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.