Comparative Functional Characterization of nst1, nst2, and nst3 in Arabidopsis thaliana Uncovers Previously Unknown Functions in Diverse Developmental Pathways Beyond Secondary Wall Formation

The regulation of secondary cell wall formation in Arabidopsis thaliana has been extensively studied with NST1, NST2, and NST3 playing key roles in secondary cell wall development in stem, anther, and silique. However, their broader impact on plant growth and development is less understood. This study investigates the phenotypes of T-DNA insertional mutants of NST1 (nst1-1), NST2, NST3 (nst3-1), and the double mutant nst1-1nst3-1 revealing their previously unknown functions in traits crucial for plant fitness. Phylogenetic analysis of the NAC gene family, based on chromosome locations, suggests that local and segmental duplication has expanded the family. NST1, NST2, and NST3 are phylogenetically close, within the same sub-clade, yet located on separate chromosomes, indicating a complex evolutionary history with functional redundancy and diversification. Loss-of-function mutants of NST1, NST2, and NST3 (i.e., nst1-1, nst2, nst3-1, and nst1-1nst3-1) displayed changes in root and hypocotyl length, rosette leaf size and area, stem diameter, vascular bundle structure, stamen characteristics, and silique. The nst3-1 single mutant exhibits the most pronounced defective phenotypes, emphasizing the pivotal role of NST3 in governing various developmental processes. Furthermore, the compounded effects observed in the nst1-1nst3-1 double mutant underscore the intricate interplay between these genes and their collective impact on Arabidopsis growth. Our findings indicate that the function of NST1, NST2, and NST3 genes extends beyond their well-established roles in secondary wall regulation to significantly influence multiple aspects of plant growth and development, enhancing our understanding of their regulatory network in Arabidopsis.