Microarray data analysis, RNA isolation, semi-qRT-PCR and qRT-PCR analyses

Tissue-specific expression data on the PtAPs were downloaded from the Populus eFP browser [72]. Probe sets corresponding to PtAP genes were identified using an online Probe Match tool available at NetAffx Analysis Center (http://www.affymetrix.com/). The heat map was generated by Heat map illustrator (HemI) with the default settings [73].

Total RNA was isolated with plant RNA extraction reagents (Bio-Flux, China). For each sample, 1 μg of total RNA was reverse-transcribed into total cDNAs using the PrimeScript RT reagent Kit (TaKaRa, China). The expression of the PtActin2 was used as an internal control of the same total cDNAs. Expression of the PtAPs in different tissues and internodes were examined by semi-qRT-PCR using ExTaq (TaKaRa, China). The reaction mixture (20 μl) consisted of 1 μl of cDNA template, 0.5 μl of each gene-specific primer (10 μM), 2 μl 10× ExTaq buffer (Mg²⁺ free), 2 μl dNTP mixture (2.5 mM each), 1.6 μl MgCl₂ and 0.1 μl Ex-Taq. The PCR parameters were 94 °C for 3 min; followed by 25–32 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s; and a final step at 72 °C for 7 min. The resulting PCR products were run on a 2% agarose gel and visualized under a UV transilluminator to verify product sizes. All primers used in the present study for semi-qRT-PCR are listed in Additional file 12. Each reaction was conducted in triplicate to ensure the reproducibility of the results.

To validate the PtAP gene expression profiles, qRT-PCR analysis of these genes were performed with the same cDNA templates and primers that were used in semi-qRT-PCR experiment above. The qRT-PCR experiments were performed with SYBR Green (TaKaRa, China) in the ABI Prism 7500 system (Applied Biosystems, USA) according to the manufacturer’s instructions. The reaction mixture (20 μl) consisted of 10 μl 2 × TB Green Premix Ex Taq II (Tli RNaseH Plus), 0.8 μl of each gene-specific primer, 0.4 μl ROX Reference Dye II, 1 μl cDNA template and 7 μl distilled deionized H₂O. The PCR parameters as follows: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 15 s, 72 °C for 30 s. PtActin2 was used as an internal control and the comparative Ct (2^-△Ct) method was used to calculate gene expression levels. Three technical replicates were done for each sample.