Mesembryanthemum crystallinum plasma membrane root aquaporins are regulated via clathrin-coated vesicles in response to salt stress

The regulation of PIP-type aquaporins in the roots of plants has been identified as an important aspect in salinity tolerance. However, the molecular and cellular details underlying this process in halophytes remain unclear. Using free flow electrophoresis and label-free proteomics, we report that the increased abundance of PIPs at the plasma membrane of Mesembryanthemum crystallinum roots under salinity conditions is regulated by clathrin-coated vesicles (CCVs). To understand this regulation, we analyzed several components of the M. crystallinum CCVs complexes: clathrin light chain (McCLC) and subunits 1 and 2 of the adaptor complex (McAP1 and McAP2). Co-localization analyses revealed the association between McPIP1;4 and McAP2 and between McPIP2;1 and McAP1, observations corroborated by mbSUS assays, suggesting that aquaporin abundance at the PM is under the control of CCVs. The ability of McPIP1;4 and McPIP2;1 to form homo- and hetero-oligomers was tested and confirmed, as well as their activity as water channels. Also, we found increased phosphorylation of McPIP2;1 only at plasma membrane in response to salt stress. Our results prompt how root PIPs from halophytes are regulated through CCVs trafficking and phosphorylation, impacting their localization, transport activity and abundance under salinity conditions. One-sentence summaryAbundance of plasma membrane aquaporins in M. crystallinum roots increases in response to salinity via a clathrin-coated vesicle-dependent mechanism.