Soil Science and Plant Nutrition (2009) 55, 277–282 doi: 10.1111/j.1747-0765.2009.00359.x

© 2009 Japanese Society of Soil Science and Plant Nutrition

Blackwell Publishing LtdBarley HvNAS1 and rice OsNAS1 promotersS. Ito et al.ORIGINAL ARTICLE/SHORT PAPER

Comparison of the functions of the barley nicotianamine synthase gene HvNAS1 and rice nicotianamine synthase gene OsNAS1 promoters in response to iron deficiency in transgenic tobacco

Satoshi ITO1, Haruhiko INOUE2,3†, Takanori KOBAYASHI2,3, Masaaki YOSHIBA1,3, Satoshi MORI2, Naoko K. NISHIZAWA2,3 and Kyoko HIGUCHI1,3

1Laboratory of Plant Production Chemistry, Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Tokyo 156-8502, 2Laboratory of Plant Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, and 3Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 102-0075, Japan

Abstract

Barley (Hordeum vulgare L.) nicotianamine synthase gene (HvNAS1) expression in barley is strongly inducedby Fe deficiency in the roots and rice (Oryza sativa L.) nicotianamine synthase gene (OsNAS1) expressionin rice is induced by Fe deficiency both in the roots and in the shoots. In dicots, NAS genes are not stronglyinduced by Fe deficiency, and they function to maintain Fe homeostasis. Rice OsNAS1promoter::GUS orbarley HvNAS1promoter::GUS was introduced into tobacco (Nicotiana tabacum L.) and tissue specificitiesand systemic regulation of their expression were compared. A split-root experiment revealed that the HvNAS1promoter exhibited functions similar to those of Fe-acquisition-related genes in tobacco roots, suggesting thatthis promoter responded to certain Fe-deficiency systemic signals and to the Fe concentration in the rhizo-sphere. The HvNAS1 promoter might harbor a type of universal system of gene expression for Fe acquisition.However, the OsNAS1 promoter did not respond to local application of Fe to the roots and induced GUSactivities in mature leaves in response to Fe deficiency. This promoter might possess numerous types ofcis-acting sequences that are involved in Fe metabolism.

Key words: barley, gene expression, iron deficiency, nicotianamine synthase, rice.

INTRODUCTION

Ferric iron (Fe3+) is generally precipitated in soil and plantbodies under aerobic or alkaline pH conditions; thus,Fe3+ exhibits low availability. In contrast, ferrous iron(Fe2+) is more likely to produce toxic radicals (Marschner1995). Therefore, plants have acquired chelation strate-gies for the solubilization or detoxification of Fe.

In Strategy I plants, which includes all dicots and non-graminaceous monocots, the reduction of ferric to ferrousions at the root surface is an essential step in Fe uptake.The ferric-chelate reductase gene FRO has been clonedfrom dicots (Robinson et al. 1999; Waters et al. 2002).Following reduction, Fe2+ is absorbed via an iron-regulatedtransporter (IRT) (Vert et al. 2002). These mechanisms areinduced by Fe deficiency. Nicotianamine, which chelatesthe divalent cation of transition metals, is constitutivelyessential for Fe distribution in Strategy I plants (Scholzet al. 1992). Grasses are Strategy II plants and use achelation mechanism for Fe uptake. Strategy II plantssynthesize mugineic acid family phytosiderophores (MAs),compounds that have a high affinity for Fe3+, and secretethese cpmpounds into the rhizosphere to chelate Fe3+.Nicotianamine is a key precursor of MAs in StrategyII plants (Shojima et al. 1990). The ferric iron–MAcomplex is absorbed via a specific transporter, namely,YS (Curie et al. 2001). These mechanisms are induced

Correspondence: K. HIGUCHI, Laboratory of Plant ProductionChemistry, Department of Applied Biological Chemistry, TokyoUniversity of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku,Tokyo 156-8502, Japan. Email: khiguchi@nodai.ac.jpPresent address: †Plant Disease Resistance Research Unit,Division of Plant Sciences, National Institute of Agrobio-logical Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.Received 31 July 2008.Accepted for publication 29 November 2008.

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by Fe deficiency. In addition, although rice has beencategorized as a Strategy II plant it takes up Fe2+ via anIRT transporter, similar to the mechanism observed inStrategy I plants (Ishimaru et al. 2006). Thus, nicotian-amine synthesized from S-adenosyl methionine by nico-tianamine synthase (NAS) (Higuchi et al. 1999) plays akey role in Fe nutrition in all higher plants. However, theexpression patterns of NAS genes in response to the Fenutritional status differ between plant species (Ito et al.2007), and this difference might be correlated with thedifference in the physiological functions of nicotianamine.Nicotianamine synthase might function in both Fe uptakeand distribution in rice, and this represents an interme-diate between Strategy I and Strategy II plants.

Recently, cis-acting elements and trans-acting factorsregulating the expression of Fe-deficiency-related geneshave been revealed. Several basic helix-loop-helix tran-scription factors, such as FER from tomato, OsIRO2from rice and FIT from Arabidopsis, have been cloned(Colangelo and Guerinot 2004; Jakoby et al. 2004; Linget al. 2002; Ogo et al. 2006), and the recognition sequenceof OsIRO2 has also been revealed (Ogo et al. 2006).The expression of OsNAS genes is affected by the levelof OsIRO2 expression in rice (Ogo et al. 2007). The reg-ulatory components of OsIRO2, Fe-deficiency-responsivecis-acting elements IDE1 and IDE2, and the IDE1-bindingand IDE2-binding trans-acting factors IDEF1 and IDEF2have been discovered (Kobayashi et al. 2003, 2007; Ogoet al. 2007, 2008). In general, IDE1 and IDE2 are foundin higher plants, and they are also found in the promoterregions of NAS genes. However, the complicated regula-tory pathways for the expression of multiple NAS geneshave not been fully elucidated.

We previously reported that the barley (Hordeum vul-gare L.) HvNAS1 and rice (Oryza sativa L.) OsNAS1 pro-moters drove gene expression differently in transgenictobacco (Nicotiana tabacum L.) (Ito et al. 2007). Whenfused to the β-glucuronidase (GUS) gene and introducedinto tobacco, the HvNAS1 promoter induced GUS activityonly in Fe-deficient roots; this is similar to its action inbarley and rice. However, the OsNAS1 promoter inducedGUS activity more strongly in Fe-deficient shoots thanin Fe-deficient roots. This differs slightly from its actionin rice; that is, the OsNAS1 promoter induced OsNAS1expression in both the roots and shoot of rice, but it wasconsiderably stronger in the roots than in the shoots(Inoue et al. 2003). This difference in the level of geneexpression might not result solely from a difference inthe affinity between cis-acting elements and the postu-lated trans-acting factors. The OsNAS1 promoter couldrespond to a regulatory system other than Fe acquisitionin tobacco.

Recently, it has been revealed that the expression ofFe-acquisition-related genes is regulated by both systemic

and local signals (Enomoto et al. 2007; Vert et al. 2003).In the present study, we utilized the HvNAS1promoter::GUS (HvNAS1pro::GUS) and OsNAS1promoter::GUS(OsNAS1pro::GUS) transgenic tobacco lines to comparethe tissue specificities and systemic regulation of theexpression patterns of the HvNAS1 and OsNAS1 pro-moters in detail in transgenic tobacco.

MATERIALS AND METHODS

Preparation of the transgenic tobaccoHomozygous T2 transformants of the HvNAS1pro::GUStransgenic tobacco plant were derived from transgenictobacco harboring a 1.7-kb promoter fragment, whichwas amplified by polymerase chain reaction (PCR) froman HvNAS1 genomic clone template (Higuchi et al.2001a). Homozygous T2 transformants of the OsNAS1pro::GUS transgenic tobacco plant were derived fromtransgenic tobacco harboring a 1.6-kb promoter fragment,which was amplified using PCR from a rice genomicDNA template (Inoue et al. 2003; Ito et al. 2007). Thesetransformants were then cultured in a growth chamberat a temperature of 25 ± 1°C under a 16-h light (300 μmols–1 m–2 μA–1)/8-h dark cycle.

Split-root experimentNon-transformants or T2 transformants were cultured onMurashige and Skoog (MS) medium containing 0.2%gellan gum. We then transplanted 14-day-old seedlingsinto Petri dishes containing the split-root system. Eachof the four compartments of the Petri dishes (100 mm× 15 mm Stackable Quad-Plate; Kord-Valmark, Bramp-ton, Canada) was filled with MS medium with or with-out 100 μmol L–1 Fe-ethylenediaminetetraacetic acid(Fig. 1). The cotyledons of the seedlings were removedand the roots were washed with sterile water. The rootswere divided into two groups and each group was placedin a different compartment (Fig. 1). The roots on themedium were covered and fixed with 1% agarose con-taining MS nutrients, except Fe. This dish containingthe plants was placed in a large, sterile, glass Petri dish(120 mm × 60 mm); the dish was covered and then sealedwith adhesive tape. The roots of two plantlets wereharvested 3, 5 or 7 days after each treatment and usedfor the GUS assay or for the northern blot analysis.

Northern blot analysisThe partial Fe(III)-chelate reductase gene fragment fromtobacco (NtFRO) was used for the northern blot analysis(Ito et al. 2007). The probe for NtFRO was labeled with[32P]dCTP using a Random Primer Labeling Kit Ver. 2(TaKaRa, Shiga, Japan). Total RNA was extracted fromthe roots and leaves of the plant using the RNeasy PlantMini Kit (Qiagen K.K., Tokyo, Japan). As described in

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Higuchi et al. (2001b), 15 μg of the total RNA wasseparated, blotted and hybridized at 65°C using theprobes. Radioactivity was then detected using a BAS-5000 image analyzer (Fuji Film, Tokyo, Japan).

Fluorometric analysis of the GUS activityThe GUS activity was assayed according to the methodof Kobayashi et al. (2003). Enzyme activity was fluoro-metrically measured using 4-methylumbelliferyl-β-d-glucuronide as the substrate, and the reaction product,namely, 4-methylumbelliferone (MU), was detected. Theprotein concentration was determined using a Bio-RadProtein Assay Kit (Bio-Rad Laboratories K.K., Tokyo,Japan).

Histochemical analysisHistochemical analysis was conducted according to themethod of Jefferson et al. (1987) modified by Kosugi et al.(1991). Fragments of the leaf or root were embedded in4% agar and then cut into 100-μm sections using a DTK-100 microslicer (Dosaka EM, Kyoto, Japan). The leaves,roots or sections of the transgenic tobacco were incubatedat 37°C for 3 h or overnight in the GUS reaction buffer,which was composed of 1 mmol L–1 5-bromo-5-chloro-3-indolyl-β-d-glucuronide, 3 mmol L–1 K5[Fe(CN)6],0.5 mmol L–1 K3[Fe(CN)6], 50 mmol L–1 sodium phos-phate buffer (pH 7.0) and 20% (v/v) methanol. Afterstaining, the plantlets were washed in 70% ethanol toremove the chlorophyll. The GUS staining was thenobserved using a BZ-8000 microscope (Keyence, Osaka,Japan).

RESULTS AND DISCUSSION

Different expression patterns of OsNAS1pro::GUS and HvNAS1pro::GUS in transgenic tobacco plants after the split-root treatmentWe carried out a split-root experiment to investigate theeffect of systemic and local Fe signals on regulation ofthe HvNAS1 and OsNAS1 promoters in tobacco (Fig. 1).First, we verified the consistency of the split-root exper-iment using northern blot analysis with the NtFRO probe,which is a fragment of the tobacco FRO gene (Ito et al.2007). The image obtained from northern blot analysisof non-transformant roots is shown in Fig. 2. NtFROmRNA accumulation was strongly induced in the rootsof –/– plants 7 days after the treatment, and in the +/–plants it was weakly induced in Fe-supplied split roots,but not in Fe-starved split roots. These results were con-sistent with the expression patterns of AtFRO2 andAtIRT1 in Arabidopsis (Vert et al. 2003). Ferric reductaseactivities in cucumber and tomato were also induced inthe Fe-supplied region of split roots, when the other regionof the root was subjected to Fe starvation (Li et al. 2000;Schikora and Schmidt 2001). Thus, the regulation of Fe-acquisition-related genes by systemic and local signals isgenerally observed in higher plants, such as tobacco.

The GUS activities of the transgenic tobacco rootssubjected to the split-root experiment were measured(Fig. 3). GUS activity of HvNAS1pro::GUS was signifi-cantly induced in Fe-supplied split roots, but not in Fe-starved split roots in the +/– treated HvNAS1pro::GUStobacco plants. This expression pattern was similar tothat of NtFRO (Fig. 2). We observed that 3–7 days afterthe treatment, GUS activities were not induced in eitherFe-supplied or Fe-starved split roots in the +/– treatedOsNAS1pro::GUS tobacco. This expression pattern wasdifferent from that of NtFRO.

Models for the regulatory mechanisms of Fe-acquisition-related genes under conditions of Fe deficiency have beenproposed: systemic signals of the Fe nutritional statusare transmitted from the shoot to the roots, followingwhich Fe-acquisition-related gene expression is inducedin the Fe-supplied roots (Enomoto et al. 2007; Grusak and

Figure 1 Apparatus for the split-root experiment. Each compartment was filled with medium with or without Fe.

Figure 2 Northern blot analysis for the detection of NtFROexpression in the roots of non-transformants 7 days after thesplit-root treatment. Lane 1, +/+ treated roots; lane 2, Fe-supplied split roots of the +/– treated plant; lane 3, Fe-deficientsplit roots of the +/– treated plant; lane 4, –/– treated roots.Each lane was loaded with 15 μg of total RNA.

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Pezeshgi 1996; Vert et al. 2003). Our results suggest thatin response to certain systemic signals and the Fe con-centration of the rhizosphere, the HvNAS1 promoterfrom barley exhibited functions similar to those of Fe-acquisition-related genes in tobacco. In contrast, theOsNAS1 promoter in transgenic tobacco plants does not

seem to respond to the Fe concentration of therhizosphere.

Localization of the GUS activity in transgenic tobaccoHistochemical staining was used to compare the tissuespecificity of the promoter activity between the HvNAS1pro::GUS and OsNAS1pro::GUS tobacco lines (Fig. 4).In our previous work, GUS activities were predominantlyobserved in the epidermis of Fe-deficient roots in HvNAS1pro::GUS tobacco plants (Higuchi et al. 2001a). In thepresent experiment, however, the cortex and endodermisof the Fe-deficient roots of HvNAS1pro::GUS tobaccowere stained after incubation for 3 h (Fig. 4A), and allthe root cells of the section were stained after overnightincubation (data not shown). Although these results arepartially inconsistent with those of our previous study(Higuchi et al. 2001a), we discuss the results obtainedin the present study with the T2 homozygous lines. GUSexpression in the Fe-supplied split roots of the +/– treatedHvNAS1pro::GUS tobacco was also dominant in thecortex (Fig. 4B). GUS expression in the Fe-supplied splitroots of the +/– treated HvNAS1pro::GUS tobacco andin the cortex and endodermis of its roots suggests thatthe HvNAS1 promoter might be regulated by mechanismssimilar to those of genes related to Fe acquisition intobacco roots.

The stele in the Fe-deficient roots of the OsNAS1pro::GUS tobacco was stained after incubation for 3 h(Fig. 4C), and all root cells in the section were stainedafter overnight incubation (data not shown). These resultsindicated that the OsNAS1 promoter did not drive GUSactivity in response to the presence of Fe in the rhizo-sphere (Fig. 3) and that it drove GUS activity in the steleof Fe-deficient roots; this suggests that the OsNAS1 pro-moter might be regulated by mechanisms similar to thoseof genes related to the long-distance transport of Fe intransgenic tobacco. The fact that the expression of endog-enous OsNAS1 is induced by Fe deficiency in the vascularbundle of the rice leaf suggests the involvement ofOsNAS1 in the long-distance transport of Fe within theplant body (Inoue et al. 2003).

The leaves of the HvNAS1pro::GUS tobacco werehardly stained regardless of the Fe nutritional status(Fig. 4D). This is consistent with data from our previousstudy (Ito et al. 2007). In contrast, the inter-vein regionof the mature leaves of OsNAS1pro::GUS tobacco wasstained and GUS expression was induced by Fe deficiency(Fig. 4E,F), that is, mature leaves were predominantlystained compared with young leaves that required moreFe. In addition, GUS activity was predominantly observedin mesophyll cells (Fig. 4G). These results suggest thatOsNAS1 promoter activity might be regulated by themechanism for the expression of Fe-retranslocation-

Figure 3 Expression of OsNAS1promoter::GUS and HvNAS1-promoter::GUS in transgenic plants after the split-root treatment.The GUS activities in the roots of the transgenic tobacco plantsare shown (A) 3 days after the treatment, (B) 5 days after thetreatment and (C) 7 days after the treatment. Os, OsNAS1pro-moter::GUS; Hv, HvNAS1promoter::GUS. The numbers indicatethe relative ratio of GUS activity to that in the +/+ treatedplants. MU, 4-methylumbelliferone.

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related genes in transgenic tobacco. Although it is wellknown that the extent of remobilization of Fe is relativelylow in higher plants, substantial retranslocation of Fe fromold leaves to young leaves has been reported in certainplant species (Maruyama et al. 2005; Uauy et al. 2006;Zhang et al. 1995). Under Fe-deficient conditions, certainactive mechanisms for Fe retranslocation from old leavesto young leaves might function in tobacco, and OsNAS1promoter activity might be regulated by such a mecha-nism. GUS activity in the stele of the Fe-deficient rootsof OsNAS1pro::GUS tobacco might also suggest thatOsNAS1 promoter activity might be regulated by someof the mechanisms that remobilize the Fe deposited inthe vascular bundle.

ConclusionsUnexpectedly, OsNAS1pro::GUS in transgenic tobaccodrove the expression of GUS in a manner that was con-siderably different from HvNAS1pro::GUS in transgenictobacco and endogenous OsNAS1 in rice, although it alsoinduced expression in response to Fe deficiency. Theexpression pattern of OsNAS1pro::GUS in transgenictobacco appears to mimic the pattern of some unknownFe-retranslocation-related genes. Both universal compo-nents and a complicated cascade in the regulatory mech-anisms for Fe-deficiency responses have been revealed inhigher plants (Kobayashi et al. 2007). The OsNAS1 pro-moter might be unique in that it possesses numeroustypes of cis sequences involved in Fe metabolism.

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