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. 2013 Dec 19;6(1):40.
doi: 10.1186/1939-8433-6-40.

Iron biofortification of rice using different transgenic approaches

Affiliations

Iron biofortification of rice using different transgenic approaches

Hiroshi Masuda et al. Rice (N Y). .

Abstract

More than 2 billion people suffer from iron (Fe) deficiency, and developing crop cultivars with an increased concentration of micronutrients (biofortification) can address this problem. In this review, we describe seven transgenic approaches, and combinations thereof, that can be used to increase the concentration of Fe in rice seeds. The first approach is to enhance the Fe storage capacity of grains through expression of the Fe storage protein ferritin under the control of endosperm-specific promoters. Using this approach, the concentration of Fe in the seeds of transformants was increased by approximately 2-fold in polished seeds. The second approach is to enhance Fe translocation by overproducing the natural metal chelator nicotianamine; using this approach, the Fe concentration was increased by up to 3-fold in polished seeds. The third approach is to enhance Fe influx to the endosperm by expressing the Fe(II)-nicotianamine transporter gene OsYSL2 under the control of an endosperm-specific promoter and sucrose transporter promoter, which increased the Fe concentration by up to 4-fold in polished seeds. The fourth approach is introduction of the barley mugineic acid synthesis gene IDS3 to enhance Fe uptake and translocation within plants, which resulted in a 1.4-fold increase in the Fe concentration in polished seeds during field cultivation. In addition to the above approaches, Fe-biofortified rice was produced using a combination of the first, second, and third approaches. The Fe concentration in greenhouse-grown T2 polished seeds was 6-fold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than in non-transgenic seeds without any reduction in yield. When the first and fourth approaches were combined, the Fe concentration was greater than that achieved by introducing only the ferritin gene, and Fe-deficiency tolerance was observed. With respect to Fe biofortification, the introduction of multiple Fe homeostasis genes is more effective than the introduction of individual genes. Moreover, three additional approaches, i.e., overexpression of the Fe transporter gene OsIRT1 or OsYSL15, overexpression of the Fe deficiency-inducible bHLH transcription factor OsIRO2, and knockdown of the vacuolar Fe transporter gene OsVIT1 or OsVIT2, may be useful to further increase the Fe concentration of seeds.

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Figures

Figure 1
Figure 1
Seven transgenic approaches to Fe biofortification of rice. The pathway inside the gray dashed-line rectangle shows the biosynthetic pathway for mugineic acid family phytosiderophores (MAs) in graminaceous plants. SAMS, S-Adenosyl-methionine synthase; NAS, NA synthase; NAAT, NA aminotransferase; DMA, 2′-deoxymugineic acid; DMAS, DMA synthase; IDS3, MA synthase (dioxygenase that catalyzes the hydroxylation of DMA and epiHDMA at the 2′ position); Ferritin, iron storage protein; OsYSL2, Fe(II)-NA and Mn(II)-NA transporter; OsIRO2, Fe deficiency-inducible bHLH transcription factor related to Fe homeostasis in rice; OsIRT1, ferric transporter; OsYSL15, Fe(III)-DMA transporter; TOM1, MA transporter. Rice lacks the two dioxygenase genes (IDS2 and IDS3) and secretes only DMA. Approach 1: Enhancing Fe accumulation in seeds by introducing the Fe storage protein, ferritin gene, SoyferH1, SoyferH2 or Pvferritin, under the control of endosperm-specific promoters. Approach 2: Enhancing Fe transport within the plant body by the overexpression of NAS. Approach 3: Enhancing Fe influx to seeds by expression of the Fe(II)-NA transporter gene OsYSL2 under the control of the OsSUT1 promoter. Approach 4: Enhancing Fe uptake and translocation by introduction of the phytosiderophore synthase gene IDS3. Approach 5: Enhanced Fe uptake from soil by overexpression of the Fe transporter gene OsIRT1 or OsYSL15. Approach 6: Enhanced Fe uptake and translocation by overexpression of the OsIRO2 gene. Approach 7: Enhanced Fe translocation from flag leaves to seeds by knockdown of the vacuolar Fe transporter gene OsVIT1 or OsVIT2. The ferritin image was kindly provided by Dr. David S. Goodsell (Scripps Research Institute, La Jolla, CA, USA) and the RCSB PDB.
Figure 2
Figure 2
Fe concentration in the Fer-NAS-YSL2 lines (Masuda et al.2012). a; The Fe concentrations in T2 polished rice seeds (Oryza sativa cv. Tsukinohikari). Bars represent the Fe concentrations in polished seeds obtained from individual transgenic or non-transgenic plants. The numbers indicate the line numbers of the independent T1 lines. The arrows and numbers above the bars show the lines that contained high levels of Fe, which are shown in b for the subsequent generation. b; Fe concentrations in the T3 polished rice seeds (Oryza sativa cv. Tsukinohikari) harvested from the paddy field. ANOVA with the Tukey–Kramer HSD test was used for each four-block dataset (n = 4). The letters above the bars indicate significant differences (P < 0.05). NT, Non-transgenic rice; AN, OsActin1 promoter–HvNAS1 transgenic rice line No. 8 (Masuda et al. 2009); Fer-NAS-YSL2, transgenic rice lines that carry the OsGlb1 promoter–SoyferH2, OsGluB1 promoter–SoyferH2, OsSUT1 promoter–OsYSL2, OsGlb1 promoter–OsYSL2, and OsActin1 promoter–HvNAS1 (Masuda et al. 2012).

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