The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana

Abstract

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs 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, are 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. 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 while it regulates rapid changes in cell growth that contribute to root gravitropism.

Introduction

Auxin rapidly inhibits root growth via a non-transcriptional signaling pathway. This rapid growth response is critical for gravitropism and is accompanied by several cellular responses such as apoplastic alkalization, membrane depolarization and very rapid Ca²⁺ influx into the cytoplasm^1,2. Among members of the TIR1/AFB auxin receptor family, AFB1 was reported to mediate the rapid root growth inhibition^3,4. Further, the loss of AFB1 alone was sufficient to result in a significant defect in rapid root growth inhibition^3,4, indicating its dominant role in this process.

Results and Discussion

Consistent with these findings, we confirmed that the afb1 mutant is resistant to auxin during the first 20 minutes of treatment whereas the tir1 mutant is similar to wild type (Fig. 1a, b). As time progressed, the level of afb1 mutant resistance decreased while that for tir1 increased (Fig. 1b). This behavior is consistent with the roles of AFB1 and TIR1 in the nongenomic and transcriptional response, respectively. To test the role of receptors other than AFB1 in the rapid response, we measured auxin-induced root growth inhibition and the dynamics of the gravitropic response in the tir1afb2 and tir1afb345 mutants. Both the mutants responded to IAA similarly to wild type (Fig 1c). Previously, the tir1afb345 mutant was shown to be resistant in the early phase³. The basis for this difference is unknown but it might be related to the highly variable phenotype of tir1afb345 seedlings. Consistent with the growth inhibition, the afb1 mutant showed a delay in early gravitropic response, however tir1afb2 and tir1afb345 mutants responded with a similar dynamics to the Col-0 control (Fig. 1d; Supp. movie 1).

Figure 1: AFB1 triggers the early phase of auxin-dependent rapid root-growth inhibition.

a) Root growth (μm) of Col-0, afb1–3, and tir1–1 seedlings in response to mock (ethanol) or IAA treatment (10 nM). Root growth was measured every 25 seconds for 20 minutes. Col-0-mock (n=11), Col-0-IAA (n=13), afb1-mock (n=7), afb1-IAA (n=11), tir1-mock (n=12), tir1-IAA (n=12). Mean ± s.d. (s.d. represented as error bars).

b) Root growth response (growth of treated roots normalized to growth of the respective mock-treated roots) to IAA (10 nM) for 20 minutes, 1 hr, 3hr and 6hr.

c) Root growth response to IAA (10nM; 15 min) of Col-0, afb1, tir1afb2, and tir1afb345 seedlings.

d) Gravitropic response of Col-0, afb1, tir1afb2, and tir1afb345 roots. Mean root tip angle ± s.d. (represented as error bars), time after a 90° gravistimulation is indicated, n=10–14 individual roots.

e) Schematics of ccvAFB1 receptor design. Highlighted amino acid is the substitution in TIR1 and AFB1 that confers cvxIAA binding.

f) In vitro pull-down assays of ccvAFB1-mScarlet by IAA7-DII peptide co-incubated with mock (m) 10 μM cvxIAA or 10 μM IAA. Input is shown (IN), ccvAFB1-mScarlet detected by anti-mCherry antibody. Estimated molecular weight of ccvAFB1-mScarlet = 92 kD. Uncropped membrane is shown in supplementary figure 4.

g) Subcellular localization of ccvAFB1-mScarlet and ccvTIR1-mScarlet (green) in Arabidopsis root tips counterstained with propidium iodide (magenta); both constructs controlled by pTIR1 promoter. Scale bars = 50 μm.

h) Root growth after 15-minute treatment with mock, IAA (10 nM), cvxIAA (500 nM) or their combination in Col-0, afb1–3, and ccvAFB1 (afb1–3). Only ccvAFB1 roots respond to cvxIAA.

Boxplots in b, c, h represent the median and the first and third quartiles, and the whiskers extend to minimum and maximum value; all data points are shown as dots. Statistical difference according to Ordinary one-way ANOVA coupled with Tukey’s multiple comparison tests (p<0.05) indicated by letters.

To exert greater control of the rapid auxin response, we prepared Arabidopsis thaliana lines expressing fluorescent protein-tagged versions of the synthetic receptor-ligand system – ccvTIR1 and ccvAFB1 controlled by the pTIR1 promoter. The ccv (concave) receptor versions are ‘blind’ to the natural auxin IAA. Instead, they bind the synthetic cvxIAA (convex IAA⁵) (Fig. 1e,f), and show similar subcellular localization to the native proteins (Fig. 1g). The ccvAFB1 protein was sufficient to trigger the rapid root growth inhibition when seedlings were treated with cvxIAA (Fig. 1h). In previous studies it was shown that the cvxIAA-ccvTIR1 pair triggers a response that is several minutes delayed in comparison to the effect of natural IAA^2,6. In our experiments, the cvxIAA – ccvTIR1 pair triggers a response that is significantly slower⁴ than the cvxIAA – ccvAFB1 system. Taken together, these results clarify previous discrepancies regarding the overlapping function of F-box receptors in the context of rapid auxin responses and suggest that AFB1 is the primary receptor for the rapid auxin responses.

One of the earliest detectable responses to auxin in the root is the influx of calcium⁷. A mutant lacking the CNGC14 calcium channel shows a delay in the gravitropic response¹ that resembles the afb1 gravitropic defect⁴, hinting at a role for AFB1 in auxin-dependent calcium influx. We therefore visualized cytosolic calcium levels in the afb1 mutant using the R-GECO1 sensor⁸ and vertical microfluidic microscopy⁴. The control line showed an almost immediate elevation in cytosolic calcium (calcium transient) in response to 150 nM IAA, as described before^1,9,10 (Fig. 2a, b). Strikingly, in afb1 roots, cytosolic calcium did not increase after auxin application; instead, a mild calcium increase occurred only ca. 240 seconds after the treatment (Fig. 2a,b, Supp. movie 2). This indicates that the AFB1 receptor is required for the auxin-induced calcium transient.

Figure 2: AFB1 controls auxin-dependent calcium transients in Arabidopsis primary roots.

a) A kymograph showing auxin-induced R-GECO1 intensity increase indicating cytosolic calcium ([Ca²⁺]_cyt) transients after application of 150 nM IAA (arrow) in Col-0 and afb1–3 root epidermal cells. Fluorescence intensity look-up table is indicated.

b-d) Quantification of R-GECO1 intensity indicating [Ca²⁺]_cyt transients. b) Response to 150 nM IAA (arrow) in Col-0 and afb1–3 root epidermal cells. c) Response of ccvAFB1-mVenus, ccvTIR1-mScarlet and Col-0 root epidermal cells to 500 nM cvxIAA. d) Response of tir1afb2 root epidermal cells to 150 nM IAA, shown with positive (Col-0) and negative control (afb1–3). b-d) Normalized mean R-GECO1 fluorescence intensity F/F₀ ± s.d. (represented as shaded areas). Auxin treatment is indicated by arrows.

To test whether AFB1 is upstream and sufficient for the calcium transient, we introduced the RGECO-1 sensor into the ccvAFB1 and ccvTIR1 lines. While in both the control and the ccvTIR1 line, the application of 500 nM cvxIAA did not elicit a detectable calcium transient (Fig.2c), the compound triggered an immediate calcium transient in the ccvAFB1 line (Fig.2c, Supp. movie 3). In contrast to these results, cvxIAA has been reported to trigger a calcium spike in a ccvTIR1 line expressing the GCaMP3 sensor¹¹. We speculate that cvxIAA might trigger a transient detectable by GCAMP. However, the difference between control, ccvTIR1 and ccvAFB1 R-GECO-1 lines clearly shows that ccvAFB1 is required and sufficient for cytosolic calcium increase (Fig.2). Finally, to determine if AFB1 is sufficient to trigger calcium influx in the case of the native receptor, we analyzed IAA-induced calcium transient in the tir1afb345 and tir1afb2 mutants. Unfortunately, the RGECO-1 construct was silenced in the tir1afb345 line. On the other hand, the tir1afb2 mutant responded to IAA treatment with a calcium transient comparable to the wild-type control (Fig. 2d, Supp. movie 4). These results show that AFB1 is upstream of the auxin-induced calcium transient that triggers the rapid growth responses including early root gravitropic responses^1,4,9. It is intriguing that AFB1 has recently be shown to have adenyl cyclase activity¹¹. Thus, it is possible that AFB1-mediated cAMP production triggers the activity of the CNGC14 channel.

Despite the apparent differences in their modes of action, TIR1 and AFB1 are the most recently diverged members of the TIR1/AFB family in Arabidopsis³. To determine if their functional specificity is related to their expression pattern, we expressed TIR1 in the AFB1 expression domain. The pAFB1:TIR1-mCitrine expression pattern mimicked the AFB1 expression pattern³ (Fig. S1a). However, the transgene failed to rescue the afb1 phenotype (Fig. S1b) even though the levels of AFB1 and TIR1 proteins were similar in these lines (Fig. S1g). As expected, the pAFB1:AFB1-mCitrine transgene (gAFB1 #1 and gAFB1 #2) complemented the afb1 phenotype. Interestingly, one of the AFB1 complementation lines, gAFB1 #2, exhibited a higher expression level than either the wild-type or gAFB1 #1 (Fig. S1c,d) and showed a hypersensitive rapid response to auxin (Fig. S1e). These data demonstrates that the functional differences between TIR1 and AFB1 are related to differences in their protein sequences, rather than expression pattern.

We previously showed that the TIR1/AFB proteins are partitioned between the cytoplasm and the nucleus. Interestingly, AFB1 is both the most abundant member of the family and highly enriched in the cytoplasm, while TIR1 is primarily nuclear³. To determine if subcellular localization is decisive for function in the rapid response, we added either a NUCLEAR LOCALIZATION SEQUENCE (NLS) or NUCLEAR EXCLUSION SEQUENCE (NES) to the AFB1-mCitrine receptor (gAFB1-NLS and gAFB1-NES). The resulting fusion proteins were highly enriched in the nucleus and cytoplasm respectively as expected (Fig. 3a). Intriguingly, only AFB1-NES rescued the phenotype of afb1 (Fig. 3b), demonstrating that AFB1 must be localized to the cytoplasm to mediate rapid root growth inhibition. In a complementary approach, we attempted to generate gTIR1-NES-mCitrine plants but failed to recover lines with significant levels of TIR1 accumulation despite the presence of high transcript levels (Fig. S1f,g). It is possible that TIR1 is particularly unstable in the cytoplasm.

Figure 3: Cytoplasmic AFB1 regulates rapid root growth.

a) Subcellular localization of AFB1-NLS-mCitrine and AFB1-NES-mCitrine (yellow) in afb1–3; roots stained with propidium iodide (magenta). Scale bars = 100 μm.

b) Root growth response (growth of treated roots normalized to growth of the respective mock-treated roots) of Col-0, afb1–3, gAFB1 (afb1–3) and gAFB1-NLS/NES #1, #2 (afb1–3) to IAA (10 nM; 20 minutes).

c) The ccvTIR1-E12K-mScarlet (Col-0) protein (green) localizes to nuclei. Stained with propidium iodide (magenta), scale bar = 50 μm.

d) Root growth rate of ccvTIR1-mScarlet (Col-0) and ccvTIR1-E12K-mScarlet (Col-0) in mock or 500 nM cvxIAA treated seedlings.

e) Subcellular localization of AFB1, AFB1(K8E), TIR1, and TIR1(E12K)-GFP in Col-0 Arabidopsis protoplasts (left) and quantification of relative nuclear localization (right), calculated as the ratio of nuclear fluorescence to total cell fluorescence. f) Root growth response of Col-0, afb1–3, cul1–6 and cul1–7 to IAA (10 nM; 20 minutes).

g) Expression pattern and subcellular localization of AFB1-mCitrine in Col-0 and cul1–6 background. Scale bar = 100 μm.

Boxplots in b, d, e, f represent the median and the first and third quartiles, and the whiskers extend to minimum and maximum value; all data points are shown as dots. Letters indicate statistical differences according to Ordinary one-way ANOVA coupled with Tukey’s multiple comparison tests (p<0.05).

It has been reported that a polymorphism within the F-box domain of AFB1 (K at position 8 rather than E) strongly reduces its ability to interact with CUL1 and assemble into an SCF complex¹². The TIR1 E12K mutation, recapitulating AFB1, significantly reduces the interaction with CUL1 and results in a strong auxin-resistant phenotype¹². To test whether the differential affinity of TIR1 and AFB1 for CUL1 determines their distinct subcellular localization and function, we prepared the E12K version of ccvTIR1 to mimic the weak binding affinity of AFB1 with CUL1. Surprisingly, the protein still localized to the nucleus (Fig. 3c), interacted with the degron domain of Aux/IAA7 in a cvxIAA-dependent manner in vitro (Fig. S2b), but was unable to inhibit root growth in response to cvxIAA (Fig. 3d). We also expressed TIR1 E12K and AFB1 K8E in Arabidopsis protoplasts; these amino acid substitutions did not impact cellular localization (Fig.3e). These results indicate that the association with the SCF complex is not required for the co-receptor assembly and does not determine cellular localization.

As AFB1 does not bind CUL1 efficiently, we tested the possibility that AFB1 functions independently of an SCF complex by examining the rapid auxin response in the cul1–6 and cul1–7 mutants. CUL1 is an essential gene during embryogenesis but these two hypomorphic alleles are viable and auxin resistant in a long-term root growth assay^13,14. Both the cul1–6 and cul1–7 mutants exhibited a normal rapid response (Fig. 3f). In addition, AFB1 localization was not altered in the cul1–6 mutant, confirming that AFB1 localization is not regulated by interaction with CUL1 (Fig. 3g). Similarly, recently published proteomic data² confirms that TIR1, but not AFB1, interact with CUL1 in an IP-MS experiment, despite the relative abundance of AFB1. In contrast, both TIR1 and AFB1 interact with ASK1 as expected since this had been shown previously¹². These results suggest that CUL1 binding and presumably SCF complex formation are not required for AFB1-triggered rapid root growth inhibition.

The well-known substrates for SCF^TIR1/AFB are the Aux/IAA transcriptional repressors. AFB1 has been shown to interact with several members of this family, either in Y2H assays or in plants¹⁵. We tested the axr2–1 and shy2-2 mutants in the rapid response assay. These two lines have mutations in the DII region of IAA7 and IAA3 respectively, that act to stabilize the protein and confer auxin resistance in long term root growth assays^16,17. Neither mutant exhibited a significant change in rapid root growth inhibition (Fig. S1h), suggesting that Aux/IAA proteins do not contribute to the rapid response. Although these results suggest that the Aux/IAAs may not be involved in the rapid response, it is important to note that there are 28 members in the family and it is possible that one or more of these have specialized function in the cytoplasm.

To identify the domains responsible for AFB1-specific localization and function, we generated a set of TIR1/AFB1 domain swap constructs under control of the AFB1 promoter and introduced them into the afb1 mutant. The chimeric proteins are named according to the origin of each segment; T for TIR1, and A for AFB1 (Fig. 4a). As shown earlier, AFB1 (or AAAA) was localized to both nucleus and cytoplasm, and rescued the afb1 phenotype, while TIR1 (TTTT), was largely localized to the nucleus and failed to restore the afb1 defect (Fig. 4b,c). Interestingly, among the 4 chimeric proteins, only TAAA was abundant in the nucleus similar to TIR1, and failed to restore the afb1 mutant sensitivity to auxin. The ATAA, AATA, and AAAT chimeric proteins localized to both the nucleus and cytoplasm (Fig. 4b) and restored auxin sensitivity to the mutant (Fig. 4c). These results indicate that the N-terminal segment of AFB1 is important for AFB1’s cytoplasmic localization and function. This region includes the F-box domain that mediates the interaction between F-box proteins, ASK proteins and CUL1, and the N-terminal part of the Leucin-rich repeat domains (LRR) that participates in auxin and/or Aux/IAA binding^15,18 (Fig. 4a). We therefore created two additional chimeric proteins. One contained the entire region 1 (iATTT) while the second contained only the F-box from AFB1 (fbATTT) (Fig. 4a) and expressed them under the control of pAFB1 and pTIR1 promoters, respectively. Note that both pTIR1 and pAFB1 are active in the epidermis³ and iATTT and fbATTT are both localized to nucleus and cytoplasm (Fig.4d). However, only iATTT rescued the afb1 phenotype (Fig. 4e), and elicited a calcium transient similar to that of wild-type (Fig. 4f,g; Supp. movie 5). The iATTT showed a patchy expression, and interestingly, we only observed calcium transients in iATTT expressing cells demonstrating the cell-autonomous nature of the auxin-triggered calcium transient. To corroborate the results, we created the ccv-fbATTT version of the receptor, which also showed cytoplasmic and nuclear localization (Fig. S2a), and interacted with the degron domain of Aux/IAA7 in a cvxIAA-dependent manner in vitro (Fig. S2b). However, like fbATTT, ccv-fbATTT failed to trigger root growth inhibition in response to cvxIAA (Fig.S2c). Finally, consistent with the previous results, the fbATTT line failed to restore the early gravitropic response of afb1 mutant, while iATTT almost completely recovered the response (Fig. 4g, Supp.movie6). The lack of full complementation can be explained by the patchy expression of iATTT in the root tip (Fig. 4f).

Figure 4. The N- terminal region of AFB1 is crucial for its role in the rapid auxin response.

a) Schematic diagram of domain swap AFB1 (A-yellow) and TIR1 (T-blue) constructs. Numbers indicate amino acid position. Chimeric iATTT protein contains the F-box domain (magenta in 3D structure) and adjacent sequences in the LRR region (yellow in 3D structure) from AFB1, while the chimeric fbATTT protein contains only the AFB1 F-box domain (magenta in 3D).

b) Expression pattern and subcellular localization of AFB1 (AAAA), TIR1 (TTTT) and chimeric proteins (TAAA, ATAA, AATA, AAAT). All domain swap proteins were regulated by the AFB1 promoter in the afb1–3 background. Scale bar=100 μm, stained with propidium iodide (magenta).

c) Root growth response of Col-0, afb1–3, and domain swap lines to IAA (10 nM, 20 minutes); growth of treated roots normalized to growth of the respective mock-treated roots.

d) Expression pattern and subcellular localization of iATTT-mCitrine and fbATTT-mScarlet proteins. The iATTT and fbATTT proteins were regulated by the AFB1 and TIR1 promoters respectively in the afb1–3 background. Scale bar=100 μm.

e) Root growth response of Col-0, afb1–3, iATTT (afb1–3) and fbATTT (afb1–3) roots to IAA (10 nM) for 20 minutes.

f) A kymograph showing auxin-induced cytosolic calcium transients (R-GECO1 intensity) during application of 150 nM IAA (arrow) in iATTT(afb1) and fbATTT(afb1) root epidermal cells. On the top, the iATTT-mCitrine channel highlights the patchy expression of the construct, note that the iATTT-expressing cell (asterisk) also shows high calcium transient. Fluorescence intensity look-up table is indicated.

g) Quantification of R-GECO1 intensity indicating [Ca²⁺]_cyt transients in iATTT and fbATTT root epidermal cells. IAA treatment (150 nM) shown by an arrow. n = 6–11

Boxplots in c,e represent the median and the first and third quartiles, and the whiskers extend to minimum and maximum value; all data points are shown as dots.

h) Quantification of the gravitropic response in Col-0 (n=20), afb1 (n=12), iATTT (n=23), and fbATTT (n=23) roots. Mean root tip angle ± s.d. (represented as error bars) is shown. Time after 90° gravistimulation is indicated.

Boxplots in c, e represent the median and the first and third quartiles, and the whiskers extend to minimum and maximum value; all data points are shown as dots. Letters indicate statistically different groups according to Ordinary one-way ANOVA coupled with Tukey’s multiple comparison tests (p<0.05).

These results show that the F-box domain determines the cytoplasmic/nuclear partitioning of the receptors; that the cytoplasmic localization of the auxin receptor is required but not sufficient for function in the rapid response; and, finally, that the sequences in the N-terminal part of the AFB1 LRR domain are required for the function in the rapid auxin response. Since a clear nuclear-localization signals (NLS) is not present in the F-box domain of TIR1, it is possible that an unknown protein interacting with the F-box domain is involved in the regulation of subcellular localization.

Since AFB1 is abundant in both the cytoplasm and nucleus, we also determined the effects of manipulating AFB1 levels on long term auxin responses that are mediated by canonical auxin signaling. In a long-term root growth assay, we found that the gAFB1-NLS line displayed significant auxin resistance, while the gAFB1-NES line was slightly hypersensitive (Fig. 5a, S3a). Auxin-hypersensitivity of the line expressing AFB1-NES suggests that the rapid AFB1-dependent pathway can also affect auxin response over a longer time frame. However, we could not exclude the possibility that cytoplasmic AFB1 also negatively affects canonical auxin signaling because this effect could be masked by the AFB1-mediated root growth inhibition.

Figure 5. AFB1 negatively regulates canonical auxin signaling.

a) Primary root length of five-day-old seedlings of Col-0, afb1–3, gAFB1 (afb1–3), and gAFB1-NLS/NES (afb1–3) lines treated with either 100 nM IAA or mock (ethanol) for 3 days.

b) Lateral root phenotype in nine-day-old seedlings of Col-0, afb1–3, gAFB1 (afb1–3), and gAFB1-NLS/NES (afb1–3) lines.

c) Number of emerged lateral roots per primary root length in Col-0, afb1–3, gAFB1 (afb1–3) and gAFB1-NLS/NES (afb1–3) lines (#1 and #2 indicates two independent lines).

d) Number of non-emerged primordia at different stages in lateral root developed expressed per primary root length in Col-0, afb1–3, gAFB1 (afb1–3) and gAFB1-NLS/NES (afb1–3) lines (#1 and #2 indicates two independent lines).

e) Expression pattern of gAFB1-mCitrine; pUBQ10:Lti6b-mCherry during lateral root development. pUBQ10:Lti6b-mCherry was used as a plasma membrane marker. Ep; Epidermis, Co; Cortex, En; Endodermis, Pe; Pericycle. Scale bar=100 μm.

Box plots represent the median and the first and third quartiles, and the whiskers extend to minimum and maximum value; all data points are shown as dots. Letters above box plots indicate statistical differences according to Ordinary one-way ANOVA coupled with Tukey’s multiple comparison tests (p<0.05).

Earlier genetic studies suggested that AFB1 may be a negative regulator of LR formation³. Here we show that the afb1 mutant produces slightly more lateral roots than wild type while two AFB1 complementation lines (gAFB1 #1 and #2) produce many fewer lateral roots, confirming this hypothesis (Fig. 5b,c). Interestingly, the lines expressing AFB1-NLS and AFB1-NES also have reduced numbers of LR (Fig. 5b,c). All three lines, gAFB1, gAFB1-NLS and gAFB1-NES, exhibited significantly increased numbers of early stage LR (stages I-II) (Fig. 5d) compared to wild type indicating that AFB1 does not affect LR initiation, but rather suppresses LR emergence, when it is present in either the cytoplasm or nucleus. In addition, we observed that all three AFB1 proteins, AFB1, AFB1-NLS and AFB1-NES are broadly expressed in both developing primordia and its overlaying tissues (Fig. 5e, S3a).

To determine if the role of AFB1 during lateral root development is associated with auxin regulated transcription we examined the expression of the auxin-responsive genes IAA5, IAA6, IAA19 and two lateral root-related genes, LBD16 and LBD29¹⁹. As expected, auxin treatment increased expression of these genes in wild-type seedlings (Fig. S3b). Intriguingly, induction of IAA5, IAA6 and LBD29 was greater in afb1, but suppressed in a dose-dependent fashion in the AFB1 complementation lines (gAFB1 #1 and gAFB1 #2). Moreover, this suppression was also observed in gAFB1-NLS (Fig. S3b). These results are consistent with the lateral root phenotype of these lines. Surprisingly, auxin induction of gene expression was also suppressed in gAFB1-NES seedlings. These data indicate that both nuclear and cytoplasmic AFB1 function as a negative regulator of auxin-mediated transcription, presumably leading to the inhibition of canonical auxin signaling during long-term development.

Taken together, we propose that while cytoplasmic AFB1 induces non-genomic rapid auxin response which is dependent on CNGC14-mediated Ca²⁺ signaling, both nuclear and cytoplasmic AFB1 inhibit canonical auxin signaling. In the case of nuclear AFB1, the protein may act as a dominant-negative in a manner similar to TIR1(E12K)¹². In contrast, how cytoplasmic AFB1 acts to suppress the canonical pathway is unknown. Regardless, this activity may serve to integrate the two auxin responses as the root responds to changing environmental conditions.