Several additional titan mutants have subsequently been recovered from a large collection of T-DNA insertion lines generated at Syngenta ( McElver et al., 2001). Two contrasting models for TTN gene function were proposed: involvement with either cell cycle control or the structural mechanics of mitosis ( Liu and Meinke, 1998). Mutant embryos contain a few giant cells ( ttn1), several small cells ( ttn2), or phenotypically normal cells ( ttn3). The common feature among these mutants is the presence of giant endosperm nuclei.

Three recessive titan mutants were identified and the corresponding genes mapped to different chromosomes ( Franzmann et al., 1995 Liu and Meinke, 1998). Our interest in SMC proteins began with a screen for mitotic cell division mutants disrupted in seed development in Arabidopsis. Loss of function of this gene results in hypersensitivity to chromosome damage following mutagenesis. One family member (MIM) required for efficient homologous recombination and related in sequence to Rad18 in yeast ( Fousteri and Lehmann, 2000) has been described in Arabidopsis ( Mengiste et al., 1999). Little is known about the structure and function of SMC proteins in plants. The SMC2/SMC4 complexes (condensins) generally function in chromosome condensation and dosage compensation whereas SMC1/SMC3 complexes (cohesins) are involved in sister chromatid cohesion and chromosome segregation. Two different classes of heterodimers (SMC2/SMC4 and SMC1/SMC3) are found in eukaryotes.

Energy provided by ATP hydrolysis enables these protein complexes to play an active role in chromosome folding. SMC proteins form antiparallel dimers with ATP and DNA binding domains at each end ( Strunnikov and Jessberger, 1999). Rods are composed of 300–400 amino acids that differ in sequence but maintain a conserved coiled-coil secondary structure. SMC proteins are myosin-like chromosomal ATPases that have a common rod-hinge-rod structure with an N-terminal ATP binding domain and a C-terminal DNA binding domain. The genetics and biochemistry of these proteins have been extensively characterized in organisms ranging from bacteria to humans ( Heck, 1997 Hirano, 1999 Holmes and Cozzarelli, 2000 Strunnikov and Jessberger, 1999). The SMC gene family encodes chromosome scaffold proteins (condensins and cohesins) that are required for structural maintenance of chromosomes in yeast ( Strunnikov et al., 1993 1995) and play a central role in chromosome condensation ( Koshland and Strunnikov, 1996), sister chromatid cohesion ( Michaelis et al., 1997), dosage compensation ( Lieb et al., 1998) and recombination repair ( Jessberger et al., 1996). Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development. The weak embryo phenotype observed in this mutant may result from expression of a related gene ( AtSMC2) with overlapping functions. A single SMC2 knockout ( ttn3) was identified and confirmed by molecular complementation. These mutants ( ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. We used forward genetics to identify duplicate T-DNA insertions in each gene. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Endosperm defects include aberrant mitoses and giant polyploid nuclei. The titan ( ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development.

Condensin protein scaffold