Toshihiro Yoshihara

Iron fortification of rice seed by the soybean ferritin gene

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.

Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds

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 bachground); 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.

Iron accumulation and enhanced growth in transgenic lettuce plants expressing the iron- binding protein ferritin

Abstract We have produced transgenic lettuce plants accumulating the iron storage protein ferritin. The integration of the ferritin gene and expression levels in leaves were examined by Southern- and Western-blot analysis, respectively. It was shown that transgenic lettuce plants contained iron levels ranging from 1.2 to 1.7 times that of the control plants, however, the manganese content in transgenic lettuce plants was similar to that in the control. Enhanced growth of transgenic lettuces was observed at the early developmental stages, resulting in weights 27–42% greater than those of control plants. Transgenic lettuce had photosynthesis rates superior to those of the controls, and grew larger and faster compared with the controls during the period of 3 months from germination. These results demonstrate the possibility of producing lettuce plants with high yield, high iron content and rapid growth rate.

Characterization of common cis-regulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family.

Glutelin is the most abundant storage protein in rice, which is expressed specifically in the endosperm of maturing seed. Glutelin is encoded by about 10 genes per haploid genome, which are clearly divided into two subfamilies (GluA and GluB). Most of them are coordinately expressed during seed maturation in spite of the remarkable divergence in the 5′-flanking regions between members of two subfamilies. In order to identify the common regulatory mechanisms responsible for the endosperm-specific expression, various cis-regulatory elements in the 5′-flanking region of the glutelin GluB-1 gene were characterized by studying the expression of chimeric genes that consisted of the sequentially deleted or mutagenized promoter and a β-glucuronidase (GUS) reporter gene in transgenic tobacco seeds. The essential cis-regulatory elements governing the spatially and temporally specific expression of the glutelin gene expression were located within the first 245 bp of the promoter region of the GluB-1 gene from the site of initiation of transcription. The AACA motif between positions −73 and −61 common to all the six genes for glutelin sequenced to date and is repeated between positions −212 and −200 is implicated in the seed-specific expression. The GCN4 motif between positions −165 and −158 and between positions −96 and −92 that is conserved at homologous sites in all the members of glutelin gene family is also involved in the seed-specific regulation. However, both are required for the high level of seed-specific expression, because deletion of the region containing one set of both elements or substitution mutation of the AACA or GCN4 motif substantially reduced the activity. As a whole, our results suggest the combinatorial interaction of the elements in regulation of the glutelin gene expression.

Crystal Structure of Plant Ferritin Reveals a Novel Metal Binding Site That Functions as a Transit Site for Metal Transfer in Ferritin

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 Glu173 and carbonyl oxygen of Thr168, which correspond, respectively, to Glu140 and Thr135 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.

Iron accumulation in tobacco plants expressing soyabean ferritin gene

High iron-content transgenic tobacco plants have been produced by transfer via Agrobacterium tumefaciens of soyabean ferritin cDNA under the control of a CaMV 35S promoter. Immunoblot analysis of protein from transgenic tobacco plants suggested mature ferritin subunits are produced by cleavage of transit peptides. The expressed ferritin was observed in the tissues of leaves and stems. The maximal iron content of transformant leaves was approximately 30% higher than leaves from non-transformants. The increased iron content of each transformant was correlated with increases in ferritin content. These results demonstrate the potential of breeding high iron content crops by introduction of the ferritin gene

Radiocesium contaminations of 20 wood species and the corresponding gamma-ray dose rates around the canopies at 5 months after the Fukushima nuclear power plant accident

Radiocesium ((134)Cs + (137)Cs) deposition from the Fukushima nuclear power plant accident was measured in 20 woody plants (12 evergreen and 8 deciduous species) grown in Abiko (approximately 200 km SSW from the NPP). Leaves (needles) and twigs were sampled from each of three foliar positions (top, middle, and bottom) in the plant canopy in early August 2011. At the time, soils around the plants were also sampled, and gamma radiation dose rates were measured at each sampling position. The average radiocesium activity in the observed leaves of the evergreen species was 7.7 times that in the leaves of the deciduous species. Among the observed evergreen coniferous species, the activity in pre-fallout-expanded leaves was 2.4 times that in the post-fallout-expanded leaves. Notably, a distinct variation in the activity among the evergreen coniferous species could be observed for the post-fallout-expanded leaves but not for the pre-fallout-expanded leaves. Although these differences depend on whether the leaves had expanded at the time of the fallout, it is probable that a considerable amount of radiocesium was translocated to newly developed leaves at a species-specific rate. In addition, it was demonstrated that dose rates around woody plants were not consistent with the prevailing prediction that general dose rates correspondingly decrease with monitoring height from the ground. Thus, the dose rates in the top foliar layer of the deciduous species decreased more than predicted, whereas those in the top foliar layer of the coniferous species did not decrease. This may be due to differences in the balance between the attenuation resulting from a shielding effect of the plant bodies and the higher radiocesium accumulation in the leaves.

A 45-bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm-specific expression of the rice storage protein glutelin gene, GluA-3.

A 45‐bp proximal region of the rice glutelin promoter (−104/−60) containing two putative cis‐elements, the AACA motif and GCN4 motifs, was fused to a truncated CaMV 35S promoter (−90/+9; −90Δ35S)/GUS. The 45‐bp fragment specifically enhanced the promoter activity in endosperm tissue of transformed tobacco. A substitution mutation of the GCN4 motif reduced the promoter activity, whereas mutation of the AACA motif increased the activity in the embryo as well as in the endosperm. These results suggest that the GCN4 motif generally enhances the promoter activity but that the combination of the two motifs confers the endosperm specificity. Furthermore, the function of the two motifs was dependent on the orientation and/or distance from a G‐box element in −90Δ35S, suggesting that synergistic interaction between the factors that recognize those motifs and the G‐box element is important for transcriptional regulation.

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency

Abstract. The effects of the Agrobacterium rhizogenesrolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.

A novel EP-involved pathway for iron release from soya bean seed ferritin

Iron in phytoferritin from legume seeds is required for seedling germination and early growth. However, the mechanism by which phytoferritin regulates its iron complement to these physiological processes remains unknown. In the present study, protein degradation is found to occur in purified SSF (soya bean seed ferritin) (consisting of H-1 and H-2 subunits) during storage, consistent with previous results that such degradation also occurs during seedling germination. In contrast, no degradation is observed with animal ferritin under identical conditions, suggesting that SSF autodegradation might be due to the EP (extension peptide) on the exterior surface of the protein, a specific domain found only in phytoferritin. Indeed, EP-deleted SSF becomes stable, confirming the above hypothesis. Further support comes from a protease activity assay showing that EP-1 (corresponding to the EP of the H-1 subunit) exhibits significant serine protease-like activity, whereas the activity of EP-2 (corresponding to the EP of the H-2 subunit) is much weaker. Consistent with the observation above, rH-1 (recombinant H-1 ferritin) is prone to degradation, whereas its analogue, rH-2, becomes very stable under identical conditions. This demonstrates that SSF degradation mainly originates from the serine protease-like activity of EP-1. Associated with EP degradation is a considerable increase in the rate of iron release from SSF induced by ascorbate in the amyloplast (pH range, 5.8-6.1). Thus phytoferritin may have facilitated the evolution of the specific domain to control its iron complement in response to cell iron need in the seedling stage.