The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.

Department of Experimental Plant Biology Faculty of Sciences Charles University Prague Czech Republic

Plant Molecular and Cellular Biology Laboratory and Integrative Biology Laboratory Salk Institute for Biological Studies La Jolla CA USA

Section of Cell and Developmental Biology University of California San Diego La Jolla CA USA

bioRxiv. 2023 Jan 04:2023.01.04.522696. doi: 10.1101/2023.01.04.522696. PubMed

Nat Plants. 2023 Aug;9(8):1171. doi: 10.1038/s41477-023-01506-w. PubMed

Zobrazit více v PubMed

Ang ACH, and Østergaard L (2023). Save your TIRs - more to auxin than meets the eye. New Phytol. 238:971–976. PubMed PMC

Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA, et al. (2017). mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14:1–12. PubMed

Calderón Villalobos LIA, Lee S, De Oliveira C, Ivetac A, Brandt W, Armitage L, Sheard LB, Tan X, Parry G, Mao H, et al. (2012). A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. Nat. Chem. Biol. 8:477–85. PubMed PMC

Dindas J, Scherzer S, Roelfsema MRG, von Meyer K, Müller HM, Al-Rasheid KAS, Palme K, Dietrich P, Becker D, Bennett MJ, et al. (2018). AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling. Nat. Commun. 9:1174. PubMed PMC

Dubey SM, Serre NBC, Oulehlová D, Vittal P, and Fendrych M (2021). No Time for Transcription-Rapid Auxin Responses in Plants. Cold Spring Harb. Perspect. Biol. 13:a039891. PubMed PMC

Evans ML, Ishikawa H, and Estelle MA (1994). Responses ofArabidopsis roots to auxin studied with high temporal resolution: Comparison of wild type and auxin-response mutants. Planta 194:215–222.

Fendrych M, Akhmanova M, Merrin J, Glanc M, Hagihara S, Takahashi K, Uchida N, Torii KU, and Friml J (2018). Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nat. plants 4:453–459. PubMed PMC

Friml J, Gallei M, Gelová Z, Johnson A, Mazur E, Monzer A, Rodriguez L, Roosjen M, Verstraeten I, Živanović BD, et al. (2022). ABP1-TMK auxin perception for global phosphorylation and auxin canalization. Nature 24. PubMed

Gilkerson J, Hu J, Brown J, Jones A, Sun T, and Callis J (2009). Isolation and Characterization of cul1–7, a Recessive Allele of CULLIN1 That Disrupts SCF Function at the C Terminus of CUL1 in Arabidopsis thaliana. Genetics 181:945–963. PubMed PMC

Han H (2018). RNA Interference to Knock Down Gene Expression. Methods Mol. Biol. 1706:293–302. PubMed PMC

Keinath NF, Waadt R, Brugman R, Schroeder JI, Grossmann G, Schumacher K, and Krebs M (2015). Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca(2+)]cyt Patterns in Arabidopsis. Mol. Plant 8:1188–200. PubMed PMC

Kepinski S (2009). Pull-down assays for plant hormone research. Methods Mol. Biol. 495:61–80. PubMed

Kurihara D, Mizuta Y, Nagahara S, and Higashiyama T (2021). ClearSeeAlpha: Advanced Optical Clearing for Whole-Plant Imaging. Plant Cell Physiol. 62:1302–1310. PubMed PMC

Li L, Verstraeten I, Roosjen M, Takahashi K, Rodriguez L, Merrin J, Chen J, Shabala L, Smet W, Ren H, et al. (2021). Cell surface and intracellular auxin signalling for H+ fluxes in root growth. Nature 599:273–277. PubMed PMC

Malamy JE, and Benfey PN (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44. PubMed

Monshausen GB, Miller ND, Murphy AS, and Gilroy S (2011). Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis. Plant J. 65:309–18. PubMed

Moon J, Zhao Y, Dai X, Zhang W, Gray WM, Huq E, and Estelle M (2007). A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis. Plant Physiol. 143:684–96. PubMed PMC

Nagpal P, Walker LM, Young JC, Sonawala A, Timpte C, Estelle M, and Reed JW (2000). AXR2 Encodes a Member of the Aux/IAA Protein Family. Plant Physiol. 123:563–574. PubMed PMC

Okushima Y, Fukaki H, Onoda M, Theologis A, and Tasaka M (2007). ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell 19:118–30. PubMed PMC

Platre M (2022). Root Walker: an automated pipeline for large scale quantification of early root growth responses at high spatial and temporal resolution. bioRxiv 2022.11.16. PubMed PMC

Prigge MJ, Platre M, Kadakia N, Zhang Y, Greenham K, Szutu W, Pandey BK, Bhosale RA, Bennett MJ, Busch W, et al. (2020). Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions. Elife 9:1–28. PubMed PMC

Qi L, Kwiatkowski M, Chen H, Hoermayer L, Sinclair S, Zou M, Del Genio CI, Kubeš MF, Napier R, Jaworski K, et al. (2022). Adenylate cyclase activity of TIR1/AFB auxin receptors in plants. Nature 611:133–138. PubMed

Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, and Orzaez D (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162:1618–31. PubMed PMC

Serre NBC, and Fendrych M (2022). ACORBA: Automated workflow to measure Arabidopsis thaliana root tip angle dynamics. Quant. Plant Biol. 3:e9. PubMed PMC

Serre NBC, Kralík D, Yun P, Slouka Z, Shabala S, and Fendrych M (2021). AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root. Nat. plants 7:1229–1238. PubMed PMC

Shih H-W, DePew CL, Miller ND, and Monshausen GB (2015). The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana. Curr. Biol. 25:3119–25. PubMed

Simonini S, Deb J, Moubayidin L, Stephenson P, Valluru M, Freire-Rios A, Sorefan K, Weijers D, Friml J, and Østergaard L (2016). A noncanonical auxin-sensing mechanism is required for organ morphogenesis in Arabidopsis. Genes Dev. 30:2286–2296. PubMed PMC

Tan X, Calderon-Villalobos L. I. a, Sharon M, Zheng C, Robinson CV, Estelle M, and Zheng N (2007). Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446:640–5. PubMed

Tian Q, and Reed JW (1999). Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126:711–21. PubMed

Tofanelli R, Vijayan A, Scholz S, and Schneitz K (2019). Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy. Plant Methods 15:120. PubMed PMC

Uchida N, Takahashi K, Iwasaki R, Yamada R, Yoshimura M, Endo TA, Kimura S, Zhang H, Nomoto M, Tada Y, et al. (2018). Chemical hijacking of auxin signaling with an engineered auxin-TIR1 pair. Nat. Chem. Biol. 14:299–305. PubMed PMC

Wang R, Himschoot E, Grenzi M, Chen J, Safi A, Krebs M, Schumacher K, Nowack MK, Moeder W, Yoshioka K, et al. (2022). Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots. J. Exp. Bot. 73:2308–2319. PubMed PMC

Weijers D, and Wagner D (2016). Transcriptional Responses to the Auxin Hormone. Annu. Rev. Plant Biol. 67:539–74. PubMed

Yoo S-D, Cho Y-H, and Sheen J (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2:1565–72. PubMed

Yu H, Zhang Y, Moss BL, Bargmann BOR, Wang R, Prigge M, Nemhauser JL, and Estelle M (2015). Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response. Nat. plants 1. PubMed PMC

RAF-like protein kinases mediate a deeply conserved, rapid auxin response