Five amino acid mismatches in the zinc-finger domains of CELLULOSE SYNTHASE 5 and CELLULOSE SYNTHASE 6 modulate their incorporation into cellulose synthase complexes in Arabidopsis

Main conclusion Different capacities for the homodimerization of CESA5 and CESA6 are modulated by five amino acid mismatches in their zinc-finger domains and motivate discriminative incorporation of these CESAs into CSCs Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R+S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5, indicating that these mismatches modulate the incorporation of both CESAs into CSCs by controlling their ability to homodimerize.