Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

正确图案化的细胞壁指定了植物中的细胞功能。分化的原木质部和后木质部导管细胞分别表现出条纹和凹坑图案的厚次生细胞壁。皮质微管以不同的模式排列以指导细胞壁沉积。支架蛋白 MIDD1 通过与后木质部血管中的 ROP GTPases 和 KINESIN-13A 相互作用促进微管消耗。在这里，我们表明 MIDD1 的相分离可以微调拟南芥原木质部导管中的细胞壁间距。与野生型相比， midd1突变体在原木质部和后木质部导管细胞的次生细胞壁中分别表现出更窄的间隙和更小的凹坑。异位诱导的原木质素血管的实时成像显示，MIDD1 沿着解聚微管形成凝结，这反过来又导致了微管的大规模灾难。 MIDD1 缩合物表现出快速周转并且对 1,6-己二醇敏感。 ROP的缺失消除了 MIDD1 的凝聚，并导致原木质素导管中的细胞壁间隙变窄。这些结果表明，MIDD1 的微管相关相分离促进微管排列，从而调节次生细胞壁间隙的大小。这项研究揭示了相分离在细胞壁图案微调中的新生物学作用。