Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants.During PCD,the degradation of genomic DNA is catalyzed by endonucleases.However,to date,no endonuclease has been shown to participate in secondary xylem development.Two novel Ca2+-dependent DNase genes,EuCaN1 and EuCaN2,were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv.,their functions were studied by DNase activity assay,in situ hybridization,protein immunolocalization and virus-induced gene silencing experiments.Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp,encoding two proteins of 328amino acids with SNase-like functional domains.The genomic DNA sequence for EuCaN1 had no introns,while EuCaN2 had 8 introns.EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca2+-dependence at neutral pH.Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus.Their activity dynamics was closely correlated with secondary xylem development.Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes.The results provide evidence that the Ca2+-dependent DNases are involved in secondary xylem development.