Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning

Abstract

Summary• Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin ⁄ indole acetic acid (Aux ⁄ IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited.• As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP) ⁄ ARF5.• This truncated version of MP, termed MPD, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPD, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux ⁄ IAA mutations. Further, in MPD leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina.• We conclude that MPD can selectively uncouple a single ARF from regulation by Aux ⁄ IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development.

Auxin depletion from leaf primordia contributes to organ patterning

“…On the contrary, expressing MPΔ in the boundary zone or in the abaxial domain did not cause polarity defects. In addition, ubiquitous MP overexpression does not lead to leaf polarity defects (34,35). It is thus conceivable that the transient low auxin concentration in the adaxial domain and the resulting auxin gradient establishes a gradient of MP activity that acts to ensure normal establishment or maintenance of adaxial cell fate.…”

Irrepressible, truncated Auxin Response Factors

“…3,19 This is remarkable not only because it presents MP as a sufficient regulator of leaf vasculature, but also because it can be used to trace genetic interactions with other Aux/IAA and ARF proteins. In yeast-two-hybrid studies, Aux/IAA proteins interact readily with MP, 20 but genetic tests go further and explore which of these potential interactions are functionally relevant in the plant.…”

“…22 This previously proposed interaction is supported by the fact that MPΔ is epistatic over bdl, causing increased vascularization in narrow leaves also in the genetic background of bdl. 3 The removal of domains III and IV from MP not only abolishes repressive interactions with Aux/IAAs, but should also eliminate ARF-ARF homo or heterodimerization, which are mediated by the same domains ( Fig. 1C and F).…”

“…Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ), 3 which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP's role in vascular patterning, a previously characterized auxin dependent process.…”

Overcoming recalcitrance - Auxin response factor functions in plant regeneration

“…While mp mutants demonstrated requirement for MP activity during de novo shoot meristem formation, a dominantly acting MP variant, lacking C-terminal protein-protein interaction domains for Aux/IAA-mediated repression (MPD), 18 demonstrated sufficiency in various genetic backgrounds. The MPD variant generally promoted de novo shoot formation also in wild type background and was even able to organize shoot meristems from tissue sources normally recalcitrant to regeneration, such as Col-0 cotyledons (Fig.…”