Claudette Perennes

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 G1/S. Consistent with its role at the G1/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 G2-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.

Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle

Abstract. In order to understand the mode of action of auxins and cytokinins in the induction of cell division, the effects of the two plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzyladenine (BA) were investigated using mesophyll protoplasts of Petunia hybrida, cultivated in either complete medium or in medium deficient in cytokinin, auxin or both. Firstly we studied DNA synthesis, using 5-bromodeoxyuridine/bisbenzimide Hoechst/propidium iodide flow cytometry analyses and by the monitoring of histone H4 transcript levels. Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was found to block the cell cycle prior to entry into the S and M phases in the cultured P. hybrida protoplasts. This suggests that in Petunia cells there is a requirement for CDK activity in order to complete the G1 and G2 phases. Further experiments using roscovitine showed that neither 2,4-D nor BA were individually able to induce cell cycle progression beyond the roscovitine G1 arrest. We also monitored the phytohormonal induction of S phase by studying variations in transcript levels of the gene for mitogenactivated protein kinase (PMEK1) and transcript levels of the cell division cycle gene cdc2Pet. Only 2,4-D, and not BA, was able to stimulate PMEK1 gene transcription; thus, the more rapid S-phase induction in 2,4-D-treated protoplasts may be attributable to the activation of this transduction pathway. In contrast, both plant growth regulators were required to induce the appearance of cdc2Pet mRNA transcripts prior to S-phase engagement.

AtE2F-a and AtDP-a, members of the E2F family of transcription factors, induce Arabidopsis leaf cells to re-enter S phase.

Abstract. In eukaryotes, transcription factors of the E2F family, in addition to having a role in cell proliferation, participate in regulating apoptosis, differentiation and development. In Arabidopsis thaliana, eight gene sequences have been identified as encoding E2F or DP homologues. DP proteins form heterodimers with E2Fs. The aim of this work was to characterize the functions of three of these factors: AtE2F-a, AtE2F-b and AtDP-a. Here we report that AtE2F-a and AtE2F-b transactivate a reporter gene via an E2F consensus cis-acting element in Arabidopsis protoplasts. AtE2F-a is a more potent activator than AtE2F-b. Furthermore, co-expression of the E2F partner AtDP-a, or the DNA binding protein AtPurα, modulates the activation of AtE2F-a. Taken together, these results suggest that AtE2F-a, AtE2F-b and AtDP-a share features characteristic of members of the E2F family of transcription factors. Moreover, over-expression of AtE2F-a and AtDP-a can induce differentiated, non-dividing, leaf cells to re-enter S-phase. We conclude that the transcription factor AtE2F-a plays an important role in progression into S phase, which probably correlates with its capacity to stimulate transcription.

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.

Relation between protoplast division, cell-cycle stage and nuclear chromatin structure

SummaryUsing different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.

G2-and early-M-specific expression of the NTCYC1 cyclin gene in Nicotiana tabacum cells.

We have previously reported the isolation of a cDNA encoding a mitotic cyclin, NTCYC1, from a tobacco cell suspension library. Here we describe the expression patterns of NTCYC1 and of Ntsuc1, a suc1 plant homologue, in synchronized tobacco cell suspensions. Furthermore, the expression pattern of this cyclin is compared to that of Ntcdc2-1, a Nicotiana tabacum homologue of cdc2. While no NTCYC1 transcript was detected in cells synchronized in the G1 and S phases, NTCYC1 expression was observed in late G2 and early M phases, disappearing in the G1′ of a new cell cycle. On the other hand, Ntsuc1 and Ntcdc2-1 exhibited a constitutive expression during the cell cycle. A functional analysis performed by microinjecting NTCYC1 mRNA into immature Xenopus oocytes, indicates that NTCYC1 could participate in the control of the G2/M transition in plant cells. Subsequently NTCYC1 expression was used to assess the status of mesophyll cells in expanded leaves of N. tabacum. Depending on leaf position along the shoot axis, a large population of mesophyll cells appeared with a 4C DNA content, suggesting a G2 arrest. It was found that leaves with such a population also contained high levels of NTCYC1 transcripts. With respect to these results concerning a naturally occurring G2-arrested cell population, the regulation of NTCYC1 expression in planta is discussed.

Cytometric analysis of growth-regulator-dependent transcription and cell-cycle progression in Petunia protoplast cultures

Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase.Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N6-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase “G1B” having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G2 frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G2 nuclei are larger than G1 nuclei, but upon activation G1 nuclei in fact diminsh in size.This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N6-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.

The effect of male sterility on protoplast division inPetunia hybrida. Cell cycle comparison by flow cytometry

SummaryAn analysis of the division ratios of leaf mesophyll protoplasts taken from two isogenicPetunia hybrida lines, one fertile and the other cytoplasmic male-sterile, reveals that cytoplasmic male sterility affects division induction. Cell cycle analysis by flow cytometry reveals that protoplasts prepared fromPetunia leaf tissues are arrested in G1, so that nuclei of both lines have identical amounts of DNA at culture initiation. Thus protoplast division rates in the two lines may be compared. DNA synthesis occurs earlier in the malefertile line. Under our experimental conditions cytoplasmic male sterility is associated with delayed DNA synthesis during the induction of division in leaf mesophyll protoplasts.

Cloning of upstream sequences responsible for cell cycle regulation of the Nicotiana sylvestris CycB1;1 gene.

To understand the mechanisms involved in the regulation of the mitotic cyclin B Nicta; CycB1;1 expression, we have cloned the Nicotiana sylvestris cyclin gene, Nicsy; CycB1;1, whose coding sequence is homologous to that of Nicta;CycB1;1 cDNA. The structure and the function of its 5′-flanking region, 1149 bp upstream of the first start codon, was analysed. By producing stably transformed cells of a synchronized culture with the Nicsy;CycB1;1 promoter/β-glucuronidase (gus) reporter gene fusion, we demonstrate that the 1149 bp promoter fragment mediates a gus transcriptional oscillation, indistinguishable from that of endogenous Nicsy;CycB1;1 cyclin B transcripts. Transient GUS activity in BY-2 protoplasts reveals that promoter activity is considerably reduced by shortening the 5′-flanking region to 538 or 320 bp. Furthermore, the 320 bp fragment no longer mediates the observed transcriptional regulation of the 1149 bp Nicsy;CycB1;1 promoter in BY-2 protoplasts isolated from synchronized cells.

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

Abstract 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.