The D-type cyclin gene (Nicta;CycD3;4) controls cell cycle progression in response to sugar availability in tobacco

D-type cyclins play key roles in the G1-to-S phase transition that occurs in response to nutrient and hormonal signals. In higher plants, sucrose is the major transported carbon source, and is likely to be a major determinant of cell division. To elucidate how sugar affects on the regulation of cell cycle machinery and plant development, we examined the role of carbon sources on the expression of cell-cycle-related genes in transgenic tobacco plants overexpressing Nicta;CycD3;4. The Nicta;CycD3;4 overexpressed transgenic plants showed accelerated growth and remarkable increase in the number of cells in the S and G2 phases in response to sucrose concentrations. Increased expressions level of Nicta;CycD3;4 gene was observed in transgenic tobacco plants grown on 1/2 strength MS medium supplemented with a high concentration of sugar. Moreover, the expression of sugar-sensing-related gene, invertase, was also maintained at a high level in transgenic tobacco plants with elevated sugar availabiliy. These findings indicate that sugar availability plays a role during the G1 phase and the transition of the G1-to-S phase of cell cycle by controlling the expression of Nicta;CycD3;4.     

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.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.     

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.     

A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis

The Arabidopsis genome encodes 10 D-type cyclins (CYCD); however, their differential role in cell cycle control is not well known. Among them, CYCD4;2 is unique in the amino acid sequence; namely, it lacks the Rb-binding motif and the PEST sequence that are conserved in CYCDs. Here, we have shown that CYCD4;2 suppressed G1 cyclin mutations in yeast and formed a kinase complex with CDKA;1, an ortholog of yeast Cdc28, in insect cells. Hypocotyl explants of CYCD4;2 over-expressing plants showed faster induction of calli than wild-type explants on a medium containing lower concentration of auxin. These results suggest that CYCD4;2 has a promotive function in cell division by interacting with CDKA;1 regardless of the unusual primary sequence.     

Analysis of the Expression of a CycD3 Gene Isolated from Nicotiana tabacum

D-type cyclins (CycD) are thought to control the onset of cell division and responses to extracellular signals during G1 phase. In this study a cyclin D3 (CycD3) gene was isolated from Nicotiana tabacum. Southern blot analysis indicated that it was present in a single-copy in the N. tabacum genome, and Northern analysis showed that it was only expressed in organs and tissues containing dividing cells. Exposure to cold and salt stresses resulted in decreased CycD3 expression supporting the view that its expression is closely related to cell growth.     

The interface between the cell cycle and plant growth regulators: a mini review

The aim was to review knowledge about the interface betweenplant growth regulators and molecular checkpoints of the cell cycle. Atwhat level of biochemical regulation of the cell cycle do plant growthregulators interface? Are there different levels of interfacingdependent on the plant growth regulator involved? As a preamble totackling these questions, we overview the eukaryotic cell cycle withparticular emphasis on checkpoints that regulate the transition fromG0-G1-S-phase and G2-M. Cytokinins feature strongly as activators ofcell division in plants both in vivo and in vitro.Recent research has shown that zeatin treatment led to the up-regulationof CycD3 in Arabidopsis. This is a D-type cyclin showing stronghomology with vertebrate D cyclins which themselves are up-regulated byextracellular growth factors. Benzyladenine treatment can also shortenthe duration of S-phase through recruitment of latent origins of DNAreplication. Kinetin is involved in the phosphoregulation of the G2-Mcheckpoint; the major cyclin-dependent kinase (Cdk) at this checkpointhas recently been shown to be dephosphorylated as a result of cytokinintreatment, an effect which can also be mimicked by the fission yeastCdc25 phosphatase. Hence, a picture emerges of a cytokinin-inducedcontinuum of cell cycle activation through the up-regulation of a plantD-type cyclin at the G1 checkpoint and the phosphoregulation of the Cdkat the G2/M checkpoint. During S-phase, we argue for a link betweencytokinins and the proteins associated with replication origins.Gibberellic acid (GA) treatment induces internode elongation. Indeepwater rice, this response is mediated, at least partly, by aGA-induced up-regulation of a cyclin-Cdk at the G2-M checkpoint. Recentevidence has also linked abscisic acid to a cyclin-dependent kinaseinhibitor. These, so-called CKIs are negative regulators of Cdks whichfits with ABA's general role in growth inhibition; we await news ofethylene interactions. We highlight two instances of plant growthregulator-cell cycle interfacing during development, arguing for aninvolvement in microtubule orientation as a prerequisite to leafinitiation, and suggest a link between IAA and the activation of celldivisions in the pericycle required for lateral root initiation. A newD-type cyclin, recently discovered in Arabidopsis, may have akey role in this process. Finally, a model is presented which features ageneralised cyclin-Cdk checkpoint exhibiting various interfaces with theplant growth regulators.     

The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.     

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.     

Transgenic tobacco plants overexpressing the Nicta; CycD3; 4 gene demonstrate accelerated growth rates

D-type cyclins control the onset of cell division and the response to extracellular signals during the G1 phase. In this study, we transformed a D-type cyclin gene, Nicta;CycD3;4, from Nicotiana tabacum using an Agrobacterium-mediated method. A predicted 1.1 kb cyclin gene was present in all of the transgenic plants, but not in wild-type. Northern analyses showed that the expression level of the Nicta;CycD3;4 gene in all of the transgenic plants was strong when compared to the wild-type plants, suggesting that Nicta;CycD3;4 gene driven by the CaMV 35S promoter was being overexpressed. Our results revealed that transgenic plants overexpressing Nicta;CycD3;4 had an accelerated growth rate when compared to wild-type plants, and that the transgenic plants exhibited a smaller cell size and a decreased cell population in young leaves when compared to wild-type plants.