Skip to main content
SpringerLink
Log in

cDNA-AFLP analysis reveals a set of new genes differentially expressed in cucumber root apexes in response to iron deficiency

  • Published:
Biologia Plantarum

Abstract

Cucumber (Cucumis sativus L.) is considered a model plant for the investigation of Fe deficiency responses, since it strongly exhibits typical strategy I, i.e. increased activities of Fe(III)-chelate reductase, H+-ATPase and iron regulated transporters. In this study, cDNA amplified fragment length polymorphism analysis was employed to identify genes differentially expressed in the root apex following Fe deficiency. The expression patterns of the most interesting transcript derived fragments were validated by semiquantitative reverse trascriptase — polymerase chain reaction. A set of new genes overexpressed under Fe deficiency, such as those coding for calmodulin, SNAP, TIM23 and V-PPase were identified. Furthermore, we also observed that calmodulin protein accumulated in Fe-deficient root apexes.

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

Similar content being viewed by others

Abbreviations

AFLP:

amplified fragment length polymorphism

BPDS:

bathophenantrolinedisulfonate

FC-R:

Fe(III)-chelate reductase

IRT:

iron regulated transporters

PVDF:

polyvinylidene difluoride

RT-PCR:

reverse trascriptase-polymerase chain reaction

TDF:

transcript derived fragments

References

  • Bachem, C.W.B., Van der Hoeven, R.S., De Brujin, S.M., Vreugdenhil, D., Zabeau, M., Visser, R.G.F.: Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. — Plant J. 9: 745–753, 1996.

    Article  PubMed  CAS  Google Scholar 

  • Bauer, M.F., Hofmann, S., Neupert, W., Brunner, M.: Protein translocation into mitochondria: the role of TIM complexes. — Trends Cell. Biol. 10: 25–31, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Beharav, A., Maras, M., Kitner, M., Šuštar-Vozlič, J., Sun, G.L., Doležalová, I., Lebeda, A., Meglič, V.: Comparison of three genetic similarity coefficients based on dominant markers from predominantly self-pollinating species. — Biol. Plant. 54: 54–60, 2010.

    Article  CAS  Google Scholar 

  • Bouche, N., Yellin, A., Snedden, W.A., Fromm, H.: Plantspecific calmodulin-binding proteins. — Annu. Rev. Plant Biol. 56: 435–466, 2005.

    Article  PubMed  CAS  Google Scholar 

  • Bradford, M.M.: A rapid and sensitive method for the quantization of micrograms quantities of protein utilizing the principle of protein-dye binding. — Anal. Biochem. 72: 248–254, 1976.

    Article  PubMed  CAS  Google Scholar 

  • Breyne, P., Dreesen, R., Cannoot, B., Rombaut, D., Vandepoele, K., Rombauts, S., Vanderhaeghen, R., Inzé, D., Zabeau, M.: Quantitative cDNA-AFLP analysis for genome-wide expression studies. — Mol. Genet. Genom. 269: 173–179, 2003.

    CAS  Google Scholar 

  • Breyne, P., Zabeau, M.: Genome-wide expression analysis of plant cell cycle modulated genes. — Curr. Opin. Plant Biol. 4: 136–142, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Buckhout, T.J., Yang, T.J., Schmidt, W.: Early iron-deficiencyinduced transcriptional changes in Arabidopsis roots as revealed by microarray analyses. — BMC Genomics 10: 147, 2009.

    Article  PubMed  Google Scholar 

  • Colangelo, E.P., Guerinot, M.L.: The essential basic helix-loophelix protein FIT1 is required for Fe deficiency response. — Plant Cell 16: 3400–3412, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Cuesta, C., Ordás, R.J., Rodríguez, A. Fernández, B.: PCRbased molecular markers for assessment of somaclonal variation in Pinus pinea clones micropropagated in vitro. — Biol. Plant. 54: 435–442, 2010.

    Article  CAS  Google Scholar 

  • Curie, C., Briat, J.F.: Iron transport and signalling in plants. — Annu. Rev. Plant Biol. 54: 183–206, 2003.

    Article  PubMed  CAS  Google Scholar 

  • De Nisi, P., Zocchi, G.: Phosphoenolpyruvate carboxylase in cucumber (Cucumis sativus L.) roots under iron deficiency: activity and kinetic characterisation. — J. exp. Bot. 51: 1903–1909, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Ditt, R.F., Nester, E.W., Comai, L.: Plant gene expression response to Agrobacterium tumefaciens. — Proc. nat. Acad. Sci. USA 98: 10954–10959, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Donnini, S., Prinsi, B., Negri, A.S., Vigani, G., Espen, L., Zocchi, G.: Proteomic characterization of iron deficiency response in Cucumis sativus L. roots. — BMC Plant Biol. 10: 268, 2010.

    Article  PubMed  CAS  Google Scholar 

  • Endo, T., Yamamoto, H., Esaki, M.: Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles. — J. cell. Sci. 116: 3259–3267, 2003.

    Article  PubMed  CAS  Google Scholar 

  • Espen, L., Dell’Orto, M., De Nisi, P., Zocchi, G.: Metabolic responses in cucumber (Cucumis sativus L.) root under Fedeficiency: a 31NMR in vivo study. — Planta 210: 985–992, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Fukumura, R., Takahashi, H., Saito, T., Tsutsumi, T., Fujimori, A., Sato, S., Tatsumi, K., Araki, R., Abe, M.: A sensitive transcriptome analysis method that can detect unknown transcripts. — Nucl. Acid Res. 31 (Suppl.): e94, 2003.

  • Hartings, H.: High resolution fingerprinting of transcribed genes by means of a modified cDNA-AFLP method. — Maydica. 44: 179–181, 1999.

    Google Scholar 

  • Ikura, M., Ames, J.B.: Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: two ways to promote multifunctionality. — Proc. nat. Acad. Sci. USA 103: 1159–1164, 2006.

    Article  PubMed  CAS  Google Scholar 

  • Jensen, R., Dunn, C.: Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocation. — Biochim. biophys. Acta. 1592: 25–34, 2002.

    Article  PubMed  CAS  Google Scholar 

  • Kang, S.-Y., Lee, K.J., Lee, G.-J., Kim, J.-B., Chung, S.-J., Song, J.Y., Lee, B.-M., Kim, D.S.: Development of AFLP and STS markers linked to a waterlogging tolerance in Korean soybean landraces. — Biol. Plant. 54: 61–68, 2010.

    Article  CAS  Google Scholar 

  • Koehler, C.M.: Protein translocation pathways of the mitochondrion. — FEBS Lett. 476: 27–31, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Landsberg, E.C.: Function of rhizodermal transfer cells in the Fe stress response mechanism of Capsicum annuum L. — Plant Physiol. 82: 511–517, 1986.

    Article  PubMed  CAS  Google Scholar 

  • Landsberg, E.C.: Transfer cell formation in sugar beet roots induced by latent Fe deficiency. — Plant Soil. 165: 197–205, 1994.

    Article  CAS  Google Scholar 

  • Li, J., Wu, X.D., Hao, S.T., Wang, X.J., Ling, H.Q.: Proteomic response to iron deficiency in tomato root. — Proteomics 8: 2299–2311, 2008.

    Article  PubMed  CAS  Google Scholar 

  • Lister, R., Chew, O., Lee, M.N., Heazlewood, J.L., Clifton, R., Parker, K., Millar, A.H., Whelan, J.: A transcriptomic and proteomic characterization of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction. — Plant Physiol. 134: 777–789, 2004.

    Article  PubMed  CAS  Google Scholar 

  • López-Millán, A.F., Morales, F., Andaluz, S., Gogorcena, Y., Abadía, A., De Las Rivas, J., Abadía, J.: Responses of sugar beet roots to iron deficiency. Changes in carbon assimilation and oxygen use. — Plant Physiol. 124: 885–897, 2000.

    Article  PubMed  Google Scholar 

  • Maeshima, M.: Vacuolar H+-pyrophosphatase. — Biochim. biophys. Acta. 1465: 37–51, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Marschner, H., Römheld, V., Ossenberg-Neuhaus, H.: Rapid method for measuring changes in pH and reducing process along roots of intact plants. — Z. Pflanzenphysiol. 105: 407–416, 1982.

    Google Scholar 

  • O’Rourke, J.A., Charlson, D.V., Gonzalez, D.O., Vodkin, L.O., Graham, M.A., Cianzio, S.R., Grusak, M.A., Shoemaker, R.C.: Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines. — BMC Genomics 8: 476, 2007.

    Article  PubMed  Google Scholar 

  • Ratajczak, R., Hinz, G., Robinson, D.G.: Localization of pyrophosphatase in membranes of cauliflower inflorescence cells. — Planta 208: 205–211, 1999.

    Article  PubMed  CAS  Google Scholar 

  • Rehling, P., Pfanner, N., Meisinger, C.: Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane — a guided tour. — J. mol. Biol. 326: 639–657, 2003.

    Article  PubMed  CAS  Google Scholar 

  • Sikdar, B., Bhattacharya, M., Mukherjee, A., Banerjee, A., Ghosh, E., Ghosh, B., Roy, S.C.: Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers. — Biol. Plant. 54: 135–140, 2010.

    Article  CAS  Google Scholar 

  • Sojikula, P., Kongsawadworakula, P., Viboonjuna, U., Thaiprasitc, J., Intawong, B., Narangajavanaa, J., Svastid, M.R.J.: AFLP-based transcript profiling for cassava genome-wide expression analysis in the onset of storage root formation. — Physiol. Plant. 140: 198–198, 2010.

    Google Scholar 

  • Stenbeck, G.: Soluble NSF-attachment proteins. — Int. J. Biochem. cell. Biol. 30: 573–577, 1998.

    Article  PubMed  CAS  Google Scholar 

  • Thimm, O., Essigmann, B., Kloska, S., Altmann, T., Buckhout, T.J.: Response of Arabidopsis to iron deficiency stress as revealed by microarray analysis. — Plant Physiol. 127: 1030–1043, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Uemura, T., Ueda, T., Ohniwa, R.L., Nakano, A., Takeyasu, K., Sato, M.H.: Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells. — Cell Struct. Funct. 29: 49–65, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Vigani, G., Maffi, D., Zocchi, G.: Iron availability affects the function of mitochondria in cucumber roots. — New Phytol. 182: 127–136, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Vigani, G., Zocchi, G.: The fate and the role of mitochondria in Fe deficient roots of Strategy I plants. — Plant Signal. Behaviour 4: 375–379, 2009.

    Article  CAS  Google Scholar 

  • Vigani, G., Zocchi, G.: Effect of Fe deficiency on mitochondrial alternative NAD(P)H dehydrogenases in cucumber roots. — J. Plant Physiol. 167: 666–669, 2010.

    Article  PubMed  CAS  Google Scholar 

  • Vuylsteke, M., Peleman, J.D., Van Eijk, M.J.T.: AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. — Nat. Protoc. 2: 1399–1413, 2007.

    Article  PubMed  CAS  Google Scholar 

  • Waters, B.M., Lucena, C., Romera, F.J., Jester, G.G., Wynn, A.N., Rojas, C.L., Alcantara, E., Perez-Vicente, R.: Ethylene involvement in the regulation of the H+-ATPase CsHA1 gene and of the new isolated ferric reductase CsFRO1 and iron transporter CsIRT1 genes in cucumber plants. — Plant Physiol. Biochem. 45: 293–301, 2007.

    Article  PubMed  CAS  Google Scholar 

  • Zocchi, G.: Metabolic changes in iron-stressed dicotyledonous plants. — In: Barton, L.L., Abadía, J. (ed.): Iron Nutrition in Plants and Rhizospheric Microorganisms. Pp 359–370. Springer, Dordrecht 2006.

    Chapter  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dipartimento di Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, I-20133, Milano, Italy

    G. Vigani, A. Chittó, P. De Nisi & G. Zocchi

Authors
  1. G. Vigani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Chittó
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. De Nisi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Zocchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Vigani.

Additional information

Acknowledgements: The authors wish to thank Dr. H. Hartings for critical reading of the manuscript, Dr. S. Donnini for technical assistance for western blot analysis and the reviewers for their valuable comments useful to improve the manuscript. The research was financially supported by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (MiUR). GV was partially supported by “Dote Ricerca”: FSE, Regione Lombardia. We thank the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF-CAAS) for the release of information about the cucurbit genome. The first two authors equally contributed to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vigani, G., Chittó, A., De Nisi, P. et al. cDNA-AFLP analysis reveals a set of new genes differentially expressed in cucumber root apexes in response to iron deficiency. Biol Plant 56, 502–508 (2012). https://doi.org/10.1007/s10535-012-0050-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10535-012-0050-1

Additional key words

Search

Navigation