A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins

Abstract

The organization of living cells is based on networks of interacting molecules. Systematic analysis of protein interactions of 3-aa loop extension (TALE) homeodomain proteins, fundamental regulators of plant meristem function and leaf development, revealed a highly connected, complex network. The network includes nine members of Arabidopsis thaliana ovate family proteins (AtOFPs), a plant-specific protein family, indicating a close functional connection to TALE homeodomain proteins. Evidence is provided that AtOFP1 is an essential pleiotropic developmental regulator. AtOFP1 and AtOFP5 are shown to associate with the cytoskeleton and to regulate subcellular localization of TALE homeodomain proteins, suggesting a previously unrecognized control mechanism in plant development.

Fig. 1.

TALE-protein interaction network. (a) Protein interactions in the TALE cluster. +, interaction; 0, no interaction;/, not determined. (b) Network of protein interactions among TALE proteins and cofactors. Green, BELL proteins; red, KNOX proteins; black, nonhomeodomain cofactors; open circles, AtOFPs. The clustering coefficients Q and C are given as a measure of the degree of local clustering. (c) Quasi-clique formed by a fully connected hexagon motif of TALE proteins (BLH1, BLH3, BLH4, BLH10, KNAT5, and KNAT6) and three AtOFPs. (d) A highly connected quasi-clique formed by seven BELL proteins and AtOFP5.

Fig. 2.

Ectopic expression of AtOFP1. Overexpression of AtOFP1 from the CaMV ³⁵S promoter causes dominant pleiotropic developmental aberrations in A. thaliana (a-f) and tobacco (g). (a and b) Stunted growth of ³⁵S::AtOFP1 plants. (c) Flowers exhibit oval petals and sepals and protruding styles. (d) Stamens have shorter filaments than do wild-type plants. (e) Leaves are lobed and asymmetric and have broader midveins than wild-type leaves. (f) Siliques are shortened and contain only a few fertile seeds. (g) Flowers, leaves, and siliques of tobacco plants overexpressing AtOFP1 are altered in a similar way as in A. thaliana. (h) RT-PCR analysis reveals decreased levels of AtGA20ox1 mRNA in vegetative leaf tissue of AtOFP1-overexpressing plants. Control amplification of RAN3 transcript indicates equal amounts of cDNA. [Scale bars: c, d, and f, 1 mm; e, 20 mm (enlargement, 4 mm); g, 20 mm.]

Fig. 3.

AtOFP1 is associated with the cytoskeleton. Localization of transiently expressed GFP-fusion proteins in N. benthamiana leaves was analyzed by confocal laser scanning microscopy. (a) AtOFP1-GFP accumulates in the nucleolus and is located at the cytoskeleton. (b) Localization of AtOFP1-GFP is not altered after 20-min treatment with the actin depolymerizing drug cytochalasin D. (c) GFP fusion of the I/LWEQ domain of talin localizes to the actin filaments. (d) MAP3-GFP associates with microtubules. (Scale bars: 20 μm.)

Fig. 4.

AtOFP1 controls subcellular localization of TALE protein BLH1. Localization of transiently expressed GFP- and RFP-fusion proteins in N. benthamiana leaves was analyzed by confocal laser scanning microscopy. (a) Overlay of transmitted and fluorescent images of a BLH1-RFP-expressing cell. BLH1-RFP is localized exclusively in the nucleus. (b) Coexpression of BLH1-RFP with GFP alone does not affect the nuclear localization of BLH1. Shown are the GFP (Left) and RFP (Right) channels. (c) Coexpression of AtOFP1-GFP and BLH1-RFP in N. benthamiana leaves. BLH1-RFP is relocated from the nucleus to the cytoplasmic space and colocalizes with AtOFP1 in punctate structures at the cytoskeleton and in the cell periphery. GFP channel (Left), RFP channel (Center), and merged image (Right) are shown. (d) Coexpression of AtOFP1-GFP does not alter nuclear localization of the noninteracting BLH7-RFP. GFP channel (Left), RFP channel (Center), and merged image (Right) are shown. (Scale bars: 20 μm.)