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.     

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.     

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