Plant materials and stress treatments
Populus
trichocarpa (clone ‘Nisqually-1’) was used for isolation ofPtrWRKY75 .
Plantlets of P. trichocarpa were cultured in vitro on
solid Lloyd and McCown’s Woody Plant Basal Salts (WPM) medium or
Murashige and Skoog (MS) medium (Song et al ., 2006). After
plantlet regeneration, the plantlets were transplanted and grown
individually in pots containing a mixture of soil and vermiculite (2:1)
at 22 °C under a 16 h/8 h (light/dark) photoperiod (150 μmol
m−2 s−1) and 70% relative humidity.
Salicylic acid (5 mM aqueous solution) was applied as a foliar spray
onto the leaves. The treated plants were immediately covered with
transparent plastic film (Jiang et al ., 2014).
For the dehydration treatment, 4-week-old seedlings were removed from
the soil and the roots were exposed to air at 50% relative humidity and
25 °C under dim light for 8 h (Ma et al ., 2010).
For each experiment, leaves were collected from the third and fifth
internodes at different time points and frozen immediately in liquid
nitrogen. We also simultaneously collected the following tissues from
2-month-old P. trichocarpa plants: young leaf, mature leaf,
senescent leaf, stem, and root. The samples were immediately frozen in
liquid nitrogen.
Plantlets of triploid white poplar (P. tomentosa ‘YiXianCiZhu
B385’) (Zhu et al . 1998) were cultured in vitro on solid
half-strength (1/2) MS medium as previously described (Wang et
al. , 2016). Leaves were incubated on substrate (pH = 5.8) containing
0.02 mg/L thidiazuron (TDZ), 0.1 mg/L α-naphthalene acetic acid (NAA),
and 0.6% (w/v) agar for shoot induction. Adventitious buds elongated on
MS medium containing 0.5 mg/L 6-benzylaminopurine (6-BA) and 0.6% (w/v)
agar. The regenerated shoots were individually separated from the callus
and transferred to rooting medium [1/2 MS medium supplemented with
0.05 mg/L NAA and 0.6% (w/v) agar] (Li et al. , 2012). The
4-week-old seedlings were acclimatized in pots and then transferred to a
greenhouse at 22 °C under a 16 h/8 h (light/dark) photoperiod and
40%–45% relative humidity.
cDNA cloning of PtrWRKY75 from Populus
trichocarpa and quantitative real-time PCR analysis
Total RNA was extracted from tissue samples using the RN38 EASYspin Plus
Plant RNA Kit (Aidlab Biotech, Beijing, China). A total of 2 µg RNA was
used for first-strand reverse transcription using M-MLV Reverse
Transcriptase and an oligo (dT) primer (Promega, Madison, WI,
USA) following the manufacturer’s instructions. The resultant cDNA was
used for PCR amplification with gene-specific primers.
Quantitative real-time PCR was performed using the ABI StepOnePlus™
Real-Time PCR System (Applied Biosystems, Inc., Carlsbad, CA, USA) in
accordance with the manufacturer’s instructions. The reaction mixture
for the qRT-PCR analysis comprised 1 µl (~100 ng)
template (the cDNA template diluted ~10-fold with
nuclease-free water), 1 µl (10 µM) forward primer, 1 µl (10 µM) reverse
primer, 7 µl RNase-free ddH2O, 12.5 µl SuperReal PreMix
Plus, and 2.5 µl ROX Reference Dye in a total volume of 25 µl. Each
experiment was based on three biological replicates of each sample and
four technical replicates of each biological replicate. The
2−∆∆C T method (Schmittgen and Livak, 2008) was
used to calculate the relative expression level of PtrWRKY75 ,
with PtrUBQ employed as the internal control. All primers used
are listed in Table S1.