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Fe-deficiency-induced expression of bHLH104 enhances Fe-deficiency tolerance of Arabidopsis thaliana


Main conclusion

Expression of bHLH104 - GFP driven by the MYB72 promoter improves plants’ tolerance to Fe deficiency and increases seed Fe concentrations.

Iron (Fe) deficiency causes reduced crop yield and quality. In humans, Fe deficiency is directly associated with Fe-deficiency anemia. Therefore, breeding Fe-deficiency tolerant and Fe-enriched plants are an ideal approach to deal with these problems. Here, different strategies were explored to generate Fe-deficiency tolerant and Fe-enriched plants. Unexpectedly, the overexpression of Fe-deficiency responsive genes (IRT1, MYB72, and bHLH100) resulted in enhanced sensitivity to Fe deficiency, including leaf chlorosis and short roots under Fe-deficiency conditions. Next, three different types of Fe-deficiency responsive promoters (Pro IRT1 , Pro MYB72, and Pro bHLH100 ) were used to drive the expression of bHLH104-GFP fusion gene in Arabidopsis. Pro IRT1 :bHLH104-GFP plants showed the enhanced sensitivity to Fe deficiency on Fe-deficient media and the reduced fertility in alkaline soil. In contrast, Pro bHLH100 :bHLH104-GFP plants displayed a slight tolerance to Fe deficiency and Pro MYB72 :bHLH104-GFP plants had a significant advantage in growth in alkaline soil, including increased root length, chlorophyll, and biomass. Further analysis revealed that the expression of Fe-deficiency responsive genes was dramatically upregulated in both Pro MYB72 :bHLH104-GFP and Pro bHLH100 :bHLH104-GFP plants under Fe-deficiency conditions. When grown in alkaline soil, Pro MYB72 :bHLH104-GFP plants greatly improved the seed yield and Fe concentration. These results are fundamental for plant manipulation approaches to modify tolerance to Fe deficiency and Fe accumulation through alterations of bHLH104 gene expression.

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  1. Barberon M, Zelazny E, Robert S, Conéjéro G, Curie C, Friml J, Vert G (2011) Monoubiquitin-dependent endocytosis of the IRON-REGULATED TRANSPORTER 1 (IRT1) transporter controls iron uptake in plants. Proc Natl Acad Sci USA 108:E450–E458

  2. Bashir K, Takahashi R, Nakanishi H, Nishizawa NK (2013) The road to micronutrient biofortification of rice: progress and prospects. Front Plant Sci 4:15

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

  4. Colangelo EP, Guerinot ML (2004) The essential bHLH protein FIT1 is required for the iron deficiency response. Plant Cell 16:3400–3412

  5. 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

  6. Curie C, Cassin G, Couch D, Divol F, Higuchi K, Le Jean M, Misson J, Schikora A, Czernic P, Mari S (2009) Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters. Ann Bot 103:1–11

  7. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282–286

  8. Inoue H, Higuchi K, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2003) Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long-distance transport of iron and differentially regulated by iron. Plant J 36:366–381

  9. Ishimaru Y, Kim S, Tsukamoto T, Oki H, Kobayashi T, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2007) Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil. Proc Natl Acad Sci USA 104:7373–7378

  10. Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T, Takahashi M, Nakanishi H, Aoki N, Hirose T, Ohsugi R, Nishizawa NK (2010) Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese. Plant J 62:379–390

  11. Ivanov R, Brumbarova T, Bauer P (2012) Fitting into the harsh reality: regulation of iron-deficiency responses in dicotyledonous plants. Mol Plant 5:27–42

  12. Jeong J, Guerinot ML (2009) Homing in on iron homeostasis in plants. Trends Plant Sci 14:280–285

  13. Kerkeb L, Mukherjee I, Chatterjee I, Lahner B, Salt DE, Connolly EL (2008) Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues. Plant Physiol 146:1964–1973

  14. Klatte M, Schuler M, Wirtz M, Fink-Straube C, Hell R, Bauer P (2009) The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses. Plant Physiol 150:257–271

  15. Kobayashi T, Nakanishi H, Takahashi M, Mori S, Nishizawa NK (2008) Generation and field trials of transgenic rice tolerant to iron deficiency. Rice 1:144–153

  16. Lee S, An G (2009) Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell Environ 32:408–416

  17. Lee S, Jeon US, Lee SJ, Kim YK, Persson DP, Husted S, Schjørring JK, Kakei Y, Masuda H, Nishizawa NK, An G (2009) Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc Natl Acad Sci USA 106:22014–22019

  18. Li X, Zhang H, Ai Q, Liang G, Yu D (2016) Two bHLH transcription factors, bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana. Plant Physiol 170:2478–2493

  19. Liang G, Zhang H, Li X, Ai Q, Yu D (2017) bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana. J Exp Bot. doi:10.1093/jxb/erx043

  20. Lucca P, Hurrell R, Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392–397

  21. Masuda H, Aung MS, Nishizawa NK (2013) Iron biofortification of rice using different transgenic approaches. Rice 6:40

  22. Nozoye T, Kim S, Kakei Y, Takahashi M, Nakanishi H, Nishizawa NK (2014) Enhanced levels of nicotianamine promote iron accumulation and tolerance to calcareous soil in soybean. Biosci Biotechnol Biochem 78:1677–1684

  23. Ogo Y, Itai RN, Nakanishi H, Kobayashi T, Takahashi M, Mori S, Nishizawa NK (2007) The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions. Plant J 51:366–377

  24. Ogo Y, Itai RN, Kobayashi T, Aung MS, Nakanishi H, Nishizawa NK (2011) OsIRO2 is responsible for iron utilization in rice and improves growth and yield in calcareous soil. Plant Mol Biol 75:593–605

  25. Palmer CM, Hindt MN, Schmidt H, Clemens S, Guerinot ML (2013) MYB10 and MYB72 are required for growth under iron-limiting conditions. PLoS Genet 9(11):e1003953

  26. Shin LJ, Lo JC, Chen GH, Callis J, Fu H, Yeh KC (2013) IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis. Plant Cell 25:3039–3051

  27. Suzuki M, Morikawa KC, Nakanishi H et al (2008) Transgenic rice lines that include barley genes have increased tolerance to low iron availability in a calcareous paddy soil. Soil Sci Plant Nutr 54:77–85

  28. Takahashi M, Nakanishi H, Kawasaki S, Nishizawa NK, Mori S (2001) Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes. Nat Biotechnol 19:466–469

  29. Trijatmiko KR, Dueñas C, Tsakirpaloglou N et al (2016) Biofortified indica rice attains iron and zinc nutrition dietary targets in the field. Sci Rep 6:19792

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

  31. Wang HY, Klatte M, Jakoby M, Bäumlein H, Weisshaar B, Bauer P (2007) Iron deficiency-mediated stress regulation of four subgroup Ib BHLH genes in Arabidopsis thaliana. Planta 226:897–908

  32. Wang N, Cui Y, Liu Y, Fan H, Du J, Huang Z, Yuan Y, Wu H, Ling HQ (2013) Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana. Mol Plant 6:503–513

  33. Yuan Y, Wu H, Wang N, Li J, Zhao W, Du J, Wang D, Ling HQ (2008) FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Res 18:385–397

  34. Zhang J, Liu B, Li M, Feng D, Jin H, Wang P, Liu J, Xiong F, Wang J, Wang HB (2015) The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant Cell 27:787–805

  35. Zhao Q, Ren YR, Wang QJ, Yao YX, You CX, Hao YJ (2016) Overexpression of MdbHLH104 gene enhances the tolerance to iron deficiency in apple. Plant Biotechnol J 14:1633–1645

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This work was supported by the Candidates of the Young and Middle Aged Academic Leaders of Yunnan Province [2015HB095], the Youth Innovation Promotion Association of CAS, and the program for Innovative Research Team of Yunnan Province [2014HC017].

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Correspondence to Diqiu Yu or Gang Liang.

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Wang, C., Yao, X., Yu, D. et al. Fe-deficiency-induced expression of bHLH104 enhances Fe-deficiency tolerance of Arabidopsis thaliana . Planta 246, 421–431 (2017). https://doi.org/10.1007/s00425-017-2703-y

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  • bHLH100
  • bHLH104
  • Fe deficiency
  • MYB7