The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro

Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein-protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy-terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.     

Plant CDK inhibitors: studies of interactions with cell cycle regulators in the yeast two-hybrid system and functional comparisons in transgenic Arabidopsis plants

. The cyclin-dependent kinase (CDK) inhibitors ICK1 and ICK2 have been shown to inhibit plant CDK activity in vitro, and the expression of ICK1 was able to inhibit cell division in the plant and modify plant growth and morphology. In order to characterize other ICK1-related inhibitor genes and understand possible differences among plant CDK inhibitors, the interactions of plant CDK inhibitors with cell cycle regulators were analysed in the yeast two-hybrid system and their functions were compared in transgenic Arabidopsis plants. Yeast two-hybrid results indicate that there are likely two groups of plant CDK inhibitors. The A-group inhibitors ICK1, ICK2, ICK6 and ICK7 interact with Cdc2a and three D-type cyclins (D1, D2 and D3), while the B-group inhibitors ICK4, ICK5 and ICKCr interact with D-type cyclins but not with Arabidopsis Cdc2a. ICK1 (A-group), and ICK4 and ICKCr (B-group) were expressed separately in transgenic Arabidopsis plants. Overexpression of the three inhibitor genes resulted in plants of a smaller size with serrated leaves and modified flowers. These plants also had reduced nuclear DNA content (polyploidy), suggesting that expression of these inhibitors affected endoreduplication. Further, there were apparent differences in the strength of effect among the inhibitors. These results provide the first evidence on the CDK inhibitory function for ICK4 and ICKCr. They also suggest that these CDK inhibitors play important roles in cell division and plant growth.     

The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization

Interactor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the first plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green fluorescent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1DeltaN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1DeltaN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1DeltaN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.     

Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology

The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.     

Arabidopsis cyclin D2 expressed in rice forms a functional cyclin-dependent kinase complex that enhances seedling growth

D-class cyclins play important roles in controlling the cell cycle in development and in response to external signals by forming the regulatory subunit of cyclin-dependent kinase (CDK) complexes. To evaluate the effects of D-class cyclins in transgenic rice plants, Arabidopsis cyclin D2 gene (CycD2) was linked to the maize ubiquitin1 promoter (Ubi1) and introduced into rice by the Agrobacterium-mediated transformation method. Genomic deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Western blot hybridizations of the Ubi1:CycD2 plants revealed copy number of transgene and its increased expression in leaf and callus cells at messenger RNA (mRNA) and/or protein levels. The H1 kinase assay using the immunoprecipitates of protein extracts from the Ubi1:CycD2 plants and nontransgenic controls demonstrated that the introduced Arabidopsis CycD2 forms a functional CycD2/CDK complex with an unidentified CDK of rice. Shoot and root growth was enhanced in the Ubi1:CycD2 seedlings compared with nontransgenic controls, together, suggesting that Arabidopsis cyclin D2 interacts with a rice cyclin-dependent kinase, consequently enhancing seedling growth.     

Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic <Emphasis Type="Italic">Brassica</Emphasis> plants

The cyclin-dependent protein kinases (CDKs) have a central role in cell cycle regulation and can be inhibited by the binding of small protein CDK inhibitors. The first plant CDK inhibitor gene ICK1 was previously identified in Arabidopsis thaliana. In comparison to known animal CDK inhibitors, ICK1 protein exhibits unique structural and functional properties. The expression of ICK1 directed by the constitutive CaMV 35S promoter was shown to inhibit cell division and plant growth. The aim of this study was to determine the effects of ICK1 overexpression on particular organs and cells. ICK1 was expressed in specific tissues or cells of Brassica napus L. plants using two tissue-specific promoters, Arabidopsis AP3 and Brassica Bgp1. Transgenic AP3-ICK1 plants were morphologically normal except for some modified flowers either without petals or with petals of reduced size. Surprisingly, petals of novel shapes such as tubular petals were also observed, indicating a profound effect of cell division inhibition on morphogenesis. The cell size in the smaller modified petals was similar to that in control petals, suggesting that the reduction of petal size is mainly due to the reduction of cell numbers and that the inhibition of cell division does not necessarily lead to an increase in cell size. Transgenic Bgp1-ICK1 plants were normal morphologically; however, dramatic decreases in seed production were observed in some plants. In those plants, the ability of pollen to germinate and pollen nuclear number were affected. These results are discussed in relation to the cell cycle and plant development.     

Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation,and organ and seed sizes in Arabidopsis

The ICK/KRP cyclin‐dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T‐DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lower‐order mutants showed no morphological phenotypes, the ick1 ick2 ick6 ick7 and ick1 ick2 ick5 ick6 ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild‐type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage‐dependent manner. Analyses also revealed increased expression of E2F‐dependent genes, and elevated RBR1 as well as an increased level of phospho‐RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.     

Modulation of CycD3;1‐CDK complexes by phytohormones and sucrose during maize germination

Maize CycD3;1 associates to CDKA or CDKB1;1 proteins during germination and the complexes formed develop kinase activity. These complexes appear to vary in size as germination proceeds, suggesting association to different sets of proteins. CycD3;1 and associated CDK proteins respond to phytohormones and sucrose. Results revealed a reduction in the CycD3;1 protein amount along germination in the presence of indoleacetic acid (IAA) or abscisic acid (ABA), although in the latter protein levels recover at the end of germination. While the levels of CDKA increase with IAA, they decrease with ABA. Both phytohormones, IAA and ABA, increase levels of CDKB1;1 only during the early germination times. CycD3;1 associated kinase activity is only reduced by both phytohormones towards the end of the germination period. On the other hand, lack of sucrose in the imbibition medium strongly reduces CycD3;1 protein levels without affecting the levels of neither CDKA nor CDKB1;1. The corresponding CycD3;1 associated kinase activity is also severely decreased. The presence of sucrose in the medium appears to stabilize the CycD3;1 protein levels.     

A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level,probably through a ubiquitin‐independent mechanism

The ICK/KRP family of cyclin‐dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope‐tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA‐ICK1 protein was observed when both the N‐terminal 1–40 sequence was removed and the SCF (SKP1–Cullin1–F‐box complex) function disrupted, suggesting the involvement of both SCF‐dependent and SCF‐independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21–30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N‐terminus or C‐terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP‐ICK1^1–40 in yeast. These results thus identify a protein‐destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF‐independent mechanism.     

Analysis of the subcellular localization, function, and proteolytic control of the Arabidopsis cyclin-dependent kinase inhibitor ICK1/KRP1

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.     

Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis

In animals, cyclin-dependent kinase inhibitors (CKIs) are important regulators of cell cycle progression. Recently, putative CKIs were also identified in plants, and in previous studies, Arabidopsis thaliana plants misexpressing CKIs were found to have reduced endoreplication levels and decreased numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act in a concentration-dependent manner and have an important function in cell proliferation as well as in cell cycle exit and in turning from a mitotic to an endoreplicating cell cycle mode. Endoreplication is usually associated with terminal differentiation; we observed, however, that cell fate specification proceeded independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells. On the one hand, this challenges plant cell cycle control with keeping CKIs locally controlled, and on the other hand this provides a possibility of linking cell cycle control in single cells with the supracellular organization of a tissue or an organ.     

Targeted degradation of the cyclin-dependent kinase inhibitor ICK4/KRP6 by RING-type E3 ligases is essential for mitotic cell cycle progression during Arabidopsis gametogenesis

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.     

Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.     

The effect of ICK1, a plant cyclin-dependent kinase inhibitor,on mitosis in living plant cells

The inhibitory activity of Arabidopsis thaliana ICK1, a plant cyclin-dependent kinase inhibitor, has previously been characterised by its effect on plant cyclin-dependent kinase activity in vitro and its effect on growth in transgenic plants. Herein, we examine cyclin-dependent kinase-driven cell-cycle events, probed by testing the sensitivity of living cells to introduced ICK1 protein. The microinjection of ICK1 into individual Tradescantia virginiana stamen hair cells during late prophase and prometaphase resulted in a clear protein-specific increase in the metaphase transit time (time from nuclear envelope breakdown to the onset of anaphase) in a manner dependent on load and injection time. The results indicate a continuing role for cyclin-dependent kinases in mitotic progression and provide in vivo evidence at the cellular level that ICK1 can restrict growth in the plant by inhibiting cell division.     

SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.