2013 Annual Report
1a.Objectives (from AD-416):
To analyze the progeny of rice plants transformed with constructs that result the over-expression and knockdown of the rice PIMT1 and PIMT2 genes.
1b.Approach (from AD-416):
Asparagine and aspartate in proteins can form isoaspartate through deamidation and isomerization in the former, and dehydration and isomerization in the latter. PROTEIN-L-ISOASPARTYL METHYLTRANSFERASE (EC 220.127.116.11) (PIMT) is a repair enzyme that regenerates isoaspartate back to aspartate. In the model plant, Arabidopsis thaliana there are two PIMT genes. PIMT1 is expressed throughout the life cycle of the plant, while PIMT2 is expressed at high levels in dormant seeds where PIMT specific activity is greatest. In addition, PIMT2 has multiple transcriptional start sites and splicing variants allowing for the production of multiple isoforms that are targeted to different subcellular organelles. Two PIMT genes have now been identified in many other plant species suggesting similar strategies in the protection of the proteome. Using sequences of the Arabidopsis thaliana PIMT1 and PIMT2, two genes with homology to the Arabidopsis proteins were identified in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor) and maize (Zea mays) EST databases. We propose to use rice as a model system for monocot analysis due to large genomic resources presently available. Analysis of the rice genomic and EST databases predict that both of the rice PIMT genes potentially encode multiple transcripts from each gene, which is different than the results observed in Arabidopsis. RT-PCR of rice total RNA from different tissues using gene specific primers has been used to amplify the transcripts from each gene. Each cDNA will be cloned into a transformation vector (pCAMBIA1301) for stable over-expression in rice. RNAi constructs will be developed for each transcript where possible to evaluate the knockout/knockdown effect on seed germination. Rice seed dormancy, germination, and seed longevity will be evaluated for each transgenic combination in collaboration with Dr. Bruce Downie (UK Department of Horticulture).
Due to the differences in the Protein L-isoaspartyl methyltransferase (PIMT) genes in monocot species compared to the model system, Arabidopsis, it is presently unknown which PIMT proteins will be the most effective in protection during dormancy and affect seed germination. Expression of the rice PIMT genes has been confirmed. Subcellular localization of the rice PIMT genes have been done using PIMT green fluorescent protein (GFP) fusion proteins transiently expressed in tobacco, maize and barley leaves. Over-expression of the rice PIMT1 gene in rice was confirmed, but due to the sabbatical leave of the cooperator for one year, transformation was not done. In addition, experiments directed at PIMT knock-down have been discontinued as recent evidence has shown that knockout of the PIMT gene through insertional mutagenesis results in lethality, thus regeneration of transgenic plants would not be possible. Since the cooperator’s sabbatical leave, direction of the cooperator’s research has changed and is no longer directed towards analysis of expression of the PIMT gene in rice. Further experiments in the area are directed towards determining potential targets of PIMT protein and how they affect seed dormancy and germination. Due to the change in direction, this project is being terminated.