Abstract text: Kinesins are ATP-driven motors transporting cargo along microtubule tracks. Their functions are well characterized in animals but remain underexplored in plants. Higher plants lack centrosomes and cytoplasmic and axonemal dynein, likely contributing to expansion of plant kinesin families, including minus-end-directed class-14 kinesins that fulfill roles in mitosis and intracellular organization.

Plant cells possess cellulose-reinforced walls whose anisotropic architecture is established by plasma-membrane-localized cellulose synthase complexes guided by cortical microtubules. This cytoskeleton-dependent process is fundamental for xylem structural integrity and development. Although kinesins have not been directly linked to Secondary Cell Wall (SCW) biosynthesis, motors such as KINESIN-13A and FRA1 regulate patterned SCW deposition in xylem vessels, crucial for water transport under negative pressure.

Several class-14 kinesins are strongly upregulated during xylem differentiation, suggesting previously unrecognized roles in protoxylem wall reinforcement. Notably, these proteins contain N-terminal calponin homology domains with predicted actin-binding capacity, indicating potential coordination of actin and microtubule arrays during SCW assembly.

Here, we investigate the structure–function relationship of the highly expressed, xylem-specific class-14 kinesin AT2G47500 during protoxylem development. Using CRISPR-Cas9 knockouts, fluorescent tagging, cytoskeletal marker crosses, and truncation analyses in-vivo and in-vitro. We aim to define its localization, dynamics, and developmental function, providing insight into cytoskeleton-guided SCW formation.