Fig. 3.

Origin of fast root gravitropism-specific PIN2 functions in seed plants. a–d In contrast to the wild type (Col), the pin2 mutant showed severe defects in root gravitropism (a). Genetic complementation experiments showing that of the A. thaliana non-canonical (PIN5 and PIN6) and canonical (PIN1, PIN2, PIN3, PIN4, and PIN7) PINs, only PIN2 rescues the defective pin2 gravitropism (b–d). Scale bars, 1 cm. e–m Interspecies complementation experiments with orthologous PIN2 genes from green alga (KfPIN) (e), marchantiophyte (MpPINZ) (f), moss (PpPINA) (g), lycophyte (SmPINR) (h), fern (CrPINJ) (i), gymnosperm (PtPINI, PtPINE, PtPINH, and PtPING) (j), Arabidopsis (AtPIN2) (k), and G. arboreum (GaPIN2) (l). Only the gymnosperm genes encoding PtPINH and PtPING (Supplementary Fig. 8), and the flowering plant genes encoding AtPIN2 and GaPIN2 from the PIN2 clade were able to rescue the pin2 defects in root gravitropism (k–l). Scale bars, 1 cm. m Quantification of VGI for the plants in e–l (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 and ns denotes P > 0.05, compared with the Col-0, respectively. n–x The subcellular localization of AtPIN2 (o), KfPIN (p), MpPINZ (q), PpPINA (r), SmPINR (s), CrPINJ (t), PtPINE (u), PtPINI (v), PtPINH (w), and PtPING (x) in Arabidopsis root epidermal cells. Only the gymnosperm genes encoding PtPINH and PtPING (w, x), and the flowering plant genes encoding AtPIN2 from the PIN2 clade (o) were able to localize to the shootward cell side. The coding sequences were fused with GFP in the central HL and expression was driven under the control of the Arabidopsis PIN2 promoter. Polarity index of the cellular localization of the PIN-GFP fusion proteins (n = 150–200 cells from ten roots) (n). Scale bars, 10 µm. Source data are provided as a Source Data file