Identification and expression pattern analysis ofPtrWRKY75
A 561-bp WRKY75 gene encoding 186 amino acids was cloned fromP. trichocarpa , whose genome has been sequenced (Tuskan et al. 2006). Homologous amino acid sequences of WRKY75 proteins inP. trichocarpa , Arabidopsis thaliana , Oryza sativa ,Zea mays , and Vitis vinifera  were obtained from the National Center for Biotechnology Information database (NCBI; https://www.ncbi.nlm.nih.gov/). A phylogenetic tree was constructed for WRKY75 orthologs from a multiple alignment of the amino acid sequences (Figure 1A). To further analyze the phylogenetic relationships of WRKY TFs in poplar, we constructed phylogenetic tree of 100 members in WRKY TF family in poplar (Figure S1). The phylogenetic reconstruction revealed that PtrWRKY75 is evolutionarily close to AtWRKY75 from A. thaliana (61% identity). The multiple sequence alignment revealed that PtrWRKY75 contains a conserved domain WRKYGQK at the N-terminus followed by a basic zinc finger motif (C-X5-C-X23-H-X1-H). These analyses confirmed that PtrWRKY75 belongs to group II of the WRKY TF gene family (Figure 1B) (Eulgem et al. 2000).
To determine the subcellular localization of PtrWRKY75, pSuper::PtrWRKY75-eGFP and pSuper::eGFP (as a negative control) fusion proteins were transiently transfected into tobacco (Nicotiana benthamiana ) leaves. The pSuper::PtrWRKY75-eGFP fusion protein was localized in the nucleus, as observed under a confocal laser scanning microscope, whereas the pSuper::eGFP fusion protein was distributed throughout the cell without specific localization (Figure 1C). These results suggested that PtrWRKY75 is a transcriptional regulator localized in the nucleus.
Quantitative real-time PCR (qRT-PCR) was applied to detect the tissue-specific abundance of PtrWRKY7 5 transcripts in different tissues ofP. trichocarpa (root, stem, young leaf, mature leaf, and senescent leaf).PtrWRKY7 5 transcript levels were higher in mature leaves and senescent leaves than in young leaves and the stem (Figure 1D). To investigate the response of PtrWRKY7 5 to water stress, P. trichocarpa plants were subjected to drought conditions and the transcript levels of PtrWRKY7 5 were quantified by qRT-PCR. The transcript level of PtrWRKY7 5 gradually increased and peaked at 6 h of the drought treatment, at a level 12.87-times higher than that in the control, and thereafter decreased (Figure 1E). The PtrWRKY7 5 transcript level increased 13.12-fold after 1 h treatment with 5 mM SA (Figure 1F). These results indicated thatPtrWRKY7 5 is expressed predominantly in the leaf, and is up-regulated by drought stress and SA treatment.
Identification ofPtrWRKY75-overexpressing transgenic poplar
To study the role of PtrWRKY75 in plants under drought stress, the pSuper::PtrWRKY75 vector was transformed into wild-type (WT) triploid white poplar (Populus tomentosa ‘YiXianCiZhu B385’) using the leaf disc method, thenPtrWRKY75 -overexpressing transgenic poplar lines (WRKY75-OE ) were generated. Thirteen transgenic lines were verified by PCR using gene-specific primers and qRT-PCR (Figure S2A, B). The levels of PtrWRKY75 transcripts differed among the transgenic lines. The highest transcript levels were in OE-8 and OE-10 (36.27 and 46.64 times higher, respectively, than those in the other transgenic lines). Therefore, we selected these two lines, hereafter referred to as the OE lines, as experimental materials for subsequent analyses.