Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Apr;24(4):1522-33.
doi: 10.1105/tpc.112.097881. Epub 2012 Apr 20.

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

Anuphon Laohavisit  1 , Zhonglin Shang, Lourdes Rubio, Tracey A Cuin, Anne-Aliénor Véry, Aihua Wang, Jennifer C Mortimer, Neil Macpherson, Katy M Coxon, Nicholas H Battey, Colin Brownlee, Ohkmae K Park, Hervé Sentenac, Sergey Shabala, Alex A R Webb, Julia M Davies
Affiliations

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

Anuphon Laohavisit et al. Plant Cell. 2012 Apr.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
ann1 Root Plasma Membrane Lacks the OH•-Activated Conductance. (A) Whole-cell patch clamp recordings from wild-type (WT; Col-0) root hair apical spheroplast PM. Left: Control currents (I) elicited by step voltage (V) changes, from a representative spheroplast. Centre: I after exposure to extracellular OH• generated by 1 mM copper and 1 mM ascorbic acid (Cu-Asc). Holding potential was 0 mV. Right: mean ± se. I-V relationships for control (open circles) and OH• exposure (n = 6). I below the V axis is cation entry into the spheroplast. (B) As in (A) but for ann1 (n = 6). Spheroplasts from the complemented mutant proved too fragile to patch clamp. (C) As in (A) but for wild-type root epidermal protoplasts (n = 6). (D) ann1 root epidermal protoplasts (n = 6). (E) Complemented mutant ann1/ANN1 (n = 6). Bathing medium (mM): 20 CaCl2, 0.1 KCl, 0.02 NaCl, and 5 MES-Tris, pH 5.6, adjusted to 270 mOsM with d-sorbitol. Pipette solution: 40 K-gluconate, 10 KCl, 0.4 CaCl2, 1 mM BAPTA, and 2 MES-Tris, pH 7.2, adjusted to 270 mOsM with d-sorbitol.
Figure 2.
Figure 2.
ann1 Root Hair Apical PM Retains the Constitutive HACC Conductance. Representative currents recorded in the whole-cell patch clamp configuration from an ann1 root hair apical spheroplast PM showing the HACC conductance that is a characteristic of the root hair apex. No OH• were used in this experiment, and Ba2+ replaced Ca2+ in the bathing medium. Current flowing below the time axis is cation entry into the protoplast. Bathing solution comprised 10 mM BaCl2 and 2 mM MES-Tris, pH 6, adjusted to 275 mOsM with d-sorbitol. Pipette solution comprised 0.5 mM CaCl2, 8.5 mM Ca(OH)2, 2 mM MgATP, 0.5 mM Tris-ATP, 10 mM BAPTA (final free Ca2+, 1 μM), and 15 mM HEPES-Tris, pH 7.3, adjusted to 275 mOsM with d-sorbitol.
Figure 3.
Figure 3.
OH•-Activated [Ca2+]cyt Increase in Root Epidermal Protoplasts Is Impaired in ann1. (A) Root epidermal protoplast [Ca2+]cyt was measured with cytosolic aequorin. Control touch response (buffer only at 35 s) was similar in the wild type (WT) and ann1. OH• was generated by 0.5 mM Cu-Asc. Data are mean ± se (n = 4). Buffer comprised 10 mM CaCl2, 0.1 mM KCl, and 2 mM Tris/MES, pH 5.8, adjusted to 270 mOsM with d-sorbitol. (B) As in (A) but protoplasts were incubated with 300 μM GdCl3 for 30 min prior to [Ca2+]cyt measurement (n = 3). (C) Summary plot of experiments from (A) and (B) showing the effect of Gd3+ on OH•-induced [Ca2+]cyt responses of wild-type and ann1 protoplasts.
Figure 4.
Figure 4.
Atgork Root Epidermal PM Retains the OH•-Activated Conductance. (A) Whole-cell recordings from wild-type (WT; Wassilewskija) root epidermal protoplasts. Left: Current traces under control conditions, elicited by step changes in voltage from a representative protoplast. Center: the same protoplast after exposure to extracellular OH• generated by 1 mM Cu/Asc. Right: Mean ± se current-voltage (I-V) relationships for control (open circles) conditions and OH• exposure (n = 6). Recording conditions are as in Figure 1. (B) As in (A) but with data gathered from gork (n = 6).
Figure 5.
Figure 5.
ann1 Perturbs Diffusion Potentials and OH•-Activated Net K+ Efflux of the Root Epidermis. (A) Representative recordings of PM potential from wild-type (WT; Col-0) and ann1 root elongation zone epidermis. Respiratory blockade was imposed by 0.1 mM NaCN-salicyl hydroxamic acid to reveal the diffusion potential (ED). Extracellular OH• were generated by 0.5 mM Cu-Asc addition, as indicated by the arrow, resulting in a hyperpolarization in the wild type (left) but not ann1 (right) followed by a similar depolarization in both lines (n = 3). Lack of arrest and reversal of the hyperpolarization in ann1 is consistent with ANN1-mediated K+ efflux (EK was estimated to be approximately −156 mV) in response to extracellular OH•. (B) Net K+ efflux recorded from the wild-type (closed circles) and ann1 (crosses) elongation zone epidermis using an extracellular vibrating K+ electrode. Extracellular OH• were generated by 1 mM Cu-Asc addition at time = 0 min. TEA+ was 20 mM (wild type, open circles; Atann1, open triangles). Negative values signify net efflux. Data are mean ± se of five trials. (C) ann1 elongation zone response from (B) at greater resolution. (D) As in (B) but values were recorded at the mature epidermis (n = 5). Recordings in the first 60 s after this addition were discarded to allow for establishment of diffusion gradients.
Figure 6.
Figure 6.
Purification of Recombinant ANN1. ANN1 was expressed in S. cerevisiae, and the protein was purified by three cycles of Ca2+-dependent binding to asolectin liposomes (Before) followed by size exclusion chromatography (After). The gel was either colloidal Coomassie blue stained (C), silver stained (S), or used to perform immunoblot analysis (W) to probe for ANN1 using anti-ANN1 peptide antibody. ANN1 is 37 kD. Overdeveloping the silver-stained gel revealed low-level contaminants that were also present in the buffer, including a predominant band at ∼60 kD, which was also found when using S. cerevisiae to produce recombinant mammalian annexins. ANN1 was not detected in preparations from an empty vector control.
Figure 7.
Figure 7.
OH•-Activated Conductance Is Reconstituted by ANN1 in PLB. (A) OH•-activated conductance mean ± se. I-V with Ca2+ as the major cation: cis-chamber (mM) 1 Ca2+, pH 6, ANN1 (3 µg); trans 200 Ca2+, 1 Cu-Asc, pH 6. Positive I is positive charge movement from cis to trans. Current was blocked by 50 μM Gd3+ trans (open symbols; n = 3). (B) OH•-activated conductance I-V with K+ as the charge carrier: cis-chamber (mM) 200 KCl, pH 6, ANN1 (3 µg); trans 50 KCl, 1 Cu-Asc, pH 6.0 (n = 4). Current was blocked by trans 50 mM TEA+ (open symbols; n = 3). (C) Representative traces from one of six experiments with cis (mM) 200 K+, 1 CaCl2, and trans 1 K+, 200 CaCl2 both pH 6. Left: ANN1 (3 µg) in the cis-chamber; V was changed in 20-mV steps. Middle: OH• (1 Cu-Asc) in trans evoked inward and outward currents. Right: Block by 50 μM Gd3+ trans. (D) I-V relationships for the OH•-activated conductance of (C), n = 5 and Gd3+ block (open symbols; n = 6). Equilibria are indicated. Expanded current traces are shown in Supplemental Figure 2 online. (E) OH•-activated conductance I-V with cis, pH 6, trans, pH 7.0, to change EH. All other conditions are as in (C); n = 4 without trans Gd3+; n = 6 with 50 μM Gd3+.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexandersson E., Saalbach G., Larsson C., Kjellbom P. (2004). Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol. 45: 1543–1556 - PubMed
    1. Balasubramanian K., Bevers E.M., Willems G.M., Schroit A.J. (2001). Binding of annexin V to membrane products of lipid peroxidation. Biochemistry 40: 8672–8676 - PubMed
    1. Benschop J.J., Mohammed S., O’Flaherty M., Heck A.J.R., Slijper M., Menke F.L.H. (2007). Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis. Mol. Cell. Proteomics 6: 1198–1214 - PubMed
    1. Burger A., Voges D., Demange P., Perez C.R., Huber R., Berendes R. (1994). Structural and electrophysiological analysis of annexin V mutants. Mutagenesis of human annexin V, an in vitro voltage-gated calcium channel, provides information about the structural features of the ion pathway, the voltage sensor and the ion selectivity filter. J. Mol. Biol. 237: 479–499 - PubMed
    1. Cano-Domínguez N., Alvarez-Delfin K., Hansberg W., Aguirre J. (2008). NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa. Eukaryot. Cell 7: 1352–1361 - PMC - PubMed

Publication types

MeSH terms

  NODES
twitter 2