interspecies compatibility of nas1 gene promoters
TRANSCRIPT
Research article
Plant Physiology and Biochemistry 45 (2007) 270e276www.elsevier.com/locate/plaphy
Interspecies compatibility of NAS1 gene promoters
Satoshi Ito a, Haruhiko Inoue b,c,1, Takanori Kobayashi b,c, Masaaki Yoshiba a,c,Satoshi Mori b, Naoko Nishizawa b,c, Kyoko Higuchi a,c,*
a Laboratory of Plant Production Chemistry, Department of Applied Biological Chemistry, Tokyo University of Agriculture,
1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japanb Laboratory of Plant Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japanc Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo, Japan
Available online 6 April 2007
Abstract
Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying theregulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-actingelements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genesand understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous(Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiencyinduced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS inducedstrong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned inArabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plantsand Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On theother hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency.In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 andOsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compat-ible with some shoot-specific Fe deficiency-related trans-factors in tobacco.� 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Dicotyledonous plants; Fe deficiency; Graminaceous plants; Heterologous expression; Nicotianamine synthase
1. Introduction
Ferric iron has an extremely low solubility in neutral or ba-sic pH conditions and is not readily available to plants [19]. In
Abbreviations: IDEs, iron deficiency-responsive elements; NAS, nicotian-
amine synthase.
* Corresponding author. Laboratory of Plant Production Chemistry, Depart-
ment of Applied Biological Chemistry, Tokyo University of Agriculture, 1-1-1
Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. Fax: þ81 3 5477 2315.
E-mail address: [email protected] (K. Higuchi).1 Present address: Plant Disease Resistance Research Unit, Division of Plant
Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai,
Tsukuba, Ibaraki 305-8602, Japan.
0981-9428/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved
doi:10.1016/j.plaphy.2007.04.001
higher plants, two mechanismsdstrategy I (employed by alldicots and non-graminaceous plants) and strategy II (em-ployed by grasses)dhave been evolved for the acquisition ofFe. In strategy I plants, the reduction of ferric iron to ferrousiron at the root surface is an essential process for Fe uptake.Grasses are strategy II plants and use a chelation mechanismfor Fe uptake. Under conditions of limited Fe availability,strategy II plants synthesize phytosiderophoresdcompoundsthat have a high affinity for ferric irondand secrete theminto the rhizosphere to chelate Fe3þ. Nicotianamine is theprecursor of phytosiderophores and is synthesized fromS-adenosyl-methionine by nicotianamine synthase (NAS)[24]. NAS is induced due to Fe deficiency in strategy II plants;Fe deficiency induces HvNAS1 in the roots of barley (Hordeum
.
271S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
vulgare L.) and OsNAS1 in the shoots and roots of rice (Oryzasativa L.) [10]. Strategy I plants also produce nicotianamine,which is essential for Fe homeostasis [23]; however, in con-trast to the amount of nicotianamine produced in grasses,that produced in strategy I plants does not fluctuate signifi-cantly with the Fe nutritional status. The AtNAS1, AtNAS2,and AtNAS4 genes are induced due to Fe deficiency in theshoots and roots of Arabidopsis thaliana L. [2]. Thus, nicotian-amine and NAS play key roles in the Fe nutrition of all higherplants, but their regulation mechanisms differ among plants.
Previously, we had proposed that a simple process of adap-tation to Fe deficiency occurs in the NAS gene family [10].Since then, it has been revealed that nicotianamine plays keyroles in Fe homeostasis in both rice and dicots [11,25]; thisfinding was accompanied by the elucidation of the yellowstripe-like (YSL) family transporters as metalenicotianaminetransporters [17,28]. Thus, accurate regulation of NAS geneexpression is necessary for both Fe acquisition anddistribution.
Recently, cis-acting elementsdthe iron deficiency-respon-sive elements IDE1 and IDE2 [14]dand the transcription fac-tors FER [18] and FIT1 [6] associated with Fe-deficiencyresponse have been investigated. Homologous sequences ofIDE1 and IDE2 are widely distributed among Fe deficiency-inducible genes, including NAS genes [15]. Therefore, weaimed to verify the interspecies compatibility of the Fe-defi-ciency response in NAS1 genes and understand the universalmechanisms that regulate the expression patterns of these Fedeficiency-inducible genes in plants. Thus, we introduced gra-minaceous (H. vulgare L. and O. sativa L.) NAS1 promoter::GUSinto dicots (Nicotiana tabacum L. and A. thaliana L.), and weexamined its heterologous expression in response to Fedeficiency.
2. Materials and methods
2.1. HvNAS1 and OsNAS1 promoter constructs
The promoter fragments of HvNAS1 [9] and OsNAS1 [11]that we had used previously were subcloned in the upstreamopen reading frame of the uidA gene; this gene encodesGUS in the pCB308 vector [29].
2.2. Plant transformation
The HvNAS1 and OsNAS1 promoter constructs were usedfor Agrobacterium tumefaciens strain C-58-mediated transfor-mation. Transformation and regeneration of N. tabacum L. cv.Petit-Havana SR1 were performed using the standard leaf-disctransformation method [7]. A. thaliana L cv. Columbia wastransformed using the floral dip method [5].
2.3. Growth conditions
Wild-type tobacco seedlings and T1 transformants weregrown on MurashigeeSkoog (MS) medium containing 3%sucrose, 0.2% gellan gum, and 100 mg L�1 kanamycin for
14 days at 25 � 1 �C under a 16-h light (300 mmol s�1 m�2
mA�1)/8-h dark cycle. Wild-type Arabidopsis seedlings andT1 transformants were grown on MS medium containing 3%sucrose, 0.2% gellan gum, and 100 mg L�1 bialaphos for10 days at 23 � 1 �C under a 14-h light (45 mmol s�1 m�2 mA)/10-h dark cycle. The plantlets were transplanted to MS me-dium without Fe for Fe-deficient treatment and with Fe forFe-sufficient treatment. For each line, the roots and leaves oftwo plantlets were harvested 3, 5, or 7 days after each treat-ment, and they were used for the GUS assay and Northernblot analysis.
2.4. Fluorometric analysis of GUS activity
GUS activity was assayed according to Kobayashi et al.[14]. Enzyme activity was fluorometrically measured using4-methylumbelliferyl-b-D-glucuronide as the substrate, andthe reaction product 4-methylumbelliferone (MU) was de-tected. The protein concentration was determined with aBio-Rad Protein Assay Kit (Bio-Rad Laboratories).
2.5. AtFRO2 quantitative RTePCR
Primer sets corresponding to AtFRO2 and UBQ10 describedin a previous study [21] were used. The genes were amplifiedusing LightCycler-RNA Amplification Kit SYBR Green I andthe LightCycler Instrument (Roche Diagnostics). Data wereanalyzed according to the manufacturer’s instructions. Serialdilutions of each total RNA sample were subjected to quanti-tative RTePCR. The quantitatively meaningful data wereadapted to the subsequent analysis. The amount of totalRNA was calibrated by UBQ10 data. To normalize the AtFRO2expression, AtFRO2 data were divided by calibrated total RNAng, and the level of AtFRO2 expression was then representedby a relative value.
2.6. NtFRO probe preparation
To obtain the Fe(III)-chelate reductase gene fragment fromtobacco (NtFRO) cDNA, a pairs of primersdlefro1-f (50-GCTGTCTTGGGATGTGTATACCT-30) and lefro1-r (50-TGTCTCATCCTTTTTGGGCCACACAC-30)dwere designed fromLeFRO1 (accession number AY224079) and used for PCR.Using the guanidine method, poly(A)þ RNA was extractedfrom Fe-deficient A. thaliana and tobacco shoots and roots[4]. cDNAs were synthesized using the Poly A Tract mRNAsystem (Promega) and a cDNA Cycle Kit (Invitrogen), andthey were used as templates for PCR. The amplified fragmentswere cloned into pT7Blue (Novagen) and sequenced usinga Big Dye Terminator v3.1 Cycle Sequencing Kit (ABIPRISM) and the DNA sequencer ABI PRISM 3100-AvantGenetic Analyzer (Applied Biosystems).
2.7. Northern blot analysis
Northern blot analysis was conducted using probes for At-FRO2 and NtFRO labeled with [32P]dCTP by using Random
272 S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
Primer Labeling Kit Ver. 2 (TaKaRa). Total RNA was ex-tracted from the plant roots and leaves by using the RNeasyPlant Mini Kit (Qiagen). As described in a previous method,15 mg of total RNA was separated, blotted, and hybridizedwith the probes at 65 �C [8]. Radioactivity was then detectedusing a BAS-5000 image analyzer (Fuji Film).
3. Results
3.1. Expression of graminaceous NAS1 promoter::GUSin transgenic Arabidopsis plants
HvNAS1 and OsNAS1 promoter::GUS fusions were intro-duced in Arabidopsis plants. The GUS activity of transgenicArabidopsis plants was fluorometrically measured 5 days afterFe-deficient or Fe-sufficient treatment (Fig. 1). The GUS ac-tivity was found to be slightly increased in some lines of thetransgenic Arabidopsis plants under Fe-deficient conditions,and there were significant differences in its induction;
Hv Os
348-2
348-4
348-6 5432NT
GU
S activity
(p
mo
l M
U m
in
–1m
g–1p
ro
tein
)
GU
S activity
(p
mo
l M
U m
in
–1m
g–
1p
ro
tein
)
Hv Os
348-2
348-4
348-65432NT
0
5
10
15
20
0
100
200
300
400
B
A
1.1
1.5
1.8
1.9 1.8
0.9
1.2
2.0
2.2
2.0
1.4
3.6 ***
2.1 **
1.2
2.4 *
2.7 **
Fig. 1. Expression of graminaceous NAS1 promoter::GUS in transgenic Arabi-
dopsis plants. The GUS activity in the shoots (A) and roots (B) of Fe-deficient
(closed rectangles) or Fe-sufficient (open rectangles) transgenic Arabidopsisplants on day 5 after Fe-deficient treatment. Data are mean � SE (n ¼ 3).
NT, non-transformant; Hv, HvNAS1 promoter::GUS; Os, OsNAS1 promoter::GUS;
MU, 4-methylumbelliferone. The numerals indicate the relative ratio of GUS
activity in Fe-deficient plants to that in Fe-sufficient plants, and the asterisks indicate
significant differences between Fe-sufficient and Fe-deficient conditions as
determined using the non-parametric Fisher test (*P < 0.05, **P < 0.01,
***P < 0.001). The numerals on the horizontal axis indicate independent trans-
genic lines.
however, this activity was only two-fold higher than that innon-transformants (Fig. 1B). The GUS activities of the trans-formants were not higher than those of the non-transformantseven 7 days after the treatment (data not shown). To confirmFe deficiency in the Arabidopsis plants, we analyzed the ex-pression of Fe deficiency-inducible AtFRO2 by using thequantitative reverse-transcription polymerase chain reaction(RTePCR). The results showed that AtFRO2 expression wasstrongly induced in the roots of Fe-deficient Arabidopsisplants (Table 1). Thus, the HvNAS1 and OsNAS1 promotersdid not induce GUS activity by inducing Fe deficiency inthe transformed Arabidopsis plants.
3.2. Expression of OsNAS1 promoter::GUS in transgenictobacco plants
We did not detect any compatibility between the gramina-ceous NAS1 promoters and Arabidopsis genes. However, wereported on the GUS activities induced by the HvNAS1 pro-moter in tobacco [9]. To further confirm the compatibilities be-tween the NAS1 promoters of tobacco and graminaceousplants, OsNAS1 promoter::GUS fusion was introduced in to-bacco plants. The GUS activities of the transgenic tobaccoplants were fluorometrically measured 3, 5, and 7 days afterFe-deficient or Fe-sufficient treatment (Fig. 2AeF). Due toFe deficiency, the GUS activity was induced in the shoots3 days after Fe-deficient treatment (Fig. 2A), but it was verylow in the roots (Fig. 2B). After 5 days of Fe-deficient treat-ment, the GUS activity was induced in the shoots (Fig. 2C)and roots of all lines (Fig. 2D). The GUS activity increasedin transgenic tobacco roots 7 days after Fe-deficient treatment(Fig. 2F); in the shoots, the activity was slightly higher thanthat compared on day 5 (Fig. 2E). Thus, the shoots are the pri-mary sites of the OsNAS1 promoter response to Fe deficiencyin transgenic tobacco plants. To confirm Fe deficiency in thetobacco plants, we performed a Northern blot analysis by us-ing the Fe deficiency-inducible putative ferric chelate reduc-tase gene NtFRO as the probe. The analysis demonstratedthat NtFRO (2.7 kbp) was induced in the roots of Fe-deficienttobacco (Fig. 2G).
3.3. Comparison of the response between the OsNAS1and HvNAS1 promoters in transgenic tobacco plants
In this study, the GUS activity was induced in both the rootsand shoots of OsNAS1 promoter::GUS tobacco plants due toFe deficiency (Fig. 2), whereas it was induced only in the rootsof HvNAS1 promoter::GUS tobacco plants [9]. We confirmed
Table 1
Relative expression level of AtFRO2 in Arabidopsis plants used in the exper-
iment depicted in Fig. 1
Shoot Roots
Fe þ Fe � Fe þ Fe �1.00 1920 1.45 209000
273S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
–7d+7d
S S R R
EtBr
NtFRO
(putative, 2.7 Kb)
G
65432NT
0
4
8
12
16
20
1.2
30.5
20.8
797
296
93.5
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)A
0
0.5
1.0
1.5
2.0
2.5
3.0
2.80.9
1.2
3.4
4.0
20.1***
B
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)
65432NT
0
4
8
12
16
20
0.8
25.1***
11.6***
43.1**
12.2***
5.2* *
C
65432NT
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)
10.5**
12.7 *
1.35.6
4.0
D
19.3**
0
0.5
1.0
1.5
2.0
2.5
3.0
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1p
ro
tein
)65432NT
0
4
8
12
16
20
1.2
42.9**
18.0***
134
47.2**
37.9 *
E
65432NT
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)
0
4
8
12
16
20
1.8
38.3***
3.4
7.4
29.3**
45.8**
F
65432NT
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)
Fig. 2. Expression of OsNAS1 promoter::GUS in transgenic tobacco plants after Fe-deficient treatment. A, C, and E: shoots; B, D, and F: roots. A and B,
day 3; C and D, day 5; E and F, day 7. NT, non-transformant; MU, 4-methylumbelliferone. The other information is the same as that in Fig. 1. G shows
the Northern blot analysis. The response of NtFRO expression in shoots (S) and roots (R) of tobacco plants 7 days after Fe-deficient (�7d) or Fe-sufficient
(þ7d) treatment.
this difference by cultivating the plants under similar cultureconditions (Fig. 3). A transgenic tobacco plant containing1760 constructs [9] was used. In the roots, the induction rateof the HvNAS1 promoter due to Fe deficiency was higherthan that of the OsNAS1 promoter (Fig. 3B). This result isconsistent with the observation that the induction of HvNAS1in Fe-deficient barley roots is higher than that of OsNAS1in rice.
4. Discussion
4.1. Compatibility between the NAS1 promotersof Arabidopsis and graminaceous plants
We introduced the OsNAS1 and HvNAS1 promoters intoArabidopsis plants (Table 2). Under Fe-deficient conditions,the GUS activity was slightly increased in some lines of the
274 S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
transgenic Arabidopsis plants (Fig. 1B). These results showedthat the compatibility of graminaceous NAS1 promoter withthe Arabidopsis gene is very low. Vasconcelos et al. introduceda genomic clone of AtFRO2 that has a 0.6-kb promoter regioninto rice. However, AtFRO2 expression was not detected byRTePCR (Table 2, [26]). This result suggests that the se-quences or disposition patterns of the cis-elements that partic-ipate in Fe deficiency-responsive regulation differ to someextent between Arabidopsis and grasses. On the other hand,the �305 IDS3 promoter from barley induced GUS activityin the roots of both Arabidopsis and tobacco transformantsunder Fe-deficient conditions [16]. Therefore, some Fe defi-ciency-inducible promoters in graminaceous plants may becompatible with those in Arabidopsis, while some othersmay not.
The OsNAS1, HvNAS1, and AtFRO2 promoter regions(Table 2) contain IDE1- or IDE2-like sequences [14]. TheFe deficiency-inducible promoters of Arabidopsis are rich inIDE1-like sequences [15]. However, 1 kb of the AtIRT1
0
2.5
5.0
7.5
10.0
0
1
2
3
4
5
x5.7**
x0.8x0.9
A
Os Hv
GU
S activity
(n
mo
l M
U m
in
–1 m
g–1 p
ro
tein
)
x63.1**
x1.0
x7.7
B
Os Hv
GU
S activity
(n
mo
l M
U m
in
–1 m
g–
1 p
ro
tein
)
NT
NT
Fig. 3. Comparison of the expression pattern between the OsNAS1 and
HvNAS1 promoters in transgenic tobacco plants. The GUS activity in the
shoots (A) and roots (B) of transgenic tobacco plants 7 days after Fe-deficient
treatment. NT, non-transformant; Hv, HvNAS1 promoter::GUS; Os, OsNAS1promoter::GUS; MU, 4-methylumbelliferone. The other information is the
same as that in Fig. 1.
promoter region that did not contain an IDE1-like sequencealso induced GUS activity in Arabidopsis under Fe-deficientconditions [14,27]. Thus, an IDE1-like sequence is not a requi-site for Fe deficiency-responsive expression in Arabidopsis.On the other hand, multiple and highly homologous IDE1-and IDE2-like sequences are found in the IDS3 promoter[16], although the OsNAS1 and HvNAS1 promoters, whichdid not induce GUS activity in Arabidopsis, do not possessthe highly homologous IDE1-like sequence (data not shown).Some Fe deficiency-related transcription factors could hetero-geneously interact with IDE1 from graminaceous plants andinduce GUS activity in Arabidopsis.
FER from tomato [3,18] and FIT1/FRU/AtbHLH29 fromArabidopsis [6,13], which are categorized as basic helixeloopehelix (bHLH) proteins, were cloned as the Fe deficiency-responsive transcription factors. These factors participate inthe transcription of AtFRO2 and AtIRT1 in Arabidopsis underFe-deficient conditions, and the E-box motif 50-CANNTG-30da potential FIT1 binding sitedis found in the promoterregions of these genes [6]. FER is highly homologous toFIT1, and both tobacco and tomato belong to the family Sol-anaceae. Thus, tobacco may possess an orthologue of FIT1.However, we demonstrated that the OsNAS1 and HvNAS1promoters functioned in tobacco but not in Arabidopsis. Re-cently, rice OsIRO2, which is also categorized as a bHLHprotein, was cloned as an Fe deficiency-responsive transcrip-tion factor. Its homologue HvIRO2 from barley was also ex-pressed under Fe-deficient conditions [22]. These proteinsbelong to a different group and recognize different sequencesfrom the FER and FIT1/FRU/AtbHLH29 proteins [22]. Sometranscription factors other than the FER-like protein could in-teract with graminaceous NAS1 promoters and induce GUSactivity in tobacco under Fe-deficient conditions. Althoughthe basic molecular mechanisms of the Fe deficiency responsein higher plants may be analogous among species, the homol-ogy of the molecules that participate in these mechanismsmay not always be high, and their compatibility may be low.
4.2. Regulation of the OsNAS1 promoter in tobacco
Under Fe-deficient conditions, the GUS activity of HvNA-S1pro::GUS tobacco roots was higher than that of OsNA-S1pro::GUS tobacco roots (Fig. 3B). This result is consistentwith the observation of higher HvNAS1 induction in Fe-defi-cient barley roots than OsNAS1 induction in rice [10]. Thismay correspond to the substantial synthesis and secretion ofthe mugineic acid family phytosiderophores in barley. How-ever, the GUS activity induced by the OsNAS1 promoter intobacco was less than that in rice (Fig. 2). It is possible thatsome Fe deficiency-responsive transcription factor is capableof interacting with the Fe deficiency-responsive cis-elementsin graminaceous NAS1 promoters, but its affinity is lowbecause of heterologous expression.
HvNAS1 is strongly expressed only in the Fe-deficientroots of barley, whereas OsNAS1 is expressed in both theleaves and roots; it is expressed weakly under Fe-sufficientconditions and strongly expressed under Fe-deficient
275S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
Table 2
Expression patterns of promoters in various plant species under Fe-sufficient or Fe-deficient conditions
Origin of promoter
(length transformed)
Host plant Barley Rice Tobacco Arabidopsis
Fe (mM) in medium 100 0 100 0 100 0 100 0
HvNAS1 Shoot � � þ þþ � � � �(1.9 kb in rice) Root � þþþ þ þþ � þþþ � �(1.7 kb and 348 b in tobacco) Detection method 1 b 1 b 2 c 2 a
(348 b in Arabidopsis)
OsNAS1 Shoot þ þþ þ þþ � �(1.6 kb) Root þþ þþþ þ þþ � �
Detection method 1 b 2 a 2 a
AtFRO2 Shoot � � � þþ(0.6 kb) Root � � � þþ
Detection method 3 d 1 a
Putative NtFRO Shoot � �Root � þþDetection method 1 a
Detection method: 1, Northern blot analysis; 2, GUS activity; 3, RTePCR.
�: GUS activity <0.1 nmol min�1 mg�1 protein or Northern signal not observed; þ: GUS activity 0.1e10 nmol min�1 mg�1 protein or weak Northern signal
observed.; þþ: GUS activity 10e50 nmol min�1 mg�1 protein or Northern signal observed; þþþ: GUS activity >50 nmol min�1 mg�1 protein or strong North-
ern signal observed.
Source: a, in this work; b, Higuchi et al. [10]; c, Higuchi et al. [9]; d, Vasconcelos et al. [26].
conditions, and its level of expression in leaves is lower thanthat in roots [10]. The expression pattern of GUS in HvNA-S1pro::GUS and OsNAS1pro::GUS tobacco plants was similarto those in barley and rice, respectively (Fig. 3, Table 2). How-ever, in OsNAS1pro::GUS tobacco plants, the primary site ofthe Fe deficiency response was the leaf, and the level ofGUS activity in the leaves was identical to that in the rootsat day 7 of Fe-deficiency treatment (Fig. 2). These resultsdid not correspond to the expression pattern of OsNAS1 inFe-deficient rice, i.e., the mRNA level of OsNAS1 in Fe-defi-cient roots was higher than that in the leaves [10]. In tobacco,some sequences that can interact with the Fe deficiency-re-sponsive transcription factors that are specific to the shootsmay exist within the OsNAS1 promoter region. Consideringall these results, it is also possible that transcription factorswith a low transcription activity heterogeneously interactwith the OsNAS1 promoter in tobacco.
4.3. Conclusion
The interspecies compatibility of the Fe-deficiency re-sponse of the NAS1 genes was not always high. The mecha-nisms of Fe-deficiency response in higher plants are of twotypes: strategy I and strategy II. Although both rice and barleyhave been categorized as plants that employ strategy II, Ishi-maru et al. reported that the uptake of Fe2þ in rice plants isessentially similar to that in plants using strategy I [12]. Mar-uyama et al. reported that the distribution and retranslocationmechanisms of internal Fe differed between rice and barley[20]. The differences in the molecular mechanisms of theFe-deficiency response may not be small even between riceand barley. Moreover, the genome project of Micro-tom, themodel plant of the Solanaceae, has revealed that many
Micro-tom clones with no homology to Arabidopsis geneswere particularly categorized as kinases, and they possessedDNA-/RNA- and protein-binding functions [1]. The differ-ences in these molecular mechanisms may not be small be-tween Arabidopsis and tobacco.
Further investigation of the interspecies compatibility of thecis-elements or trans-factors involved in the Fe deficiency-responsive molecular mechanisms may reveal that some ofthese mechanisms are common ancestral mechanisms, whileeach plant species has also evolutionally acquired distinctmechanisms.
References
[1] K. Aoki, K. Yano, K. Suda, T. Suzuki, Y. Kaneko, T. Tsugane,
M. Watanabe, N. Sakurai, H. Suzuki, D. Shibata, Full-length cDNA
clones from tomato leaf and fruit: a comparison with Arabidopsis genes,
Plant Cell Physiol. 45 suppl. (2006) s83 237 (2aA01).
[2] P. Bauer, T. Thiel, M. Klatte, Z. Bereczky, T. Brumbarova, R. Hell,
I. Grosse, Analysis of sequence, map position, and gene expression
reveals conserved essential genes for iron uptake in Arabidopsis and
tomato, Plant Physiol. 136 (2004) 4169e4183.
[3] T. Brumbarova, P. Bauer, Iron-mediated control of the basic helix-loop-
helix protein FER, a regulator of iron uptake in tomato, Plant Physiol.
137 (2005) 1018e1026.
[4] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid
guanidinium thiocyanatephenolchloroform extraction, Anal. Biochem.
162 (1987) 156e159.
[5] S.J. Clough, A.F. Bent, Floral dip: a simplified method for Agrobacte-
rium-mediated transformation of Arabidopsis thaliana, Plant J. 16
(1998) 735e743.
[6] E.P. Colangelo, M.L. Guerinot, The essential basic helix-loop-helix pro-
tein FIT1 is required for the iron deficiency response, Plant Cell 16
(2004) 3400e3412.
[7] G. Helmer, M. Casadaban, M. Bevan, L. Kayes, M.D. Chilton, A new
chimeric gene as a marker for plant transformation: the expression of
276 S. Ito et al. / Plant Physiology and Biochemistry 45 (2007) 270e276
Escherichia coli beta-galactosidase in sunflower and tobacco cells, Bio/
Technology 2 (1984) 520e527.
[8] K. Higuchi, K. Suzuki, H. Nakanishi, H. Yamaguchi, N.K. Nishizawa,
S. Mori, Cloning of nicotianamine synthase genes, novel genes involved
in the biosynthesis of phytosiderophores, Plant Physiol. 119 (1999)
471e479.
[9] K. Higuchi, M. Tani, H. Nakanishi, T. Yoshihara, F. Goto,
N.K. Nishizawa, S. Mori, The expression of a barley HvNAS1 nicotian-
amine synthase gene promoter-gus fusion gene in transgenic tobacco is
induced by Fe-deficiency in roots, Biosci. Biotechnol. Biochem. 65
(2001) 1692e1696.
[10] K. Higuchi, S. Watanabe, M. Takahashi, S. Kawasaki, H. Nakanishi,
N.K. Nishizawa, S. Mori, Nicotianamine synthase gene expression dif-
fers in barley and rice under Fe-deficient conditions, Plant J. 25 (2001)
159e167.
[11] H. Inoue, K. Higuchi, M. Takahashi, H. Nakanishi, S. Mori,
N.K. Nishizawa, Three rice nicotianamine synthase genes, OsNAS1,
OsNAS2, and OsNAS3 are expressed in cells involved in long-distance
transport of iron and differentially regulated by iron, Plant J. 36 (2003)
366e381.
[12] Y. Ishimaru, M. Suzuki, T. Tsukamoto, K. Suzuki, M. Nakazono,
T. Kobayashi, Y. Wada, S. Watanabe, S. Matsuhashi, M. Takahashi,
H. Nakanishi, S. Mori, N.K. Nishizawa, Rice plants take up iron as an
Fe3þ phytosiderophore and as Fe2þ, Plant J. 45 (2006) 335e346.
[13] M. Jakoby, H.Y. Wang, W. Reidt, B. Weisshaar, P. Bauer, FRU
(BHLH029) is required for induction of iron mobilization genes in
Arabidopsis thaliana, FEBS Lett. 577 (2004) 528e534.
[14] T. Kobayashi, Y. Nakayama, R.N. Itai, H. Nakanishi, T. Yoshihara,
S. Mori, N.K. Nishizawa, Identification of novel cis-acting elements,
IDE1 and IDE2, of the barley IDS2 gene promoter conferring iron-
deficiency-inducible, root-specific expression in heterogeneous tobacco
plants, Plant J. 36 (2003) 780e793.
[15] T. Kobayashi, M. Suzuki, H. Inoue, R.N. Itai, M. Takahashi,
H. Nakanishi, S. Mori, N.K. Nishizawa, Expression of iron-acquisition-
related genes in iron-deficient rice is co-ordinately induced by partially
conserved iron-deficiency-responsive elements, J. Exp. Bot. 56 (2005)
1305e1316.
[16] T. Kobayashi, T. Yoshihara, R.N. Itai, H. Nakanishi, M. Takahashi,
S. Mori, N.K. Nishizawa, Promoter analysis of iron-deficiency-inducible
barley IDS3 gene in Arabidopsis and tobacco plants, Plant Physiol.
Biochem. 45 (2007) 262e269.
[17] S. Koike, H. Inoue, D. Mizuno, M. Takahashi, H. Nakanishi, S. Mori,
N.K. Nishizawa, OsYSL2 is a rice metal-nicotianamine transporter that
is regulated by iron and expressed in the phloem, Plant J. 39 (2004)
415e424.
[18] H.Q. Ling, P. Bauer, Z. Bereczky, B. Keller, M. Ganal, The tomato Fer
gene encoding a bHLH protein controls iron-uptake responses in roots,
Proc. Natl. Acad. Sci. USA 99 (2002) 13938e13943.
[19] H. Marschner, Mineral Nutrition of Higher Plants, 2nd Edition,
Academic Press, New York, 1995.
[20] T. Maruyama, K. Higuchi, M. Yoshiba, T. Tadano, Comparison of iron
availability in leaves of barley and rice, Soil Sci. Plant Nutr. 51 (2005)
1035e1042.
[21] I. Mukherjee, N.H. Campbell, J.S. Ash, E.L. Connolly, Expression
profiling of the Arabidopsis ferric chelate reductase (FRO) gene family
reveals differential regulation by iron and copper, Planta. 223 (2006)
1178e1190.
[22] Y. Ogo, R.N. Itai, H. Nakanishi, H. Inoue, T. Kobayashi, M. Suzuki,
M. Takahashi, S. Mori, N.K. Nishizawa, Isolation and characterization
of IRO2, a novel iron-regulated bHLH transcription factor in gramina-
ceous plants, J. Exp. Bot. 57 (2006) 2867e2878.
[23] G. Scholz, R. Becker, A. Pich, U.W. Stephan, Nicotianamineda
common constituent of strategies I and II of iron-acquisition by plants:
a review, J. Plant Nutr. 15 (1992) 1647e1665.
[24] S. Shojima, N.K. Nishizawa, S. Fushiya, S. Nozoe, T. Irifune, S. Mori,
Biosynthesis of phytosiderophores: in vitro biosynthesis of 20-deoxymu-
gineic acid from l-methionine and nicotianamine, Plant Physiol. 93
(1990) 1497e1503.
[25] M. Takahashi, Y. Terada, I. Nakai, H. Nakanishi, E. Yoshimura, S. Mori,
N.K. Nishizawa, Role of nicotianamine in the intracellular delivery of
metals and plant reproductive development, Plant Cell 15 (2003)
1263e1280.
[26] M. Vasconcelos, V. Musetti, C.M. Li, S.K. Datta, M.A. Grusak, Func-
tional analysis of transgenic rice (Oryza sativa, L.) transformed with
an Arabidopsis thaliana ferric reductase (AtFRO2), Soil Sci. Plant
Nutr. 50 (2004) 1151e1157.
[27] G. Vert, N. Grotz, F. Dedaldechamp, F. Gaymard, M.L. Guerinot,
J.F. Briat, C. Curie, IRT1, an Arabidopsis transporter essential for iron
uptake from the soil and for plant growth, Plant Cell 14 (2002) 1223e
1233.
[28] E.L. Walker, Functional analysis of the Arabidopsis yellow stripe-like
(YSL) family. Heavy metal transport and partitioning via metal-nicotian-
amine (NA) complexes, Plant Physiol. 129 (2002) 431e432.
[29] C. Xiang, P. Han, I. Lutziger, K. Wang, D.J. Oliver, A mini binary
vector series for plant transformation, Plant Mol. Biol. 40 (1999)
711e717.