Auxin responsiveness of the MONOPTEROS-BODENLOS module in primary root initiation critically depends on the nuclear import kinetics of the Aux/IAA inhibitor BODENLOS

Abstract

Primary root formation in early embryogenesis of Arabidopsis thaliana is initiated with the specification of a single cell called hypophysis. This initial step requires the auxin-dependent release of the transcription factor MONOPTEROS (MP, also known as ARF5) from its inhibition by the Aux/IAA protein BODENLOS (BDL, also known as IAA12). Auxin-insensitive bdl mutant embryos and mp loss-of-function embryos fail to specify the hypophysis, giving rise to rootless seedlings. A suppressor screen of rootless bdl mutant seedlings yielded a mutation in the nuclear import receptor IMPORTIN-ALPHA 6 (IMPα6) that promoted primary root formation through rescue of the embryonic hypophysis defects, without causing additional phenotypic changes. Aux/IAA proteins are continually synthesized and degraded, which is essential for rapid transcriptional responses to changing auxin concentrations. Nuclear translocation of bdl:3×GFP was slowed down in impα6 mutants as measured by fluorescence recovery after photobleaching (FRAP) analysis, which correlated with the reduced inhibition of MP by bdl in transient expression assays in impα6 knock-down protoplasts. The MP-BDL module acts like an auxin-triggered genetic switch because MP activates its own expression as well as the expression of its inhibitor BDL. Using an established simulation model, we determined that the reduced nuclear translocation rate of BDL in impα6 mutant embryos rendered the auxin-triggered switch unstable, impairing the fast response to changes in auxin concentration. Our results suggest that the instability of the inhibitor BDL necessitates a fast nuclear uptake in order to reach the critical threshold level required for auxin responsiveness of the MP-BDL module in primary root initiation.

Keywords: Arabidopsis thaliana; BODENLOS; IMPORTIN-ALPHA 6; MONOPTEROS; auxin; import.