**6. Conclusions**

cAMP is the object of intense scientific interest, both in animal systems, where much more progress was achieved in defining its role, and in plants, becoming lately the centre of a bustling research. cAMP is nowadays recognised as a relevant signalling molecule in plant development as well as in responses to environmental stimuli, of both biotic and abiotic nature. As cAMP-signalling networks and their spatial and temporal regulation are extremely complex, future research must deal with the nature of cAMP signals in terms of strength, duration and frequency, considering also the crosstalk between this second messenger and other intracellular regulators [139]. Since the existence of cAMP-regulated processes in plants and the first evidence of compartmentalised cAMP signals in animals, the need for reliable cAMP detection methods able to reveal cAMP waves in living systems arose. Recent advances in modern biotechnologies and synthetic biology, alongside newly developed detection methods and instrumentations, o ffer a wide range of possibilities to unravel cAMP role in living cells.

The cAMP-sponge represents a cutting-edge genetically encoded tool, used to exploit cAMP fluctuations for the first time in living plant organisms and specific cell compartments. It overcomes major concerns on biochemical assays and pharmacological studies performed so far in plants [31–33]. Other developed genetically-encoded tools employed in bacteria and isolated plant cells are the promoter reporter systems, based on the plant protein Oligopeptide TransporterX promoter, which measure alterations in downstream gene expression following changes in intracellular levels of cyclic nucleotides. Unfortunately, this system cannot discriminate between cGMP and cAMP [140].

Taking advantage of the progress reached in animal systems, many other strategies and their combination may help in elucidating cAMP signalling in plant systems. Indeed, optogenetic approaches and genetically encoded fluorescent biosensors are e ffectively used to monitor and modulate cAMP levels [141,142]. Photoactivated ACs and light-regulated PDEs, or even their association, are successfully used in animal cells [143,144]. The generation of stable plant lines, expressing the combination of optimised sensors for cAMP and concomitant or downstream messengers, may provide a comprehensive view of the signalling event investigated.

Another important requirement is a clear identification and functional characterisation of cAMP-binding proteins involved in the signalling of this second messenger. Nowadays, many lines of evidence indicate that, in plants, the conversion of cAMP into Ca2+ signals via CNGCs is the main signalling mechanism of this cyclic nucleotide. However, although indications for bona fide PKA are lacking, its presence in plants cannot be excluded. New bioinformatics algorithms and molecular tools may provide opportunities to extend the presently scarce knowledge of cAMP-dependent protein kinases [16,23]. Moreover, studies on cAMP-dependent changes in transcriptomes, proteomes and phosphoproteomes, as well as metabolomes, will improve the understanding of cAMP involvement in plant physiological processes, along with acclimation to adverse environmental conditions.

**Author Contributions:** Conceptualisation, M.C.d.P. and E.B.; writing—original draft preparation, E.B., S.F., L.V. and M.C.d.P.; writing—review and editing, E.B., S.F., L.V. and M.C.d.P.; visualisation, S.F.; supervision, M.C.d.P. funding acquisition, E.B. and M.C.d.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by CNR, gran<sup>t</sup> number FOE-2019 DBA.AD003.139, and by University of Bari Aldo Moro, gran<sup>t</sup> number H95E10000710005.

**Acknowledgments:** The authors thank M. Hofer (Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School of Massachusetts, USA) for kindly providing "cAMP-sponge".

**Conflicts of Interest:** The authors declare no conflict of interest.
