Mutation of a conserved motif of PP2C.D phosphatases confers SAUR immunity and constitutive activity

The phytohormone auxin promotes the growth of plant shoots by stimulating cell expansion via plasma membrane (PM) H⁺- ATPase activation, which facilitates cell wall loosening and solute uptake. Mechanistic insight was recently obtained by demonstrating that auxin-induced SMALL AUXIN UP RNA (SAUR) proteins inhibit D-CLADE TYPE 2C PROTEIN PHOSPHATASE (PP2C.D) activity, thereby trapping PM H1-ATPases in the phosphorylated, activated state, but how SAURs bind PP2C.D proteins and inhibit their activity is unknown. Here, we identified a highly conserved motif near the C-terminal region of the PP2C.D catalytic domain that is required for SAUR binding in Arabidopsis (Arabidopsis thaliana). Missense mutations in this motif abolished SAUR binding but had no apparent effect on catalytic activity. Consequently, mutant PP2C.D proteins that could not bind SAURs exhibited constitutive activity, as they were immune to SAUR inhibition. In planta expression of SAUR-immune pp2c.d2 or pp2c.d5 derivatives conferred severe cell expansion defects and corresponding constitutively low levels of PM H⁺-ATPase phosphorylation. These growth defects were not alleviated by either auxin treatment or 35S:StrepII-SAUR19 coexpression. In contrast, a PM H1-ATPase gain-of-function mutation that results in a constitutively active H⁺ pump partially suppressed SAUR-immune pp2c.d5 phenotypes, demonstrating that impaired PM H⁺- ATPase function is largely responsible for the reduced growth of the SAUR-immune pp2c.d5 mutant. Together, these findings provide crucial genetic support for SAUR-PP2C.D regulation of cell expansion via modulation of PM H1-ATPase activity. Furthermore, SAUR-immune pp2c.d derivatives provide new genetic tools for elucidating SAUR and PP2C.D functions and manipulating plant organ growth.