Auxin, indole-3-acetic acid (IAA), is a key phytohormone with diverse morphogenic roles in land plants, but its function and transport mechanisms in algae remain poorly understood. We therefore aimed to explore the role of IAA in a complex, streptophyte algae Chara braunii. Here, we described novel responses of C. braunii to IAA and characterized two homologs of PIN auxin efflux carriers: CbPINa and CbPINc. We determined their localization in C. braunii using epitope-specific antibodies and tested their function in heterologous land plant models. Further, using phosphoproteomic analysis, we identified IAA-induced phosphorylation events. The thallus regeneration assay showed that IAA promotes thallus elongation and side branch development. Immunolocalization of CbPINa and CbPINc confirmed their presence on the plasma membrane of vegetative and generative cells of C. braunii. However, functional assays in tobacco BY-2 cells demonstrated that CbPINa affects auxin transport, whereas CbPINc does not. The IAA is effective in the acceleration of cytoplasmic streaming and the phosphorylation of evolutionary conserved targets such as homolog of RAF-like kinase. These findings suggest that, although canonical PIN-mediated auxin transport mechanisms might not be fully conserved in Chara, IAA is involved in morphogenesis and fast signaling processes.

ARC Centre of Excellence for Plant Success in Nature and Agriculture Monash University Melbourne 3800 Vic Australia

Department of Experimental Plant Biology Faculty of Science Charles University Viničná 5 Prague 2 128 44 Czech Republic

Department of Plant Cell Biology Green Life Sciences Cluster Swammerdam Institute for Life Sciences University of Amsterdam Amsterdam 1098XH the Netherlands

Faculty of Biotechnology University of Wroclaw Joliot Curie 14a Wroclaw 50 383 Poland

Institute of Science and Technology Austria Klosterneuburg 3400 Austria

Laboratory of Biochemistry Wageningen University Stippeneng 4 Wageningen 6708 SP the Netherlands

Laboratory of Hormonal Regulations in Plants Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 Prague 6 165 02 Czech Republic

School of Biological Sciences Monash University Melbourne 3800 Vic Australia

Zobrazit více v PubMed

Abas L, Kolb M, Stadlmann J, Janacek DP, Lukic K, Schwechheimer C, Sazanov LA, Mach L, Friml J, Hammes UZ. 2021. Naphthylphthalamic acid associates with and inhibits PIN auxin transporters. Proceedings of the National Academy of Sciences, USA 118: e2020857118. PubMed PMC

Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. 2015. GROMACS: High performance molecular simulations through multi‐level parallelism from laptops to supercomputers. SoftwareX 1–2: 19–25.

An G. 1985. High efficiency transformation of cultured tobacco cells 1. Plant Physiology 79: 568–570. PubMed PMC

Barbier FF, Dun EA, Kerr SC, Chabikwa TG, Beveridge CA. 2019. An update on the signals controlling shoot branching. Trends in Plant Science 24: 220–236. PubMed

Bayly‐Jones C, Lupton CJ, Keen AC, Dong S, Mastos C, Luo W, Qian C, Jones GD, Venugopal H, Chang Y‐G PubMed PMC

Becker B, Marin B. 2009. Streptophyte algae and the origin of embryophytes. Annals of Botany 103: 999–1004. PubMed PMC

Beilby MJ, Turi CE, Baker TC, Tymm FJ, Murch SJ. 2015. Circadian changes in endogenous concentrations of indole‐3‐acetic acid, melatonin, serotonin, abscisic acid and jasmonic acid in Characeae ( PubMed PMC

Bennett T, Brockington SF, Rothfels C, Graham SW, Stevenson D, Kutchan T, Rolf M, Thomas P, Wong GK‐S, Leyser O PubMed PMC

Best RB, Zhu X, Shim J, Lopes PEM, Mittal J, Feig M, MacKerell AD. 2012. Optimization of the additive CHARMM all‐atom protein force field targeting improved sampling of the backbone φ, ψ and side‐chain χ1 and χ2 dihedral angles. Journal of Chemical Theory and Computation 8: 3257–3273. PubMed PMC

Bierenbroodspot MJ, Pröschold T, Fürst‐Jansen JMR, de Vries S, Irisarri I, Darienko T, de Vries J. 2024. Phylogeny and evolution of streptophyte algae. Annals of Botany 134: 385–400. PubMed PMC

Blake TJ, Reid DM, Rood SB. 1983. Ethylene, indoleacetic acid and apical dominance in peas: a reappraisal. Physiologia Plantarum 59: 481–487.

Boot KJM, Libbenga KR, Hille SC, Offringa R, van Duijn B. 2012. Polar auxin transport: an early invention. Journal of Experimental Botany 63: 4213–4218. PubMed PMC

Bowman JL. 2022. The origin of a land flora. Nature Plants 8: 1352–1369. PubMed

Bowman JL, Flores Sandoval E, Kato H. 2021. On the evolutionary origins of land plant auxin biology. Cold Spring Harbor Perspectives in Biology 13: a040048. PubMed PMC

Bulychev AA, Cherkashin AA, Rubin AB, Vredenberg WJ, Zykov VS, Müller SC. 2001. Comparative study on photosynthetic activity of chloroplasts in acid and alkaline zones of PubMed

Buschmann H. 2020. Into another dimension: how streptophyte algae gained morphological complexity. Journal of Experimental Botany 71: 3279–3286. PubMed

Carrillo‐Carrasco VP, Hernandez‐Garcia J, Mutte SK, Weijers D. 2023. The birth of a giant: evolutionary insights into the origin of auxin responses in plants. EMBO Journal 42: e113018. PubMed PMC

Causse HB. 1993. Marchantia L: the European and African Taxa. Berlin, Germany: J Cramer.

Chau R, Bisson MA, Siegel A, Elkin G, Klim P, Straubinger RM. 1994. Distribution of charasomes in chara: re‐establishment and loss in darkness and correlation with banding and inorganic carbon uptake. Functional Plant Biology 21: 113–123.

Cheng S, Xian W, Fu Y, Marin B, Keller J, Wu T, Sun W, Li X, Xu Y, Zhang Y PubMed

Clabeaux BL, Navarro DAG, Aga DS, Bisson MA. 2011. Cd tolerance and accumulation in the aquatic macrophyte, Chara australis: potential use for charophytes in phytoremediation. Environmental Science & Technology 45: 5332–5338. PubMed

Delbarre A, Muller P, Imhoff V, Guern J. 1996. Comparison of mechanisms controlling uptake and accumulation of 2,4‐dichlorophenoxy acetic acid, naphthalene‐1‐acetic acid, and indole‐3‐acetic acid in suspension‐cultured tobacco cells. Planta 198: 532–541. PubMed

Dibb‐Fuller JE, Morris DA. 1992. Studies on the evolution of auxin carriers and phytotropin receptors: transmembrane auxin transport in unicellular and multicellular Chlorophyta. Planta 186: 219–226. PubMed

Donoghue PCJ, Harrison CJ, Paps J, Schneider H. 2021. The evolutionary emergence of land plants. Current Biology 31: R1281–R1298. PubMed

Edgar RC. 2004. muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797. PubMed PMC

Feng X, Zheng J, Irisarri I, Yu H, Zheng B, Ali Z, de Vries S, Keller J, Fürst‐Jansen JMR, Dadras A PubMed PMC

Fisher TJ, Flores‐Sandoval E, Alvarez JP, Bowman JL. 2023. PIN‐FORMED is required for shoot phototropism/gravitropism and facilitates meristem formation in Marchantia polymorpha. New Phytologist 238: 1498–1515. PubMed

Flores‐Sandoval E, Eklund DM, Bowman JL. 2015. A simple auxin transcriptional response system regulates multiple morphogenetic processes in the liverwort marchantia polymorpha. PLoS Genetics 11: e1005207. PubMed PMC

Foissner I, Sommer A, Hoeftberger M. 2015. Photosynthesis‐dependent formation of convoluted plasma membrane domains in Chara internodal cells is independent of chloroplast position. Protoplasma 252: 1085–1096. PubMed PMC

Friml J. 2022. Fourteen stations of auxin. Cold Spring Harbor Perspectives in Biology 14: a039859. PubMed PMC

Friml J, Gallei M, Gelová Z, Johnson A, Mazur E, Monzer A, Rodriguez L, Roosjen M, Verstraeten I, Živanović BD PubMed

Gamborg OL, Miller RA, Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50: 151–158. PubMed

Gao X, Nagawa S, Wang G, Yang Z. 2008. Cell polarity signaling: focus on polar auxin transport. Molecular Plant 1: 899–909. PubMed PMC

Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE. 2018. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Science 27: 14–25. PubMed PMC

Godlewski M. 1980. Auxin as a factor regulating initiation, of mitosis in antheridial filaments of Chara vulgaris L. Biochemie und Physiologie der Pflanzen 175: 314–321.

Hackenberg D, Pandey S. 2014. Heterotrimeric G‐proteins in green algae: an early innovation in the evolution of the plant lineage. Plant Signaling & Behavior 9: e28457. PubMed PMC

Harrison J. 2017. Development and genetics in the evolution of land plant body plans. Philosophical Transactions of the Royal Society, B: Biological Sciences 372: 20150490. PubMed PMC

Hernández‐García J, Carrillo‐Carrasco VP, Rienstra J, Tanaka K, De RM, Dipp‐Álvarez M, Freire‐Ríos A, Crespo I, Boer DR, van Den BWAM PubMed PMC

Heß D, Heise CM, Schubert H, Hess WR, Hagemann M. 2023a. The impact of salt stress on the physiology and the transcriptome of the model streptophyte green alga PubMed

Heß D, Holzhausen A, Hess WR. 2023b. Insight into the nodal cells transcriptome of the streptophyte green alga PubMed

Holzhausen A, Stingl N, Rieth S, Kühn C, Schubert H, Rensing SA. 2022. Establishment and optimization of a new model organism to study early land plant evolution: germination, cultivation and oospore variation of PubMed PMC

Hori K, Maruyama F, Fujisawa T, Togashi T, Yamamoto N, Seo M, Sato S, Yamada T, Mori H, Tajima N PubMed PMC

Ishizaki K, Chiyoda S, Yamato KT, Kohchi T. 2008. Agrobacterium‐mediated transformation of the haploid liverwort PubMed

Ishizaki K, Nishihama R, Ueda M, Inoue K, Ishida S, Nishimura Y, Shikanai T, Kohchi T. 2015. Development of gateway binary vector series with four different selection markers for the liverwort marchantia polymorpha. PLoS ONE 10: e0138876. PubMed PMC

Jahnke E, Libbert E. 1964. Indol‐3‐essigsäure Als Einziges Natives Auxin Bei Chara‐Arten. Plant Signaling & Behavior 52: 283–290.

Janacek DP, Kolb M, Schulz L, Mergner J, Kuster B, Glanc M, Friml J, Tusscher K, Schwechheimer C, Hammes UZ. 2024. Transport properties of canonical PIN‐FORMED proteins from Arabidopsis and the role of the loop domain in auxin transport. Developmental Cell 59: 3259–3271. PubMed

Jin Q, Scherp P, Heimann K, Hasenstein KH. 2008. Auxin and cytoskeletal organization in algae. Cell Biology International 32: 542–545. PubMed

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A PubMed PMC

Klämbt D, Knauth B, Dittmann I. 1992. Auxin dependent growth of rhizoids of

Kuhn A, Roosjen M, Mutte S, Dubey SM, Carrillo Carrasco VP, Boeren S, Monzer A, Koehorst J, Kohchi T, Nishihama R PubMed PMC

Kurtović K, Schmidt V, Nehasilová M, Vosolsobě S, Petrášek J. 2024. Rediscovering Chara as a model organism for molecular and evo‐devo studies. Protoplasma 261: 183–196. PubMed

Laskowski RA, Swindells MB. 2011. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. Journal of Chemical Information and Modeling 51: 2778–2786. PubMed

Liang Z, Geng Y, Ji C, Du H, Wong CE, Zhang Q, Zhang Y, Zhang P, Riaz A, Chachar S PubMed PMC

Luschnig C, Friml J. 2024. Over 25 years of decrypting PIN‐mediated plant development. Nature Communications 15: 9904. PubMed PMC

Lv S, Wang L, Zhang X, Li X, Fan L, Xu Y, Zhao Y, Xie H, Sawchuk M, Scarpella E PubMed

Métraux JP, Richmond PA, Taiz L. 1980. Control of cell elongation in nitella by endogenous cell wall pH gradients: MULTIAXIAL EXTENSIBILITY AND GROWTH STUDIES 1. Plant Physiology 65: 204–210. PubMed PMC

Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. 2022. colabfold: making protein folding accessible to all. Nature Methods 19: 679–682. PubMed PMC

Mravec J, Skůpa P, Bailly A, Hoyerová K, Křeček P, Bielach A, Petrášek J, Zhang J, Gaykova V, Stierhof Y‐D PubMed

Müller K, Hošek P, Laňková M, Vosolsobě S, Malínská K, Čarná M, Fílová M, Dobrev PI, Helusová M, Hoyerová K PubMed

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497.

Mutte SK, Kato H, Rothfels C, Melkonian M, Wong GK‐S, Weijers D. 2018. Origin and evolution of the nuclear auxin response system. eLife 7: e33399. PubMed PMC

Naramoto S. 2017. Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport. Current Opinion in Plant Biology 40: 8–14. PubMed

Nishiyama T, Sakayama H, de Vries J, Buschmann H, Saint‐Marcoux D, Ullrich KK, Haas FB, Vanderstraeten L, Becker D, Lang D PubMed

Pan L, Fonseca De Lima CF, Vu LD, De Smet I. 2021. A comprehensive phylogenetic analysis of the MAP4K family in the green lineage. Frontiers in Plant Science 12: 2541. PubMed PMC

Petrášek J, Černá A, Schwarzerová K, Elčkner M, Morris DA, Zažímalová E. 2003. Do phytotropins inhibit auxin efflux by impairing vesicle traffic? Plant Physiology 131: 254–263. PubMed PMC

Petrášek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertová D, Wiśniewska J, Tadele Z, Kubeš M, Čovanová M PubMed

Pollak B, Cerda A, Delmans M, Álamos S, Moyano T, West A, Gutiérrez RA, Patron NJ, Federici F, Haseloff J. 2019. Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytologist 222: 628–640. PubMed

Ravindranath PA, Forli S, Goodsell DS, Olson AJ, Sanner MF. 2015. AutoDockFR: advances in protein‐ligand docking with explicitly specified binding site flexibility. PLoS Computational Biology 11: e1004586. PubMed PMC

Sauer M, Kleine‐Vehn J. 2019. PIN‐FORMED and PIN‐LIKES auxin transport facilitators. Development 146: dev168088. PubMed

Schmidt V, Skokan R, Depaepe T, Kurtović K, Haluška S, Vosolsobě S, Vaculíková R, Pil A, Dobrev PI, Motyka V PubMed PMC

Schmölzer PM, Höftberger M, Foissner I. 2011. Plasma membrane domains participate in pH banding of PubMed PMC

Skokan R, Medvecká E, Viaene T, Vosolsobě S, Zwiewka M, Müller K, Skůpa P, Karady M, Zhang Y, Janacek DP PubMed

Su N, Zhu A, Tao X, Ding ZJ, Chang S, Ye F, Zhang Y, Zhao C, Chen Q, Wang J PubMed

Sztein AE, Cohen JD, Cooke TJ. 2000. Evolutionary patterns in the auxin metabolism of green plants. International Journal of Plant Sciences 161: 849–859.

Takahashi K, Hayashi K, Kinoshita T. 2012. Auxin activates the plasma membrane H PubMed PMC

Tyanova S, Temu T, Cox J. 2016a. The MaxQuant computational platform for mass spectrometry‐based shotgun proteomics. Nature Protocols 11: 2301–2319. PubMed

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J. 2016b. The Perseus computational platform for cosmprehensive analysis of (prote)omics data. Nature Methods 13: 731–740. PubMed

Ung KL, Schulz L, Stokes DL, Hammes UZ, Pedersen BP. 2023. Substrate recognition and transport mechanism of the PIN‐FORMED auxin exporters. Trends in Biochemical Sciences 48: 937–948. PubMed PMC

Ung KL, Winkler M, Schulz L, Kolb M, Janacek DP, Dedic E, Stokes DL, Hammes UZ, Pedersen BP. 2022. Structures and mechanism of the plant PIN‐FORMED auxin transporter. Nature 609: 605–610. PubMed PMC

Vieten A, Vanneste S, Wiśniewska J, Benková E, Benjamins R, Beeckman T, Luschnig C, Friml J. 2005. Functional redundancy of PIN proteins is accompanied by auxin‐dependent cross‐regulation of PIN expression. Development 132: 4521–4531. PubMed

Vosolsobě S, Petrášek J, Schwarzerová K. 2017. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins. Biochimica et Biophysica Acta (BBA) ‐ Biomembranes 1859: 686–697. PubMed

Vosolsobě S, Skokan R, Petrášek J. 2020. The evolutionary origins of auxin transport: what we know and what we need to know. Journal of Experimental Botany 71: 3287–3295. PubMed

Wasteneys GO, Jablonsky PP, Williamson RE. 1989. Assembly of purified brain tubulin at cortical and endoplasmic sites in perfused internodal cells of the alga

Weller B, Zourelidou M, Frank L, Barbosa ICR, Fastner A, Richter S, Jürgens G, Hammes UZ, Schwechheimer C. 2017. Dynamic PIN‐FORMED auxin efflux carrier phosphorylation at the plasma membrane controls auxin efflux‐dependent growth. Proceedings of the National Academy of Sciences, USA 114: E887–E896. PubMed PMC

Wiśniewska J, Xu J, Seifertová D, Brewer PB, Růžička K, Blilou I, Rouquié D, Benková E, Scheres B, Friml J. 2006. Polar PIN localization directs auxin flow in plants. Science 312: 883. PubMed

Yang Z, Xia J, Hong J, Zhang C, Wei H, Ying W, Sun C, Sun L, Mao Y, Gao Y PubMed PMC

Żabka A, Polit JT, Winnicki K, Paciorek P, Juszczak J, Nowak M, Maszewski J. 2016. PIN2‐like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in PubMed PMC

Zeng HY, Deng S, Jin C, Shang Z, Chang L, Wang J, Han X, Wang A, Jin D, Wang Y PubMed PMC

Zhang S, de Boer AH, van Duijn B. 2016. Auxin effects on ion transport in PubMed

Zhang Y, Xiao G, Wang X, Zhang X, Friml J. 2019. Evolution of fast root gravitropism in seed plants. Nature Communications 10: 3480. PubMed PMC

Zhao Y, Stoffler D, Sanner M. 2006. Hierarchical and multi‐resolution representation of protein flexibility. Bioinformatics 22: 2768–2774. PubMed

Zuo J, Niu Q‐W, Chua N‐H. 2000. An estrogen receptor‐based transactivator XVE mediates highly inducible gene expression in transgenic plants. The Plant Journal 24: 265–273. PubMed