3.1 Phylogenetic analysis of Al tolerance-associated genes/ gene families
In order to investigate the origin of Al tolerance in plants, we performed the genetic similarity and phylogenetic analysis of Al tolerance-associated predicted protein families (Figure 1; Table S2). multidrug and toxic compound extrusion (MATEs) and aluminum-activated malate transporter (ALMTs) mediate Al-induced organic acid secretions from roots (Sasaki et al., 2004; Wang et al., 2007), showed relatively lower protein sequence similarity among the tested species than other membrane transporters related to Al tolerance (Figures S1; Table S3). Moreover, Al-tolerance responsive transcription factors such as sensitive to proton rhizotoxicity (STOP1/2), and calmodulin-binding transcription activators (CAMTAs) (Kobayashi et al., 2014; Tokizawa et al., 2015) also showed relatively lower genetic similarity between Arabidopsis and other species and a possible origin from streptophyte algae (i.e. Klebsormidium flaccidum ) (Figure 1; Table S3). Moreover, cell wall modification enzymes, pectin methylesterase (PMEs) and xyloglucan endotransglucosylase/hydrolase (XTHs) (Yang et al., 2008; Zhu et al., 2012) were originated from streptophyte algae, and their numbers increased rapidly in land plants (Table S4). Some phytohormones-related protein families relevant to Al tolerance, such as ARFs (Auxin Response Factor), tryptophan-pyruvate aminotransferase (TAAs), flavin-containing monooxygenase (YUCs), and coronatine insensitive (COIs) (Yang et al., 2014; Yang et al., 2017), were largely absent in algae, but were highly conserved in land plants from hornworts to angiosperms (Figure 1; Tables S2-4).
Glycolysis was reported to provide the energy for plant to tolerate Al stress (Dai et al., 2013). Most of the predicted protein families in glycolysis, except hexokinase (HXKs), were highly conserved in green plants and algae (Figure 1). It is worth noting that predicted protein families of glycolysis (52%-81%) showed higher average genetic similarity to the Arabidopsis than those in other protein families (26%-75%) (Figures 1, S2; Table S2). SPX-MFSs and PHT1s are tonoplast and plasma membrane localized Pi transporters, respectively, which play vital roles in cellular P homeostasis (Rae et al., 2003; Wang et al., 2012; Wang et al., 2015). Importantly, most of predicted protein families/sub-families of the Pi transporters, including PHT1s, PHT2s, PHT3s, PHT4s and SPX-MFSs, were highly conserved in land plants and streptophyte algae (Figure 1).
To further investigate the origin and evolution of SPX-MFSs and PHT1s, we performed phylogenetic analysis of their predicted protein sequences in land plants and algae using OneKP database and published genome assemblies (Figures 2a, 2b, S3). The genetic similarity of SPX-MFSs and PHT1s to the reference species Arabidopsis was low in Rhodophyta (34% and 29%), Chromista (32% and 31%) and chlorophyte algae (30% and 32%), but it increased significantly in streptophyte algae to 43% and 56%, respectively (Figure 2c). Moreover, genetic similarity of PHT1s in streptophyte algae were closer to the land plants - hornworts, liverworts, mosses, and other plant lineages (Figure 2C; Table S3). Interestingly, the average gene number of PHT1s (but not SPX-MFSs) increased gradually in chromista (1), chlorophyte algae (2), streptophyte algae (3), bryophytes (6), lycophytes (7), ferns (12), conifers (15), and angiosperms (17) (Figure 2d). In addition, the analysis of conserved domains of SPX-MFSs and PHT1s demonstrated high amino acid conservation from streptophyte algae (e.g.Klebsormidium flaccidum ) to angiosperms (Figures S4, S5). Taken together, it is thus suggested that streptophyte algae are likely to be a key transitional clade in the evolution of gene families for the SPX-MFS and PHT1 Pi transporters.