Fumiyuki Goto

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.

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

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.

Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48–200 Fe2+/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe3+ from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site

Ferritins are ubiquitous iron storage proteins. Recently, we identified a novel metal-binding site, transit site, in the crystal structure of phytoferritin. To elucidate the function of the transit site in ferritin from other species, we prepared transit-site-deficient mutants of human H ferritin, E140A and E140Q, and their iron oxidation kinetics was analyzed. The initial velocities of iron oxidization were reduced in the variants, especially in E140Q. The crystal structure of E140Q showed that the side chain of the mutated Gln140 was fixed by a hydrogen bond, whereas that of native Glu140 was flexible. These results suggest that the conserved transit site also has a function to assist with the metal ion sequestration to the ferroxidase site in ferritins from vertebrates.

Nicotinate/nicotinamide mononucleotide adenyltransferase-mediated regulation of NAD biosynthesis protects guard cells from reactive oxygen species in ABA-mediated stomatal movement in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.

Long-Distance Signaling of Iron Deficiency in Plants

In a recent issue of the Planta, we established two points regarding the long-distance signal of iron status in tobacco (Nicotiana tabacum L.). One is that the long-distance signal generated in iron deficient tissues is a major factor in positively regulating the expressions of iron uptake genes in tobacco. The expression of a ferric chelate reductase gene (NtFRO1) and an iron-regulated transporter gene (NtIRT1) in roots decreased by cutting off the leaves grown under the iron-deficient condition. Conversely, the leaf-excision did not cause upregulation of the genes under the iron-sufficient condition. These results indicated that signals sent from shoots regulate iron uptake in roots under the iron-deficient condition. The second point regarding the long-distance signals is that the strength of the long-distance signals depends on the size of plant including roots. Both genes expressed in proportion to the weight of the remaining leaves, until a certain threshold. The gene expressions were observed also in hairy roots cultured under the iron deficient condition. In this paper, we discuss the long distance signals of iron status in plants, using a newly obtained data.