Characterization of the de novo pyrimidine biosynthetic pathway in Arabidopsis thaliana

Abstract

<p>We have characterized the enzyme cytidine deaminase. The full length cDNA was expressed in E. coli and purified to apparent homogeneity using a Ni-NTA agarose column. The Km and Vmax for cytidine were determined to be 226uM and 39.7 umoles/min respectively. The enzyme was also able to use 2'-deoxycytidine as substrate with an apparent Km of 49 uM and Vmax of 24 umoles/min.;We have also developed a single tube semi-quantitative relative reverse transcription polymerase chain reaction (RT-PCR) assay. We have used this assay to quantitate the relative transcript levels of the UMP synthase gene and the proximal SKIP5 F-box protein. We have determined that the two genes are coordinately transcriptionally up-regulated in Arabidopsis under conditions of pyrimidine starvation.;We have used our quantitative PCR method to analyze transcript levels of the entire de novo pathway of pyrimidine biosynthesis relative to the 18S rRNA. We have synthesized an oligonucleotide that is modified on its 3' end and cannot be used by the polymerase in template extension. This attentuates the level of amplification of the 18S transcript. Under conditions of pyrimidine starvation ATCase and UMP synthase show the highest level of transcriptional up-regulation at 20 fold and 5 fold, respectively. During abiotic stress conditions of phosphate starvation, saline and flooding the pathway is relatively unaffected. Under completely anaerobic conditions DHOase, DHODH and UMP synthase transcript levels are severely reduced, while ATCase is unaffected. During ORMV infection DHODH and UMP synthase are both up-regulated by two and four fold, respectively.;We have generated lines of transgenic Arabidopsis plants containing the promoters of each member of the de novo biosynthetic pathway driving beta-glucuronidase expression. Two promoter GUS fusions were constructed for both ATCase and DHOase. The introns within the 5' UTR of both these genes were required for expression within roots of our transgenic plants. Further, the DHOase intron is required for the highest levels of expression within aerial portions of the plants. The DHODH and UMP synthase promoters were both relatively weak in comparison to the ATCase and DHOase genes. Regions within the UMP synthase coding region conferred greatly increased GUS expression.</p>