Epsilon Open Archive

Abstract

Cytokinins are plant hormones, and their relative abundance in proportion to auxin determines whether shoots or roots are formed in callus tissue. The cytokinin to auxin ratio is also believed to regulate apical dominance in plants. The suggested biosynthetic route for cytokinin involves formation of the nucleotide iPMP as the first metabolite synthesized, which is then subsequently hydroxylated to form the nucleotide ZMP. In the work underlying this thesis the potential role of an iPMP-independent biosynthetic route, with direct formation of the nucleotide ZMP was explored. Using an inducible IPT line of Arabidopsis, and in vivo deuterium labelling coupled to analysis with LC-MS/MS, we concluded that ZMP could indeed be formed independently of iPMP. In a similar experiment with wild type Arabidopsis, ZMP was also formed independently of iPMP. Furthermore, the two plant hormones cytokinin and auxin have been shown to regulate each other’s metabolism. In experiments using the IAA analogue NAA, a very rapid decrease in the biosynthesis and pool size of cytokinins was observed. The effect of NAA was mediated through the iPMP-independent pathway. A negative effect of cytokinin on auxin biosynthesis and pool size was also detected. This regulation was found to be slower, and probably related to alterations in development. Arabidopsis and tobacco leaves and roots were separately incubated in media containing 2H2O. Cytokinin biosynthesis capacity was detected in all tissues, and small leaves seemed to possess the highest de novo biosynthesis capability. An Arabidopsis mutant defective in production of the ADK enzyme responsible for conversion of adenosine to adenosine 5´monophosphate and the corresponding cytokinins was found to contain elevated levels of cytokinins, while its IAA levels were lower than in wild type. The increased cytokinin content was due to increased cytokinin biosynthesis. Finally, a LC-MS/MS method capable of quantifying central cytokinin metabolites in sample sizes <50mg FW including bases, ribosides and intact nucleotides at a rate of 70 samples/day was developed.