The phytohormone auxin (Aux) is a principal endogenous developmental signal in plants. It mediates transcriptional reprogramming by a well-established canonical signalling mechanism. TIR1/AFB auxin receptors are F-box subunits of an ubiquitin ligase complex; after auxin perception, they associate with Aux/IAA transcriptional repressors and ubiquitinate them for degradation, thus enabling the activation of auxin response factor (ARF) transcription factors^1–3. Here we revise this paradigm by showing that without TIR1 adenylate cyclase (AC) activity⁴, auxin-induced degradation of Aux/IAAs is not sufficient to mediate the transcriptional auxin response. Abolishing the TIR1 AC activity does not affect auxin-induced degradation of Aux/IAAs but renders TIR1 non-functional in mediating transcriptional reprogramming and auxin-regulated development, including shoot, root, root hair growth and lateral root formation. Transgenic plants show that local cAMP production in the vicinity of the Aux/IAA–ARF complex by unrelated AC enzymes bypasses the need for auxin perception and is sufficient to induce ARF-mediated transcription. These discoveries revise the canonical model of auxin signalling and establish TIR1/AFB-produced cAMP as a second messenger essential for transcriptional reprograming.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of many plant responses. Here, we show that an amino acid search motif based on annotated adenylate cyclases (ACs) identifies 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3 and At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally with two different methods. Given that the in vitro activity is low (fmoles cAMP pmol⁻¹ protein min⁻¹), but highly reproducible, we term the enzyme a crypto-AC. Our results are consistent with a role for ACs with low activities in multi-domain moonlighting proteins that have at least one other distinct molecular function, such as catalysis or ion channel activation. We propose that crypto-ACs be examined from the perspective that considers their low activities as an innate feature of regulatory ACs embedded within multi-domain moonlighting proteins. It is therefore conceivable that crypto-ACs form integral components of complex plant proteins participating in intra-molecular regulatory mechanisms, and in this case, potentially linking cAMP to ABA synthesis.

A variety of plant cellular activities are regulated through mechanisms controlling the level of signal molecules, such as cyclic nucleotides (cNMPs, e.g., cyclic adenosine 3′:5′-monophosphate, cAMP, and cyclic guanosine 3′:5′- monophosphate, cGMP) and calcium ions (Ca2⁺). The mechanism regulating cNMP levels affects their synthesis, degradation, efflux and cellular distribution. Many transporters and the spatiotemporal pattern of calcium signals, which are transduced by multiple, tunable and often strategically positioned Ca2⁺-sensing elements, play roles in calcium homeostasis. Earlier studies have demonstrated that while cNMPs and Ca2⁺ can act separately in independent transduction pathways, they can interact and function together. Regardless of the context, the balance between Ca2⁺ and cNMP is the most important consideration. This balance seems to be crucial for effectors, such as phosphodiesterases, cyclic nucleotide gated channels and cyclase activity. Currently, a wide range of molecular biology techniques enable thorough analyses of cellular cross talk. In recent years, data have indicated relationships between calcium ions and cyclic nucleotides in mechanisms regulating specific signaling pathways. The purpose of this study is to summarize the current knowledge on nucleotide-calcium cross talk in plants.

Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein.