Abstract
Background and aims: The master transcription factor NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3), also known as SND1, plays a pivotal role in regulating secondary cell wall (SCW) development in interfascicular and xylary fibres in Arabidopsis thaliana. Despite progress in understanding SCW assembly in xylem vessel-like cells, the mechanisms behind its assembly across different cell types remain unclear. Overexpression of NST3 or its homologue NST1 leads to reduced fertility, posing challenges for studying their impact on secondary wall formation. This study aimed to develop a tightly regulated dexamethasone (DEX)-inducible expression system for NST3 and NST1 to elucidate the structure and assembly of diverse SCWs. Methods: Using the DEX-inducible system, we characterized ectopically formed SCWs for their diverse patterns, mesoscale organization, cellulose microfibril orientation and molecular composition using spinning disc confocal microscopy, field emission scanning electron microscopy, vibrational sum-frequency generation spectroscopy, and histochemical staining and time-of-flight secondary ion mass spectrometry, respectively. Key results: Upon DEX treatment, NST3 and NST1 transgenic hypocotyls underwent time-dependent transdifferentiation, progressing from protoxylem-like to metaxylem-like cells. NST3-induced plants exhibited normal growth but had rough secondary wall surfaces with delaminating S2 and S3 layers. Mesoscale examination of induced SCWs in epidermal cells revealed that macrofibril thickness and orientation were comparable to xylem vessels, while wall thickness resembled that of interfascicular fibres. Additionally, induced epidermal cells formed SCWs with altered cellulose and lignin contents. Conclusions: These findings suggest NST3 and/or NST1 induce SCWs with shared characteristics of both xylem and fibre-like cells forming loosely arranged cell wall layers and cellulose organized at multiple angles relative to the cell growth axis and with varied cellulose and lignin abundance. This inducible system opens avenues to explore ectopic SCWs for bioenergy and bioproducts, offering valuable insights into SCW patterning across diverse cell types and developmental stages.