The transcriptional expression patterns of all of the BpeAQPs from various plant organs and, subsequently, in leaves undergoing cold stress were recorded from the plant samples that were previously studied in two experimental assays on B. pendula [33]. The transcriptomic data are available in the NCBI SRA (Sequence Read Archive) database with accession numbers PRJNA535361 and PRJNA532995, respectively.

Samples were collected in triplicate from the roots, young leaves, female inflorescences, and xylem (upper stem, about 10th nodes) of healthy two-year-old birch planted in the experimental field of the Northeast Forestry University (Harbin, China). Two biological replicates per sample were sequenced, and each biological replicate consisted of a pool of three plant RNAs. The Northeast Forestry University experimental field is situated at 45.72° north latitude and 126.63° east longitude. The average daylight is 14.25 h and the nighttime is 9.75 h, the mean temperature is 17 °C to 27 °C, and the average relative humidity about was 76% at the date of sampling.

Concerning the cold stress assay, samples were taken from young leaves from two-month-old B. pendula plants grown in the greenhouse at a constant temperature of 25 °C with a photoperiod of 16 h of light and 8 h of dark. A total of six 28W sunlamps were used for illumination, but the intensity of illumination was not measured specifically. For the experimentation, the plants were exposed to cold stress (6 °C) for 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, and 3 h. Plants left at 25 °C were used as the control (0 h). Two biological replicates for time points of 1 h, 2 h, 2.5 h, and 3 h, control plants, and three biological replicates for time points of 0.5 h and 1.5 h were generated.

Total RNA was extracted using the CTAB (Cetyltrimethylammonium Bromide) method [154]. The constructed cDNA libraries were subjected to paired-end sequencing using the Illumina HiSeq platform at Biomarker Technologies Corporation (Beijing, China). The clean reads of each sample were obtained by filtering out any reads of low quality, and then aligned to the B. pendula reference genome using Bowtie2 [155]. The RNA-sequencing data were analyzed using the RNA-seq by Expectation-Maximization (RSEM) pipeline [156]. RSEM could compute transcript abundance, estimating the number of RNA-seq fragments corresponding to each gene, and normalized expression values as TMM (trimmed mean of M-values). Every methodological step is detailed in [33].

Statistical analyses were performed with R (version 3.6.3). The following key packages were used: bestNormalize, version 1.6.1 [157]; broom, version 0.7.4 [158]; FactoMiner, version 2.4 [159]; factoextra, version 1.0.7 [160]; ggplot2, version 3.3.3 [161]; janitor, version 2.1.0 [162]; multcomp, version 1.4-16 [163]; pander, version 0.6.3 [164]; rstatix, version 0.6.0 [165]; and tidyverse, version 1.3.0 [166].

AQPs with zero expression values were considered as non-available data, when distribution analysis showed non-normal distribution of such zero values. Normality and homoscedasticity were checked before analysis of variance (tested at 5% risk level) (Figures S10, S11 and S12). A post-hoc Tukey Honestly Significant Difference (HSD) test was performed at 5% risk level and a Student’s T test was performed at 5% risk level with Benjamini and Hochberg FDR adjustments for all AQPs p-values [167]. Principal component analysis was carried out on the first two dimensions and calculated using the first five dimensional variables as parameterized by default in FactoMineR package. The models of variance considered cold stress (CS), time duration of the cold stress (6 °C), and the organ type for the samples analyzed in the different libraries of the organs sampled at 25 °C.

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