Root gravitropism, the downward reorientation of root growth, is mediated in flowering plants by a three-step module: gravity perception by sedimenting starch-filled amyloplasts (statoliths) in columella cells of the root apex, polar redistribution of the hormone auxin via AUX1/LAX influx carriers and PIN (PIN-FORMED) efflux carriers, and differential elongation driven by the resulting auxin gradient. Zhang et al. (2019) showed comparatively that the fast, efficient form of this module is a seed-plant trait, assembled from two concomitant innovations: confinement of sedimenting statoliths to the root apex, and PIN proteins with shootward (apical) subcellular polarity that carry auxin from the perception zone to the elongation zone, while lycophyte and fern roots show only slow, rudimentary bending. What remains open is the completion of this evolutionary account: whether the shootward-localized, gravitropism-mediating PIN function arose once in the seed-plant ancestor (gymnosperm PING/PINH as co-orthologs of angiosperm PIN2) or convergently in gymnosperms and flowering plants, given conflicting protein-level versus nucleotide-level and domain-modular phylogenetic signals; which sequence changes in the PIN transmembrane domains and hydrophilic loop causally produced the two inferred functional steps, and whether positive selection drove them; whether PING/PINH proteins perform this transport function natively in gymnosperm roots (all current evidence is heterologous, in Arabidopsis); and when and how the PINE/PIN3 clade (the gymnosperm clade PINE and the angiosperm clade PIN3 of Bennett et al. 2014, whose proposed correspondence is itself contested) implicated in rapid lateral PIN relocalization during gravistimulation originated, including whether expanded monocot PINs replaced PIN3 function.

Background

Land plants (embryophytes) span the bryophytes (mosses and liverworts, which lack true roots and anchor themselves with root-hair-like rhizoid filaments), the lycophytes (spikemosses and allies), the ferns, and the seed plants, which comprise the gymnosperms (conifers and relatives) and the angiosperms (flowering plants). Roots are not a single ancestral organ: comparative anatomy and fossils indicate that roots evolved stepwise, and at least twice independently, once in the lycophyte lineage and once in the fern-plus-seed-plant (euphyllophyte) lineage (Hetherington and Dolan 2018). Any complex physiological module of roots, such as gravity-guided growth, is therefore either a heritage of deep shared ancestry or a product of repeated, independent assembly, and distinguishing the two is a central evo-devo question.

Gravitropism is the directional growth response by which plants align their organs with the gravity vector; roots typically bend downward (positive gravitropism). In the model flowering plant Arabidopsis thaliana the response is understood as a three-step module. First, perception: in the columella cells (specialized gravity-sensing cells, or statocytes) of the root cap, dense starch-filled plastids called amyloplasts sediment under gravity and act as statoliths, literally 'gravity stones'. Second, transmission: sedimentation is converted into lateral redistribution of the plant hormone auxin (indole-3-acetic acid), whose directed cell-to-cell movement requires plasma-membrane efflux carriers of the PIN-FORMED (PIN) family, whose polar subcellular localization determines the direction of transport, together with AUX1/LAX influx carriers. Third, response: auxin accumulating on the lower side of the elongation zone inhibits elongation there, so the root bends. Within the root apex, the PIN2 efflux carrier localizes to the shootward (upper, toward-the-shoot) side of epidermal cells and carries auxin from the perception zone in the cap to the growth zone, while the related PIN3 and PIN7 carriers, sitting at the bottom of statocytes, relocate laterally within minutes of gravistimulation to set up the initial auxin asymmetry (Friml et al. 2002). Canonical PIN proteins consist of two transmembrane-domain (TMD) bundles flanking a central hydrophilic loop (HL) that carries phosphorylation motifs controlling PIN trafficking, polarity, and transport activity.

Until 2019 these mechanisms were known almost exclusively from flowering plants. Zhang et al. (2019) performed the first systematic cross-lineage comparison, quantifying gravitropic bending kinetics in a moss rhizoid system (Physcomitrium patens), a lycophyte (Selaginella moellendorffii), a fern (Ceratopteris richardii), a gymnosperm (Pinus taeda, loblolly pine), and three flowering plants, together with statolith anatomy, amyloplast-sedimentation assays, radioactive auxin-transport measurements, heterologous PIN-complementation in Arabidopsis pin2 mutants, and PIN domain-swap experiments. They found that non-seed plant roots (and moss rhizoids) bend only slowly and inefficiently after reorientation whereas seed-plant roots bend fast; that apex-confined, sedimenting amyloplast statoliths and efficient, NPA-sensitive (NPA is an inhibitor of PIN-dependent auxin transport) shootward auxin transport occur only in seed plants; that two pine PIN genes, PtPING and PtPINH, but not other gymnosperm or non-seed PINs, localize shootward in Arabidopsis root cells and rescue the pin2 gravitropism defect; and that domain swaps attribute the capacity for shootward PIN localization to a TMD-level change acquired with land plants and an HL-level change acquired in seed plants. They concluded that fast root gravitropism evolved in seed plants through two concomitant innovations: anatomical confinement of gravity sensing to the root apex, and PIN proteins with shootward polarity that connect the now-separated perception and growth zones via a mobile auxin signal.

After 2019, the downstream transduction step inside flowering-plant statocytes was resolved: sedimenting amyloplasts trigger LAZY/LZY (also called NGR, for 'negative gravitropic response of roots') proteins to translocate from the statoliths to the adjacent plasma membrane, where they recruit regulators — RLD proteins (a small family of membrane-trafficking regulators of PIN localization) and the D6 protein kinase (D6PK, a kinase whose phosphorylation of PIN carriers stimulates auxin transport) — that re-polarize and activate PIN-mediated auxin fluxes (Nishimura et al. 2023; Chen et al. 2023; Kulich et al. 2024). The evolutionary questions that Zhang et al. (2019) explicitly left open, however, remain unanswered: the homology-versus-convergence status of the gravitropism-mediating PIN function, the causal sequence changes and selective pressures behind the two-step PIN2 innovation, the native function of gymnosperm PING/PINH (tested only heterologously in Arabidopsis), and the origin of the PINE/PIN3 clade. Related but distinct from these open points, the mechanism of the slow gravitropism that non-seed plant roots nevertheless display also remains uncharacterized.

Problem Statement

Complete the evolutionary account of the fast root gravitropic mechanism in land plants by resolving three linked issues that Zhang et al. (2019) left open.

(1) Homology versus convergence: determine whether the shootward-localized, gravitropism-mediating PIN function was present in the most recent common ancestor of extant seed plants — making gymnosperm PING/PINH co-orthologs of angiosperm PIN2 — or evolved independently in the gymnosperm and angiosperm lineages, resolving the standing conflict between protein-level phylogenies that group PING/PINH with PIN2 and nucleotide-level plus hydrophilic-loop-modular analyses that favor convergence (Bennett et al. 2014).

(2) Causal sequence changes and selection: identify the specific historical sequence changes in the transmembrane domains and the central hydrophilic loop that produced (i) the competence for polar plasma-membrane localization acquired at the origin of land plants and (ii) the hydrophilic-loop innovation enabling shootward localization and fast gravitropism, inferred to have occurred with seed plants — a single ancestral change under the shared-descent outcome of (1), or the corresponding independent gymnosperm and angiosperm changes under the convergence outcome — and establish whether positive selection acted on these changes.

(3) Native function and clade history: establish whether PING/PINH proteins natively mediate the efficient, inhibitor-sensitive shootward auxin transport from root apex to elongation zone in gymnosperm roots — all current gene-linked functional evidence is heterologous (rescue of the Arabidopsis pin2 mutant and shootward localization in Arabidopsis root cells), while the native NPA-sensitive shootward transport measured in pine root tips is not linked to the responsible gene product — and determine when and how the PINE/PIN3 clade, implicated in the rapid lateral relocalization of PINs during gravistimulation, originated and diversified, including whether expanded monocot PIN clades functionally replaced PIN3 in PIN3-clade-lacking grasses.

References

  1. Yuzhou Zhang, Guanghui Xiao, Xiaojuan Wang, Xixi Zhang, Jiří Friml. Evolution of fast root gravitropism in seed plants. Nature Communications 10:3480 (2019). DOI 10.1038/s41467-019-11471-8. https://doi.org/10.1038/s41467-019-11471-8
  2. Tom Bennett, Samuel F. Brockington, Carl Rothfels, Sean W. Graham, Dennis Stevenson, Toni Kutchan, Megan Rolf, Philip Thomas, Gane Ka-Shu Wong, Ottoline Leyser, Beverley J. Glover, C. Jill Harrison. Paralogous Radiations of PIN Proteins with Multiple Origins of Noncanonical PIN Structure. Molecular Biology and Evolution 31:2042–2060 (2014). DOI 10.1093/molbev/msu147. https://doi.org/10.1093/molbev/msu147
  3. Yuzhou Zhang, Lesia Rodriguez, Lanxin Li, Xixi Zhang, Jiří Friml. Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants. Science Advances 6:eabc8895 (2020). DOI 10.1126/sciadv.abc8895. https://doi.org/10.1126/sciadv.abc8895
  4. Yuzhou Zhang, Corinna Hartinger, Xiaojuan Wang, Jiří Friml. Directional auxin fluxes in plants by intramolecular domain–domain coevolution of PIN auxin transporters. New Phytologist 227:1406–1416 (2020). DOI 10.1111/nph.16629. https://doi.org/10.1111/nph.16629
  5. Han Tang, Kuan-Ju Lu, YuZhou Zhang, You-Liang Cheng, Shih-Long Tu, Jiří Friml. Divergence of trafficking and polarization mechanisms for PIN auxin transporters during land plant evolution. Plant Communications 5:100669 (2024). DOI 10.1016/j.xplc.2023.100669. https://doi.org/10.1016/j.xplc.2023.100669
  6. Han Tang, Adrijana Smoljan, Minxia Zou, Yuzhou Zhang, Kuan-Ju Lu, Jiří Friml. The miniW Domain Directs Polarized Membrane Localization of Non-Canonical PINs in Marchantia polymorpha. Plant, Cell & Environment 49:1505–1508 (2025). DOI 10.1111/pce.70295. https://doi.org/10.1111/pce.70295
  7. Takeshi Nishimura, Shogo Mori, Hiromasa Shikata, Moritaka Nakamura, Yasuko Hashiguchi, Yoshinori Abe, Takuma Hagihara, Hiroshi Y. Yoshikawa, Masatsugu Toyota, Takumi Higaki, Miyo Terao Morita. Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane. Science 381:1006–1010 (2023). DOI 10.1126/science.adh9978. https://doi.org/10.1126/science.adh9978
  8. Jiayue Chen, Renbo Yu, Na Li, Zhaoguo Deng, Xinxin Zhang, Yaran Zhao, Chengfu Qu, Yanfang Yuan, Zhexian Pan, Yangyang Zhou, Kunlun Li, Jiajun Wang, Zhiren Chen, Xiaoyi Wang, Xiaolian Wang, Shu-Nan He, Juan Dong, Xing Wang Deng, Haodong Chen. Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants. Cell 186:4788–4802.e15 (2023). DOI 10.1016/j.cell.2023.09.014. https://doi.org/10.1016/j.cell.2023.09.014
  9. Ivan Kulich, Julia Schmid, Anastasia Teplova, Linlin Qi, Jiří Friml. Rapid translocation of NGR proteins driving polarization of PIN-activating D6 protein kinase during root gravitropism. eLife 12:RP91523 (2024). DOI 10.7554/eLife.91523. https://doi.org/10.7554/eLife.91523
  10. Mimi Hashimoto-Sugimoto, Takuya Norizuki, Shoji Segami, Yusaku Ohta, Noriyuki Suetsugu, Takashi Ueda, Miyo Terao Morita. Amyloplasts are necessary for full gravitropism in thallus of Marchantia polymorpha. Journal of Experimental Botany 76:6741–6757 (2025). DOI 10.1093/jxb/eraf375. https://doi.org/10.1093/jxb/eraf375
  11. Dorina P. Janacek, Martina Kolb, Lukas Schulz, Julia Mergner, Bernhard Kuster, Matouš Glanc, Jiří Friml, Kirsten ten Tusscher, Claus Schwechheimer, Ulrich Z. Hammes. Transport properties of canonical PIN-FORMED proteins from Arabidopsis and the role of the loop domain in auxin transport. Developmental Cell 59:3259–3271.e4 (2024). DOI 10.1016/j.devcel.2024.09.020. https://doi.org/10.1016/j.devcel.2024.09.020
  12. Roman Skokan, Eva Medvecká, Tom Viaene, Stanislav Vosolsobě, Marta Zwiewka, Karel Müller, Petr Skůpa, Michal Karady, Yuzhou Zhang, Dorina P. Janacek, Ulrich Z. Hammes, Karin Ljung, Tomasz Nodzyński, Jan Petrášek, Jiří Friml. PIN-driven auxin transport emerged early in streptophyte evolution. Nature Plants 5:1114–1119 (2019). DOI 10.1038/s41477-019-0542-5. https://doi.org/10.1038/s41477-019-0542-5
  13. Katarina Kurtović, Stanislav Vosolsobě, Daniel Nedvěd, Karel Müller, Petre Ivanov Dobrev, Vojtěch Schmidt, Piotr Piszczek, Andre Kuhn, Adrijana Smoljan, Tom J. Fisher, Dolf Weijers, Jiří Friml, John L. Bowman, Jan Petrášek. The role of indole-3-acetic acid and characterization of PIN transporters in complex streptophyte alga Chara braunii. New Phytologist 246:1066–1083 (2025). DOI 10.1111/nph.70019. https://doi.org/10.1111/nph.70019
  14. Christian Luschnig, Jiří Friml. Over 25 years of decrypting PIN-mediated plant development. Nature Communications 15:9904 (2024). DOI 10.1038/s41467-024-54240-y. https://doi.org/10.1038/s41467-024-54240-y
  15. Alexander J. Hetherington, Liam Dolan. Stepwise and independent origins of roots among land plants. Nature 561:235–238 (2018). DOI 10.1038/s41586-018-0445-z. https://doi.org/10.1038/s41586-018-0445-z
  16. Jiří Friml, Justyna Wiśniewska, Eva Benková, Kurt Mendgen, Klaus Palme. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415:806–809 (2002). DOI 10.1038/415806a. https://doi.org/10.1038/415806a
  17. Sjoerd Woudenberg, Melissa Dipp Alvarez, Juriaan A. Rienstra, Victor Levitsky, Victoria Mironova, Enrico Scarpella, Andre Kuhn, Dolf Weijers. Analysis of auxin responses in the fern Ceratopteris richardii identifies the developmental phase as a major determinant for response properties. Development 151:dev203026 (2024). DOI 10.1242/dev.203026. https://doi.org/10.1242/dev.203026
  18. De-Liang Xiang, Gui-Sheng Li. Control of leaf development in the water fern Ceratopteris richardii by the auxin efflux transporter CrPINMa in the CRISPR/Cas9 analysis. BMC Plant Biology 24:322 (2024). DOI 10.1186/s12870-024-05009-4. https://doi.org/10.1186/s12870-024-05009-4
  19. Victoria M. R. Spencer, Lucy Bentall, C. Jill Harrison. Diverse branching forms regulated by a core auxin transport mechanism in plants. Development 150:dev201209 (2023). DOI 10.1242/dev.201209. https://doi.org/10.1242/dev.201209
  20. Yuzhou Zhang, Lanxin Li, Jiří Friml. Evaluation of Gravitropism in Non-seed Plants. Methods in Molecular Biology (Plant Gravitropism), pp. 43–51 (2022). DOI 10.1007/978-1-0716-1677-2_2. https://doi.org/10.1007/978-1-0716-1677-2_2
  21. Miyo Terao Morita, Takeshi Nishimura, Hiromasa Shikata. Gravity Sensing for Gravitropism. Annual Review of Plant Biology 77:425–448 (2026). DOI 10.1146/annurev-arplant-070225-032745. https://doi.org/10.1146/annurev-arplant-070225-032745
  22. Abdellah Lakehal, Edward Farrar, Bipin K. Pandey, Craig J. Sturrock, Tom Beeckman, Malcolm J. Bennett. Evolution of root systems in land plants. Current Biology 36:R565–R576 (2026). DOI 10.1016/j.cub.2026.04.002. https://doi.org/10.1016/j.cub.2026.04.002