annexins: multifunctional components of growth and adaptation

Journal of Experimental Botany, Vol. 59, No. 3, pp. 533–544, 2008

doi:10.1093/jxb/erm344 Advance Access publication 10 February, 2008

REVIEW ARTICLE

Annexins: multifunctional components of growth andadaptation

Jennifer C. Mortimer1, Anuphon Laohavisit1, Neil Macpherson1, Alex Webb1, Colin Brownlee2,

Nicholas H. Battey3 and Julia M. Davies1,*

1 Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK2 Marine Biological Association, The Laboratory, Citadel Hill, Plymouth Pl1 2PB, UK3 School of Biological Sciences, Plant Science Laboratories, University of Reading, Whiteknights, Reading RG66AS, UK

Received 10 October 2007; Revised 3 December 2007; Accepted 4 December 2007

Abstract

Plant annexins are ubiquitous, soluble proteins cap-

able of Ca2+-dependent and Ca2+-independent binding

to endomembranes and the plasma membrane. Some

members of this multigene family are capable of

binding to F-actin, hydrolysing ATP and GTP, acting

as peroxidases or cation channels. These multifunc-

tional proteins are distributed throughout the plant and

throughout the life cycle. Their expression and intra-

cellular localization are under developmental and

environmental control. The in vitro properties of

annexins and their known, dynamic distribution pat-

terns suggest that they could be central regulators or

effectors of plant growth and stress signalling. Poten-

tially, they could operate in signalling pathways in-

volving cytosolic free calcium and reactive oxygen

species.

Key words: Annexin, calcium, channel, GTP, peroxide, stress.

Introduction

Plants sense and respond to a range of environmental,metabolic, and developmental signals. Operation andcontrol of interacting signal transduction pathways willinvolve cell and endomembranes, and integral, peripheral,and soluble proteins. Downstream responses may requireremodelling of the cytoskeleton and changes to exocytoticmachinery and walls. One family of plant proteins appearsto have the capacity to function at all of those levels—theannexins. What are they? First discovered in plants

(tomato) by Boustead et al. (1989), research interest inplant annexins has been spasmodic. In contrast, animalannexins have been studied extensively. Animal annexinsare involved in signal transduction, free cytosolic Ca2+

([Ca2+]cyt) homeostasis, exo- and endocytosis, membraneorganization, cytoskeletal dynamics, cell cycle control,and water permeability (Gerke and Moss, 2002; Hill et al.,2003; Gerke et al., 2005). Analogous functions in plantscould place annexins centre stage in signalling andadaptation. They are already implicated in cold, oxidative,saline, and abscisic acid (ABA) stress responses. What dowe know about them and could they be importantcomponents of signalling and response? Here, plantannexin proteins, their localization, and possible roles arereviewed.

Annexins of the plant kingdom

Annexins are a multigene, multifunctional family ofsoluble proteins with a broad taxonomic distribution. Over200 unique annexin sequences have been described in >65species covering plants, fungi, protists, higher vertebrates,and recently a prokaryote (reviewed by Gerke and Moss,2002; Hofmann, 2004; Moss and Morgan, 2004; Morganet al., 2006). Most of what we know comes from studieson mammalian annexins. Little is known about thephylogenetically distinct plant annexins (Fig. 1). Theyhave been found in all monocot and dicot plants tested todate (reviewed by Delmer and Potikha, 1997; Hofmann,2004), including the model plant Arabidopsis (Clarket al., 2001) and the model legume Medicago (Kovacset al., 1998; de Carvalho-Niebel et al., 1998, 2002).

* To whom correspondence should be addressed. E-mail: [email protected]

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Genome sequencing has revealed seven annexin genesin Arabidopsis (with an eighth evident; Cantero et al.,2006) and nine in rice (Moss and Morgan, 2004).Annexins are expressed throughout the body and lifespanof the plant; embryo (Yu et al., 2005), seedlings (Clarket al., 1992, 2001; Proust et al., 1996; Cantero et al.,2006), roots and tubers (Gidrol et al., 1996; Carroll et al.,1998; de Carvalho-Niebel et al., 1998; Kovacs et al., 1998;Lim et al., 1998; Clark et al., 2001, 2005a, b; Bassaniet al., 2004; Hoshino et al., 2004; Bauw et al., 2006;Cantero et al., 2006), stems, hypocotyls, and coleoptiles(Blackbourn et al., 1991; Thonat et al., 1997; Kovacset al., 1998; Hoshino et al., 2004; Cantero et al., 2006),cotyledons and leaves (Kovacs et al., 1998; Santoni et al.,1998; Hofmann et al., 2000; Seigneurin-Berny et al.,2000; Hoshino et al., 2004; Cantero et al., 2006),inflorescence (Blackbourn et al., 1992; Kovacs et al.,1998), and fruit (Wilkinson et al., 1995; Proust et al.,1996; Hofmann et al., 2000). In addition to expression inthe vasculature (Clark et al., 2001), annexin proteins havebeen found in phloem sap (Barnes et al., 2004; Giavaliscoet al., 2006). It is estimated that annexins can comprise0.1% of plant cell protein (Blackbourn et al., 1991).Proteomic studies now show that plant and oomyceteannexins exist in the cell wall as well as the cytoplasm(Kwon et al., 2005; Meijer et al., 2006). To date, plantstudies have focused on annexin structure and in vitroprotein function. Such studies have revealed a capacity forannexins to be multifunctional and point towards possiblein vivo roles.

Plant annexin structure differs from that ofanimal annexins

Plant annexins have a molecular weight in the region of32–42 kDa and, although sharing a common evolutionaryancestor, differ structurally from their animal counterparts.Animal annexins consist of a conserved a-helical core anda variable N-terminal region. Their annexin core isconstructed from annexin domains (usually found re-peated four times within the protein) each comprising fiveshort a-helices. The annexin domain, of ;70 amino acids,contains the conserved endonexin fold (K-G-X-G-T-{38}-D/E) and is able to bind Ca2+ (Fig. 2). Structurally, eachannexin domain forms a slightly curved disc. The convexside contains the Ca2+-binding sites (described as type IIand type III) and faces the membrane surface when anannexin is associated with lipid (Gerke and Moss, 2002).Ca2+ ions form a bridge between the annexin andnegatively charged membrane phospholipids. The concaveside faces towards the cytosol, and is available forinteractions both with other parts of the protein and withother molecules within the cytosol (Fig. 3A). Mammalianannexins show great variability in the length and sequenceof the N-terminal region. It is thought that this region

Fig. 1. Phylogenetic tree containing annexins from both plant andanimal species. Plant annexins form a distinct monophyletic group.Identifiers after species names refer to the common annexin designation.AnxD nomenclature for plant annexins (where established) is includedin parentheses. Multiple sequence alignment was carried out usingClustal X (Thompson et al., 1997) and the tree was drawn withTreeView (Page, 1996). NCBI identifiers are as follows (RefSeq whereavailable, otherwise GI): A. thaliana AnxAt1, NP_174810.1; A.thaliana AnxAt2, NP_201307.1; A. thaliana AnxAt3, NP181410.1;A. thaliana AnxAt4, NP_181409.1; A. thaliana AnxAt5, NP_564920.1;A. thaliana AnxAt6, NP_196584.1; A. thaliana AnxAt7, NP_196585.1; A.thaliana AnxAt8, NP_568271.2; Z. mays AnxZm33, 6272285; Z. maysAnxZm35, 1370603; Fragaria3ananassa AnxFa4, 6010777; O. sativaAnxOs1, NP_001061839; T. aestivum AnxTa1, 38606205; C. annuumAnxCa24, 75319682; M. truncatula AnxMt1, 3176098; C. elegans Nex-1,NP_497903; H. sapiens AnxA1, NP_000691; H. sapiens AnxA5,NP_001145; H. sapiens AnxA6, NP_001146; M. musculus AnxA1,NP_034860; M. musculus AnxA2, NP_031611; M. musculusAnxA3, NP_038498; M. musculus AnxA11, NP_038497; M. musculusAnxA13, NP_081487; D. rerio AnxA1a, NP_861423; R. norvegicus AnxA1,NP_037036; R. norvegicus AnxA2, NP_063970; R. norvegicus AnxA3,NP_036055.

534 Mortimer et al.


regulates the stability of different annexin conformations,determines interactions with other proteins, and hence isresponsible for the functional diversity of mammalianannexins (Gerke and Moss, 2002). It is the site ofsecondary modification, including phosphorylation, nitro-sylation, S-glutathiolation, and N-myristoylation, indica-tive of regulation by several distinct signalling pathways(reviewed by Gerke and Moss, 2002; Gerke et al., 2005).

For plant annexins, typically only the first and fourthrepeated domains have the characteristic endonexin

sequence (Fig. 2). Crystal structures have been described(Hofmann et al., 2000, 2003). Plant annexins have, onaverage, a larger surface area than mammalian annexins(Clark et al., 2001). This is due to extra grooves andclefts, perhaps suggesting a wide range of interactionpartners and a broad range of roles within the cell.However, in contrast to their animal counterparts, theN-terminal region of all known plant annexins is short(;10 amino acids). The crystal structure of bell pepperannexin (AnxCa32) revealed that the short N-terminal

Fig. 2. Multiple sequence alignment of a selection of plant annexins. Sequences in the alignment, and the highlighted features, are discussed in thetext. Sequence shading: red box and white character, strict identity; red box and red character, similarity in a group; blue box, similarity acrossgroups. Features described in the text are highlighted. Green triangle, His40 key peroxidase residue; blue square, IRI actin-binding motif; yellow star,GXXXXGKT, DXXG putative GTP-binding motif; purple square, KGXGT-38-D/E Ca2+-binding sites; black triangle, putative S3 cluster; turquoisecircle, conserved tryptophan required for membrane binding. GenBank accession numbers: AnxZm33, CAA66900; AnxZm35, CAA66901;AnxCa32, CAA10210; AnxGh1, AAC33305; AnxAt1, NP_174810; AnxAt2, NP_201307; AnxLe35, AAC97493; AnxMt1, CAA75308. Sequencealignment was generated using Clustal X (Thompson et al., 1997; default settings). Features were added using ESPript (Gouet et al., 2003).

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region interacts with the annexin core, suggesting thatsome regulatory function of this region is conserved inplant annexins (Hofmann et al., 2000).

Ca2+-dependent and -independent membranebinding

Characteristically, both animal and plant annexins bindCa2+ and, in the presence of (micromolar) Ca2+, will bindto negatively charged phospholipids including phosphati-

dylserine, phosphatidylinositol, and phosphatidic acid(Blackbourn et al., 1991; Balasubramanian et al., 2001).Binding can be reversed by addition of Ca2+ chelators. Anannexin may be membrane associated or even membraneinserted, depending on the [Ca2+]cyt, pH, lipid composi-tion, and voltage (reviewed by Gerke and Moss, 2002).Certain annexins such as AnxB12 from Hydra have thecapacity to be soluble, peripheral, and integral proteins(Ladokhin and Haigler, 2005). Using molecular simulationand site-directed mutagenesis, Montaville et al. (2002)identified a consensus phosphatidylserine-binding site ([R/K]XXXK-BC-helices-[R/K]XXXXDXXS[D/E]+Ca2+) inmammalian annexins. Found in domain I and sometimesadditionally in domain II (but never in domains III or IV),the site overlaps the Ca2+-binding domains.

Sequence alignment with mammalian annexinsrevealed that the phosphatidylserine-binding site is onlypoorly conserved in plant annexins (Hofmann et al.,2000). Despite divergence in amino acid sequence,phosphatidylserine-binding activity is conserved in atleast some plant annexins including maize, bell pepper,and cotton (Blackbourn et al., 1991; Hofmann et al.,2000; Dabitz et al., 2005). Strict sequence conservationdoes not appear to be necessary for membrane-bindingfunction. This is supported by the observation that bothplant and animal annexins bind to a range of negativelycharged phospholipids in addition to phosphatidylserine,including phosphatidylinositol, phosphatidic acid, andmalonaldehyde-conjugated lipids (Blackbourn et al.,1991; Balasubramanian et al., 2001). In plants, hydro-phobic interactions are also involved in membranebinding. AnxCa32 attachment to membranes involvesthe hydrogen bonding of several amino acid residues tothe phospholipid headgroup and glycerol backbone(Dabitz et al., 2005). Site-directed mutagenesis ofrecombinant tomato annexin p35 (AnxLe35) revealedthat the fourth repeat of the core domain was critical tolipid binding (Lim et al., 1998).

Although Ca2+ is required for membrane binding atneutral pH, at acidic pH (<pH 6.0) some animal annexinsbind to membranes independently of Ca2+ (Kohler et al.,1997; Langen et al., 1998; Rosengrath et al., 1998;Golczak et al., 2004). Plant annexins also appear capableof Ca2+-independent membrane binding. Recently, it hasbeen reported that ;20% of the annexin protein (AnxGh1and AnxCa32) remains bound to lipid vesicles in theabsence of Ca2+ at neutral pH, and the proteins can bereleased following addition of detergent (Dabitz et al.,2005). However, rather than Ca2+-independent membranebinding, as suggested by Dabitz et al. (2005), this mayrepresent a proportion of the population undergoingmembrane insertion, hence the requirement for detergentto release the protein. In addition to promotingCa2+-independent binding, acidic pH can reduce the Ca2+

requirement for phosphatidylserine binding (Blackbourn

Fig. 3. Annexin structure and localization. (A) Animal annexin (blue)membrane association in the presence of Ca2+ ions (green). Each of thefour conserved annexin domains (I–IV) is predicted to bind a singleCa2+ ion, forming a slightly concave disc. In plants, typically only thefirst and fourth repeated domains have the characteristic endonexinsequence. The N-terminal region (N) is the site of secondary modi-fication and in plants is only ;10 amino acids long. (B) Subcellularlocalization of plant annexin proteins (blue circles) in an idealized plantcell. The localization depends on many factors, including plant species,tissue type, and [Ca2+]cyt, as described in the text. A, actin;C, chloroplast; CW, cell wall; G, Golgi; M, mitochondria; N, nucleus;P, peroxisome; PM, plasma membrane; R-ER, rough endoplasmicreticulum; S-ER, smooth endoplasmic reticulum; V, vacuole.

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et al., 1991). The mechanism of plant annexinCa2+-independent membrane binding is still unclear, al-though a pair of conserved tryptophans (W35/W107) isinvolved (Dabitz et al., 2005). Additionally, it has beensuggested that Ca2+-independent membrane bindingserves as a platform for an annexin population whosemembrane binding is Ca2+ dependent. Given that sequen-tial and co-operative binding of annexins has been shown,the two modes of membrane binding may be intimatelylinked (Dabitz et al., 2005).

Although the majority of plant annexins tend to befound in the cytosol (Blackbourn et al., 1992; Clark et al.,1992, 1994; Thonat et al., 1997), they are also foundbound to (or in some cases inserted into) both plasmamembranes and endomembranes (Fig. 3B). Annexins canlocalize to the plant vacuolar membrane (Seals et al.,1994; Seals and Randall, 1997; Carter et al., 2004), theGolgi, and Golgi-derived vesicles (Clark et al., 1992).Plant annexins can also cause aggregation of liposomesand secretory vesicles, implicating them in membraneorganization (Blackbourn and Battey, 1993). Medicagotruncatula annexin1 (AnxMt1; de Carvalho-Niebel et al.,1998) localizes to the nuclear membrane, and M. sativa(the model legume) AnxMs2 has been shown to localizein the nucleolus under stress conditions even though theprotein shows no typical nuclear localization signal(Kovacs et al., 1998). A nuclear localization has also beenreported for pea annexin (Clark et al., 1998). A putativespinach annexin has been identified in chloroplast envel-ope membranes (Seigneurin-Berny et al., 2000). Arabi-dopsis AnxAt1 has been found in chloroplasts (Peltieret al., 2002, 2006; Friso et al., 2004; Kleffmann et al.,2004; Renaut et al., 2006) but also as a tonoplast protein(Carter et al., 2004) and an integral plasma membraneprotein ostensibly under non-stressed conditions (Santoniet al., 1998; Alexandersson et al., 2004). Its membraneassociation is prompted by salinity stress (Lee et al.,2004). Arabidopsis AnxAt4 has also been identified asa plasma membrane protein (Alexandersson et al., 2004).An annexin from Bryonia diocia relocates from thecytoplasm to the plasma membrane following mechano-stimulation (Thonat et al., 1997). In wheat exposed to lowtemperatures, two wheat annexins accumulate in theplasma membrane (Breton et al., 2000). Moreover, theyare integral membrane proteins, which cannot be releasedby addition of Ca2+ chelators (Breton et al., 2000). Thatannexin association with or insertion into membranes canbe dynamic and responsive to environmental change isconsistent with their involvement in signalling andadaptation.

Actin binding

Actin filaments help shape a cell, are essential for thedevelopment of certain plant cell types, and act in

signalling (reviewed by Drøbak et al., 2004). Actinbinding is limited to a small number of animal annexinsand, as a generalization, actin–annexin interaction appearsto be restricted to regions close to animal membranes(Hayes et al., 2004). The molecular mechanism of actinbinding is poorly understood, but a C-terminal motif(LLYLCGGDD) has been implicated; this is not presentin plant annexins (reviewed by Hayes et al., 2004;Konopka-Postupolska, 2007). Evidence for binding ofplant annexins to actin is mixed, and appears to be speciesspecific. Tomato and mimosa annexins both undergoCa2+-dependent F-actin binding in vitro (Calvert et al.,1996; Hoshino et al., 2004). Two plasma membrane-associated annexins from zucchini bind to zucchini-derived F-actin (Hu et al., 2000). Mimosa annexinorganizes F-actin into thick bundles in the presence of2 mM Ca2+ in vitro (Hoshino et al., 2004). Cotton, bellpepper, and maize annexins have all been extensivelytested and show no affinity for actin, in either the presenceor absence of calcium (Blackbourn et al., 1992; Delmerand Potikha, 1997; Hoshino et al., 2004). However, thelatter all possess the IRI motif (needed in F-actin bindingto mysosin) implicated in actin binding by Lim et al.(1998) for tomato annexin. This suggests that thestructural requirement for actin binding is more complex.As yet annexins from two of the most studied species,Arabidopsis and Medicago, have not been tested forF-actin binding, although Arabidopsis sequences containa full or partially conserved F-actin-binding motif (Clarket al., 2001). The functional significance of annexin–actininteraction is unknown, but has been postulated to be in-volved in exocytosis and signalling (Konopka-Postupolska,2007).

Peroxidase activity

The crystal structure of cotton annexin AnxGh1 revealedtwo adjacent cysteine residues which, in combination witha nearby methionine residue, form an S3 cluster. Althoughno function has been demonstrated for this cluster, it hasbeen suggested that it may have a role in transfer ofelectrons to an oxidizing molecule, potentially a reducedreactive oxygen species (ROS) (Hofmann et al., 2003).Although experimental studies have been limited toa single Arabidopsis annexin, it is possible that severalplant annexins may be able to act as peroxidases.Heterologous expression of Arabidopsis annexin AnxAt1in the Escherichia coli DoxyR (peroxide-sensitive) mutantor overexpression in mammalian cells afforded protectionagainst peroxide-mediated oxidative stress (Gidrol et al.,1996; Kush and Sabapathy, 2001). Peroxidase activitywas demonstrated in vitro using both recombinantAnxAt1 and AnxAt1 purified from Arabidopsis tissue(Gidrol et al., 1996; Gorecka et al., 2005). Post-translationalmodification of AnxAt1 may be required for peroxidase

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activity as recombinant AnxAt1 purified from E. coli hadlower activity than that purified from Nicotiana benthami-ana, the activity of which was decreased by dephosphor-ylation (Gorecka et al., 2005). Indeed, secondarymodification of plant annexins is suggested by SDS–PAGE analysis. For example, the theoretical size andisoelectric point of AnxAt1 are 36 kDa and pI 5.2,respectively, whereas following two-dimensional electro-phoresis Lee et al. (2004) found two microsomal AnxAt1spots: 40 kDa, pI 5.2; and 40 kDa, pI 5.3. Santoni et al.(1998) reported two plasma membrane forms of AnxAt1with the following migration characteristics: 39 kDa, pI5.0; and 34 kDa and pI 5.1. Additionally, annexin nitro-sylation has been observed in Arabidopsis (Lindemeyret al., 2000).

The peroxidase activity of AnxAt1 is suggested to bedue to a region of the first annexin domain in theN-terminal region (centring on a conserved histidine residue;His40) which has a strong sequence similarity to the ;30amino acid haem-binding motif of plant peroxidases,typified by horseradish peroxidase (Clark et al., 2001;Gorecka et al., 2005). Consistent with this idea, mutationof the conserved histidine to alanine substantially reducedin vitro peroxidase activity (Gorecka et al., 2005). Theputative haem-binding motif is conserved in several plantannexins including those of maize, cotton, and bell pepper(Fig. 2). Haem-containing peroxidases catalyse the oxida-tion of a substrate by the removal of a single electron andreduce hydrogen peroxide to water. However, to datethere have been no reports of haem binding to plantannexins to confer peroxidase function. Neither are thepotential targets of annexin peroxidase activity identifiedas yet. It remains feasible that in vitro peroxidase activityis a consequence of metal-binding ability generatingartificial catalytic sites. It is feasible that in common withother types of peroxidases, annexins are capable ofgenerating ROS. However, the sheer numbers of perox-idases in plants and potential for redundancy may makethis aspect of annexin physiology particularly difficult toelucidate.

ATPase and GTPase activity

Binding to ATP and GTP is a common feature of animalannexins even though they do not contain the WalkerA and B motifs. Rather, the nucleotide-binding motif isthought to be FXXKYD/EKSL (Bandorowicz-Pikulaet al., 2003). Plant annexins not only bind purinenucleotides but also hydrolyse them (maize, McClunget al., 1994; tomato, Calvert et al., 1996; Lim et al., 1998;cotton, Shin and Brown, 1999). In contrast to animalannexins, nucleotide binding and hydrolysis may dependon a Walker A motif (GXXXXGKT/S) and a GTP-binding motif typical of the GTPase superfamily (DXXG).These sequences have been found in the fourth repeat of

AnxGh1; C-terminal deletion and loss of the fourth repeatabolished its GTPase activity (Shin and Brown, 1999). InArabidopsis, the greatest similarity is in the fourth repeatof AnxAt2 and AnxAt7 (Clark et al., 2001).

Both maize and tomato annexins are able to hydrolyseATP and GTP at a similar rate, but GTP is the preferredsubstrate for cotton annexin AnxGh1. Tomato annexinGTPase activity still proceeds when the protein is boundto actin (Calvert et al., 1996), suggesting that cytoskeletalassociation may specifically locate the annexin’s GTPasefunction in the cell. Ca2+ has an inhibitory effect on cottonannexin GTPase activity but not the ATPase/GTPaseactivity of maize annexin AnxZm33/35 (McClung et al.,1994; Shin and Brown, 1999). Alignments of the primarysequence of cotton annexin, AnxAt2, and AnxZm33/35showed that the GTP-binding motifs overlap theCa2+-binding motif of the fourth endonexin domain(McClung et al., 1994; Shin and Brown, 1999). Ca2+ andGTP may therefore compete for binding. Ca2+-mediatedphospholipid binding has been shown to inhibit hydrolyticactivity of tomato annexin (Calvert et al., 1996). Muta-genesis of the Ca2+-binding sites does not impair GTPaseactivity of the soluble form (Lim et al., 1998), demon-strating that membrane binding prevents GTP from reach-ing its catalytic site. Overall, modulation of enzymeactivity by Ca2+ and membrane binding may afford spatio-temporal definition of annexin function. That tomato,maize, and cotton annexins have different requirements,despite catalysing the same reaction, may reflect thediverse roles that plant annexins fulfil.

Sensor and channel activity

Ca2+ binding is a defining annexin characteristic, butstudies on animal annexins show them to be capable ofsensing and regulating free cytosolic calcium (Hawkinset al., 2000; Watson et al., 2004). Mammalian AnxA6 hasbeen shown to act as a Ca2+ sensor, mediating membraneassociation of a GTPase-activating protein (GAP) thatthen regulates a monomeric GTPase (Grewal et al., 2005).In addition to controlling trafficking of ion transporters totheir target membranes and regulating their activity(reviewed by Gerke et al., 2005), animal annexins canalso form Ca2+-permeable ion channels themselves. Thisability was first demonstrated with bovine AnxA7 whichforms a highly selective, voltage-gated Ca2+ channel inphosphatidylserine bilayers (Pollard and Rojas, 1988).Since then, many other animal annexins have been shownto form ion channels, although their properties (particu-larly selectivity) differ (reviewed by Hawkins et al., 2000;Kourie and Wood, 2000). ATP, GTP (Kirilenko et al.,2006), cAMP, hydrogen peroxide (Kubista et al., 1999),and low pH (Kohler et al., 1997; Langen et al., 1998;Rosengarth et al., 1998) have all been shown to regulateannexin channel activity. Animal annexins can cause Ca2+

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influx across animal plasma membrane and mobilize Ca2+

from internal stores (Kubista et al., 1999; Watson et al.,2004) placing them at the core of Ca2+-mediated signaltransduction and Ca2+ homeostasis.

A number of models have been proposed to explain themechanism whereby animal annexin proteins (which aretoo small to span a membrane) form ion channels. Atacidic pH, the short helices of the monomer may join toform longer helices to allow spanning of the membrane.In invertebrates this has been observed for AnxB12,which undergoes large-scale conformational changes underconditions that result in membrane insertion (Langenet al., 1998; Isas et al., 2000). Recently, freeze-fractureelectron microscopy revealed images of AnxB12 as anintegral membrane protein at pH 4 but not at higher pH(Hegde et al., 2006). This membrane-inserted form ofAnxB12 appears to be monomeric, unlike the trimericmembrane-attached form (Ladokhin and Haigler, 2005).Another model suggests that an annexin monomerassociates with the membrane and causes an electrostaticdisruption which allows passage of ions through thecentral pore of the protein, although it is not clear howthis pore would be selective for Ca2+ (reviewed by Kourieand Wood, 2000). Regardless of the mechanism, annexinion channels challenge the view of ion channels as solelyintegral membrane proteins. As unusual as this is,membrane insertion of ion channels from the aqueousphase is not unprecedented. In addition to annexinchannels, an animal chloride channel (CLIC-1; Tulket al., 2002) also moves directly from the cytosol intoa membrane.

An ability of plant annexins to form or regulate Ca2+

channels in plasma and endomembranes would enablesignal transduction and amplification (Kovacs et al., 1998;White et al., 2002). The putative pore region of the humanAnxA5 channel contains two salt bridges (Asp92–Arg117and Glu112–Arg271) that regulate selectivity for calciumand channel opening in response to voltage (Liemannet al., 1996). Both salt bridges are quite well conserved inplant annexins. It has been proposed that plant annexinscould act as the plasma membrane Ca2+-permeablechannels that mediate Ca2+ entry into the cell at verynegative (hyperpolarized) membrane voltage (White et al.,2002). Such channels are strongly implicated in growthand signalling. To date, AnxCa32 has been shown tomediate passive Ca2+ flux in fura-2-loaded vesicles(Hofmann et al., 2000), supporting the general concept ofchannel function. Maize annexins AnxZm33/35 containthe putative salt bridges, and a partially purified prepara-tion formed hyperpolarization-activated Ca2+-permeablechannels in planar lipid bilayers (Nichols, 2005). As onlyone gene has been verified as encoding a plant Ca2+

channel (TPC1 encoding a vacuolar channel; Peiter et al.,2005), it will be of great interest to see whether plantannexins purified to homogeneity support Ca2+ channel

activity. Of the eight Arabidopsis annexins, AnxAt1 hasbeen found to form K+-permeable channels in bilayers,with channel formation favoured at low pH (Goreckaet al., 2007). In common with AnxA5 and AnxB12, acidicpH increases the hydrophobicity of AnxAt1 and promotesthe oligomerization thought necessary for transport activity.The Ca2+ permeability of the AnxAt1 channel remains tobe determined, as does its in vivo function, but AnxAt1illustrates clearly the multifunctional nature of annexins—potentially a peroxidase and a channel in one.

Function in exocytosis, growth, and development

Annexin expression is dynamic even under normal growthconditions. Transcript levels of annexin genes in Arabi-dopsis vary depending on tissue type and age, suggestingspecific purposes at different developmental stages indifferent tissue (Clark et al., 2001, 2005a; Hoshino et al.,2004; Cantero et al., 2006). Annexin expression increasesduring fruit ripening and gall ontogeny, implying hor-monal control (Wilkinson et al., 1995; Proust et al., 1996;Vandeputte et al., 2007). Nod factors induce M. trunca-tula annexin1 (AnxMt1) expression, and co-localizationstudies using AnxMt1–GFP (green fluorescent protein)have suggested that it may be involved in the early stagesof cell division required for nodule formation (deCarvalho-Niebel et al., 2002).

The in vitro ability to bind membranes, Ca2+, purinenucleotides, and actin predicts critical roles for bothanimal and plant annexins in membrane trafficking andsignal transduction. Animal annexins are clearly involvedin exo- and endocytosis, and the targeting of proteins tospecific membrane sites (reviewed by Gerke and Moss,2002; Gerke et al., 2005). The clearest demonstration todate of annexin function in planta has been the stimula-tion of Ca2+-dependent vesicle fusion to the plasmamembrane of maize root cap protoplasts by AnxZm33and 35 (Carroll et al., 1998). Plant annexins are abundantand underlie the plasma membrane in cells associated withhigh secretion rates. Annexins are prominent at the apexof cells undergoing polar elongation, such as root hairs,pollen tubes, and fern rhizoids (Blackbourn et al., 1992;Blackbourn and Battey, 1993; Clark et al., 1995, 2001,2005a). Annexin expression has been detected in the rootelongation zone of maize (Carroll et al., 1998; Bassaniet al., 2004) and Arabidopsis (Clark et al., 2005a, b). Aswell as possibly being involved in primary and root hairelongation growth, a specific annexin in Arabidopsis(AnxAt2) is implicated in lateral root development (Clarket al., 2005a), implying upstream regulation by growthregulators. Cotton annexin (AnxGh1) mRNA is up-regulated during cotton fibre elongation, and the proteinmay be associated with Golgi-derived coated vesiclesmediating fibre elongation (Shin and Brown, 1999; BulakArpat et al., 2004).

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Recent work on a Saprolegnia annexin has revealed anability to stimulate (1–3)-b-D-glucan synthase activity,implying a role in the regulation of wall synthesis(Bouzenenza et al., 2006). This is in contrast to theinhibitory effect of cotton annexin AnxGh1 (Andrawiset al., 1993) and suggests that different annexins playdistinct regulatory roles. Arabidopsis AnxAt7 expression isup-regulated by oxylipin treatment that induces calloseformation and causes wavy roots and lateral root in-hibition (Vellosillo et al., 2007). It will be interesting tosee whether this annexin regulates glucan synthase. Inaddition to roles in exocytosis and wall synthesis, in-dividual annexins could be acting as Ca2+ or GTP sensorsto co-ordinate growth. A role as a Ca2+ sensor has beenproposed for vacuole-associated annexins (Seals et al.,1994). Vacuolar biogenesis is a key component of cellexpansion, and expression of the vacuole-associatedtobacco annexin VCaB42 correlates with vacuolar bio-genesis in expanding cells (Seals and Randall, 1997). Theannexin is associated with a ROP GTPase (Lin et al.,2005), a type of protein viewed as master regulators ofgrowth. Addition of GTP inhibited annexin-mediatedexocytosis in root cap protoplast (Carroll et al., 1998),suggesting that annexin function is co-ordinated by localGTP and Ca2+ levels.

Light response, nyctinastic movement, andgravitropism

As well as being linked to growth and development,annexin expression and distribution can also change inresponse to environmental stimuli. Light affects theexpression of certain annexins in Arabidopsis (Canteroet al., 2006). In hypocotyls, AnxAt5 expression is in-creased by red light and this is reversible by application offar red light; in cotyledons, AnxAt6 has a similar red/farred response (Cantero et al., 2006). These results point toannexin expression being downstream of phytochrome A,and further dissection of this relationship is awaited.Phytochrome action has previously been implicated in theregulation of polarized annexin distribution in fernrhizoids (Clark et al., 1995). Nyctinastic (night time)movement of the Mimosa pulvinus provides a beautifulexample of temporal regulation of annexin abundance andpositioning. The amount of annexin is more significant atnight (when the leaf droops) and the protein is largelycytosolic, whilst in the morning (when the leaf is held up)it has redistributed to the outermost periphery of the motorcells in the pulvinus (Hoshino et al., 2004). Quite whatthe annexin does in either position remains unknown butits abundance is positively regulated by ABA (Hoshinoet al., 2004), which suggests that annexins link stressresponses with nyctinastic and possibly seismonastic(touch-induced) movement. Both types of movementinvolve Ca2+ influx from the apoplast (Campbell and

Thomson, 1977), and perhaps this is involved in annexinrelocation. Mimosa annexin binds F-actin in vitro (Hoshinoet al., 2004). Actin filaments in Mimosa are thought tocontrol pulvinus movement, in conjunction with alteredosmotic pressures, through decreased tyrosine phosphory-lation of the actin, leading to tissue bending. However,since Mimosa annexin distribution does not preciselyfollow actin distribution in vivo, the relationship betweenthe two remains unclear (Hoshino et al., 2004; Kanzawaet al., 2006).

Although there are no reports as yet for involvement ofannexins in gravisensing, they are implicated in mediatingthe resultant differential growth response. Prior to a grav-itational stimulus, annexins are asymmetrically distributedin the direction of gravity at the periphery of cells justbelow the apical meristem of etiolated pea plumules(Clark et al., 2000). Using immunofluorescence, annexinswere found to redistribute within 15 min of a gravitationalstimulus (and prior to the onset of plumule curvature),occupying a more evenly distributed peripheral position(Clark et al., 2000). This has been interpreted in terms ofan annexin involvement in redirecting materials forgrowth (Clark et al., 2000). In gravistimulated Arabidop-sis roots, the abundance of AnxAt1 increases in roots(Kamada et al., 2005) and predominates in epidermal cellsthat would undergo the greatest growth rate to bend theroot (Clark et al., 2005b). This distribution largelymatched that of polysaccharide secretion, supporting a rolefor annexins in the gravistimulated growth response(Clark et al., 2005b). In gravistimulated hypocotyls,AnxAt2 was detected preferentially in the epidermis thatwould grow the fastest (Clark et al., 2005b). Determina-tion of how these distributions are caused and link togravistimulated changes in [Ca2+]cyt, actin, and ROS isincreasingly within the technical range of plant biologists.Overall, the ability to bind membrane, GTP, and actinsuggests the involvement of annexins in (differential)growth and places them downstream of a wide range ofsignal transduction pathways.

Responding to stress stimuli and pathogens

The mechanical stress caused by wind results in short,bushy plants (thigmomorphogenesis). Annexins respondto mechanical stress in B. dioica internodes by redistribut-ing from the cytosol to the plasma membrane inparenchyma cells (sampled 30 min after the stimulus;Thonat et al., 1997). The chain of events leading to thisremains to be determined. However, mechanical stress isknown to elevate [Ca2+]cyt and this could stimulateannexin–plasma membrane association. The significanceof annexin relocation is not understood, but as regulatorsof growth they may govern the radial expansion thatresults from mechanical stress or could be ‘conditioning’the plasma membrane for further stress responses.

540 Mortimer et al.


Cold causes increased annexin expression in poplarleaves (Renault et al., 2006). In wheat, the cold-inducedaccumulation of annexins p39 and p22.5 and theirinsertion into the plasma membrane could be involved insensing or transducing Ca2+ signals or in regulating[Ca2+]cyt during signalling or acclimation (Breton et al.,2000). Annexin expression, abundance, and cellularposition can respond to osmotic stress, salinity, drought,and ABA (Watkinson et al., 2003; Lee et al., 2004;Buitnik et al., 2006; Vandeputte et al., 2007). Alfalfaannexin AnxMs2 mRNA increased during tissue develop-ment, but the amount of transcripts was elevated signif-icantly upon NaCl treatment or exogenous ABAapplication (Kovacs et al., 1998). The level of ArabidopsisAnxAt1 protein changes as the plant is subjected toosmotic stress, even though its transcript is not affected(Lee et al., 2004). However, in contrast, Cantero et al.(2006) found that AnxAt1 mRNA abundance is increasedby salinity stress. The temporary annexin relocation tomembranes in response to ABA and saline stress (Leeet al., 2004) could reflect a role in signalling, or representtheir regulation of proteins already in the membrane orannexin-mediated insertion of new proteins to cope withadverse conditions. AnxAt1 transcript accumulates inresponse to phosphate deprivation (Muller et al., 2007)and in the presence of H2O2 (Gidrol et al., 1996). AsH2O2 accumulates in response to phosphate deprivation(Shin et al., 2005), this result suggests that AnxAt1operates downstream in this stress response. AnxAt1expression is also up-regulated by salicylic acid, whichimplicates this annexin in pathogen defence responses(Gidrol et al., 1996). Expression of Arabidopsis AnxAt4,tomato AnxLe34, and tobacco AnxNt12 also increasesduring pathogen attack but the functional significanceremains unknown (Xiao et al., 2001; Truman et al., 2007;Vandeputte et al., 2007). Salinity and ABA also induceAnxNt12 expression, but application of H2O2 does not,suggesting control by distinct signalling pathways andspecific annexin function (Vandeputte et al., 2007).

Annexins and reactive oxygen species

Production of ROS is implicated in control of plant(tropic) growth and development, in some cases control-ling [Ca2+]cyt (reviewed by Gapper and Dolan, 2006).Stress conditions that cause ROS generation (such aspathogen attack, drought, salinity, cold, hypoxia, andnutritional restriction) also prompt annexin accumulationor relocation to membranes. However, it is as yet unclearwhether ROS or elevated [Ca2+]cyt trigger annexinresponses. Membrane oxidation increases membrane bind-ing of animal annexins (Balasubramanian et al., 2001).Peroxide can induce the channel-forming vertebrateAnxA5 to be inserted into membranes in vitro, andperoxide-induced Ca2+ influx in vivo in DT40 pre-B cells

requires AnxA5 (Kubista et al., 1999). From this itfollows that channel-forming plant annexins (such asAnxAt1) are candidates for the ROS-activated channelsidentified in several plant cells (Foreman et al., 2003).

Recently, annexins have been identified as proteincomponents of an M. truncatula plasma membrane lipidraft alongside signalling and redox proteins (Lefebvreet al., 2007). They could, in common with raft-associatedanimal annexins (Babiychuk and Draeger, 2000), anchorrafts to the actin cytoskeleton (Konopka-Postupolska,2007). Alternatively, conceivably, raft-associated annexinscould function as channels or peroxidases operating ina localized ROS signalling ‘hub’. AnxAt1 is present at theroot hair apex (which is thought to harbour lipid rafts;Jones et al., 2006) and as a peroxidase could help regulatethe intracellular peroxide generated during polar growth(Foreman et al., 2003). As peroxidases, annexins couldregulate peroxide generated as an inter- or intracellularmessenger or relay/terminate a signal through peroxide-dependent oxidation. A protective role can also beenvisaged. Peroxidase activity of annexins associated withchloroplast RNA polymerase could protect newly synthe-sized transcripts from oxidation (Pfannschmidt et al.,2000).

Future perspectives

Sensors? Skeletal regulators? Channels? Peroxidases?Plant annexins are potentially multifunctional proteinsin vivo involved in membrane dynamics, actin modelling,and [Ca2+]cyt and ROS regulation. Swifter resolution ofstimulus-induced annexin relocation to membranes (par-ticularly in single cell paradigms such as the root hair orguard cell) will help secure definitions of function, as willthe increasing availability of mutants.

Acknowledgements

This work was supported by the BBSRC and the CambridgeOverseas Trust.

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annexins: multifunctional components of growth and adaptation

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