Comparative Molecular and Functional Analyses of the Tobacco Cyclin-Dependent Kinase Inhibitor NtKIS1a and Its Spliced Variant NtKIS1b

Abstract

In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.The precise control of cell cycle progression is critical for coherent development because perturbation of this control leads to phenomena such as oncogenesis in animals. However, developmental programs adopted by plants differ markedly from those of animals. Plant development is mostly postembryonic, and throughout their life, plants form new organs by virtue of meristematic cells that are permanently able to divide. However, key cell cycle regulators discovered in yeast and animals appear remarkably conserved in plants (Huntley and Murray, 1999;Mironov et al., 1999;den Boer and Murray, 2000). Thus, in all eukaryotes, the core of the cell cycle machinery consists of complexes composed of a catalytic subunit, a cyclin-dependent kinase (CDK), and a regulatory subunit, a cyclin. Two major types of plant CDKs are revealed by sequence analysis, yeast complementation assays and expression patterns. Whereas canonical PSTAIR-containing A-type CDKs resemble CDK1 and CDK2 from mammals and CDC28/cdc2 ϩ from budding and fission yeast, respectively, B-type CDKs have plant specific features, notably a cell cycle phase-specific expression that usually peaks in the G2 phase (Segers et al., 1997;Mironov et al., 1999;Joubes et al., 2000)...

An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid Division[W]

“…In 35S:AtSulA-GFP plants, transcripts accumulate at very high levels, whereas the AtSulA-GFP protein does not, indicating a translational or posttranslational regulation of the transgene expression. Moreover, we have shown previously that overexpression of the NtKIS cell-cycle inhibitor fused to GFP results in homogeneous GFP staining in all cells throughout plant development (Jasinski et al, 2002). Therefore, the heterogeneity of AtSulA-GFP accumulation between cells does not result from variation of transgene transcription from one cell to another.…”

The Cyclin-Dependent Kinase Inhibitor Orysa;KRP1 Plays an Important Role in Seed Development of Rice

“…It should be noted that the proteins were numbered sequentially and, consequently, that the KRP members from rice and other species should not be regarded as systematic orthologs. Sequence similarity between the KRPs of rice and other plants is mostly concentrated in a region of 40 amino acids located at the extreme C-terminal end of each KRP, which encompasses the conserved motifs 1, 2, and 3 of plant ICK/KRPs (De Veylder et al, 2001;Jasinski et al, 2002). Conserved motifs 1 and 2 include the CDKbinding box, whereas motif 3 corresponds to the CYCD-binding box (Wang et al, 1998).…”

“…In tobacco (Nicotiana tabacum), a KRP-like inhibitor designated NtKIS1a was isolated by a yeast two-hybrid screen. Overexpression of this gene in Arabidopsis (Arabidopsis thaliana) reduced CDK kinase activity (Jasinski et al, 2002). Also, the tomato (Lycopersicon esculentum) LeKRP1 has recently been shown to inhibit CDK/CYC kinase activities in endoreduplicating cells of the gel tissue (Bisbis et al, 2006).…”

“…Additionally, transgenic plants have been generated that overexpress KRP genes by means of a constitutive promoter De Veylder et al, 2001;Jasinski et al, 2002;Zhou et al, 2002a). In all cases, drastic developmental abnormalities were reported, such as overall reduced growth evidenced by smaller/ shorter vegetative and reproductive organs.…”

Balance between cell division and differentiation during plant development

“…Overexpression of KRP1 or KRP2 inhibits cell division and changes morphogenesis of leaves that contain less but larger cells (Wang, et al, 2000;De Veylder, et al, 2001), an effect partially reversed by overexpression of an inducer of cell division, such as CYCD3 (Zhou, et al, 2003). Likewise, overexpression of tobacco CDK inhibitor (KIS1) reverses the altered leaf phenotype of plants expressing CYCD3 ectopically (Jasinski, et al, 2002). This indicates that a proper balance of cell division activity and cell cycle arrest is required to develop a correct morphogenetic pattern.…”