Plant Cell Cycle Control Research Articles

Identification and expression analysis of the E2F/DP genes under salt stress in Medicago truncatula

Cell cycle control in plants converges on the Rb/E2F/DP pathway, which is regulated by cyclin-dependent kinases. Plants can coordinate their cell cycles during salt stress to benefit their growth and development. However, the mechanism underlying cell cycle control under salt stress is still unclear. Here, we identified five predicted E2F/DP genes in the Medicago truncatula genome, including three E2Fs, one DP, and one DEL. The conserved domains of the E2F/DP proteins were relatively well conserved with those of Arabidopsis thaliana and Oryza sativa. Intron/exon organization analyses indicated that Medtr;E2Fc and Medtr;DPa gained/lost introns in the conserved domains during recent evolutionary process. Furthermore, an expression analysis showed that these genes were expressed with varied transcription levels in all of the tissues tested. Contrasting gene expression changes in response to salt treatment in salt-tolerant versus salt-sensitive genotypes indicated that Medtr;DPa might be a candidate gene underlying the salt tolerance. This study will contribute to the understanding of the E2F/DP transcription factors in M.truncatula and of the mechanism organizing cell cycle regulation and salt stress.

The plant cell cycle in context.

Plants have a distinctive mode of continuous development, involving the repeated initiation and growth of new organs throughout the lifespan of the organism. Their sessile nature means that this growth must be responsive not only to developmental cues but also to changes in the prevailing conditions. Since almost all growth ultimately derives from the process of cell division, the control of the cell cycle is at the heart of understanding patterning, growth, and development in plants. The unique features of plant development are reflected in distinct modes and molecular mechanisms of cell cycle control, using underpinning conserved control modules in distinct ways. The development of our understanding of these distinct aspects of plant cell cycle control is reviewed here.

Control of the meiotic cell division program in plants.

While the question of why organisms reproduce sexually is still a matter of controversy, it is clear that the foundation of sexual reproduction is the formation of gametes with half the genomic DNA content of a somatic cell. This reduction in genomic content is accomplished through meiosis that, in contrast to mitosis, comprises two subsequent chromosome segregation steps without an intervening S phase. In addition, meiosis generates new allele combinations through the compilation of new sets of homologous chromosomes and the reciprocal exchange of chromatid segments between homologues. Progression through meiosis relies on many of the same, or at least homologous, cell cycle regulators that act in mitosis, e.g., cyclin-dependent kinases and the anaphase-promoting complex/cyclosome. However, these mitotic control factors are often differentially regulated in meiosis. In addition, several meiosis-specific cell cycle genes have been identified. We here review the increasing knowledge on meiotic cell cycle control in plants. Interestingly, plants appear to have relaxed cell cycle checkpoints in meiosis in comparison with animals and yeast and many cell cycle mutants are viable. This makes plants powerful models to study meiotic progression and allows unique modifications to their meiotic program to develop new plant-breeding strategies.

A high-throughput bimolecular fluorescence complementation protein-protein interaction screen identifies functional Arabidopsis CDKA/B-CYCD4/5 complexes

The eukaryotic cell cycle is a process controlled by protein assemblies, of which the key subunits are serine-threonine cyclin-dependent kinases (CDKs). Timely association and dissociation of these assemblies ensure that the cell division program is executed correctly. The challenge to unravel the rules of the plant cell cycle results from the multiplicity of the process-regulating genes that emerged through genome duplications during the evolution of flowering plants. Despite the increasing knowledge on the plant cell cycle control, little is known about the composition of the different CDK-Cyclin complexes and their spatio-temporal occurrence. The binary interactions of the previously annotated 58 Arabidopsis thaliana core cell cycle proteins were tested in two high-throughput protein-protein interaction (PPI) assays: the bimolecular fluorescence complementation (BiFC) and the yeast two-hybrid. The resulting PPI network was integrated with available cycle phase-dependent gene expression data and subcellular localization information, revealing distinct cell cycle clusters acting at different cell division stages. Additionally, the BiFC assay revealed that three D-type cyclins, CYCD4;1, CYCD4;2 and CYCD5;1, form active kinase complexes with CDKA;1 and CDKB1;1 in vivo because they induce cell divisions in differentiated tobacco (Nicotiana benthamiana) epidermal cells. We demonstrate that these complexes promote cell proliferation in Arabidopsis and we discuss their putative mode of action in plant development.

Activity of the 5′ regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.

Proteomic Analysis of CDK‐Cyclin Complexes

Cyclin-dependent kinases (CDKs), in complex with cyclins and other cellular proteins, regulate progression of cell division in eukaryotic organisms. Characterization of the interactions between CDKs and other proteins is particularly important in plants as the cell cycle is closely associated with plant growth and development. Two main groups of CDKs, CDKA and CDKB, are involved in plant cell cycle control, while 49 cyclins have been identified in Arabidopsis. It is poorly understood which cyclins interact with which CDKs, and very little is known about the substrates of different CDK-cyclin complexes. To better understand the various CDK-cyclin interactions and to identify putative substrates of these complexes, we carried out protein complex isolation by two different methods: affinity purification using the CDK-binding yeast scaffolding protein suc1 as a ligand, as well as immunopurifications using anti-sera raised against plant CDKA and CDKB. By tandem mass spectrometry, we identified 75 proteins from duplicate suc1 purifications, 51 proteins from CDKA immunopurifications, and 63 proteins from CDKB purifications. Some putative substrates of the CDK complexes were validated using yeast-two-hybrid assays. We show that protein complexes can be isolated independently using different affinity approaches and that interacting proteins identified by these methods do interact directly with CDKs.

Switching the Cell Cycle. Kip-Related Proteins in Plant Cell Cycle Control

During the development of multicellular organisms, many different cell types are created. These display characteristic cell cycle programs that can radically change during the organism's lifetime ([Jakoby and Schnittger, 2004][1]; [Fig. 1][2]). Usually, in younger and less differentiated tissues, a

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.

The developmental context of cell-cycle control in plants

Plant growth is characterised both by continued growth and organogenesis throughout development, as well as by environmental influences on the rate and pattern of these processes. This necessitates a close relationship between cell cycle control, differentiation and development that can be readily observed and studied. The sequencing of the Arabidopsis genome has revealed the full complexity of cell cycle regulators in plants, creating a challenge to understand how these genes control plant growth and differentiation, and how they are integrated with intrinsic and external signals. Here, we review the control of the cell cycle and examine how it is integrated with proliferative activity within meristems, and during the differentiation processes leading to leaf and lateral root formation.

Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control

Arabidopsis has over 80 genes encoding conserved and plant-specific core cell cycle regulators, but in most cases neither their timing of expression in the cell cycle is known nor whether they represent redundant and/or tissue-specific functions. Here we identify novel cell cycle regulators, including new cyclin-dependent kinases related to the mammalian galactosyltransferase-associated protein kinase p58, and new classes of cyclin-like and CDK-like proteins showing strong tissue specificity of expression. We analyse expression of all cell cycle regulators in synchronized Arabidopsis cell cultures using multiple approaches including Affymetrix microarrays, massively parallel signature sequencing and real-time reverse transcriptase polymerase chain reaction, and in plant material using the results of over 320 microarray experiments. These global analyses reveal that most core cell cycle regulators are expressed across almost all tissues and more than 85% are expressed at detectable levels in the cell suspension culture, allowing us to present a unified model of transcriptional regulation of the plant cell cycle. Characteristic patterns of D-cyclin expression in early and late G1 phase, either limited to the re-entry cycle or continuously oscillating, suggest that several CYCD genes with strong oscillatory regulation in late G1 may play the role of cyclin E in plants. Alone amongst the six groups of A and B type cyclins, members of CYCA3 peak in S-phase suggest it is a major component of S-phase kinases, whereas others show a peak in G2/M. 82 genes share this G2/M regulatory pattern, about half being new candidate mitotic genes of previously unknown function.

Cell Cycle Regulation through Ubiquitin/Proteasome-Mediated Proteolysis in Plants

Understanding the mechanism of cell cycle control in plants is important for agricultural technology enabling enhancement of growth rate and then crop yield. Progression of the cell cycle requires the cell cycle-dependent appearance of cell cycle regulatory proteins such as cyclin-dependent kinases (CDKs) and cyclins. These regulatory proteins are controlled at several levels, including expression, phosphorylation, and interaction with other regulatory proteins. Recently, it has become clear that the controlled and timed destruction of the proteins plays an essential role in cell cycle regulation and the 26S proteasome is involved in the destruction. For example, Cyc (cyclin) A, CycB, CycD, and B-type CDK are degraded by 26S proteasome in a cell cycle-dependent manner. In this review, recent advances in our understanding of cell cycle regulation through ubiquitin/proteasome-mediated proteolysis in plants are described.

Cyclin-Dependent Protein Kinases and Their Regulators in Plants.

A unique feature of plant cell cycle control is that quiescent meristematic and differentiated cells of plants are capable of re-entering the cell cycle. The activation of cell division and transitions between different phases of cell cycle are controlled by a family of cyclin-dependent serine/threonine protein kinases (CDKs). Post-translational regulation of CDKs plays an important role in the maintenance and activation of cell division, and thus controls plastic development of plant tissues. Here I review CDKs and cyclins in plants and describe the current view of CDK regulation by phosphorylation.

A cell cycle role for a plant sucrose nonfermenting-1-related protein kinase (SnRK1) is indicated by expression in yeast

Plant sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRK1s) have been shown to restore carbon catabolite derepression of gene expression in the yeast Saccharomyces cerevisiae when expressed in snf1 mutants. SNF1 has been implicated in the mediation of cell cycle control in response to nutrient levels and, in the present study, we show that expression of the rye (Secale cereale) SnRK1, RKIN1, in a yeast snf1 mutant has a dramatic effect on the size of cells growing on a minimal medium where SNF1 function is essential. The mean volume of the yeast cells which were expressing RKIN1 was two-thirds that of the whi1 mutant, the smallest viable cells known in S. cerevisiae, and the cells died after 3 days unless rescued onto complex medium. This is the first experimental evidence of a role for SnRK1s in plant cell cycle control.

Retinoblastoma proteins in plants.

The retinoblastoma protein Rb is part of a conserved pathway that controls the activation of cell division in animals. Rb represses cell cycle transcription factors of the E2F family, and thereby prevents uncontrolled cell proliferation. Rb itself is inactivated when phosphorylated by cyclin-dependent kinases, and the D-type cyclin kinases are particularly important in this process during the reactivation of cell division in quiescent cells. In addition, Rb has important developmental roles in controlling the onset of cellular differentiation in a number of cell types. The recent discovery in plants of both Rb proteins and other components of the Rb pathway suggests that, far from being restricted to the animal kingdom, Rb may have a conserved role in allowing multicellular organisms to develop complex body plans consisting of many different cell types. This review assesses the potential roles of Rb proteins in plant cell cycle control and development.

Phosphorylation by a cyclin-dependent kinase modulates DNA binding of the Arabidopsis heat-shock transcription factor HSF1 in vitro.

Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription factors in yeast and mammalian cells. Here we describe partial purification, identification, and characterization of a protein kinase that phosphorylates the Arabidopsis heat-shock factor AtHSF1 at multiple serine residues. The HSF1 kinase forms a stable complex with AtHSF1, which can be detected by kinase pull-down assays using a histidine-tagged AtHSF1 substrate. The HSF1 kinase interacts with the cell-cycle control protein Suc1p and is immunoprecipitated by an antibody specific for the Arabidopsis cyclin-dependent CDC2a kinase. Phosphorylation by CDC2a in vitro inhibits DNA binding of AtHSF1 to the cognate heat-shock elements, suggesting a possible regulatory interaction between heat-shock response and cell-cycle control in plants.

Regulation of Cell Division in Arabidopsis

The study of cell cycle control in plants is expected to contribute to the understanding of plants' unique developmental features. The principal regulators of the eukaryotic cell cycle, namely, cyclin-dependent kinases (CDKs) and cyclins, are also conserved in plants. This review is concerned with our present knowledge on cell cycle regulation in Arabidopsis thaliana, which is widely accepted as a model plant for the study of a broad range of biological questions. Up to the present, 2 CDKs and 11 cyclins have been identified in Arabidopsis. While the expression of one of these CDKs has been found to be positively correlated with the competence of cells to divide, cyc1A1 expression of the cyclin has been almost exclusively confined to dividing cells. Although much remains to be studied concerning upstream regulators of these genes, the successful introduction of mutant CDKs into plants demonstrates the potential of using such an approach to intentionally modulate the plant cell cycle and development.