Targeted Knockouts of Selected GDSL Esterases and Class III Peroxidases In The Moss Physcomitrella patens Reveal Their Roles in Spore Wall Formation and Germination

Abstract

Early land plants evolved a number of key innovations to invade the land and diversify. One such innovation was a robust spore wall containing sporopollenin. Sporopollenin is the main lipidic component of spore and pollen walls and has been proposed to be the decisive factor for the successful terrestrialization by early plants. Until now, no enzyme involved in sporopollenin metabolism (polymerization and degradation) has been studied at the genetic and biochemical levels. I hypothesized that sporopollenin metabolizing enzymes might be homologs of cutin and lignin metabolizing enzymes belonging to GDSL esterases and class III peroxidase families, respectively. In this study, two GDSL esterases (CUSL and CUTL) and three class III peroxidases (PRX38, PRX39 and PRX47) of a model plant, Physcomitrella patens, were selected as candidate enzymes in sporopollenin metabolism based on gene expression profiles and phylogenetic analysis. Targeted knockout experiments were performed, and stable single (cutl, cusl and prx47) and double knockout (prx38 prx39) plants were generated. Stable knockout lines were confirmed by gDNA PCR. Single targeted gene replacement in the cutl and cusl lines was confirmed by Southern blot and RT-PCR. Then the stable knockout lines were phenotypically analyzed. Cutl and prx47 spores showed delayed germination, whereas early germination was observed with cusl spores as compared to control spores. In addition, cusl and prx38 prx39 spores showed augmented osmolysis and cusl spores were more susceptible to alkaline hydrolysis than control spores. These results suggest that we may have discovered novel GDSL esterases and class III peroxidases involved in spore wall formation (CUSL, PRX38 and/or PRX39) and degradation (CUTL and PRX47) in Physcomitrella. In addition, treatment of developing sporophytes with ROS scavengers provided support for the involvement of oxidative cross-linking in spore wall development, including sporopollenin polymerization and/or deposition, as well as a role for ROS in intine/aperture development. This research contributes to our understanding of the metabolism of sporopollenin and its evolution in land plants, and may have direct applications involving this biopolymer in the fields of biomedicine, biotechnology and green chemistry.