Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

42Plants are the tallest organisms on Earth; a feature sustained by solute-transporting 43 xylem vessels in the plant vasculature. The xylem vessels are supported by strong 44 cell walls that are assembled in intricate patterns. Cortical microtubules direct wall 45 deposition and need to rapidly re-organize during xylem cell development. We 46 established long-term live-cell imaging of single Arabidopsis cells undergoing proto-47 xylem trans-differentiation, resulting in spiral wall patterns, to investigate the 48 microtubule re-organization. The initial disperse microtubule array rapidly readjusted 49 into well-defined microtubule bands, which required local de-stabilization of individual 50 microtubules in band-interspersing gap regions. Using extensive microtubule 51 simulations, we could recapitulate the process in silico and found that local 52 recruitment of microtubule-bound nucleation is critical for pattern formation, which we 53 confirmed in vivo. Our simulations further indicated that the initial microtubule 54 alignment impact microtubule band patterning. We confirmed this prediction using 55 katanin mutants, which have microtubule organization defects, and uncovered active 56 KATANIN recruitment to the forming microtubule bands. Our combination of 57 quantitative microscopy and modelling outlines a framework towards a 58 comprehensive understanding of microtubule re-organization during wall pattern 59 formation. 60 61 62The plant vasculature contains xylem cells that are organised in interconnected 63 tubular networks to enable efficient water distribution to plant organs, and that 64 support plant stature (Myburg et al. 2001). All plant cells are surrounded by primary 65 cell walls, which dictate growth direction. Xylem cells are reinforced by an additional 66 wall layer, referred to as a secondary wall, that is deposited in local thickenings that 67 form highly ordered spatial patterns (Turner et al. 2007). Xylem cells subsequently 68 undergo programmed cell death, which leads to the clearing of their cytoplasmic 69 content and the resulting formation of a hollow tube that provides the water-70 conducting capacity of vascular plants (Meents et al. 2018).71 The major load-bearing component of plant cell walls is cellulose; a β-1,4-72 linked glucan. Cellulose is synthesised by cellulose synthase (CESA) complexes 73 (CSCs) that span the plasma membrane (Schneider et al. 2016). Nascent cellulose 74 chains coalesce via hydrogen bonds, get entangled in the cell wall and further 75 synthesis thus forces the CSCs to move in the membrane (Diotallevi and Mulder 76 4 2007). The CSC delivery to, and locomotion within, the plasma membrane is guided 77 by cortical microtubules that presumably are associated with the plasma membrane 78 (Paradez et al. 2006; Crowell et al. 2009; Gutierrez et al. 2009; Watanabe et al. 79 2015). Cortical microtubules thus directionally and spatially template the cellulose 80 synthesis machinery during cell wall deposition. 81Microtubules undergo dynamic re-organizati...