The AtMYB2 downstream targets AtMYB48 and AtbHLH68 redundantly regulate cambial cell proliferation and xylem...

Xylem formation involves a series of processes, including vascular cambium cell division, 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 reduces cambial cell numbers and increases xylem cell numbers, whereas loss of AtMYB2 produces 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 that positively regulate cambial cell proliferation and negatively regulate xylem differentiation. Single mutants of AtMYB48 or AtbHLH68 exhibit only mild phenotypic alterations, whereas the atmyb48 atbhlh68 double mutant shows enhanced defects in xylem development. Notably, overexpression of either gene alone is sufficient to rescue the double-mutant phenotype, demonstrating the 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 formation: AtMYB48 primarily regulates genes associated with SCW deposition and PCD, whereas AtbHLH68 predominantly regulates genes involved in cambial cell proliferation. These findings reveal the redundant and partially distinct functions of AtMYB48 and AtbHLH68 downstream of AtMYB2 and provide new insights into the transcriptional network that controls xylem formation.