Plant cyclic AMP comes in from the cold

Whether or not plant cells contain the growth factor cyclic AMP has been a source of controversy for over 25 years. Now, characterization of an enzyme that produces cAMP and mediates the action of the plant hormone auxin marks a decisive defeat for the majority view.

Cyclic AMP is a key growth factor and signalling molecule. Produced by the enzyme adenylyl cyclase, it mediates the action of many other growth factors and hormones. Yet despite the numerous growth regulators in plant cells — and an evident need for growth regulation during development — it has been dogma for 25 years that higher plants do not possess adenylyl cyclase, do not use cAMP and are, thus, unique among living organisms.

Bitter controversy in the 1970s surrounded the initial claims that cAMP is found in plant cells. Two camps rapidly separated — sceptics who could find only vanishingly small quantities, and believers who reported biologically meaningful micromolar concentrations, albeit with weaker technology. At meetings to resolve the issue, heated and vitriolic exchanges often took place between believers and sceptics: “Does Dr ⃛ really choose to join those who incorrectly report the presence of cAMP in plant tissues?” is a statement I recall from one such altercation. Made by one of the most powerful figures in US plant physiology at the time, such attitudes rapidly relegated believers to obscure journals and the likelihood of failed tenure or promotion expectations, as opinion swung into line behind the dominant hierarchy. The dogma that plants contained either no cAMP or, at best, biologically irrelevant concentrations, rapidly assumed the rule of law and textbook status1,2. Believers, few in number, would make occasional forays to semi-respectable journals. But they were met with repulse by the sceptics, who again reported that cAMP, if it was there, “ ⃛ was below the detection limit ⃛ ” of very sensitive radioimmune assays3. What the believers lacked — which would have resolved the issue firmly in their favour — was a convincing demonstration that plant cells contain a functioning adenylyl cyclase.

Over the past decade, however, cracks have appeared in the sceptical edifice4: sequences similar to proteins that bind cAMP and regulate gene expression have turned up in plant libraries. Moreover, in guard cells (which regulate gas exchange into and out of the leaf), potassium channels have been found to respond to cAMP-dependent phosphorylation. But perhaps the most telling information originated with plant phosphoinositide studies. Attempts to measure the concentrations of phosphatidylinositol mono- and bisphosphates — the precursors of inositol-1,4,5-trisphosphate, Ins(1,4,5)P3— could detect only vanishingly small quantities in plant tissues. However, using loaded, caged Ins(1,4,5)P3to release the compound in plant cells, activity was found in guard cells, protoplasts from etiolated leaves, and growing pollen tubes. Not only did Ins(1,4,5)P3initiate the release of intracellular Ca2+, but it caused direct physiological changes5,6.These plant cells respond easily to many signals, and all have now been shown to have functional phosphoinositide systems.

In many plant tissues, massive downregulation of signalling constituents is thought to accompany tissue maturation. Could downregulation of adenylyl cyclase account for the missing cAMP5,6? On page 698 of this issue, Ichikawa et al.7 have decisively answered this question. A team led by Rick Walden has not only identified adenylyl cyclase in regeneration of tobacco protoplasts but, by overexpression in bacteria and complementation in yeast, has left no doubt as to its biological activity. When they treated protoplasts with forskolin (which activates adenylyl cyclase) or cAMP, protoplast regeneration occurred in the absence of the endogenous plant growth regulator auxin (indoleacetic acid). So cAMP is clearly part of the auxin signal-transduction chain.

The growth of stems, roots, flowers and fruits is controlled by a variety of growth factors, among them, auxin. In concert with other growth regulators (notably the adenine-based cytokinins), auxin also regulates the growth and division of cultured cells, protoplast regeneration and callus formation. With callus of some species, high ratios of auxin to cytokinin in the medium induce the formation of roots, whereas low ratios induce shoot formation.

Now that Ichikawa et al. have shown that auxin signals are transduced through cAMP (and, from other studies8, probably through cytosolic Ca2+ as well), we can see that auxin follows a familiar transduction pattern. Auxin signals activate protein kinases, which regulate the electrogenic (H++K+)ATPase and gene expression through a mitogenactivated protein (MAP) kinase cascade (Fig. 1). No doubt protein kinase A will find a prominent place in this sequence. And with regulation of cell-wall vesicle secretion by cytosolic Ca2+, enabling increased growth of the cell wall, the pieces of the growth jigsaw are beginning to fall into place.

Figure 1: Transduction sequences of auxins and cytokinins.
figure1

Ichikawa et al.7 have laid to rest the debate about whether plant cells contain cyclic AMP, by identifying functional adenylyl cyclase (the enzyme that synthesizes cAMP) and showing that it can mediate the actions of the plant hormone auxin. The growth-regulating effect of auxin is pre-eminent, and the main role of cytokinins is to attenuate this growth. So whereas auxin can regulate growth in the absence of other growth regulators, cytokinins do so only in the presence of auxin. The cyi peptide is suggested to join the two mitogen-activated protein (MAP) kinase signal-transduction cascades11. The diagram includes only some of the known transduction information.

In the absence of other growth factors, auxin induces highly polarized cell growth, exemplified by elongation of cells from the coleoptile (the sheath that protects growing shoot tips). Overexpression of cytokinins in tobacco reduces stem height and increases stem thickness. Cytokinins, the receptor for which is now thought to be a histidine kinase9, seem to be transduced only through MAP kinase cascades, with no involvement of Ca2+ or cAMP. In this case, then, cytokinins simply attenuate auxin-generated growth, and redirect cell-growth polarity to a more isodiametric shape.

Cell shape often underpins overall tissue form: a high ratio of auxin to cytokinin will encourage the regeneration of long, thin tissues (such as adventitious and lateral roots) from callus, whereas a low auxin to cytokinin ratio ensures the regeneration of the thicker, more isodiametric tissue forms that we associate with the shoot. Recently identified cyi peptides10 may mediate the cytokinin attenuation and induce the expression of cyclins and cyclin kinases11. These enable plant cells to grow and divide in the presence of both auxin and cytokinin.

Knowledge of the transduction sequence of auxin brings us much closer to the holy grail of understanding morphogenesis — tissue form. But the solution to one puzzle highlights equally pressing questions. For example, other growth factors (such as gibberellins, abscisins and ethylene) also control the shape and division of plant cells. Will these regulate growth and tissue form through adenylyl cyclase and Ca2+, or will they, like cytokinin, simply attenuate the effects of the master controller, auxin?

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Trewavas, A. Plant cyclic AMP comes in from the cold. Nature 390, 657–658 (1997). https://doi.org/10.1038/37720

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