SummaryProtein phosphatase 2A (PP2A), a heterotrimeric serine/ threonine-speci®c protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wildtype seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of lightgrown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18-to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.Methanosarcina is the most metabolically diverse genus among the methanogens (12). Whereas most methanogenic Archaea grow by CO 2 reduction with H 2 , these species grow by the dismutation of acetate, by methylotrophic catabolism of methanol, methylated amines, and dimethylsulfide, and in some cases also by CO 2 reduction with H 2 . The aceticlastic methanogens exhibit a hierarchy of substrate preference coinciding with the free-energy yields of the substrates. Previous reports indicate that acetate catabolism is regulated at the transcriptional level in Methanosarcina spp. in response to alternative substrates, but the mechanism of catabolic gene regulation by these Archaea is currently unknown (3,11,15). With the complete sequencing of genomes from two methanosarcinal species (4, 5) and partial completion of a third species (http://genome.jgi-psf.org), this genus is well poised for genetic studies on archaeal gene expression and physiological pathways.One of the difficulties encountered when studying the Archaea has been the dearth of genetic techniques for transferring DNA into cells and selecting mutants. Although gene transfer and recombination techniques are straightforward in many bacterial and eukaryal systems, application of these techniques in the methanogenic Archaea has been problematic and is only in the developing stages. A genetic system developed for the Methanosarcina spp. is among the most advanced available for the methanogenic Archaea. Clonal colonies of Methanosarcina spp. can be grown on solidified medium by streaking or replica plating (1, 13). Development of an efficient transformation system for transfer of both plasmid and linear DNA into Methanosarcina spp. (9), combined with transposon-mediated random mutagenesis (19) and directed gene disruption (20), provides approaches for constructing and isolating mutants. The recent report of a markerless genetic exchange system for Methanosarcina spp. overcomes the current problem of limited selectable markers by allowing the marker to be removed and reused after gene disruption (10).A genetic system is required for functional analysis of the available methanosarcinal genomic da...