Phylogenetic and stress-responsive expression analysis of 20 WRKY genes in Populus simonii × Populus nigra

WRKY transcription factors play important roles in regulating biotic and abiotic stress responses in plants. Although a plethora of studies have revealed the functions and mechanisms of some WRKYs in various plants, the studies of WRKYs in woody plants especially tree species under different abiotic and biotic stress conditions are still not well characterized. In this study, we selected 20 Populus simonii×Populus nigra WRKY genes based on our previous transcriptome study, and characterized these genes by phylogenetic analysis to investigate their evolutionary relations, then studied their expression patterns under NaCl, NaHCO3, PEG6000, CdCl2 and Alternaria alternata (Fr.) Keissl treatments that mimic the salt, alkalinity, drought, heavy metal and fungal infection conditions. The phylogenetic analysis showed that these 20 genes can be divided into five clades (Groups I, IIa, IIb, IIc and III) and all of their WRKY domains are conserved except for an N-terminal single amino acid mutation in PsnWRKY8. Before conducting quantitative real time PCR calculation, we evaluated five candidate reference genes under different stress treatments, and chose At4g33380-like as the reference gene for salt stress, Actin for alkalinity stress, UBQ for drought stress, TUA for heavy metal stress, and 18S rRNA for pathogen infection stress. The final qRT-PCR analysis indicated that 20/20, 20/20, and 15/20 PsnWRKYs were downregulated under salt, alkali and drought stresses, and 14/20 and 19/20 PsnWRKYs were upregulated under heavy metal and pathogen stresses. Members from the same clade tended to present similar expression patterns. In addition, we observed noticeable changes in the expression of PsnWRKY11 (increased by 41 times) and PsnWRKY20 (increased by 141 times) under pathogen infection condition, implying that these two genes are potentially important for the disease resistance of P. simonii × P. nigra.