Minisymposium 27: Temperature

Abs # 48003: Mutants in the Arabidopsis SAC9 gene are constitutively cold-acclimated and overaccumulate PtdIns(4,5)P₂

Presenter:	Williams, Mary E., Mary_Williams@hmc.edu
Authors	Williams, Mary E. (A)   Torabinejad, Javad  (B) (C)  Parker, Katherine  (A)   Cohick, Evan  (A)   Hortter, Michelle  (A)   Nguyen, Vi  (A)   Thompson, James E. (B)   DeWald, Daryll B. (B)
Affiliations:	(A): Biology Department, Harvey Mudd College (B): Department of Biology, Utah State University (C): Department of Biochemistry, Virginia Tech Univeristy

We isolated an Arabidopsis mutant with characteristics of a constitutive cold-acclimation response including dwarfism, a prostrate growth habit and freezing tolerance. The mutant accumulates anthocyanin, which together with its prostrate growth suggests it is light-sensitive. Growth in dim light reverses the anthocyanin accumulation and prostrate growth, but not the dwarfism. At room temperature the mutant expresses several cold-induced genes including CBF1 and CBF2. Isolated leaves from the mutant show resistance to freezing damage as assayed by electrolyte leakage. However, a whole-plant freezing tolerance test gives conflicting results. When grown under bright light conditions (120 µmol/m²/sec), the mutant plants are less freezing tolerant than wild-type plants, but when grown under dim light conditions (35 µmol/m²/sec), the mutant and wild-type plants are identical in freezing tolerance. Starting with our original EMS-allele, we mapped the mutation to an interval contained on two BACs, and then screened through T-DNA insert lines for genes in this interval. We identified a plant homozygous for a T-DNA insertion in the gene At3g59770 that exactly resembles the original mutant, and showed that the EMS and T-DNA alleles are in the same gene. A second T-DNA allele was also found, and we believe all three are functionally null alleles. The affected gene, SAC9, encodes a protein with an N-terminal SAC domain and a long C-terminal extension of unknown function. SAC domain-containing proteins from other organisms have been shown to have phosphoinositide phosphatase activity. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein-interaction domain within the SAC domain and a substitution of a conserved residue in the catalytic domain. The rice and Arabidopsis SAC9 proteins are closely related, but no SAC9-like proteins have been found in non-plant genomes. Because the mutated gene putatively encodes a phosphoinositide phosphatase, we labeled plants with [³H] myo-inositol, then extracted and analyzed phosphoinositides by HPLC. These studies showed that sac9 roots accumulate nearly 40 times more PtdIns (4,5)P₂ than wild-type roots, but no difference was detected between wild-type and sac9 shoots. When wild-type Arabidopsis plants were incubated at 0°C for one hour, PtdIns(4,5)P₂ levels in the roots increased four-fold. These results suggest that PtdIns (4,5)P₂ has a role in the cold-signaling pathway.