NtKIS2, a novel tobacco cyclin-dependent kinase inhibitor is differentially expressed during the cell cycle and plant development

The precise control of cell cycle progression is critical for coherent development. In all eukaryotes, the cell cycle is controlled by complexes composed of a cyclin-dependent kinase (CDK) and a cyclin. CDK activity is controlled at multiple levels, including association with CDK inhibitory proteins called CKIs. Here, we report the isolation and characterisation of a novel Nicotiana tabacum CKI, named NtKIS2, revealing the existence of a CKI family in tobacco. Like NtKIS1a, the tobacco CKI we previously identified, the NtKIS2 protein interacts with A-type CDK and D-type cyclins; is localised in the nucleus; and its overexpression strongly impairs plant development. Furthermore, our results show that NtKIS2 is a cell division inhibitor in planta and suggest that this CKI acts mainly in G1 phase. However, NtKIS2 shows clear differences to NtKIS1a in its expression patterns both during the cell cycle and plant development. Finally, to understand the developmental modifications seen in planta, the links between cell division inhibition and stomata determination or chloroplast division are explored.     

CycD1, a putative G1 cyclin from Antirrhinum majus, accelerates the cell cycle in cultured tobacco BY-2 cells by enhancing both G1/S entry and progression through S and G2 phases

A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells. To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2. Green fluorescent protein:CycD1 is located in the nucleus throughout interphase. Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries. We examined the effect of induced expression at different stages of the cell cycle. Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis. Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins. The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression. Continuous expression of CycD1 led to moderate increases in growth rate. Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression. This indicates that D cyclin function may have diverged between plants and animals.     

<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.     

OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation

The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc finger domains and have antagonistic effects on plant programmed cell death (PCD). In this study, a rice (Oryza sativa) functional homolog of LSD1, designated OsLSD1, was identified. The expression of OsLSD1 was light-induced or dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus 35S promoter accelerated callus differentiation in transformed rice tissues and increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice plants exhibited lesion mimic phenotype, increased expression of PR-1 mRNA, and an accelerated hypersensitive response when inoculated with avirulent isolates of blast fungus. Both sense and antisense transgenic rice plants conferred significantly enhanced resistance against a virulent isolate of blast fungus. Moreover, ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum) enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin. OsLSD1 green fluorescent protein fusion protein was located in the nucleus of tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating plant PCD, whereas it plays a positive role in callus differentiation.     

Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro

The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro. Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.     

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.     

IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation

Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin alpha proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin alpha members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed.     

Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants

Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco (Nicotiana tabacum) and rice (Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta;NAP1;1 and Orysa;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.     

Increased leakiness of the tetracycline-inducible Triple-Op promoter in dividing cells renders it unsuitable for high inducible levels of a dominant negative CDC2aAt gene

A tetracycline-inducible promoter system was used to generate transgenic tobacco plants that confer inducible expression of the wild type or a dominant negative allele of the gene coding for the cyclin-dependent kinase (CDK) of Arabidopsis thaliana CDC2aAt. Although the total extractable CDK activity was doubled, the induced expression of the wild-type CDC2aAt did not correlate with any change of the cell cycle kinetics. An increase of CDK activity upon CDC2aAt expression was only seen in dividing cell populations, demonstrating that CDC2aAt expression itself is not sufficient to induce CDK activation. Induced expression of the dominant negative CDC2aAt.N146 correlated with a reduction of CDK activity to 66% of the level found in non-induced cells. This decrease was not sufficient to block cell division. The isolation of plants showing only low inducible levels of CDC2aAt.N146 suggests that a counterselection against strong inducible lines had occurred. Accordingly, Triple-Op promoter activity was found in dividing cells in the absence of tetracycline.     

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.     

The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg2?-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.     

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.     

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.     

Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.