Functional divergence of plant expansins as revealed by genetic complementation of root-hair tip growth in Arabidopsis thaliana.

Plant cell wall expansion is essential for plant growth, with expansins promoting wall creep during diffuse growth. Expansin's role in tip growth, such as root hair elongation, is less clear. Here, we used CRISPR/Cas9 to knock out root hair-specific α-expansins EXPA7 and EXPA18 in Arabidopsis thaliana, which abolished root hair elongation. Complementation with expansin genes from various clades (driven by the EXPA7 promoter) revealed functional differences: Some fully restored root hair growth, others only partially restored growth, while others failed, notably EXPA13 (clade-VIII) and EXPA20 (clade-IX)-both lacking a conserved Asp considered essential, but not sufficient, for expansin-induced wall enlargement. Phylogenetic analysis suggests loss of this Asp may have predated angiosperms. Mutation of this Asp in EXPA7 confirmed its necessity for wall loosening, but its restoration in EXPA13 did not restore activity. Other expansin families (EXPB, EXLA, and EXLB) failed to restore root hair growth. Chimeric fusions of EXPAs with mCherry revealed differences in trafficking patterns and wall binding among EXPA clades. These results demonstrate functional differences among EXPA clades and among expansin families, establishing a genetic platform to analyze functionalities (trafficking, binding, activity) of EXPA and other wall-modifying proteins during root-hair tip growth.