Rhizobacterium Arthrobacter agilis UMCV2 increases organ-specific expression of FRO genes in conjunction with genes associated with the systemic resistance pathways of Medicago truncatula

  • Vicente Montejano-Ramírez
  • Ramiro Martínez-Cámara
  • Ernesto García-Pineda
  • Eduardo Valencia-CanteroEmail author
Original Article


Ferric-chelate reductase (FRO) genes are essential for iron uptake in strategy I plants. In this study, Medicago truncatula plants were grown in a greenhouse under iron-sufficient and iron-deficient conditions with inoculation of the beneficial rhizobacterium Arthrobacter agilis UMCV2. The expression of five MtFRO genes and the marker genes MtDef2.1 and MtPR1—involved in systemic resistance pathways—was quantified by RT-qPCR in plant organs. MtFRO1 and MtFRO3 were expressed in roots, and MtFRO2 was expressed in leaves, flowers, and pods. MtFRO4 was expressed in leaves and roots, and MtFRO5 was expressed in roots, stems, and leaves. A. agilis UMCV2 and iron deficiency induced the expression of all MtFRO genes and systemic defense marker genes. The defense genes exhibited expression profiles similar to those of the MtFRO genes, and strong synergistic gene induction was observed in A. agilis UMCV2-inoculated plants grown under iron-deficient conditions. Our data supported the existence of a relationship between systemic defense responses and systemic iron deficiency responses.


Medicago truncatula Arthrobacter agilis UMCV2 MtFRO ISR SAR 



We thank the Valencia-Macias foundation (México, Grant 2.1) and the Coordinación de la Investigación Científica UMSNH (México, Grant 2.22) for providing financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Andaluz S, Rodríguez-Chelma J, Abadía A, Abadía J, López-Milán AF (2009) Time course induction of several key enzymes in Medicago truncatula roots in response to Fe deficiency. Plant Physiol Biochem 47:1082–1088. CrossRefPubMedGoogle Scholar
  2. Aviles-Garcia ME, Flores-Cortez I, Hernández-Soberano C, Santoyo G, Valencia-Cantero E (2016) The plant growth-promoting rhizobacterium Arthrobacter agilis UMCV2 endophytically colonizes Medicago truncatula.. Rev Argent Microbiol 48:342–346. PubMedCrossRefGoogle Scholar
  3. Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plant stress signaling. J Exp Bot 65:1229–1240. CrossRefPubMedGoogle Scholar
  4. Berendsen RL, Pieterse CM, Bakker PA (2012) The rhizosphere microbiome and plant health. Trends Plant Sci 17:478–486. CrossRefPubMedGoogle Scholar
  5. Castulo-Rubio DY, Alejandre-Ramírez N, Orozco-Mosqueda MC, Santoyo G, Macias-Rodríguez LI, Valencia-cantero E (2015) Volatile organic compounds produced by the rhizobacterium Arthrobacter agilis UMCV2 modulate Sorghum bicolor (strategy II plant) morphogenesis and SbFRO1 transcription in vitro. J Plant Growth Regul 34:611–623. CrossRefGoogle Scholar
  6. Conn VM, Walker AR, Franco CMM (2008) Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana. Mol Plant Microbe Interact 21:208–218. CrossRefPubMedGoogle Scholar
  7. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Curie C, Briat JF (2003) Iron transport and signaling in plants. Annu Rev Plant Biol 54:183–206. CrossRefPubMedGoogle Scholar
  9. Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409:346–349. CrossRefPubMedGoogle Scholar
  10. De Nobili M, Contin M, Mondini C, Brookes P (2001) Soil microbial biomass is triggered into activity by trace amounts of substrate. Soil Biol Biochem 33:1163–1170. CrossRefGoogle Scholar
  11. de Santiago A, García-López AM, Quintero JM, Avilés M, Delgado A (2013) Effect of Trichoderma asperellum strain T34 and glucose addition on iron nutrition in cucumber grown on calcareous soils. Soil Biol Biochem 57:598–605. CrossRefGoogle Scholar
  12. Eide D, Broderius M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci 93:5624–5628. CrossRefPubMedGoogle Scholar
  13. Farag MA, Zhang H, Ryu CM (2013) Dynamic chemical communication between plants and bacteria through airborne signals: induced resistance by bacterial volatiles. J Chem Ecol 39:1007–1018. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Feng H, An F, Zhang S, Ji Z, Ling HQ, Zuo J (2006) Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)-chelate reductase gene AtFRO6. Plant Physiol 140:1345–1354. CrossRefPubMedPubMedCentralGoogle Scholar
  15. García-Gutiérrez L, Zeriouh H, Romero D, Cubero J, Vicente A, Pérez-García A (2013) The antagonistic strain Bacillus subtilis UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate-and salicylic acid-dependent defence responses. Microb Biotechnol 6:264–274. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM (2005) Defensin gene family in Medicago truncatula: structure, expression and induction by signal molecules. Plant Mol Biol 58:385–399. CrossRefPubMedGoogle Scholar
  17. Hernández-Calderón E, Aviles-Garcia MA, Castulo-Rubio DY, Macías-Rodríguez L, Montejano-Ramírez V, Santoyo G, López-Bucio J, Valencia-Cantero E (2017) Volatile compounds from beneficial or pathogenic bacteria differentially regulate root exudation, transcription of iron transporters, and defense signaling pathways in Sorghum bicolor. Plant Mol Biol 96:291–304. CrossRefGoogle Scholar
  18. Hoagland DR, Arnon D (1950) The water-culture method for growing plants without soil. Calif Agric Expt Stn Circ 1950:347Google Scholar
  19. Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2006) Rice plants take up Fe as an Fe3+-phytosiderophore and as Fe2+. Plant J 45:335–346. CrossRefPubMedGoogle Scholar
  20. Ito H, Gray WM (2006) A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides. Plant Physiol 142:63–74. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Jeong J, Cohu C, Kerkeb L, Pilon M, Connolly EL, Guerinot ML (2008) Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions. Proc Natl Acad Sci USA 105:10619–10624. CrossRefPubMedGoogle Scholar
  22. Johnson DC, Dean DR, Smith AD, Johnson MK (2005) Structure, function, and formation of biological iron-sulfur clusters. Annu Rev Biochem 74:247–281. CrossRefPubMedGoogle Scholar
  23. Kobayashi T, Itai RN, Senoura T, Oikawa T, Ishimaru Y, Ueda M, Nakanishi H, Nishizaw NK (2016) Jasmonate signaling is activated in the very early stages of iron deficiency responses in rice roots. Plant Mol Biol 4:533–547. CrossRefGoogle Scholar
  24. Koen E, Trapet P, Brulé D, Kulik A, Klinguer A, Atauri-Miranda L, Meunier-Priest R, Boni G, Glauser G, Mauch-Mani B, Wendehenne D, Besson-Bard A (2014) β-Aminobutyric acid (BABA)-induced resistance in Arabidopsis thaliana: link with iron homeostasis. Mol Plant Microbe Interact 27:1226–1240. CrossRefPubMedGoogle Scholar
  25. Li L, Cheng X, Ling HQ (2004) Isolation and characterization of Fe(III)-chelate reductase gene LeFRO1 in tomato. Plant Mol Biol 54:125–136. CrossRefPubMedGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  27. Mahuku SG (2004) A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA. Plant Mol Biol Rep 22:71–81. CrossRefGoogle Scholar
  28. Martínez-Medina A, VanWees SCM, Pieterse CMJ (2017) Airborne signals from Trichoderma fungi stimulate iron uptake responses in roots resulting in priming of jasmonic acid-dependent defences in shoots of Arabidopsis thaliana and Solanum lycopersicum. Plant Cell Environ 40:2691–2705. CrossRefPubMedGoogle Scholar
  29. Masalha J, Kosegarten H, Elmaci O, Mengel K (2000) The central role of microbial activity for iron acquisition in maize and sunflower. Biol Fertil Soils 30:433–4399. CrossRefGoogle Scholar
  30. Mikami Y, Saito A, Miwa E, Higuchi K (2011) Allocation of Fe and ferric chelate reductase activities in mesophyll cells of barley and sorghum under Fe-deficient conditions. Plant Physiol Biochem 49:513–519. CrossRefPubMedGoogle Scholar
  31. Mimmo T, Del Buono D, Terzano R, Tomasi N, Vigani G, Crecchio R, Pinton R, Zocchi G, Cesco S (2014) Rhizospheric organic compounds in the soil-microorganism-plant system: their role in iron availability. Eur J Soil Sci 65:629–642. CrossRefGoogle Scholar
  32. Morrissey J, Guerinot ML (2009) Iron uptake and transport in plants: the good, the bad, and the ionome. Chem Rev 109:4553–4567. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Mukherjee I, Campbell NH, Ash JS, Connolly EL (2006) Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper. Planta 223:1178–1190. CrossRefPubMedGoogle Scholar
  34. Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H, Nishizawa NK (2011) Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J Biol chem 286:5446–5454. CrossRefPubMedGoogle Scholar
  35. Orozco-Mosqueda MC, Santoyo G, Farías-Rodríguez R, Macías-Rodríguez LI, Valencia-Cantero E (2012) Identification and expression analysis of multiple FRO gene copies in Medicago truncatula.. Genet Mol Res 11:4402–4410. CrossRefGoogle Scholar
  36. Orozco-Mosqueda MC, Macías-Rodríguez LI, Santoyo G, Flores Cortez I, Farías-Rodríguez R, Valencia-Cantero E (2013a) Medicago truncatula increases its Fe-uptake mechanisms in response to volatile organic compounds produced by Sinorhizobium meliloti. Folia Microbiol 58:579–585. CrossRefGoogle Scholar
  37. Orozco-Mosqueda MC, Velázquez-Becerra C, Macías-Rodríquez LI, Santoyo G, Flores-Corez I, Alfaro-Cuevas R, Valencia-Cantero E (2013b) Arthrobacter agilis UMCV2 induces iron acquisition in Medicago truncatula (strategy I plant) in vitro via dimethylhexadecylamine emission. Plant Soil 362:51–66. CrossRefGoogle Scholar
  38. Peleg-Grossman S, Melamed-Book N, Levine A (2012) ROS production during symbiotic infection suppresses pathogenesis-related gene expression. Plant Signal Behav 7:409–415. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Pieterse CM, Zamioudis C, Berendsen RL, Weller DM, Van Wees SC, Bakker PA (2014) Induced systemic resistance by beneficial microbes. Ann Rev Phytopathol 52:347–375. CrossRefGoogle Scholar
  40. Pii Y, Mimmo T, Tomasi N, Terzano R, Cesco S, Crecchio C (2015a) Microbial interactions in the rhizosphere: beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. a review. Biol Fertil Soils 51:403–415. CrossRefGoogle Scholar
  41. Pii Y, Penn A, Terzano R, Crecchio C, Mimmo T, Cesco S (2015b) Plant-microorganism-soil interactions influence the Fe availability in the rhizosphere of cucumber plants. Plant Physiol Biochem 87:45–52. CrossRefPubMedGoogle Scholar
  42. Radhamani R, Kannan R, Rakkiyappan P (2016) Leaf Chlorophyll meter readings as an indicator for sugarcane yield under iron deficient typic haplustert. Sugar Tech 18:61–66. CrossRefGoogle Scholar
  43. Raya-González J, Velázquez-Becerra C, Barrera-Ortíz S, López-Bucio J, Valencia-Cantero E (2017) N,N-dimethyl hexadecylamine and related amines regulate root morphogenesis via jasmonic acid signaling in Arabidopsis thaliana. Protoplasma 254:1399–1410. CrossRefPubMedGoogle Scholar
  44. Robinson NJ, Procter CM, Connolly EL, Guerinot ML (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397:694–697. CrossRefPubMedGoogle Scholar
  45. Sánchez M, Sabio L, Gálvez N, Capdevila M, Dominguez-Vera JM (2017) Iron chemistry at the service of life. IUBMB Life 6:382–388. CrossRefGoogle Scholar
  46. Scagliola M, Pii Y, Mimmo T, Cesco S, Ricciuti P, Crecchio C (2016) 20Characterization of plant growth promoting traits of bacterial isolates from the rhizosphere of barley (Hordeum vulgare L.) and tomato (Solanum lycopersicon L.) grown under Fe sufficiency and deficiency. Plant Physiol Biochem 107:187–196. CrossRefPubMedGoogle Scholar
  47. Solti Á, Müller B, Czech V, Sárvári É, Fodor F (2014) Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme. New Phytol 202:920–928. CrossRefPubMedGoogle Scholar
  48. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Terrazas RA, Giles C, Paterson E, Robertson-Albertyn S, Cesco S, Mimmo T, Pii‖ Y, Bulgarelli D, Plant–microbiota interactions as a driver of the mineral turnover in the rhizosphere. Adv Appl Microbiol 95:1–67.
  50. Valencia-Cantero E, Hernández-Calderón E, Velázquez-Becerra C, López-Meza JE, Alfaro-Cuevas R, López-Bucio J (2007) Role of dissimilatory fermentative Fe-reducing bacteria in Fe uptake by common bean (Phaseolus vulgaris L.) plants grown in alkaline soil. Plant Soil 291:263–273. CrossRefGoogle Scholar
  51. Van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453–483. CrossRefPubMedGoogle Scholar
  52. Velázquez-Becerra C, Macías-Rodríguez LI, López-Bucio J, Altamirano-Hernández J, Flores-Cortez I, Valencia-Cantero E (2011) A volatile organic compound analysis from Arthrobacter agilis identifies dimethylhexadecylamine, an amino-containing lipid modulating bacterial growth and Medicago sativa morphogenesis in vitro. Plant Soil 339:329–340. CrossRefGoogle Scholar
  53. Vigani G, Zocchi G, Bashir K, Philippar K, Briat JF (2013) Signal from chloroplasts and mitochondria for iron homeostasis regulation. Trends Plant Sci 18:305–311. CrossRefPubMedGoogle Scholar
  54. Wang J, Hou Q, Li P, Yang L, Sun X, Benedito VA, Wen J, Chen B, Mysore KS, Zhao J (2017) Diverse functions of multidrug and toxin extrusion (MATE) transporters in citric acid efflux and metal homeostasis in Medicago truncatula. Plant J 90:79–95. CrossRefPubMedGoogle Scholar
  55. Waters BM, Lucena C, Romera FJ (2007) 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. CrossRefPubMedGoogle Scholar
  56. Wu AC, Lesperance L, Bernstein H (2002) Screening for iron deficiency. Pediatr Rev 23:171–178. CrossRefPubMedGoogle Scholar
  57. Wu H, Li L, Du J, Yuan Y, Cheng X, Ling HQ (2005) Molecular and biochemical characterization of the Fe(III) chelate reductase gene family in Arabidopsis thaliana. Plant Cell Physiol 46:1505–1514. CrossRefPubMedGoogle Scholar
  58. Yang J, Kloepper JW, Ryu CM (2009) Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci 14:1–4. CrossRefPubMedGoogle Scholar
  59. Ye L, Li L, Wang L, Wang S, Li S, Du J, Zhang S, Shou H (2015) MPK3/MPK6 are involved in iron deficiency-induced ethylene production in Arabidopsis. Front Plant Sci 6:1–10. CrossRefGoogle Scholar
  60. Zhang H, Sun Y, Xie X, Kim MS, Dowd SE, Paré PW (2009) A soil bacterium regulates plant acquisition of iron via deficiency-inducible mechanisms. Plant J 58:568–577. CrossRefPubMedGoogle Scholar
  61. Zhao L, Wang F, Zhang Y, Zhang J (2014) Involvement of Trichoderma asperellum strain T6 in regulating iron acquisition in plants. J Basic Microbiol 54:S115–S124. CrossRefPubMedGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2018

Authors and Affiliations

  1. 1.Instituto de Investigaciones Químico-BiológicasUniversidad Michoacana de San Nicolás de HidalgoMoreliaMexico

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