Evolution of fast root gravitropism in seed plants

Abstract

An important adaptation during colonization of land by plants is gravitropic growth of roots, which enabled roots to reach water and nutrients, and firmly anchor plants in the ground. Here we provide insights into the evolution of an efficient root gravitropic mechanism in the seed plants. Architectural innovation, with gravity perception constrained in the root tips along with a shootward transport route for the phytohormone auxin, appeared only upon the emergence of seed plants. Interspecies complementation and protein domain swapping revealed functional innovations within the PIN family of auxin transporters leading to the evolution of gravitropism-specific PINs. The unique apical/shootward subcellular localization of PIN proteins is the major evolutionary innovation that connected the anatomically separated sites of gravity perception and growth response via the mobile auxin signal. We conclude that the crucial anatomical and functional components emerged hand-in-hand to facilitate the evolution of fast gravitropic response, which is one of the major adaptations of seed plants to dry land.

Fig. 1

Slow and fast mechanisms of root gravitropism during plant evolution. Slow gravitropic bending of the rhizoids of the moss P. patens, roots of the lycophyte S. moellendorffii, and the fern C. richardii after a 90° reorientation of the seedlings. Much faster response of the gymnosperm P. taeda, the dicots G. arboreum and A. thaliana, and the monocot O. sativa after gravistimulation. Scale bars, 1 mm

Fig. 2

Exclusive root apex-specific amyloplast localization in seed plants. a Living representative species from different plant lineages included in the analysis (from left to right): K. flaccidum (green alga), P. patens (moss), S. moellendorffii (lycophyte), C. richardii (fern), P. taeda (gymnosperm), G. arboreum, and A. thaliana (dicots), and O. sativa (monocot). b Lugol’s staining of the rhizoid (P. patens). c–h mPS-PI staining of the root tips from S. moellendorffii (c), C. richardii (d), P. taeda (e), G. arboreum (f), A. thaliana (g), and O. sativa (h). The blue arrows indicate root hair initiation. The yellow arrows indicate the apical cell (QC-like cell) in the fern C. richardii and the QC in seed plants. The dashed red rectangles indicate the zone with amyloplasts. Scale bars, 20 µm

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

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

Fig. 5

Origin of fast root gravitropism facilitated seed plant adaption to the dry land. A schematic diagram representing an evolution of fast root gravitropism that can be detected only in seed plants. It depicts (i) the root anatomical innovation with evolution of the apex-specific gravity perception that is spatially separated from the elongation zone where differential growth occurs and (ii) the functional innovations of the auxin transporter PIN2 that occurred in two disparate plant evolution stages and in different parts of the PIN2 protein sequence ultimately leading to the shootward subcellular localization of PIN2. This novel PIN2 property endowed roots with the ability to transport auxin shootwards enabling the auxin-based signaling from the place of gravity perception to the zone of growth regulation. The fast root gravitropism has evolved as a result of these concomitant anatomical and functional innovations exclusively in seed plants as one of the important adaptions to dry land