Abstract
The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.
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Data availability
Data supporting the findings of this study are available in the paper and its Supplementary Information. Source data are provided with this paper.
References
Friml, J. Fourteen stations of auxin. Cold Spring Harb. Perspect. Biol. 14, a039859 (2021).
Parry, G. & Estelle, M. Auxin receptors: a new role for F-box proteins. Curr. Opin. Cell Biol. 18, 152–156 (2006).
Quint, M. & Gray, W. M. Auxin signaling. Curr. Opin. Plant Biol. 9, 448–453 (2006).
Gray, W. M. et al. Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. Genes Dev. 13, 1678–1691 (1999).
Gray, W. M., Kepinski, S., Rouse, D., Leyser, O. & Estelle, M. Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 414, 271–276 (2001).
Dharmasiri, N., Dharmasiri, S. & Estelle, M. The F-box protein TIR1 is an auxin receptor. Nature 435, 441–445 (2005).
Kepinski, S. & Leyser, O. The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435, 446–451 (2005).
Tan, X. et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007).
Prigge, M. J. et al. Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions. eLife 9, e54740 (2020).
Kubes, M. & Napier, R. Non-canonical auxin signalling: fast and curious. J. Exp. Bot. 70, 2609–2614 (2019).
Gallei, M., Luschnig, C. & Friml, J. Auxin signalling in growth: Schrodinger’s cat out of the bag. Curr. Opin. Plant Biol. 53, 43–49 (2020).
Fendrych, M. et al. Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nat. Plants 4, 453–459 (2018).
Li, L. et al. Cell surface and intracellular auxin signalling for H+ fluxes in root growth. Nature 599, 273–277 (2021).
Serre, N. B. C. et al. AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root. Nat. Plants 7, 1229–1238 (2021).
Shih, H. W., DePew, C. L., Miller, N. D. & Monshausen, G. B. The cyclic nucleotide-gated channel CNGC14 regulates root gravitropism in Arabidopsis thaliana. Curr. Biol. 25, 3119–3125 (2015).
Dindas, J. et al. AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling. Nat. Commun. 9, 1174 (2018).
Gehring, C. Adenyl cyclases and cAMP in plant signaling—past and present. Cell Commun. Signal. 8, 15 (2010).
Wong, A., Gehring, C. & Irving, H. R. Conserved functional motifs and homology modeling to predict hidden moonlighting functional sites. Front. Bioeng. Biotechnol. 3, 82 (2015).
Swiezawska, B. et al. Molecular cloning and characterization of a novel adenylyl cyclase gene, HpAC1, involved in stress signaling in Hippeastrum x hybridum. Plant Physiol. Biochem. 80, 41–52 (2014).
Al-Younis, I., Wong, A. & Gehring, C. The Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre. FEBS Lett. 589, 3848–3852 (2015).
Al-Younis, I. et al. The Arabidopsis thaliana K+-uptake permease 5 (AtKUP5) contains a functional cytosolic adenylate cyclase essential for K+ transport. Front. Plant Sci. 9, 1645 (2018).
Chatukuta, P. et al. An Arabidopsis clathrin assembly protein with a predicted role in plant defense can function as an adenylate cyclase. Biomolecules 8, 15 (2018).
Bianchet, C. et al. An Arabidopsis thaliana leucine-rich repeat protein harbors an adenylyl cyclase catalytic center and affects responses to pathogens. J. Plant Physiol. 232, 12–22 (2019).
Prigge, M. J., Lavy, M., Ashton, N. W. & Estelle, M. Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway. Curr. Biol. 20, 1907–1912 (2010).
Johnstone, T. B., Agarwal, S. R., Harvey, R. D. & Ostrom, R. S. cAMP signaling compartmentation: adenylyl cyclases as anchors of dynamic signaling complexes. Mol. Pharmacol. 93, 270–276 (2018).
Parry, G. et al. Complex regulation of the TIR1/AFB family of auxin receptors. Proc. Natl Acad. Sci. USA 106, 22540–22545 (2009).
Uchida, N. et al. Chemical hijacking of auxin signaling with an engineered auxin–TIR1 pair. Nat. Chem. Biol. 14, 299–305 (2018).
Li, L., Gallei, M. & Friml, J. Bending to auxin: fast acid growth for tropisms. Trends Plant Sci. 27, 440–449 (2022).
Beavo, J. A. & Brunton, L. L. Cyclic nucleotide research–still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3, 710–718 (2002).
Trewavas, A. J. Plant cyclic AMP comes in from the cold. Nature 390, 657–658 (1997).
Kwezi, L. et al. The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants. J. Biol. Chem. 286, 22580–22588 (2011).
Wheeler, J. I. et al. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling. Plant J. 91, 590–600 (2017).
Dharmasiri, N. et al. Plant development is regulated by a family of auxin receptor F box proteins. Dev. Cell 9, 109–119 (2005).
Toyota, M. et al. Glutamate triggers long-distance, calcium-based plant defense signaling. Science 361, 1112–1115 (2018).
Longair, M. H., Baker, D. A. & Armstrong, J. D. Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27, 2453–2454 (2011).
Li, L., Krens, S. F. G., Fendrych, M. & Friml, J. Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana hypocotyls. Bio. Protoc. 8, e2685 (2018).
Lee, S. et al. Defining binding efficiency and specificity of auxins for SCFTIR1/AFB–Aux/IAA co-receptor complex formation. ACS Chem. Biol. 9, 673–682 (2014).
Quareshy, M. et al. The tetrazole analogue of the auxin indole-3-acetic acid binds preferentially to TIR1 and not AFB5. ACS Chem. Biol. 13, 2585–2594 (2018).
Van Damme, T. et al. Wounding stress causes rapid increase in concentration of the naturally occurring 2′,3′-isomers of cyclic guanosine- and cyclic adenosine monophosphate (cGMP and cAMP) in plant tissues. Phytochemistry 103, 59–66 (2014).
Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
Hanwell, M. D. et al. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J. Cheminform. 4, 17 (2012).
O’Boyle, N. M. et al. Open Babel: an open chemical toolbox. J. Cheminform. 3, 33 (2011).
Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Sanner, M. F., Olson, A. J. & Spehner, J. C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996).
Barbez, E., Dunser, K., Gaidora, A., Lendl, T. & Busch, W. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 114, E4884–E4893 (2017).
Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).
Acknowledgements
This research was supported by the Lab Support Facility (LSF) and the Imaging and Optics Facility (IOF) of IST Austria. We thank C. Gehring for suggestions and advice; and K. U. Torii and G. Stacey for seeds and plasmids. This project was funded by a European Research Council Advanced Grant (ETAP-742985). M.F.K. and R.N. acknowledge the support of the EU MSCA-IF project CrysPINs (792329). M.K. was supported by the project POWR.03.05.00-00-Z302/17 Universitas Copernicana Thoruniensis in Futuro–IDS “Academia Copernicana”. CIDG acknowledges support from UKRI under Future Leaders Fellowship grant number MR/T020652/1.
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Contributions
L.Q. and J.F. conceived and designed the experiments. L.Q. carried out most of the experiments. M.K. and K.J. performed most of the protein purification, in vitro AC activity assay and LC–MS/MS analysis. H.C. performed the vRootchip experiments. L.H. assisted with the root growth tracking with the vertical microscope. M.Z. did the root gravitropism assay. S.S. originally tested relationship between auxin and eATP signalling. M.F.K. and R.N. assisted with TIR1 expression in insect cells. C.I.d.G. performed the molecular docking. L.Q. and J.F. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Additional data to support the AC activity of TIR1/AFBs.
a-b, E. coli complementation assay showing that AFB1 and AFB5 have AC activity. The AC deficient SP850 strain was complemented with the empty vector (pGEX-4T-1), the positive control (HpAC1), and TIR1/AFBs. Red colour of the MacConkey Agar indicates the presence of AC activity (a). Western blot result shows that only AFB1 and AFB5 can be visibly detected among the 6 members of TIR1/AFBs. Ponceau staining of the same membrane was used as the loading control. For gel source data, see Supplementary Fig. 1. The experiments were repeated twice independently with similar results. c, Michaelis-Menten kinetics for the AC activity of GST-AFB1 purified from E. coli. cAMP level after reaction was quantified by LC-MS/MS. S, substrate; V, velocity. For each data point, means ± SD from 3 biological replicates are shown. d, Representative LC-MS/MS spectrum showing the detection of cAMP after the in vitro AC activity assay for His-GFP-FLAG-TIR1 purified from Sf9 insect cells.
Extended Data Fig. 2 Gel images showing the purity of all the proteins used.
a, GST-AFB5. b, GST-AFB1. c, His-GFP-FLAG-TIR1. d, GST-PpAFB1. e, GST-PpAFB2. f, GST-PpAFB3. g, GST-PpAFB4. h, GST-AFB5ACm1/m2/m3. i, GST-TIR1ΔNT. j, GST-TIR1ΔNT-ACm1/m2/m3. k, AFB5 after cleavage of GST tag. l, IAA7 after cleavage of GST tag. m, IAA17 after cleavage of GST tag. His-GFP-FLAG-TIR1 was purified from Sf9 insect cells. All the other proteins were purified from BL-21 E. coli cells. Proteins were separated on SDS-PAGE gels and the gels were stained with Coomassie Brilliant Blue. The experiment was repeated twice with similar results.
Extended Data Fig. 3 AC activity is conserved in TIR1/AFBs orthologues from Physcomitrella.
a, Alignment of the C-terminal protein sequences of TIR1/AFBs together with their orthologues from Physcomitrella. Note the AC motif is highly conserved in all the sequences. Only the first amino acid is a bit more relaxed. b, TIR1/AFBs orthologues from Physcomitrella have AC activity. GST-tagged PpAFBs were purified from E. coli. In vitro AC activity assay was performed with GST as the negative control. cAMP level after reaction was quantified using LC-MS/MS. The values shown are means ± SD from 3 biological replicates. One-way ANOVA and Dunnett’s multiple comparisons test were performed. Asterisks indicate significant difference between the corresponding group and the negative control (GST). ** p ≤ 0.01 (p = 0.0034); **** p ≤ 0.0001.
Extended Data Fig. 4 Protein structure of TIR1-IAA-Aux/IAA complex and ribbon structure showing ATP docking.
a, Protein structure of TIR1-IAA-Aux/IAA complex showing the spatial position of the C-terminal AC motif. Different parts were labelled as different colours. Dark green, ASK1; Red, TIR1; White, C-terminal AC motif; Blue, IAA7 peptide; Yellow, InsP6 (inositol hexakisphosphate); Green, IAA. b, Ribbon structure showing the interaction of ATP with the key amino acids of the AC motif. AC center was labelled in magenta. E554 is the residue identified for m1 in Fig. 1a, R566 for m2, and D568 for m3. Note that V84 from the Aux/IAA degron restricts the space available to ATP.
Extended Data Fig. 5 Auxin perception enhances the AC activity of AFB5.
In vitro AC activity assay for AFB5 in the presence of 10 µM IAA, IAA7, IAA17 and the indicated combinations, followed by cAMP quantification using LC-MS/MS. Tag-cleaved clean proteins were used for this experiment. Data are presented as means ± SD from 3 biological replicates. One-way ANOVA and Tukey’s multiple comparisons test were performed. Asterisks indicate significant difference between the corresponding group and Control. ** p ≤ 0.01 (p = 0.0022); **** p ≤ 0.0001.
Extended Data Fig. 6 Delayed requirement of the AC activity in root growth regulation.
a, Loss of AC activity does not affect cvxIAA/ccvTIR1-induced root growth inhibition within the first hour. A vRootchip experiment was performed with the transgenic lines indicated, and the images were captured with a time interval of 1 min. Mock medium was changed to medium containing 500 nM of cvxIAA at 40 min. Root growth rate was normalized to the starting point of the respective group. The values shown are means + SD. n = 4 or 5 seedlings. The experiment was repeated twice independently with similar results. b, Resistance of ccvTIR1ACm1 to cvxIAA-triggered root growth inhibition occurs only after 1 h of treatment. Vertical scanner growth assay was performed to track the root growth dynamics. Five-days-old seedlings of the indicated genotypes were transferred to either Mock medium or medium containing 200 nM of cvxIAA. Images were taken every 30 min. Root growth rate was measured. The values shown are means + SD. n = 10 seedlings. The experiment was repeated twice independently with similar results.
Extended Data Fig. 7 TIR1 AC activity is not crucial for rapid auxin responses.
a, cvxIAA triggers similar Ca2+ spikes in the ccvTIR1 and ccvTIR1ACm1 lines. The calcium sensor GCaMP3 was crossed to the indicated transgenic lines. Five-days-old F1 seedlings were used for vRootchip experiment, and the images were captured with a time interval of 15 s. Mock medium was changed to medium containing 500 nM of cvxIAA at 10 min. The fluorescence signal in the epidermal cells of root elongation zone was quantified, and was normalized to the average value of time points before treatment. The values shown are means + SD. n = 4 seedlings. The experiment was repeated twice independently with similar results. b, Auxin-induced apoplastic alkalinisation is not changed in the TIR1ACm1 line. Five-days-old seedlings of the indicated genotypes were used for vRootchip experiment. Mock medium was changed to medium containing 10 nM of IAA at 11 min. Ratiometric (488 nm/405 nm) imaging of HPST staining was used to measure apoplastic pH in the epidermal cells of root elongation zone. The values shown are means + SD normalized to the average of those time points before treatment. n = 3 or 4 seedlings. The experiment was repeated twice independently with similar results.
Supplementary information
Supplementary Figure 1
Gel source data.
Supplementary Table 1
Primers used in this study.
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Qi, L., Kwiatkowski, M., Chen, H. et al. Adenylate cyclase activity of TIR1/AFB auxin receptors in plants. Nature 611, 133–138 (2022). https://doi.org/10.1038/s41586-022-05369-7
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DOI: https://doi.org/10.1038/s41586-022-05369-7
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