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Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development
BBRC Biochemical and Biophysical Research Communications 330 (2005) 887–890 www.elsevier.com/locate/ybbrc
Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development Woong Han a, Hae-Ik Rhee a, Jeong Woo Cho b, Maurice S.B. Ku c, Pill Soon Song b, Myeong-Hyeon Wang a,* a
Division of Biotechnology, Kangwon National University, Chuncheon, Kangwon-do 200-701, Republic of Korea Kumho Life and Environmental Science Laboratory, Oryongdong, Bukgu, Kwangju 500-712, Republic of Korea School of Biological Sciences and Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-4236, USA b
c
Received 3 March 2005 Available online 18 March 2005
Abstract Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division. 2005 Elsevier Inc. All rights reserved. Keywords: CDK inhibitor; Transgenic Arabidopsis; ACK1; Plant growth; ICK; Leaf morphology
Cell division and subsequent differentiation are highly organized processes that are regulated by members of the cyclin dependent kinase family [1,2]. Cyclin–cyclin-dependent kinase (CDK) complexes can regulate cell cycle progression positively or negatively [3,4], and the activity and specificity of a CDK depends on its association with cyclins and other regulatory proteins [5,6]. Plants with reduced levels of CDK activity are altered in cell division and growth [7]. CDKs exert their effects by phosphorylating serine/threonine residues on specific substrates involved in signal transduction pathways [8]. CDK inhibitors (ICKs) are low molecular-weight proteins that bind to cyclin–CDK
*
Corresponding author. Fax: +82 33 241 6480. E-mail address: [email protected] (M.-H. Wang).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.056
complexes and also act as assembly factors for CDKs and cyclins [3,9,10]. The cell cycle is an integral part of plant growth and development [11,12], and is regulated by CDKs [13]. The role of the cell cycle is being approached in many studies of the conserved mechanisms controlling cell division and development [14,15]. These mechanisms depend on interactions between intrinsic developmental programs and environmental signals [16] and require proper control of the cell division cycle [17,18]. It is likely that signals intrinsic to plant growth and development are integrated through a variety of pathways and channeled into the regulation of cell proliferation and differentiation. The present study addresses the link between cell division, morphogenesis and plant growth by investigating the functions of a plant CDK inhibitor. We report the characterization of an ACK1
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W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
gene by overexpressing it in Arabidopsis. The results provide evidence of how ACK1 is involved in plant growth and development.
Materials and methods Plant materials. Plants of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) were grown either in soil or in MS medium, at a constant temperature of 22 C under 16/8 h day/night cycle. cDNA clone and transformation. The ACK1 cDNA was identified in a yeast two-hybrid screen, as described previously [19]. The primers used were based on sequence information (Accession No: AF106705) and the ACK1 cDNA was amplified from total RNA using the ThermoScript RT-PCR system (Life Technologies). The plasmid contained ACK1 gene introduced into Agrobacterium tumefaciens strain LBA4404 and used to transform Arabidopsis plants by vacuum infiltration [20]. Seeds (T1) from infiltrated plants were harvested and selected on MS medium containing 50 mg/L kanamycin. The resulting kanamycin-resistant plants were transferred to soil in pots and grown in chambers. RT-PCR analysis. Total RNA was isolated with Trizol (GibcoBRL) according to the manufacturerÕs instructions and used for RT-PCR with forward primer: 5 0 GAGAAAAGACTTGTTCGA TGGTTCTCATA3 0 and reverse primer: 5 0 TGAATTGCTTCTT CTTATCGTCTTGACTC3 0 . RT reaction mixtures (20 ll) contained 5 U AMV reverse transcriptase (Life Sciences), 20 U RNase inhibitor, 5 mM MgCl2, 2 ll of 10· RNA PCR buffer, 4 mM dNTPs, 1 lg total RNA, and 1 lM of random primer, and were incubated for 30 min at 50 C. For synthesis of the first strand cDNA, 50 ll reaction mixtures contained 3 ll of 25 mM MgCl2, 4 ll of 10· PCR buffer, 2.5 U Taq DNA polymerase, and 1 ll RT mixture. The PCR protocol consisted of 30 cycles of 92 C for 1 min, 42 C for 30 s, and 72 C for 1 min 30 s, followed by extension at 72 C for 7 min. Ten microliter samples of the PCR products were analyzed by agarose gel electrophoresis and stained by ethidium bromide. Immunoblotting. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes. The blots were blocked with 5% milk in TBS-T and probed with anti-ACK1 antibody followed by HRP-conjugated a-Ig secondary antibody (Amersham). Evaluation of plant growth. Transgenic Arabidopsis plants were examined by microscope for alterations in cell size and shape. Overall plant architecture was also compared with control plants.
Results and discussion Characterization of ACK1 The identification of CDK regulators is important for understanding how CDK activity is regulated in plant development and during plant responses to environmental changes. Using the yeast two-hybrid system, we identified and cloned several putative genes encoding proteins interacting with CDKs in Arabidopsis and one of these genes encoded ACK1 (Accession No. AF106705). Four identical clones of a 0.9 kb ACK1 cDNA encoded a putative 196 amino acid protein. Phylogenetic analysis showed a close relationship between ACK1 and ICK1, ICK3, ICK5, and particularly ICK7 (Fig. 1), suggesting that it is an ICK-related protein. ICK1, ICK2, ICK6, and ICK7 interact with both Cdc2a
Fig. 1. The ACK/ICK genes in plants. A phylogenetic tree showing the relationship between ACK1 and ICK proteins. ACK1 (AF 106705) was compared with ICK1 (ATU94772), ICK3 (NM-124259), ICK5 (NM-113393), and ICK7 (NM-103850) using CLUSTAL W with tree construction parameters.
Fig. 2. Structure T-DNA tagging. (A) The T-DNA tagged position in the mutant alleles (B) Southern blot analysis indicating T-DNA tagged mutant alleles produce smaller size than the wild type. m, TDNA insertion site. Wt, wild type (ecotype Columbia). M, mutant alleles. (C) RT-PCR showing that the ACK1 gene is knocked out in the transgenic plants. Thirty cycles of PCR were used to amplify ACK1 and actin. Tissues were collected from 20-day-old wild type and transgenic plants, and RNA was isolated. Wt, wild type and M, mutant plants.
and D-type cyclins, whereas ICK4 and ICK5 interact only with D-type cyclins [6]. Plants contain a small family of CDK inhibitors consisting of two members, ICK1 and ICK2, which inhibit CDK activity [21]. Clearly the ACK1 product is structurally related to CDK inhibitors. The ACK1 transgenic plants generated harbored a selectable marker for kanamycin resistance and Southern blot analysis showed that the T-DNA tagged pool yielded DNA fragments of different sizes from those of wild type plant, as would be expected (Fig. 2B). (RT)PCR confirmed that ACK1 was deleted in transgenic plants (Fig. 2C). Effect of overexpression of ACK1 on plant cells and plant morphology To determine whether the ACK1 transgenic plants had fewer cells due to inhibition of cell division or of cell growth, we compared the size and structure of the cells
W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
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Fig. 3. Structural characterization of cells in wild type (Wt) and ACK1 transgenic plants by light microscopy. The ACK1 transgenic plants have irregular leaf veins. All scale bars = 100 lm.
of the ACK1 transgenic plants with those of wild type plants. The cells of ACK1 transgenic tissues were more irregular than the corresponding cells in wild type plants (Fig. 3). There was also a reduction in leaf size, resulting in altered leaf margin serration. These characteristic features were observed in the leaves of all ACK1 transgenic plants, reflecting strong growth inhibition. There were profound changes in the morphology of organs such as leaves and roots in the transgenic plants (Fig. 4); overexpression of ACK1 exerted a strong inhibitory effect on leaf and root growth, suggesting a lack of cell division in the marginal region (Fig. 4B). There were also serious defects in leaf formation, as well as overall growth retardation (Fig. 4C). We have presented strong in vivo evidence of ACK1 in growth and development, suggesting that ACK1 will play a negative role in cell proliferation. Similar effects were observed in ICK1 transgenic Arabidopsis plants in an earlier study [22]. Overexpression of ICK genes resulted in highly lobed leaves due to changes in the cell cycle [23]. Phenotypic changes in leaf morphology were observed in Arabidopsis plants transformed with each of the plant CDK inhibitors [23], indicating that precise regulation of terminal cell division is very important for tissue architecture and function. An ACK1 expression in the Arabidopsis transgenic plants resulted in growth retardation and the severity of retardation in different transgenic lines. The ACK1 transgenic plants were successfully rescued by cross-pollination of wild type (Fig. 4D). The expressions of ACK1 protein are visualized by Western blot using ACK1 transgenic plant lines (Fig. 4E). Plant growth and development is controlled via an intricate network of extracellular and intracellular signaling pathways [24]. Mutant analysis has provided insight into organ formation during cell division and growth in plants [25]. However, we still do not understand the role of ACK1 proteins in coordinating cell division with growth and development. We conclude that overall plant growth rate is directly related to cell division rate. ACK1 probably reduces cell growth by inhibiting cell division. It is clear that overexpression of ACK1, as a cell cycle regulator, interferes with plant growth and morphogenesis. Therefore, it
Fig. 4. Phenotypes of ACK1 transgenic and wild type plants. Ten-dayold wild type and ACK1 transgenic plants (A), and 20-day-old wild type and ACK1 transgenic plants (B,C). The ACK1 transgenic plants were growth-retarded and showed poor leaf and root growth. (D) Phenotypes of ACK1 transgenic plants cross-pollinated with wild type grown at 20 days. (E) Levels of ACK1 protein in different transgenic lines shown by Western blotting with a monospecific polyclonal antibody.
may have an important function in the control of plant growth by regulating the cell cycle, thus providing a mechanism for integrating multiple signals affecting meristem activity. However, comprehensive studies of cell cycle genes are required to provide a thorough understanding of the regulation of plant growth and development.
Acknowledgments We are grateful for financial support from the Korea Research Foundation (grant 0802006-1-1) and to John H. Doonan and Julian Gross for their critical reading of the manuscript.
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W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
References [1] R.P. Huntley, J.A. Murray, The plant cell cycle, Curr. Opin. Plant Biol. 2 (1999) 440–446. [2] V. Mironov, L. De Veylder, M. Van Montagu, D. Inze, Cyclin dependent kinases and cell division in plants—the nexus, Plant Cell 11 (1999) 509–522. [3] J. Pines, Four-dimensional control of the cell cycle, Nat. Cell Biol. 1 (1999) 73–79. [4] K. Swaminathan, Y. Yang, N. Grotz, L. Campisi, T. Jack, An enhancer trap line associated with a D-class cyclin gene in Arabidopsis, Plant Physiol. 124 (2000) 1658–1667. [5] R.M. Barroco, L. De Veylder, Z. Magyar, G. Engler, D. Inze, V. Mironov, Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division, Cell Mol. Life Sci. 60 (2003) 401–412. [6] Y. Zhou, G. Li, F. Brandizzi, L.C. Fowke, H. Wang, The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition, protein instability and nuclear localization, Plant J. 35 (2003) 476–489. [7] H. Lui, H. Wang, C. DeLong, L.C. Fowke, W.L. Crosby, P.R. Fobert, The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin dependent kinase activity in vitro, Plant J. 21 (2000) 379–385. [8] H. Stals, P. Casteels, M. Van Montagu, D. Inze, Regulation of cyclin-dependent kinases in Arabidopsis thaliana, Plant Mol. Biol. 43 (2000) 583–593. [9] M. Cheng, P. Olivier, J.A. Diehl, M. Fero, M.F. Roussel, J.M. Roberts, C.J. Sherr, The p21(Cip1) and p27(Kip1) CDK ÔinhibitorsÕ are essential activators of cyclin D-dependent kinases in murine fibroblasts, EMBO J. 18 (1999) 1571–1583. [10] C.J. Sherr, J.M. Roberts, CDK inhibitors: positive and negative regulators of G1-phase progression, Genes Dev. 13 (1999) 1501– 1512. [11] Schnittger, C. Weinl, D. Bouyer, U. Schobinger, M. Hulskamp, Misexpression of the cyclin-dependent kinase inhibitor ICK1/ KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death, Plant Cell 15 (2003) 303–315. [12] Y. Zhou, H. Wang, S. Gilmer, S. Whitwill, L.C. Fowke, Effects of co-expressing the plant CDK inhibitor ICK1 and D-type cyclin genes on plant growth, cell size and ploidy in Arabidopsis thaliana, Planta 216 (2003) 604–613.
[13] S. Jasinski, C. Riou-Khamlichi, O. Roche, C. Perennes, C. Bergounioux, N. Glab, The CDK inhibitor NtKIS1a is involved in plant development, endoreduplication and restores normal development of cyclin D3; 1-overexpressing plants, J. Cell Sci. 115 (2002) 973–982. [14] G.T. Beemster, F. Fiorani, D. Inze, Cell cycle: the key to plant growth control?, Trends Plant Sci. 8 (2003) 154–158. [15] R. Huntley, S. Healy, D. Freemen, P. Lavender, S. de Jager, J. Greenwood, J. Makker, E. Walker, M. Jackman, Q. Xie, The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf development and displays conserved interactions with G1/S regulator and plant cyclin D(CycD) protein, Plant Mol. Biol. 37 (1998) 155–169. [16] C. Fankhauser, J. Chory, Light control of plant development, Annu. Rev. Cell Dev. Biol. 13 (1997) 203–229. [17] D. Inze, Why should we study the plant cell cycle?, J. Exp. Bot. 54 (2003) 1125–1126. [18] M. Meijer, J.A. Murray, Cell cycle controls and the development of plant form, Curr. Opin. Plant Biol. 4 (2001) 44–49. [19] J.W. Cho, S.C. Park, E.A. Shin, C.K. Kim, W. Han, S.I. Sohn, P.S. Song, M.H. Wang, Cyclin D1 and p22ack1 play opposite roles in plant growth and development, Biochem. Biophys. Res. Commun. 324 (2004) 52–57. [20] N. Bechtold, G. Pelletier, In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration, Methods Mol. Biol. 82 (1998) 259–266. [21] H. Wang, L.C. Fowke, W.L. Crosby, A plant cyclin-dependent kinase inhibitor gene, Nature 386 (1997) 451–452. [22] H. Wang, Y. Zhou, S. Gilmer, S. Whitwill, L.C. Fowke, Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology, Plant J. 24 (2000) 613–623. [23] Y. Zhou, L.C. Fowke, H. Wang, Plant CDK inhibitors: studies of interactions with cell cycle regulators in the yeast two-hybrid system and functional comparisons in transgenic Arabidopsis plants, Plant Cell Rep. 20 (2002) 967–975. [24] C.E. Cockcroft, B.G.W. den Boer, Healy, J.M.S. Healy, J.A.H. Murray, Cyclin D control of growth rate in plants, Nature 405 (2000) 575–579. [25] C. Lincoln, I. Long, J. Yamaguchi, K. Serikawa, S. Hake, A knotted 1 like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants, Plant Cell 6 (1994) 1859–1876.
Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development Woong Han a, Hae-Ik Rhee a, Jeong Woo Cho b, Maurice S.B. Ku c, Pill Soon Song b, Myeong-Hyeon Wang a,* a
Division of Biotechnology, Kangwon National University, Chuncheon, Kangwon-do 200-701, Republic of Korea Kumho Life and Environmental Science Laboratory, Oryongdong, Bukgu, Kwangju 500-712, Republic of Korea School of Biological Sciences and Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-4236, USA b
c
Received 3 March 2005 Available online 18 March 2005
Abstract Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division. 2005 Elsevier Inc. All rights reserved. Keywords: CDK inhibitor; Transgenic Arabidopsis; ACK1; Plant growth; ICK; Leaf morphology
Cell division and subsequent differentiation are highly organized processes that are regulated by members of the cyclin dependent kinase family [1,2]. Cyclin–cyclin-dependent kinase (CDK) complexes can regulate cell cycle progression positively or negatively [3,4], and the activity and specificity of a CDK depends on its association with cyclins and other regulatory proteins [5,6]. Plants with reduced levels of CDK activity are altered in cell division and growth [7]. CDKs exert their effects by phosphorylating serine/threonine residues on specific substrates involved in signal transduction pathways [8]. CDK inhibitors (ICKs) are low molecular-weight proteins that bind to cyclin–CDK
*
Corresponding author. Fax: +82 33 241 6480. E-mail address: [email protected] (M.-H. Wang).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.056
complexes and also act as assembly factors for CDKs and cyclins [3,9,10]. The cell cycle is an integral part of plant growth and development [11,12], and is regulated by CDKs [13]. The role of the cell cycle is being approached in many studies of the conserved mechanisms controlling cell division and development [14,15]. These mechanisms depend on interactions between intrinsic developmental programs and environmental signals [16] and require proper control of the cell division cycle [17,18]. It is likely that signals intrinsic to plant growth and development are integrated through a variety of pathways and channeled into the regulation of cell proliferation and differentiation. The present study addresses the link between cell division, morphogenesis and plant growth by investigating the functions of a plant CDK inhibitor. We report the characterization of an ACK1
888
W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
gene by overexpressing it in Arabidopsis. The results provide evidence of how ACK1 is involved in plant growth and development.
Materials and methods Plant materials. Plants of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) were grown either in soil or in MS medium, at a constant temperature of 22 C under 16/8 h day/night cycle. cDNA clone and transformation. The ACK1 cDNA was identified in a yeast two-hybrid screen, as described previously [19]. The primers used were based on sequence information (Accession No: AF106705) and the ACK1 cDNA was amplified from total RNA using the ThermoScript RT-PCR system (Life Technologies). The plasmid contained ACK1 gene introduced into Agrobacterium tumefaciens strain LBA4404 and used to transform Arabidopsis plants by vacuum infiltration [20]. Seeds (T1) from infiltrated plants were harvested and selected on MS medium containing 50 mg/L kanamycin. The resulting kanamycin-resistant plants were transferred to soil in pots and grown in chambers. RT-PCR analysis. Total RNA was isolated with Trizol (GibcoBRL) according to the manufacturerÕs instructions and used for RT-PCR with forward primer: 5 0 GAGAAAAGACTTGTTCGA TGGTTCTCATA3 0 and reverse primer: 5 0 TGAATTGCTTCTT CTTATCGTCTTGACTC3 0 . RT reaction mixtures (20 ll) contained 5 U AMV reverse transcriptase (Life Sciences), 20 U RNase inhibitor, 5 mM MgCl2, 2 ll of 10· RNA PCR buffer, 4 mM dNTPs, 1 lg total RNA, and 1 lM of random primer, and were incubated for 30 min at 50 C. For synthesis of the first strand cDNA, 50 ll reaction mixtures contained 3 ll of 25 mM MgCl2, 4 ll of 10· PCR buffer, 2.5 U Taq DNA polymerase, and 1 ll RT mixture. The PCR protocol consisted of 30 cycles of 92 C for 1 min, 42 C for 30 s, and 72 C for 1 min 30 s, followed by extension at 72 C for 7 min. Ten microliter samples of the PCR products were analyzed by agarose gel electrophoresis and stained by ethidium bromide. Immunoblotting. Proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes. The blots were blocked with 5% milk in TBS-T and probed with anti-ACK1 antibody followed by HRP-conjugated a-Ig secondary antibody (Amersham). Evaluation of plant growth. Transgenic Arabidopsis plants were examined by microscope for alterations in cell size and shape. Overall plant architecture was also compared with control plants.
Results and discussion Characterization of ACK1 The identification of CDK regulators is important for understanding how CDK activity is regulated in plant development and during plant responses to environmental changes. Using the yeast two-hybrid system, we identified and cloned several putative genes encoding proteins interacting with CDKs in Arabidopsis and one of these genes encoded ACK1 (Accession No. AF106705). Four identical clones of a 0.9 kb ACK1 cDNA encoded a putative 196 amino acid protein. Phylogenetic analysis showed a close relationship between ACK1 and ICK1, ICK3, ICK5, and particularly ICK7 (Fig. 1), suggesting that it is an ICK-related protein. ICK1, ICK2, ICK6, and ICK7 interact with both Cdc2a
Fig. 1. The ACK/ICK genes in plants. A phylogenetic tree showing the relationship between ACK1 and ICK proteins. ACK1 (AF 106705) was compared with ICK1 (ATU94772), ICK3 (NM-124259), ICK5 (NM-113393), and ICK7 (NM-103850) using CLUSTAL W with tree construction parameters.
Fig. 2. Structure T-DNA tagging. (A) The T-DNA tagged position in the mutant alleles (B) Southern blot analysis indicating T-DNA tagged mutant alleles produce smaller size than the wild type. m, TDNA insertion site. Wt, wild type (ecotype Columbia). M, mutant alleles. (C) RT-PCR showing that the ACK1 gene is knocked out in the transgenic plants. Thirty cycles of PCR were used to amplify ACK1 and actin. Tissues were collected from 20-day-old wild type and transgenic plants, and RNA was isolated. Wt, wild type and M, mutant plants.
and D-type cyclins, whereas ICK4 and ICK5 interact only with D-type cyclins [6]. Plants contain a small family of CDK inhibitors consisting of two members, ICK1 and ICK2, which inhibit CDK activity [21]. Clearly the ACK1 product is structurally related to CDK inhibitors. The ACK1 transgenic plants generated harbored a selectable marker for kanamycin resistance and Southern blot analysis showed that the T-DNA tagged pool yielded DNA fragments of different sizes from those of wild type plant, as would be expected (Fig. 2B). (RT)PCR confirmed that ACK1 was deleted in transgenic plants (Fig. 2C). Effect of overexpression of ACK1 on plant cells and plant morphology To determine whether the ACK1 transgenic plants had fewer cells due to inhibition of cell division or of cell growth, we compared the size and structure of the cells
W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
889
Fig. 3. Structural characterization of cells in wild type (Wt) and ACK1 transgenic plants by light microscopy. The ACK1 transgenic plants have irregular leaf veins. All scale bars = 100 lm.
of the ACK1 transgenic plants with those of wild type plants. The cells of ACK1 transgenic tissues were more irregular than the corresponding cells in wild type plants (Fig. 3). There was also a reduction in leaf size, resulting in altered leaf margin serration. These characteristic features were observed in the leaves of all ACK1 transgenic plants, reflecting strong growth inhibition. There were profound changes in the morphology of organs such as leaves and roots in the transgenic plants (Fig. 4); overexpression of ACK1 exerted a strong inhibitory effect on leaf and root growth, suggesting a lack of cell division in the marginal region (Fig. 4B). There were also serious defects in leaf formation, as well as overall growth retardation (Fig. 4C). We have presented strong in vivo evidence of ACK1 in growth and development, suggesting that ACK1 will play a negative role in cell proliferation. Similar effects were observed in ICK1 transgenic Arabidopsis plants in an earlier study [22]. Overexpression of ICK genes resulted in highly lobed leaves due to changes in the cell cycle [23]. Phenotypic changes in leaf morphology were observed in Arabidopsis plants transformed with each of the plant CDK inhibitors [23], indicating that precise regulation of terminal cell division is very important for tissue architecture and function. An ACK1 expression in the Arabidopsis transgenic plants resulted in growth retardation and the severity of retardation in different transgenic lines. The ACK1 transgenic plants were successfully rescued by cross-pollination of wild type (Fig. 4D). The expressions of ACK1 protein are visualized by Western blot using ACK1 transgenic plant lines (Fig. 4E). Plant growth and development is controlled via an intricate network of extracellular and intracellular signaling pathways [24]. Mutant analysis has provided insight into organ formation during cell division and growth in plants [25]. However, we still do not understand the role of ACK1 proteins in coordinating cell division with growth and development. We conclude that overall plant growth rate is directly related to cell division rate. ACK1 probably reduces cell growth by inhibiting cell division. It is clear that overexpression of ACK1, as a cell cycle regulator, interferes with plant growth and morphogenesis. Therefore, it
Fig. 4. Phenotypes of ACK1 transgenic and wild type plants. Ten-dayold wild type and ACK1 transgenic plants (A), and 20-day-old wild type and ACK1 transgenic plants (B,C). The ACK1 transgenic plants were growth-retarded and showed poor leaf and root growth. (D) Phenotypes of ACK1 transgenic plants cross-pollinated with wild type grown at 20 days. (E) Levels of ACK1 protein in different transgenic lines shown by Western blotting with a monospecific polyclonal antibody.
may have an important function in the control of plant growth by regulating the cell cycle, thus providing a mechanism for integrating multiple signals affecting meristem activity. However, comprehensive studies of cell cycle genes are required to provide a thorough understanding of the regulation of plant growth and development.
Acknowledgments We are grateful for financial support from the Korea Research Foundation (grant 0802006-1-1) and to John H. Doonan and Julian Gross for their critical reading of the manuscript.
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W. Han et al. / Biochemical and Biophysical Research Communications 330 (2005) 887–890
References [1] R.P. Huntley, J.A. Murray, The plant cell cycle, Curr. Opin. Plant Biol. 2 (1999) 440–446. [2] V. Mironov, L. De Veylder, M. Van Montagu, D. Inze, Cyclin dependent kinases and cell division in plants—the nexus, Plant Cell 11 (1999) 509–522. [3] J. Pines, Four-dimensional control of the cell cycle, Nat. Cell Biol. 1 (1999) 73–79. [4] K. Swaminathan, Y. Yang, N. Grotz, L. Campisi, T. Jack, An enhancer trap line associated with a D-class cyclin gene in Arabidopsis, Plant Physiol. 124 (2000) 1658–1667. [5] R.M. Barroco, L. De Veylder, Z. Magyar, G. Engler, D. Inze, V. Mironov, Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division, Cell Mol. Life Sci. 60 (2003) 401–412. [6] Y. Zhou, G. Li, F. Brandizzi, L.C. Fowke, H. Wang, The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition, protein instability and nuclear localization, Plant J. 35 (2003) 476–489. [7] H. Lui, H. Wang, C. DeLong, L.C. Fowke, W.L. Crosby, P.R. Fobert, The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin dependent kinase activity in vitro, Plant J. 21 (2000) 379–385. [8] H. Stals, P. Casteels, M. Van Montagu, D. Inze, Regulation of cyclin-dependent kinases in Arabidopsis thaliana, Plant Mol. Biol. 43 (2000) 583–593. [9] M. Cheng, P. Olivier, J.A. Diehl, M. Fero, M.F. Roussel, J.M. Roberts, C.J. Sherr, The p21(Cip1) and p27(Kip1) CDK ÔinhibitorsÕ are essential activators of cyclin D-dependent kinases in murine fibroblasts, EMBO J. 18 (1999) 1571–1583. [10] C.J. Sherr, J.M. Roberts, CDK inhibitors: positive and negative regulators of G1-phase progression, Genes Dev. 13 (1999) 1501– 1512. [11] Schnittger, C. Weinl, D. Bouyer, U. Schobinger, M. Hulskamp, Misexpression of the cyclin-dependent kinase inhibitor ICK1/ KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death, Plant Cell 15 (2003) 303–315. [12] Y. Zhou, H. Wang, S. Gilmer, S. Whitwill, L.C. Fowke, Effects of co-expressing the plant CDK inhibitor ICK1 and D-type cyclin genes on plant growth, cell size and ploidy in Arabidopsis thaliana, Planta 216 (2003) 604–613.
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