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.