ETTIN (ARF3) physically interacts with KANADI proteins to form a functional complex essential for integument development and polarity determination in Arabidopsis

Abstract

KANADI (KAN) transcription factors promote abaxial cell fate throughout plant development and are required for organ formation during embryo, leaf, carpel and ovule development. ABERRANT TESTA SHAPE (ATS, or KAN4) is necessary during ovule development to maintain the boundary between the two ovule integuments and to promote inner integument growth. Yeast two-hybrid assays identified ETTIN (ETT, or AUXIN RESPONSE FACTOR 3) as a transcription factor that could physically interact with ATS. ATS and ETT were shown to physically interact in vivo in transiently transformed tobacco epidermal cells using bimolecular fluorescence complementation. ATS and ETT were found to share an overlapping expression pattern during Arabidopsis ovule development and loss of either gene resulted in congenital fusion of the integuments and altered seed morphology. We hypothesize that in wild-type ovules a physical interaction between ATS and ETT allows these proteins to act in concert to define the boundary between integument primordia. We further show protein-protein interaction in yeast between ETT and KAN1, a paralog of ATS. Thus, a direct physical association between ETT and KAN proteins underpins their previously described common role in polarity establishment and organogenesis. We propose that ETT-KAN protein complex(es) constitute part of an auxin-dependent regulatory module that plays a conserved role in a variety of developmental contexts.

Fig. 1.

ATS and ETT are nuclear localized and physically interact in vivo in a BiFC assay. (A-D) ATS-eGFP (A,B) and ETT-eGFP (C,D) are nuclear localized. (E,F) Fluorescence indicates direct interaction in vivo between ATS and ETT that is restricted to nuclei of transformed tobacco cells. (G,H) Co-transformation of ATS and a different ARF protein, MP, fails to show fluorescence complementation. Scale bars: 20 μm in A-D; 50 μm in E-H.

Fig. 2.

ATS and ETT display overlapping mRNA accumulation patterns during ovule development. (A-D) ATS (A,B) and ETT (C,D) in situ hybridizations on young (A,C) and mature (B,D) wild-type Arabidopsis ovules show an inner integument-specific signal. f, funiculus; n, nucellus; ii, inner integument; oi, outer integument. Scale bars: 5 μm in A,C; 10 μm in B,D.

Fig. 3.

Loss of ATS or ETT results in integument fusion and altered seed morphology. (A-I) Wild-type (A,E,I), ats (B,F,J) ett (C,G,K) and ats ett (D,H,L) ovules (A-H) and seeds (I-L). Wild-type ovules initiate two distinct integument primordia (A). Integument formation in ats, ett and ats ett leads to a single integument primordium (B-D). The outer integument grows over the inner integument in wild-type ovules (E), whereas the fused integuments in ats, ett and ats ett mutants grow in a single plane (F-H). Abnormal integument formation in ats, ett and ats ett mutants gives rise to aberrant seed morphology (compare J-L with I). n, nucellus; ii, inner integument; oi, outer integument; i, integument. Scale bars: 5 μm in A-D; 10 μm in E-H; 200 μm in I-L.

Fig. 4.

Model for ATS-ETT action during ovule development. (A,B) Benkova et al. (Benkova et al., 2003) have shown that PIN1-mediated auxin transport in an ovule primordium initially creates an auxin maximum at the nucellar tip (A) and subsequently two auxin maxima in the chalaza corresponding to the two integument primordia (B). Inner integument initiation coincides with the formation of an active ATS-ETT complex, which we hypothesize restricts the domain of PIN1 and thus enables the formation of the two auxin maxima at the chalaza (B). (C) In the absence of an active ATS-ETT complex (i.e. in ats or ett mutant ovules) PIN1 fails to be restricted and auxin remains distributed across the chalaza. (D) Formation of ATS-ETT heterodimers restricts PIN1 activity, which controls auxin efflux, while auxin positively regulates both ETT and PIN1 function.