• [Ca2+]cyt signalling toolkit;
  • calcium (Ca);
  • evolution;
  • photosynthetic eukaryotes;
  • plants;
  • signalling


  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References


  • Summary 1

  • I. 
    Introduction 1
  • II. 
    Homology vs homoplasy 2
  • III. 
    The structure and variation of [Ca2+]cyt signalling pathways 7
  • IV. 
    A putative course of descent for plant [Ca2+]cyt signalling 9
  • V. 
    Conclusion 13
  •  Acknowledgements 14

  • References 14


It is likely that cytosolic Ca2+ elevations have played a part in eukaryotic signal transduction for about the last 2 Gyr, being mediated by a group of molecules which are collectively known as the [Ca2+]cyt signalling toolkit. Different eukaryotes often display strikingly similar [Ca2+]cyt signalling elevations, which may reflect conservation of toolkit components (homology) or similar constraints acting on different toolkits (homoplasy). Certain toolkit components, which are presumably ancestral, are shared by plants and animals, but some components are unique to photosynthetic organisms. We propose that the structure of modern plant [Ca2+]cyt signalling toolkits may be explained by their modular adaptation from earlier pathways.

I. Introduction

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

Opinion is divided over how many species of eukaryote share the planet with us but, no matter how obscure the species which we consider, the Ca2+ ion has always been found to play a role in their signal transduction (Fig. 1). Photosynthetic organisms are no exception to this rule, and many excellent reviews exist to lead the forgetful through the intricacies of plant Ca2+ signalling (Sanders et al., 1999; Rudd & Franklin-Tong, 2001; White & Broadley, 2003; Hetherington & Brownlee, 2004). These authors concur that an elevation in the cytosolic Ca2+ concentration ([Ca2+]cyt) is required in a number of developmental and physiological pathways, and is generated by the [Ca2+]cyt signalling ‘toolkit’ (Berridge et al., 2000). Broadly speaking, eukaryotic toolkit components fall into one of four categories – stimuli change the rates of influx and efflux pathways to give [Ca2+]cyt elevations which are interpreted by various [Ca2+]cyt sensors (Box 1). The range of stimuli invoking [Ca2+]cyt changes, and the sheer variety of spatio-temporal patterns these changes can take, have lead to speculation over whether specific responses are encoded by specific patterns of [Ca2+]cyt elevations (Scrase-Field & Knight, 2003). It is not our intention to add significantly to this debate. Instead, we would like to focus on an aspect of [Ca2+]cyt signalling which such functional explanations often leave to one side: the relationship between pathway morphology and evolutionary descent.


Figure 1. The ubiquity of eukaryotic [Ca2+]cyt signalling. This unrooted tree shows the organisms mentioned in this review which have been shown to use [Ca2+]cyt signalling. Distances are roughly indicative of phylogenetic separation, and were calculated by J. H. F. Bothwell using data taken from Baldauf (1999), Yoon et al. (2002), Sanderson (2003), Funes et al. (2004) and Bhattacharya et al. (2004).

Download figure to PowerPoint

When faced with any characteristic of a living organism, we tend to ask what purpose it serves. The underlying assumption is that the characteristic has survived millennia of natural selection because it affords the most efficient way of performing a particular task. However, biological systems are not usually considered to have been designed in advance, being pieced together over evolutionary timescales in a manner which has famously led to natural selection being described as a tinkerer, rather than an engineer (Jacob, 1977). This means that a characteristic does not reflect an optimal solution to environmental challenges, but an optimal adaptation to those challenges given the organism's predecessors. Such a characteristic is called an adaptive trait, or said to show adaptation.

Acknowledgement that [Ca2+]cyt signalling toolkits show adaptation is of particular relevance for two reasons. Firstly, consideration of how [Ca2+]cyt signalling has developed in response to changing selection pressures may help to explain how other complex traits arise. Secondly, understanding that [Ca2+]cyt signalling toolkits have been constructed from a limited set of possible components may shed light on a number of apparent imperfections in [Ca2+]cyt signalling. Without such an outlook, it would be hard to see why some plant kinases have degenerate Ca2+-binding subunits which are no longer able to bind the Ca2+ ion (Hrabak et al., 2003). It would be hard to see why a system based on the precise control of [Ca2+]cyt should mediate Ca2+ influx through nonselective channels (White & Broadley, 2003). Indeed, it would sometimes be hard to see why [Ca2+]cyt should be adopted as a signal at all, when a more rapidly diffusing metabolite could presumably do the job as well, if not better. We must, of course, remember that a seemingly ill-adapted process more often reflects faults in our understanding than in evolution. Nevertheless, we shall see that the more we look at plant [Ca2+]cyt signalling, the more we find traces of Jacob's tinkerer.

II. Homology vs homoplasy

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

Patterns of [Ca2+]cyt changes are often remarkably similar across species. For example, when treated with 20 mm caffeine under certain conditions, the green alga Eremosphaera viridis displays [Ca2+]cyt oscillations. These reach [Ca2+]cyt peaks of c. 700 nm, display a period of c. 1 min and are dependent on extracellular Ca2+ (Bauer et al., 1997). They thus bear a remarkable similarity to the response of rat neuronal cells to 5 mm caffeine (Fig. 2), which also show Ca2+ext-dependent [Ca2+]cyt oscillations with periods of c. 1 min, although the peak [Ca2+]cyt is only c. 200 nm (Usachev & Thayer, 1999).


Figure 2. Interspecific comparison of caffeine-induced [Ca2+]cyt oscillations. Rat dorsal root ganglion neurones (top) were ester-loaded with Indo-1 AM (© Blackwell Science Ltd. Reproduced, with permission, from Usachev & Thayer, 1999). Eremosphaera viridis (bottom) was microinjected with fura-2 10 kDa dextran (Reproduced, with permission, from Bauer et al., 1997).

Download figure to PowerPoint

inline image

Two extremes bound any explanation for such similarities. The first extreme is homology, in which two patterns in two different organisms are similar because they are generated by the same pathway. This pathway may have been inherited by each organism from a common ancestor, in which case it is called orthologous, or it may have been independently constructed in each organism from the same components, a process which often reflects developmental constraints and which is called parallelism. The second extreme is homoplasy, also known as convergence, in which the efficacy of natural selection in moulding an optimal characteristic leads to the creation of similar patterns from different pathways (Hall, 2003).

1. Probable [Ca2+]cyt signalling homology: nodulation in legumes

Around 30–60 Myr ago (Wikström et al., 2001), one eudicot clade of flowering plants developed the ability to host nitrogen-fixing bacteria in a symbiosis characterized by the formation of distinctive nodules on the root hairs of infected plants (Soltis et al., 1995). Their descendants include the legumes, a group of crop plants, which are commercially important because the nitrogen-fixing nodules allow them to be grown on poor soils. The formation of nodules is triggered by a bacterial invitation to prospective hosts, in which the bacteria release lipo-chito-oligosaccharide chains, called Nod (for nodulation) factors, and the plants respond by diverting the normal process of root hair development to encompass their bacterial allies.

As part of this diversion of normal development, root hairs display a variety of [Ca2+]cyt elevations which are thought to trigger some, or all, of the necessary changes in metabolism and gene expression. It is currently unclear whether all these [Ca2+]cyt elevations are produced by a single Nod-factor-activated pathway, or whether multiple pathways operate in parallel (Wais et al., 2000; Esseling et al., 2004). Nonetheless, challenging root hairs of Medicago sativa (Ehrhardt et al., 1996), Phaseolus vulgaris (Cárdenas et al., 1999), Medicago truncatula (Wais et al., 2000; Shaw & Long, 2003), Pisum sativum (Walker et al., 2000) or Lotus japonicus (Harris et al., 2003) with nanomolar amounts of Nod factors results in a lag of c. 10 min followed by the onset of ‘[Ca2+]cyt spiking’: nuclear localized [Ca2+]cyt elevations which recur, with periods of between 0.5 and 3 min, for a couple of hours (Fig. 3a). In some cases, a distinct second response (Cárdenas et al., 1999; Shaw & Long, 2003) results in [Ca2+]cyt elevations near the root hair tip (Fig. 3b).


Figure 3. Interspecific comparison of Nod factor-induced [Ca2+]cyt spiking. Phaseolus vulgaris root hairs (on the right) were microinjected with fura-2 70 kDa dextran (© Blackwell Science Ltd. Reproduced, with permission, from Cárdenas et al., 1999); Medicago truncatula root hairs (on the left) were microinjected with Calcium Green-1 10 kDa dextran and Texas Red 10 kDa dextran (© American Society of Plant Biologists. Reproduced, with permission, from Shaw & Long, 2003). Each root hair is approx. 10 µm wide. (a) About 10 min after application of 10 nm Nod factor, [Ca2+]cyt spiking is seen in the perinuclear region of root hairs, and oscillations can persist for several hours. (b) Within 10 min of application of 10 nm Nod factor, [Ca2+]cyt elevations are sometimes seen in the tips of root hairs (Ehrhardt et al., 1996; Walker et al., 2000).

Download figure to PowerPoint

This conservation of [Ca2+]cyt patterns seems to be matched by conservation of the toolkit components identified so far, which suggests that these responses are homologous. Work on L. japonicus nodulation mutants has shown that a pair of Nod factor receptor kinases, NFR1 (Radutoiu et al., 2003) and NFR5 (Madsen et al., 2003), are required to trigger nodulation. LYK3 and LYK4, the LysM domain-containing receptor-like kinases of M. truncatula, are NFR1/5 orthologues (Limpens et al., 2003), and, again, without these nodulation will not occur. The identity between NFR1/5 and LYK3/4 is not exact; NFR5 has three LysM domains (Madsen et al., 2003), but NFR1 (Madsen et al., 2003), LYK3 and LYK4 (Limpens et al., 2003) have only two. Given that the LysM domain is thought to be responsible for oligosaccharide binding (Radutoiu et al., 2003), such variation presumably underlies receptor specificity, and may explain why chitin oligomers whose structures are closely related to those of Nod factors elicit [Ca2+]cyt spiking in P. sativum (Walker et al., 2000) and M. truncatula (Oldroyd et al. 2001) but not in P. vulgaris (Cárdenas et al., 1999).

A more contentious analysis of [Ca2+]cyt-spiking mutants (Wais et al., 2000 vs Esseling et al., 2004) has revealed three further loci named DMI1-3, which do not make bacterial infections and which have orthologues in all legumes studied to date. DMI1 is a novel protein which may form a cation channel and probably interacts with other proteins (Anéet al., 2004). DMI2, which is also referred to as SYMRK (symbiosis receptor-like kinase) or NORK (nodulation receptor kinase), is a membrane-bound serine/threonine protein kinase (Endre et al., 2002; Kistner & Parniske, 2002; Stracke et al., 2002); DMI3 is an orthologue of the Pisum sym9 gene (Mitra et al., 2004), and a member of the [Ca2+]cyt sensor-responder family known as the Ca2+ and calmodulin-dependent protein kinases, or CCaMKs (Lévy et al., 2004).

Exactly which [Ca2+]cyt signalling pathway DMI1-3 belong to is still unclear. Until recently, DMI1-3 were thought to be components in the nuclear [Ca2+]cyt-spiking pathway (Wais et al., 2000), but this view may need revision, following the discovery that DMI mutants are able, when handled extremely delicately, to display root hair deformation (Esseling et al., 2004). DMI1-3 may be involved in the tip-high [Ca2+]cyt pathway, rather than the nuclear one, although this remains to be confirmed either way (Shaw & Long, 2003; Esseling et al., 2004).

2. Probable [Ca2+]cyt signalling homoplasy: Ca2+ release from endomembrane stores

While homology is often an intuitively appealing explanation for any similarities which exist between stimulus-evoked [Ca2+]cyt patterns, convergent evolution of complex traits is not unknown in either the animal or plant world, explaining, among other things, similarities between transcriptional regulation in bacteria and yeast (Conant & Wagner, 2003) and intelligence in animals (Emery & Clayton, 2004).

It is likely that a further example of convergence occurs in the mechanisms by which [Ca2+]cyt elevations are propagated. There is little common morphological ground to be found between metazoa and plants, which diverged between 1 and 2 Gyr ago (Feng et al., 1997; Sanderson, 2003), but the early development of both begins with a sperm fertilizing an egg. In metazoa, sperm entry is accompanied by a [Ca2+]cyt fertilization wave, whose speed is conserved at c. 10 µm s−1 in all species studied to date (Jaffe, 2002). A ten-fold slower [Ca2+]cyt fertilization wave has been seen in Zea mays (Digonnet et al., 1997; Antoine et al., 2000), and no wave at all can be seen in the stramenopile Fucus serratus (Roberts et al., 1994) (Fig. 4).


Figure 4. Interspecific comparison of [Ca2+]cyt elevations during fertilization. Fucus serratus eggs (© Company of Biologists Ltd. Adapted, with permission, from Roberts et al., 1994) and metazoan Ciona intestinalis eggs were microinjected with Calcium Green-1 dextran. Zea mays eggs were ester loaded with fluo-3 AM (© Company of Biologists Ltd. Adapted, with permission, from Digonnet et al., 1997). Figs have been adapted to fit on the same timescale, with t = 0 being the time of sperm entry into the egg.

Download figure to PowerPoint

The elucidation of the machinery by which the metazoan fertilization waves, and other metazoan [Ca2+]cyt elevations, are propagated is one of the most elegant achievements of signal transduction research and readers are referred elsewhere for a more detailed description (Berridge et al., 2003). Briefly, metazoan cells have a number of endomembrane Ca2+ stores, and agonists can cause Ca2+ to be released from any of these by the activation of three main pathways. In the first pathway, agonists bind G protein-coupled receptors. The G proteins then stimulate one of a variety of phospholipase C (PLC) isoforms, which cleave phosphatidylinositol 4,5 bisphosphate (PIP2), generating inositol-1,4,5-trisphosphate (IP3), which binds to the IP3 receptor (IP3R) on the endoplasmic reticulum (ER). In the second pathway, agonists cause intracellular generation of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), both of which are synthesized by the same enzyme, ADP ribosyl cyclase. In the third pathway, [Ca2+]cyt elevation causes activation of ryanodine receptors (RyR) on the ER. Activation of IP3R, the NAADP receptor, or RyR will cause increases in their Ca2+ permeablility, and hence release of Ca2+ from the ER and other endomembrane stores (Berridge et al., 2003).

The interplay between Ca2+ influx and efflux determines the spatio-temporal [Ca2+]cyt pattern, so early attempts to mathematically simulate metazoan [Ca2+]cyt elevations tended to focus on describing the behaviour of the IP3R using the so-called De Young/Keizer, or DYK, model (De Young & Keizer, 1992). This assumes that the IP3R consists of three subunits, each having stimulatory IP3- and Ca2+-binding sites, as well as inhibitory Ca2+-binding sites. When the DYK approximation is combined with terms for Ca2+ efflux through a nonlinear pump and Ca2+ diffusion through a homogenous cytosol, model predictions match experiment extremely well. Varying the intracellular IP3 concentration ([IP3]cyt) gives rise to different [Ca2+]cyt patterns: a low [IP3]cyt gives a steady low [Ca2+]cyt; increasing [IP3]cyt gives solitary [Ca2+]cyt waves or oscillations; and high [IP3]cyt leads to cessation of waves and a steady high [Ca2+]cyt (De Young & Keizer, 1992).

Since there is evidence that NAADP (Navazio et al., 2000), cADPR (Allen et al., 1995; Leckie et al., 1998) and IP3 (MacRobbie, 2000) can also stimulate [Ca2+]cyt elevations in plants, it is often assumed that plants and metazoan use the same internal Ca2+-release pathways. Indeed, many of the proteins involved in the metazoan response, such as PLC (Koyanagi et al., 1998; Mueller-Roeber & Pical, 2002) and the IP3-removing inositol phosphatases (Berdy et al., 2001; Xiong et al., 2001; Ercetin & Gillaspy, 2004) have been found in plants, and their expression is both affected by plant hormones (Hunt & Gray, 2001; Ercetin & Gillaspy, 2004) and required for certain Ca2+-dependent pathways such as nodulation (Engstrom et al., 2002) and the stomatal closure response (Sánchez & Chua, 2001; Xiong et al., 2001; Hunt et al., 2003).

Despite these similarities, there are caveats which make us suggest that Ca2+ release from endomembrane stores is an example of homoplasy, and not homology. While cADPR is generated in plants, no orthologue to the metazoan ADP-ribosyl cyclase has been found in plant genomes (Sánchez et al., 2004). Similarly, no study has ever shown the existence of orthologues of IP3R or RyR in plants or algae (Nagata et al., 2004). To complicate matters further, the PLCβ isoform which is activated by a variety of G proteins in metazoa (Hartweck et al., 1997) is not found in sequenced plant genomes, which encode the PLCδ isoform and only one Gα protein (Mueller-Roeber & Pical, 2002). In fact, although it is not seriously doubted that phosphoinositides are important regulators of plant cell physiology, the exact species responsible for plant [Ca2+]cyt elevations have yet to be pinned down. Although a rise in IP3 has been observed to follow hormonal and stress signalling (Hunt & Gray, 2001), and addition of IP3 to isolated vacuoles stimulated Ca2+-permeable channels (Allen et al., 1995; Muir & Sanders, 1997), more recent work suggests that IP6 is also competent to stimulate Ca2+ release from endomembrane stores (Lemtiri-Chlieh et al., 2003), which has lead to the proposal that IP3 is converted to IP6 and is active in that form. For the moment we can only say that if IP3 is involved in plant endomembrane Ca2+ release, it probably isn't generated or perceived as it is in metazoa, which may explain much current confusion among plant physiologists.

It is, however, important to realize that spatio-temporal patterns of [Ca2+]cyt elevations are not inextricably linked to certain molecules. Bearing this in mind, it is instructive to look at a mathematical model which was developed to look at [Ca2+]cyt oscillations in cardiac myocytes, in which the Ca2+ release sites are not IP3 receptors, but the poorly characterized RyR. This is the Fire-Diffuse-Fire (FDF) model (Keizer et al., 1998), in which a cluster of IP3 receptors is replaced by a release unit which is activated when [Ca2+]cyt rises above a certain threshold. All the biology of the receptor model is then approximated by the choice of threshold, and once the threshold is passed, Ca2+ release occurs. No assumptions need to be made about the identity of the Ca2+ release agent or the Ca2+ release site, yet the FDF model is also able to simulate a wide variety of [Ca2+]cyt patterns, including waves (Ponce-Dawson et al., 1999) and oscillations (Keizer et al., 1998).

So, although interspecific stimulus-evoked [Ca2+]cyt patterns are often extremely similar, perhaps best exemplified by the caffeine-induced [Ca2+]cyt oscillations seen in both green algae and mammals, these similarities may not necessarily reflect conserved mechanisms of generation but could easily result from the similarity in wiring between two systems, each composed of very different components. Having persuaded ourselves of this, we now turn to see how the components of [Ca2+]cyt signalling toolkits are organised and may vary.

III. The structure and variation of [Ca2+]cyt signalling pathways

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

[Ca2+]cyt signalling pathways are complex phenomena. To describe them properly, the traditional reductionist, bottom-up approach, in which metabolites and proteins are individually characterized, is increasingly being complemented by a top-down, systems biology approach, in which mathematical models of information transfer are used to make sense of large datasets, especially the relevant genomes and proteomes.

Between them, these approaches are revealing that [Ca2+]cyt signalling toolkits consist of a number of levels, each of which forms a discrete combinatorial system (Fig. 5). On the bottom rung lie amino acids, which are held in common by most cellular life. These may be combined to form a number of polypeptide domains, such as the Ca2+-binding EF hand (Kretsinger & Nockolds, 1973), which are, again, broadly conserved across living organisms. Domains may then be combined to form proteins, and it is here that we begin to see major interspecific differences between toolkit components.


Figure 5. Levels of the [Ca2+]cyt signalling tookit. Each level consists of a number of discrete components which may be combined in many ways to create new components for the next level of organization. This allows the generation of a diverse modular system, which is then subject to natural selection.

Download figure to PowerPoint

1. Protein variation

Orthologous proteins do not necessarily behave the same way in all eukaryotes. For example, SOS2, which is a plant calcineurin B like (CBL) sensor relay, binds a kinase, SOS3, in contrast to yeast CBLs (Gong et al., 2004) which bind phosphatases. Those plant Ca2+-binding proteins which do interact with kinases activate serine/threonine kinases, rather than the tyrosine kinases prevalent in metazoa (Reddy & Reddy, 2004). Plant Ca2+-permeable glutamate receptor channels seem to be activated by glycine, whereas orthologous metazoan channels are activated mainly by glutamate (Dubos et al., 2003).

How does such variation arise? A single gene may be altered by recombination, nucleotide substitution, insertion or deletion, but all these can often disrupt the function of the gene product to the detriment of the whole organism, as seen in most point mutations in [Ca2+]cyt signalling toolkit components (e.g. Schumacher et al., 1999). Every once in a while, however, a mutation in a [Ca2+]cyt toolkit component can lead to a subtle enough alteration of function to hint at new possibilities whilst allowing survival. As examples, we proffer both the single amino acid alteration in the yeast VCX1 transporter which results in a shift in specificity from Ca2+ towards Mn2+ (Del Pozo et al., 1999) and the gradual coevolution of symbiont and host in rhizobial nodulation (Aguilar et al., 2004).

Such examples notwithstanding, it is more likely that an organism will survive if a mutation retains the old function while creating a new one. While this can happen through developmental variation and differential expression/splicing, as in some plant Ca2+-binding proteins (Persson et al., 2003; Kolukisaoglu et al., 2004), it can also happen through gene duplication, in which one of the two resulting genes will be made redundant, and so freed from a functional role. A duplicated gene, also known as a paralogue, may then evolve in one of three ways: one copy acquires a new function, while the other maintains the old function (neofunctionalization); both copies share the work of the original (subfunctionalization); or one copy is lost (degeneration). Individual and local tandem duplications of many components of the [Ca2+]cyt handling toolkit are seen in the available plant genomes (Navazio et al., 1998; Felleisen et al., 2000; Baxter et al., 2003; Vandepoele et al., 2003; Kolukisaoglu et al., 2004), often occuring through the actions of transposons (Zhang & Wessler, 2004).

inline image

2. Module variation

Duplication is not limited to single genes. Fragments of chromosomes, or even whole genomes, may also be duplicated (Anderson & Stebbins, 1954; Lawton-Rauh, 2003), with Arabidopsis having undergone a couple of rounds of polyploidy (Blanc et al., 2000; Vision et al., 2000; The Arabidopsis Genome Initiative, 2000; Bowers et al., 2003). Natural selection will tend to favour such duplication if a group of proteins on the duplicated region work efficiently together, and can be adapted for use in a variety of pathways. Such groups are known as ‘modules’ (Hartwell et al., 1999) or ‘cassettes’ (Mori & Schroeder, 2004) and may be considered to form the next level of toolkit construction.

There is little doubt that plant [Ca2+]cyt signalling toolkits employ modules. At its simplest, selection over time for efficient functional interactions between proteins creates gene fusions, such as those which have given rise to many varieties of Ca2+-binding protein kinases (Hrabak et al., 2003). However, there are tantalising hints of larger [Ca2+]cyt signalling toolkit modules in the Arabidopsis thaliana genome, most notably in the repetitive activation of hyperpolarization-activated Ca2+-permeable plasma membrane cation channels by reactive oxygen species (Mori & Schroeder, 2004) and the CaM/ACA4/CPK14 gene cluster on chromosome 2 which is duplicated as CaM/ACA11/CPK32 on chromosome 3 (J. H. F. Bothwell, unpublished data).

3. Network variation

Recent mathematical topological analysis (Strogatz, 2001) suggests that individual proteins and protein modules (Jeong et al., 2000; Almaas et al., 2004; Tong et al., 2004) fit together to form networks which exhibit so-called ‘small-world’ properties (Box 2). We may consider a random network (Box 2) as the way in which random variation throws up the substrate for evolution but, if we filter this through natural selection, preferential survival will lead to preferential attachment. A system growing in this way will thus tend to become a small-world network (Barabási & Albert, 1999; Lenski et al., 2003).

Explaining how a complex system might vary poses an intuitive problem. It has been argued that, in a complex system, components are so well adapted to each other that they resist further change (von Mering et al., 2003; Fernández et al., 2004). Altering any component causes the system to fail, so no further change will occur. Such reasoning has been used explicitly to explain the conservation of [Ca2+]cyt wave speeds in metazoa and plants as reflecting the conservation of the propagating machinery (Jaffe, 2002) and is implicit in many studies which look for plant homologues of metazoan [Ca2+]cyt signalling phenomena.

Although the existence of such a mechanism, which would effectively limit the power of Darwinian adaptation, is hotly contested (Dennett, 1995), there is nonetheless a way out of any theoretical impasse. The recent ‘toolkit remodelling hypothesis’ (Berridge et al., 2003) points out that many [Ca2+]cyt elevations are able to regulate the activities and transcription of [Ca2+]cyt signalling toolkit components (Yang & Poovaiah, 2002) and suggests that this is one way in which the effects of altering individual components may be buffered. If correct, this would allow [Ca2+]cyt patterns to remain constant while toolkit components were changed and, eventually, even replaced. Homology could thus be turned into homoplasy.

IV. A putative course of descent for plant [Ca2+]cyt signalling

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

We have briefly described how [Ca2+]cyt signalling toolkits are constructed across several levels (Fig. 5) to produce a complex network. Understanding how such complexity arises has always required an investment in counterintuition, which painstaking work on the ‘perfect and complex eye’ which so troubled Darwin (Darwin, 1859; Goldsmith, 1990), and similar ‘organs of extreme perfection’, has repaid. Darwin's speculation that random variation, filtered by natural selection, can generate complex traits in a series of small, gradual steps has been shown to be retrospectively possible through analysis of fossil records and comparative anatomy (Goldsmith, 1990), and prospectively possible in computer simulations of development in digital organisms (Lenski et al., 2003). Can we now imagine creating a [Ca2+]cyt signalling response in a series of finely graded intermediate forms, each one useful to its possessor?

1. Prevention of [Ca2+]cyt toxicity in early cells

Evidence for biogenic CO2 fixation in 3.8-Gyr-old rocks from west Greenland hints at the presence of self-replicating organisms (Rosing, 1999). The exact conditions in which these organisms formed are unknown (Nisbet & Sleep, 2001), but given the composition of environments (Krasnov et al., 1995) which are presumed to resemble the prebiotic Earth (Nisbet & Sleep, 2001), it seems reasonable to suppose that then, as now, Ca2+ would have been one of the major cations present. Energy derived from redox reactions involving other cations, especially the Fe2+/Fe3+ pair, is thought to have driven the replication of early organisms (Martin & Russell, 2003), and the control of cation concentrations in early cells would thus have been of prime importance. Consequently the cation pumping ATPases which effect this control are ubiquitous to cellular life (Gogarten et al., 1989; Palmgren & Axelsen, 1998), and are some of the few proteins found in early cells that are not involved in nucleic acid replication (Martin & Russell, 2003).

Fine control of [Ca2+]cyt is particularly vital because Ca2+ has an unusually large radius for a divalent cation. The electrostatic attraction which the Ca2+ nucleus holds for anions is thus relatively weak, so anions which coordinate with Ca2+ are not forced into rigidly packed complexes around the Ca2+ nucleus, as they are when coordinating with, say, the smaller Mg2+ nucleus (Levine & Williams, 1982). In practical terms, this means that even low [Ca2+]cyt is able to cross-link, and thereby precipitate, flexible molecules carrying a negative charge, which is clearly something to be avoided in a cell consisting of self-replicating organic anions. To avoid such Ca2+ toxicity, there seems to have been early adaptation of one family of cation pumps and one family of cation antiporters for Ca2+ removal. The pump family became the P2 ATPases, and it is an indication of their importance that, of the five P-type ATPase families found in living organisms, only these and the P1 family are ubiquitous (Palmgren & Axelsen, 1998). The antiporter family became the Ca2+/H+ exchangers, such as the Arabidopsis CAX (Cai & Lytton, 2004).

In addition to moving Ca2+ across the cell membrane as a final sink, early cells developed intracellular Ca2+-binding proteins, which acted to buffer [Ca2+]cyt prior to its removal. It is unlikely that our ancestors appreciated the irony, but those same properties of Ca2+ which predispose it to precipitating organic salts also form its Achilles’ heel. A large divalent cation may be specifically bound in a flexible, weakly anionic pocket. Flexibility excludes smaller, highly charged cations, which cannot form complexes of the correct shape, and the weak anionic charge allows rapid removal of Ca2+ from the pocket, while excluding monovalent cations of similar size, for which the electrostatic interaction is not strong enough (Levine & Williams, 1982).

This structure has been seen in the P2 ATPases (Toyoshima et al., 2000), and in the motif called the helix-loop-helix, or EF hand. As their synonym suggests, EF hands consist of two α helices connected by a flexible loop, which helps to give them remarkable Ca2+ specificity (Kretsinger & Nockolds, 1973). Short polypeptides containing four EF hands are found in all domains of life (Babu et al., 1985), suggesting that they came to predominate as [Ca2+]cyt buffers before the separation of the bacterial, archaeal, and eukaryotic lineages, possibly striking an opimal balance between Ca2+-carrying capacity and diffusion rate. Whatever the reason for their success, this loose group of polypeptides, which are today called the calmodulins, remain the major bacterial and archaeal [Ca2+]cyt buffers, although they have been supplemented in eukaryotes.

2. [Ca2+]cyt elevations as indicators of stress in early cells

The selection advantage that avoiding calcium phosphate precipitation would confer on any system of self-replicating, phosphate-bound nucleotides is clear enough that we presume it to have driven the development of Ca2+-ATPases and the calmodulins. It has been suggested that one consequence of the resultant [Ca2+]cyt handling mechanism would be the maintenance of a large inward [Ca2+] gradient, which could be adapted to form the basis of a highly sensitive signalling pathway (Sanders et al., 1999). This predisposition of the [Ca2+]cyt handling machinery to become adapted for [Ca2+]cyt signalling is not, in itself, sufficient for [Ca2+]cyt signalling to develop. Although studies on E. coli expressing the Ca2+-sensitive photoprotein, aequorin, have shown that they are able to regulate [Ca2+]cyt (Jones et al., 1999), there is little evidence that [Ca2+]cyt signalling in bacteria or archaea is as important as it is in eukaryotes (Norris et al., 1996; Herbaud et al., 1998).

Nonetheless, anything tending to compromise the integrity of a cell membrane would result in a steep increase in [Ca2+]cyt. It therefore seems likely that the first step towards the adaptation of the existing [Ca2+]cyt handling pathway into a [Ca2+]cyt signalling pathway involved [Ca2+]cyt acting as an indicator of membrane damage. This could simply involve a mutation which allowed the interaction of a Ca2+-bound calmodulin with a stress response protein, giving an opportunistic stress response pathway. Some studies have indeed implicated [Ca2+]cyt in bacterial heat shock responses (Freestone et al., 1998; Nazarenko et al., 2003), although the elucidation of [Ca2+]cyt functions in bacteria has not progressed to the stage where we could, with confidence, claim that this finding supports our hypothesis.

3. Recruitment of [Ca2+]cyt elevations as integrators in the first eukaryotes

The events underlying the appearance of eukaryotes sometime after 2.7 Gyr ago (Martin & Russell, 2003) are a matter of contention (Katz, 1999; Pennisi, 2004). Although current dogma maintains that at least one endosymbiosis (Katz, 1999), including the formation of mitochondria by the endosymbiotic engulfment of bacteria, was crucial to the establishment of eukaryotic cells, it is debatable whether the eukaryotic lineage predated this endosymbiosis (Cavalier-Smith, 2002; Hartman & Fedorov, 2002; Baluška et al., 2004) or not (Martin & Russell, 2003; Rivera & Lake, 2004).

Whatever the exact history, there is a marked diversification of [Ca2+]cyt signalling toolkit components which appears to accompany the appearance of eukaryotes. The P2-ATPases split into the P2A and P2B subfamilies (Palmgren & Axelsen, 1998); the calmodulins, with four EF hands, are joined by the calcineurins, which have three, and the penta-EF hand family, which have five (Maki et al., 2002); and the β-sheet C2 domains became adapted for Ca2+-handling, possibly from an earlier binding function, joining the α-helical EF hand (Rizo & Südhof, 1998).

The sudden expansion of the [Ca2+]cyt signalling toolkit suggests that eukaryotes made more of the opportunities offered by the pre-existing [Ca2+] gradient than prokaryotes. In the absence of any obvious candidates for a eukaryotic cell without endosymbiotic organelles, it is often assumed that endosymbiosis confers a selection advantage (Williams & Fraústo Da Silva, 2003). However, if this is the case, why has primary endosymbiosis been so rare an event?

We suggest endosymbiosis to be an optimal resolution to conflict, rather than an optimizing of reproductive efficiency. The idea that a characteristic of eukaryotes may have evolved to resolve biotic conflict, rather than to optimize the utilization of abiotic resources, has already been mooted to explain sex (Hurst, 1995), and we believe that the struggle for resources which would follow an endosymbiotic event is best resolved in a similar fashion, by the creation of a system for organizing the subsystems which comprise the eukaryotic cell (Box 3). Given the high concentration of Ca2+ in the early ocean (Brennan et al., 2004), the ancestral [Ca2+]cyt handling system is ideally – some would say inevitably (Williams & Fraústo Da Silva, 2003) – placed for adaptation into such an integrative system. Not only does the large inward [Ca2+] gradient confer sensitivity (Sanders et al., 1999) but, due to the large variations in metabolic pathways found in bacteria and archaea (Martin & Russell, 2003), the [Ca2+]cyt stress response mechanism would be one of the few shared by host and symbiont, allowing for easier integration.

We make a clear distinction here between [Ca2+]cyt as a carrier of information and [Ca2+]cyt as an organizer of information. As we have seen, in some bacterial pathways, [Ca2+]cyt seems to act as a signal, being causally linked to the conditions it reports, but in many eukaryotic responses [Ca2+]cyt acts largely as an organizer, being a seemingly arbitrary agent by which different cellular pathways may communicate. We do not expect [Ca2+]cyt to be the only organizing entity, and suppose that messengers such as NO would behave in a similar way. The extent to which [Ca2+]cyt elevations act in tandem with these other integrating systems through crosstalk may help explain contradictory findings in different studies, a point which has been well argued already (Scrase-Field & Knight, 2003).

4. Intracellular Ca2+ stores

One feature of all modern eukaryotic cells is the presence of intracellular compartments, and we suppose that these would also have been a feature of the first eukaryotes. Soon after the establishment of eukaryotes, protein targeting modifications allowed the incorporation of [Ca2+]cyt handling components into the ER, creating the endomembrane [Ca2+]cyt sink (Navazio et al., 1998; Felleisen et al., 2000). The release of Ca2+ from endomembrane stores thus became another source of Ca2+ for [Ca2+]cyt elevations, and this has been observed in small, unicellular eukaryotes such as yeast (Denis & Cyert, 2002) as well as larger, multicellular ones.

Why should internal Ca2+ release become so entrenched? We proffer two possible explanations. Firstly, the more sources of Ca2+ release are present in a cell, the more variety of [Ca2+]cyt elevations are possible, and the more flexible the [Ca2+]cyt signalling system can be. Secondly, the small-world networks adopted by complex biological systems are, in general, resilient to the effect of random mutations (Jeong et al., 2000; Li et al., 2004), but are especially vulnerable to loss of the more highly interacting components (Albert et al., 2000), and it might be supposed that Ca2+ would be such a central agent. Thus the development of internal and external stores may introduce a certain amount of redundancy, guarding against such loss of function, as shown by findings in which external Ca2+ entry is up-regulated following depletion of intracellular stores (Csutora et al., 1999).

inline image

5. An increase in eukaryotic cell size would require rapidly diffusing messengers

We have argued that the successful endosymbiosis required for the establishment of eukaryotes was favoured by the creation of a system for mediating the conflict between host and symbiont. Thus we suppose the existence of [Ca2+]cyt signalling to be unrelated to cell size. Nevertheless, the nature of the eukaryotic cell rendered an increase in cell size possible, and such increases seem to have happened early. Most prokaryotic cells have a maximum dimension of < 5 µm, but the earliest putative eukaryotic fossils are an order of magnitude larger. 1.8-Gyr-old acritarchs – microfossils with organic walls but a phylogeny so uncertain that their identification as eukaryotes is not definite – have been found with diameters of 40–200 µm (Zhang, 1986), and larger fossils resembling algae have been claimed to be 2.1 Gyr old (Han & Runnegar, 1992). 1.5-Gyr-old fossils which are more definitely eukaryotic can be > 200 µm in diameter (Javaux et al., 2001).

What changes would such an increase in size bring to an information organizing system, such as signal transduction? The key to a successful signal transduction pathway is the precise and timely interaction of components. Early cells, like modern bacteria and archaea, would have been small enough for the diffusion of proteins to maintain a suitable rate of information transfer. The predominant form of signal transduction may thus be effected by protein–protein interactions, exemplified by prokaryotic two-component signalling (Bray, 1998).

The speed at which a protein can move through a cell is directly proportional to its diffusion coefficient, D, which, for most proteins, varies between c. 1 and 10 × 10−7 cm2 s−1 (Swaminathan et al., 1997; Bray, 1998; Dayel et al., 1999). This is comparable to the effective D for [Ca2+]cyt which, because of the constant binding of Ca2+ to intracellular buffers, lies between 1.3 and 6.5 × 10−7 cm2 s−1 (Allbritton et al., 1992). This is too low to allow rapid signal transduction in cells larger than c. 20 µm in diameter (Allbritton et al., 1992; Batada et al., 2004). Since any modification which increases the rate at which the slower-diffusing components are able to interact will tend to enhance the speed of signal transduction, slowly diffusing components, such as proteins and [Ca2+]cyt, tend to be localized, and smaller, faster-diffusing components communicate between them (Batada et al., 2004), which explains why the speed of [Ca2+]cyt wave propagation is constrained by the speed at which IP3 can diffuse (Gromada et al., 1993).

We find therefore that many slow-diffusing components of a single signalling pathway will tend to cluster together in complexes with other components. These complexes form at certain locations on cell membranes or the cytoskeleton, and are held together by scaffold proteins (Weng et al., 1999; Forgacs et al., 2004). Such complexes, of course, bear more than a superficial resemblance to manifestations of the modules (Hartwell et al., 1999) upon which natural selection may act. Examples of such [Ca2+]cyt signalling complexes exist in metazoa (Berridge et al., 2003), but we await stronger evidence that plant [Ca2+]cyt signalling toolkit complexes exist. This looks most likely to come from proteomic analysis, but we note that components similar to those involved in metazoan [Ca2+]cyt signalling have already been found in plant signalling complexes. For example, KAPP (Kistner & Parniske, 2002) and POLTERGEIST (Yu & Clark, 2003), which are protein phosphatase 2Cs similar to those required by guard cell [Ca2+]cyt signalling and encoded by the abi loci (Allen et al., 1999), form multiprotein complexes with the CLAVATA signalling system, and it would not be surprising if they formed similar complexes in their role in guard cell [Ca2+]cyt signalling.

We turn now to faster-diffusing metabolites. Eukaryotes use a number of these, including NAADP (D∼ 1 × 10−6 cm2 s−1; Churchill & Galione, 2000), IP3 (D∼ 2.83 × 10−6 cm2 s−1; Allbritton et al., 1992), cAMP (D∼ 2.7 × 10−6 cm2 s−1; Chen et al., 1999) and NO (D∼ 3.3 × 10−5 cm2 s−1; Malinski et al., 1993), but we have already suggested (in Section II.2) that their perception may have evolved separately in different clades. Indeed, if the faster-diffusing metabolites are reserved for covering larger distances, then we might imagine NO, with its extremely high diffusion coefficient, to have developed as a signal in response to the independent adoption of large size by plants and metazoa, as there are no reports of it functioning as a signal in protists or bacteria.

We propose, then, that [Ca2+]cyt is an integrative signal, but not necessarily the means of its own transmission. We also suggest that early single-celled eukaryotes had a basic [Ca2+]cyt signalling pathway before the divergence of plants and animals. As cell size increased, a number of small metabolites were co-opted to speed up [Ca2+]cyt signalling, although we cannot tell at what stage these were adopted. Although fungi, as well as plants, show IP3-dependent [Ca2+]cyt elevations (Silverman-Gavrila & Lew, 2001), the canonical IP3R has only been found in the metazoa. Given the absence of IP3 receptor homologues in fungi and plants, either IP3 acted as a Ca2+ release agent in early eukaryotes, and the initial receptor for IP3 was later supplanted in metazoa by the canonical IP3R, or IP3 was independently adopted as a Ca2+ release agent by plants, fungi and metazoa. Further speculation must await the identification of the receptors for IP3 in plants and fungi.

The discovery of ultrasmall eukaryotes which are comparable in size to bacteria (Baldauf, 2003) allows, in theory, a proving ground for our hypothesis that endomembrane signalling by [Ca2+]cyt, but not its mediation by IP3, is essential in eukaryotes. Ostreococcus tauri, with a cell diameter of c. 1 µm, is a promising avenue (Khadaroo et al., 2004) and it is interesting that endomembrane Ca2+ release from Saccharomyces cerevisiae, which is small, does not appear to involve IP3 (Denis & Cyert, 2002).

6. Plastids

The next major expansion of the [Ca2+]cyt signalling toolkit seems to have followed the endosymbiotic events which first gave rise to, and then shuffled, photosynthetic plastids (Fig. 1). This is presumed to have begun with the engulfment of a cyanobacterial symbiont by a eukaryotic host, which has been estimated to have occurred c. 1.6 Gyr ago (Yoon et al., 2004), and was followed c. 1.3 Gyr ago by secondary endosymbiosis of one eukaryotic cell by another, in which the symbiont was already acting as a host for a cyanobacterium (Yoon et al., 2004).

Following these endosymbioses, gene fusions created novel Ca2+-handling proteins (Nagata et al., 2004). In the protist lineage leading to fungi and metazoa, the interaction of calmodulin-binding domains and tyrosine protein kinases has led to the creation of the Ca2+/calmodulin protein kinases, the CaMKs, which are largely absent from green plant genomes. The distribution of [Ca2+]cyt sensor-responders is less clearly associated with lineages in the plantae. The interaction of Ca2+-binding EF hands and serine/threonine protein kinases has given rise to the creation of monophyletic calcium-dependent protein kinases, the CDPKs, in both apicomplexans and the viridiplantae (Zhang & Choi, 2001; Billker et al., 2004). However, the heterokont (stramenopile) diatom Thalassiosira pseudonana and the recently released genome of the red alga, Cyanidioschyzon merolae (Matsuzaki et al., 2004) appear to have, in addition to a calmodulin gene, CaMK homologues, rather than CDPK homologues (J. H. F. Bothwell, unpublished data).

While we await elucidation of whether CaMKs of Thalassiosira, C. merolae and metaozoa are monophyletic, two complicating phenomena, gene loss and lateral gene transfer, may help to explain this mismatch between sensor-responder phylogeny and lineage phylogeny. Loss of gene families is not unknown (Shiu & Li, 2004), so we might imagine that CaMKs, CDPKs or both were present in the last common ancestor of photosynthetic organisms, but were differentially lost or retained by various clades. Alternatively, if the multiple independent creation of either CaMKs or CDPKs is too improbable, we must invoke lateral gene transfer. It is known that the secondary endosymbiosis which gave rise to the chromalveolate ancestor resulted in the transfer of a large number of genes from the symbiont to the host (Bhattacharya et al., 2004; Funes et al., 2004). Similarly, in flowering plants, real time gene transfer has been seen in tobacco between plastids and the nucleus (Huang et al., 2004), while up to one-fifth of the Arabidopsis genome has been estimated to have derived from the chloroplast progenitor (Martin et al., 2002), including the two-component signalling histidine kinases (McCarty & Chory, 2000). Thus it is not too great a leap of the imagination to suppose that CDPKs were among the genes transferred, which may explain, for example, the monophyly of CDPKs in viridiplantae and apicomplexans.

7. Multicellularity and toolkit diversification in flowering plants

We come at last to the viridiplantae and flowering plants. We would like to be able to compare the numbers of [Ca2+]cyt signalling toolkit components in red algae and viridiplantae to see whether the appearance of green plants was accompanied by a [Ca2+]cyt toolkit expansion, but we have only one red algal genome, which is of the highly reduced C. merolae, and is thus of limited use.

We do, however, see toolkit diversification. Interaction among Ca2+-binding EF hands, calmodulin-binding domains and serine/threonine protein kinases has resulted in the CCaMKs, which possess separate binding sites for calmodulin and Ca2+, and which are only present in c. 80% of flowering plants (Hrabak et al., 2003). In addition, an intriguing class of kinases offer a possible snapshot of adaptation. As their name suggests, the CDPK-related kinases (CRKs) are similar to CDPKs, but their EF hands do not bind Ca2+ (Hrabak et al., 2003). Since it has been demonstrated that EF hands can bind other molecules (Ermilov et al., 2001), this may be a case of previously Ca2+-binding kinases adapting over evolutionary timescales to mediate novel signal transduction pathways.

Tantalising traces of such adaptation may still be glimpsed in other [Ca2+]cyt signalling pathways. The DMI1-3 genes involved in Nod-factor symbiosis, and discussed in Section II.1, are also thought to mediate the more ancient arbuscular mycorrhizal symbiosis pathway, suggesting their recruitment from one symbiotic pathway to another (Lévy et al., 2004; Parniske, 2004). More definitely, orthologous reactive oxygen generating respiratory burst oxidase homologues (RBOH) signal to Ca2+ channels in a number of tissues (Mori & Schroeder, 2004). The more ancestral RBOHD and RBOHF are active in plant defence (Torres et al., 2002) and development (Kwak et al., 2003), and the younger RBOHC is involved in root hair growth (Foreman et al., 2003), suggesting that ancestral stress response pathways have been adapted to drive development, as we argue in Section IV.3.

In this light, a particularly interesting observation has recently been made concerning natural selection following gene duplications. While not directly involved in [Ca2+]cyt signalling, the receptor-like kinases (RLKs) of the viridiplantae have independently expanded in Arabidopsis (through whole genome duplications and tandem repeats) and rice (mainly through tandem repeats). It may be of note that the RLKs involved in defense/disease resistance have been expanded more than those involved in development (Shiu et al., 2004). Given the plasticity required of plant signalling responses, similar selection pressures may have acted to increase the number of CDPKs in flowering plants, and we might expect that a majority of these are also involved in stress response pathways, rather than developmental ones.

V. Conclusion

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

The evolutionary dissection of any trait should, ideally, be founded on a wealth of comparative interspecific observations. Unfortunately, such data are limited for [Ca2+]cyt signalling, especially in photosynthetic organisms. Although the current boom in genome sequences will allow useful comparisons to be made (Gutman & Niyogi, 2004), they will not stand alone, and we add our voice to those already calling for more comparative physiological studies (Kellogg, 2004).

Given the environmental and physical constraints under which cells develop, it is to be expected that large eukaryotic cells develop a [Ca2+]cyt based signalling network which keeps [Ca2+]cyt at a steady level of c. 100 nm and certainly below 1 µm, which makes use of diffusible components and membrane-bound protein complexes, and whose topology can be described as a small-world network. Given this template, the observed patterns of [Ca2+]cyt elevation will follow.

We hypothesize that [Ca2+]cyt signalling developed as a system to minimize biotic conflict between the host and symbiont during endosymbiosis. This organizing system might subsequently have been adapted to respond to external stimuli, whether biotic or abiotic, and then adapted once more for development. It is not to be expected that such adaptation could be achieved without the aid of other signalling systems. We thus expect that [Ca2+]cyt signalling should involve cross-talk with other signalling components, as [Ca2+]cyt elevations probably did not originate to answer the uses to which they are now put.

We have argued that many of the basic mechanisms for propagating [Ca2+]cyt elevations have developed independently in metazoa and in plants. Thus there is no reason to expect conservation of components, or to invoke evolutionary relationships to explain similarities. A move away from comparative genomics is overdue, and we eagerly await the development of novel techniques for the identification of pathway components. The rewards of such an evolutionary appreciation of pathway structure are great, and it is to be hoped that [Ca2+]cyt signalling, and signal transduction in general, stay in the forefront of research into the evolution of the complexity of living organisms.


  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References

JHFB would like to thank Judy Armitage (Microbiology, University of Oxford) for information on prokaryotic signal transduction, Steve Coombes (School of Mathematics, University of Nottingham) for explaining mathematical modelling, and Kathryn Stevens (St. John's College, Oxford) for help with the bibliography. JHFB would also like to thank Colin Brownlee (Marine Biological Association, Plymouth), Luis Cárdenas (UNAM, Mexico), Christian Dumas (ENS, Lyon), Gerald Schönknecht (Oklahoma State, USA), Sidney Shaw (Stanford, USA), and Stanley Thayer (University of Minnesota, USA) for permission to reproduce data. Particular thanks are reserved for three anonymous referees and Colin Brownlee, without whom this would have been more unreadable than it is already.


  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Homology vs homoplasy
  5. III. The structure and variation of [Ca2+]cyt signalling pathways
  6. IV. A putative course of descent for plant [Ca2+]cyt signalling
  7. V. Conclusion
  8. Acknowledgements
  9. References
  • Aguilar OM, Riva O, Peltzer E. 2004. Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification. Proceedings of the National Academy of Sciences, USA 101: 1354813553.
  • Albert R, Jeong H, Barabási AL. 2000. Error and attack tolerance of complex networks. Nature 406: 378382.
  • Allbritton NL, Meyer T, Stryer L. 1992. Range of messenger action of calcium ion and Inositol 1,4,5-trisphosphate. Science 258: 18121815.
  • Allen GJ, Kuchitsu K, Chu SP, Murata Y, Schroeder JI. 1999. Arabidopsis abi1–1 and abi2–1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Plant Cell 11: 17851798.
  • Allen GJ, Muir SR, Sanders D. 1995. Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose. Science 268: 735737.
  • Almaas E, Kovács B, Vicsek T, Oltvai ZN, Barabási AL. 2004. Global organization of metabolic fluxes in the bacterium Escherichia coli. Nature 427: 839843.
  • Anderson E, Stebbins GL. 1954. Hybridization as an evolutionary stimulus. Evolution 8: 378388.
  • Ané JM, Kiss GB, Riely BK, Penmetsa RV, Oldroyd GED, Ayax C, Lévy J, Debellé F, Baek JM, Kalo P, Rosenberg C, Roe BA, Long SR, Denarié J, Cook DR. 2004. Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science 303: 13641367.
  • Antoine AF, Faure JE, Cordeiro S, Dumas C, Rougier M, Feijó JA. 2000. A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants. Proceedings of the National Academy of Sciences, USA 97: 1064310648.
  • Babu YS, Sack JS, Greenhough TJ, Bugg CE, Means AR, Cook WJ. 1985. Three-dimensional structure of calmodulin. Nature 315: 3740.
  • Baldauf SL. 1999. A search for the origins of animals and fungi: comparing and combining molecular data. American Naturalist 154: S178S188.
  • Baldauf SL. 2003. The deep roots of eukaryotes. Science 300: 17031706.
  • Baluška F, Volmann D, Barlow PW. 2004. Cells and their cell bodies: cell theory revisited. Annals of Botany 94: 932.
  • Barabási AL, Albert R. 1999. Emergence of scaling in random networks. Science 286: 509512.
  • Batada NN, Shepp LA, Siegmund DO. 2004. Stochastic model of protein–protein interaction: why signaling proteins need to be colocalized. Proceedings of the National Academy of Sciences, USA 101: 64456449.
  • Bauer CS, Plieth C, Hansen UP, Sattelmacher B, Simonis W, Schönknecht G. 1997. Repetitive Ca2+ spikes in a unicellular green alga. FEBS Letters 405: 390393.
  • Baxter I, Tchieu J, Sussman MR, Boutry M, Palmgren MG, Gribskov M, Harper JF, Axelsen KB. 2003. Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice. Plant Physiology 132: 618628.
  • Berdy SE, Kudla J, Gruissem W, Gillaspy GE. 2001. Molecular characterization of At5PTase1, an inositol phosphatase capable of terminating inositol trisphosphate signaling. Plant Physiology 126: 801810.
  • Berridge MJ, Bootman MD, Roderick HL. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nature Reviews: Molecular Cell Biology 4: 517528.
  • Berridge MJ, Lipp P, Bootman MD. 2000. The versatility and universality of calcium signalling. Nature Reviews: Molecular Cell Biology 1: 1121.
  • Bhattacharya D, Yoon HS, Hackett JD. 2004. Photosynthetic eukaryotes unite: endosymbiosis connects the dots. Bioessays 26 1: 5060.
  • Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B, Brinkmann V. 2004. Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 117: 503514.
  • Blanc G, Barakat A, Guyot R, Cooke R, Delseny M. 2000. Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell 12: 10931101.
  • Bowers JE, Chapman BA, Rong J, Paterson AH. 2003. Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature 422: 433438.
  • Bray D. 1998. Signaling complexes: biophysical constraints on intracellular communication. Annual Review of Biophysics BioMolecular Struct 27: 5975.
  • Brennan ST, Lowenstein TK, Horita J. 2004. Seawater chemistry and the advent of biocalcification. Geology 32: 473476.
  • Cai XJ, Lytton J. 2004. Molecular cloning of a sixth member of the K+-dependent Na+/Ca2+ exchanger gene family, NCKX6. Journal of Biological Chemistry 279: 58575876.
  • Cárdenas L, Feijó JA, Kunkel JG, Sánchez F, Holdaway-Clarke T, Hepler PK, Quinto C. 1999. Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs. Plant Journal 19: 347352.
  • Cavalier-Smith T. 2002. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. International Journal of Systematic Evolution Microbiology 52: 297354.
  • Chen CH, Nakamura T, Koutalos Y. 1999. Cyclic AMP diffusion coefficient in frog olfactory cilia. Biophysics Journal 76: 28612867.
  • Churchill GC, Galione A. 2000. Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients. Journal of Biological Chemistry 275: 3868738692.
  • Conant GC, Wagner A. 2003. Convergent evolution of gene circuits. Nature Genetics 34: 264266.
  • Csutora P, Su Z, Kim HY, Bugrim A, Cunningham KW, Nuccitelli R, Keizer JE, Hanley MR, Blalock JE, Marchase RB. 1999. Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores. Proceedings of the National Academy of Sciences, USA 96: 121126.
  • Darwin C. 1859. On the Origin of Species by Means of Natural Selection. London, UK: John Murray.
  • Day IS, Reddy VS, Ali GS, Reddy ASN. 2002. Analysis of EF-hand-containing proteins in Arabidopsis. Genome Biology 3: 56.156.24.
  • Dayel MJ, Hom EFY, Verkman AS. 1999. Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum. Biophysics Journal 76: 28432851.
  • De Young GW, Keizer J. 1992. A single-pool inositol 1,4,5-trisphosphate receptor-based model for agonist-stimulated oscillations in Ca2+ concentration. Proceedings of the National Academy of Sciences, USA 89: 98959899.
  • Del Pozo L, Osaba L, Corchero J, Jimenez A. 1999. A single nucleotide change in the MNR1 (VCX1/HUM1) gene determines resistance to manganese in Saccharomyces cerevisiae. Yeast 15: 371375.
  • Denis V, Cyert MS. 2002. Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue. Journal of Cell Biology 156: 2934.
  • Dennett DC. 1995. Darwin's Dangerous Idea. London, UK: Penguin.
  • Digonnet C, Aldon D, Leduc N, Dumas C, Rougier M. 1997. First evidence of a calcium transient in flowering plants at fertilization. Development 124: 28672874.
  • Dubos C, Huggins D, Grant GH, Knight MR, Campbell MM. 2003. A role for glycine in the gating of plant NMDA-like receptors. Plant Journal 35: 800810.
  • Ehrhardt DW, Wais R, Long SR. 1996. Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85: 673681.
  • Emery NJ, Clayton NS. 2004. The mentality of crows: convergent evolution of intelligence in corvids and apes. Science 306: 19031907.
  • Endre G, Kereszt A, Kevei Z, Mihacea S, Kaló P, Kiss GB. 2002. A receptor kinase gene regulating symbiotic nodule development. Nature 417: 962966.
  • Engstrom EM, Ehrhardt DW, Mitra RM, Long SR. 2002. Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling. Plant Physiology 128: 13901401.
  • Ercetin ME, Gillaspy GE. 2004. Molecular characterization of an Arabidopsis gene encoding a phospholipid-specific inositol polyphosphate 5-phosphatase. Plant Physiology 135: 938946.
  • Ermilov AN, Olshevskaya EV, Dizhoor AM. 2001. Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase. Journal of Biological Chemistry 276: 4814348148.
  • Esseling JJ, Lhuissier FGP, Emons AMC. 2004. A nonsymbiotic root hair tip growth phenotype in NORK-mutated legumes: implications for nodulation factor-induced signaling and formation of a multifaceted root hair pocket for bacteria. Plant Cell 16: 933944.
  • Felleisen RS, Hemphill A, Ingold K, Gottstein B. 2000. Conservation of calnexin in the early branching protozoan Tritrichomonas suis. Molecular Biochemistry and Parasitology 108: 109117.
  • Feng DF, Cho G, Doolittle RF. 1997. Determining divergence times with a protein clock: update and reevaluation. Proceedings of the National Academy of Sciences, USA 94: 1302813033.
  • Fernández A, Scott R, Berry RS. 2004. The nonconserved wrapping of conserved protein folds reveals a trend toward increasing connectivity in proteomic networks. Proceedings of the National Academy of Sciences, USA 101: 28232827.
  • Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JDG, Davies JM, Dolan L. 2003. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422: 442446.
  • Forgacs G, Yook SH, Janmey PA, Jeong H, Burd CG. 2004. Role of the cytoskeleton in signaling networks. Journal of Cell Science 117: 27692775.
  • Freestone P, Grant S, Trinei M, Onoda T, Norris V. 1998. Protein phosphorylation in Escherichia coli L. form NC-7. Microbiology 144: 32893295.
  • Funes S, Reyes-Prieto A, Perz-Martinez X, Gonzalez-Halphen D. 2004. On the evolutionary origins of apicoplasts: revisiting the rhodophyte vs. chlorophyte controversy. Microbes Infection 6: 305311.
  • Gogarten JP, Kibak H, Dittrich P, Taiz L, Bowman EJ, Bowman BJ, Manolson MF, Poole RJ, Date T, Oshima T, Konishi J, Denda K, Yoshida M. 1989. Evolution of the vacuolar H+-ATPase: implications for the origin of eukaryotes. Proceedings of the National Academy of Sciences, USA 86: 66616665.
  • Goldsmith TH. 1990. Optimization, constraint, and history in the evolution of eyes. Quarterly Review of Biology 65: 281322.
  • Gong D, Guo Y, Schumaker KS, Zhu JK. 2004. The SOS3 family of calcium sensors and SOS2 family of protein kinases in Arabidopsis. Plant Physiology 134: 919926.
  • Gromada J, Jorgensen TD, Tritsaris K, Nauntofte B, Dissing S. 1993. Ca2+ signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-trisphosphate and its role in the release of Ca2+. Cell Calcium 14: 711723.
  • Gutman, BL, Niyogi KK. 2004. Chlamydomonas and Arabidopsis. A dynamic duo. Plant Physiology 135: 607610.
  • Hall BK. 2003. Descent with modification: the unity underlying homology and homoplasy as seen through an analysis of development and evolution. Biology Review of Camb Philosophical Society 78: 409433.
  • Han TM, Runnegar B. 1992. Megascopic eukaryotic algae from the 2.1-billion-year-old Negaunee iron-formation, Michigan. Science 257: 232235.
  • Harris JM, Wais R, Long SR. 2003. Rhizobium-induced calcium spiking in Lotus japonicus. Molecular Plant Microbe Internation 16: 335341.
  • Hartman H, Fedorov A. 2002. The origin of the eukaryotic cell: a genomic investigation. Proceedings of the National Academy of Sciences, USA 99: 14201425.
  • Hartweck LM, Llewellyn DJ, Dennis ES. 1997. The Arabidopsis thaliana genome has multiple divergent forms of phosphoinositol-specific phospholipase C. Gene 202: 151156.
  • Hartwell LH, Hopfield JJ, Leibler S, Murray AW. 1999. From molecular to modular cell biology. Nature (Suppl.: Impacts of Foreseeable Science) 402: C47– C52.
  • Herbaud ML, Guiseppi A, Denizot F, Haiech J, Kilhoffer MC. 1998. Calcium signalling in Bacillus subtilis. Biochimica Biophysica Acta 1448: 212226.
  • Hetherington AM, Brownlee C. 2004. The generation of Ca2+ signals in plants. Annual Review of Plant Biology 55: 401427.
  • Hrabak EM, Chan CWM, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK, Harmon AC. 2003. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiology 132: 666680.
  • Huang CY, Ayliffe MA, Timmis JN. 2004. Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco. Proceedings of the National Academy of Sciences, USA 101: 97109715.
  • Hunt L, Gray JE. 2001. ABA signalling: a messenger's FIERY fate. Current Biology 11: R968R970.
  • Hunt L, Mills LN, Pical C, Leckie CP, Aitken FL, Kopka J, Mueller-Roeber B, McAinsh MR, Hetherington AM, Gray JE. 2003. Phospholipase C is required for the control of stomatal aperture by ABA. Plant Journal 34: 4755.
  • Hurst LD. 1995. Selfish genetic elements and their role in evolution – the evolution of sex and some of what that entails. Philosophical Transactions of the Royal Society of London B 349: 321332.
  • Jacob F. 1977. Evolution and tinkering. Science 196: 11611166.
  • Jaffe LF. 2002. On the conservation of fast calcium wave speeds. Cell Calcium 32: 217229.
  • Javaux EJ, Knoll AH, Walter MR. 2001. Morphological and ecological complexity in early eukaryotic ecosystems. Nature 412: 6669.
  • Jeong H, Tombor B, Albert R, Oltvai ZN, Barabási AL. 2000. The Large-Scale Organization of Metabolic Networks. Nature 407: 651654.
  • Jones HE, Holland IB, Baker HL, Campbell AK. 1999. Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium. Cell Calcium 25: 265274.
  • Katz LA. 1999. The tangled web: gene genealogies and the origin of eukaryotes. American Naturalist 154: S137S145.
  • Keizer J, Smith GD, Ponce-Dawson S, Pearson JE. 1998. Saltatory propagation of Ca2+ waves by Ca2+ sparks. Biophysics Journal 75: 595600.
  • Kellogg EA. 2004. Evolution of developmental traits. Current Opinion Plant Biology 7: 9298.
  • Khadaroo B, Robbins S, Ferraz C, Derelle E, Eychenie S, Cooke R, Peaucellier G, Delseny M, Demaille J, Van De Peer Y, Picard A, Moreau H. 2004. The first green lineage cdc25 dual-specificity phosphatase. Cell Cycle 3: 513518.
  • Kistner C, Parniske M. 2002. Evolution of signal transduction in intracellular symbiosis. Trends in Plant Science 7: 511518.
  • Kolukisaoglu Ü, Weinl S, Blazevic D, Batistic O, Kudla J. 2004. Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and Rice CBL-CIPK signaling networks. Plant Physiology 134: 4358.
  • Koyanagi M, Ono K, Suga H, Iwabe N, Miyata T. 1998. Phospholipase C cDNAs from sponge and hydra: antiquity of genes involved in the inositol phospholipid signaling pathway. FEBS Letters 439: 6670.
  • Krasnov SG, Cherkashev GA, Stepanova TV, Batuyev BN, Ktotov AG, Malin BV, Maslov MN, Markov VF, Poroshina IM, Samovarov MS, Ashadze AM, Lazareva LI, Ermolayev IK. 1995. Detailed geological studies of hydrothermal fields in the North Atlantic. In: ParsonLM, WalkerCL, DixonDR, eds. Geological Society Special Publication: Hydrothermal vents and processes. 87: 4364.
  • Kretsinger RH, Nockolds CE. 1973. Carp muscle calcium-binding protein. II. Structure determination and general description. Journal of Biological Chemistry 248: 33133326.
  • Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI. 2003. NAPDH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO Journal 22: 26232633.
  • Lawton-Rauh A. 2003. Evolutionary dynamics of duplicated genes in plants. Molecular Phylogenetics and Evolution 29: 396409.
  • Leckie CP, McAinsh MR, Allen GJ, Sanders D, Hetherington AM. 1998. Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose. Proceedings of the National Academy of Sciences, USA 95: 1583715842.
  • Lemtiri-Chlieh F, MacRobbie EAC, Webb AAR, Manison NF, Brownlee C, Skepper JN, Chen J, Prestwich GD, Brearley CA. 2003. Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proceedings of the National Academy of Sciences, USA 100: 1009110095.
  • Lenski RE, Ofria C, Pennock RT, Adami C. 2003. The evolutionary origin of complex features. Nature 423: 139144.
  • Levine BA, Williams RJP. 1982. The chemistry of calcium ion and its biological relevance. In: Anghileri, LJ Anghileri, AMT, eds. The Role of Calcium in Biology Systems, Vol. 1. Boca Raton, FL, USA: CRC Press.
  • Lévy J, Bres C, Geurts R, Chalhoub B, Kulikova O, Duc G, Journet EP, Ané JM, Lauber E, Bisseling T, Dénarié J, Rosenberg C, Debellé F. 2004. A putative Ca2+ and calmodulin-dependent proteinkinase required for bacterial and fungal symbioses. Science 303: 13611364.
  • Li F, Long T, Lu Y, Ouyang Q, Tang C. 2004. The yeast cell cycle network is robustly designed. Proceedings of the National Academy of Sciences, USA 101: 47814786.
  • Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R. 2003. LysM domain receptor kinases regulating rhizobial nod factor-induced infection. Science 302: 630633.
  • MacRobbie EAC. 2000. ABA activates multiple Ca2+ fluxes in stomatal guard cells, triggering vacuolar K+ (Rb+) release. Proceedings of the National Academy of Sciences, USA 97: 1236112368.
  • Madsen EB, Madsen LH, Radutoiu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J. 2003. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425: 637640.
  • Maki M, Kitaura Y, Satoh H, Ohkouchi S, Shibata H. 2002. Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins. Biochimica Biophysica Acta 1600: 5160.
  • Malinski T, Taha Z, Grunfeld S, Patton S, Kapturczak M, Tomboulian P. 1993. Diffusion of nitric oxide in the aorta wall monitored in situ by porphyrinic microsensors. Biochemistry and Biophysics Research Communications 193: 10761082.
  • Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M, Penny D. 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proceedings of the National Academy of Sciences, USA 99: 1224612251.
  • Martin W, Russell MJ. 2003. On the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells. Philosophical Transactions of the Royal Society of London B 358: 5985.
  • Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K, Yagisawa F, Nishida K, Yoshida Y, Nishimura Y, Nakao S, Kobayashi T, Momoyama Y, Higashiyama T, Minoda A, Sano M, Nomoto H, Oishi K, Hayashi H, Ohta F, Nishizaka S, Haga S, Miura S, Morishita T, Kabeya Y, Terasawa K, Suzuki Y, Ishii Y, Asakawa S, Takano H, Ohta N, Kuroiwa H, Tanaka K, Shimizu N, Sugano S, Sato N, Nozaki H, Ogasawara N, Kohara Y, Kuroiwa T. 2004. Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428: 653657.
  • Maynard Smith J, Price GR. 1973. The logic of animal conflict. Nature 246: 1618.
  • McCarty DR, Chory J. 2000. Conservation and innovation in plant signalling pathways. Cell 103: 201209.
  • Von Mering C, Zdobnov EM, Tsoka S, Ciccarelli FD, Pereira-Leal JB, Ouzounis CA, Bork P. 2003. Genomic evolution reveals biochemical networks and functional modules. Proceedings of the National Academy of Sciences, USA 100: 1542815433.
  • Mitra RM, Gleason CA, Edwards A, Hadfield J, Downie JA, Oldroyd GE, Long SR. 2004. A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development. Proceedings of the National Academy of Sciences, USA 101: 47014705.
  • Mori IC, Schroeder JI. 2004. Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiology 135: 702708.
  • Mueller-Roeber B, Pical C. 2002. Inositol phospolipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. Plant Physiology 130: 2246.
  • Muir SR, Sanders D. 1997. Inositol 1,4,5-trisphosphate-sensitive Ca2+ release across nonvacuolar membranes in cauliflower. Plant Physiology 114: 15111521.
  • Nagata T, Iizumi S, Satoh K, Ooka H, Kawai J, Carninci P, Hayashizaki Y, Otomo Y, Murakami K, Matsubara K, Kikuchi S. 2004. Comparative analysis of plant and animal calcium signal transduction element using plant full-length cDNA data. Molecular Biology and Evolution 21: 18551870.
  • Navazio L, Bewell MA, Siddiqua A, Dickinson GD, Galione A, Sanders D. 2000. Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate. Proceedings of the National Academy of Sciences, USA 97: 86938698.
  • Navazio L, Nardi MC, Pancaldi S, Dainese P, Baldan B, Fitchette-Laine AC, Faye L, Meggio F, Martin W, Mariani P. 1998. Functional conservation of calreticulin in Euglena gracilis. Journal of Eukaryotic Microbiology 45: 307313.
  • Nazarenko LV, Andreev IM, Lyukevich AA, Pisareva TV, Los DA. 2003. Calcium release from Synechocystis cells induced by depolarization of the plasma membrane: MscL as an outward Ca2+ channel. Microbiology 149: 11471153.
  • Nisbet EG, Sleep NH. 2001. The habitat and nature of early life. Nature 409: 10831091.
  • Norris V, Grant S, Freestone P, Canvin J, Sheikh FN, Toth I, Trinei M, Modha K, Norman RI. 1996. Calcium signalling in bacteria. Journal of Bacteriology 178: 36773682.
  • Oldroyd GED, Mitra RM, Wais RJ, Long SR. 2001. Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway. Plant Journal 28: 191199.
  • Palmgren MG, Axelsen KB. 1998. Evolution of P-type ATPases. Biochimica et Biophysica Acta 1365: 3745.
  • Parniske M. 2004. Molecular genetics of arbuscular mycorrhizal symbiosis. Current Op Plant Biology 7: 414421.
  • Pennisi E. 2004. The birth of the nucleus. Science 305: 766768.
  • Persson S, Rosenquist M, Svensson K, Galvão R, Boss WF, Sommarin M. 2003. Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants. Plant Physiology 133: 13851396.
  • Ponce-Dawson S, Keizer J, Pearson JE. 1999. Fire-diffuse-fire model of dynamics of intracellular calcium waves. Proceedings of the National Academy of Sciences, USA 96: 60606063.
  • Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J. 2003. Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425: 585592.
  • Reddy VS, Reddy ASN. 2004. Proteomics of calcium-signaling components in plants. Phytochemistry 65: 17451746.
  • Rivera MC, Lake JA. 2004. The ring of life provides evidence for a genome fusion origin of eukaryotes. Nature 431: 152155.
  • Rizo J, Südhof TC. 1998. C2-domains, structure and function of a universal Ca2+-binding domain. New Phytologist 153: 371386.
  • Roberts SK, Gillot I, Brownlee C. 1994. Cytoplasmic calcium and Fucus egg activation. Development 120: 155163.
  • Rosing MT. 1999. 13C-depleted carbon microparticles in > 3700-Ma sea-floor sedimentary rocks from West Greenland. Science 283: 674676.
  • Rudd JJ, Franklin-Tong V. 2001. Unravelling response-specificity in Ca2+ signalling pathways in plant cells. New Phytologist 151: 733.
  • Sánchez JP, Chua NH. 2001. Arabidopsis PLC1 is required for secondary responses to abscisic acid signals. Plant Cell 13: 11431154.
  • Sánchez JP, Duque P, Chua NH. 2004. ABA activates ADPR cyclase and cADPR induces a subset of ABA-responsive genes in Arabidopsis. Plant Journal 38: 381395.
  • Sanders D, Brownlee C, Harper JF. 1999. Communicating with calcium. Plant Cell 11: 691706.
  • Sanderson MJ. 2003. Molecular data from 27 proteins do not support a Precambrain origin of land plants. American Journal of Botany 90: 954956.
  • Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J. 1999. The Arabidopsis det3 mutants reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes and Development 13: 32593270.
  • Scrase-Field SAMG, Knight MR. 2003. Calcium: just a chemical switch? Current Opinion in Plant Biology 6: 500506.
  • Shaw SL, Long SR. 2003. Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells. Plant Physiology 131: 976984.
  • Shiu SH, Karlowski WM, Pan R, Tzeng YH, Mayer KF, Li WH. 2004. Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16: 12201234.
  • Shiu SH, Li WH. 2004. Origins, lineage-specific expansions, and multiple losses of tyrosine kinases in eukaryotes. Molecular Biology and Evolution 21: 828840.
  • Silverman-Gavrila LB, Lew RR. 2001. Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa. European Journal of Cell Biology 80: 379390.
  • Soltis DE, Soltis PS, Morgan DR, Swensen SM, Mullin BC, Dowd JM, Martin PG. 1995. Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms. Proceedings of the National Academy of Sciences, USA 92: 26472651.
  • Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J, Szczyglowski K, Parniske M. 2002. A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature 417: 959962.
  • Strogatz SH. 2001. Exploring complex networks. Nature 410: 268276.
  • Swaminathan R, Hoang CP, Verkman AS. 1997. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophysics Journal 72: 19001907.
  • The Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796815.
  • Tong AHY, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, Chen Y, Cheng X, Chua G, Friesen H, Goldberg DS, Haynes J, Humphries C, He G, Hussein S, Ke L, Krogan N, Li Z, Levinson JN, Lu H, Ménard P, Munyana C, Parsons AB, Ryan O, Tonikian R, Roberts T, Sdicu AM, Shapiro J, Sheikh B, Suter B, Wong SL, Zhang LV, Zhu H, Burd CG, Munro S, Sander C, Rine J, Greenblatt J, Peter M, Bretscher A, Bell G, Roth FP, Brown GW, Andrews B, Bussey H, Boone C. 2004. Global mapping of the yeast genetic interaction network. Science 303: 808813.
  • Torres MA, Dangl JL, Jones JDG. 2002. Arabidopsis gp91 (phox) homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proceedings of the National Academy of Sciences, USA 99: 517522.
  • Toyoshima C, Nakasako M, Nomura H, Ogawa H. 2000. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405: 647655.
  • Usachev YM, Thayer SA. 1999. Ca2+ influx in resting rat sensory neurones that regulates and is regulated by ryanodine-sensitive Ca2+ stores. Journal of Physiology 519: 115130.
  • Vandepoele K, Simillion C, Van de Peer Y. 2003. Evidence that rice and other cereals are ancient aneuploids. Plant Cell 15: 21922202.
  • Vision TJ, Brown DG, Tanksley SD. 2000. The origins of genomic duplications in Arabidopsis. Science 290: 21142117.
  • Wais RJ, Galera C, Oldroyd G, Catoira R, Penmetsa RV, Cook D, Gough C, Dénarié J, Long SR. 2000. Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. Proceedings of the National Academy of Sciences, USA 97: 1340713412.
  • Walker SA, Viprey V, Downie JA. 2000. Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by Nod factors and chitin oligomers. Proceedings of the National Academy of Sciences, USA 97: 1341313418.
  • Weng G, Bhalla US, Iyengar R. 1999. Complexity in biological signaling systems. Science 284: 9296.
  • White PJ, Broadley MR. 2003. Calcium in plants. Annals of Botany 92: 487511.
  • Wikström N, Savolainen V, Chase MW. 2001. Evolution of the angiosperms: calibrating the family tree. Proceedings of the Royal Society of London B 268: 22112220.
  • Williams RJP, Fraústo Da Silva JJR. 2003. Evolution was chemically constrained. Journal of Theoretical Biology 220: 323343.
  • Xiong L, Lee BH, Ishitani M, Lee H, Zhang C, Zhu JK. 2001. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signalling in Arabidopsis. Genes and Development 15: 19711984.
  • Yang T, Poovaiah BW. 2002. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. Journal of Biological Chemistry 277: 4504945058.
  • Yoon HS, Hackett JD, Pinto G, Bhattacharya D. 2002. The single, ancient origin of chromist plastids. Proceedings of the National Academy of Sciences, USA 99: 1550715512.
  • Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D. 2004. A molecular timeline for the origin of photosynthetic eukaryotes. Molecular Biology and Evolution 21: 809818.
  • Yu LP, Miller AK, Clark SE. 2003. POLTERGEIST encodes a protein phosphatase 2C that regulates CLAVATA pathways controlling stem cell identity at Arabidopsis shoot and flower meristems. Current Biology 13: 179188.
  • Zhang Z. 1986. Clastic facies microfossils from the Chaunlingguo formation (1800 Ma) near Jixian, North China. Journal of Micropalaeontology 5: 916.
  • Zhang XS, Choi JH. 2001. Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists. Journal of Molecular Evolution 53: 214224.
  • Zhang X, Wessler SR. 2004. Genome-wide comparative analysis of the transposable elements in the related species Arabidopsis thaliana and Brassica oleracea. Proceedings of the National Academy of Sciences, USA 101: 55895594.