Auxin (indole-3-acetic acid, IAA), as one of the most important plant hormones, regulates almost all important processes in plants from embryonic development to reproduction. The study of auxin signal transduction has always been at the forefront of botany and has provided clues for revealing other signaling pathways.
The classic auxin signal transduction model written into textbooks has dominated research in this field for nearly two decades since its establishment: the model believes that IAA binds to and promotes the interaction between the receptor TIR1/AFB and the co-receptor Aux/IAA, and the E3 ubiquitin ligase activity of TIR1 promotes the ubiquitination and degradation of Aux/IAA, relieves its inhibitory effect on ARF transcription factors, and ultimately activates downstream gene expression.
This classic theoretical model believes that the degradation of Aux/IAA protein is a sufficient and necessary condition for mediating downstream auxin transcriptional responses.
Although the academic community has discovered non-classical mechanisms such as the ABP1/ABLs-TMKs-mediated extracellular auxin signaling pathway and IAA directly regulating the ARF3 transcription factor, the TIR1-Aux/IAA-ARF pathway is still widely considered to be the core pathway that mediates most auxin effects. This classic theoretical model has remained largely unchanged in the past two decades.
In 2022, Jiří Friml's team at the Institute of Science and Technology Austria (ISTA) revealed that the TIR1/AFB receptor family has adenylate cyclase (AC) activity, which can regulate root growth inhibition and gravitropism by catalyzing ATP to generate cyclic adenosine monophosphate (cAMP), opening up a new research direction for the study of auxin transcriptional regulation pathways. This previous study reported for the first time the AC activity of TIR1/AFB and its importance to its transcriptional regulation function.
However, the relationship between the AC activity of TIR1/AFB and the E3 ubiquitin ligase activity is not clear, and whether the product of AC activity, cAMP, does play the role of a second messenger in the auxin signal transduction pathway remains to be confirmed.
Recently, Jiří Friml's team published a research paper titled "TIR1-produced cAMP as a second messenger in transcriptional auxin signalling" in Nature. The results of this study revised the classic auxin signal transduction model, indicating that the degradation of Aux/IAA is not a sufficient and necessary condition for mediating downstream transcriptional regulation, and TIR1 AC activity and its product cAMP play an indispensable role in auxin signal transduction. This study also used genetic evidence to confirm the historical controversy that cAMP is the second messenger of plant cells.
Since TIR1/AFB has both ubiquitin ligase activity and AC activity, does the AC activity mutation affect the ubiquitin ligase activity of TIR1/AFB?
To clarify this point, the authors used TIR1 AC mutants (TIR1ACm1 and TIR1ACm3, which eliminated AC activity in vitro) and introduced them into the cvxIAA/ccvTIR1 engineering system. The engineered IAA analog cvxIAA specifically binds to the modified ccvTIR1 receptor, circumventing cross-interference of endogenous TIR1/AFB. The authors crossed the R2D2 reporter strain used to indicate Aux/IAA degradation into the ccvTIR1 and its ccvTIR1ACm mutant transgenic lines to track the TIR1 ubiquitin ligase activity.
The results showed that cvxIAA treatment had no effect on pTIR1::TIR1, but led to specific degradation of the DII signal in the pTIR1::ccvTIR1 line. Importantly, cvxIAA-induced DII degradation occurred normally in pTIR1::ccvTIR1ACm1 and pTIR1::ccvTIR1ACm3 lines. This shows that the AC activity of TIR1/AFB is independent of the SCFTIR1 E3 ubiquitin ligase activity. The normal degradation of Aux/IAA also directly indicates that TIR1ACm1 and TIR1ACm3 mutations do not affect TIR1's perception of IAA, IAA-induced TIR1-Aux/IAA interaction, and SCFTIR1 complex assembly.
These results indicate that the AC activity of TIR1/AFB is not required for IAA-induced Aux/IAA degradation, and also indicate that Aux/IAA degradation is not a sufficient condition for mediating downstream auxin transcriptional responses, suggesting that cAMP may also play an important role in this process.
The authors further examined the effect of Aux/IAA on the AC activity of TIR1/AFB. AFB5, co-receptors AXR2 (IAA7) and AXR3 (IAA17) and their gain-of-function DII domain point mutants (axr2 and axr3) were recombinantly expressed and purified. Wild-type AXR2/3 and IAA synergized to significantly enhance the AC activity of AFB5, while the axr2/axr3 mutants completely lost their ability to activate AC activity due to the inability to form a functional TIR1/AFB-Aux/IAA complex.
This phenomenon suggests that Aux/IAA not only serves as a degradation substrate for SCFTIR1, but its dynamic binding to TIR1/AFB itself is a necessary condition for activating AC activity.
For in vivo verification, the authors constructed an inducible XVE>>axr3-mCherry transgenic line. Previous studies have shown that the accumulation of axr3-mCherry protein not only inhibits the transcription level of endogenous Aux/IAA through a dominant negative effect, but also directly leads to the complete disappearance of the DII-Venus reporter signal, indicating that the induction of axr3-mCherry expression results in a state of endogenous Aux/IAA protein deficiency.
Correspondingly, with the accumulation of axr3-mCherry, IAA-induced cAMP production is significantly inhibited, proving that the enhancement of AC activity depends on the functional interaction between Aux/IAA and TIR1.
It is worth noting that this finding provides a new explanation for traditional genetic observations: the severe developmental defects caused by typical Aux/IAA gain-of-function mutants (such as shy2 (IAA3), bdl (IAA12) and axr3 (IAA17)) are not only due to the continuous inhibition of ARF transcription factor activity by Aux/IAA, but also likely involve the systemic blockade of the TIR1 AC-cAMP signaling pathway.
These results also reveal the dual role of Aux/IAA: it is both a core component of the ubiquitination degradation pathway and a key switch for regulating AC activity.
Figure 1. TIR1 AC activity is not required for IAA-induced Aux/IAA degradation. (Chen, et al., 2025)
Since Aux/IAA can be degraded normally, what effect will the TIR1 AC mutant that cannot produce cAMP have on IAA-mediated transcriptional regulation?
To answer this question, the authors introduced the DR5::Luc reporter system used to indicate auxin transcriptional response into pTIR1::TIR1, pTIR1::ccvTIR1 and their AC mutant strains. The results showed that cvxIAA activated DR5::Luc signal only in pTIR1::ccvTIR1, but was significantly weakened in pTIR1::ccvTIR1ACm1 and completely disappeared in pTIR1::ccvTIR1ACm3.
RNA-seq and RT-qPCR also further confirmed that TIR1 AC activity is indispensable for global transcriptional reprogramming. Crucially, under the condition that the Aux/IAA degradation pathway operates normally (verified by systems such as R2D2), the loss of TIR1 AC activity still leads to a comprehensive blockage of the auxin transcriptional response, proving that cAMP is an essential molecular mediator that connects TIR1 with ARF-mediated transcriptional reprogramming.
Jiří Friml's team previously reported that the TIR1 AC activity is involved in regulating root growth inhibition and gravitropism. Here, the authors further explored whether the TIR1 AC activity is widely involved in various growth and development mediated by IAA and whether it is universal.
The authors used the complementation line of the tir1 afb2 mutant (pTIR1::TIR1 and its AC mutant) to explore the effect of AC activity on IAA-regulated growth and development. The experiment showed that pTIR1::TIR1ACm could not restore the lateral root formation and root hair development defects of the tir1 afb2 mutant.
Further phenotypic analysis clarified that the TIR1 AC activity is not only the key to mediating root growth inhibition, but also necessary for classic auxin responses such as lateral root formation, root hair growth and hypocotyl elongation, suggesting that the role of cAMP produced by AC activity in all developmental functions mediated by auxin transcriptional regulation may be universal.
Even if Aux/IAA is degraded normally, the loss of TIR1 AC activity still leads to transcriptional reprogramming and plant growth and development defects. So what role does cAMP play in the auxin signaling pathway? Is it a key second messenger independent of Aux/IAA degradation, and can it directly activate auxin signaling?
To verify this hypothesis, the authors genetically fused the characterized active AC domain (KUP5/LRRAC1) that is not related to the auxin signaling pathway with axr3 (a dominant suppressor mutant of IAA17 that escapes degradation due to its inability to interact with TIR1 but still retains the ability to bind to and inhibit ARF transcriptional activity) to construct an inducible transgenic strain. The experimental data showed that induced expression of axr3-KUP5 or axr3-LRRAC1 can specifically activate the DR5::Luc reporter system, mimicking the transcriptional response effect induced by IAA, while axr3-KUP5m fused with the AC inactive mutant has no such effect.
Further phenotypic analysis showed that axr3-KUP5 could restore the defects of root formation, lateral root development and root hair growth caused by axr3 accumulation, while axr3-KUP5m had no such effect and showed consistent growth defects with axr3. Fusion of LRRAC1 caused axr3 to be partially localized in the cytoplasm, weakening the inhibitory effect of axr3 on auxin signaling, but axr3-LRRAC1 with AC activity could still promote lateral root development.
These results confirmed that local cAMP production can bypass the perception of IAA by TIR1 receptors and the degradation pathway of Aux/IAA, directly activate ARF-mediated transcriptional programs, confirm the core role of cAMP as an independent second messenger, and prove that Aux/IAA degradation is not a necessary condition for auxin transcriptional regulation response.
In summary, this study revised the traditional auxin signaling model: cAMP produced by TIR1/AFB receptors through their AC activity is the core mediator of signal transduction, and simple Aux/IAA protein degradation is not sufficient to fully activate downstream transcriptional responses.
Key evidence shows that when the Aux/IAA degradation pathway is intact, the TIR1 mutant with AC activity loss still cannot mediate auxin-induced transcriptional activation and growth and development phenotypes.
In vivo experiments confirmed that the production of cAMP near Aux/IAA-ARF by inducing the expression of artificially constructed fusion proteins can bypass TIR1/AFB's perception of IAA and the degradation pathway of Aux/IAA, directly activate ARF transcription factor-mediated transcriptional programming, and achieve the biological effect of auxin simulation.
Figure 2. New insights into the auxin signaling pathway. (Chen, et al., 2025)
This study not only confirmed the biological status of cAMP as a second messenger in plants through genetic evidence, but also revised the theoretical framework of the classical auxin signal transduction pathway. It is worth noting that the core argument in the traditional model that "Aux/IAA degradation is a necessary and sufficient condition for mediating transcriptional responses" has thus become a scientific issue that needs to be re-examined, opening up a new theoretical exploration direction for subsequent research.
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