To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L. cv. Kita-ake) by Agrobacterium-mediated transformation. The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis. Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing. The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.
To answer the question whether iron accumulation in transgenic rice seeds depends on the expression level of exogenous soybean ferritin, we generated two kinds of ferritin hyper-expressing rice lines by introducing soybean ferritin SoyferH-1 gene under the control of the rice seed storage glutelin gene promoter, GluB-1 and the rice seed storage globulin gene promoter, Glb-1, (GluB-1/SoyferH-1 and Glb-1/SoyferH-1, DF lines), and by introducing the SoyferH-1 gene under the control of Glb-1 promoter alone (Glb-1/SoyferH-1, OF lines). Ferritin expression was restricted to the endosperm in both lines and protein levels determined by western blot analysis were up to 13-fold higher than in a construct previously reported FK22 (GluB-1/SoyferH-1, in genetically Kitaake background); [corrected] however, the maximum iron concentrations in seeds of both of the new lines were only about 30% higher than FK22. The maximum iron concentration in the OF and DF lines was about threefold higher than in the non-transformant. The mean Fe concentration in leaves of ferritin over-expressing lines decreased to less than half of the non-transformant while that the plant biomasses and seed yields of the ferritin-transformed lines were not significantly different from those of the non-transformant, suggesting that accumulation of Fe in seeds of hyper-expression ferritin rice did not always depend on the expression level of exogenous ferritin but may have been limited by Fe uptake and transport. No obvious differences were observed for other divalent-metal concentrations (Ca, Cd, Cu, Mg, Mn and Zn) in the seeds among all experimental lines and non-transformant.
Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-Å resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu 173 and carbonyl oxygen of Thr 168 , which correspond, respectively, to Glu 140 and Thr 135 of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.
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