Every putative BpeAQP sequence was carefully scrutinized, including expected AQP motifs (NPA, ar/R selectivity filter, and the FPs) and the prediction of the transmembrane topology, with Interproscan from EMBL (http://www.ebi.ac.uk/Tools/pfa/iprscan/; accessed on 2 July 2021) and the NCBI’s conserved domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; accessed on 2 July 2021). Motifs were identified by multiple sequence alignment and performed using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/; accessed on 2 July 2021) [145] with the default parameters. Specificity-determining positions (SDP1–9; [21]), which were essential to the transport of non-aqua substrates, were identified from multiple sequence alignments as described by [146]. Molecular weight, theoretical isoelectric point, and the grand average of hydropathicity (GRAVY) of the BpeAQPs were analyzed by the ExPaSy compute pI/Mw tool (https://web.expasy.org/protparam/; accessed on 2 July 2021). The transmembrane regions were predicted using the TMHMM-2.0 software tool (www.cbs.dtu.dk/services/TMHMM/; accessed on 2 July 2021) [147], SOSUI tools (http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html; accessed on 2 July 2021) [148], and were, when necessary, manually corrected with heterologous AtAQP, SlAQP and OleaAQP sequences. The subcellular localizations of the BpeAQPs were predicted using the online tools WoLF PSORT (http://wolf-psort.hgc.jp; accessed on 2 July 2021) and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/; accessed on 2 July 2021) [149]. The phylogenetic studies were conducted with the BpeAQP-deduced peptide sequences, and then with a total of 221 amino acid sequences, including BpeAQPs, AtAQPs, OleaAQPs, PoptrAQPs, and SlAQPs. Alignments were performed using the ClustalW progressive alignment method, and the phylogenetic trees were inferred using the maximum parsimony method. Maximum parsimony analyses were conducted using the subtree-pruning-regrafting (SPR) algorithm, and were bootstrapped with 5000 replicates. All analyses were performed using the MEGA X program. The graphical representation and the edition of the phylogenetic trees were performed with iTOL software (https://itol.embl.de/; accessed on 2 July 2021) [150]. The intron and exon structures of BpeAQP genes were analyzed by online software GSDS 2.0 (http://gsds.gao-lab.org/; accessed on 2 July 2021) [151]. Conserved motifs of the BpeAQP proteins were identified by MEME Suite 4.11.1 (Multiple Expectation Maximization for Motif Elicitation; http://meme-suite.org/tools/meme; accessed on 2 July 2021) [144]. The motif discovery mode was selected as a classic mode by using the default settings, i.e., the zero or one occurrence per sequence (ZOOPS) site distribution per sequence, of a minimum width of 6, a maximum width of 50 amino acid motifs, and a maximum number of motifs to find which was set to 10.

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