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

Abstract

SUMMARYKANADI (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. ETT cDNA without the stop codon was amplified from leaf cDNA and cloned into pENTR/D-Topo to create pDK74. pAA29 was Gateway cloned into pDEST-GBKT7 (Rossignol et al., 2007) creating pAA36 (BD-KAN2). pDK132 was Gateway cloned into pDEST-GADT7 creating plasmid pDK136 (AD-ETT). ETT cDNA was cloned as an NdeI/XhoI fragment from pDK136 into pGBKT7 using NdeI/SalI to create pDK137 (BD-ETT). ARF4 cDNA was amplified from Col leaf cDNA and cloned into pENTR/D-Topo, creating pAA30. pAA30 was Gateway cloned into pDEST-GADT7 to create pAA42 (AD-ARF4). KAN1 cDNA was amplified and inserted as a BamHI/PstI fragment into pLITMUS28 to create pLMK37 and transferred as a BamHI/PstI fragment into: (1) pGAD424, creating pLMK44 (AD-KAN1); and (2) pAS2, creating pLMK46 (BD-KAN1).A 700 bp subclone of ETT (pDK73) cDNA was generated from pAS13 as described (Sessions et al., 1997). ATS-eGFP (pDK77) was created by Gateway cloning pAA34 into pDH51-GW-eGFP (Zhong et al., 2008). ETT-eGFP (pDK80) was created by Gateway cloning pDK74 into pDH51-GW-eGFP. Subclones for ATS-YFPc (pCG51) and ETT-YFPn (pCG54) were created by amplifying ATS or ETT cDNA without stop codons and inserting the resulting fragments into pJET1.2 (Fermentas) to form pCG45 and pCG46, respectively. The cDNAs were inserted into 2X35S-SPYCE or 2X35S-SPYNE vectors (Walter et al., 2004), respectively, as XhoI/XmaI fragments into these same sites forming pCG47 and pCG50. The resulting expression cassettes were transferred into pMLBART (Gleave, 1992) as NotI fragments, producing pCG51and pCG54. Gateway reactions were performed using LR Clonase II (Invitro...

RiceLHS1/OsMADS1Controls Floret Meristem Specification by Coordinated Regulation of Transcription Factors and Hormone Signaling Pathways

“…Interestingly, these phenotypes are also seen in kan1 kan2 double mutants (Pekker et al, 2005). Recently, physical interactions between KANADI4 (KAN4/ ATS) and ETTIN were shown to coordinate ovule and integument development (Kelley et al, 2012). Our finding that gene homologs of Arabidopsis KANADI2, ATS, and ETTIN (Supplemental Table S1) are directly regulated by OsMADS1 in rice florets brings new light on how OsMADS1 can contribute to lateral organ development.…”

Genetic Dissection of Leaf Development in Brassica rapa Using a Genetical Genomics Approach

“…ER encodes a Leu-rich repeat receptor-like kinase, with pleiotropic effects on many traits, including morphological differences, leaf chlorophyll, and tolerance to drought and salt stresses in transgenic tomato plants (Keurentjes et al, 2007;Seo et al, 2012;Villagarcia et al, 2012). In Arabidopsis, Kelley et al (2012) provided evidence that KAN and ARF3 proteins formed a functional complex active in leaf development. copQTL23 on A10 (PL, LI, LC, LL, LW, BL, PB, and Ft) at the BrLNG1 locus colocalized with a cis-eQTL for BrLNG1, a trans-eQTL for BrKAN2, and a cis-eQTL for the Ft gene BrCO.…”

The ASYMMETRIC LEAVES Complex Employs Multiple Modes of Regulation to Affect Adaxial-Abaxial Patterning and Leaf Complexity

“…Additionally, repression of TAS3A may result from cooperativity between ARF3/ARF4 and factors that are misexpressed only in as2. One such candidate is KAN1, which physically interacts with ARF3 (Kelley et al, 2012), and whose expression extends into the adaxial side of as2 leaves (Wu et al, 2008). The fact that TAS3A expression persists in as2 seems to favor the requirement of a cofactor during the ARF3/ARF4-mediated repression of TAS3A.…”

“…Much like the examples described above, the increase in leaf complexity derives from the misregulation of their shared target ARF3 and the presence of additional factor(s) normally repressed by AS1-AS2. Candidates of particular interest include the AS1-AS2 target YAB5, which promotes outgrowth at the leaf margin (Sarojam et al, 2010); KAN1, which physically interacts with ARF3 (Kelley et al, 2012); and the KNOX targets, which already have a well-established role in compound leaf development (Bharathan et al, 2002;Barkoulas et al, 2008). Interestingly, reduced auxin signaling deepens serrations of as1 leaves in a BPdependent manner .…”