Targeted knockout of genes involved in phytohormone signaling

The moss Physcomitrella patens offers the possibility to investigate the basal mechanisms of phytohormone action, taking advantage of the high degree of similarity to higher plants on the molecular level. As almost nothing is known about auxin distribution and sensitivity of cells during moss development, we started to characterize it with the help of an auxin inducible reporter gene system . Therefore we produced stable transgenic plants with the b-glucuronidase gene (gus) driven by the auxin inducible promoter elements GH3 and DR5, respectively. The transgene copy number of several plants was estimated by southern blot analysis. The GUS staining pattern in protonema, buds and gametophores revealed that both auxin inducible elements are functional in Physcomitrella but behave differently concerning staining intensity and response time. The staining was predominantly visible in buds and stems of gametophores. Characterized plants will be used as platforms for knock-outs of genes encoding proteins involved in auxin transport like the auxin efflux carrier PIN. We found an EST representing a putative Physcomitrella pin homologue. A molecular analysis of putative knock-out plants is carried out at the moment. In addition, we isolated two different gh3 homologues expressed in Physcomitrella protonema (PpGH3 1 and PpGH3 2). A possible third homologue is existing as an EST found in a gametophore specific library. We knocked out PpGH3 1 and the phenotype is currently under investigation. One of the key cell-cycle regulatory points is the G1/S transition controlled by the so called Rb pathway. In this pathway expression of cycD and cdkA, that build a Rb phosphorylating kinase in a binary complex, is controlled by cytokinin and auxin, respectively 2 and by sugar availability. We isolated the Physcomitrella homologues for the essential members of this pathway. This is the first time that all important members of this pathway have been found in such an evolutionary old organism. We proved the G1/S regulating function of the Physcomitrella CycD homologue by complementation of a yeast mutant, which is deficient for G1/S cyclin genes and knocked out this gene in Physcomitrella. The knockout mutants showed accelerated caulonema development and enhanced and quicker formation of buds in the presence of cytokinin. Furthermore, knockout mutants for the Rb homologue have been created, which are currently under investigation.