Fig. 4.

Functional innovations of PIN2 during plant evolution. a–e Hybrid PIN (X1) with the central hydrophilic loop (HL) of green alga KfPIN substituted by the Arabidopsis AtPIN2 HL fused with GFP (HL-GFP) failed to rescue the pin2 mutant (a, middle panel). Hybrid PIN proteins (X2-X4) with the central HL of marchantiophyte MpPINZ, lycophyte SmPINR, and fern CrPINJ replaced by the Arabidopsis AtPIN2 HL-GFP were able to rescue the defective root gravitropism phenotype of the Arabidopsis pin2 mutant (b, c, middle panels). The PtPING and AtPIN2 proteins with GFP fused with the central HL can complement the pin2 mutant phenotype (d, middle panel). Scale bars, 1 cm. The hybrid PIN protein (X5) with the central HL of CrPINJ replaced with the gymnosperm PtPING HL fused with GFP successfully complemented the Arabidopsis pin2 mutant phenotype (e, middle panel). The cellular localization of hybrid PIN proteins (X1–X5) and the PIN2-GFP and PtPING-GFP fusion proteins in Arabidopsis root epidermal cells with expression driven by the AtPIN2 promoter (a–e, lower panels). All and only PIN2 chimeric proteins that were able to localize to the shootward cell side also rescued the pin2 gravitropism. Scale bars, 10 µm. The red and blue dashed lines indicate the separate two functional innovations at different plant evolution stages. f Quantification of vertical growth index (VGI) for the transgenic lines in a–e (n ≥ 10 roots). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Student’s t-test, ***P < 0.001, compared with the pin2;pPIN2::X1-GFP, respectively. g Polarity index of the cellular localization of the PINs in epidermal cells in (a–e, lower panels) (n = 150 to 200 cells from ten roots). h Schematic showing the contributions of the TMDs and the HL domain to PIN2 functions. Source data are provided as a Source Data file