AtMYB2 downstream targets AtMYB48 and AtbHLH68 redundantly regulate cambial cell proliferation and xylem differentiation in Arabidopsis thaliana

Xylem formation involves a series of processes, including cell division of the vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death (PCD). In this study, we identified AtMYB2, AtMYB48, and AtbHLH68 as key regulators of cambial cell proliferation and xylem differentiation in Arabidopsis. Overexpression of AtMYB2 decreases cambial cell numbers and increases xylem cell numbers, whereas loss of AtMYB2 results in the opposite phenotype, indicating that AtMYB2 negatively regulates cambial cell proliferation while positively regulating xylem cell differentiation during xylem formation. Both AtMYB48 and AtbHLH68 are direct targets of AtMYB2 and function as positive regulators of cambial cell proliferation and negative regulators of xylem differentiation. Single mutants of AtMYB48 or AtbHLH68 exhibit only mild phenotypic alterations, whereas the atmyb48 atbhlh68 double mutant displays enhanced defects in xylem development. Notably, overexpression of either gene alone is sufficient to rescue the double mutant phenotype, demonstrating functional redundancy between AtMYB48 and AtbHLH68. Although these two proteins can physically interact, this interaction is dispensable for their regulatory functions in xylem development. Despite their redundancy, AtMYB48 and AtbHLH68 exhibit distinct regulatory preferences in xylem differentiation: AtMYB48 primarily regulates genes associated with SCW deposition and PCD, whereas AtbHLH68 predominantly regulates genes involved in cambial cell proliferation. These findings reveal redundant and partially distinct functions of AtMYB48 and AtbHLH68 downstream of AtMYB2 and provide new insights into the transcriptional network controlling xylem formation.