Results and Discussion

3.1. Characterization of nodule cell cycle activity. To reveal cell cycle activity we have been studying the expression pattern of several cell cycle marker genes during nodule development by in situ hybridization, RT-PCR analysis and by construction of transgenic M. truncatula plants carrying cell cycle gene promoter -GUS fusions. We identified a set of genes that were induced during dedifferentiation/reactivation of cortical cells (Figure 1). They included Medsa;cycA2 whose cell cycle function was described recently (Roudier et al. 2000), eyeD3-I (F. Foucher, unpublished), two E2F transcription factors required for S phase entry and function (J. Gyorgyey, unpublished), the S-phase-specific histone H3 and Medsa;cycB2 (Savoure et al 1995). All of these genes, except cycD3-I that was repressed when cell proliferation started, exhibited a constitutive expression during nodule development. Although the transcripts were present, their spatial distribution displayed differences. For example, expression of cycA2 was restricted to proliferating cells (F. Roudier, unpublished) while expression of the other genes was detected both in the proliferating cells (nodule primordium, meristem) as well as in nodule zone II. Expression of histone H3 exhibited a spotty pattern in zone II and marked S-phase cells undergoing DNA replication. In zone II, a novel variant of cycD3 (II) was switched on that likely contributes to Gl-S progression during endocycles (F. Foucher, unpublished). In the symbiotic, nitrogen-fixing nodule zone III, all these genes were repressed reflecting the inactivation of the cell cycle. In M. sativa and M. truncatula nodules, expression of these cell cycle genes in nodule zone II coincided with the progressive maturation of symbiotic cells along 10-12 cell layers where cells more distal to the meristem became larger and contained larger nuclei and more symbiosomes. The cell and nuclear volumes in the terminally differentiated cells of nodule zone III were constant. These data on the expression pattern of cell cycle genes allowed to show that cells undergo endoreduplication in nodule zone II and inter-zone II-III.

3.2. Conversion of mitotic cycles to endocycles. Recently by screening a young nodule cDNA library for genes involved in nodule organogenesis in M. sativa, we identified a cell cycle switch gene, ccs52 that controls transition from mitotic cycles to differentiation programs and conversion of mitotic cycles to endocycles (Cebolla et al. 1999). This cell cycle switch gene exhibited highly stimulated expression in the nodules compared to roots and encoded a 52 kDa protein. The protein, containing seven WD40-repeats, several CDK phosphorylation sites and conserved oligopeptide motifs, exhibited homology to regulatory proteins involved in ubiquitin-dependent proteolysis of mitotic cyclines. The effect of ccs52 on cell cycle was first studied in fission yeast. Overexpression of the ccs52 gene resulted in growth arrest, elongation and enlargement of cells. These cells contained large polyploid nuclei measured by flow cytometry as a consequence of repeated rounds of endoreduplication cycles. We have shown that production of the CCS52 protein resulted in the degradation of the fission yeast mitotic cyclin, CDC 13 and thereby inhibition of the M-phase has led to the conversion of mitotic cycle to endoreduplication cycles

NODULE NODULE

Cell cycle Cell Primordia Differentiation

CycD3-E2F

enod40 -

ccs5

CycD3-

Figure 1. Expression of cell cycle genes and enod40 during nodule development

In Medicago nodules, expression of ccs52 was localized by in situ hybridization in zone I where cells exit from proliferation and in zone II which is the major site for endoreduplication and cell differentiation. RT-PCR analysis of the ccs52 expression during nodule development revealed activation of the genes 4-5 days after Rhizobium inoculation. To study temporal and spatial expression patterns of ccs52, the genomic region was isolated and the promoter region was cloned in front of the uidA reporter gene (J. Vinardell et al., unpublished). This construct was introduced in M. truncatula by Agrobacterium tumefaciens-mc&\ ated transformation and the ccs52 promoter-driven GUS activity was tested in the transgenic roots as well as during different stages of nodule development. The GUS staining was absent in the growing primordia whereas gene expression was switched on prior to differentiation of primordia to the different the nodule zones (J. Vinardell et al., unpublished). The ccs52 promoter-driven GUS activity and expression pattern were consistent with the in situ hybridization experiments and with the predicted function of ccs52 in mitosis arrest and endocycles.

M. truncatula displays a systemic endoploidy with the exception of leaves which are diploid. Northern analysis demonstrated that the ccs52 transcripts were present also in other tissues. This indicated that the ccs52 function might not be restricted to nodulation and could be involved in the development of other polyploid tissues and organs. To test this function in M. truncatula, transgenic plants were regenerated that carried the ccs52 cDNA either in sense or antisense orientation downstream of the 35S promoter. By testing more than 30 transgenic plants in to independent transformations, no overexpression of the sense ccs52 cDNA was found in the transgenic plants. This result indicates that the inhibitory effect of ccs52 on cell proliferation likely interferes with callus formation and somatic embryogenesis. The antisense expression in three lines out of 75

transgenic plants resulted in slight downregulation of ccs52. The lack of knock-out phenotypes or significant reduction of ccs52 transcript levels suggests that absence of gene function might be lethal and the level of gene expression might vary within a narrow window. However, even a 40% reduction in the mRNA level compared to the wild type was sufficient to reduce the degree of ploidy in the polyploid organs such as the petioles, roots and hypocotyls. Moreover, the reduced ploidy correlated with the formation of smaller cells demonstrating that expression level of ccs52 affects directly both the degree of endoploidy and the size of the cell volume. Down-regulation of ccs52 expression affected also nodule development. Though nodule primordia were formed with the same kinetics and number as in the control plants, later stages of nodule development halted. The ploidy level was significantly reduced, invasion of plant cells was less efficient and maturation of symbiotic cells were not completed that induced early senescence and cell death.

These results indicate that formation of highly polyploid cells is an integral part of nodule development. CCS52 appears to act as a key regulator of endocycles. Though its highest expression level correlates with the highest ploidy level in the nodule, ccs52 is likely to have a general role in plant development, particularly in organs containing polyploid cells. CCS52, like its orthologs in yeast and animals, acts as substrate-specific activator of the Anaphase-Promoting Complex (APC), a cell cycle regulated ubiquitin ligase. These WD40-repeat proteins may target a great variety of proteins carrying D-box and KEN-box motifs in different stages of the cell cycle to APC for ubiquitination and then degradation by the 26S proteasome. Moreover, plants compared to other eukaryotes have evolved a multigenic family for these WD40-repeat proteins that likely regulate cell function and fate by fast and irreversible destruction of regulatory proteins.

4. References

Bisseling T et al. (1977) J. Gen. Microbiol. 101, 79-84 Cebolla A et al. (1999) EMBO J. 18, 4476-4484 Kondorosi E et al. (2000) Curr. Opin. Plant Biol. 3,488-492 Roudier F et al. (2000) Plant J. 23, 73-83 Savouré A et al. (1995) Plant Mol. Biol. 27, 1059-1070 Trinh TH et al. (1998) Plant Cell Rep. 17, 345-355 Truchet G (1978) Ann. Sci. Nat. Bot. Paris 19, 3-38

IDENTIFICATION OF TRANS-ACTING FACTORS REGULATING NODULE DEVELOPMENT

A.C. Hansen, C. Johansson, H. Busk, A. Jepsen, D. Jensen, L. Frasmohs, E.O. Jensen

Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Denmark

1. Introduction

Functional studies of nodulin gene promoters in transgenic legumes have identified a number of cis-regulatory elements important for nodule specific expression. One example is the pea enodl2 promoter which was investigated in details in transgenic Vicia hirsuta plants (Figure 1). By analyzing 50 promoter deletions and hybrid promoters fused to the GUS reporter gene a tissue specific element was identified in the Psenodl2 promoter (Vijn et al. 1995; Christiansen et al. 1996; Hansen et al. 1999).

TATAA

Figure 1. Schematic representation of the rfs-regulatory element m the pea enodl2 promoter. TSE: tissue specific element

2. Identification of a putative transcription factor, ENBP1

Rather than searching for a single protein interacting with a specific sequence in the promoter, e.g. the TSE element, we decided to go for all proteins interacting with the proximal promoter region, since all the cis-iegulatory elements required for nodule specific expression are located on this part. A set of 10 double-stranded overlapping oligonucleotides were synthesized covering the promoter region from -200 to +1 (Figure 2). Each of the oligonucleotides were then concatenated before they were used as probes.

Figure 2. Positions of the oligonucleotides used as probes for the South-Western screening

Using a cocktail of all ten oligonucleotides 200,000 pfu were screened from an un-amplified Vicia hirsuta nodule A,gtll cDNA library. The screening yielded 4 positive clones and the corresponding phages were purified. The clones were hybridized to each other and it turned out that 2 of the clones represented the same mRNA. The two clones encoded a putative transcription factor ENBP1 (Early Nodulin Binding Protein 1) that binds to the Psenodl2B promoter in vitro (Figure 3). A bacterially expressed fusion protein, ATh, covering amino acids 188 to 728 of VsENBPl, bound to polypeptide includes the six AT-hooks present in VsENBPl and constitutes the sequence-specific DNA binding domain of double-stranded oligonucleotides with VsENBPl. DNasel sequences, corresponding to Psenodl2B promoter regions -45 to -139 and -160 to -206. The ATh footprinting revealed interactions at the sequences AATAA and TTATT present at position -78 to -82 and -92 to -96, respectively, in the Psenodl2B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal dependent way.

1641 amino acids

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