Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction

Abstract

To understand how proteinase inhibitor genes, as a part of plant defense system, are activated following wounding, we have developed a molecular genetic approach to directly select for mutants blocked in wound response in Arcibidopsis thaliana. The wound-inducible promoter from the potato Proteinase Inhibitor II gene {pinl) was linked to a bacterial negative marker gene codA encoding for cytosine deaminase and transformed into Arcibidopsis thaliana. The cytosine deaminase activity was detected in the leaves of the transgenic plants (Tr349) induced by sucrose, ABA, methyl jasmonate and mechanical wounding. Methyl jasmonate was the best for the pinl induction in the seedlings among all the wound signal chemicals tested. Wild type seedlings had a dose-dependent sensitivity to 5FU. The seedlings were completely killed at a level of 100 |ig/mL and 130 |ig/mL 5FU for Arcibidopsis and N. tabacLim, respectively. 5FC at a concentration of 1 mg/mL had no effect on the seedlings of wild type of Arabidopsis or N. tabacum, or on Arabidopsis transgenic Tr349 seedlings without pinl induction. However, 5FC had a dose-dependent effect on the seedlings of transgenic Tr349 with the pinl induction by methyl jasmonate. No seedlings could survive above the concentration of 500 |J.g/mL 5FC. Homozygous Tr349 seeds were mutagenized by EMS. M2 seeds were collected and plated out on selective media containing 50 |J.g/mL kanamycin + 25 |jM methyl jasmonate + 500 |ig/mL 5FC. The 110 seedlings from M2 seeds survived on selective media. M3 seeds were collected and rescreened on selective media to eliminate false positives. Four M2 mutant lines that lost cytosine deaminase activity in M2 and M3 plants have been isolated. Because of the long timeframes involved in the work with transgenic plants several smaller projects were also conducted. Due to the success of these side projects, they are included in this thesis as separate chapters. To test whether the UMP synthase gene from D. discoideum functions in plants or not, the construct pm2-UMPS'^'' was prepared and transformed to N. tabacum. The expression of the UMPS^'' in the leaves of transgenic tobacco was detected in a wound-inducible manner in both mRNA and enzyme activity. The UMP/CMP kinase cDNA from Arabidopsis thaliana was isolated, expressed in E. coli and characterized. Site-directed mutagenesis studies showed that a glycine-rich conserved sequence (P-Ioop) played a role in ATP-binding and enzyme catalysis.