Action at a distance: Defects in division plane positioning in the root meristematic zone affect cell organization in the differentiation zone

Abstract

Cell division requires spatial coordination to properly position the division plane. How division plane positioning contributes to plant growth remains unknown. Two unrelated microtubule binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9), are together required for normal Arabidopsis growth and division. tan1 air9 double mutants have synthetic growth and division plane orientation defects while single mutants lack obvious defects. We show that the first 132 amino acids of TAN1 (TAN11-132) rescue the tan1 air9 double mutant and localize to the division site during telophase. Loss of both rescue and division-site localization occurred when interaction between TAN1 and PHRAGMOPLAST ORIENTING KINESIN1 (POK1) was disrupted by replacing six amino acid residues with alanines in TAN11-132. However, full-length TAN1 with the same alanine substitutions significantly rescued the tan1 air9 double mutant and remained at the division site throughout mitosis, although its accumulation was reduced and phragmoplast positioning defects occurred. POK1 often fails to accumulate at the division site in tan1 air9 mutants, suggesting that both TAN1 and AIR9 stabilize POK1 there. Finally, a mitosis specific promoter driving TAN1 rescued the tan1 air9 double mutant phenotypes indicating that defects seen in the root differentiation zone reflect the loss of mitotic-specific TAN1 activity.One sentence summarySpecific amino acids within TAN1 are required for its correct localization and function partially through interaction with POK1; both TAN1 and AIR9 mediate POK1 division site localization.

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

“…CFP – TAN1(28–33A) and YFP–POK1 colocalized with the PPB in 41% of cells ( n = 32/79), which is significantly less frequent when compared to 72% of cells ( n = 58/82 cells, 20 plants) with PPBs in the tan1 air9 mutant containing unaltered CFP – TAN1 and YFP–POK1 ( Figure 6, A and I ; Fisher’s exact test, P = 0.0001, similar to Lipka et al (2014) and Schaefer et al, 2017 ). Unaltered CFP – TAN1 fully rescued the tan1 air9 double mutant ( Mills and Rasmussen, 2022 ), and served here as a control. Unaltered CFP – TAN1 and YFP–POK1 localized and were maintained at the division site similar to in the WT during metaphase ( Figure 6B , n = 13/13), while CFP – TAN1(28–33A) and YFP–POK1 in the tan1 air9 mutant were sometimes absent from the division site during metaphase with only 58% of metaphase cells maintaining both proteins at the division site ( n = 11/19 cells, Figure 6, F and I ).…”

“…TAN1 remains at the division site throughout cell division, while AIR9 is lost from the division site upon PPB disassembly and then reappears at the division site during cytokinesis when the phragmoplast contacts the cortex. When full length TAN1 fused to YELLOW FLUORESCENT PROTEIN (TAN1-YFP) and driven either by the constitutive viral cauliflower mosaic CaMV35S promoter (p35S:TAN1-YFP) or the native promoter with the fluorescent protein as either N-or C-terminal fusion (pTAN1:TAN1-YFP or pTAN1:CFP-TAN1) was transformed into the tan1 air9 double mutant, the phenotype was rescued such that plants looked similar to and grew as well as wild-type plants (Mir et al, 2018;Mills and Rasmussen, 2022). TAN1 is an intrinsically disordered protein with no well-defined domains.…”

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

“…CFP-TAN1(28-33A) and YFP-POK1 colocalized with the PPB in 41% of cells (n = 32/79) which is significantly less frequent when compared to 72% of cells (n=58/82 cells, 20 plants) with PPBs in tan1 air9 mutants containing unaltered CFP-TAN1 and YFP-POK1 (Figure 6A, Table 2; Fisher’s exact test, P-value = 0.0001). Unaltered CFP-TAN1 fully rescued the tan1 air9 double mutant (Mills and Rasmussen, 2022), and serves here as a control. Unaltered CFP-TAN1 and YFP-POK1 localized and were maintained at the division site similar to wild type in metaphase (Figure 6B, n=13/13), while CFP-TAN1(28-33A) and YFP-POK1 in tan1 air9 mutants were sometimes absent from the division site in metaphase with only 58% of metaphase cells maintaining both proteins at the division site (n = 11/19 cells, Figure 6F, Table 2).…”

“…TAN1 remains at the division site throughout cell division, while AIR9 is lost from the division site upon PPB disassembly and then reappears at the division site during cytokinesis when the phragmoplast contacts the cortex. When full length TAN1 fused to YELLOW FLUORESCENT PROTEIN ( TAN1-YFP ) and driven either by the constitutive viral cauliflower mosaic CaMV35S promoter ( p35S:TAN1-YFP ) or the native promoter with the fluorescent protein as either N- or C-terminal fusion ( pTAN1:TAN1-YFP or pTAN1:CFP-TAN1 ) was transformed into the tan1 air9 double mutant, the phenotype was rescued such that plants looked similar to and grew as well as wild-type plants (Mir et al, 2018; Mills and Rasmussen, 2022).…”