Transcriptional Profiling of PtDef in Populus

To investigate PtDef transcription in different tissues, total RNA was isolated from mature leaves, young leaves, petioles, roots, and the upper and lower regions of stems of P. trichocarpa. P. trichocarpa seedlings treated with 200 mM NaCl, 200 μm abscisic acid (ABA), 200 μm SA, 200 μm JA, and 2 mM H₂O₂ were sampled at 0, 2, 4, 6, 8, 12, 24, and 48 h, and those subjected to 4 °C cold stress and 10% PEG 6000 were collected after 1, 2, 3, 4, 5, 6, and 7 days. In addition, a group of P. trichocarpa seedlings were wounded with S. populiperda and sampled at 0, 1, 3, 6, 9, 12, 24, 48, and 72 h. Following these treatments, leaves were immediately subjected to RNA extraction using a plant-specific RNA kit (Invitrogen). The housekeeping gene actin (XM_006370951.1) was used as an internal control for each reaction^{41 (link)–44 (link)}. Triplicate measurements were used to determine the mean values for each parameter.