The cAMP second messenger in auxin signalling

Auxin is one of the most important phytohormones, playing a crucial role in plant growth and development. Genetic studies in Arabidopsis have identified core components of auxin signalling and established the classical transcriptional regulation mechanism. This classical paradigm recognizes TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN SIGNALLING F-BOX (AFB) proteins as auxin receptors possessing E3 ubiquitin ligase activity. Auxin functions as a "molecular glue" enhancing TIR1/AFB-Aux/IAA (AUXIN/INDOLE-3-ACETIC ACID) inhibitor interactions, leading to Aux/IAAs’ ubiquitination and degradation. This process releases AUXIN RESPONSE FACTORs (ARFs) from repression, enabling transcriptional activation of auxin-responsive genes. Notably, the discovery of adenylate cyclase (AC) activity in TIR1/AFBs, catalysing cAMP production from ATP, presents novel transcriptional regulatory possibilities, though cAMP's mechanistic role in auxin signalling remains unclear. Beyond transcriptional regulation (slow response) a non transcriptional pathway (rapid response) mediated by AFB1 also exists, as auxin triggers rapid root growth inhibition within 30 seconds. How TIR1 and AFB1, with their distinct subcellular localizations, differentially regulate transcriptional and non-transcriptional processes remains unclear. To address these two key questions, this study conducted the following investigations. TIR1 and AFB1 display distinct subcellular localization patterns, with AFB1 specifically mediating auxin-induced rapid root growth inhibition. To examine whether subcellular localization differences contribute to functional divergence between TIR1 and AFB1, we utilized microfluidic systems combined with genetic complementation assays. Nuclear export signal-fused TIR1 failed to rescue the rapid response defects observed in afb1 mutants. In contrast, nuclear localization signal-fused AFB1 exhibited dual functional deficiencies: it neither restored the rapid response impairment in afb1 mutants nor compensated for transcriptional regulation defects in tir1 mutants which limitation stemmed from AFB1's intrinsic incapacity to assemble functional SCF complexes. These results collectively establish that the functional specialization of TIR1 and AFB1 is governed by both their subcellular compartmentalization and inherent biochemical characteristics. The mechanism by which cAMP derived from the auxin receptor TIR1/AFB modulates transcriptional regulation remains undefined. To address this, we genetically dissociated TIR1 AC activity from its E3 ubiquitin ligase function to investigate cAMP's signalling role. AC inactivating mutations preserved TIR1-mediated Aux/IAAs’ degradation but abolished ARF i dependent transcriptional activation. tir1afb2 mutants expressing AC-deficient TIR1 variants failed to restore transcriptional responses. Crucially, compartmentalized cAMP accumulation within Aux/IAA - ARF complexes vicinity triggered auxin signalling independently of both auxin perception and Aux/IAAs’ degradation, confirming its secondary messenger identity. Further mechanistic analysis demonstrated cAMP directly interact with ARF. cAMP directly promotes ARF dissociation from Aux/IAA repressors and enhances ARF homodimerization. Genetic complementation assays revealed that ARF mutants lacking conserved cAMP-binding motifs exhibited partial functional recovery, providing direct evidence for cAMP-ARF physical interaction during signal propagation. These findings collectively challenge the established model of auxin signalling and define a novel TIR1-cAMP-ARF regulatory axis, substantially advancing our comprehension of auxin-mediated physiological regulation.