Hydrolases of the ILR1-like family of Arabidopsis thaliana modulate auxin response by regulating auxin homeostasis in the endoplasmic reticulum

Amide-linked conjugates of indole-3-acetic acid (IAA) have been identified in most plant species. They function in storage, inactivation or inhibition of the growth regulator auxin. We investigated how the major known endogenous amide-linked IAA conjugates with auxin-like activity act in auxin signaling and what role ILR1-like proteins play in this process in Arabidopsis. We used a genetically encoded auxin sensor to show that IAA-Leu, IAA-Ala and IAA-Phe act through the TIR1-dependent signaling pathway. Furthermore, by using the sensor as a free IAA reporter, we followed conjugate hydrolysis mediated by ILR1, ILL2 and IAR3 in plant cells and correlated the activity of the hydrolases with a modulation of auxin response. The conjugate preferences that we observed are in agreement with available in vitro data for ILR1. Moreover, we identified IAA-Leu as an additional substrate for IAR3 and showed that ILL2 has a more moderate kinetic performance than observed in vitro. Finally, we proved that IAR3, ILL2 and ILR1 reside in the endoplasmic reticulum, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis which results in activation of auxin signaling.