The Unexpected Membrane Targeting of Marchantia Short PIN Auxin Exporters Illuminates Sequence Determinants and Evolutionary Significance

The plant hormone auxin and its directional transport are crucial for growth and development. PIN auxin transporters, on account of their polarized distribution, are instrumental in guiding auxin flow across tissues. Based on protein length and subcellular localization, the PIN family is classified into two groups: plasma membrane (PM)-localized long PINs and endoplasmic reticulum (ER)-localized short PINs. The origin of PINs was traced to the alga Klebsormidium, with a single PM-localized long KfPIN. Bryophytes, the earliest land plant clade, represent the initial clade harboring the short PINs. We tracked the evolutionary trajectory of the short PINs and explored their function and localization in the model bryophyte Marchantia polymorpha, which carries four short and one long PIN. Our findings reveal that all short MpPINs can export auxin, and they are all PM-localized with MpPINX and MpPINW exhibiting asymmetric distribution. We identified a unique miniW domain within the MpPINW hydrophilic loop region, which is sufficient for its PM localization. Phosphorylation site mutations within the miniW domain abolish the PM localization. These findings not only identify the essential sequence determinant of PINs' PM localization but also provide a unique insight into the evolution of ER-localized PINs. Short MpPINW, which is evolutionarily positioned between the ancestral long PINs and contemporary short PINs, still preserves the critical region essential for its PM localization. We propose that throughout land plant evolution, the unique miniW domain has been gradually lost thus converting the PM-localized short PINs in bryophytes to ER-localized short PINs in angiosperms.