Whereas the tips of WT gametophores showed a clear reorientation toward the light stimulus ( Figure 6D), pinA pinB colonies subjected to the same light stimulus continued to
grow in a disoriented manner, showing no clear tropic growth toward the light stimulus ( Figure 6D). These data suggest conservation of PIN-dependent, auxin transport-driven gravitropism and phototropism pathways between mosses and angiosperms and again highlight the importance of auxin transport-driven processes in Physcomitrella gametophore development. For reasons outlined in the introduction, this study has principally targeted recent controversy surrounding the roles of auxin transport in Physcomitrella gametophore development. However, as auxin transport has previously been detected in moss sporophytes and application of transport inhibitors perturbs Selleck Roxadustat sporophyte development [ 32], we also tested the hypothesis that PIN-mediated auxin transport regulates sporophyte development. We detected sporophytic expression of PINA and PINB ( Figure S4B) and grew WT and pin mutant sporophytes to evaluate their phenotypes. Cultures were grown on four peat plugs in continuous
light at 23°C for 6 weeks before INK 128 purchase transfer to a short-day 16°C regime for induction, and all the sporophytes present were harvested 4 weeks after induction. Whereas gametangia appeared normal ( Figure 7A), PINA and PINB contributed synergistcially to fertility and development ( Figures 7B and S6). Sporophytic defects were detected with variable penetrance: a low proportion (6 out of 208) on our GH3:GUS WT line had duplicated sporangia or dead sporophytes. Whereas pinA mutants had no obvious defects (1 out of 115 had duplicated sporangia; 3 out of 115 had an enlarged sporangium), a significant proportion of pinB mutants had duplicated sporangia (19 out of 89; 6 out of 89 were dead or had other defects), and around half of pinA pinB mutants had severe, sometimes lethal, developmental defects (5 out of 34 had duplicated sporangia; check 7 out of 34 were dead
or had other defects). The results suggest that PIN-mediated auxin transport regulates sporophytic shoot development, with a stronger contribution from PINB than from PINA. On the basis of heterologous gene expression assays in tobacco, previous work suggested that Physcomitrella PINs A and D localize at the ER and cytosol, respectively, and land plant PINs were therefore postulated to have an ancestral role in regulating intracellular auxin homeostasis rather than intercellular transport [ 34 and 35]. However, we have recently shown that Physcomitrella PINA–PINC are canonical, sharing sequence motifs that are required for plasma membrane targeting with Arabidopsis canonical PINs [ 45]. Our work suggested that canonical PINs are one ancestral type within the land plants and that Physcomitrella PINs A–C should have a capacity for plasma membrane targeting [ 45].