Plants develop important regulatory networks to adapt to the frequently-changing availability of copper (Cu).However,little is known about miRNA-associated Cu-regulatory networks in plant species other than Arabidopsis.Here,we report that Cu-responsive miRNAs in Populus trichocarpa (Torr.& Gray)include not only conserved miR397,miR398 and miR408,but also Populus-specific miR1444,suggesting the conservation and diversity of Cu-responsive miRNAs in plants.Copper-associated suppression of mature miRNAs is in company with the up-regulation of their target genes encoding Cu-containing proteins in Populus.The targets include miR397-targeted PtLAC5,PtLAC6 and PtLAC110a,miR398-targeted PtCSD1,PtCSD2a and PtCSD2b,miR408-targeted PtPCL1,PtPCL2,PtPCL3 and PtLAC4,and miR1444-targeted PtPPO3 and PtPPO6.Consistently,P.trichocarpa miR408 promoter-directed GUS gene expression is down-regulated by Cu in transgenic tobacco plants.Cu-response elements (CuREs) are found in the promoters of Cu-responsive miRNA genes.We identified 34 SQUAMOSA-promoter binding protein-like (SPL) genes,of which 17 are full-length PtSPL proteins or partial sequences with at least 300 amino acids.Phylogenetic analysis indicates that PtSPL3 and PtSPL4 are CuRE-binding proteins controlling Cu-responsive gene expression.Cu appears to be not involved in the regulation of these transcription factors because neither PtSPL3 nor PtSPL4 is Cu-regulated and no CuRE exists in their promoters.