Skip to main content
SpringerLink
Log in

Characterisation of Arabidopsis calnexin 1 and calnexin 2 in the endoplasmic reticulum and at plasmodesmata

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Calnexin (CNX) is a highly conserved endoplasmic reticulum (ER) chaperone protein. Both calnexin and the homologous ER-lumenal protein, calreticulin, bind calcium ions and participate in protein folding. There are two calnexins in Arabidopsis thaliana, CNX1 and CNX2. GUS expression demonstrated that these are expressed in most Arabidopsis tissues throughout development. Calnexin transfer DNA (T-DNA) mutant lines exhibited increased transcript abundances of a number of other ER chaperones, including calreticulins, suggesting a degree of redundancy. CNX1 and CNX2 localised to the ER membrane including that within plasmodesmata, the intercellular channels connecting plant cells. This is comparable with the previous localisations of calreticulin in the ER lumen and at plasmodesmata. However, from green fluorescent protein (GFP) diffusion studies in single and double T-DNA insertion mutant lines, as well as overexpression lines, we found no evidence that CNX1 or CNX2 play a role in intercellular transport through plasmodesmata. In addition, calnexin T-DNA mutant lines showed no change in transcript abundance of a number of plasmodesmata-related proteins. CNX1 and CNX2 do not appear to have a specific localisation or function at plasmodesmata—rather the association of calnexin with the ER is simply maintained as the ER passes through plasmodesmata.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Baluska F, Samaj J, Napier R, Volkmann D (1999) Maize calreticulin localizes preferentially to plasmodesmata in root apex. Plant J 19:481–488

    Article  CAS  PubMed  Google Scholar 

  • Bendahmane A, Querci M, Kanyuka K, Baulcombe DC (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J 21:73–81

    Article  CAS  PubMed  Google Scholar 

  • Benitez-Alfonso Y, Faulkner C, Pendle A, Miyashima S, Helariutta Y, Maule A (2013) Symplastic intercellular connectivity regulates lateral root patterning. Dev Cell 26:136–147

    Article  CAS  PubMed  Google Scholar 

  • Bergeron JJM, Brenner MB, Thomas DY, Williams DB (1994) Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci 19:124–128

    Article  CAS  PubMed  Google Scholar 

  • Bhuja P, McLachlan K, Stephens J, Taylor G (2004) Accumulation of 1,3-β-d-glucans, in response to aluminum and cytosolic calcium in Triticum aestivum. Plant Cell Physiol 45:543–549

    Article  CAS  PubMed  Google Scholar 

  • Borgese N, Francolini M, Snapp E (2006) Endoplasmic reticulum architecture: structures in flux. Curr Opin Cell Biol 18:358–364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boulaflous A, Saint-Jores-Dupas C, Herranz-Gordo M-C, Pagny-Salehabadi S, Plasson C, Garidou F, Kiefer-Meyer M-C, Ritzenthaler C, Faye L, Gomord V (2009) Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER. BMC Plant Biol 9:144–165

    Article  PubMed  PubMed Central  Google Scholar 

  • Christensen A, Svensson K, Thelin L, Zhang W, Tintor N, Prins D, Funke N, Michalak M, Schulze-Lefert P, Saijo Y, Sommarin M, Widell S, Persson S (2010) Higher plant calreticulins have acquired specialized functions in Arabidopsis. PLoS ONE 5, e11342

    Article  PubMed  PubMed Central  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Coughlan SJ, Hastings C, Winfrey R (1997) Cloning and characterization of the calreticulin gene from Ricinus communis L. Plant Mol Biol 34:897–911

    Article  CAS  PubMed  Google Scholar 

  • Crawford KM, Zambryski PC (2000) Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport. Curr Biol 10:1032–1040

    Article  CAS  PubMed  Google Scholar 

  • Crofts AJ, Denecke J (1998) Calreticulin and Calnexin in plants. Trends Plant Sci 3:396–399

    Article  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Del Bem L (2011) The evolutionary history of calreticulin and Calnexin genes in green plants. Genetica 139:255–259

    Article  PubMed  Google Scholar 

  • Delmer DP, Amor Y (1995) Cellulose biosynthesis. Plant Cell 7:987–1000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Delmer DP, Volokita M, Solomon M, Fritz U, Delphendahl W, Herth W (1993) A monoclonal antibody recognizes a 65 kDa higher plant membrane polypeptide which undergoes cation-dependent association with callose synthase in vitro and co-localizes with sites of high callose deposition in vivo. Protoplasma 176:33–42

    Article  CAS  Google Scholar 

  • Dunkley TPJ, Watson R, Griffin JL, Dupree P, Lilley KS (2004) Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics 3:1128–1134

    Article  CAS  PubMed  Google Scholar 

  • Dyachok YV, Dmitriev AP, Grodzinskii DM (1997) Ca2+ as a second messenger in the elicitation of phytoalexin and callose synthesis in Allium cepa cell culture. Russ J Plant Physiol 44:333–338

    CAS  Google Scholar 

  • Ehtesham NZ, Phan T-N, Gaikwad A, Sopory SK, Tuteja N (1999) Calnexin from Pisum sativum: cloning of the cDNA and characterization of the encoded protein. DNA Cell Biol 18:853–862

    Article  CAS  PubMed  Google Scholar 

  • Flynn CR, Heinze M, Schumann U, Gietl C, Trelease RN (2005) Compartmentalization of the plant peroxin, AtPex10p, within subdomain(s) of ER. Plant Sci 168:635–652

    Article  CAS  Google Scholar 

  • Gao C, Cai Y, Wang Y, Kang B-H, Aniento F, Robinson DG, Jiang L (2014) Retention mechanisms for ER and Golgi membrane proteins. Trends Plant Sci 19:508–515

    Article  CAS  PubMed  Google Scholar 

  • Gething M-J (1999) Role and regulation of the ER chaperone BiP. Semin Cell Dev Biol 10:465–472

    Article  CAS  PubMed  Google Scholar 

  • Greenfield J, High S (1999) The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment. J Cell Sci 112:1477–1486

    CAS  PubMed  Google Scholar 

  • Guenoune-Gelbart D, Elbaum M, Sagi G, Levy A, Epel BL (2008) Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana. MPMI 21:335–345

    Article  CAS  PubMed  Google Scholar 

  • Hasenfratz MP, Jeltsch JM, Michalak M, Durst F (1997) Cloning and characterization of a wounding-induced analog of the chaperone Calnexin from Helianthus tuberosus. Plant Physiol Biochem 35:553–564

    CAS  Google Scholar 

  • Holmes-Davis R, Tanaka CK, Vensel WH, Hurkman WJ, McCormick S (2005) Proteome mapping of mature pollen of Arabidopsis thaliana. Proteomics 5:4864–4884

    Article  CAS  PubMed  Google Scholar 

  • Hong SM, Bahn SC, Lyu A, Jung HS, Ahn JH (2010) Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis. Plant Cell Physiol 51:1694–1706

    Article  CAS  PubMed  Google Scholar 

  • Huang L, Franklin A, Hoffman N (1993) Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein. J Biol Chem 268:6560–6566

    CAS  PubMed  Google Scholar 

  • Irons SL, Evans DE, Brandizzi F (2003) The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelope in plants. J Exp Bot 54:943–950

    Article  CAS  PubMed  Google Scholar 

  • Ishiguro S, Watanabe Y, Ito N, Nonaka H, Takeda N, Sakai T, Kanaya H, Okada K (2002) SHEPHERD is the Arabidopsis GRP94 responsible for the formation of functional CLAVATA proteins. EMBO J 21:898–908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jia X-Y, He L-H, Jing R-L, Li R-Z (2009) Calreticulin: conserved protein and diverse functions in plants. Physiol Plant 136:127–138

    Article  CAS  PubMed  Google Scholar 

  • Jin Y, Zhuang M, Hendershot LM (2008) ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues. Biochemistry 48:41–49

    Article  Google Scholar 

  • Jin H, Hong Z, Su W, Li J (2009) A plant-specific calreticulin is a key retention factor for a defective brassinosteroid receptor in the endoplasmic reticulum. PNAS 106:13612–13617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kwiatkowski BA, Zielinska-Kwiatkowska AG, Migdalski A, Kleczkowski LA Wasilewska LD (1995) Cloning of two cDNAs encoding Calnexin-like and calreticulin-like proteins from maize (Zea mays) leaves: identification of potential calcium-binding domains. Gene 165:219–222

    Article  CAS  PubMed  Google Scholar 

  • Lenartowska M, Lenartowski R, Smoliński D, Wróbel B, Niedojadło J, Jaworski K, Bednarska E (2009) Calreticulin expression and localization in plant cells during pollen–pistil interactions. Planta 231:67–77

    Article  CAS  PubMed  Google Scholar 

  • Levy A, Erlanger M, Rosenthal M, Epel BL (2007) A plasmodesmata-associated β-1,3-glucanase in Arabidopsis. Plant J 49:669–682

    Article  CAS  PubMed  Google Scholar 

  • Liu J-X, Howell SH (2010) Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants. Plant Cell 22:2930–2942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Malhotra JD, Kaufman RJ (2007) The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 18:716–731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matheson LA, Hanton SL, Brandizzi F (2006) Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond. Curr Opin Plant Biol 9:601–609

    Article  CAS  PubMed  Google Scholar 

  • Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M (2009) Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 417:651–666

    Article  CAS  PubMed  Google Scholar 

  • Nardi M, Feron R, Navazio L, Mariani P, Pierson E, Wolters-Arts M, Knuiman B, Mariani C, Derksen J (2006) Expression and localization of calreticulin in tobacco anthers and pollen tubes. Planta 223:1263–1271

    Article  CAS  PubMed  Google Scholar 

  • Navazio L, Miuzzo M, Royle L, Baldan B, Varotto S, Merry AH, Harvey DJ, Dwek RA, Rudd PM, Mariani P (2002) Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis. Biochemistry 41:14141–14149

    Article  CAS  PubMed  Google Scholar 

  • Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136

    Article  CAS  PubMed  Google Scholar 

  • Niehl A, Amari K, Gereige D, Brandner K, Mély Y, Heinlein M (2012) Control of Tobacco mosaic virus movement protein fate by CELL-DIVISION-CYCLE protein48. Plant Physiol 160:2093–2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat Protoc 2:2910–2917

    Article  PubMed  Google Scholar 

  • Overall RL, Wolf J, Gunning BES (1982) Intercellular communication in Azolla roots I. Ulstrastructure of plasmodesmata. Protoplasma 111:134–150

    Article  Google Scholar 

  • Pagny S, Lerouge P, Faye L, Gomord V (1999) Signals and mechanisms for protein retention in the endoplasmic reticulum. J Exp Bot 50:157–164

    Article  CAS  Google Scholar 

  • Persson S, Rosenquist M, Svensson K, Galvao R, Boss WF, Sommarin M (2003) Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants. Plant Physiol 133:1385–1396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pittman JK, Hirschi KD (2003) Don’t shoot the (second) messenger: endomembrane transporters and binding proteins modulate cytosolic Ca2+ levels. Curr Opin Plant Biol 6:257–262

    Article  CAS  PubMed  Google Scholar 

  • Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66

    Article  CAS  PubMed  Google Scholar 

  • Robards AW, Lucas WJ (1990) Plasmodesmata. Annu Rev Plant Physiol Plant Mol Biol 41:369–419

    Article  Google Scholar 

  • Runions J, Brach T, Kuhner S, Hawes C (2006) Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane. J Exp Bot 57:43–50

    Article  CAS  PubMed  Google Scholar 

  • Sanders D, Pelloux J, Brownlee C, Harper JF (2002) Calcium at the crossroads of signaling. Plant Cell 14:S401–S417

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schott A, Ravaud S, Keller S, Radzimanowski J, Viotti C, Hillmer S, Sinning I, Strahl S (2010) Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem 285:18113–18121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schrag JD, Bergeron JJM, Li Y, Borisova S, Hahn M, Thomas DY, Cygler M (2001) The structure of Calnexin, an ER chaperone involved in quality control of protein folding. Mol Cell 8:633–644

    Article  CAS  PubMed  Google Scholar 

  • Simpson C, Thomas C, Findlay K, Bayer E, Maule AJ (2009) An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell 21:581–594

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sparkes I, Runions J, Hawes C, Griffing L (2009) Movement and remodeling of the endoplasmic reticulum in nondividing cells of tobacco leaves. Plant Cell 21:3937–3949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sparkes I, Tolley N, Aller I, Svozil J, Osterrieder A, Botchway S, Mueller C, Frigerio L, Hawes C (2010) Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties. Plant Cell 22:1333–1343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large number of discrete functional domains. Plant J 11:1151–1165

    Article  CAS  PubMed  Google Scholar 

  • Tung C-W, Dwyer KG, Nasrallah ME, Nasrallah JB (2005) Genome-wide identification of genes expressed in Arabidopsis pistils specifically along the path of pollen tube growth. Plant Physiol 138:977–989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Urade R (2007) Cellular response to unfolded proteins in the endoplasmic reticulum of plants. FEBS J 274:1152–1171

    Article  CAS  PubMed  Google Scholar 

  • Vitale A, Boston RS (2008) Endoplasmic reticulum quality control and the unfolded protein response: insights from plants. Traffic 9:1581–1588

    Article  CAS  PubMed  Google Scholar 

  • Vitale A, Denecke J (1999) The endoplasmic reticulum—gateway of the secretory pathway. Plant Cell 11:615–628

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJ, Louvard D, Bell AW, Dignard D, Thomas DY, Bergeron JJ (1991) SSR alpha and associated Calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 266:19599–19610

    CAS  PubMed  Google Scholar 

  • Wang D, Weaver ND, Kesarwani M, Dong X (2005) Induction of protein secretory pathway is required for systemic acquired resistance. Science 308:1036–1040

    Article  CAS  PubMed  Google Scholar 

  • Zerangue N, Schwappach B, Jan YN, Jan LY (1999) A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22:537–548

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

DYTL was supported by a Grains Research and Development Corporation scholarship. We thank Will Armour for assistance with statistical analysis and Patrick Loughlin for assistance with real-time PCR analyses. We also thank three anonymous reviewers for their helpful comments and suggestions during the revision process.

Author information

Author notes

  1. Danny Y. T. Liu

    Present address: Learning and Teaching Centre, Macquarie University, Building C3B 417, Sydney, NSW, 2109, Australia

  2. David A. Day

    Present address: School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia

Authors and Affiliations

  1. School of Biological Sciences, University of Sydney, Macleay Building A12, Sydney, NSW, 2006, Australia

    Danny Y. T. Liu, Penelope M. C. Smith, Deborah A. Barton, David A. Day & Robyn L. Overall

Authors
  1. Danny Y. T. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Penelope M. C. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Deborah A. Barton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David A. Day
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robyn L. Overall
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robyn L. Overall.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Handling Editor: Manfred Heinlein

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

(DOCX 70 kb)

Table S2

(DOCX 102 kb)

Fig. S1

Growth of calnexin T-DNA insertion mutant plants. a Wild-type and mutant plants at 25 days after sowing. There was no obvious difference in morphology. b Growth curves of the plants over 18 days of measurable growth. Error bars represent the standard error; n = 28 (wild-type), 20 (cnx1 and cnx2) and 27 (cnx1cnx2). Scale bar represents 20 mm. (GIF 142 kb)

High-resolution image (TIF 1825 kb)

Fig. S2

Calnexin T-DNA insertions and physiology of a tsn1 T-DNA insertion mutant. a, b T-DNA insertions in CNX1 and CNX2. Gene schematics show untranslated regions (grey), exons (deep blue) and introns (light blue). The locations of T-DNA insertion screening primers (Table S1) are shown (blue half-arrows). Locations of T-DNA insertions are shown in red, and the orientation and approximate location of the T-DNA left border primer (LB) binding sites are located by a yellow arrowhead. a There was a double insertion in the third exon of CNX1 and b a single insertion in exons of the CNX2 and TSN1 genes. c Transcript levels of TSN1 and TSN2 in cnx2 mutant and tsn1 mutant lines. Two wild-type background ecotypes, Columbia-0 (Col-0) and Wassilewskija (Ws) were included. The TSN1 transcript was absent in the tsn1 mutant and all mutants containing the cnx2 T-DNA insertion allele. Error bars represent standard error. d, e Diffusion of cytoplasmic GFP from transformed leaf epidermal cells (arrowheads) in d tsn1 and e Ws wild-type plants. f The average number of cell layers into which GFP diffused from transformed cells. Error bars represent standard error; n = 46 (tsn1) or 58 (Ws wt). g, h Callose accumulation at plasmodesmata (arrows) along the anticlinal walls of leaf epidermal cells in g tsn1 or h Ws wild-type plants. i Average fluorescence intensity of aniline blue stained callose deposits at plasmodesmata. Error bars represent the standard error; n = 236 (tsn1) or 203 (Ws wt). Scale bars represent 100 μm d, e or 10 μm g, h (GIF 177 kb)

High-resolution image (TIF 1904 kb)

Movie S1

Confocal z-stack of CNX1-GFP punctae co-localising with callose at plasmodesmata in an Arabidopsis leaf epidermal cell. Both CNX1 (green) and callose (magenta) are at plasmodesmata (white). Images were collected with a pinhole set to 1 Airy unit. Scale bar represents 5 μm. (AVI 575 kb)

Movie S2

A z-stack of confocal images showing CNX2-GFP co-localisation with callose at plasmodesmata in an Arabidopsis leaf epidermal cell. Both CNX2 (green) and callose (magenta) are located at plasmodesmata (white). Images were collected with a pinhole set to 1 Airy unit. Scale bar represents 5 μm. (AVI 684 kb)

Movie S3

A z-stack through an Arabidopsis leaf epidermal cell showing ER-GFP located close to plasmodesmata. There is some co-localisation of ER-GFP (green) and callose (magenta) at plasmodesmata (white). Images were collected with a pinhole set to 1 Airy unit. Scale bar represents 5 μm. (AVI 694 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, D.Y.T., Smith, P.M.C., Barton, D.A. et al. Characterisation of Arabidopsis calnexin 1 and calnexin 2 in the endoplasmic reticulum and at plasmodesmata. Protoplasma 254, 125–136 (2017). https://doi.org/10.1007/s00709-015-0921-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-015-0921-3

Keywords

Search

Navigation