Distinct functions of TIR1 and AFB1 receptors in auxin signaling

Abstract

Auxin is the major plant hormone regulating growth and development (Friml, 2022Friml J. Fourteen Stations of Auxin.Cold Spring Harbor Perspect. Biol. 2022; 14a039859https://doi.org/10.1101/cshperspect.a039859Crossref PubMed Scopus (28) Google Scholar). Forward genetic approaches have identified major components of auxin signaling and established the canonical mechanism mediating transcriptional and thus developmental reprogramming in Arabidopsis thaliana. In this textbook view, TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) proteins are auxin receptors, which act as F-box subunits determining the substrate specificity of the Skp1–Cullin1–F box protein (SCF) type E3 ubiquitin ligase complex. Auxin acts as a “molecular glue,” increasing the affinity between TIR1/AFBs and the Auxin/Indole-3-Acetic Acid (Aux/IAA) repressors. Subsequently, Aux/IAAs are ubiquitinated and degraded, thus releasing auxin transcription factors from their repression and making them free to mediate transcription of auxin response genes (Yu et al., 2022Yu Z. Zhang F. Friml J. Ding Z. Auxin signaling: Research advances over the past 30 years.J. Integr. Plant Biol. 2022; 64: 371-392https://doi.org/10.1111/jipb.13225Crossref PubMed Scopus (54) Google Scholar). Nonetheless, accumulating evidence suggests the existence of rapid, non-transcriptional responses downstream of TIR1/AFBs such as auxin-induced cytosolic calcium (Ca2+) transients, plasma membrane depolarization, and apoplast alkalinization, all converging on the process of root growth inhibition and root gravitropism (Li et al., 2022Li L. Gallei M. Friml J. Bending to auxin: fast acid growth for tropisms.Trends Plant Sci. 2022; 27: 440-449https://doi.org/10.1016/j.tplants.2021.11.006Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Particularly, these rapid responses are mostly contributed by predominantly cytosolic AFB1, while the long-term growth responses are mediated by mainly nuclear TIR1 and AFB2–AFB5 (Prigge et al., 2020Prigge M.J. Platre M. Kadakia N. Zhang Y. Greenham K. Szutu W. Pandey B.K. Bhosale R.A. Bennett M.J. Busch W. et al.Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions.Elife. 2020; 9e54740https://doi.org/10.7554/eLife.54740Crossref Scopus (79) Google Scholar; Li et al., 2021Li L. Verstraeten I. Roosjen M. Takahashi K. Rodriguez L. Merrin J. Chen J. Shabala L. Smet W. Ren H. et al.Cell surface and intracellular auxin signalling for H(+) fluxes in root growth.Nature. 2021; 599: 273-277https://doi.org/10.1038/s41586-021-04037-6Crossref PubMed Scopus (70) Google Scholar; Serre et al., 2021Serre N.B.C. Kralík D. Yun P. Slouka Z. Shabala S. Fendrych M. AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root.Native Plants. 2021; 7: 1229-1238https://doi.org/10.1038/s41477-021-00969-zCrossref PubMed Scopus (30) Google Scholar). How AFB1 conducts auxin-triggered rapid responses and how it is different from TIR1 and AFB2–AFB5 remains elusive. Here, we compare the roles of TIR1 and AFB1 in transcriptional and rapid responses by modulating their subcellular localization in Arabidopsis and by testing their ability to mediate transcriptional responses when part of the minimal auxin circuit is reconstituted in yeast. One prominent difference between TIR1 and AFB1 is their subcellular localization. TIR1 primarily localizes to the nucleus while AFB1 to the cytoplasm (Figure 1A and Supplemental Figure 1) (Prigge et al., 2020Prigge M.J. Platre M. Kadakia N. Zhang Y. Greenham K. Szutu W. Pandey B.K. Bhosale R.A. Bennett M.J. Busch W. et al.Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions.Elife. 2020; 9e54740https://doi.org/10.7554/eLife.54740Crossref Scopus (79) Google Scholar). To test whether their specific localization is a necessary prerequisite for their function in either transcriptional or rapid responses, we fused Venus report gene combined with nuclear exporting signal (NES) or nuclear localization signal (NLS) at the C termini of TIR1 (TIR1–NES–Venus) and AFB1 (AFB1–NLS–Venus), respectively. We showed that the majority of TIR1–NES–Venus is shifted to cytosol, while AFB1–NLS–Venus mostly concentrates in the nucleus (Figure 1B and Supplemental Figure 1). To characterize the importance of nuclear versus cytosolic localization of TIR1 and AFB1 in auxin-mediated transcription, we tested if they can rescue the mutant phenotype in a sustained root growth inhibition after auxin treatment for 6 days. We introduced the cytosolic-localized TIR1–NES–Venus into a tir1 mutant background and found that all TIR1 constructs were able to completely restore auxin sensitivity of root growth (Figures 1C and 1D and Supplemental Figures 1 and 2). This can be explained by either of the following: (i) the residual TIR1 present in the nucleus is sufficient to conduct full transcriptional activity, or (ii) cytosolic TIR1 may still degrade Aux/IAAs, releasing the ARFs from their inhibition. Besides, we introduced the nuclear-localized AFB1–NLS–Venus into tir1 afb2 mutants but did not observe any rescue of the auxin-insensitive phenotype in root growth inhibition, root gravitropism, lateral root formation, and root hair elongation (Figures 1C and 1D and Supplemental Figures 1 and 2). This implies that AFB1, even when localized to the nucleus, cannot functionally replace TIR1 for its transcriptional regulation and related development. The predominantly cytosolic AFB1 seems to be the major receptor for the rapid auxin effects (Prigge et al., 2020Prigge M.J. Platre M. Kadakia N. Zhang Y. Greenham K. Szutu W. Pandey B.K. Bhosale R.A. Bennett M.J. Busch W. et al.Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions.Elife. 2020; 9e54740https://doi.org/10.7554/eLife.54740Crossref Scopus (79) Google Scholar). Therefore, we introduced our mistargeted TIR1 and AFB1 versions into the afb1 mutant background (Figures 1A and 1B and Supplemental Figure 1) and tested their effect in auxin-induced rapid root growth inhibition in a microfluidic vRootchip system. AFB1, when targeted to the nucleus, could no longer mediate the rapid auxin effect on root growth (Figure 1E). On the other hand, TIR1, despite being present in the cytosol, could not rescue the afb1 mutant (Figure 1F). This reveals that cytosolic AFB1 is necessary for its function but that cytosolic TIR1 cannot replace or supplement the AFB1 function. The observations that nuclear AFB1 cannot functionally replace TIR1 and that cytosolic TIR1 cannot functionally replace AFB1 show that TIR1 and AFB1 have distinct functional properties unrelated to their subcellular localization. To confirm this, we made use of the minimal auxin signaling pathway reconstructed in yeast (Pierre-Jerome et al., 2014Pierre-Jerome E. Jang S.S. Havens K.A. Nemhauser J.L. Klavins E. Recapitulation of the forward nuclear auxin response pathway in yeast.Proc. Natl. Acad. Sci. USA. 2014; 111: 9407-9412https://doi.org/10.1073/pnas.1324147111Crossref PubMed Scopus (63) Google Scholar). In this system, only TIR1, not AFB1, regardless of their subcellular localization, was able to mediate the auxin effect on transcription as monitored by the fluorescence intensity of P3_Venus transcriptional auxin reporter (Figure 1G). To understand why AFB1 cannot mediate transcriptional signaling, we tested its ability to form an SCF complex using the yeast two-hybrid approach. Only TIR1, not AFB1, was able to interact with CUL1 (Cullin1), the key component of the ubiquitin ligase complex (Figure 1H). This is consistent with the available coimmunoprecipitation/mass spectrometry data, where all SCF components were detected to interact with TIR1; however, for AFB1, no or only an extremely weak interaction with CUL1 was detected (Supplemental Figure 3) (Yu et al., 2015Yu H. Zhang Y. Moss B.L. Bargmann B.O.R. Wang R. Prigge M. Nemhauser J.L. Estelle M. Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.Native Plants. 2015; 114030https://doi.org/10.1038/nplants.2014.30Crossref PubMed Google Scholar; Li et al., 2021Li L. Verstraeten I. Roosjen M. Takahashi K. Rodriguez L. Merrin J. Chen J. Shabala L. Smet W. Ren H. et al.Cell surface and intracellular auxin signalling for H(+) fluxes in root growth.Nature. 2021; 599: 273-277https://doi.org/10.1038/s41586-021-04037-6Crossref PubMed Scopus (70) Google Scholar). The reason why AFB1 does not interact with CUL1 might be the natural mutation of the glutamic 8 site in AFB1 (Yu et al., 2015Yu H. Zhang Y. Moss B.L. Bargmann B.O.R. Wang R. Prigge M. Nemhauser J.L. Estelle M. Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.Native Plants. 2015; 114030https://doi.org/10.1038/nplants.2014.30Crossref PubMed Google Scholar). The absence of an interaction with the SCF components will prevent AFB1 from conducting E3 ubiquitin ligase activity, thus failing to mediate Aux/IAA degradation (Yu et al., 2015Yu H. Zhang Y. Moss B.L. Bargmann B.O.R. Wang R. Prigge M. Nemhauser J.L. Estelle M. Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.Native Plants. 2015; 114030https://doi.org/10.1038/nplants.2014.30Crossref PubMed Google Scholar) and transcriptional regulation (Figure 1G). This also explains why AFB1, even when artificially targeted to the nucleus, still cannot replace the TIR1 function. Our observations also imply that CUL1 is not essential for rapid auxin responses. This requires further clarification on the role of SCF components as well as Aux/IAA ubiquitination and degradation in rapid auxin responses. A recent study revealed the novel function of TIR1/AFBs in producing 3′,5′-cyclic adenosine monophosphate (cAMP), a prominent second messenger in animals. Though this activity of TIR1 specifically seems not important for rapid auxin responses (Qi et al., 2022Qi L. Kwiatkowski M. Chen H. Hoermayer L. Sinclair S. Zou M. Del Genio C.I. Kubeš M.F. Napier R. Jaworski K. et al.Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.Nature. 2022; 611: 133-138https://doi.org/10.1038/s41586-022-05369-7Crossref PubMed Scopus (27) Google Scholar), it is still possible that AFB1-mediated cAMP production in the cytosol is. However, whether and how the adenylate cyclase activity of AFB1 contributes to rapid auxin responses remain unknown. In summary, we demonstrated that TIR1 and AFB1 have distinct functions, with the predominantly nuclear TIR1 mediating slow responses and cytosolic AFB1 conducting rapid responses. This functional divergence is not, however, simply due to the differential subcellular localization of these auxin receptors. The function of TIR1 in mediating slow/transcriptional responses seems to be independent of its predominant localization. In contrast, the function of AFB1 in rapid responses necessitates both its localization in the cytosol and the specific AFB1 protein properties themselves. Furthermore, cytosolic AFB1 mediates rapid auxin responses without forming SCF machinery, leaving the mechanism of AFB1-mediated rapid responses an exciting topic for future investigations. This project was funded by the European Research Council Advanced Grant (ETAP-742985).

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