Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency

Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency Rice (2008) 1:144–153 DOI 10.1007/s12284-008-9011-x REVIEW Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency Takanori Kobayashi & Hiromi Nakanishi & Michiko Takahashi & Satoshi Mori & Naoko K. Nishizawa Received: 5 March 2008 /Accepted: 1 August 2008 /Published online: 24 September 2008 Springer Science + Business Media, LLC 2008 Abstract Iron deficiency is a major cause of reduced crop Introduction yields worldwide, particularly in calcareous soils. Unlike barley, rice is highly susceptible to iron deficiency because Iron (Fe) is essential for most living organisms including of a low capacity to secrete mugineic acid family plants. Although abundant in mineral soils, Fe is sparingly phytosiderophores (MAs), which are iron chelators secreted soluble under aerobic conditions at high soil pH, especially by graminaceous plants. We present an approach toward the in calcareous soils, which account for about 30% of the generation along with field trials of transgenic rice lines world’s cultivated soils. Fe deficiency is a widespread exhibiting increased tolerance to iron deficiency. Cloning agricultural problem that reduces plant growth and crop barley genes that encode biosynthetic enzymes for MAs yields [33, 36]. To take up and utilize Fe from the enabled us to produce transgenic rice plants by introducing rhizosphere, higher plants have evolved two major strate- barley MAs biosynthesis-related genes. We tested three gies [34]: reduction (strategy I) and chelation (strategy II). transgenic lines possessing barley genomic fragments The strategy I mechanism, utilized by nongraminaceous responsible for MAs biosynthesis in a paddy field exper- plants, includes induction of ferric-chelate reductase to iment on calcareous soil, which revealed tolerance of these reduce Fe at the root surface to the more soluble ferrous lines to low iron availability. We also applied new form and transport the ferrous ions generated across the approaches to generate iron-deficiency-tolerant rice lines, root plasma membrane. In contrast, the strategy II mecha- including the introduction of an engineered ferric-chelate nism, which is specific to graminaceous plants, is mediated reductase gene and manipulation of transcription factor by natural Fe chelators, the mugineic acid family phytosi- genes regulating the iron deficiency response. derophores (MAs). Graminaceous plants synthesize and secrete MAs from their roots to solubilize Fe(III) in the . . Keywords Iron deficiency Field trial rhizosphere [59], and the resulting Fe(III)–MAs complexes Mugineic acid family phytosiderophores are taken up by roots through a specific transporter in the Transgenic rice plants plasma membrane [5, 59]. In calcareous soils, the strategy II mechanism is more efficient than that of strategy I [33]. Tolerance to Fe deficiency is divergent among graminaceous plants and is thought to be dependent on the amount and kinds of MAs that they secrete. Rice, sorghum, and maize secrete only : : : : small amounts of 2’-deoxymugineic acid (DMA) among T. Kobayashi H. Nakanishi M. Takahashi S. Mori N. K. Nishizawa (*) the MAs and thus are susceptible to low Fe availability. In Graduate School of Agricultural and Life Sciences, contrast, barley secretes large amounts of MAs, including The University of Tokyo, mugineic acid (MA), 3-epihydroxy-2’-deoxymugineic acid 1-1-1 Yayoi, Bunkyo-ku, (epiHDMA), and 3-epihydroxymugineic acid (epiHMA), in Tokyo 113-8657, Japan e-mail: annaoko@mail.ecc.u-tokyo.ac.jp addition to DMA, under Fe deficiency; therefore, it is more Rice (2008) 1:144–153 145 145 tolerant to Fe deficiency than other graminaceous plants transport of various micronutrients including Fe and zinc [32, 37, 52, 59]. (Zn) [9, 62]. NA aminotransferase (NAAT) catalyzes the We therefore hypothesized that introducing barley genes first step specific to graminaceous plants: transamination of responsible for MAs biosynthesis into rice would lead to NA to produce the 3’’-oxo intermediate [20, 54]. DMA enhanced MAs production and tolerance in calcareous soils. synthase (DMAS) subsequently reduces the 3’’-oxo form to We successfully produced various transgenic rice lines DMA [2]. All MAs share their biosynthetic pathway from showing enhanced tolerance to low Fe availability by methionine to DMA, which is then hydroxylated to form introducing barley MAs biosynthesis genes. Using selected other MAs in some species, including barley. lines, we carried out field trials on calcareous soil that revealed Attempts to isolate the genes responsible for MAs tolerance of these lines to low Fe availability. We also biosynthesis have included “direct” approaches via enzyme produced Fe-deficiency-tolerant rice lines using two other purification and “indirect” approaches through screening the strategies: introduction of a reconstructed ferric-chelate genes and proteins specifically induced in Fe-deficient roots. reductase gene and manipulation of transcription factor genes The former approach was applied to NAS and NAAT. NAS controlling the expression of Fe-deficiency-induced genes. genes were first isolated from barley (HvNAS1–7)through Future perspectives on generating further favorable and the establishment of a NAS activity assay [11] and enzyme commonly acceptable transformants are described. purification from Fe-deficient barley roots [13]. Two barley NAAT genes, HvNAAT-A and HvNAAT-B,were also cloned through the establishment of an enzyme activity assay [46] Generation of Fe-deficiency-tolerant transgenic rice and enzyme purification [20, 60]. Expression of HvNAS1, by introducing barley MAs biosynthesis genes HvNAAT-A, and HvNAAT-B is strongly induced by Fe deficiency and occurs almost exclusively in the roots [13, Identification of genes responsible for MAs biosynthesis 60], suggesting direct involvement in MAs biosynthesis for the acquisition of Fe from the rhizosphere. Detection of NAS The biosynthetic pathway of MAs (Fig. 1) has been and NAAT enzyme activities in Fe-deficient roots of various identified through extensive biochemical and physiological graminaceous species revealed that NAS and NAAT activ- studies [21, 30, 32, 37, 54]. Methionine is the precursor of ities are positively correlated with both the amounts of MAs MAs [37] and is adenosylated by S-adenosylmethionine secreted and Fe-deficiency tolerance [12, 19]. (SAM) synthetase. Nicotianamine synthase (NAS) cata- In the latter “indirect” approach, the differential hybrid- lyzes the trimerization of SAM to nicotianamine (NA) [11]. ization method was applied with mRNA from Fe-deficient All higher plants, including nongraminaceous plants, have and Fe-sufficient barley roots. We cloned iron-deficiency- the biosynthetic pathway to synthesize NA [29, 42], which specific (IDS) genes specifically expressed in Fe-deficient serves as a common metal chelator involved in the internal barley roots [39, 49, 50]. Among these, IDS2 and IDS3 are Fig. 1 Biosynthesis pathway of MAs mugineic acid family phytosi- COOH COOH COOH COOH COOH COOH derophores (MAs). SAMS, S- DMAS N NH O adenosylmethionine synthetase; N NH OH NAS, nicotianamine synthase; 3''-oxo form NAAT, nicotianamine amino- DMA transferase; DMAS, deoxymugi- NAAT IDS2 neic acid synthase; IDS2, iron- IDS3 deficiency-specific clone no. 2; COOH COOH COOH IDS3, iron-deficiency-specific COOH COOH COOH COOH COOH COOH N NH NH clone no. 3; DMA,2’-deoxy- N NH OH mugineic acid; MA, mugineic nicotianamine(NA) HO N NH OH OH acid; HMA, 3-hydroxymugineic epiHDMA MA acid; epiHDMA, 3-epihydroxy- NAS 2’-deoxymugineic acid; epi- IDS2 HMA, 3-epihydroxymugineic S-adenosyl- IDS3 acid. L-methionine (SAM) COOH COOH COOH COOH COOH COOH SAMS N NH OH HO N NH OH HO COOH OH OH H C SNH epiHMA HMA L-methionine 146 Rice (2008) 1:144–153 homologous to 2-oxoglutarate-dependent dioxygenases, which the HvNAAT-A and HvNAAT-B genes are tandemly suggesting their possible involvement in the hydroxylation located [61], designated “gNAAT”; (c) a 20-kb genome of MAs. By interspecies correlation between the expression fragment of the IDS3 gene [23], designated “gIDS3”;(d) a of IDS2–IDS3 and the capacity to secrete hydroxylated 7.6-kb genome fragment of HvNAS1 plus an 11-kb genome MAs, we deduced that IDS3 is the enzyme that hydrox- fragment containing the HvNAAT-A and HvNAAT-B genes, ylates the C-2’ positions of DMA and epiHDMA, while designated “gNAS1-gNAAT”;(e) an HvAPT cDNA frag- IDS2 hydroxylates the C-3 positions of DMA and MA ment fused downstream of the 2.2-kb IDS3 promoter; (f) (Fig. 1;[40]). IDS3 was further confirmed to be the “MA HvNAS1, HvNAAT-A,and HvAPT cDNA fragments each synthase” by introducing the barley IDS3 gene into rice fused downstream of the 2.2-kb IDS3 promoter; and (g) an [23]: transgenic rice plants secreted MAs in addition to IDS3 cDNA fragment fused downstream of the cauliflower DMA, while nontransformants secreted only DMA. mosaic virus 35S promoter [23]. Expression analysis We also compared proteins of Fe-sufficient and Fe- revealed that the Fe-deficiency-induced expression was deficient barley roots using two-dimensional polyacryl- strongly conferred by genome fragments of the HvNAS1, amide gel electrophoresis. Peptide sequencing of the HvNAAT-A, HvNAAT-B,or IDS3 genes [14, 23, 61], induced proteins revealed that formate dehydrogenase confirming the potency of the barley promoter elements (FDH) and adenine phosphoribosyltransferase (APRT), as included in the genome fragments to drive Fe-deficiency- well as the IDS3 protein, were induced in Fe-deficient roots induced expression in rice plants. Although the barley [55]. The corresponding genes, HvFDH and HvAPT, were promoters exhibit induction almost exclusively in Fe- subsequently cloned [18, 55]. Both FDH and APRT are deficient roots in native barley, they induced moderate thought to function in scavenging the by-products (formate expression in Fe-deficient leaves and prominent expression and adenine) that are released during the methionine cycle in Fe-deficient roots when introduced into rice. [36], thus supporting the production of MAs. Indeed, the To examine whether the transformants have enhanced methionine cycle works vigorously in roots to meet the tolerance to low Fe availability, the plants were cultured in increased demand for methionine in the synthesis of MAs pots filled with calcareous soils (pH 8.5–9.0; [61]) under [31]. We also applied a revised differential hybridization controlled conditions in a greenhouse. Of the 36 gNAAT screening, identifying iron-deficiency-induced (IDI) genes lines evaluated, ten showed remarkable tolerance to from barley roots [70–72]. IDI1 and IDI2 putatively encode calcareous soils [61]. Nontransformants exhibited reduced enzymes catalyzing steps in the methionine cycle [26, 56]. growth and severe leaf chlorosis caused by Fe deficiency, Recent application of microarray techniques reconfirmed whereas the gNAAT lines had greener and larger shoots. At the induction of the abovementioned genes involved in harvest, the gNAAT lines possessed 4.2 and 4.1 times MAs biosynthesis in Fe-deficient barley roots [41, 56]. The higher shoot dry weight and grain yield per pot than the microarray approach also resulted in cloning of DMAS nontransformants [61]. We also examined tolerance in genes from rice (OsDMAS1), barley (HvDMAS1), wheat calcareous soils for the other transformants. We found rice (TaDMAS1), and maize (ZmDMAS1). All of the lines showing some tolerance to calcareous soils from all corresponding encoding proteins were confirmed to possess transgenes (a)–(g). In these lines, increased amounts or the reductase activity to produce DMA [2]. kinds of MAs secreted were thought to have contributed to enhanced Fe availability under Fe-limiting conditions. Introduction of barley genes responsible for MAs biosynthesis into rice Field trials of Fe-deficiency-tolerant rice lines To produce transgenic rice plants with enhanced tolerance to Fe deficiency by increasing MAs production capacity, we Approval for using transgenics in field trials introduced barley HvNAS1, HvNAAT-A, HvNAAT-B,and/or IDS3 genes using either their genomic fragments or the IDS3 For the field experiment, we first selected one line from each gene promoter to confer inducibility to Fe deficiency. To of (a) to (f) described above. Prior to culture in a quarantine introduce barley genomic fragments, we utilized the field in Japan, we needed approval for Type 1 Use Regulations pBIGRZ1 vector [1], which was developed as a modified for living modified organisms from the Ministry of Agricul- binary vector capable of transferring large-size DNA frag- ture, Forestry, and Fisheries in Japan. For that purpose, we ments into the rice genome. Rice cultivar Tsukinohikari was performed evaluation tests on their priority in competition, subjected to Agrobacterium-mediated transformation [10]. possible production of harmful substances, and influence on The transformants included those introduced with (a) a 13.5- biological diversity in relation to interspecific crossing. We kb genome fragment of the HvNAS1 gene [14], designated performed the following tests on six lines of transgenic rice “gNAS1”; (b) an 11-kb genome fragment of HvNAAT in and the nontransformant: growth comparison in Andosol, Rice (2008) 1:144–153 147 147 stability of the transgene and expression beyond the first boundary adjacent to the transgenic gIDS3 rice plants generation, determination of the interspecific crossing rate (Fig. 3d), which suggests that NTs utilized MAs secreted by from transformants to nontransformants, evaluation of soil the transgenic rice. microorganism populations, evaluation of the residual effect From 16 to 42 DAT, plant height and the SPAD value (leaf of harmful compounds in postharvest soil, plowing-under color) of the three transformant lines were higher than those of effect of the dead transformants, carryover of Agrobacterium, NTs. In addition, the number of tillers per plant was higher in tests on pollen shape, fertility, and dispersal distance, and gIDS3 than in the other lines. By 42 DAT, however, all lines tests on the germination rates of the seeds and tolerance to had about 15 tillers per plant. After 42 DAT, when soil Eh fell low temperature during early growth. below 0 mV, all plant lines recovered their leaf color, and, We confirmed stable inheritance of every transgene over consequently, the SPAD value of NT plants rose up to levels at least three generations and found no harmful impacts on similar to that of transformants. The decrease in soil redox the environment in any of the abovementioned tests. potential with time is thought to have resulted in the Moreover, we detected no interspecific crossing from absorption of generated ferrous ion via the ferrous transporter transformants to nontransformants. Based on these results, OsIRT1 (“Introducing an engineered ferric-chelate reductase the transformants were approved for the quarantine field gene”;[16]). trials following the Type 1 Use Regulations. At the time of grain harvest, the number of grains, 1,000- grain weights, and the grain yield of gNAS1 were higher Field trials of the selected lines than those of the NT and other lines. Plant height and the proportion of fully matured grains showed no significant A calcareous subsoil from Toyama Prefecture containing difference among the lines. Timing of the decrease in soil fossil shells (pH ∼9.2; [38]) was used to establish a paddy redox potential might account for the relatively small field in the quarantine area of the Field Science Center of differences in grain parameters between transformants and Tohoku University (Osaki, Miyagi, Japan; 38° 44’ N; 140° NTs, despite the clearly inferior performance of NTs during 45’ E). The paddy field in the first-year experiment was early growth. Indeed, in a prior experiment, NT seedlings 7 m long, 14 m wide, and 0.5 m deep, with the external grown in the same calcareous paddy field showed severe ridges completely covered with a vinyl sheet to avoid chlorosis, and many seedlings died in the early stages contamination from the surrounding Andosol at the site. before the Eh fell below 0 mV [38]. Therefore, it is crucial The first-year experiment was conducted from April to for rice in calcareous paddy fields to survive the early October 2005, using the six transformant lines (a)–(f) and stages of growth, when enhanced MAs production greatly nontransformants (cv. Tsukinohikari). The following year, supports Fe acquisition. from April to October 2006, the second-year experiment Interestingly, the concentrations of Fe and Zn in the rice was performed using the three most promising lines: grains of gIDS3 were significantly higher than those of NTs gNAS1 (a), gIDS3 (c), and gNAS1–gNAAT (d). Experi- and the other lines, suggesting that MA synthesized by IDS3 mental procedures of the second-year experiment were contributed not only to improved Fe uptake from the soil but described by Suzuki et al. [57]. The paddy field in the also to increased translocation to the grain. MAs have been second-year experiment was 6 m long and 4 m wide, and suggested to be involved in long-distance transport of Fe and the experimental plots were arranged in a completely Zn inside rice plants [15, 58]. Since more hydroxylated MAs randomized design (Fig. 2b) including the three transgenic exhibit higher stability under mildly acidic conditions [67], rice lines (gNAS1, gIDS3, and gNAS1–gNAAT) and MA synthesized by IDS3 would have been favorable for nontransformants (NTs). Germinated seeds were grown internal translocation of Fe and Zn. for 45 days in a greenhouse, and seedlings were then In conclusion, our field trial of the transformants demon- transplanted (three per hill) into the calcareous paddy field. strated that a transgenic approach to increase the tolerance of Sixteen days after transplanting (DAT), chlorosis and rice to low Fe availability is practical for improving growth retardation began to appear. By 42 DAT, the three agricultural productivity in calcareous paddy soils. transgenic rice lines were clearly superior to the NTs (Fig. 2a) both in leaf color and growth, although differences in performance were observed in individual plots. Using Production of other transgenic rice plants tolerant to Fe gIDS3 as an example, we saw no evident difference from deficiency NT on 16 DAT (Fig. 3a); chlorotic symptoms appeared in NTs but not in gIDS3 at 30 DAT (Fig. 3b). The clearest Introducing an engineered ferric-chelate reductase gene difference between gIDS3 and NTs was evident at 42 DAT (Fig. 3c). One week later (50 DAT), leaf chlorosis began to In strategy I plants, Fe uptake from the rhizosphere is disappear, especially in NT plants close to the plot mediated by ferrous ion transporters. Eide et al. [7] isolated 148 Rice (2008) 1:144–153 (a) Fig. 2 a Photograph (42 DAT) of the rice lines tested in a paddy field in the quarantine area of the Field Science Center of Tohoku University (Osaki, Miyagi, Japan) and b field lay- out. Each population contained five 1.2-m-long rows of rice with 20 cm between rows and 15 cm between hills. The box in the upper left indicates the two plots photographed on several occasions (Fig. 3). NT, non- transformant. Original figure: Suzuki et al. [57]. (b) 1.0 m 5 rows 1.2 m gNAS1 NT gIDS3 gNAS1 8 plants gNAAT 0.4 m gNAS1 gNAS1 gNAS1 NT gNAAT gNAS1 NT gIDS3 gNAS1 gNAAT gNAS1 gNAS1 NT gIDS3 the Arabidopsis IRT1 gene, which is the dominant ferrous To take up ferrous ion directly using OsIRT1, without transporter in the Fe-uptake process [65]. Rice, in spite of reducing ferric chelates, seems to be a consequence of being a strategy II plant, possesses homologs of the adaptation of rice to waterlogged soils, in which the concen- Arabidopsis IRT1 gene, OsIRT1 and OsIRT2, the ferrous tration of soluble ferrous iron increases with the decrease in soil transport capacity of which was demonstrated by functional redox potential [16, 57]. Because of the presence of OsIRT1, complementation in yeast [3, 16]. OsIRT1 expression is severe Fe deficiency is relatively rare in irrigated rice systems. strongly induced in Fe-deficient roots, and OsIRT2 is Nevertheless, rice plants grown in calcareous soils exhibit Fe expressed similarly but at lower levels. Promoter β- deficiency symptoms even under waterlogged conditions as glucuronidase (GUS) analysis indicated that OsIRT1 is noted previously because of their inability to induce ferric- mainly expressed in the epidermis, exodermis, and inner chelate reductase and their low capacity to synthesize MAs. layer of the cortex in Fe-deficient roots, as well as in Therefore, we hypothesized that introducing ferric-chelate companion cells of shoots. Moreover, an analysis using a reductase into rice would enhance Fe deficiency tolerance, positron-emitting tracer imaging system (PETIS) revealed creating a complete strategy I system in addition to the rice 2+ that rice is able to take up both Fe(III)–DMA and Fe . endogenous strategy II. Thus, rice plants possess a system other than the MAs- For functional expression in plants, we modified and based strategy II for Fe uptake [16]. In contrast to their completely reconstructed the yeast ferric reductase gene, ferrous-transporting ability, Fe-deficient rice roots do not FRE1,to produce refre1 (reconstructed FRE1;[47]). Since induce ferric-chelate reductase activity [16], which is a ferric-chelate reductase activity is inhibited by high pH, we hallmark of the strategy I response. then screened reductases with improved enzymatic activity at Rice (2008) 1:144–153 149 149 own promoter but observed no transgene mRNA expres- sion. In Arabidopsis, expression of ferric-chelate reductase FRO2 and ferrous transporter IRT1 is similarly and coordinately regulated at transcriptional and posttranscrip- tional levels [4, 66]. Therefore, we chose the promoter of the rice ferrous transporter gene OsIRT1 to drive the exogenous ferric-chelate reductase gene refre1/372 [17]. Transgenic rice plants with the introduced OsIRT1 promoter connected to refre1/372, successfully induced ferric-chelate reductase expression and activity in Fe- deficient roots, leading to higher Fe uptake than by vector controls, as revealed by a PETIS analysis. The transformants exhibited enhanced tolerance to low Fe availability in both hydroponic culture and calcareous soil (Fig. 4a). When grown in calcareous soil until harvest, the transformants had a 7.9 times higher grain yield than vector controls (Fig. 4b,c; [17]), demonstrating that creating a complete strategy I system in rice by enhancing ferric-chelate reductase activity is extremely effective in improving Fe deficiency tolerance. Manipulating transcription factors regulating the Fe deficiency response The above studies have shown that introduction of only a single or a few genes is effective in conferring Fe deficiency tolerance if appropriate promoter(s) and gene(s) are utilized. However, further enhancement of Fe availability might be achieved by engineering multiple genes in a coordinated manner. The genetic enhancement of a wide range of related genes requires manipulation of basal regulatory systems, including transcription factors. Therefore, we also aimed to clarify the regulation mechanism controlling the Fe deficiency response in graminaceous plants. Under low Fe availability, graminaceous plants induce various genes, many of which are involved in Fe acquisition and utilization [2, 26, 28, 36, 41]. Despite the number of Fe-deficiency-inducible genes isolated, little is known about the regulation of gene expression in response Fig. 3 Visual comparison between gIDS3 (left) and NT (right) from to Fe deficiency. Therefore, we applied a stepwise strategy 16 to 50 DAT, as illustrated in Fig. 2 but photographed from the to identify the molecular components regulating the opposite direction. a 16 DAT, b 30 DAT, c 42 DAT, and d 50 DAT. Original figure: Suzuki et al. [57]. expression of Fe-deficiency-responsive genes: establish- ment of a promoter assay system, identification of cis- high pH [48]. Through screening of randomly mutagenized acting elements, and identification of trans-acting factors refre1 derivatives, we obtained a variant designated refre1/ that interact with the elements. 372, whose encoding protein maintained strong reductase We introduced the promoter region of the barley IDS2 gene activity at pH 8–9. Transgenic tobacco plants with the connected to the GUS gene as a reporter into tobacco plants introduced refre1/372 under control of the 35S promoter [73]. Transgenic tobacco plants induced GUS expression in exhibited enhanced ferric-chelate reductase activity in roots Fe-deficient roots, basically reflecting the regulation pattern and better growth when grown in calcareous soils [48]. in native barley. Precise deletion and mutation analyses using Another concern in relation to the introduction of numerous lines of transgenic tobacco identified the novel Fe- exogenous reductase genes into rice was the choice of an deficiency-responsive cis-acting elements, iron-deficiency- appropriate promoter. Vasconcelos et al. [64] introduced the responsive element 1 and 2 (IDE1 and IDE2; [24]); these are Arabidopsis ferric-chelate reductase gene FRO2 with its the first identified elements related to micronutrient deficien- 150 Rice (2008) 1:144–153 (a) (b) (c) TF V V TF V Calcareous Bonsol TF V Fig. 4 Tolerance to Fe deficiency in transformants with the (normal cultivated soil). b Transformant (TF, left) and vector control introduced OsIRT1 promoter refre1/372 grown in calcareous soil. a (V, right) after 17 weeks of growth in calcareous soil. c Grain yield Transformants (TF, left) and vector controls (V, center) after 4 weeks after cultivation for 17 weeks in calcareous soil. Original figure: of growth in a calcareous soil; vector controls (V, right) in bonsol Ishimaru et al. [17]. cies in plants. IDE1 and IDE2 synergistically induce Fe- In an attempt to improve Fe deficiency tolerance by deficiency-responsive expression in tobacco roots. When modulating IDEF1 expression, we introduced IDEF1 introduced into rice, the pair IDE1 and IDE2 is able to cDNA fused to either the constitutive 35S promoter or the induce Fe-deficiency-responsive expression both in roots and Fe-deficiency-inducible IDS2 promoter. Transgenic rice leaves [25]. Sequences similar to IDE1 or IDE2 were found in various Fe-deficiency-inducible promoters of barley, rice, Fe-deficiency signal tobacco, and Arabidopsis [6, 24, 26]. This suggests that gene regulation mechanisms involving IDEs are not only con- served among graminaceous (strategy II) plants but are also IDEF1 IDEF2 IDEF2 functional in nongraminaceous (strategy I) plant species. Fe-deficiency- Next, we searched for transcription factors that interact IDE2-like responsive genes with IDEs. Very recently, we successfully identified two rice transcription factors, IDE-binding factor 1 (IDEF1) and Fe-deficiency- IDE1-like IDE1-like IDEF2, which specifically bind to IDE1 and IDE2, respec- OsIRO2 responsive genes tively [27, 45]. IDEF1 and IDEF2 belong to uncharacterized OsIRO2 branches of plant-specific transcription factor families ABI3/ VP1 and NAC, respectively, and exhibit novel properties of Fe-deficiency- CACGTGG sequence recognition. IDEF1 recognizes the CATGC se- responsive genes quence within IDE1, whereas IDEF2 predominantly recog- nizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the AP2-domain CACGTGG OsNAC4 CACGTGG transcription factor core binding site. Both IDEF1 and IDEF2 transcripts are NAC4 AP2 constitutively expressed in rice roots and leaves. Fe-deficiency- unknown Fe-deficiency- unknown responsive genes responsive genes element element Fig. 6 Proposed regulatory network for the induction of Fe- deficiency-responsive genes via IDEF1, IDEF2, and OsIRO2. Under Fe-deficient conditions, IDEF1 and IDEF2 transactivate the expres- sion of Fe-deficiency-responsive genes by binding to the IDE1-like and IDE2-like elements, respectively [27, 45]. OsIRO2, which is induced by Fe deficiency and is positively regulated by IDEF1, binds to the CACGTGG element to activate another subset of Fe-deficiency- responsive genes, including two transcription factor genes: OsNAC4 NT and the AP2 domain-containing gene. These transcription factors may Fig. 5 Tolerance to Fe deficiency in seedlings with the introduced then regulate Fe-deficiency-responsive genes lacking IDEs and IDS2 promoter IDEF1 germinated in a calcareous soil (lines 9, 12, and CACGTGG in their promoter regions [44]. The induced expression 13) compared to nontransformants (NT) 17 days after sowing. Original of IDEF1 in transgenic rice plants would effectively strengthen the figure: Kobayashi et al. [27]. overall regulatory pathway to confer tolerance to Fe deficiency. Line9 Line12 Line13 Grain weight / plant (g) Rice (2008) 1:144–153 151 151 seedlings with the introduced 35S promoter IDEF1 showed (Figs. 2, 3). Availability of Fe in rice fields is severely severe growth retardation during early growth, while those affected by soil type and redox potential, as well as carrying the IDS2 promoter IDEF1 showed healthy growth. numerous other environmental factors. An elaborate combi- Notably, the IDS2 promoter IDEF1 transformants exhibited nation of previously adopted or new strategies will be needed slower progression of leaf chlorosis in Fe-free hydroponic to produce rice lines with even more tolerance to low Fe culture and also showed better growth when germinated on availability in problematic soils without loss of favorable calcareous soil (Fig. 5;[27]). agricultural traits. Manipulation of DMAS genes, which were To clarify the molecular mechanisms that regulate Fe recently cloned and thus have not been genetically modified, acquisition, we also characterized Fe-deficiency-induced in the steps of MAs biosynthesis [2] would be of special transcription factors. Microarray analyses revealed the upre- interest. In addition, further clarification of the underlying gulation of several transcription factor genes in barley and rice mechanisms involved in Fe homeostasis is extremely [41, 43], among which a bHLH transcription factor gene, important, including expressional regulation, secretion of IRO2, is of particular interest because of its pronounced MAs, and metal translocation inside the plants. transcriptional upregulation by Fe deficiency in shoots and Understanding metal homeostasis also paves the way to roots of barley and rice [43]. The core sequence for OsIRO2 fortifying rice grains with Fe and Zn. Previous efforts to binding was determined to be CACGTGG [43]. enhance Fe in grains were performed by overexpressing We produced transgenic rice plants with enhanced or ferritin, a common Fe storage protein in rice grain [8, 51, 63]. repressed OsIRO2 expression by introducing the 35S- Our field trials revealed that the gIDS3 line is capable of OsIRO2 cassette or using the RNA interference technique accumulating more Fe in grains in both calcareous and [44]. In Fe-deficient hydroponic culture, OsIRO2-over- Andosol paddy fields [35, 57]. Production and characteriza- expressing lines showed enhanced MAs secretion and tion of transgenic rice lines with introduced biosynthetic genes slightly better growth compared to nontransformants, for MAs and ferritin genes in combination to enhance both Fe whereas OsIRO2-repressed lines resulted in lower MAs uptake and storage is in progress (Masuda et al. unpublished). secretion and hypersensitivity to Fe deficiency. Microarray Other advanced applications of our knowledge on Fe nutrition and Northern blot analyses revealed that the expression include the production of novel antihypertensive substrates. level of OsIRO2 is positively related to various Fe- NA, the precursor of MAs, inhibits angiotensin-I-converting deficiency-induced genes in roots, including those respon- enzyme in humans and consequently reduces high blood sible for MAs biosynthesis (OsNAS1, OsNAS2, OsNAAT1, pressure [22, 53]. We produced a yeast strain that highly OsDMAS1, and various genes involved in the methionine accumulates NA by introducing the Arabidopsis NAS gene, cycle) and Fe(III)–MAs uptake (OsYSL15). OsIRO2 also AtNAS2 [69]. Production and selection of rice lines with affects the expression of some Fe-deficiency-inducible elevated levels of NA in grain by introducing the HvNAS1 transcription factor genes that possess OsIRO2-binding gene under the control of a seed-specific promoter of the rice core sequences in their promoter regions [44]. Importantly, glutelin gene is now under way [68]. OsIRO2 itself possesses multiple IDEF1-binding core Public acceptance of genetically modified organisms is sequences in its promoter region and is positively regulated still low. As a technical way to improve public acceptance, by IDEF1 [27]. Based on these results, a sequential link in we modified the “marker-free vector” of the Cre/loxP DNA the Fe deficiency response involving IDEF1, IDEF2, excision system [74] to construct a high-capacity binary OsIRO2, and its downstream Fe-deficiency-inducible tran- vector for the transformation of rice, from which the scription factors is proposed (Fig. 6;[27, 44, 45]). sequence sandwiched between two loxP sites (including In contrast to growth retardation observed in the 35S the selectable marker) can be removed by 17β-estradiol promoter IDEF1 transformants, the 35S promoter OsIRO2 administration [68]. Many other approaches may aid public transformants were healthy, not exhibiting any obvious acceptance of transgenic plants, which have such high defects. These differences in phenotypes of the trans- potential to increase food production, preserve the environ- formants are thought to be related to the distinct nature of ment, and improve human health. the two transcription factors. References Future perspectives 1. Akiyama K, Nakamura S, Suzuki T, Wisniewska I, Sasaki N, Kawasaki S. Development of a system of rice transformation with We produced various lines of transgenic rice plants with long genome inserts for their functional analysis for positional enhanced tolerance to low Fe availability. Among these, cloning. Plant Cell Physiol Supplement 1997;38:s94. tolerance of three selected lines (gNAS1, gIDS3, and gNAS1– 2. Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori gNAAT) in calcareous soil was demonstrated in field trials S, et al. Cloning and characterization of deoxymugineic acid 152 Rice (2008) 1:144–153 synthase genes from graminaceous plants. J Biol Chem. 22. Kinoshita E, Yamakoshi J, Kikuchi M. Purification and identifi- 2006;43:32395–402. cation of an angiotensin I-converting enzyme inhibitor from soy 3. Bughio N, Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S. sauce. Biosci Biotechnol Biochem. 1993;57:1107–10. Cloning an iron-regulated metal transporter from rice. J Exp Bot 23. Kobayashi T, Nakanishi H, Takahashi M, Kawasaki S, Nishizawa 2002;53:1677–82. NK, Mori S. In vivo evidence that Ids3 from Hordeum vulgare 4. Connolly EL, Campbell NH, Grotz N, Prichard CL, Guerinot ML. encodes a dioxygenase that converts 2’-deoxymugineic acid to Overexpression of the FRO2 ferric chelate reductase confers mugineic acid in transgenic rice. Planta 2001;212:864–71. tolerance to growth on low iron and uncovers posttranscriptional 24. Kobayashi T, Nakayama Y, Itai RN, Nakanishi H, Yoshihara T, control. Plant Physiol. 2003;133:1102–10. Mori S, et al. Identification of novel cis-acting elements, IDE1 and 5. Curie C, Panavience Z, Loulergue C, Dellaporta SL, Briat JF, IDE2, of the barley IDS2 gene promoter conferring iron- Walker EL. Maize yellow stripe1 encodes a membrane protein deficiency-inducible, root-specific expression in heterogeneous directly involved in Fe(III) uptake. Nature 2001;409:346–9. tobacco plants. Plant J. 2003;36:780–93. 6. Ducos E, Fraysse ÅS, Boutry M. NtPDR3, an iron-deficiency 25. Kobayashi T, Nakayama Y, Takahashi M, Inoue H, Nakanishi H, inducible ABC transporter in Nicotiana tabacum. FEBS Lett. Yoshihara T, et al. Construction of artificial promoters highly 2005;579:6791–5. responsive to iron deficiency. Soil Sci Plant Nutr. 2004;50:1167– 7. Eide D, Broderius M, Fett J, Guerinot ML. A novel iron-regulated 75. metal transporter from plants identified by functional expression 26. Kobayashi T, Suzuki M, Inoue H, Itai RN, Takahashi M, in yeast. Proc Natl Acad Sci USA. 1996;93:5624–8. Nakanishi H, et al. Expression of iron-acquisition-related genes 8. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F. Iron in iron-deficient rice is co-ordinately induced by partially fortification of rice seed by the soybean ferritin gene. Nat conserved iron-deficiency-responsive elements. J Exp Bot. Biotechnol 1999;17:282–6. 2005;56:1305–16. 9. Hell R, Stephan UW. Iron uptake, trafficking and homeostasis in 27. Kobayashi T, Ogo Y, Itai RN, Nakanishi H, Takahashi M, Mori S, plants. Planta 2003;216:541–51. et al. The novel transcription factor IDEF1 regulates the response 10. Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation to and tolerance of iron deficiency in plants. Proc Natl Acad Sci of rice (Oryza sativa L.) mediated by Agrobacterium and USA. 2007;104:19150–5. sequence analysis of the boundaries of the T-DNA. Plant J. 28. Kobayashi T, Nishizawa NK. Regulation of iron and zinc uptake 1994;6:271–82. and translocation in rice. In: Hirano HY, Hirai A, Sano Y, Sasaki 11. Higuchi K, Kanazawa K, Nishizawa NK, Chino M, Mori S. T, editors. Biotechnology in agriculture and forestry 62. Rice Purification and characterization of nicotianamine synthase from biology in the genomics era. Meppel: Springer; 2008. p. 321–35. Fe deficient barley roots. Plant Soil. 1994;165:173–9. 29. Ling HQ, Koch G, Bäumlein H, Ganal MW. Map-based cloning 12. Higuchi K, Kanazawa K, Nishizawa NK, Mori S. The role of of chloronerva, a gene involved in iron uptake of higher plants nicotianamine synthase in response to Fe nutrition status in encoding nicotianamine synthase. Proc Natl Acad Sci USA. Gramineae. Plant Soil. 1996;178:171–7. 1999;96:7098–710. 13. Higuchi K, Suzuki K, Nakanishi H, Yamaguchi H, Nishizawa NK, 30. Ma JF, Nomoto K. Two related biosynthetic pathways of mugineic Mori S. Cloning of nicotianamine synthase genes, novel genes acids in gramineous plants. Plant Physiol. 1993;102:373–8. involved in the biosynthesis of phytosiderophores. Plant Physiol 31. Ma JF, Shinada T, Matsuda C, Nomoto K. Biosynthesis of 1999;119:471–9. phytosiderophores, mugineic acids, associated with methionine 14. Higuchi K, Watanabe S, Takahashi M, Kawasaki S, Nakanishi H, cycling. J Biol Chem. 1995;270:16549–54. Nishizawa NK, et al. Nicotianamine synthase gene expression 32. Ma JF, Taketa S, Chang YC, Iwashita T, Matsumoto H, Takeda K, differs in barley and rice under Fe-deficient conditions. Plant J. et al. Genes controlling hydroxylations of phytosiderophores are 2001;25:159–67. located on different chromosomes in barley (Hordeum vulgare L.). 15. Inoue H, Takahashi M, Kobayashi T, Suzuki M, Nakanishi H, Mori S, Planta 1999;207:590–6. et al. Identification and localisation of the rice nicotianamine 33. Marschner H. Mineral nutrition of higher plants, 2nd edn. aminotransferase gene OsNAAT1 expression suggests the site of London: Academic; 1995. phytosiderophore synthesis in rice. Plant Mol Biol. 2008;66:193–203. 34. Marschner H, Römheld V, Kissel M. Different strategies in higher 16. Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, plants in mobilization and uptake of iron. J Plant Nutr. 3+ Kobayashi T, et al. Rice plants take up iron as an Fe - 1986;9:695–713. 2+ phytosiderophore and as Fe . Plant J. 2006;45:335–46. 35. Masuda H, Suzuki M, Morikawa KC, Kobayashi T, Nakanishi H, 17. Ishimaru Y, Kim S, Tsukamoto T, Oki H, Kobayashi T, Watanabe Takahashi M, et al. Increase in iron and zinc concentrations in rice S, et al. Mutational reconstructed ferric chelate reductase confers grains via the introduction of barley genes involved in phytosi- enhanced tolerance in rice to iron deficiency in calcareous soil. derophore synthesis. Rice 2008. doi:10.1007/s12284-008-9007-6. Proc Natl Acad Sci USA. 2007;104:7373–8. 36. Mori S. Iron acquisition by plants. Curr Opin Plant Biol. 18. Itai R, Suzuki K, Yamaguchi H, Nakanishi H, Nishizawa NK, 1999;2:250–3. Yoshimura E, et al. Induced activity of adenine phosphoribosyl- 37. Mori S, Nishizawa N. Methionine as a dominant precursor of transferase (APRT) in Fe-deficient barley roots: a possible role for phytosiderophores in Graminaceae plants. Plant Cell Physiol phytosiderophore production. J Exp Bot. 2000;51:1179–88. 1987;28:1081–92. 19. Kanazawa K, Higuchi K, Nishizawa NK, Fushiya S, Chino M, 38. Morikawa CK, Saigusa M, Nakanishi H, Nishizawa NK, Mori S. Nicotianamine aminotransferase activities are correlated Hasegawa K, Mori S. Co-situs application of controlled-release to the phytosiderophore secretions under Fe-deficient conditions fertilizers to alleviate iron chlorosis of paddy rice grown in in Gramineae. J Exp Bot. 1994;45:1903–6. calcareous soil. Soil Sci Plant Nutr. 2004;50:1013–21. 20. Kanazawa K, Higuchi K, Nishizawa NK, Fushiya S, Mori S. 39. Nakanishi H, Okumura N, Umehara Y, Nishizawa NK, Chino M, Detection of two distinct isozymes of nicotianamine aminotrans- Mori S. Expression of a gene specific for iron deficiency (Ids3) ferase in Fe-deficient barley roots. J Exp Bot. 1995;46:1241–4. in the roots of Hordeum vulgare. Plant Cell Physiol 1993;34:401– 21. Kawai S, Takagi S, Sato Y. Mugineic acid-family phytosider- 10. ophores in root-secretions of barley, corn and sorghum varieties. J 40. Nakanishi H, Yamaguchi H, Sasakuma T, Nishizawa NK, Mori S. Plant Nutr. 1988;11:633–42. Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are Rice (2008) 1:144–153 153 153 involved in the biosynthesis of mugineic acid family phytosider- increased tolerance to low iron availability in a calcareous paddy ophores. Plant Mol Biol. 2000;44:199–207. soil. Soil Sci Plant Nutr. 2008;54:77–85. 41. Negishi T, Nakanishi H, Yazaki J, Kishimoto N, Fujii F, Shimbo 58. Suzuki M, Tsukamoto T, Inoue H, Watanabe S, Matsuhashi S, K, et al. cDNA microarray analysis of gene expression during Fe- Takahashi M, et al. Deoxymugineic acid increases Zn transloca- deficiency stress in barley suggests that polar transport of vesicles tion in Zn-deficient rice plants. Plant Mol Biol 2008;66:609–17. is implicated in phytosiderophore secretion in Fe-deficient barley 59. Takagi S. Naturally occurring iron-chelating compounds in oat- roots. Plant J. 2002;30:83–94. and rice-root washings. Soil Sci Plant Nutr. 1976;22:423–33. 42. Noma M, Noguchi M. Occurrence of nicotianamine in higher 60. Takahashi M, Yamaguchi H, Nakanishi H, Shioiri T, Nishizawa plants. Phytochemistry 1976;15:1701–2. NK, Mori S. Cloning two genes for nicotianamine aminotransfer- 43. Ogo Y, Itai RN, Nakanishi H, Inoue H, Kobayashi T, Suzuki M, et ase, a critical enzyme in iron acquisition (strategy II) in gramina- al. Isolation and characterization of IRO2, a novel iron-regulated ceous plants. Plant Physiol. 1999;121:947–56. bHLH transcription factor in graminaceous plants. J Exp Bot. 61. Takahashi M, Nakanishi H, Kawasaki S, Nishizawa NK, Mori S. 2006;57:2867–78. Enhanced tolerance of rice to low iron availability in alkaline soils 44. Ogo Y, Itai RN, Nakanishi H, Kobayashi T, Takahashi M, Mori S, using barley nicotianamine aminotransferase genes. Nat Biotech- et al. The rice bHLH protein OsIRO2 is an essential regulator of nol. 2001;19:466–9. the genes involved in Fe uptake under Fe-deficient conditions. 62. Takahashi M, Terada Y, Nakai I, Nakanishi H, Yoshimura E, Mori Plant J. 2007;51:366–77. S, et al. Role of nicotianamine in the intracellular delivery of 45. Ogo Y, Kobayashi T, Itai RN, Nakanishi H, Kakei Y, Takahashi metals and plant reproductive development. Plant Cell M, et al. A novel NAC transcription factor IDEF2 that recognizes 2003;15:1263–80. the iron deficiency-responsive element 2 regulates the genes 63. Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L, involved in iron homeostasis in plants. J Biol Chem 2008; Krishnan S, et al. Enhanced iron and zinc accumulation in 283:13407–17. transgenic rice with the ferritin gene. Plant Sci. 2003;164:371–8. 46. Ohata T, Kanazawa K, Mihashi S, Nishizawa NK, Fushiya S, 64. Vasconcelos M, Musetti V, Li CM, Datta SK, Grusak MA. Nozoe S, et al. Biosynthetic pathway of phytosiderophores in Functional analysis of transgenic rice (Oryza sativa L.) trans- iron-deficient Graminaceous plants. Development of an assay formed with an Arabidopsis thaliana ferric reductase (AtFRO2). system for the detection of nicotianamine aminotransferase Soil Sci Plant Nutr. 2004;50:1151–7. activity. Soil Sci Plant Nutr. 1993;39:745–9. 65. Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, 47. Oki H, Yamaguchi H, Nakanishi H, Mori S. Introduction of the Briat JF, et al. IRT1, an Arabidopsis transporter essential for iron reconstructed yeast ferric reductase gene, refre1, into tobacco. uptake from the soil and plant growth. Plant Cell 2002;14:1223– Plant Soil 1999;215:211–20. 33. 48. Oki H, Kim S, Nakanishi H, Takahashi M, Yamaguchi H, Mori S, 66. Vert G, Briat JF, Curie C. Dual regulation of the Arabidopsis high- et al. Directed evolution of yeast ferric reductase to produce plants affinity root iron uptake system by local and long-distance signals. with tolerance to iron deficiency in alkaline soils. Soil Sci Plant Plant Physiol 2003;132:796–804. Nutr. 2004;50:1159–65. 67. von Wirén N, Khodr H, Hider RC. Hydroxylated phytosider- 49. Okumura N, Nishizawa NK, Umehara Y, Mori S. An iron ophore species possess an enhanced chelate stability and affinity deficiency-specific cDNA from barley roots having two homolo- for iron(III). Plant Physiol. 2000;124:1149–57. gous cysteine-rich MT domains. Plant Mol Biol 1991;17:531–3. 68. Wada Y, Ishimaru Y, Takahashi M, Nakanishi H, Mori S, 50. Okumura N, Nishizawa NK, Umehara Y, Ohata T, Nakanishi H, Nishizawa NK. Engineering of hypotensive rice contain high Yamaguchi H, et al. A dioxygenase gene (Ids2) expressed under amounts of nicotianamine for functional foods. In: Li CJ, et al., iron deficiency conditions in the roots of Hordeum vulgare. Plant editors. Plant nutrition for food security, human health and Mol Biol 1994;25:705–19. environmental protection. Beijing: Tsinghua University Press; 51. Qu LQ, Yoshihara T, Ooyama A, Goto F, Takaiwa F. Iron 2005. p. 424–5. accumulation does not parallel the high expression level of ferritin 69. Wada Y, Kobayashi T, Takahashi M, Nakanishi H, Mori S, in transgenic rice seeds. Planta 2005;222:225–33. Nishizawa NK. Metabolic engineering of Saccharomyces cerevi- 52. Römheld V, Marschner H. Evidence for a specific uptake system siae producing nicotianamine: potential for industrial biosynthesis for iron phytosiderophores in roots of grasses. Plant Physiol. of a novel antihypertensive substrate. Biosci Biotechnol Biochem. 1986;80:175–80. 2006;70:1408–15. 53. Shimizu E, Hayashi A, Takahashi R, Aoyagi Y, Murakami T, 70. Yamaguchi H, Nakanishi H, Nishizawa NK, Mori S. Induction of Kimoto K. Effects of angiotensin I-converting enzyme inhibitor the IDI1 gene in Fe-deficient barley roots: a gene encoding from ashitaba (Angelica keiskei) on blood pressure of spontane- putative enzyme that catalyses the methionine salvage pathway for ously hypertensive rats. J Nutr Sci Vitaminol. 1999;45:375–83. phytosiderophore production. Soil Sci Plant Nutr. 2000;46:1–9. 54. Shojima S, Nishizawa NK, Fushiya S, Nozoe S, Irifune T, Mori S. 71. Yamaguchi H, Nakanishi H, Nishizawa NK, Mori S. Isolation and Biosynthesis of phytosiderophores: in vitro biosynthesis of 2’- characterization of IDI2, a new Fe-deficiency induced cDNA from deoxymugineic acid from L-methionine and nicotianamine. Plant barley roots, which encodes a protein related to the a subunit of Physiol. 1990;93:1497–503. eukaryotic initiation factor 2B (eIF2B a). J Exp Bot. 55. Suzuki K, Itai R, Suzuki K, Nakanishi H, Nishizawa NK, 2000;51:2001–7. Yoshimura E, et al. Formate dehydrogenase, an enzyme of 72. Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S. IDI7, a new anaerobic metabolism, is induced by Fe-deficiency in barley iron-regulated ABC transporter from barley roots, localizes to the roots. Plant Physiol. 1998;116:725–32. tonoplast. J Exp Bot. 2002;53:727–235. 56. Suzuki M, Takahashi M, Tsukamoto T, Watanabe S, Matsuhashi 73. Yoshihara T, Kobayashi T, Goto F, Masuda T, Higuchi K, S, Yazaki J, et al. Biosynthesis and secretion of mugineic acid Nakanishi H, et al. Regulation of the iron-deficiency responsive family phytosiderophores in zinc-deficient barley. Plant J. gene, Ids2, of barley in tobacco. Plant Biotechnol. 2003;20:33–41. 2006;48:85–97. 74. Zuo J, Niu QW, Møller SG, Chua NH. Chemical-regulated, site- 57. Suzuki M, Morikawa KC, Nakanishi H, Takahashi M, Saigusa M, specific DNA excision in transgenic plants. Nat Biotechnol. Mori S, et al. Transgenic rice lines that include barley genes have 2001;19:157–61. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rice Springer Journals

Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency

Loading next page...
 
/lp/springer-journals/generation-and-field-trials-of-transgenic-rice-tolerant-to-iron-YnHtrdceH1

References (79)

Publisher
Springer Journals
Copyright
Copyright © Springer Science + Business Media, LLC 2008
Subject
Life Sciences; Plant Sciences; Plant Genetics & Genomics; Plant Breeding/Biotechnology; Agriculture; Plant Ecology
ISSN
1939-8425
eISSN
1939-8433
DOI
10.1007/s12284-008-9011-x
Publisher site
See Article on Publisher Site

Abstract

Rice (2008) 1:144–153 DOI 10.1007/s12284-008-9011-x REVIEW Generation and Field Trials of Transgenic Rice Tolerant to Iron Deficiency Takanori Kobayashi & Hiromi Nakanishi & Michiko Takahashi & Satoshi Mori & Naoko K. Nishizawa Received: 5 March 2008 /Accepted: 1 August 2008 /Published online: 24 September 2008 Springer Science + Business Media, LLC 2008 Abstract Iron deficiency is a major cause of reduced crop Introduction yields worldwide, particularly in calcareous soils. Unlike barley, rice is highly susceptible to iron deficiency because Iron (Fe) is essential for most living organisms including of a low capacity to secrete mugineic acid family plants. Although abundant in mineral soils, Fe is sparingly phytosiderophores (MAs), which are iron chelators secreted soluble under aerobic conditions at high soil pH, especially by graminaceous plants. We present an approach toward the in calcareous soils, which account for about 30% of the generation along with field trials of transgenic rice lines world’s cultivated soils. Fe deficiency is a widespread exhibiting increased tolerance to iron deficiency. Cloning agricultural problem that reduces plant growth and crop barley genes that encode biosynthetic enzymes for MAs yields [33, 36]. To take up and utilize Fe from the enabled us to produce transgenic rice plants by introducing rhizosphere, higher plants have evolved two major strate- barley MAs biosynthesis-related genes. We tested three gies [34]: reduction (strategy I) and chelation (strategy II). transgenic lines possessing barley genomic fragments The strategy I mechanism, utilized by nongraminaceous responsible for MAs biosynthesis in a paddy field exper- plants, includes induction of ferric-chelate reductase to iment on calcareous soil, which revealed tolerance of these reduce Fe at the root surface to the more soluble ferrous lines to low iron availability. We also applied new form and transport the ferrous ions generated across the approaches to generate iron-deficiency-tolerant rice lines, root plasma membrane. In contrast, the strategy II mecha- including the introduction of an engineered ferric-chelate nism, which is specific to graminaceous plants, is mediated reductase gene and manipulation of transcription factor by natural Fe chelators, the mugineic acid family phytosi- genes regulating the iron deficiency response. derophores (MAs). Graminaceous plants synthesize and secrete MAs from their roots to solubilize Fe(III) in the . . Keywords Iron deficiency Field trial rhizosphere [59], and the resulting Fe(III)–MAs complexes Mugineic acid family phytosiderophores are taken up by roots through a specific transporter in the Transgenic rice plants plasma membrane [5, 59]. In calcareous soils, the strategy II mechanism is more efficient than that of strategy I [33]. Tolerance to Fe deficiency is divergent among graminaceous plants and is thought to be dependent on the amount and kinds of MAs that they secrete. Rice, sorghum, and maize secrete only : : : : small amounts of 2’-deoxymugineic acid (DMA) among T. Kobayashi H. Nakanishi M. Takahashi S. Mori N. K. Nishizawa (*) the MAs and thus are susceptible to low Fe availability. In Graduate School of Agricultural and Life Sciences, contrast, barley secretes large amounts of MAs, including The University of Tokyo, mugineic acid (MA), 3-epihydroxy-2’-deoxymugineic acid 1-1-1 Yayoi, Bunkyo-ku, (epiHDMA), and 3-epihydroxymugineic acid (epiHMA), in Tokyo 113-8657, Japan e-mail: annaoko@mail.ecc.u-tokyo.ac.jp addition to DMA, under Fe deficiency; therefore, it is more Rice (2008) 1:144–153 145 145 tolerant to Fe deficiency than other graminaceous plants transport of various micronutrients including Fe and zinc [32, 37, 52, 59]. (Zn) [9, 62]. NA aminotransferase (NAAT) catalyzes the We therefore hypothesized that introducing barley genes first step specific to graminaceous plants: transamination of responsible for MAs biosynthesis into rice would lead to NA to produce the 3’’-oxo intermediate [20, 54]. DMA enhanced MAs production and tolerance in calcareous soils. synthase (DMAS) subsequently reduces the 3’’-oxo form to We successfully produced various transgenic rice lines DMA [2]. All MAs share their biosynthetic pathway from showing enhanced tolerance to low Fe availability by methionine to DMA, which is then hydroxylated to form introducing barley MAs biosynthesis genes. Using selected other MAs in some species, including barley. lines, we carried out field trials on calcareous soil that revealed Attempts to isolate the genes responsible for MAs tolerance of these lines to low Fe availability. We also biosynthesis have included “direct” approaches via enzyme produced Fe-deficiency-tolerant rice lines using two other purification and “indirect” approaches through screening the strategies: introduction of a reconstructed ferric-chelate genes and proteins specifically induced in Fe-deficient roots. reductase gene and manipulation of transcription factor genes The former approach was applied to NAS and NAAT. NAS controlling the expression of Fe-deficiency-induced genes. genes were first isolated from barley (HvNAS1–7)through Future perspectives on generating further favorable and the establishment of a NAS activity assay [11] and enzyme commonly acceptable transformants are described. purification from Fe-deficient barley roots [13]. Two barley NAAT genes, HvNAAT-A and HvNAAT-B,were also cloned through the establishment of an enzyme activity assay [46] Generation of Fe-deficiency-tolerant transgenic rice and enzyme purification [20, 60]. Expression of HvNAS1, by introducing barley MAs biosynthesis genes HvNAAT-A, and HvNAAT-B is strongly induced by Fe deficiency and occurs almost exclusively in the roots [13, Identification of genes responsible for MAs biosynthesis 60], suggesting direct involvement in MAs biosynthesis for the acquisition of Fe from the rhizosphere. Detection of NAS The biosynthetic pathway of MAs (Fig. 1) has been and NAAT enzyme activities in Fe-deficient roots of various identified through extensive biochemical and physiological graminaceous species revealed that NAS and NAAT activ- studies [21, 30, 32, 37, 54]. Methionine is the precursor of ities are positively correlated with both the amounts of MAs MAs [37] and is adenosylated by S-adenosylmethionine secreted and Fe-deficiency tolerance [12, 19]. (SAM) synthetase. Nicotianamine synthase (NAS) cata- In the latter “indirect” approach, the differential hybrid- lyzes the trimerization of SAM to nicotianamine (NA) [11]. ization method was applied with mRNA from Fe-deficient All higher plants, including nongraminaceous plants, have and Fe-sufficient barley roots. We cloned iron-deficiency- the biosynthetic pathway to synthesize NA [29, 42], which specific (IDS) genes specifically expressed in Fe-deficient serves as a common metal chelator involved in the internal barley roots [39, 49, 50]. Among these, IDS2 and IDS3 are Fig. 1 Biosynthesis pathway of MAs mugineic acid family phytosi- COOH COOH COOH COOH COOH COOH derophores (MAs). SAMS, S- DMAS N NH O adenosylmethionine synthetase; N NH OH NAS, nicotianamine synthase; 3''-oxo form NAAT, nicotianamine amino- DMA transferase; DMAS, deoxymugi- NAAT IDS2 neic acid synthase; IDS2, iron- IDS3 deficiency-specific clone no. 2; COOH COOH COOH IDS3, iron-deficiency-specific COOH COOH COOH COOH COOH COOH N NH NH clone no. 3; DMA,2’-deoxy- N NH OH mugineic acid; MA, mugineic nicotianamine(NA) HO N NH OH OH acid; HMA, 3-hydroxymugineic epiHDMA MA acid; epiHDMA, 3-epihydroxy- NAS 2’-deoxymugineic acid; epi- IDS2 HMA, 3-epihydroxymugineic S-adenosyl- IDS3 acid. L-methionine (SAM) COOH COOH COOH COOH COOH COOH SAMS N NH OH HO N NH OH HO COOH OH OH H C SNH epiHMA HMA L-methionine 146 Rice (2008) 1:144–153 homologous to 2-oxoglutarate-dependent dioxygenases, which the HvNAAT-A and HvNAAT-B genes are tandemly suggesting their possible involvement in the hydroxylation located [61], designated “gNAAT”; (c) a 20-kb genome of MAs. By interspecies correlation between the expression fragment of the IDS3 gene [23], designated “gIDS3”;(d) a of IDS2–IDS3 and the capacity to secrete hydroxylated 7.6-kb genome fragment of HvNAS1 plus an 11-kb genome MAs, we deduced that IDS3 is the enzyme that hydrox- fragment containing the HvNAAT-A and HvNAAT-B genes, ylates the C-2’ positions of DMA and epiHDMA, while designated “gNAS1-gNAAT”;(e) an HvAPT cDNA frag- IDS2 hydroxylates the C-3 positions of DMA and MA ment fused downstream of the 2.2-kb IDS3 promoter; (f) (Fig. 1;[40]). IDS3 was further confirmed to be the “MA HvNAS1, HvNAAT-A,and HvAPT cDNA fragments each synthase” by introducing the barley IDS3 gene into rice fused downstream of the 2.2-kb IDS3 promoter; and (g) an [23]: transgenic rice plants secreted MAs in addition to IDS3 cDNA fragment fused downstream of the cauliflower DMA, while nontransformants secreted only DMA. mosaic virus 35S promoter [23]. Expression analysis We also compared proteins of Fe-sufficient and Fe- revealed that the Fe-deficiency-induced expression was deficient barley roots using two-dimensional polyacryl- strongly conferred by genome fragments of the HvNAS1, amide gel electrophoresis. Peptide sequencing of the HvNAAT-A, HvNAAT-B,or IDS3 genes [14, 23, 61], induced proteins revealed that formate dehydrogenase confirming the potency of the barley promoter elements (FDH) and adenine phosphoribosyltransferase (APRT), as included in the genome fragments to drive Fe-deficiency- well as the IDS3 protein, were induced in Fe-deficient roots induced expression in rice plants. Although the barley [55]. The corresponding genes, HvFDH and HvAPT, were promoters exhibit induction almost exclusively in Fe- subsequently cloned [18, 55]. Both FDH and APRT are deficient roots in native barley, they induced moderate thought to function in scavenging the by-products (formate expression in Fe-deficient leaves and prominent expression and adenine) that are released during the methionine cycle in Fe-deficient roots when introduced into rice. [36], thus supporting the production of MAs. Indeed, the To examine whether the transformants have enhanced methionine cycle works vigorously in roots to meet the tolerance to low Fe availability, the plants were cultured in increased demand for methionine in the synthesis of MAs pots filled with calcareous soils (pH 8.5–9.0; [61]) under [31]. We also applied a revised differential hybridization controlled conditions in a greenhouse. Of the 36 gNAAT screening, identifying iron-deficiency-induced (IDI) genes lines evaluated, ten showed remarkable tolerance to from barley roots [70–72]. IDI1 and IDI2 putatively encode calcareous soils [61]. Nontransformants exhibited reduced enzymes catalyzing steps in the methionine cycle [26, 56]. growth and severe leaf chlorosis caused by Fe deficiency, Recent application of microarray techniques reconfirmed whereas the gNAAT lines had greener and larger shoots. At the induction of the abovementioned genes involved in harvest, the gNAAT lines possessed 4.2 and 4.1 times MAs biosynthesis in Fe-deficient barley roots [41, 56]. The higher shoot dry weight and grain yield per pot than the microarray approach also resulted in cloning of DMAS nontransformants [61]. We also examined tolerance in genes from rice (OsDMAS1), barley (HvDMAS1), wheat calcareous soils for the other transformants. We found rice (TaDMAS1), and maize (ZmDMAS1). All of the lines showing some tolerance to calcareous soils from all corresponding encoding proteins were confirmed to possess transgenes (a)–(g). In these lines, increased amounts or the reductase activity to produce DMA [2]. kinds of MAs secreted were thought to have contributed to enhanced Fe availability under Fe-limiting conditions. Introduction of barley genes responsible for MAs biosynthesis into rice Field trials of Fe-deficiency-tolerant rice lines To produce transgenic rice plants with enhanced tolerance to Fe deficiency by increasing MAs production capacity, we Approval for using transgenics in field trials introduced barley HvNAS1, HvNAAT-A, HvNAAT-B,and/or IDS3 genes using either their genomic fragments or the IDS3 For the field experiment, we first selected one line from each gene promoter to confer inducibility to Fe deficiency. To of (a) to (f) described above. Prior to culture in a quarantine introduce barley genomic fragments, we utilized the field in Japan, we needed approval for Type 1 Use Regulations pBIGRZ1 vector [1], which was developed as a modified for living modified organisms from the Ministry of Agricul- binary vector capable of transferring large-size DNA frag- ture, Forestry, and Fisheries in Japan. For that purpose, we ments into the rice genome. Rice cultivar Tsukinohikari was performed evaluation tests on their priority in competition, subjected to Agrobacterium-mediated transformation [10]. possible production of harmful substances, and influence on The transformants included those introduced with (a) a 13.5- biological diversity in relation to interspecific crossing. We kb genome fragment of the HvNAS1 gene [14], designated performed the following tests on six lines of transgenic rice “gNAS1”; (b) an 11-kb genome fragment of HvNAAT in and the nontransformant: growth comparison in Andosol, Rice (2008) 1:144–153 147 147 stability of the transgene and expression beyond the first boundary adjacent to the transgenic gIDS3 rice plants generation, determination of the interspecific crossing rate (Fig. 3d), which suggests that NTs utilized MAs secreted by from transformants to nontransformants, evaluation of soil the transgenic rice. microorganism populations, evaluation of the residual effect From 16 to 42 DAT, plant height and the SPAD value (leaf of harmful compounds in postharvest soil, plowing-under color) of the three transformant lines were higher than those of effect of the dead transformants, carryover of Agrobacterium, NTs. In addition, the number of tillers per plant was higher in tests on pollen shape, fertility, and dispersal distance, and gIDS3 than in the other lines. By 42 DAT, however, all lines tests on the germination rates of the seeds and tolerance to had about 15 tillers per plant. After 42 DAT, when soil Eh fell low temperature during early growth. below 0 mV, all plant lines recovered their leaf color, and, We confirmed stable inheritance of every transgene over consequently, the SPAD value of NT plants rose up to levels at least three generations and found no harmful impacts on similar to that of transformants. The decrease in soil redox the environment in any of the abovementioned tests. potential with time is thought to have resulted in the Moreover, we detected no interspecific crossing from absorption of generated ferrous ion via the ferrous transporter transformants to nontransformants. Based on these results, OsIRT1 (“Introducing an engineered ferric-chelate reductase the transformants were approved for the quarantine field gene”;[16]). trials following the Type 1 Use Regulations. At the time of grain harvest, the number of grains, 1,000- grain weights, and the grain yield of gNAS1 were higher Field trials of the selected lines than those of the NT and other lines. Plant height and the proportion of fully matured grains showed no significant A calcareous subsoil from Toyama Prefecture containing difference among the lines. Timing of the decrease in soil fossil shells (pH ∼9.2; [38]) was used to establish a paddy redox potential might account for the relatively small field in the quarantine area of the Field Science Center of differences in grain parameters between transformants and Tohoku University (Osaki, Miyagi, Japan; 38° 44’ N; 140° NTs, despite the clearly inferior performance of NTs during 45’ E). The paddy field in the first-year experiment was early growth. Indeed, in a prior experiment, NT seedlings 7 m long, 14 m wide, and 0.5 m deep, with the external grown in the same calcareous paddy field showed severe ridges completely covered with a vinyl sheet to avoid chlorosis, and many seedlings died in the early stages contamination from the surrounding Andosol at the site. before the Eh fell below 0 mV [38]. Therefore, it is crucial The first-year experiment was conducted from April to for rice in calcareous paddy fields to survive the early October 2005, using the six transformant lines (a)–(f) and stages of growth, when enhanced MAs production greatly nontransformants (cv. Tsukinohikari). The following year, supports Fe acquisition. from April to October 2006, the second-year experiment Interestingly, the concentrations of Fe and Zn in the rice was performed using the three most promising lines: grains of gIDS3 were significantly higher than those of NTs gNAS1 (a), gIDS3 (c), and gNAS1–gNAAT (d). Experi- and the other lines, suggesting that MA synthesized by IDS3 mental procedures of the second-year experiment were contributed not only to improved Fe uptake from the soil but described by Suzuki et al. [57]. The paddy field in the also to increased translocation to the grain. MAs have been second-year experiment was 6 m long and 4 m wide, and suggested to be involved in long-distance transport of Fe and the experimental plots were arranged in a completely Zn inside rice plants [15, 58]. Since more hydroxylated MAs randomized design (Fig. 2b) including the three transgenic exhibit higher stability under mildly acidic conditions [67], rice lines (gNAS1, gIDS3, and gNAS1–gNAAT) and MA synthesized by IDS3 would have been favorable for nontransformants (NTs). Germinated seeds were grown internal translocation of Fe and Zn. for 45 days in a greenhouse, and seedlings were then In conclusion, our field trial of the transformants demon- transplanted (three per hill) into the calcareous paddy field. strated that a transgenic approach to increase the tolerance of Sixteen days after transplanting (DAT), chlorosis and rice to low Fe availability is practical for improving growth retardation began to appear. By 42 DAT, the three agricultural productivity in calcareous paddy soils. transgenic rice lines were clearly superior to the NTs (Fig. 2a) both in leaf color and growth, although differences in performance were observed in individual plots. Using Production of other transgenic rice plants tolerant to Fe gIDS3 as an example, we saw no evident difference from deficiency NT on 16 DAT (Fig. 3a); chlorotic symptoms appeared in NTs but not in gIDS3 at 30 DAT (Fig. 3b). The clearest Introducing an engineered ferric-chelate reductase gene difference between gIDS3 and NTs was evident at 42 DAT (Fig. 3c). One week later (50 DAT), leaf chlorosis began to In strategy I plants, Fe uptake from the rhizosphere is disappear, especially in NT plants close to the plot mediated by ferrous ion transporters. Eide et al. [7] isolated 148 Rice (2008) 1:144–153 (a) Fig. 2 a Photograph (42 DAT) of the rice lines tested in a paddy field in the quarantine area of the Field Science Center of Tohoku University (Osaki, Miyagi, Japan) and b field lay- out. Each population contained five 1.2-m-long rows of rice with 20 cm between rows and 15 cm between hills. The box in the upper left indicates the two plots photographed on several occasions (Fig. 3). NT, non- transformant. Original figure: Suzuki et al. [57]. (b) 1.0 m 5 rows 1.2 m gNAS1 NT gIDS3 gNAS1 8 plants gNAAT 0.4 m gNAS1 gNAS1 gNAS1 NT gNAAT gNAS1 NT gIDS3 gNAS1 gNAAT gNAS1 gNAS1 NT gIDS3 the Arabidopsis IRT1 gene, which is the dominant ferrous To take up ferrous ion directly using OsIRT1, without transporter in the Fe-uptake process [65]. Rice, in spite of reducing ferric chelates, seems to be a consequence of being a strategy II plant, possesses homologs of the adaptation of rice to waterlogged soils, in which the concen- Arabidopsis IRT1 gene, OsIRT1 and OsIRT2, the ferrous tration of soluble ferrous iron increases with the decrease in soil transport capacity of which was demonstrated by functional redox potential [16, 57]. Because of the presence of OsIRT1, complementation in yeast [3, 16]. OsIRT1 expression is severe Fe deficiency is relatively rare in irrigated rice systems. strongly induced in Fe-deficient roots, and OsIRT2 is Nevertheless, rice plants grown in calcareous soils exhibit Fe expressed similarly but at lower levels. Promoter β- deficiency symptoms even under waterlogged conditions as glucuronidase (GUS) analysis indicated that OsIRT1 is noted previously because of their inability to induce ferric- mainly expressed in the epidermis, exodermis, and inner chelate reductase and their low capacity to synthesize MAs. layer of the cortex in Fe-deficient roots, as well as in Therefore, we hypothesized that introducing ferric-chelate companion cells of shoots. Moreover, an analysis using a reductase into rice would enhance Fe deficiency tolerance, positron-emitting tracer imaging system (PETIS) revealed creating a complete strategy I system in addition to the rice 2+ that rice is able to take up both Fe(III)–DMA and Fe . endogenous strategy II. Thus, rice plants possess a system other than the MAs- For functional expression in plants, we modified and based strategy II for Fe uptake [16]. In contrast to their completely reconstructed the yeast ferric reductase gene, ferrous-transporting ability, Fe-deficient rice roots do not FRE1,to produce refre1 (reconstructed FRE1;[47]). Since induce ferric-chelate reductase activity [16], which is a ferric-chelate reductase activity is inhibited by high pH, we hallmark of the strategy I response. then screened reductases with improved enzymatic activity at Rice (2008) 1:144–153 149 149 own promoter but observed no transgene mRNA expres- sion. In Arabidopsis, expression of ferric-chelate reductase FRO2 and ferrous transporter IRT1 is similarly and coordinately regulated at transcriptional and posttranscrip- tional levels [4, 66]. Therefore, we chose the promoter of the rice ferrous transporter gene OsIRT1 to drive the exogenous ferric-chelate reductase gene refre1/372 [17]. Transgenic rice plants with the introduced OsIRT1 promoter connected to refre1/372, successfully induced ferric-chelate reductase expression and activity in Fe- deficient roots, leading to higher Fe uptake than by vector controls, as revealed by a PETIS analysis. The transformants exhibited enhanced tolerance to low Fe availability in both hydroponic culture and calcareous soil (Fig. 4a). When grown in calcareous soil until harvest, the transformants had a 7.9 times higher grain yield than vector controls (Fig. 4b,c; [17]), demonstrating that creating a complete strategy I system in rice by enhancing ferric-chelate reductase activity is extremely effective in improving Fe deficiency tolerance. Manipulating transcription factors regulating the Fe deficiency response The above studies have shown that introduction of only a single or a few genes is effective in conferring Fe deficiency tolerance if appropriate promoter(s) and gene(s) are utilized. However, further enhancement of Fe availability might be achieved by engineering multiple genes in a coordinated manner. The genetic enhancement of a wide range of related genes requires manipulation of basal regulatory systems, including transcription factors. Therefore, we also aimed to clarify the regulation mechanism controlling the Fe deficiency response in graminaceous plants. Under low Fe availability, graminaceous plants induce various genes, many of which are involved in Fe acquisition and utilization [2, 26, 28, 36, 41]. Despite the number of Fe-deficiency-inducible genes isolated, little is known about the regulation of gene expression in response Fig. 3 Visual comparison between gIDS3 (left) and NT (right) from to Fe deficiency. Therefore, we applied a stepwise strategy 16 to 50 DAT, as illustrated in Fig. 2 but photographed from the to identify the molecular components regulating the opposite direction. a 16 DAT, b 30 DAT, c 42 DAT, and d 50 DAT. Original figure: Suzuki et al. [57]. expression of Fe-deficiency-responsive genes: establish- ment of a promoter assay system, identification of cis- high pH [48]. Through screening of randomly mutagenized acting elements, and identification of trans-acting factors refre1 derivatives, we obtained a variant designated refre1/ that interact with the elements. 372, whose encoding protein maintained strong reductase We introduced the promoter region of the barley IDS2 gene activity at pH 8–9. Transgenic tobacco plants with the connected to the GUS gene as a reporter into tobacco plants introduced refre1/372 under control of the 35S promoter [73]. Transgenic tobacco plants induced GUS expression in exhibited enhanced ferric-chelate reductase activity in roots Fe-deficient roots, basically reflecting the regulation pattern and better growth when grown in calcareous soils [48]. in native barley. Precise deletion and mutation analyses using Another concern in relation to the introduction of numerous lines of transgenic tobacco identified the novel Fe- exogenous reductase genes into rice was the choice of an deficiency-responsive cis-acting elements, iron-deficiency- appropriate promoter. Vasconcelos et al. [64] introduced the responsive element 1 and 2 (IDE1 and IDE2; [24]); these are Arabidopsis ferric-chelate reductase gene FRO2 with its the first identified elements related to micronutrient deficien- 150 Rice (2008) 1:144–153 (a) (b) (c) TF V V TF V Calcareous Bonsol TF V Fig. 4 Tolerance to Fe deficiency in transformants with the (normal cultivated soil). b Transformant (TF, left) and vector control introduced OsIRT1 promoter refre1/372 grown in calcareous soil. a (V, right) after 17 weeks of growth in calcareous soil. c Grain yield Transformants (TF, left) and vector controls (V, center) after 4 weeks after cultivation for 17 weeks in calcareous soil. Original figure: of growth in a calcareous soil; vector controls (V, right) in bonsol Ishimaru et al. [17]. cies in plants. IDE1 and IDE2 synergistically induce Fe- In an attempt to improve Fe deficiency tolerance by deficiency-responsive expression in tobacco roots. When modulating IDEF1 expression, we introduced IDEF1 introduced into rice, the pair IDE1 and IDE2 is able to cDNA fused to either the constitutive 35S promoter or the induce Fe-deficiency-responsive expression both in roots and Fe-deficiency-inducible IDS2 promoter. Transgenic rice leaves [25]. Sequences similar to IDE1 or IDE2 were found in various Fe-deficiency-inducible promoters of barley, rice, Fe-deficiency signal tobacco, and Arabidopsis [6, 24, 26]. This suggests that gene regulation mechanisms involving IDEs are not only con- served among graminaceous (strategy II) plants but are also IDEF1 IDEF2 IDEF2 functional in nongraminaceous (strategy I) plant species. Fe-deficiency- Next, we searched for transcription factors that interact IDE2-like responsive genes with IDEs. Very recently, we successfully identified two rice transcription factors, IDE-binding factor 1 (IDEF1) and Fe-deficiency- IDE1-like IDE1-like IDEF2, which specifically bind to IDE1 and IDE2, respec- OsIRO2 responsive genes tively [27, 45]. IDEF1 and IDEF2 belong to uncharacterized OsIRO2 branches of plant-specific transcription factor families ABI3/ VP1 and NAC, respectively, and exhibit novel properties of Fe-deficiency- CACGTGG sequence recognition. IDEF1 recognizes the CATGC se- responsive genes quence within IDE1, whereas IDEF2 predominantly recog- nizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the AP2-domain CACGTGG OsNAC4 CACGTGG transcription factor core binding site. Both IDEF1 and IDEF2 transcripts are NAC4 AP2 constitutively expressed in rice roots and leaves. Fe-deficiency- unknown Fe-deficiency- unknown responsive genes responsive genes element element Fig. 6 Proposed regulatory network for the induction of Fe- deficiency-responsive genes via IDEF1, IDEF2, and OsIRO2. Under Fe-deficient conditions, IDEF1 and IDEF2 transactivate the expres- sion of Fe-deficiency-responsive genes by binding to the IDE1-like and IDE2-like elements, respectively [27, 45]. OsIRO2, which is induced by Fe deficiency and is positively regulated by IDEF1, binds to the CACGTGG element to activate another subset of Fe-deficiency- responsive genes, including two transcription factor genes: OsNAC4 NT and the AP2 domain-containing gene. These transcription factors may Fig. 5 Tolerance to Fe deficiency in seedlings with the introduced then regulate Fe-deficiency-responsive genes lacking IDEs and IDS2 promoter IDEF1 germinated in a calcareous soil (lines 9, 12, and CACGTGG in their promoter regions [44]. The induced expression 13) compared to nontransformants (NT) 17 days after sowing. Original of IDEF1 in transgenic rice plants would effectively strengthen the figure: Kobayashi et al. [27]. overall regulatory pathway to confer tolerance to Fe deficiency. Line9 Line12 Line13 Grain weight / plant (g) Rice (2008) 1:144–153 151 151 seedlings with the introduced 35S promoter IDEF1 showed (Figs. 2, 3). Availability of Fe in rice fields is severely severe growth retardation during early growth, while those affected by soil type and redox potential, as well as carrying the IDS2 promoter IDEF1 showed healthy growth. numerous other environmental factors. An elaborate combi- Notably, the IDS2 promoter IDEF1 transformants exhibited nation of previously adopted or new strategies will be needed slower progression of leaf chlorosis in Fe-free hydroponic to produce rice lines with even more tolerance to low Fe culture and also showed better growth when germinated on availability in problematic soils without loss of favorable calcareous soil (Fig. 5;[27]). agricultural traits. Manipulation of DMAS genes, which were To clarify the molecular mechanisms that regulate Fe recently cloned and thus have not been genetically modified, acquisition, we also characterized Fe-deficiency-induced in the steps of MAs biosynthesis [2] would be of special transcription factors. Microarray analyses revealed the upre- interest. In addition, further clarification of the underlying gulation of several transcription factor genes in barley and rice mechanisms involved in Fe homeostasis is extremely [41, 43], among which a bHLH transcription factor gene, important, including expressional regulation, secretion of IRO2, is of particular interest because of its pronounced MAs, and metal translocation inside the plants. transcriptional upregulation by Fe deficiency in shoots and Understanding metal homeostasis also paves the way to roots of barley and rice [43]. The core sequence for OsIRO2 fortifying rice grains with Fe and Zn. Previous efforts to binding was determined to be CACGTGG [43]. enhance Fe in grains were performed by overexpressing We produced transgenic rice plants with enhanced or ferritin, a common Fe storage protein in rice grain [8, 51, 63]. repressed OsIRO2 expression by introducing the 35S- Our field trials revealed that the gIDS3 line is capable of OsIRO2 cassette or using the RNA interference technique accumulating more Fe in grains in both calcareous and [44]. In Fe-deficient hydroponic culture, OsIRO2-over- Andosol paddy fields [35, 57]. Production and characteriza- expressing lines showed enhanced MAs secretion and tion of transgenic rice lines with introduced biosynthetic genes slightly better growth compared to nontransformants, for MAs and ferritin genes in combination to enhance both Fe whereas OsIRO2-repressed lines resulted in lower MAs uptake and storage is in progress (Masuda et al. unpublished). secretion and hypersensitivity to Fe deficiency. Microarray Other advanced applications of our knowledge on Fe nutrition and Northern blot analyses revealed that the expression include the production of novel antihypertensive substrates. level of OsIRO2 is positively related to various Fe- NA, the precursor of MAs, inhibits angiotensin-I-converting deficiency-induced genes in roots, including those respon- enzyme in humans and consequently reduces high blood sible for MAs biosynthesis (OsNAS1, OsNAS2, OsNAAT1, pressure [22, 53]. We produced a yeast strain that highly OsDMAS1, and various genes involved in the methionine accumulates NA by introducing the Arabidopsis NAS gene, cycle) and Fe(III)–MAs uptake (OsYSL15). OsIRO2 also AtNAS2 [69]. Production and selection of rice lines with affects the expression of some Fe-deficiency-inducible elevated levels of NA in grain by introducing the HvNAS1 transcription factor genes that possess OsIRO2-binding gene under the control of a seed-specific promoter of the rice core sequences in their promoter regions [44]. Importantly, glutelin gene is now under way [68]. OsIRO2 itself possesses multiple IDEF1-binding core Public acceptance of genetically modified organisms is sequences in its promoter region and is positively regulated still low. As a technical way to improve public acceptance, by IDEF1 [27]. Based on these results, a sequential link in we modified the “marker-free vector” of the Cre/loxP DNA the Fe deficiency response involving IDEF1, IDEF2, excision system [74] to construct a high-capacity binary OsIRO2, and its downstream Fe-deficiency-inducible tran- vector for the transformation of rice, from which the scription factors is proposed (Fig. 6;[27, 44, 45]). sequence sandwiched between two loxP sites (including In contrast to growth retardation observed in the 35S the selectable marker) can be removed by 17β-estradiol promoter IDEF1 transformants, the 35S promoter OsIRO2 administration [68]. Many other approaches may aid public transformants were healthy, not exhibiting any obvious acceptance of transgenic plants, which have such high defects. These differences in phenotypes of the trans- potential to increase food production, preserve the environ- formants are thought to be related to the distinct nature of ment, and improve human health. the two transcription factors. References Future perspectives 1. Akiyama K, Nakamura S, Suzuki T, Wisniewska I, Sasaki N, Kawasaki S. Development of a system of rice transformation with We produced various lines of transgenic rice plants with long genome inserts for their functional analysis for positional enhanced tolerance to low Fe availability. Among these, cloning. Plant Cell Physiol Supplement 1997;38:s94. tolerance of three selected lines (gNAS1, gIDS3, and gNAS1– 2. Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori gNAAT) in calcareous soil was demonstrated in field trials S, et al. Cloning and characterization of deoxymugineic acid 152 Rice (2008) 1:144–153 synthase genes from graminaceous plants. J Biol Chem. 22. Kinoshita E, Yamakoshi J, Kikuchi M. Purification and identifi- 2006;43:32395–402. cation of an angiotensin I-converting enzyme inhibitor from soy 3. Bughio N, Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S. sauce. Biosci Biotechnol Biochem. 1993;57:1107–10. Cloning an iron-regulated metal transporter from rice. J Exp Bot 23. Kobayashi T, Nakanishi H, Takahashi M, Kawasaki S, Nishizawa 2002;53:1677–82. NK, Mori S. In vivo evidence that Ids3 from Hordeum vulgare 4. Connolly EL, Campbell NH, Grotz N, Prichard CL, Guerinot ML. encodes a dioxygenase that converts 2’-deoxymugineic acid to Overexpression of the FRO2 ferric chelate reductase confers mugineic acid in transgenic rice. Planta 2001;212:864–71. tolerance to growth on low iron and uncovers posttranscriptional 24. Kobayashi T, Nakayama Y, Itai RN, Nakanishi H, Yoshihara T, control. Plant Physiol. 2003;133:1102–10. Mori S, et al. Identification of novel cis-acting elements, IDE1 and 5. Curie C, Panavience Z, Loulergue C, Dellaporta SL, Briat JF, IDE2, of the barley IDS2 gene promoter conferring iron- Walker EL. Maize yellow stripe1 encodes a membrane protein deficiency-inducible, root-specific expression in heterogeneous directly involved in Fe(III) uptake. Nature 2001;409:346–9. tobacco plants. Plant J. 2003;36:780–93. 6. Ducos E, Fraysse ÅS, Boutry M. NtPDR3, an iron-deficiency 25. Kobayashi T, Nakayama Y, Takahashi M, Inoue H, Nakanishi H, inducible ABC transporter in Nicotiana tabacum. FEBS Lett. Yoshihara T, et al. Construction of artificial promoters highly 2005;579:6791–5. responsive to iron deficiency. Soil Sci Plant Nutr. 2004;50:1167– 7. Eide D, Broderius M, Fett J, Guerinot ML. A novel iron-regulated 75. metal transporter from plants identified by functional expression 26. Kobayashi T, Suzuki M, Inoue H, Itai RN, Takahashi M, in yeast. Proc Natl Acad Sci USA. 1996;93:5624–8. Nakanishi H, et al. Expression of iron-acquisition-related genes 8. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F. Iron in iron-deficient rice is co-ordinately induced by partially fortification of rice seed by the soybean ferritin gene. Nat conserved iron-deficiency-responsive elements. J Exp Bot. Biotechnol 1999;17:282–6. 2005;56:1305–16. 9. Hell R, Stephan UW. Iron uptake, trafficking and homeostasis in 27. Kobayashi T, Ogo Y, Itai RN, Nakanishi H, Takahashi M, Mori S, plants. Planta 2003;216:541–51. et al. The novel transcription factor IDEF1 regulates the response 10. Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation to and tolerance of iron deficiency in plants. Proc Natl Acad Sci of rice (Oryza sativa L.) mediated by Agrobacterium and USA. 2007;104:19150–5. sequence analysis of the boundaries of the T-DNA. Plant J. 28. Kobayashi T, Nishizawa NK. Regulation of iron and zinc uptake 1994;6:271–82. and translocation in rice. In: Hirano HY, Hirai A, Sano Y, Sasaki 11. Higuchi K, Kanazawa K, Nishizawa NK, Chino M, Mori S. T, editors. Biotechnology in agriculture and forestry 62. Rice Purification and characterization of nicotianamine synthase from biology in the genomics era. Meppel: Springer; 2008. p. 321–35. Fe deficient barley roots. Plant Soil. 1994;165:173–9. 29. Ling HQ, Koch G, Bäumlein H, Ganal MW. Map-based cloning 12. Higuchi K, Kanazawa K, Nishizawa NK, Mori S. The role of of chloronerva, a gene involved in iron uptake of higher plants nicotianamine synthase in response to Fe nutrition status in encoding nicotianamine synthase. Proc Natl Acad Sci USA. Gramineae. Plant Soil. 1996;178:171–7. 1999;96:7098–710. 13. Higuchi K, Suzuki K, Nakanishi H, Yamaguchi H, Nishizawa NK, 30. Ma JF, Nomoto K. Two related biosynthetic pathways of mugineic Mori S. Cloning of nicotianamine synthase genes, novel genes acids in gramineous plants. Plant Physiol. 1993;102:373–8. involved in the biosynthesis of phytosiderophores. Plant Physiol 31. Ma JF, Shinada T, Matsuda C, Nomoto K. Biosynthesis of 1999;119:471–9. phytosiderophores, mugineic acids, associated with methionine 14. Higuchi K, Watanabe S, Takahashi M, Kawasaki S, Nakanishi H, cycling. J Biol Chem. 1995;270:16549–54. Nishizawa NK, et al. Nicotianamine synthase gene expression 32. Ma JF, Taketa S, Chang YC, Iwashita T, Matsumoto H, Takeda K, differs in barley and rice under Fe-deficient conditions. Plant J. et al. Genes controlling hydroxylations of phytosiderophores are 2001;25:159–67. located on different chromosomes in barley (Hordeum vulgare L.). 15. Inoue H, Takahashi M, Kobayashi T, Suzuki M, Nakanishi H, Mori S, Planta 1999;207:590–6. et al. Identification and localisation of the rice nicotianamine 33. Marschner H. Mineral nutrition of higher plants, 2nd edn. aminotransferase gene OsNAAT1 expression suggests the site of London: Academic; 1995. phytosiderophore synthesis in rice. Plant Mol Biol. 2008;66:193–203. 34. Marschner H, Römheld V, Kissel M. Different strategies in higher 16. Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, plants in mobilization and uptake of iron. J Plant Nutr. 3+ Kobayashi T, et al. Rice plants take up iron as an Fe - 1986;9:695–713. 2+ phytosiderophore and as Fe . Plant J. 2006;45:335–46. 35. Masuda H, Suzuki M, Morikawa KC, Kobayashi T, Nakanishi H, 17. Ishimaru Y, Kim S, Tsukamoto T, Oki H, Kobayashi T, Watanabe Takahashi M, et al. Increase in iron and zinc concentrations in rice S, et al. Mutational reconstructed ferric chelate reductase confers grains via the introduction of barley genes involved in phytosi- enhanced tolerance in rice to iron deficiency in calcareous soil. derophore synthesis. Rice 2008. doi:10.1007/s12284-008-9007-6. Proc Natl Acad Sci USA. 2007;104:7373–8. 36. Mori S. Iron acquisition by plants. Curr Opin Plant Biol. 18. Itai R, Suzuki K, Yamaguchi H, Nakanishi H, Nishizawa NK, 1999;2:250–3. Yoshimura E, et al. Induced activity of adenine phosphoribosyl- 37. Mori S, Nishizawa N. Methionine as a dominant precursor of transferase (APRT) in Fe-deficient barley roots: a possible role for phytosiderophores in Graminaceae plants. Plant Cell Physiol phytosiderophore production. J Exp Bot. 2000;51:1179–88. 1987;28:1081–92. 19. Kanazawa K, Higuchi K, Nishizawa NK, Fushiya S, Chino M, 38. Morikawa CK, Saigusa M, Nakanishi H, Nishizawa NK, Mori S. Nicotianamine aminotransferase activities are correlated Hasegawa K, Mori S. Co-situs application of controlled-release to the phytosiderophore secretions under Fe-deficient conditions fertilizers to alleviate iron chlorosis of paddy rice grown in in Gramineae. J Exp Bot. 1994;45:1903–6. calcareous soil. Soil Sci Plant Nutr. 2004;50:1013–21. 20. Kanazawa K, Higuchi K, Nishizawa NK, Fushiya S, Mori S. 39. Nakanishi H, Okumura N, Umehara Y, Nishizawa NK, Chino M, Detection of two distinct isozymes of nicotianamine aminotrans- Mori S. Expression of a gene specific for iron deficiency (Ids3) ferase in Fe-deficient barley roots. J Exp Bot. 1995;46:1241–4. in the roots of Hordeum vulgare. Plant Cell Physiol 1993;34:401– 21. Kawai S, Takagi S, Sato Y. Mugineic acid-family phytosider- 10. ophores in root-secretions of barley, corn and sorghum varieties. J 40. Nakanishi H, Yamaguchi H, Sasakuma T, Nishizawa NK, Mori S. Plant Nutr. 1988;11:633–42. Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are Rice (2008) 1:144–153 153 153 involved in the biosynthesis of mugineic acid family phytosider- increased tolerance to low iron availability in a calcareous paddy ophores. Plant Mol Biol. 2000;44:199–207. soil. Soil Sci Plant Nutr. 2008;54:77–85. 41. Negishi T, Nakanishi H, Yazaki J, Kishimoto N, Fujii F, Shimbo 58. Suzuki M, Tsukamoto T, Inoue H, Watanabe S, Matsuhashi S, K, et al. cDNA microarray analysis of gene expression during Fe- Takahashi M, et al. Deoxymugineic acid increases Zn transloca- deficiency stress in barley suggests that polar transport of vesicles tion in Zn-deficient rice plants. Plant Mol Biol 2008;66:609–17. is implicated in phytosiderophore secretion in Fe-deficient barley 59. Takagi S. Naturally occurring iron-chelating compounds in oat- roots. Plant J. 2002;30:83–94. and rice-root washings. Soil Sci Plant Nutr. 1976;22:423–33. 42. Noma M, Noguchi M. Occurrence of nicotianamine in higher 60. Takahashi M, Yamaguchi H, Nakanishi H, Shioiri T, Nishizawa plants. Phytochemistry 1976;15:1701–2. NK, Mori S. Cloning two genes for nicotianamine aminotransfer- 43. Ogo Y, Itai RN, Nakanishi H, Inoue H, Kobayashi T, Suzuki M, et ase, a critical enzyme in iron acquisition (strategy II) in gramina- al. Isolation and characterization of IRO2, a novel iron-regulated ceous plants. Plant Physiol. 1999;121:947–56. bHLH transcription factor in graminaceous plants. J Exp Bot. 61. Takahashi M, Nakanishi H, Kawasaki S, Nishizawa NK, Mori S. 2006;57:2867–78. Enhanced tolerance of rice to low iron availability in alkaline soils 44. Ogo Y, Itai RN, Nakanishi H, Kobayashi T, Takahashi M, Mori S, using barley nicotianamine aminotransferase genes. Nat Biotech- et al. The rice bHLH protein OsIRO2 is an essential regulator of nol. 2001;19:466–9. the genes involved in Fe uptake under Fe-deficient conditions. 62. Takahashi M, Terada Y, Nakai I, Nakanishi H, Yoshimura E, Mori Plant J. 2007;51:366–77. S, et al. Role of nicotianamine in the intracellular delivery of 45. Ogo Y, Kobayashi T, Itai RN, Nakanishi H, Kakei Y, Takahashi metals and plant reproductive development. Plant Cell M, et al. A novel NAC transcription factor IDEF2 that recognizes 2003;15:1263–80. the iron deficiency-responsive element 2 regulates the genes 63. Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L, involved in iron homeostasis in plants. J Biol Chem 2008; Krishnan S, et al. Enhanced iron and zinc accumulation in 283:13407–17. transgenic rice with the ferritin gene. Plant Sci. 2003;164:371–8. 46. Ohata T, Kanazawa K, Mihashi S, Nishizawa NK, Fushiya S, 64. Vasconcelos M, Musetti V, Li CM, Datta SK, Grusak MA. Nozoe S, et al. Biosynthetic pathway of phytosiderophores in Functional analysis of transgenic rice (Oryza sativa L.) trans- iron-deficient Graminaceous plants. Development of an assay formed with an Arabidopsis thaliana ferric reductase (AtFRO2). system for the detection of nicotianamine aminotransferase Soil Sci Plant Nutr. 2004;50:1151–7. activity. Soil Sci Plant Nutr. 1993;39:745–9. 65. Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, 47. Oki H, Yamaguchi H, Nakanishi H, Mori S. Introduction of the Briat JF, et al. IRT1, an Arabidopsis transporter essential for iron reconstructed yeast ferric reductase gene, refre1, into tobacco. uptake from the soil and plant growth. Plant Cell 2002;14:1223– Plant Soil 1999;215:211–20. 33. 48. Oki H, Kim S, Nakanishi H, Takahashi M, Yamaguchi H, Mori S, 66. Vert G, Briat JF, Curie C. Dual regulation of the Arabidopsis high- et al. Directed evolution of yeast ferric reductase to produce plants affinity root iron uptake system by local and long-distance signals. with tolerance to iron deficiency in alkaline soils. Soil Sci Plant Plant Physiol 2003;132:796–804. Nutr. 2004;50:1159–65. 67. von Wirén N, Khodr H, Hider RC. Hydroxylated phytosider- 49. Okumura N, Nishizawa NK, Umehara Y, Mori S. An iron ophore species possess an enhanced chelate stability and affinity deficiency-specific cDNA from barley roots having two homolo- for iron(III). Plant Physiol. 2000;124:1149–57. gous cysteine-rich MT domains. Plant Mol Biol 1991;17:531–3. 68. Wada Y, Ishimaru Y, Takahashi M, Nakanishi H, Mori S, 50. Okumura N, Nishizawa NK, Umehara Y, Ohata T, Nakanishi H, Nishizawa NK. Engineering of hypotensive rice contain high Yamaguchi H, et al. A dioxygenase gene (Ids2) expressed under amounts of nicotianamine for functional foods. In: Li CJ, et al., iron deficiency conditions in the roots of Hordeum vulgare. Plant editors. Plant nutrition for food security, human health and Mol Biol 1994;25:705–19. environmental protection. Beijing: Tsinghua University Press; 51. Qu LQ, Yoshihara T, Ooyama A, Goto F, Takaiwa F. Iron 2005. p. 424–5. accumulation does not parallel the high expression level of ferritin 69. Wada Y, Kobayashi T, Takahashi M, Nakanishi H, Mori S, in transgenic rice seeds. Planta 2005;222:225–33. Nishizawa NK. Metabolic engineering of Saccharomyces cerevi- 52. Römheld V, Marschner H. Evidence for a specific uptake system siae producing nicotianamine: potential for industrial biosynthesis for iron phytosiderophores in roots of grasses. Plant Physiol. of a novel antihypertensive substrate. Biosci Biotechnol Biochem. 1986;80:175–80. 2006;70:1408–15. 53. Shimizu E, Hayashi A, Takahashi R, Aoyagi Y, Murakami T, 70. Yamaguchi H, Nakanishi H, Nishizawa NK, Mori S. Induction of Kimoto K. Effects of angiotensin I-converting enzyme inhibitor the IDI1 gene in Fe-deficient barley roots: a gene encoding from ashitaba (Angelica keiskei) on blood pressure of spontane- putative enzyme that catalyses the methionine salvage pathway for ously hypertensive rats. J Nutr Sci Vitaminol. 1999;45:375–83. phytosiderophore production. Soil Sci Plant Nutr. 2000;46:1–9. 54. Shojima S, Nishizawa NK, Fushiya S, Nozoe S, Irifune T, Mori S. 71. Yamaguchi H, Nakanishi H, Nishizawa NK, Mori S. Isolation and Biosynthesis of phytosiderophores: in vitro biosynthesis of 2’- characterization of IDI2, a new Fe-deficiency induced cDNA from deoxymugineic acid from L-methionine and nicotianamine. Plant barley roots, which encodes a protein related to the a subunit of Physiol. 1990;93:1497–503. eukaryotic initiation factor 2B (eIF2B a). J Exp Bot. 55. Suzuki K, Itai R, Suzuki K, Nakanishi H, Nishizawa NK, 2000;51:2001–7. Yoshimura E, et al. Formate dehydrogenase, an enzyme of 72. Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S. IDI7, a new anaerobic metabolism, is induced by Fe-deficiency in barley iron-regulated ABC transporter from barley roots, localizes to the roots. Plant Physiol. 1998;116:725–32. tonoplast. J Exp Bot. 2002;53:727–235. 56. Suzuki M, Takahashi M, Tsukamoto T, Watanabe S, Matsuhashi 73. Yoshihara T, Kobayashi T, Goto F, Masuda T, Higuchi K, S, Yazaki J, et al. Biosynthesis and secretion of mugineic acid Nakanishi H, et al. Regulation of the iron-deficiency responsive family phytosiderophores in zinc-deficient barley. Plant J. gene, Ids2, of barley in tobacco. Plant Biotechnol. 2003;20:33–41. 2006;48:85–97. 74. Zuo J, Niu QW, Møller SG, Chua NH. Chemical-regulated, site- 57. Suzuki M, Morikawa KC, Nakanishi H, Takahashi M, Saigusa M, specific DNA excision in transgenic plants. Nat Biotechnol. Mori S, et al. Transgenic rice lines that include barley genes have 2001;19:157–61.

Journal

RiceSpringer Journals

Published: Dec 1, 2008

Keywords: Iron deficiency; Field trial; Mugineic acid family phytosiderophores; Transgenic rice plants

There are no references for this article.