High throughput metabolic screen of Physcomitrella transformants

A large-scale metabolic screen was performed for 51,180 targeted knockout mutants of the haploid moss Physcomitrella patens (Hedw.) Bruch & Schimp. The growth ability of each mutant was compared to the wild type. Plants were cultured on a minimal medium which contained only macroelements, as well as on a supplemented medium, additionally containing microelements, glucose, vitamins, ammonium tartrate, adenine, peptone and Na-palmitic acid. The screen resulted in the identification of 20 (0.04 %%) auxotrophs. Medium supplementation tests were performed for five auxotrophs, which showed no growth on minimal medium, but were rescued on supplemented medium. One vitamin-deficient mutant was identified as p-aminobenzoic acid auxotroph, three plants were nitrate assimilation deficient mutants and one transformant showed undefined growth requirements. The screen resulted further in the identification of two physiological mutants, exhibiting an albino phenotype on minimal medium but a green wild type phenotype on the supplemented medium. The culture of both albinos under low light intensities could not prevent bleaching, revealing that the missing production of chlorophyll was not caused by light sensitivity. Astonishingly, the supplementation of the minimal medium with selected compounds of the supplemented medium did also not prevent bleaching. Moreover, both mutants produced green protonemata, even if single substances of the supplemented medium were lacking. It was concluded that a complex network of interactions related to photosynthesis in Physcomitrella was disturbed. This is the first detailed study of auxotrophic and albino Physcomitrella mutants produced by transformation-mediated gene disruption. The described physiological mutants provide valuable resources for the identification of essential gene functions of plant metabolism.