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

Abstract

Plant cell wall expansion is essential for plant growth, with expansins promoting wall loosening and creep during diffuse growth. Expansins role in tip growth, such as root hair elongation, is less clear. Here we used CRISPR/Cas9 to knock out root-hair specific -expansins EXPA7 and EXPA18 in Arabidopsis thaliana, abolishing 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 two EXPA clades [clade-VIII (EXPA13) and clade-IX (EXPA20)] lacking a conserved Asp considered essential for expansin-induced wall enlargement. Phylogenetic analysis indicated loss of this Asp predated angiosperms. Mutation of this Asp in EXPA7 confirmed its necessity for wall loosening. Members of the 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 expansin families and among EXPA clades and demonstrate a genetic platform to analyze functionalities (trafficking, wall binding, wall extension) of EXPA and other wall-modifying proteins during root-hair tip growth. Significance StatementPlant cells can enlarge >100-fold during growth, a process controlled by proteins called expansins. Plants have many expansin proteins that are difficult to study in isolation and whose specific activities and properties have remained elusive. Here, we engineered a root-hairless Arabidopsis mutant as a genetic platform to assess expansin activity in vivo. By introducing various expansins with a fluorescent tag into this mutant, we uncovered an unexpected functional diversity across the expansin superfamily. While some expansins successfully restored root hair growth, others failed, potentially due to differences in protein trafficking, cell wall binding, or evolutionary loss of critical residues required for wall loosening. These findings point to a divergence of expansin functions during >140 million years of plant evolution.