The localization of PHRAGMOPLAST ORIENTING KINESIN1 at the division site depends on the microtubule-binding proteins TANGLED1 and AUXIN-INDUCED IN ROOT CULTURES9 in Arabidopsis.

Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11-132), we assessed the localization and function of TAN11-132 in the tan1 air9 double mutant. TAN11-132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11-132, which disrupted the POK1-TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN11-132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1-POK1 interactions.

Division Plane Orientation Defects Revealed by a Synthetic Double Mutant Phenotype.

TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOTS9 (AIR9) are microtubule-binding proteins that localize to the division site in plants. Their function in Arabidopsis (Arabidopsis thaliana) remained unclear because neither tan1 nor air9 single mutants have a strong phenotype. We show that tan1 air9 double mutants have a synthetic phenotype consisting of short, twisted roots with disordered cortical microtubule arrays that are hypersensitive to a microtubule-depolymerizing drug. The tan1 air9 double mutants have significant defects in division plane orientation due to failures in placing the new cell wall at the correct division site. Full-length TAN1 fused to yellow fluorescent protein, TAN1-YFP, and several deletion constructs were transformed into the double mutant to assess which regions of TAN1 are required for its function in root growth, root twisting, and division plane orientation. TAN1-YFP expressed in tan1 air9 significantly rescued the double mutant phenotype in all three respects. Interestingly, TAN1 missing the first 126 amino acids, TAN1-ΔI-YFP, failed to rescue the double mutant phenotype, while TAN1 missing a conserved middle region, TAN1-ΔII-YFP, significantly rescued the mutant phenotype in terms of root growth and division plane orientation but not root twisting. We use the tan1 air9 double mutant to discover new functions for TAN1 and AIR9 during phragmoplast guidance and root morphogenesis.