Biologia plantarum 2015, 59:654-660 | DOI: 10.1007/s10535-015-0543-9

Characterization of transgenic Poncirus trifoliata overexpressing the ferric chelate reductase gene CjFRO2 from Citrus junos

A. H. Peng1, X. F. Liu1, Y. R. He1, L. Z. Xu1, T. G. Lei1, L. X. Yao1, L. Cao1, S. C. Chen1,*
1 Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing, P.R. China

Iron deficiency chlorosis occurs frequently in calcareous soils. The transformation of plants with ferric chelate reductase genes (FROs) provides a potential strategy to alleviate plant chlorosis under iron deficiency. A CjFRO2 gene isolated from Citrus junos Sieb. ex Tanaka was introduced into Poncirus trifoliata (L.) Raf via Agrobacterium-mediated transformation. The transgene integration and expression were confirmed by PCR, Southern blot, and real-time PCR analyses. Hydroponic- and soil-grown transgenic plants were tested for their tolerance to iron deficiency. Compared with nontransgenic (NT) P. trifoliata plants, a rhizosphere acidification capacity in the transgenic lines increased, and a ferric chelate reductase activity in roots was up to 3.39- and 2.93-fold higher in a hydroponic solution and soil, respectively. A transgenic line TO-8, which reacted similarly in hydroponics and soil, appeared tolerant to the iron deficiency. Its leaf chlorophyll and ferrous ion content was significantly higher than in NT. These results indicate that tolerance to the iron deficiency in P. trifoliata could be improved through the genetic engineering.

Keywords: chlorosis; iron deficiency; real-time PCR; rhizosphere acidification; Southern blot
Subjects: transgenic plants; chlorosis; iron deficiency; rhizosphere acidification; Southern blot; ferric chelate reductase; trifoliate orange
Species: Poncirus trifoliata; Citrus junos

Received: September 5, 2014; Revised: March 6, 2015; Accepted: March 16, 2015; Published: December 1, 2015Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Peng, A.H., Liu, X.F., He, Y.R., Xu, L.Z., Lei, T.G., Yao, L.X., Cao, L., & Chen, S.C. (2015). Characterization of transgenic Poncirus trifoliata overexpressing the ferric chelate reductase gene CjFRO2 from Citrus junos. Biologia plantarum, 59(4), 654-660. doi: 10.1007/s10535-015-0543-9.
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)

    • Open full article

    References

    1. Brand, J.D., Tang, C., Rathjen, A.J.: Screening rough-seeded lupins (Lupinus pilosus Murr. and Lupinus atlanticus Glads.) for tolerance to calcareous soils. - Plant Soil. 245: 261-275, 2002. Go to original source...
    2. Cao, L., Peng, L.Z., Peng, A.H., Ling, L.L., Jiang, D., Wang, F.S., Fan, D., Wang, J.Z., Ma, X.H., Zhang, W.W., Chang, Q.H., Wang, X.X.: [Preliminary identification of alkaliresistance hybrid seedlings of Junos and Trifoliate orange.] - J. Fruit Sci. 28: 20-25, 2011. [In Chin.]
    3. Chaney, R.L., Brown, J.C., Tiffin, L.O.: Obligatory reduction of ferric chelates in iron uptake by soybean. - Plant Physiol. 50: 208-213, 1972. Go to original source...
    4. Chaney, R.L., Coulombe, B.A., Bell, P.F., Angle, J.S.: Detailed method to screen dicot cultivars for resistance to Fechlorosis using FeDTPA and bicarbonate in nutrient solutions. - J. Plant Nutr. 15: 2063-2083, 1992. Go to original source...
    5. Connolly, E.L., Campbell, N.H., Grotz, N., Prichard, C.L., Guerinot, M.L.: Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers post-transcriptional control. - Plant Physiol. 133: 1102-1110, 2003. Go to original source...
    6. Dancis, A., Klausner, R.D., Hinnebusch, A.G., Barriocanal, J.G.: Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae. - Mol. cell. Biol. 10: 2294-2301, 1990. Go to original source...
    7. Dancis, A., Roman, D.G., Anderson, G.J., Hinnebusch, A.G., Klausner, R.D.: Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron. - Proc. nat. Acad. Sci. USA. 89: 3869-3876, 1992. Go to original source...
    8. Dutt, M., Grosser, J.W.: Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. - Plant Cell Tissue Organ Cult. 98: 331-340, 2009. Go to original source...
    9. Egilla, J.N., Byrne, D.H., Reed, D.W.: Iron stress response of three peach rootstock cultivars: ferric-iron reduction capacity. - J. Plant Nutr. 17: 2079-2103, 1994. Go to original source...
    10. Hiscox, J.C., Israelstam, G.F.: A method for the extraction of chlorophyll from tissue without maceration. - Can. J. Bot. 57: 1332-1334, 1979. Go to original source...
    11. Li, L.: [Physiological and molecular basis for resistance to iron chlorosis in Malus xiaojinensis and Citrus junos.] - PhD Thesis. Southwest Agriculture University, Chongqing 2002. [In Chin.]
    12. Li, L., Fan, Y.H., Luo, X.Y., Pei, Y., Zhou, Z.Y.: [Expression of ferric chelate reductase gene in Citrus junos and Poncirus trifoliata tissues.] - Acta bot. sin. 44: 771-774, 2002. [In Chin.]
    13. Li, L., Fan, Y.H., Luo, X.Y., Zhou, Z.Y., Pei, Y.: [Study on ferric chelate reductase of Citrus junos and Poncirus trifoliata.] - J. southwest agr. Univ. 23: 435-437, 2001. [In Chin.]
    14. Li, L., Zhou, Z.Y., Pei, Y.: [Physiological reaction on resistance to iron chlorosis in Malus xiaojinensis and M. Rockii.] - Acta hort. sin. 30: 639-642, 2003. [In Chin.]
    15. Li, L.H., Cheng, X.D., Ling, H.Q.: Isolation and characterization of Fe(III)-chelate reductase gene LeFRO1 in tomato. - Plant mol. Biol. 54: 125-136, 2004.
    16. Li, Y.L., Qiu, Y.C., Dian, S.Y., Guo, C.H.: Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to irondeficiency chlorosis. - Mol. Biol. Rep. 38: 3605-3613, 2011. Go to original source...
    17. Liu, T., Li, Y., Ren, J., Zhang, C., Kong, M., Song, X., Zhou, J., Hou, X.: Over-expression of BcFLC1 from non-heading Chinese cabbage enhances cold tolerance in Arabidopsis. - Biol. Plant. 57: 262-266, 2013. Go to original source...
    18. Meng, X., Yin, B., Feng, H.L., Zhang, S., Liang, X.Q., Meng Q.W.: Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress. - Biol. Plant. 58: 121-130, 2014. Go to original source...
    19. M'sehli, W., Youssfi, S., Donnini, S., Dell'Orto, M., Nisi, P.D., Zocchi, G., Abdelly, C., Gharsalli, M.: Root exudation and rhizosphere acidification by two lines of Medicago ciliaris in response to lime-induced iron deficiency. - Plant Soil. 312: 151-162, 2008.
    20. Murashige, T., Skoog, F.: A revised medium for rapid growth and bio assays with tobacco tissue cultures. - Physiol. Plant. 15: 473-497, 1962. Go to original source...
    21. Nikolic, M., Römheld, V.: Mechanism of Fe uptake by the leaf symplast: is Fe inactivation in leaf a cause of Fe deficiency chlorosis? - Plant Soil 215: 229-237, 1999. Go to original source...
    22. Nikolic, M., Römheld, V.: Does high bicarbonate supply to roots change availability of iron in the leaf apoplast? - Plant Soil 241: 67-74, 2002. Go to original source...
    23. Pal, A.K., Acharya, K., Vats, S.K., Kumar, S., Ahuja, P.S.: Over-expression of PaSOD in transgenic potato enhances photosynthetic performance under drought. - Biol. Plant. 57: 359-364, 2013. Go to original source...
    24. Ren, L.X., Zuo, Y.M., Jiang, R.F., Zhang, F.S.: [Mechanisms of bicarbonate induced iron-deficiency chlorosis of peanut on calcareous soils.] - Acta ecol. sin. 25: 795-801, 2005. [In Chin.]
    25. Robinson, N.J., Procter, C.M., Connolly, E.L., Guerinot, M.L.: A ferric-chelate reductase for iron uptake from soils. - Nature 397: 694-697, 1999. Go to original source...
    26. Samuelsen, A.I., Martin, R.C., Mok, D.W.S., Mok, M.C.: Expression of the yeast FRE genes in transgenic tobacco. - Plant Physiol. 118: 51-58, 1998. Go to original source...
    27. Susín, S., Abadía, A., González-Reyes, J.A., Lucena, J.J., Abadía, J.: The pH requirement for in vivo activity of the iron-deficiency-induced "Turbo" ferric chelate reductase: a comparison of the iron-deficiency-induced iron reductase activities of intact plants and isolated plasma membrane fractions in sugar beet. - Plant Physiol. 110: 111-123, 1996. Go to original source...
    28. Vasconcelos, M., Eckert, H., Arahana, V., Graef, G., Grusak, M.A., Clemente, T.: Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2. - Planta 224: 1116-1128, 2006. Go to original source...
    29. Waters, B.M., Blevins, D.G., Eide, D.J.: Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition. - Plant Physiol. 129: 85-94, 2002. Go to original source...
    30. Yang, J., Liu, Y., Liu, Y., Zhang, W., Huang, J., Li, J., Du, C.C., Sui, Z., Wang-Pruski, G.: Characterization of chlorosis-resistant apple mutants of transgenic FRO2. - Acta Hort. 829: 251-257, 2009. Go to original source...

    Return to the content