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.     

Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases

In animals and fungi, a group of proteins called the cyclin-dependent kinase inhibitors play a key role in cell cycle regulation. However, comparatively little is known about the role of these proteins in plant cell cycle regulation. To gain insight into the mechanisms by which the plant cell cycle is regulated, we studied the cyclin-dependent kinase inhibitor KRP1 in Arabidopsis. KRP1 interacts with the CDKA;1/CYCD2;1 complex in planta and functions in the G1–S transition of the cell cycle. Furthermore, we show that KRP1 is a likely target of the ubiquitin/proteasome pathway. Two different ubiquitin protein ligases, SCF^SKP2 and the RING protein RKP, contribute to its degradation. These results suggest that SCF^SKP2b and RPK play an important role in the cell cycle through regulating KRP1 protein turnover.     

<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.     

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.     

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.     

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.     

Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.     

The Arabidopsis D-type cyclin CYCD2;1 and the inhibitor ICK2/KRP2 modulate auxin-induced lateral root formation

The integration of cell division in root growth and development requires mediation of developmental and physiological signals through regulation of cyclin-dependent kinase activity. Cells within the pericycle form de novo lateral root meristems, and D-type cyclins (CYCD), as regulators of the G₁-to-S phase cell cycle transition, are anticipated to play a role. Here, we show that the D-type cyclin protein CYCD2;1 is nuclear in Arabidopsis thaliana root cells, with the highest concentration in apical and lateral meristems. Loss of CYCD2;1 has a marginal effect on unstimulated lateral root density, but CYCD2;1 is rate-limiting for the response to low levels of exogenous auxin. However, while CYCD2;1 expression requires sucrose, it does not respond to auxin. The protein Inhibitor-Interactor of CDK/Kip Related Protein2 (ICK2/KRP2), which interacts with CYCD2;1, inhibits lateral root formation, and ick2/krp2 mutants show increased lateral root density. ICK2/KRP2 can modulate the nuclear levels of CYCD2;1, and since auxin reduces ICK2/KRP2 protein levels, it affects both activity and cellular distribution of CYCD2;1. Hence, as ICK2/KRP2 levels decrease, the increase in lateral root density depends on CYCD2;1, irrespective of ICK2/CYCD2;1 nuclear localization. We propose that ICK2/KRP2 restrains root ramification by maintaining CYCD2;1 inactive and that this modulates pericycle responses to auxin fluctuations.     

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.     

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.     

Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity

Synchronized suspension cultures are powerful tools in plant cell-cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry. We propose that ICK2 (KRP2) may have a specific role in this process.     

Involvement of C4 Protein of Beet Severe Curly Top Virus (Family Geminiviridae) in Virus Movement

Background

Beet severe curly top virus (BSCTV) is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction.

Methods and Findings

To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties.

Conclusions

Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.     

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.     

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

? Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. ? The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. ? Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. ? The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.     

BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis

Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.     

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.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

Autonomy of cell proliferation and developmental programs during Arabidopsis aboveground organ morphogenesis

Elaboration of size and shape in multicellular organisms involves coordinated cell division and cell growth. In higher plants, continuity of cell layer structures exists from the shoot apical meristem (SAM), where organ primordia arise, to mature aboveground organs. To unravel the extent of inter-cell layer coordination during SAM and aboveground organ development, cell division in the epidermis was selectively restricted by expressing two cyclin-dependent kinase inhibitor genes, KRP1/ICK1 and KRP4, driven by the L1 layer-specific AtML1 promoter. The transgenes conferred reduced plant size with striking, distorted lateral organ shape. While epidermal cell division was severely inhibited with compensatory cell size enlargement, the underlying mesophyll/cortex layer kept normal cell numbers and resulted in small, packed cells with disrupted cell files. Our results demonstrate the autonomy of cell number checkpoint in the underlying tissues when epidermal cell division is restricted. Finally, the L1 layer-specific expression of both KRP1/ICK1 and KRP4 showed no effects on the structure and function of the SAM, suggesting that the effects of these cyclin-dependent kinase inhibitors are context dependent.     

ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid

Cyclin-dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene ICK1 was recently identified from Arabidopsis thaliana . Although the C-terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27^Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors. In vitro assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA). ICK1 was expressed at a relatively low level in the tissues surveyed. However, ICK1 was induced by ABA, and along with ICK1 induction there was a decrease in Cdc2-like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA. ICK1 clones were also identified from independent yeast two-hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by in vitro binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.