the sula factor

First published online June 18, 2004; 10.1105/tpc.022335

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The Plant Cell 16:1801-1811 (2004)
© 2004 American Society of Plant Biologists

An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid Division

Cécile Raynaud^a,1, Corinne Cassier-Chauvat^b, Claudette Perennes^a and Catherine Bergounioux^a

^a Institut de Biotechnologie des Plantes, Equipe Cycle Cellulaire Unité Mixte de Recherche 8618 Centre National de la Recherche Scientifique, Orsay Cedex, France
^b Service de Bioénergétique, Unité de Recherche Associée 2096, Centre National de la Recherche Scientifique, Centre d'Etudes Atomiques, Saclay 91191, Gif-Sur-Yvette Cedex, France

¹ To whom correspondence should be addressed. E-mail raynaud@ibp.u-psud.fr; fax 33-1-69-15-344.

	ABSTRACT

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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.

	INTRODUCTION

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Plastids arose through endosymbiosis between a cyanobacterial ancestor and a eukaryotic host cell (McFadden, 1999). These semiautonomous organelles can divide, and their division is crucial for their maintenance after mitosis. Moreover, this seems to be a tightly regulated process because plastid number depends on the cell type (in the Landsberg ecotype of Arabidopsis thaliana, meristematic cells contain 10 to 15 plastids, whereas mesophyll cells contain 120) (Marrison et al., 1999) and is correlated with the cell area in mesophyll cells (Pyke and Leech, 1991).

In the past decade, several proteins involved in plastid division have been identified, many of which are of bacterial origin. Indeed, the first plastid division proteins to be isolated were homologs of the bacterial division protein FtsZ (Osteryoung et al., 1998). FtsZ is a tubulin-related protein (Erickson, 1997) that polymerizes at midcell, forming a ring that shrinks until division is completed (Rothfield and Justice, 1997). Involvement of FtsZ homologs in plastid division has been clearly established in Arabidopsis (AtFtsZ1-1 and AtFtsZ2-1) and the moss Physcomitrella patens (Osteryoung et al., 1998; Strepp et al., 1998; McAndrews et al., 2001).

The identification of homologs of bacterial division proteins in the Arabidopsis genome and the analysis of the accumulation and replication of chloroplasts (arc) mutants (Pyke and Leech, 1992; Marrison et al., 1999) allowed Osteryoung and Nunnari (2003) to build a model for chloroplast division. The first step seems to be the polymerization of AtFtsZ at the division site, forming the Z-ring (Vitha et al., 2001), probably stabilized by ARC6, a DnaJ domain protein (Vitha et al., 2003). Like in bacteria (Justice et al., 2000), establishment of the proper division site is mediated by AtMinD and AtMinE (Colletti et al., 2000; Itoh et al., 2001). Then, sequential assembly of the inner and outer plastid division rings (PD ring) follows. This protein scaffold was discovered by microscopic observation of dividing plastids (Miyagishima et al., 2001), but none of its components has been identified until now. The Z-ring and the PD-ring shrink, and constriction of the plastid begins. Finally, cytoplasmic dynamin related proteins, such as ARC5, are recruited at the division site on the outer membrane of the plastid (Gao et al., 2003; Miyagishima et al., 2003b). In addition, a translocase named ARTEMIS also required for plastid division and for cell division in cyanobacteria (Fulgosi et al., 2002) has been hypothesized to function in assembly of the division apparatus. Hence, plastid division is a complex phenomenon requiring cooperation between proteins of eukaryotic origin (dynamin related protein) and of bacterial origin (FtsZ, Min, and ARC6) that are now encoded by the nuclear genome and imported into plastids posttranslationally (Soll, 2002).

A search for homologs of known bacterial cell division proteins allowed us to identify a potential regulator of plastid division, AtSulA. This gene was named on the basis of its sequence similarity to bacterial cell division inhibitors. In Escherichia coli, transcription of SulA is induced by the SOS response (Huisman et al., 1984), and the corresponding protein inhibits FtsZ polymerization (Justice et al., 2000), delaying cell division until DNA damage is repaired. The function of SulA homologs was investigated in Arabidopsis and Synechocystis. This cyanobacteria is a valuable model to study plastid division. First, cyanobacteria are closely related to the ancestors of plastids: components of the plastid division machinery, namely ARC6 and ARTEMIS, can be found in cyanobacteria but not in other prokaryotes. Second, Synechocystis is neither a rod-like nor a filamentous bacterium but resembles chloroplasts by its spherical shape. Our results show that SSulA, the cyanobacterial homolog of AtSulA, is required for cell division in Synechocystis. Moreover, overexpression of AtSulA inhibits plastid division in planta and rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, indicating that AtSulA may interact with AtFtsZ1-1 and AtFtsZ2-1 during plastid division. Finally, comparison of plants overexpressing these plastid division proteins shows that the effect of the overexpression is stochastic, independent of developmental stage, tissue, or cell type. In 35S:AtSulA-GFP (green fluorescent protein) plants, this heterogeneous phenotype correlates with unexpectedly poor accumulation of the AtSulA-GFP protein. These data suggest that as for components of the photosynthesis machinery (Choquet and Wollman, 2002), the stability of the various components involved in plastid division depends upon the correct assembly of the complex.

	RESULTS

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Molecular Characterization of AtSulA and SSulA
A bioinformatic search for cell-cycle inhibitors drew our interest to At2g21280. The corresponding protein shares similarities with several bacterial and cyanobacterial proteins annotated as cell division inhibitors, homologous to the E. coli cell division inhibitor SulA. This gene was therefore named AtSulA. It is a unique gene in Arabidopsis conserved in many plant species (corresponding ESTs can be found in the Chlamydomonas, maize [Zea mays], soybean [Glycine max], alfalfa [Medicago sativa], and rice [Oryza sativa] databases). The AtSulA protein shows 50% identity with the slr1223 protein of Synechocystis (SSulA) (Figure 1).