Annexins

Annexins are multifunctional lipid-binding proteins. Plant annexins are expressed throughout the life cycle and are under environmental control. Their association or insertion into membranes may be governed by a range of local conditions (Ca(2+), pH, voltage or lipid identity) and nonclassical sorting motifs. Protein functions include exocytosis, actin binding, peroxidase activity, callose synthase regulation and ion transport. As such, annexins appear capable of linking Ca(2+), redox and lipid signalling to coordinate development with responses to the biotic and abiotic environment. Significant advances in plant annexin research have been made in the past 2 yr. Here, we review the basis of annexin multifunctionality and suggest how these proteins may operate in the life and death of a plant cell.     

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

Heme-independent soluble and membrane-associated peroxidase activity of a Zea mays annexin preparation

Annexins are cytosolic proteins capable of reversible, Ca²⁺-dependent membrane binding or insertion. Animal annexins form and regulate Ca²⁺-permeable ion channels and may therefore participate in signaling. Zea mays (maize) annexins (ZmANN33 and ZmANN35) have recently been shown to form a Ca²⁺-permeable conductance in planar lipid bilayers and also exhibit in vitro peroxidase activity. Peroxidases form a superfamily of intra- or extracellular heme-containing enzymes that use H₂O₂ as the electron acceptor in a number of oxidative reactions. Maize annexin peroxidase activity appears independent of heme and persists after membrane association, the latter suggesting a role in reactive oxygen species signaling.Key words: annexin, calcium, C2, lipoxygenase, maize, peroxidasePeroxidases may guard cells against the toxicity of reactive oxygen species (ROS), and are thought to be involved in stress signaling, auxin regulation and wall remodelling.¹ Recombinant Arabidopsis thaliana annexin 1 (AtANN1) has peroxidase activity in vitro,^2,3 as does an annexin4 from Brassica juncea BjANN1 and Capsicum annum⁵ CaANN24. Annexin peroxidase activity^2,3,6,7 appears to rely on a region (including a conserved His residue; His40) which is similar to the ∼30 amino acid heme-binding domain of plant peroxidases such as horse radish peroxidase (HRP). Mutagenesis of His40 in AtANN1 abolishes peroxidase activity.³In vitro peroxidase activity was also evident in a maize annexin preparation in which ZmANN33/35 were the predominant components but traces of a lipoxygenase and a novel C2-domain-containing protein were also evident.⁸ Peroxidase activity of this maize annexin preparation has Michaelis-Menten kinetics (Fig. 1A). K_m values (mean ± se estimated from Lineweaver-Burk plots) were 15 ± 5 µM and 31 ± 8 µM (n = 3) for maize annexin preparation and HRP respectively (Fig. 1B–D). The mean ± se maximum velocity (V_max) was 76 ± 14 µmole s⁻¹ µg⁻¹ protein compared with 2424 ± 115 µmole s⁻¹ µg⁻¹ for HRP. The K_m of the annexin preparation (15 µM) is within range of the soluble cytosolic heme-containing ascorbate peroxidases (APXs; pea⁹ 20 µM; tea¹⁰ APX1, 30 µM and APX2, 80 µM; rice¹¹ APX1, 33 µM and APX2, 76 µM). APXs are usually associated with tightly regulating the redox status of the cell.¹²Open in a separate windowFigure 1Peroxidase activity of maize annexin preparation measured using Amplex Red. (A) Effect of substrate concentration. Initial rate of Amplex Red oxidation to resorufin by annexin preparation (25 µg/ml) in the presence of a range of [H₂O₂] shows standard Michaelis-Menten behaviour (mean ± se from 3 independent trials). (B) Lineweaver-Burk analysis of the data in (A). (C) Comparison with HRP. Initial rate of Amplex Red oxidation by HRP (0.095 µg/ml) (mean ± se from 3 independent trials). (D) Lineweaver-Burk analysis of HRP reaction rates. Maize annexin preparation was isolated and assayed as described previously.⁸The previous study on the maize annexin preparation failed to detect heme.⁸ The putative heme-binding motif is present in maize annexins but the maize annexin preparation supporting peroxidase activity did not exhibit the Soret peak (in spectral analysis) that is characteristic of the presence of heme.⁸ As it was feasible that a heme moiety could have dissociated, protein was recovered from the Amplex Red peroxidase assay (pH 7.4 and 1 mM H₂O²) and re-tested for peroxidase activity using luminol-based detection. The annexin preparation still displayed peroxidase activity (Fig. 2A) therefore the null result for heme association was unlikely to be the consequence of experimental procedures. In separate tests, staining of native PAGE gels with 3,3′,5,5′ tetramethylbenzidine (TMB) revealed the presence of a heme moiety for as little as 0.095 ng (HRP) but yielded a null result for 10 µg annexin preparation (Fig. 2B). Thus it appears that heme is not essential for peroxidase activity of the annexin preparation.Open in a separate windowFigure 2Heme detection. (A) Peroxidase activity of annexin preparation recovered from spectral analysis with bovine serum albumen (BSA) as a negative control. Peroxidase activity was analysed using an ECL dot blot. Annexin, HRP (“proteins”; 5 µg and 2 ng respectively) and BSA (5 µg; “control”) were dotted onto nitrocellulose. The dot blot was treated with ECL solutions (Amersham) and used to expose photographic film for 5 minutes. (B) Staining of native PAGE gels reveals the presence of heme in 0.095 ng HRP but not in 10 µg annexin preparation. Native PAGE gels were stained for heme.²⁰ After incubation for 45 min in 1.25 mM 3,3′,5,5′tetramethylbenzidine (TMB); 30% (v/v) methanol, 175 mM sodium acetate (pH 5.0), gels were rinsed and incubated for up to 1 hour in 30% (v/v) isopropanol, 175 mM sodium acetate (pH 5.0). Results are representative of 3 determinations.The physiological significance of annexin peroxidase activity could depend on whether the protein is cytosolic or membrane-bound. However, the effects of Ca²⁺ and lipid binding have not been quantified to date. Addition of 10 mM Ca²⁺ caused a significant increase (29%, p = 5.52 × 10⁻⁵; n= 3, Student''s t-test) in peroxidase activity of the maize annexin preparation (1 mM H₂O₂) but had no significant effect on HRP (13 Addition of PS:PC liposomes depressed activity of both annexin preparation and HRP (with no Ca²⁺, by 65% for annexin and 22% for HRP). However, in the presence of liposomes, activity of the annexin preparation was still stimulated by addition of 10 mM Ca²⁺ (79%, p = 5.52 × 10⁻⁵; n = 3) while HRP was unresponsive.

Table 1

Peroxidase activity supported by maize annexin preparation or HRP

Voltage,reactive oxygen species and the influx of calcium

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca²⁺ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca²⁺ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca²⁺ influx, suggesting that these enzymes are involved in sensing Ca²⁺ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) encodes plant cell signals.¹ Reactive oxygen species (ROS) are potent regulators of the PM Ca²⁺ channels implicated in signalling and developmental increases in [Ca²⁺]_cyt.^1,2 Plasma membrane (PM) voltage (V_m) also plays a significant part in generating specific [Ca²⁺]_cyt elevations through the opening of voltage-gated Ca²⁺-permeable channels, allowing Ca²⁺ influx.^1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca²⁺ channels (HACCs),⁴ ROS-activated HACCs⁵ and depolarisation-activated Ca²⁺ channels (DACCs).⁶ The DACC characterisation pointed to the presence of a Cl⁻-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.⁶ This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.⁷ Previous studies have shown that root hair PM also harbors K⁺ channels (mediating inward or outward flux)^8–10 and a H⁺-ATPase.¹¹ A key problem to address now is how these transporters interact to generate and be influenced by PM V_m, thus gating and in turn being regulated by their companion Ca²⁺ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between V_m and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.¹² In the “pump state” the H⁺-ATPase predominates, there is net H⁺ efflux and the hyperpolarized V_m is negative of the equilibrium potential for K⁺ (E_K). In the “K state”, K⁺ permeability predominates but there is still net H⁺ efflux and V_m = E_K. In the third state, there is net H⁺ influx and V_m > E_K. In this depolarized H⁺-influx state, the H⁺-ATPase is thought to be inactive. Oscillations in PM V_m and H⁺ flux may be more profound in growing cells^13,14 and oscillations between these states may explain the temporal changes in H⁺ flux recently observed at the apex of growing Arabidopsis root hairs.¹⁵ Peaks of H⁺ influx may reflect a depolarized V_m that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC^4–6 and because resting V_m is usually found to be hyperpolarized. In a growing cell, with a V_m oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca²⁺ influx it permits could be amplified through intracellular release.The PM H⁺-ATPase traditionally lies at the core of models of voltage and ionic flux^14,16 but in terms of [Ca²⁺]_cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.⁵ AtrbohC is implicated in the transition to polar growth at normal extracellular pH⁵ and also osmoregulation.¹⁷ NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e⁻ efflux systems,¹⁸ their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.¹⁹ Two H⁺ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e⁻ efflux by animal NADPH oxidases is fairly constant over negative V_m and decreases at very depolarized V_m.²⁰ AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H⁺ flux oscillations.¹⁵ The latter suggests that AtrbohC function does in some way affect V_m, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.²¹NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency^22,23 to signal stress conditions. Blockade of PM Ca²⁺ channels by lanthanides increases superoxide production in tobacco suspension cells.²⁴ This suggests that NADPH oxidases are involved in sensing the cell''s Ca²⁺ status and the prediction would be that extracellular Ca²⁺ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).^25,26 Lowering extracellular Ca²⁺ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca²⁺ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca²⁺]_cyt²⁷ (Fig. 1). However, reduction of Ca²⁺ influx should promote voltage hyperpolarisation (just as block of K⁺ influx causes hyperpolarisation in root hairs²⁸) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity⁵ to increase Ca²⁺ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl₂, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187,³⁰ to increase [Ca²⁺]_cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to V_m, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca²⁺]_cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing V_m and [Ca²⁺]_cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.²⁹ While there is still much to do on the “inventory” of PM transporters involved in Ca²⁺ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action.     

Exploiting algal NADPH oxidase for biophotovoltaic energy

Photosynthetic microbes exhibit light‐dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light‐dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild‐type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light‐dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.