Conservation and Diversity of MicroRNA‐associated Copper‐regulatory Networks in Populus trichocarpa F

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.