Skip to main content
SpringerLink
Log in

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2

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

Abstract

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe+3 reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 μM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 μM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

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
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Abadía J (1992) Leaf responses to Fe deficiency: a review. J Plant Nutr 15:1655–1713

    Google Scholar 

  • Abadía J, Monge E, Montañes L, Heras L (1984) Extraction of iron from plant leaves by Fe(II) chelators. J Plant Nutr 7:777–784

    Google Scholar 

  • Álvarez-Fernández A, García-Laviña P, Fidalgo C, Abadía J, Abadía A (2004) Foliar fertilization to control iron chlorosis in pear (Pyrus communis L.) trees. Plant and Soil 263:5–15

    Article  Google Scholar 

  • Bollivar DW, Beale SI (1996) The chlorophyll biosynthetic enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. Plant Physiol 112:105–114

    PubMed  CAS  Google Scholar 

  • Bruggemann W, Maas-Kantel K, Moog PR (1993) Iron uptake by leaf mesophyll cells: the role of the plasma membrane-bound ferric-chelate reductase. Planta 190:151–155

    Article  Google Scholar 

  • Callis J, Fromm ME, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Develop 1:1183–1200

    Article  PubMed  CAS  Google Scholar 

  • Cantera RG, Zamarreño AM, García-Mina JM (2002) Characterization of commercial iron chelates and their behavior in an alkaline and calcareous soil. J Agric Food Chem 50:7609–7615

    Article  PubMed  CAS  Google Scholar 

  • Carrington JC, Freed DD (1990) Cap-independent enhancement of translation by a plant potyvirus 5’ nontranslated region. J Virol 64:1590–1597

    PubMed  CAS  Google Scholar 

  • Cataldo DA, McFadden KM, Garland TR, Wildung RE (1988) Organic constituents and complexation of nickel (II), iron (III), cadmium (II), and plutonium (IV) in soybean xylem exudates. Plant Physiol 86:734–739

    PubMed  CAS  Google Scholar 

  • Chaney RL, Coulombe BA, Bell PF, Angle JS (1992) Detailed method to screen dicot cultivars for resistance to Fe-chlorosis using FeDTPA and bicarbonate in nutrient solutions. J Plant Nutr 15:2063–2083

    CAS  Google Scholar 

  • Charlson DV, Cianzio SR, Shoemaker RC (2003) Associating SSR markers with soybean resistance to iron deficiency chlorosis. J Plant Nutrition 26:2267–2276

    Article  CAS  Google Scholar 

  • Connolly EL, Campbell NH, Grotz N, Prichard CL, Guerinot ML (2003) Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers posttranscriptional control. Plant Physiol 133:1102–1110

    Article  PubMed  CAS  Google Scholar 

  • Connolly EL, Fett JP, Guerinot ML (2002) Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell 14:1347–1357

    Article  PubMed  CAS  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Reptr 1:19–21

    Article  CAS  Google Scholar 

  • Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351

    Article  PubMed  CAS  Google Scholar 

  • Fehr WR, Caviness CE (1979) Stages of development. Cooperative Extension Service: Iowa State University 80:1–12

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:152–158

    Article  Google Scholar 

  • Goos RJ, Germain S (2001) Solubility of twelve iron fertilizer products in alkaline soils. Commun Soil Sci Plant Anal 32:2317–2323

    Article  CAS  Google Scholar 

  • Goos RJ, Johnson BE (2000) A comparison of three methods for reducing iron-deficiency chlorosis in soybean. Agron J 92:1135–1139

    Article  CAS  Google Scholar 

  • Green LS, Rogers EE (2004) FDR3 controls iron localization in Arabidopsis. Plant Physiol 136:2523–2531

    Article  PubMed  CAS  Google Scholar 

  • Grusak MA, Welch RM, Kochian LV (1990) Does iron deficiency in Pisum sativum enhance the activity of the root plasmalemma iron transport protein? Plant Physiol 94:1353–1357

    PubMed  CAS  Google Scholar 

  • Guerinot ML (2001) Improving rice yields–ironing out the details. Nat Biotechnol 19:417–418

    Article  PubMed  CAS  Google Scholar 

  • Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  PubMed  CAS  Google Scholar 

  • Halliwell B, Gutteridge JMC (1992) Biologically relevant metal ion dependent hydroxyl radical generation. FEBS Lett 307:108–112

    Article  PubMed  CAS  Google Scholar 

  • Hansen NC, Schmitt MA, Anderson JE, Strock JS (2003) Iron deficiency in soybean in the upper midwest and associated soil properties. Agron J 95:1595–1601

    Article  CAS  Google Scholar 

  • Hell R, Stephan UW (2003) Iron uptake, trafficking and homeostasis in plants. Planta 216:541–551

    PubMed  CAS  Google Scholar 

  • Hood EE, Helmer GL, Fraley RT, Chilton M-D (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

    PubMed  CAS  Google Scholar 

  • Kobayashi T, Nakayama Y, Itai R-N, Nakanishi H, Yoshihara T, Mori S, Nishizawa NK (2003) Identification of novel cis-acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter conferring iron-deficiency-inducible, root-specific expression in heterogeneous tobacco plants. Plant J 36:780–793

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi T, Suzuki M, Inoue H, Itai RN, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2005) Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements. J Exp Bot 56:1305–1316

    Article  PubMed  CAS  Google Scholar 

  • Libiakova G, Jorgensen B, Palmgren G, Ulvskov P, Johansen E (2001) Efficacy of an intron-containing kanamycin resistance gene as a selectable marker in plant transformation. Plant Cell Rep 20:610–615

    Article  CAS  Google Scholar 

  • Lin S-F, Baumer JS, Ivers D, de Cianzio SR, Shoemaker RC (1998) Field and nutrient solution tests measure similar mechanisms controlling iron deficiency chlorosis in soybean. Crop Sci 38:254–259

    Article  CAS  Google Scholar 

  • Lin SF, Grant D, Cianzio S, Shoemaker R (2000) Molecular characterization of iron deficiency chlorosis in soybean. J Plant Nutr 23:1591–1606

    Google Scholar 

  • Lucena JJ (2000) Effects of bicarbonate, nitrate and other environmental factors on iron deficiency chlorosis. A review. J Plant Nutr 23:1591–1606

    Article  CAS  Google Scholar 

  • Maas C, Laufs J, Grant S, Korfhage C, Werr W (1991) The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold. Plant Mol Biol 16:199–207

    Article  PubMed  CAS  Google Scholar 

  • Miller GW, Huang IJ, Welkie GW, Pushnik JC (1995) Function of iron in plants with special emphasis on chloroplasts and photosynthetic activity. In: Abadia J (ed) Iron nutrition in soils and plants. Kluwer, Netherlands, pp 19–28

    Google Scholar 

  • Mok MC, Samuelsen AI, Martin RC, Mok DWS (2000) Fe(III) reductase, the FRE genes, and FRE-transformed tobacco. J Plant Nutr 23:1941–1951

    CAS  Google Scholar 

  • Moseley J, Quinn J, Eriksson M, Merchant S (2000) The Crd1gene encodes a putative di-iron enzyme required for photosystem 1 accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii. EMBO 19:2139–2151

    Article  CAS  Google Scholar 

  • Nasrulhaq-Boyce A, Griffiths WT, Jones OTG (1987) The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring. Biochem J 243:23–30

    PubMed  CAS  Google Scholar 

  • Robinson NJ, Procter CM, Connolly EL, Guerinot ML (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397:694–697

    Article  PubMed  CAS  Google Scholar 

  • Samuelsen AI, Martin RC, Mok DWS, Mok MC (1998) Expression of the yeast FRE genes in transgenic tobacco. Plant Physiol 118:51–58

    Article  PubMed  CAS  Google Scholar 

  • Schmidt W (1999) Review: mechanisms and regulation of reduction-based iron uptake in plants. New Phytol 141:1–26

    Article  CAS  Google Scholar 

  • Spiller S, Castlefranco A, Castlefranco P (1982) Effects on iron and oxygen on chlorophyll biosynthesis 1: In vivo observations on iron and oxygen deficient plants. Plant Physiol 69:107–111

    PubMed  CAS  Google Scholar 

  • Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO 6:2519–2523

    CAS  Google Scholar 

  • Tottey S, Block M, Allen M, Westergren T, Albrieux C, Scheller H, Merchant S, Jensen P (2003) Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proc Natl Acad Sci USA 100:16119–16124

    Article  PubMed  CAS  Google Scholar 

  • Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250

    Article  PubMed  CAS  Google Scholar 

  • Varsano T, Wolf SG, Pick U (2006) A chlorophyll a/b binding protein homolog which is induced by iron deficiency is associated with enlarged photosytem I units in the eukaryotic alga Dunaliella salina. J Biol Chem 281(15):10305–10315

    Article  PubMed  CAS  Google Scholar 

  • Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat J-F, Curie C (2002) IRT1, an Arabidopsis transporter essential for iron uptake from soil and plant growth. Plant Cell 14:1223–1233

    Article  PubMed  CAS  Google Scholar 

  • Waters BM, Blevins DG, Eide DJ (2002) Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition. Plant Physiol 129:85–94

    Article  PubMed  CAS  Google Scholar 

  • Xing A, Zhang Z, Sato S, Staswick P, Clemente T (2000) The use of the two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Biol 36:456–463

    Article  CAS  Google Scholar 

  • Zhang Z, Xing A, Staswick PE, Clemente TE (1999) The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tiss Org Cult 56:37–46

    Article  CAS  Google Scholar 

  • Zouari M, Abadía A, Abadía J (2001) Iron is required for the induction of root ferric chelate reductase activity in iron-deficient tomato. J Plant Nutr 24:383–396

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Nebraska Research Initiative, Center for Biotechnology (TC), the Nebraska Soybean Board (TC), the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-6250-1-001 (MAG), and the HarvestPlus Biofortification Project under Agreement Number 58-6250-4-F029 (MAG). The contents of this publication neither necessarily reflect the views or policies of the US Department of Agriculture, nor mention trade names, commercial products, or organizations that imply endorsement by the US Government. Plasmid pELC202 was a gift from Nigel Robinson. This is journal series paper no. 15104 from the Nebraska Agriculture Research Division.

Author information

Authors and Affiliations

  1. USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

    Marta Vasconcelos & Michael A. Grusak

  2. Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, USA

    Helene Eckert, Venancio Arahana, George Graef & Tom Clemente

  3. Center for Biotechnology/Plant Science Initiative, University of Nebraska-Lincoln, Lincoln, NE, USA

    Tom Clemente

Authors
  1. Marta Vasconcelos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helene Eckert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Venancio Arahana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George Graef
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael A. Grusak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tom Clemente
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tom Clemente.

Additional information

Authors Marta Vasconcelos and Helene Eckert contributed equally to this project.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vasconcelos, M., Eckert, H., Arahana, V. et al. Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2 . Planta 224, 1116–1128 (2006). https://doi.org/10.1007/s00425-006-0293-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-006-0293-1

Keywords

Search

Navigation