Rheostat control of xylem differentiation by ATHB8/ACL5-BUD2 transcription module

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS 5 (ACL5) and BUSHY AND DWARF 2 (BUD2)is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of the homeodomain leucine zipper (HD-Zip) III and key auxin signalling geens. This mechanism involves the posititve regulation of thermospermine biosynthesis by the HD-Zip III protein ARABIDOPSIS THALIANA HOMEOBOX tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-Zip III protein REVOLUTA contributes to the increased leaf vascularizaton and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signalling genes such as LIKE AUXIN RESISTANT 2 (LAX2) and (LAX3). Through this work we present a model of positive and negative feedback between auxin and HD-Zip gene expressions that tunes the rate of xylem differentiation.