been described previously (Prymakowska-Bosak et al., 1996). Briefly, plasmid vectors containing the gene for histone H1 were incorporated into tobacco using the method of leaf disc transformation by Agrobacterium, according to the procedure of Horsch et al. (1986). Leaf discs were placed on solid Murashige and Skoog (1962) medium with agar and the time of regeneration was measured from that moment. Two-week-old plantlets grown from the somatic embryos were placed in soil and cultured in a growth chamber under controlled light, temperature and humidity. Leaves of 13-week-old transgenic H1-overexpressing plants were used for microscopical studies. As a control we used transgenic plants of the same age obtained by the same transformation and regeneration procedure, that did not express the transgene. Squares of 1–2 mm were cut from the middle part of leaves and fixed in 2 % glutaraldehyde in 0n1 cacodylate buffer pH 7n2 for 2 h, followed by additional fixation in 2 % OsO for 2 h at 4 mC. The material % was dehydrated in a graded ethanol series, washed in propylene oxide and embedded in a mixture of Epon and Spurr. Semi-thin (1–2 µm) and ultrathin (80 nm) sections were cut with an LKB ultramicrotome (Sweden). Semi-thin
sections were stained with 0n1 % toluidine blue in 1 % borax and leaf structure was analysed in a light microscope (Nikon). Ultrathin sections were contrasted in water solutions of uranyl acetate (30 min) and lead citrate (30 min) according to Reynolds (1963). Sections were investigated in a transmission electron microscope (JEOL JEM-1200EX, 90 kV). Electron micrographs were taken on 60i90 mm negative film (TN-12, Foton), and used for the analysis of leaf ultrastructure as well as the frequency and size of chloroplasts and starch grains in H1-overexpressing and control plants. RESULTS Growth and morphological appearance of H1oerexpressing plants The present work concerns characterization of transgenic tobacco that contained about 2n5-times more H1 than normal plants, and displayed a distinct mutant phenotype. A detailed description of the construction and the complete molecular and phenotypic characterization of transgenic plants are given in Prymakowska-Bosak et al. (1996). There were no distinct differences in growth rate and shape of control (TRANS H1k) and H1-overexpressing (TRANS H1j) plants in the first phase of development. From the ninth week of regeneration, growth of TRANS H1j plants with severe mutant phenotypes was slowed. This was reflected by a gradual and progressive shortening of stem internodes. TRANS H1j plants achieved their final size after 12–13 weeks of development, producing 14 leaves. Due to almost complete inhibition of the uppermost internodes, the youngest leaves were closely adjacent one to another creating a characteristic palm form. Because TRANS H1k plants grew normally until the twenty-first week there were visible differences in size and shape between control and TRANS H1j plants : at 21 weeks TRANS H1k plants were at least 30 % larger than TRANS H1j plants and had abundant flowers. The TRANS H1j plants did not flower even after 36 weeks, when their lowest leaves were already drying.
Anatomical and ultrastructural differences in leaes Despite differences in leaf size between control and H1overexpressing plants, the overall shape, colour and appearance of leaves were similar. There was also no distinct difference in anatomical structure, which was typical for dicotyledonous plants, with palisade and spongy mesophyll layers and upper and lower epidermal layers (Figs 1 and 2). In both types of plants stomata were present in the two epidermal layers, although they were more frequent in the lower layer, and hairs were present on both epidermal layers. However, in transverse section, the leaf lamella of TRANS H1j plants was much thinner (almost 20 %) than that of control TRANS H1k plants (Figs 1 and 2). The difference was hardly visible in the epidermis (the upper epidermis was 4n6 % thinner and the lower one 2n1 %), and resulted mostly from differences in thickness of the palisade and spongy mesophylls. These were thinner in TRANS
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331
F. 1. Transverse section through the leaf of a control (TRANS Hk) plant. Semi-thin section stained with toluidine blue. Bar l 20 µm. F. 2. Transverse section through the leaf of an H1-overexpressing (TRANS Hj) plant. Bar l 20 µm. F. 3. Typical eu- and heterochromatin dispersion in the nuclei of palisade mesophyll in TRANS H1k plants. Bar l 0n5 µm. F. 4. Maximal heterochromatization in cell nuclei of palisade mesophyll in TRANS H1j plants. Bar l 1 µm. PM, Palisade mesophyll ; SM, spongy mesophyll ; UE, upper epidermis ; LE, lower epidermis.
H1j plants by 26n5 and 21n1 %, respectively (Table 1). The difference in thickness of the mesophyll tissue was the result of slower growth in TRANS H1j plants. As a consequence, cells of the palisade mesophyll of TRANS H1j plants were 28n8 % shorter and 27 % thinner, while cells of the spongy mesophyll were almost 30 % shorter and 36 % thinner (Table 2) compared to corresponding cells in TRANS H1k plants.
Additionally, leaf epidermal cells of TRANS H1j plants differed from those in TRANS H1k plants by the presence of structures of electron-grey and sometimes even black consistency (Figs 1 and 2). There were also visible differences in the number and size of cell organelles especially chloroplasts, which were larger and more frequent in TRANS H1j cells (up to 100 % more frequent in cells of the palisade mesophyll and 80 % more frequent in cells of
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T 1. Aerage (n l 40) thickness of leaf lamella, palisade and spongy mesophylls and upper and lower epidermal layers of control (TRANS H1k) and H1-oerexpressing (TRANS H1j) plants
in epidermal cells (Figs 5 and 6). In these cells the extent of dispersion and the quantitative proportions of heterochromatin and euchromatin regions were the same as in control TRANS H1k plants.
TRANS H1j TRANS H1k Difference (µm) (µm) (% of control) Upper epidermis Palisade mesophyll Spongy mesophyll Lower epidermis Leaf blade
20n9 61n1 83n7 18n1 183n8
19n9 44n9 66n8 17n7 149n3
D I S C U S S I ON Histone H1 is a key element of the structure of chromosomes. In this work we investigated the influence on the anatomy and ultrastructure of leaves of the 2n5-fold increase over the physiological level of the major somatic form of histone H1 in chromatin of tobacco, caused by the overexpression of the histone H1 gene in the transgenic plants. As reported previously, the plants in which the physiological proportion of H1 : DNA was so drastically disturbed were able to develop and grow (Prymakowska-Bosak et al., 1996), suggesting that histone H1 is not functioning as a general repressor of transcription acting in a dose-dependent manner. Nevertheless, there were several interesting changes in the H1-overexpressing plants that could point to a more specific function of linker histones. The dwarf size and inability to flower were the most characteristic phenotypic changes observed in plants overexpressing H1. These morphological changes were accompanied by profound and characteristic heterochromatinization of the nuclei of the mesophyll cells. However, the normal anatomy of the leaves and unchanged number of cells in H1-overexpressing plants indicate that the ability to differentiate the basic anatomical structures and to continue cell division was not disturbed. This could mean that the extra dose of histone H1 is deposited in chromatin in a rather selective manner, not interfering with the chromosome regions containing important housekeeping and developmental genes. On the other hand, the decrease in the size of the mesophyll cells shows that normal growth had been disturbed in certain cell-types. One possible
95n2 73n5 79n8 97n7 81n2
the spongy mesophyll ; Table 3). Starch grains were also slightly larger in TRANS H1j plant cells and their number was almost 100 % higher than in control cells. Condensation of chromatin was observed in the nuclei on semi-thin sections of TRANS H1j leaf mesophyll. This feature of the nuclei was also clearly visible in TEM (Figs 3–7). The nuclei of control plants consisted mostly of fibres of dispersed chromatin (euchromatin) immersed in karyolymph (Fig. 3). Numerous heterochromatin areas had a thick granular structure but were not very condensed. The edges of heterochromatin were partly diffused, changing into euchromatin fibres (Figs 3 and 6). The nuclei of TRANS H1j plants looked totally different. Their heterochromatin areas were much larger and denser, and showed characteristic lient holes (Figs 4 and 7). The heterochromatin regions had less contact with euchromatin fibres. These fibres were not so frequent and in consequence the transparency of karyolymph was higher. Highly heterochromatinized nuclei were common in mesophyll cells (Figs 4 and 7) especially in the vascular mesophyll of TRANS H1j plants (compare Figs 8 and 10 with 9 and 11). Interestingly, heterochromatinized nuclei were absent
T 2. Aerage length and width of palisade and spongy mesophyll cells in leaes of TRANS H1k and TRANS H1j plants TRANS H1k
Palisade mesophyll Spongy mesophyll
TRANS H1j
Differences (% of control)
Length (µm)
Width (µm)
Length (µm)
Width (µm)
Length
Width
63n1 36n0
15n4 21n4
44n9 25n3
11n3 13n7
71n2 70n3
73n3 64
Data are means of 500 measurements on ten sections.
T 3. Number of chloroplasts and starch grains in palisade and spongy mesophyll cells of TRANS H1- and TRANS H1j plants TRANS H1k
Palisade mesophyll Spongy mesophyll
TRANS H1j
Differences (% of control)
Chloroplasts
Starch grains
Chloroplasts
Starch grains
Chloroplasts
Starch grains
13n6 10n2
58n5 31n3
28n2 18n3
114n4 53n9
207n3 179n4
207n3 172n2
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F. 5. Nucleus with dispersed heterochromatin in a lower epidermal (LE) cell and with highly condensed heterochromatin in the mesophyll cell of a TRANS H1j plant. Bar l 1 µm. SM, Spongy mesophyll. F. 6. Fragment of epidermal cell nucleus of TRANS H1j plant with typical decondensed euchromatin and heterochromatin. Bar l 0n5 µm. F. 7. Fragment of cell nucleus from subepidermal spongy mesophyll with typical heterochromatin and low electron density of karyolymph. Bar l 0n5 µm.
explanation is aberration of the cytoskeleton. In this context it is worth noting that histone H1 has been shown to stabilize microtubular structures in animal cells and green algae (Multigner, Gagnon and van Doisselaer, 1992). This
raises an intriguing possibility that some of the observed phenotypic effects of the overdose of H1 in transgenic tobacco could be due to its interaction with cytoskeletal rather than chromosomal structures.
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F 8 and 10. Dispersion of chromatin in cell nucleus of vascular mesophyll in TRANS H1k plants. Bar l 0n5 µm. F 9 and 11. Maximal heterochromatization in cell nuclei of vascular mesophyll of TRANS H1j plants. Note the decrease in the number of euchromatin fibrils and increased electron transparency of karyolymph. Bar l 0n5 µm.
The noticeable changes in H1-overexpressing plants also concerned the number of chloroplasts. This could be an indication of the disturbed interactions between nuclear and organeller genomes. Interestingly, we recently obtained data showing that the decrease in the amount of major somatic variants of H1 in tobacco results in flower phenotypes strikingly similar to those caused by mutations in mitochondrial DNA (unpubl. res.). The possible link between
H1-dependent chromatin structure and organellar function could provide the basis for identification of new genes responsible for nuclear-organellar interactions. Characteristically, the overexpression of histone H1 did not cause any distinct differences in the epidermal cells. The reason for this is difficult to explain at present. Different reactions to overdoses of H1 could either reflect differences in structural organization of chromatin between epidermal
ST lusarczyk et al.—Oerexpressing Histone H1 and mesophyll cells or the specific inhibition of transgenic H1 expression in epidermal cells. Whatever the reason, this observation is an interesting new clue for the investigation of the mechanisms of cellular differentiation. Summarizing, studies of the ultrastructural organization of plants with strong overexpression of histone H1 confirmed that H1 is not a general inhibitor of genes regulating basic cell functions, including cell division and differentiation. However, the documented anomalies in the growth rate and size of the cells and in nuclei\chloroplast proportions in histone H1-overexpressing plants indicate that the H1 : DNA ratio could influence some specialized cellular functions involving the cytoskeleton, and nuclear\organellar interactions which are of importance for the normal development of a plant. A C K N O W L E D G E M E N TS This research was supported by Howard Hughes Medical Institute grant 79195-543403 (A. J.) and by Polish Committee of Scientific Research grant 6PO4A 02913 (A. J.). L I T E R A T U R E C I T ED Bouvet P, Dimitrov S, Wolffe AP. 1994. Specific regulation of Xenopus chromosomal 5s ribosomal-RNA gene transcription in io by histone H1. Genes and Deelopment 8 : 1147–1159. Dasso M, Dimitrov S, Wolffe AP. 1994. Nuclear assembly is independent of linker histones. Proceedings of the National Academy of Sciences USA 91 : 12477–12481. Horsch H, Klee J, Stachel S, Winans SC, Nester W, Rogers SG, Fraley RT. 1986. Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proceedings of the National Academy of Sciences USA 83 : 2571–2575.
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Kandolf H. 1994. The H1A histone variant of the in io repressor of oocyte-type 5S gene transcription in Xenopus leais embryos. Proceedings of the National Academy of Sciences USA 91 : 7257–7260. Multigner L, Gagnon J, van Doisselaer A, Job D. 1992. Stabilization of sea urchin flagellar microtubules by histone. Nature 360 : 33–39. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15 : 473–497. Ohsumi K, Katagiri C, Kishimoto T. 1993. Chromosome condensation in Xenopus mitotic extracts without histone H1. Science 262 : 2033–2035. Paranjape SM, Kamakaka RT, Kadanoga JT. 1994. Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annual Reiew of Biochemistry 63 : 265–297. Prymakowska-Bosak M, Przewloka M, Iwkiewicz J, Egierszdorff S, Kuras! M, Chaubet N, Gigot C, Spiker S, Jerzmanowski A. 1996. Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions. Proceedings of the National Academy of Sciences USA 93 : 10250–10255. Reynolds ES. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. Journal of Cell Biology 17 : 208–212. Shen X, Gorovsky MA. 1996. Linker histone H1 regulates specific gene expression but not global transcription in io. Cell 86 : 475–483. Shen X, Yu L, Weir JW, Gorovsky MA. 1995. Linker histones are not essential and affect chromatin condensation in io. Cell 82 : 47–56. Steinbach OC, Wolffe AP, Rupp RAW. 1997. Somatic linker histones cause loss of mesodermal competence in Xenopus. Nature 389 : 395–399. van Holde KE. 1989. Chromatin. New York : Springer. van Holde K, Zlatanova J. 1996. What determines the folding of the chromatin fibre ? Proceedings of the National Academy of Sciences USA 93 : 10548–10555. Wolffe AP. 1994. Regulation of chromatin structure and function. Austin : R. G. Landes.
Ultrastructural Organization of Leaves of Transgenic Tobacco Overexpressing Histone H1 from Arabidopsis thaliana J O A N N A S! L U S A R C Z Y K*, M A R T A P R Y M A K O W S KA-B O S AK†, M A R C I N P R Z E W Ł O K A†, A N D R Z E J J E R Z M A N O W S KI† ‡ and M I E C Z Y S Ł A W K U R A S! §R * Department of Ecology and Enironmental Protection, Pedagogical School, ul.Konopnickiej 15, Kielce, Poland, † Laboratory of Plant Molecular Biology, Warsaw Uniersity, ul. PawinT skiego 5a, Warsaw, Poland, ‡ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul.PawinT skiego 5a, 02-106 Warsaw, Poland and § Department of Plant Morphogenesis, Warsaw Uniersity, ul. Banacha 2, Warsaw, Poland Received : 8 January 1999
Returned for revision : 19 March 1999
Accepted : 13 May 1999
We investigated the anatomical and ultrastructural features of transgenic tobacco plants that overexpressed a gene of histone H1 from Arabidopsis thaliana. The overexpression of the heterologous gene resulted in more than a 2n5fold increase over the physiological level of the histone H1 : DNA ratio in chromatin. H1-overexpressing plants had a distinct mutant phenotype characterized by dwarf appearance and severely hampered flowering. These changes were accompanied by extensive and unusual heterochromatinization of nuclei occurring in all leaf parenchymal cells but not in leaf epidermal cells. The observed anomalies in the growth rate and size of the cells and in nuclei\chloroplast proportions in histone H1-overexpressing plants suggest that the H1 : DNA ratio can influence some specialized cellular functions involving the cytoskeleton, and nuclear\organellar interactions which are of importance for the normal development of a plant. # 1999 Annals of Botany Company Key words : Transgenic tobacco plant, histone H1 overexpression, heterochromatinization.
INTRODUCTION Eukaryotic DNA is assembled into chromatin in which it is packed by histone proteins into nucleosomes (van Holde, 1989 ; Wolffe, 1994). The inner (core) particle of the nucleosome is formed by an octamer of core histones : H2A, H2B, H3 and H4, around which is wrapped 146 base pairs of DNA. The application, in 1989, of the mutational analysis of core histone genes in yeast revealed for the first time that apart from a purely architectural role in packing DNA, the core histones are involved in many regulatory functions, predominantly concerning transcription. In particular, it was shown that the flexible N terminal tails of histone H3 and H4 participate in activation and repression of transcription of the defined genes (reviewed in Paranjape, Kamakaka and Kadanoga, 1994). In addition to core histones, a complete nucleosome contains a molecule of another type of protein, classified as linker or H1-class histone, which is bound to a variable length linker DNA extending between neighbouring core particles. There are usually several sequence variants of histone H1 in a single multicellular eukaryotic organism. In some organisms, characteristic changes in the profile of H1 variants were found to accompany development. However, in contrast to the abundance of data on the function of core histones, the role in chromatin of histone H1 is far less clear. Typical metazoan linker histone has a three-dimensional structure with a central globular domain flanked by mostly unstructured N- and C-terminal tails. For a long time the presence of linker histones in chromatin was thought 0305-7364\99\090329j07 $30.00\0
essential for folding of the chromatin fibre and histone H1 was considered a general repressor of transcription. The current view, based on analysis of the condensation properties of H1-depleted chromatin, is that linker histones while able to facilitate and guide the proper folding of a nucleosomal fibre, are not the cause of the folding per se (van Holde and Zlatanowa, 1996). As regards the functional role of H1, a key finding was the demonstration that the ciliated protozoan Tetrahymena in which linker histones have been knocked-out by genetic manipulation, grew normally (Shen et al., 1995). This suggested that linker histones are not essential for critical functions such as replication, transcription and protein synthesis. It was later shown that in a histone H1 knock-out strain of Tetrahymena the number of mature RNAs produced by genes transcribed by polymerases I and III and most genes transcribed by polymerase II remained unchanged (Shen and Gorovsky, 1996). These data were supplemented by the demonstration that Xenopus egg extracts depleted of linker histones assemble normal nuclei that are capable of initiating replication and condensing their chromosomes (Ohsumi, Katagiri and Kishimoto, 1993 ; Dasso, Dimitrov and Wolffe, 1994). The inhibition by H1-targeted ribozyme of the accumulation of somatic H1 in the developing embryo of Xenopus did not lead to activation of the vast majority of genes whose expression is required for viability of the embryonic cells (Kandolff, 1994 ; Bouvet, Dimitrov and Wolffe, 1994). In laboratory experiments with transgenic plants, it was shown that overexpression of a somatic-type of histone H1 in tobacco that led to an over two-fold # 1999 Annals of Botany Company
330
ST lusarczyk et al.—Oerexpressing Histone H1
elevation of the physiological H1 : DNA ratio in chromatin and a marked increase of the heterochromatinization of nuclei, had little effect on basal cellular functions (Prymakowska et al., 1996). This indicated that the saturation of chromatin sites with H1 does not lead to marked changes in expression of constitutive genes. Thus, the data from different organisms strongly support the view that linker histones are not general repressors of transcription in io. However, there is evidence that H1 can be involved in highly selective regulation of specific classes of genes. In the H1 knock-out strain of Tetrahymena, two genes, ngoA, a gene of unknown function, and CyP, which encodes a cysteine protease, were shown to be regulated by histone H1 in opposite directions (Shen and Gorovsky, 1996). In an analysis of the early stages of tissue differentiation in Xenopus, it was shown that the accumulation of somatic H1 in chromatin is linked to a selective transcriptional silencing of regulatory genes required for mesodermal differentiation (Steinbach, Wolffe and Rupp, 1997). In our work on tobacco we also noticed that the overexpression of histone H1 in plants, while not hampering the ability of transgenic plants to grow and differentiate, exerted a rather selective effect at the stage of flowering. In the most severe phenotypes, flowers were not formed or were discarded at the early flower bud stage. In order to investigate further the possible ultrastructural background of these specific effects, we subjected transgenic tobacco plants characterized by a high level of transgenic H1 protein to detailed cytological analysis. We hoped to define the anomalies that could become the target of more focused molecular investigation.
MATERIALS AND METHODS Leaves of transgenic tobacco (Nicotiana tabacum sp.) containing the gene for Arabidopsis thaliana L. histone H1 were used. Methods of obtaining and cultivating the plants have been described previously (Prymakowska-Bosak et al., 1996). Briefly, plasmid vectors containing the gene for histone H1 were incorporated into tobacco using the method of leaf disc transformation by Agrobacterium, according to the procedure of Horsch et al. (1986). Leaf discs were placed on solid Murashige and Skoog (1962) medium with agar and the time of regeneration was measured from that moment. Two-week-old plantlets grown from the somatic embryos were placed in soil and cultured in a growth chamber under controlled light, temperature and humidity. Leaves of 13-week-old transgenic H1-overexpressing plants were used for microscopical studies. As a control we used transgenic plants of the same age obtained by the same transformation and regeneration procedure, that did not express the transgene. Squares of 1–2 mm were cut from the middle part of leaves and fixed in 2 % glutaraldehyde in 0n1 cacodylate buffer pH 7n2 for 2 h, followed by additional fixation in 2 % OsO for 2 h at 4 mC. The material % was dehydrated in a graded ethanol series, washed in propylene oxide and embedded in a mixture of Epon and Spurr. Semi-thin (1–2 µm) and ultrathin (80 nm) sections were cut with an LKB ultramicrotome (Sweden). Semi-thin
sections were stained with 0n1 % toluidine blue in 1 % borax and leaf structure was analysed in a light microscope (Nikon). Ultrathin sections were contrasted in water solutions of uranyl acetate (30 min) and lead citrate (30 min) according to Reynolds (1963). Sections were investigated in a transmission electron microscope (JEOL JEM-1200EX, 90 kV). Electron micrographs were taken on 60i90 mm negative film (TN-12, Foton), and used for the analysis of leaf ultrastructure as well as the frequency and size of chloroplasts and starch grains in H1-overexpressing and control plants. RESULTS Growth and morphological appearance of H1oerexpressing plants The present work concerns characterization of transgenic tobacco that contained about 2n5-times more H1 than normal plants, and displayed a distinct mutant phenotype. A detailed description of the construction and the complete molecular and phenotypic characterization of transgenic plants are given in Prymakowska-Bosak et al. (1996). There were no distinct differences in growth rate and shape of control (TRANS H1k) and H1-overexpressing (TRANS H1j) plants in the first phase of development. From the ninth week of regeneration, growth of TRANS H1j plants with severe mutant phenotypes was slowed. This was reflected by a gradual and progressive shortening of stem internodes. TRANS H1j plants achieved their final size after 12–13 weeks of development, producing 14 leaves. Due to almost complete inhibition of the uppermost internodes, the youngest leaves were closely adjacent one to another creating a characteristic palm form. Because TRANS H1k plants grew normally until the twenty-first week there were visible differences in size and shape between control and TRANS H1j plants : at 21 weeks TRANS H1k plants were at least 30 % larger than TRANS H1j plants and had abundant flowers. The TRANS H1j plants did not flower even after 36 weeks, when their lowest leaves were already drying.
Anatomical and ultrastructural differences in leaes Despite differences in leaf size between control and H1overexpressing plants, the overall shape, colour and appearance of leaves were similar. There was also no distinct difference in anatomical structure, which was typical for dicotyledonous plants, with palisade and spongy mesophyll layers and upper and lower epidermal layers (Figs 1 and 2). In both types of plants stomata were present in the two epidermal layers, although they were more frequent in the lower layer, and hairs were present on both epidermal layers. However, in transverse section, the leaf lamella of TRANS H1j plants was much thinner (almost 20 %) than that of control TRANS H1k plants (Figs 1 and 2). The difference was hardly visible in the epidermis (the upper epidermis was 4n6 % thinner and the lower one 2n1 %), and resulted mostly from differences in thickness of the palisade and spongy mesophylls. These were thinner in TRANS
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F. 1. Transverse section through the leaf of a control (TRANS Hk) plant. Semi-thin section stained with toluidine blue. Bar l 20 µm. F. 2. Transverse section through the leaf of an H1-overexpressing (TRANS Hj) plant. Bar l 20 µm. F. 3. Typical eu- and heterochromatin dispersion in the nuclei of palisade mesophyll in TRANS H1k plants. Bar l 0n5 µm. F. 4. Maximal heterochromatization in cell nuclei of palisade mesophyll in TRANS H1j plants. Bar l 1 µm. PM, Palisade mesophyll ; SM, spongy mesophyll ; UE, upper epidermis ; LE, lower epidermis.
H1j plants by 26n5 and 21n1 %, respectively (Table 1). The difference in thickness of the mesophyll tissue was the result of slower growth in TRANS H1j plants. As a consequence, cells of the palisade mesophyll of TRANS H1j plants were 28n8 % shorter and 27 % thinner, while cells of the spongy mesophyll were almost 30 % shorter and 36 % thinner (Table 2) compared to corresponding cells in TRANS H1k plants.
Additionally, leaf epidermal cells of TRANS H1j plants differed from those in TRANS H1k plants by the presence of structures of electron-grey and sometimes even black consistency (Figs 1 and 2). There were also visible differences in the number and size of cell organelles especially chloroplasts, which were larger and more frequent in TRANS H1j cells (up to 100 % more frequent in cells of the palisade mesophyll and 80 % more frequent in cells of
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T 1. Aerage (n l 40) thickness of leaf lamella, palisade and spongy mesophylls and upper and lower epidermal layers of control (TRANS H1k) and H1-oerexpressing (TRANS H1j) plants
in epidermal cells (Figs 5 and 6). In these cells the extent of dispersion and the quantitative proportions of heterochromatin and euchromatin regions were the same as in control TRANS H1k plants.
TRANS H1j TRANS H1k Difference (µm) (µm) (% of control) Upper epidermis Palisade mesophyll Spongy mesophyll Lower epidermis Leaf blade
20n9 61n1 83n7 18n1 183n8
19n9 44n9 66n8 17n7 149n3
D I S C U S S I ON Histone H1 is a key element of the structure of chromosomes. In this work we investigated the influence on the anatomy and ultrastructure of leaves of the 2n5-fold increase over the physiological level of the major somatic form of histone H1 in chromatin of tobacco, caused by the overexpression of the histone H1 gene in the transgenic plants. As reported previously, the plants in which the physiological proportion of H1 : DNA was so drastically disturbed were able to develop and grow (Prymakowska-Bosak et al., 1996), suggesting that histone H1 is not functioning as a general repressor of transcription acting in a dose-dependent manner. Nevertheless, there were several interesting changes in the H1-overexpressing plants that could point to a more specific function of linker histones. The dwarf size and inability to flower were the most characteristic phenotypic changes observed in plants overexpressing H1. These morphological changes were accompanied by profound and characteristic heterochromatinization of the nuclei of the mesophyll cells. However, the normal anatomy of the leaves and unchanged number of cells in H1-overexpressing plants indicate that the ability to differentiate the basic anatomical structures and to continue cell division was not disturbed. This could mean that the extra dose of histone H1 is deposited in chromatin in a rather selective manner, not interfering with the chromosome regions containing important housekeeping and developmental genes. On the other hand, the decrease in the size of the mesophyll cells shows that normal growth had been disturbed in certain cell-types. One possible
95n2 73n5 79n8 97n7 81n2
the spongy mesophyll ; Table 3). Starch grains were also slightly larger in TRANS H1j plant cells and their number was almost 100 % higher than in control cells. Condensation of chromatin was observed in the nuclei on semi-thin sections of TRANS H1j leaf mesophyll. This feature of the nuclei was also clearly visible in TEM (Figs 3–7). The nuclei of control plants consisted mostly of fibres of dispersed chromatin (euchromatin) immersed in karyolymph (Fig. 3). Numerous heterochromatin areas had a thick granular structure but were not very condensed. The edges of heterochromatin were partly diffused, changing into euchromatin fibres (Figs 3 and 6). The nuclei of TRANS H1j plants looked totally different. Their heterochromatin areas were much larger and denser, and showed characteristic lient holes (Figs 4 and 7). The heterochromatin regions had less contact with euchromatin fibres. These fibres were not so frequent and in consequence the transparency of karyolymph was higher. Highly heterochromatinized nuclei were common in mesophyll cells (Figs 4 and 7) especially in the vascular mesophyll of TRANS H1j plants (compare Figs 8 and 10 with 9 and 11). Interestingly, heterochromatinized nuclei were absent
T 2. Aerage length and width of palisade and spongy mesophyll cells in leaes of TRANS H1k and TRANS H1j plants TRANS H1k
Palisade mesophyll Spongy mesophyll
TRANS H1j
Differences (% of control)
Length (µm)
Width (µm)
Length (µm)
Width (µm)
Length
Width
63n1 36n0
15n4 21n4
44n9 25n3
11n3 13n7
71n2 70n3
73n3 64
Data are means of 500 measurements on ten sections.
T 3. Number of chloroplasts and starch grains in palisade and spongy mesophyll cells of TRANS H1- and TRANS H1j plants TRANS H1k
Palisade mesophyll Spongy mesophyll
TRANS H1j
Differences (% of control)
Chloroplasts
Starch grains
Chloroplasts
Starch grains
Chloroplasts
Starch grains
13n6 10n2
58n5 31n3
28n2 18n3
114n4 53n9
207n3 179n4
207n3 172n2
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F. 5. Nucleus with dispersed heterochromatin in a lower epidermal (LE) cell and with highly condensed heterochromatin in the mesophyll cell of a TRANS H1j plant. Bar l 1 µm. SM, Spongy mesophyll. F. 6. Fragment of epidermal cell nucleus of TRANS H1j plant with typical decondensed euchromatin and heterochromatin. Bar l 0n5 µm. F. 7. Fragment of cell nucleus from subepidermal spongy mesophyll with typical heterochromatin and low electron density of karyolymph. Bar l 0n5 µm.
explanation is aberration of the cytoskeleton. In this context it is worth noting that histone H1 has been shown to stabilize microtubular structures in animal cells and green algae (Multigner, Gagnon and van Doisselaer, 1992). This
raises an intriguing possibility that some of the observed phenotypic effects of the overdose of H1 in transgenic tobacco could be due to its interaction with cytoskeletal rather than chromosomal structures.
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F 8 and 10. Dispersion of chromatin in cell nucleus of vascular mesophyll in TRANS H1k plants. Bar l 0n5 µm. F 9 and 11. Maximal heterochromatization in cell nuclei of vascular mesophyll of TRANS H1j plants. Note the decrease in the number of euchromatin fibrils and increased electron transparency of karyolymph. Bar l 0n5 µm.
The noticeable changes in H1-overexpressing plants also concerned the number of chloroplasts. This could be an indication of the disturbed interactions between nuclear and organeller genomes. Interestingly, we recently obtained data showing that the decrease in the amount of major somatic variants of H1 in tobacco results in flower phenotypes strikingly similar to those caused by mutations in mitochondrial DNA (unpubl. res.). The possible link between
H1-dependent chromatin structure and organellar function could provide the basis for identification of new genes responsible for nuclear-organellar interactions. Characteristically, the overexpression of histone H1 did not cause any distinct differences in the epidermal cells. The reason for this is difficult to explain at present. Different reactions to overdoses of H1 could either reflect differences in structural organization of chromatin between epidermal
ST lusarczyk et al.—Oerexpressing Histone H1 and mesophyll cells or the specific inhibition of transgenic H1 expression in epidermal cells. Whatever the reason, this observation is an interesting new clue for the investigation of the mechanisms of cellular differentiation. Summarizing, studies of the ultrastructural organization of plants with strong overexpression of histone H1 confirmed that H1 is not a general inhibitor of genes regulating basic cell functions, including cell division and differentiation. However, the documented anomalies in the growth rate and size of the cells and in nuclei\chloroplast proportions in histone H1-overexpressing plants indicate that the H1 : DNA ratio could influence some specialized cellular functions involving the cytoskeleton, and nuclear\organellar interactions which are of importance for the normal development of a plant. A C K N O W L E D G E M E N TS This research was supported by Howard Hughes Medical Institute grant 79195-543403 (A. J.) and by Polish Committee of Scientific Research grant 6PO4A 02913 (A. J.). L I T E R A T U R E C I T ED Bouvet P, Dimitrov S, Wolffe AP. 1994. Specific regulation of Xenopus chromosomal 5s ribosomal-RNA gene transcription in io by histone H1. Genes and Deelopment 8 : 1147–1159. Dasso M, Dimitrov S, Wolffe AP. 1994. Nuclear assembly is independent of linker histones. Proceedings of the National Academy of Sciences USA 91 : 12477–12481. Horsch H, Klee J, Stachel S, Winans SC, Nester W, Rogers SG, Fraley RT. 1986. Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proceedings of the National Academy of Sciences USA 83 : 2571–2575.
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