Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division

Abstract

Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced cytokinins. Levels of immunoreactive cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type cytokinins were the most abundant cytokinins in Physcomitrella, whereas zeatin-type cytokinins, the major native cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in cytokinin metabolism.