Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.

The proliferating cell nuclear antigen (PCNA) functions as a sliding clamp for DNA polymerase, and is thus a key actor in DNA replication. It is also involved in DNA repair, maintenance of heterochromatic regions throughout replication, cell cycle regulation and programmed cell death. Identification of PCNA partners is therefore necessary for understanding these processes. Here we identify two Arabidopsis SET-domain proteins that interact with PCNA: ATXR5 and ATXR6. A truncated ATXR5Deltaex2, incapable of interacting with PCNA, also occurs in planta. ATXR6, upregulated during the S phase, is upregulated by AtE2F transcription factors, suggesting that it is required for S-phase progression. The two proteins differ in their subcellular localization: ATXR5 has a dual localization in plastids and in the nucleus, whereas ATXR6 is solely nuclear. This indicates that the two proteins may play different roles in plant cells. However, overexpression of either ATXR5 or ATXR6 causes male sterility because of the degeneration of defined cell types. Taken together, our results suggest that both proteins may play a role in the cell cycle or DNA replication, and that the activity of ATXR5 may be regulated via its subcellular localization.

Cell and plastid division are coordinated through the prereplication factor AtCDT1.

The cell division cycle involves nuclear and cytoplasmic events, namely organelle multiplication and distribution between the daughter cells. Until now, plastid and plant cell division have been considered as independent processes because they can be uncoupled. Here, down-regulation of AtCDT1a and AtCDT1b, members of the prereplication complex, is shown to alter both nuclear DNA replication and plastid division in Arabidopsis thaliana. These data constitute molecular evidence for relationships between the cell-cycle and plastid division. Moreover, the severe developmental defects observed in AtCDT1-RNA interference (RNAi) plants underline the importance of coordinated cell and organelle division for plant growth and morphogenesis.

An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division.

Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1.

A CDC45 homolog in Arabidopsis is essential for meiosis, as shown by RNA interference-induced gene silencing.

CDC45 is required for the initiation of DNA replication in yeast and cell proliferation in mammals and functions as a DNA polymerase alpha loading factor in Xenopus. We have cloned a CDC45 homolog from Arabidopsis whose expression is upregulated at the G1/S transition and in young meiotic flower buds. One-third of Arabidopsis 35S:CDC45 T1 RNA interference lines are partially to completely sterile, and the proportion of sterile plants is increased by using a dmc1 promoter. T1 plants have decreased levels of the CDC45 transcript and contain 21- to 23-bp RNA fragments specific to the CDC45 gene. T2 transgenic lines, in which small RNA fragments are still present, were used to analyze S-phase entry by 5-bromodeoxyuridine incorporation, which was not altered compared with that in the wild type. However, microarray data show that other cell cycle genes are upregulated or downregulated. T2 plants also have highly reduced fertility. The severity of the phenotype is correlated with the levels of the CDC45 transcript and small RNA fragments. Severe chromosome fragmentation arising during meiosis, which is not the result of a defect in the repair of SPO11-induced double strand breaks, leads to abnormal chromosome segregation and defective pollen and ovule development.

Comparative molecular and functional analyses of the tobacco cyclin-dependent kinase inhibitor NtKIS1a and its spliced variant NtKIS1b.

In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.

Characterization of cis-acting element involved in cell cycle phase-independent activation of Arath;CycB1;1 transcription and identification of putative regulatory proteins.

Progression through the cell cycle is driven by cyclin-dependent kinases (CDKs) whose activity is controlled by regulatory subunits called cyclins. The expression of cyclins is subject to numerous controls at multiple levels, not least at the level of transcription. As a first step to unravel the mechanisms that regulate expression of B-cyclins in plants, we undertook the identification of the required promoter elements of the Arath;CycB1;1 gene. A detailed analysis of different promoter fragments consisted in analysing their ability to mediate cell cycle-dependent transcriptional oscillations of the gus reporter gene in transformed BY-2 cell lines. We showed that different promoter regions took part in transcriptional activation. Furthermore, 202 bp upstream of the ATG were sufficient to induce M-phase-specific expression. This region contains an 18 bp sequence including a Myb-binding core (AACGG) which is able to activate reporter gene without leading to M-phase-specific expression. Electrophoretic mobility shift assays showed that this 18 bp sequence specifically binds protein complexes from Arabidopsis cell suspension enriched either in G1 or G2 phase. Furthermore, the Myb core, AACGG, was characterized as necessary for the binding of proteins. DNA affinity purification of the complexes bound to the 18 bp sequence allowed the isolation of three different complexes and two proteins from these complexes were identified by mass spectrometry analyses. A new putative Myb transcription factor and a hypothetical protein, HYP containing with a leucine zipper and Myc-type dimerization domains were identified. When over-expressed in plants, HYP factor is able to trans-activate the expression of gus reporter gene downstream from the -202 promoter fragment as well as the endogenous CycB1;1 gene.

Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression.

The commitment to DNA replication is a key step in cell division control. The Arabidopsis MCM3 homologue forms part of the mini chromosome maintenance (MCM) complex involved in the initiation of DNA replication at the transition G(1)/S. Consistent with its role at the G(1)/S transition we show that the AtMCM3 gene is transcriptionally regulated at S phase. The 5' region of this gene contains several E2F consensus binding sites, two of which match the human consensus closely and whose roles have been studied here. The identity of the two sequences as E2F binding sites has been confirmed by electrophoretic mobility shift assay analyses. Furthermore the promoter is activated by AtE2F-a and AtDP-a factors in transient expression studies. One of the E2F binding sites is shown to be responsible for the G(2)-specific repression of the promoter in synchronized cell suspension cultures. In contrast, the second E2F binding site has a role in meristem-specific expression in planta as deletion of this site eliminates the expression of a reporter gene in root and apical meristems. Thus two highly similar E2F binding sites in the promoter of the MCM3 gene are responsible for different cell cycle regulation or developmental expression patterns depending on the cellular environment.

The CDK inhibitor NtKIS1a is involved in plant development, endoreduplication and restores normal development of cyclin D3; 1-overexpressing plants.

Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs). Acting in opposition to CDKs are CDK inhibitors (CKIs). The first tobacco CKI (NtKIS1a) identified was shown to inhibit in vitro the kinase activity of CDK/cyclin complexes and to interact with CDK and D-cyclins. However, these features, which are common to other plant and animal CKIs already characterised, did not provide information about the function of NtKIS1a in plants. Thus, to gain insight into the role of NtKIS1a and especially its involvement in cell proliferation during plant development, we generated transgenic Arabidopsis thaliana plants that overexpress NtKIS1a. These plants showed reduced growth with smaller organs that contained larger cells. Moreover, these plants displayed modifications in plant morphology. These results demonstrated that plant organ size and shape, as well as organ cell number and cell size, might be controlled by modulation of the single NtKIS1a gene activity. Since in mammals, D-cyclins control cell cycle progression in a CDK-dependent manner but also play a CDK independent role by sequestering the CKIs p27(Kip1) and p21(Cip1), we tested the significance of cyclin D-CKI interaction within a living plant. With this aim, NtKIS1a and AtCycD3;1 were overexpressed simultaneously in plants by two different methods. Our results demonstrated that overexpression of the CKI NtKIS1a restores essentially normal development in plants overexpressing AtCycD3;1, providing the first evidence of cyclin D-CKI co-operation within the context of a living plant.