19Expansins facilitate cell expansion via mediating pH-dependent cell wall (CW) loosening. 20However, the role of expansins in the control of biomechanical CW properties in the tissue and 21 organ context remains elusive. We determined hormonal responsiveness and specificity of 22 expression and localization of expansins predicted to be direct targets of cytokinin signalling. We 23 found EXPA1 homogenously distributed throughout the CW of columella/ lateral root cap, while 24 EXPA10 and EXPA14 localized predominantly at the three-cell boundaries of epidermis/cortex in 25 various root differentiation zones. Expression of EXPA15, revealing cell type-specific localization 26 pattern, overlaps with higher CW stiffness measured via Brillouin light scattering microscopy. As 27 indicated by both higher Brillouin frequency shift and AFM-measured Youngs' modulus, EXPA1 28 overexpression upregulated CW stiffness, associated with shortening of the root apical meristem 29 and root growth arrest. We propose that root growth in Arabidopsis requires delicate orchestration 30 of biomechanical CW properties via tight regulation of various expansins' localization to specific 31 cell types and extracellular domains. 32

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at three-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell type-specific combination of homogenous vs. three-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young’s modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone. The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier transform infrared spectroscopy in the root transition zone. The EXPA1-induced CW remodeling associated with shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.