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Ectopic Expression of the NtSET1 Histone Methyltransferase Inhibits Cell Expansion, and Affects Cell Division and Differentiation in Tobacco Plants

Ectopic Expression of the NtSET1 Histone Methyltransferase Inhibits Cell Expansion, and Affects... Abstract The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases. Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth. Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation. We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth. Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development. (Received April 30, 2004; Accepted August 27, 2004) In eukaryotes, nuclear DNA is wrapped around a histone octamer that consists of two molecules each of histone proteins H2A, H2B, H3 and H4 (Luger et al. 1997). The histones, particularly their N-terminal tails, are subject to various covalent modifications, including acetylation, methylation, phosphorylation, ubiquitination and ADP ribosylation (reviewed in Ausio et al. 2001). These distinct modifications, proposed as ‘histone codes’ (Strahl and Allis 2000, Jenuwein and Allis 2001), could serve as binding sites for regulatory proteins or protein complexes that participate in certain downstream nuclear processes including chromosomal condensation and segregation, gene transcription, DNA replication and repair. Only recently, functions of histone methylation have started to be uncovered and this is primarily due to the findings that SET-domain proteins possess histone methyltransferase (HMTase) activity (reviewed in Lachner et al. 2003, Loidl 2004). The evolutionarily conserved SET domain of approximately 130 amino acids length in, which was first identified and named after three Drosophila genes [Su(var), E(z) and Trithorax] involved in epigenetic processes (Tschiersch et al. 1994), forms a knot-like structure and constitutes the catalytic core of the enzyme (reviewed in Marmorstein 2003). The enzyme activity and specificity for different histone lysine residues depend not only on the SET domain but also on its surrounding sequences. In general, methylation of histone H3 lysine 4, lysine 36 and lysine 79 correlates with transcriptional activation, whereas methylation of histone H3 lysine 9 (H3K9), H3 lysine 27 and H4 lysine 20 associates with transcriptional repression. At least 29 SET-domain proteins have been identified in Arabidopsis, but only a few of them have so far been characterized (reviewed in Loidl 2004). ArabidopsisKYP encodes a H3K9 HMTase and its loss-of-function reduces H3K9 methylation but without causing phenotypic abnormalities even after extensive inbreeding (Jackson et al. 2002). We previously identified the tobacco NtSET1 as a gene encoding a SET-domain protein that binds chromatin (Shen 2001). More recently, we showed that NtSET1, like KYP, functions as a H3K9 HMTase and that ectopic expression of NtSET1–GFP (NtSET1 fused to the coding sequence of green fluorescent protein) increases H3K9 methylation and causes chromatin segregation defects in tobacco suspension-cultured cells (Yu et al. 2004). Here we analysed cellular effects of increased H3K9 methylation on tobacco plant growth by ectopic overexpression of NtSET1–GFP. Transgenic tobacco plants expressing GFP (control) and NtSET1–GFP under the control of the dexamethasone (DEX) inducible promoter have been described previously (Shen 2001). In order to test the role of the SET domain, a construct containing SET domain deletion of NtSET1–GFP (NtSET1D1–GFP) under the control of the DEX-inducible promoter was obtained by subcloning the XhoI–SpeI fragment from pSKNtSET1D1–GFP (Yu et al. 2004) into the pTA7002 (Aoyama and Chua 1997) vector. The transgenes encoding NtSET1–GFP and NtSET1D1–GFP are schematically shown in Fig. 1A. Transformation of tobacco leaf discs was performed as described (Shen 2001) and 14 independent transformants were regenerated for the NtSET1D1–GFP construct. Growth tests on F2 progeny revealed that none of the NtSET1D1–GFP transgenic lines manifested a phenotype upon DEX treatment. However, Northern analysis of F2 progeny on five lines revealed that two of them, NtSET1D1–GFP OE3 and OE5, showed good DEX-induced expression of NtSET1D1–GFP (Fig. 1B), to levels comparable to those of NtSET1–GFP found in the previously described NtSET1–GFP OE1 to OE3 transgenic lines (Shen 2001). Microscopic examination also detected high levels of GFP fluorescence in these lines (data not shown). When grown on the same plate containing the medium in the presence of DEX, NtSET1D1–GFP OE3 and OE5 plants did not show significant differences compared with the control plants (Fig. 1C) whereas NtSET1–GFP OE1 and OE3 plants were, as previously reported (Shen 2001), strongly inhibited. Together these results establish a crucial role for the catalytic SET domain in NtSET1-mediated inhibition of tobacco plant growth. To analyse the growth inhibition at the cellular level, we first performed an induction of transgene expression by spraying DEX onto greenhouse-grown plants at the four-leaf stage. This treatment inhibited growth of NtSET1–GFP OE3 plants, visible by small size leaves as early as 4 d after treatment (Fig. 2B), whereas the growth of NtSET1D1–GFP OE5 plants was not affected (Fig. 2A). Microscopic observation revealed that the leaf epidermal pavement cells in the NtSET1–GFP OE3 plants (Fig. 2D) are significantly smaller than those in the control plants (Fig. 2C). The cell areas of NtSET1–GFP OE3 and control pavement cells were 3,215±1,604 µm2 (n = 29) and 7,038±2,305 µm2 (n = 30), respectively. Reduced cell expansion is possibly the primary cause of the small size of the treated leaves. An inhibitory effect of NtSET1–GFP expression on cell expansion was also observed in in vitro cultured plants. Root hairs, tubular single cells that form from certain root epidermal cells, are much shorter in NtSET1–GFP OE3 plants (Fig. 2F) than in control plants (Fig. 2E). Previous kinetic studies showed that the DEX-induced transgene transcripts reach a maximal level at 24 h and decrease to a barely detectable level at 96 h after DEX application (Aoyama and Chua 1997). In order to study the effects of continuous induction of NtSET1–GFP expression on plant growth, we germinated seeds on sterile filter paper wetted in liquid culture medium. The culture medium containing DEX was renewed by a freshly prepared solution every third day. Under this continuous induction, NtSET1–GFP OE3 plants showed growth inhibition as early as after germination and dramatically modified phenotypes at later growth stages (Fig. 3A). Narrow and shrinking leaves (Fig. 3B) as well as leaf fusions (Fig. 3C) were observed. Longitudinal sections revealed distortions of the shoot apex as well as defects of tissue differentiation of the leaf (Fig. 3D). The epidermal and the mesophyll cells within the leaf or parts of the leaf were small and dense (Fig. 3F), indicating a less-differentiated state, as compared with the normal fully differentiated epidermal and mesophyll cells containing large vacuoles (Fig. 3E). Consistent with previous results obtained in tobacco BY2 suspension cells (Yu et al. 2004), abnormal nuclear divisions (resulting in poly-/micro-nuclei) were observed in about 1% of cells within the leaf (Fig. 3G, H), indicating ectopic effects of NtSET1–GFP on chromatin segregation. In contrast to NtSET1–GFP OE3 plants, NtSET1D1–GFP OE5 plants as well as control plants did not show abnormal phenotypes under the same treatment conditions. Together our results demonstrate that ectopic overexpression of the NtSET1 H3K9 HMTase dramatically affects cell expansion, division and differentiation in tobacco plants. This occurs in a catalytic SET-domain-dependent manner, suggesting that H3K9 methylation plays a crucial role in the regulation of plant development. The pleiotropic phenotype observed in plants overexpressing NtSET1–GFP is consistent with our previous data showing that NtSET1–GFP protein is broadly localized within chromatin (Shen 2001, Yu et al. 2004), suggesting that NtSET1 targets a large number of genes. Although the HMTase activity has not been studied, ectopic overexpression of the rice SET-domain gene OsSET1 in Arabidopsis also impaired shoot development (Liang et al. 2003). Detailed analyses of cellular effects of OsSET1, however, had not been performed. Distinct from overexpression studies, knockout of KYP decreases H3K9 methylation but without affecting Arabidopsis plant growth (Jackson et al. 2002). Nevertheless, several loci were affected by reduced DNA methylation and gene silencing in the kyp mutants (Jackson et al. 2002, Malagnac et al. 2002). Arabidopsis contains an additional eight KYP-related genes that are all expressed (Baumbusch et al. 2001). Their function, however, is currently unknown. This amplification of genes encoding potential H3K9 HMTases also occurs in other plant species (Springer et al. 2003, Zhao and Shen 2004), suggesting crucial roles of H3K9 methylation in plant development. More studies will be necessary to further analyse the roles of up- and down-regulation of H3K9 methylation in the control of plant growth, and more importantly, to identify downstream molecular mechanisms involved in the regulation of cell expansion, division and differentiation. 1 Corresponding author: E-mail, Wen-Hui.Shen@ibmp-ulp.u-strasbg.fr; Fax, +33-3-88-61-44-42. View largeDownload slide Fig. 1 Ectopic overexpression of NtSET1–GFP but not of NtSET1D1–GFP inhibits tobacco plant growth. (A) Diagram of the transgenes encoding NtSET1–GFP and NtSET1D1–GFP under the control of the DEX inducible promoter. The NtSET1 containing the SET- and RING finger-associated domain (SRA), the cysteine-rich domain (Cys) and the SET domain (SET) was fused to the N-terminus of GFP. (B) Northern blot analysis. Total RNA was prepared from 3-week-old seedlings, separated by electrophoresis on a formaldehyde–agarose gel, blotted onto membrane and hybridized successively with radioactively labelled GFP and 18S rRNA probes. Three NtSET1–GFP (OE 1–3) and five NtSET1D1–GFP (OE 1–5) transgenic lines ware analysed together with the wild-type seedlings (W). The closed and open arrowheads indicate the position of the NtSET1–GFP and the NtSET1D1–GFP transcripts, respectively. (C) Seeds of F2 progeny of the transgenic plants were germinated on the medium in the presence of 5 µM DEX. Two 3-week-old seedlings representing each of the indicated transgenic lines are shown in the photograph. View largeDownload slide Fig. 1 Ectopic overexpression of NtSET1–GFP but not of NtSET1D1–GFP inhibits tobacco plant growth. (A) Diagram of the transgenes encoding NtSET1–GFP and NtSET1D1–GFP under the control of the DEX inducible promoter. The NtSET1 containing the SET- and RING finger-associated domain (SRA), the cysteine-rich domain (Cys) and the SET domain (SET) was fused to the N-terminus of GFP. (B) Northern blot analysis. Total RNA was prepared from 3-week-old seedlings, separated by electrophoresis on a formaldehyde–agarose gel, blotted onto membrane and hybridized successively with radioactively labelled GFP and 18S rRNA probes. Three NtSET1–GFP (OE 1–3) and five NtSET1D1–GFP (OE 1–5) transgenic lines ware analysed together with the wild-type seedlings (W). The closed and open arrowheads indicate the position of the NtSET1–GFP and the NtSET1D1–GFP transcripts, respectively. (C) Seeds of F2 progeny of the transgenic plants were germinated on the medium in the presence of 5 µM DEX. Two 3-week-old seedlings representing each of the indicated transgenic lines are shown in the photograph. View largeDownload slide Fig. 2 Cell expansion is inhibited by ectopic overexpression of NtSET1–GFP. Four-leaf-stage greenhouse-grown plants were induced for transgene expression by spraying with a solution containing 10 µM DEX and 0.01% (w/v) Tween-20. (A) and (B) show a representative plant of NtSET1D1–GFP OE5 and of NtSET1–GFP OE3, 4 d after the induction, respectively. (C) and (D) show the differential interference contrast images of epidermal cells of the third leaf of the NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants, respectively. (E) and (F) show root zones carrying mature hairs from NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants cultured in a medium containing 5 µM DEX, respectively. Bars = 20 µm in (C, D) and 100 µm in (E, F). View largeDownload slide Fig. 2 Cell expansion is inhibited by ectopic overexpression of NtSET1–GFP. Four-leaf-stage greenhouse-grown plants were induced for transgene expression by spraying with a solution containing 10 µM DEX and 0.01% (w/v) Tween-20. (A) and (B) show a representative plant of NtSET1D1–GFP OE5 and of NtSET1–GFP OE3, 4 d after the induction, respectively. (C) and (D) show the differential interference contrast images of epidermal cells of the third leaf of the NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants, respectively. (E) and (F) show root zones carrying mature hairs from NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants cultured in a medium containing 5 µM DEX, respectively. Bars = 20 µm in (C, D) and 100 µm in (E, F). View largeDownload slide Fig. 3 Continuous induction of NtSET1–GFP expression dramatically affects plant development. Representative plants from control and NtSET1–GFP OE3, 6 weeks after culture in the liquid medium containing 5 µM DEX that was refreshed every third day, are shown on the photograph in (A). NtSET1–GFP OE3 plants show abnormal phenotypes, including narrow and shrinking leaves (B) and leaf fusions (indicated by arrow in C). Longitudinal section through the central part of the shoot apex and a leaf of an NtSET1–GFP OE3 plant is shown in (D). Note that the shoot apical dome is deformed and that the leaf displays mosaic tissue differentiation, with the middle part containing small and less differentiated cells. (E) and (F) show close-ups of a fully differentiated and a less differentiated section of the leaf, respectively. The nuclei are lightened by staining of the sections with 4′,6-diamidino-2-phenylindole (DAPI). The darker smaller granules correspond to chloroplasts. Different layers of cells within the leaf are labelled: ‘AdE’ for the adaxial epidermis, ‘Me’ for the mesophyll, ‘Xy’ for the xylem, ‘Ph’ for the phloem and ‘AbE’ for the abaxial epidermis. (G) and (H) show poly-/micro-nuclei within two single cells observed in leaf sections of NtSET1–GFP OE3 plants, indicating cell division defects. Bars = 50 µm in (E, F) and 10 µm in (G, H). View largeDownload slide Fig. 3 Continuous induction of NtSET1–GFP expression dramatically affects plant development. Representative plants from control and NtSET1–GFP OE3, 6 weeks after culture in the liquid medium containing 5 µM DEX that was refreshed every third day, are shown on the photograph in (A). NtSET1–GFP OE3 plants show abnormal phenotypes, including narrow and shrinking leaves (B) and leaf fusions (indicated by arrow in C). Longitudinal section through the central part of the shoot apex and a leaf of an NtSET1–GFP OE3 plant is shown in (D). Note that the shoot apical dome is deformed and that the leaf displays mosaic tissue differentiation, with the middle part containing small and less differentiated cells. (E) and (F) show close-ups of a fully differentiated and a less differentiated section of the leaf, respectively. The nuclei are lightened by staining of the sections with 4′,6-diamidino-2-phenylindole (DAPI). The darker smaller granules correspond to chloroplasts. Different layers of cells within the leaf are labelled: ‘AdE’ for the adaxial epidermis, ‘Me’ for the mesophyll, ‘Xy’ for the xylem, ‘Ph’ for the phloem and ‘AbE’ for the abaxial epidermis. (G) and (H) show poly-/micro-nuclei within two single cells observed in leaf sections of NtSET1–GFP OE3 plants, indicating cell division defects. Bars = 50 µm in (E, F) and 10 µm in (G, H). Abbreviations DAPI 4′,6-diamidino-2-phenylindole DEX dexamethasone HMTase histone methyltransferase GFP green fluorescent protein H3K9 histone 3 lysine 9 OE overexpression SET Su(var)-E(z)-Trithorax associated domain. References Aoyama, T. and Chua, N.-H. ( 1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J.  11: 605–612. Google Scholar Ausio, J., Abbott, D.W., Wang, X. and Moore, S.C. ( 2001) Histone variants and histone modifications: a structural perspective. Biochem. Cell Biol.  79: 693–708. Google Scholar Baumbusch, L.O., Thorstensen, T., Krauss, V., Fischer, A., Naumann, K., Assalkhou, R., Schulz, I., Reuter, G. and Aalen, R.B. ( 2001) The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes. Nucleic Acids Res.  29: 4319–4333. Google Scholar Jackson, J.P., Lindroth, A.M., Cao, X. and Jacobsen, S.E. ( 2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature  416: 556–560. Google Scholar Jenuwein, T. and Allis, C.D. ( 2001) Translating the histone code. Science  293: 1074–1080. Google Scholar Lachner, M., O’Sullivan, R.J. and Jenuwein, T. ( 2003) An epigenetic road map for histone lysine methylation. J. Cell Sci.  116: 2117–2124. Google Scholar Liang, Y.K., Wang, Y., Zhang, Y., Li, S.G., Lu, X.C., Li, H., Zou, C., Xu, Z.H. and Bai, S.N. ( 2003) OsSET1, a novel SET-domain-containing gene from rice. J. Exp. Bot.  54: 1995–1996. Google Scholar Loidl, P. ( 2004) A plant dialect of the histone language. Trends Plant Sci.  9: 84–90. Google Scholar Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. and Richmond, T.J. ( 1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature  389: 251–260. Google Scholar Malagnac, F., Bartee, L. and Bender, J. ( 2002) An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation. EMBO J.  21: 6842–6852. Google Scholar Marmorstein, R. ( 2003) Structure of SET domain proteins: a new twist on histone methylation. Trends Biochem. Sci.  28: 59–62. Google Scholar Shen, W.-H. ( 2001) NtSET1, a member of a newly identified subgroup of plant SET-domain-containing proteins, is chromatin-associated and its ectopic overexpression inhibits tobacco plant growth. Plant J.  28: 371–383. Google Scholar Springer, N.M., Napoli, C.A., Selinger, D.A., Pandey, R., Cone, K.C., Chandler, V.L., Kaeppler, H.F. and Kaeppler, S.M. ( 2003) Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots. Plant Physiol.  132: 907–925. Google Scholar Strahl, B.D. and Allis, D. ( 2000) The language of covalent histone modifications. Nature  403: 41–45. Google Scholar Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G. and Reuter, G. ( 1994) The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3–9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J.  13: 3822–3831. Google Scholar Yu, Y., Dong, A. and Shen, W.-H. ( 2004). Molecular characterization of the tobacco SET-domain protein NtSET1 unravels its role in histone methylation, chromatin binding and segregation. Plant J.  (online publication date: 5-Oct-2004 doi: 10.1111/j.1365-313X.2004.02240.X) (in press). Google Scholar Zhao, Z. and Shen, W.-H. ( 2004) Plants contain a high number of proteins showing sequence similarity to the animal SUV39H family histone-methyltransferases. Ann. N. Y. Acad. Sci.  1030 (in press). Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Ectopic Expression of the NtSET1 Histone Methyltransferase Inhibits Cell Expansion, and Affects Cell Division and Differentiation in Tobacco Plants

Plant and Cell Physiology , Volume 45 (11) – Nov 15, 2004

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Publisher
Oxford University Press
ISSN
0032-0781
eISSN
1471-9053
DOI
10.1093/pcp/pch184
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15574848
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Abstract

Abstract The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases. Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth. Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation. We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth. Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development. (Received April 30, 2004; Accepted August 27, 2004) In eukaryotes, nuclear DNA is wrapped around a histone octamer that consists of two molecules each of histone proteins H2A, H2B, H3 and H4 (Luger et al. 1997). The histones, particularly their N-terminal tails, are subject to various covalent modifications, including acetylation, methylation, phosphorylation, ubiquitination and ADP ribosylation (reviewed in Ausio et al. 2001). These distinct modifications, proposed as ‘histone codes’ (Strahl and Allis 2000, Jenuwein and Allis 2001), could serve as binding sites for regulatory proteins or protein complexes that participate in certain downstream nuclear processes including chromosomal condensation and segregation, gene transcription, DNA replication and repair. Only recently, functions of histone methylation have started to be uncovered and this is primarily due to the findings that SET-domain proteins possess histone methyltransferase (HMTase) activity (reviewed in Lachner et al. 2003, Loidl 2004). The evolutionarily conserved SET domain of approximately 130 amino acids length in, which was first identified and named after three Drosophila genes [Su(var), E(z) and Trithorax] involved in epigenetic processes (Tschiersch et al. 1994), forms a knot-like structure and constitutes the catalytic core of the enzyme (reviewed in Marmorstein 2003). The enzyme activity and specificity for different histone lysine residues depend not only on the SET domain but also on its surrounding sequences. In general, methylation of histone H3 lysine 4, lysine 36 and lysine 79 correlates with transcriptional activation, whereas methylation of histone H3 lysine 9 (H3K9), H3 lysine 27 and H4 lysine 20 associates with transcriptional repression. At least 29 SET-domain proteins have been identified in Arabidopsis, but only a few of them have so far been characterized (reviewed in Loidl 2004). ArabidopsisKYP encodes a H3K9 HMTase and its loss-of-function reduces H3K9 methylation but without causing phenotypic abnormalities even after extensive inbreeding (Jackson et al. 2002). We previously identified the tobacco NtSET1 as a gene encoding a SET-domain protein that binds chromatin (Shen 2001). More recently, we showed that NtSET1, like KYP, functions as a H3K9 HMTase and that ectopic expression of NtSET1–GFP (NtSET1 fused to the coding sequence of green fluorescent protein) increases H3K9 methylation and causes chromatin segregation defects in tobacco suspension-cultured cells (Yu et al. 2004). Here we analysed cellular effects of increased H3K9 methylation on tobacco plant growth by ectopic overexpression of NtSET1–GFP. Transgenic tobacco plants expressing GFP (control) and NtSET1–GFP under the control of the dexamethasone (DEX) inducible promoter have been described previously (Shen 2001). In order to test the role of the SET domain, a construct containing SET domain deletion of NtSET1–GFP (NtSET1D1–GFP) under the control of the DEX-inducible promoter was obtained by subcloning the XhoI–SpeI fragment from pSKNtSET1D1–GFP (Yu et al. 2004) into the pTA7002 (Aoyama and Chua 1997) vector. The transgenes encoding NtSET1–GFP and NtSET1D1–GFP are schematically shown in Fig. 1A. Transformation of tobacco leaf discs was performed as described (Shen 2001) and 14 independent transformants were regenerated for the NtSET1D1–GFP construct. Growth tests on F2 progeny revealed that none of the NtSET1D1–GFP transgenic lines manifested a phenotype upon DEX treatment. However, Northern analysis of F2 progeny on five lines revealed that two of them, NtSET1D1–GFP OE3 and OE5, showed good DEX-induced expression of NtSET1D1–GFP (Fig. 1B), to levels comparable to those of NtSET1–GFP found in the previously described NtSET1–GFP OE1 to OE3 transgenic lines (Shen 2001). Microscopic examination also detected high levels of GFP fluorescence in these lines (data not shown). When grown on the same plate containing the medium in the presence of DEX, NtSET1D1–GFP OE3 and OE5 plants did not show significant differences compared with the control plants (Fig. 1C) whereas NtSET1–GFP OE1 and OE3 plants were, as previously reported (Shen 2001), strongly inhibited. Together these results establish a crucial role for the catalytic SET domain in NtSET1-mediated inhibition of tobacco plant growth. To analyse the growth inhibition at the cellular level, we first performed an induction of transgene expression by spraying DEX onto greenhouse-grown plants at the four-leaf stage. This treatment inhibited growth of NtSET1–GFP OE3 plants, visible by small size leaves as early as 4 d after treatment (Fig. 2B), whereas the growth of NtSET1D1–GFP OE5 plants was not affected (Fig. 2A). Microscopic observation revealed that the leaf epidermal pavement cells in the NtSET1–GFP OE3 plants (Fig. 2D) are significantly smaller than those in the control plants (Fig. 2C). The cell areas of NtSET1–GFP OE3 and control pavement cells were 3,215±1,604 µm2 (n = 29) and 7,038±2,305 µm2 (n = 30), respectively. Reduced cell expansion is possibly the primary cause of the small size of the treated leaves. An inhibitory effect of NtSET1–GFP expression on cell expansion was also observed in in vitro cultured plants. Root hairs, tubular single cells that form from certain root epidermal cells, are much shorter in NtSET1–GFP OE3 plants (Fig. 2F) than in control plants (Fig. 2E). Previous kinetic studies showed that the DEX-induced transgene transcripts reach a maximal level at 24 h and decrease to a barely detectable level at 96 h after DEX application (Aoyama and Chua 1997). In order to study the effects of continuous induction of NtSET1–GFP expression on plant growth, we germinated seeds on sterile filter paper wetted in liquid culture medium. The culture medium containing DEX was renewed by a freshly prepared solution every third day. Under this continuous induction, NtSET1–GFP OE3 plants showed growth inhibition as early as after germination and dramatically modified phenotypes at later growth stages (Fig. 3A). Narrow and shrinking leaves (Fig. 3B) as well as leaf fusions (Fig. 3C) were observed. Longitudinal sections revealed distortions of the shoot apex as well as defects of tissue differentiation of the leaf (Fig. 3D). The epidermal and the mesophyll cells within the leaf or parts of the leaf were small and dense (Fig. 3F), indicating a less-differentiated state, as compared with the normal fully differentiated epidermal and mesophyll cells containing large vacuoles (Fig. 3E). Consistent with previous results obtained in tobacco BY2 suspension cells (Yu et al. 2004), abnormal nuclear divisions (resulting in poly-/micro-nuclei) were observed in about 1% of cells within the leaf (Fig. 3G, H), indicating ectopic effects of NtSET1–GFP on chromatin segregation. In contrast to NtSET1–GFP OE3 plants, NtSET1D1–GFP OE5 plants as well as control plants did not show abnormal phenotypes under the same treatment conditions. Together our results demonstrate that ectopic overexpression of the NtSET1 H3K9 HMTase dramatically affects cell expansion, division and differentiation in tobacco plants. This occurs in a catalytic SET-domain-dependent manner, suggesting that H3K9 methylation plays a crucial role in the regulation of plant development. The pleiotropic phenotype observed in plants overexpressing NtSET1–GFP is consistent with our previous data showing that NtSET1–GFP protein is broadly localized within chromatin (Shen 2001, Yu et al. 2004), suggesting that NtSET1 targets a large number of genes. Although the HMTase activity has not been studied, ectopic overexpression of the rice SET-domain gene OsSET1 in Arabidopsis also impaired shoot development (Liang et al. 2003). Detailed analyses of cellular effects of OsSET1, however, had not been performed. Distinct from overexpression studies, knockout of KYP decreases H3K9 methylation but without affecting Arabidopsis plant growth (Jackson et al. 2002). Nevertheless, several loci were affected by reduced DNA methylation and gene silencing in the kyp mutants (Jackson et al. 2002, Malagnac et al. 2002). Arabidopsis contains an additional eight KYP-related genes that are all expressed (Baumbusch et al. 2001). Their function, however, is currently unknown. This amplification of genes encoding potential H3K9 HMTases also occurs in other plant species (Springer et al. 2003, Zhao and Shen 2004), suggesting crucial roles of H3K9 methylation in plant development. More studies will be necessary to further analyse the roles of up- and down-regulation of H3K9 methylation in the control of plant growth, and more importantly, to identify downstream molecular mechanisms involved in the regulation of cell expansion, division and differentiation. 1 Corresponding author: E-mail, Wen-Hui.Shen@ibmp-ulp.u-strasbg.fr; Fax, +33-3-88-61-44-42. View largeDownload slide Fig. 1 Ectopic overexpression of NtSET1–GFP but not of NtSET1D1–GFP inhibits tobacco plant growth. (A) Diagram of the transgenes encoding NtSET1–GFP and NtSET1D1–GFP under the control of the DEX inducible promoter. The NtSET1 containing the SET- and RING finger-associated domain (SRA), the cysteine-rich domain (Cys) and the SET domain (SET) was fused to the N-terminus of GFP. (B) Northern blot analysis. Total RNA was prepared from 3-week-old seedlings, separated by electrophoresis on a formaldehyde–agarose gel, blotted onto membrane and hybridized successively with radioactively labelled GFP and 18S rRNA probes. Three NtSET1–GFP (OE 1–3) and five NtSET1D1–GFP (OE 1–5) transgenic lines ware analysed together with the wild-type seedlings (W). The closed and open arrowheads indicate the position of the NtSET1–GFP and the NtSET1D1–GFP transcripts, respectively. (C) Seeds of F2 progeny of the transgenic plants were germinated on the medium in the presence of 5 µM DEX. Two 3-week-old seedlings representing each of the indicated transgenic lines are shown in the photograph. View largeDownload slide Fig. 1 Ectopic overexpression of NtSET1–GFP but not of NtSET1D1–GFP inhibits tobacco plant growth. (A) Diagram of the transgenes encoding NtSET1–GFP and NtSET1D1–GFP under the control of the DEX inducible promoter. The NtSET1 containing the SET- and RING finger-associated domain (SRA), the cysteine-rich domain (Cys) and the SET domain (SET) was fused to the N-terminus of GFP. (B) Northern blot analysis. Total RNA was prepared from 3-week-old seedlings, separated by electrophoresis on a formaldehyde–agarose gel, blotted onto membrane and hybridized successively with radioactively labelled GFP and 18S rRNA probes. Three NtSET1–GFP (OE 1–3) and five NtSET1D1–GFP (OE 1–5) transgenic lines ware analysed together with the wild-type seedlings (W). The closed and open arrowheads indicate the position of the NtSET1–GFP and the NtSET1D1–GFP transcripts, respectively. (C) Seeds of F2 progeny of the transgenic plants were germinated on the medium in the presence of 5 µM DEX. Two 3-week-old seedlings representing each of the indicated transgenic lines are shown in the photograph. View largeDownload slide Fig. 2 Cell expansion is inhibited by ectopic overexpression of NtSET1–GFP. Four-leaf-stage greenhouse-grown plants were induced for transgene expression by spraying with a solution containing 10 µM DEX and 0.01% (w/v) Tween-20. (A) and (B) show a representative plant of NtSET1D1–GFP OE5 and of NtSET1–GFP OE3, 4 d after the induction, respectively. (C) and (D) show the differential interference contrast images of epidermal cells of the third leaf of the NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants, respectively. (E) and (F) show root zones carrying mature hairs from NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants cultured in a medium containing 5 µM DEX, respectively. Bars = 20 µm in (C, D) and 100 µm in (E, F). View largeDownload slide Fig. 2 Cell expansion is inhibited by ectopic overexpression of NtSET1–GFP. Four-leaf-stage greenhouse-grown plants were induced for transgene expression by spraying with a solution containing 10 µM DEX and 0.01% (w/v) Tween-20. (A) and (B) show a representative plant of NtSET1D1–GFP OE5 and of NtSET1–GFP OE3, 4 d after the induction, respectively. (C) and (D) show the differential interference contrast images of epidermal cells of the third leaf of the NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants, respectively. (E) and (F) show root zones carrying mature hairs from NtSET1D1–GFP OE5 and NtSET1–GFP OE3 plants cultured in a medium containing 5 µM DEX, respectively. Bars = 20 µm in (C, D) and 100 µm in (E, F). View largeDownload slide Fig. 3 Continuous induction of NtSET1–GFP expression dramatically affects plant development. Representative plants from control and NtSET1–GFP OE3, 6 weeks after culture in the liquid medium containing 5 µM DEX that was refreshed every third day, are shown on the photograph in (A). NtSET1–GFP OE3 plants show abnormal phenotypes, including narrow and shrinking leaves (B) and leaf fusions (indicated by arrow in C). Longitudinal section through the central part of the shoot apex and a leaf of an NtSET1–GFP OE3 plant is shown in (D). Note that the shoot apical dome is deformed and that the leaf displays mosaic tissue differentiation, with the middle part containing small and less differentiated cells. (E) and (F) show close-ups of a fully differentiated and a less differentiated section of the leaf, respectively. The nuclei are lightened by staining of the sections with 4′,6-diamidino-2-phenylindole (DAPI). The darker smaller granules correspond to chloroplasts. Different layers of cells within the leaf are labelled: ‘AdE’ for the adaxial epidermis, ‘Me’ for the mesophyll, ‘Xy’ for the xylem, ‘Ph’ for the phloem and ‘AbE’ for the abaxial epidermis. (G) and (H) show poly-/micro-nuclei within two single cells observed in leaf sections of NtSET1–GFP OE3 plants, indicating cell division defects. Bars = 50 µm in (E, F) and 10 µm in (G, H). View largeDownload slide Fig. 3 Continuous induction of NtSET1–GFP expression dramatically affects plant development. Representative plants from control and NtSET1–GFP OE3, 6 weeks after culture in the liquid medium containing 5 µM DEX that was refreshed every third day, are shown on the photograph in (A). NtSET1–GFP OE3 plants show abnormal phenotypes, including narrow and shrinking leaves (B) and leaf fusions (indicated by arrow in C). Longitudinal section through the central part of the shoot apex and a leaf of an NtSET1–GFP OE3 plant is shown in (D). Note that the shoot apical dome is deformed and that the leaf displays mosaic tissue differentiation, with the middle part containing small and less differentiated cells. (E) and (F) show close-ups of a fully differentiated and a less differentiated section of the leaf, respectively. The nuclei are lightened by staining of the sections with 4′,6-diamidino-2-phenylindole (DAPI). The darker smaller granules correspond to chloroplasts. Different layers of cells within the leaf are labelled: ‘AdE’ for the adaxial epidermis, ‘Me’ for the mesophyll, ‘Xy’ for the xylem, ‘Ph’ for the phloem and ‘AbE’ for the abaxial epidermis. (G) and (H) show poly-/micro-nuclei within two single cells observed in leaf sections of NtSET1–GFP OE3 plants, indicating cell division defects. Bars = 50 µm in (E, F) and 10 µm in (G, H). Abbreviations DAPI 4′,6-diamidino-2-phenylindole DEX dexamethasone HMTase histone methyltransferase GFP green fluorescent protein H3K9 histone 3 lysine 9 OE overexpression SET Su(var)-E(z)-Trithorax associated domain. References Aoyama, T. and Chua, N.-H. ( 1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J.  11: 605–612. Google Scholar Ausio, J., Abbott, D.W., Wang, X. and Moore, S.C. ( 2001) Histone variants and histone modifications: a structural perspective. Biochem. Cell Biol.  79: 693–708. 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Journal

Plant and Cell PhysiologyOxford University Press

Published: Nov 15, 2004

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