Identification of Putative Peroxisomal Division Proteins in Arabidopsis.

Peroxisomes are valuable organelles that play many metabolic roles in plants such as synthesis of plant hormones and metabolism of hydrogen peroxide.  To this end, we searched for Arabidopsis proteins that could be linked to peroxisome division. We chose six putative peroxisomal proteins showing sequences similar to two established yeast peroxisomal proteins, Caf4p and Mdv1p.  The putative orthologs were chosen based on their similarities with the WD-40 domain, an important motif for protein-protein interaction in Caf4p and Mdv1p. After obtaining the cDNA clone of these orthologs, gateway cloning system was used to fuse them to a yellow fluorescent protein to determine there localization to peroxisomes. After about 48 hours of incubation on the bench, epifluorescence pictures of the leaf cells were taken to determine protein localization. Confocal images of the leaves confirmed the results, which showed that two out of six proteins were localized to peroxisomes, which are shown by the marker protein CFP-PTS1, a peroxisome targeting signal. As a result, we then proceeded to identify Arabidopsis knockout mutants of the putative orthologs to study gene function. Seeds of the mutants were ordered from the Arabidopsis Stock Center and genomic DNA was isolated from leaves of two-week-old plants.  PCR was used to amplify the DNA and genotypes of each plant was recorded by examining the gel. The wild type plants showed bands at about 1000 base pairs, while the mutant plants, homozygous and heterozygous, showed bands at about 500-700 base pairs.  A total of 28 homozygous plants for five genes were identified, which were transplanted for further studies. In summary, I identified two proteins that were localized to peroxisomes and 28 homozygous mutants for these proteins.  Future research will be done on these mutants to observe the peroxisomal and plant growth phenotypes.  [This study was supported by a grant from NSF awarded to Dr. Jianping Hu Ph.D., Associate Professor of the Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312 and USDA Capacity Building/NSF HBCU-UP grants awarded to Dr. Sarwan Dhir Ph.D. Associate Professor & Director, NSF HBCU-UP, S-STEM & REU Site Programs , Center for Biotechnology, Fort Valley State University, Fort Valley, GA 31030].