Allocation of Fe and ferric chelate reductase activities in mesophyll cells of barley and sorghum under Fe-deficient conditions

Plant Physiology and Biochemistry 49 (2011) 513e519

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Research article

Allocation of Fe and ferric chelate reductase activities in mesophyll cells of barley and sorghum under Fe-deficient conditions Yuichiro Mikami, Akihiro Saito 1, Eitaro Miwa, Kyoko Higuchi* Laboratory of Plant Production Chemistry, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 October 2010 Accepted 6 January 2011 Available online 14 January 2011

Although the photosynthetic apparatus requires large amounts of Fe, the adaptive mechanisms of mesophyll cells for Fe acquisition under Fe-deficient conditions are unknown. Barley and sorghum, which are tolerant and susceptible to Fe deficiency, respectively, have similar Fe and chlorophyll contents in their leaves. However, the Fe-deficient barley photosynthetic apparatus was functional while that of sorghum was not. We show that barley preferentially allocates Fe to thylakoid membranes under Fe-deficient conditions. On the other hand, in sorghum, the proportion of leaf Fe allocated to thylakoids was not altered by Fe deficiency. The relationship between the maintenance of photosynthesis and light-dependent ferric chelate reductase activity on plasma membranes and chloroplast envelopes is also discussed. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Barley Chloroplast Fe deficiency Ferric chelate reductase Sorghum Thylakoid

1. Introduction Fe is usually in insoluble forms in both soil and plant tissues, and is not available for biological processes under oxidant conditions. Photosynthesis requires large amounts of Fe, and a large proportion of leaf Fe is localized in chloroplasts [1,2]. Fe deficiency largely disrupts photosynthesis [3]; thus, plant chloroplasts must be equipped with mechanisms for tolerating Fe deficiency. However, the process by which Fe is supplied to chloroplasts is only beginning to be elucidated; meanwhile, the molecular mechanisms of Fe acquisition and transportation from soil to shoot are better clarified [4]. In Arabidopsis, it is known that ferric chelate reductase (FCR), AtFRO7 [5], and a permease, PIC1 [6], are involved in chloroplast Fe acquisition. Recently, we found that barley, which is tolerant to Fe deficiency, not only secretes a large amount of mugineic acid family phytosiderophores for the efficient acquisition of Fe [7] but also protects chloroplasts from serious damage caused by prolonged Fe deficiency [8]. Consequently, photosynthetic performance in barley is decreased by Fe deficiency, but photosynthesis is not completely stopped. However, in addition to the protection of photosynthetic Abbreviations: FCR, ferric chelate reductase; ETR, electron transfer rate; NPQ, nonphotochemical quenching. * Corresponding author. Tel./fax: þ81 3 5477 2315. E-mail address: [email protected] (K. Higuchi). 1 Present address: Laboratory of Plant Nutrition and Fertilizers, Department of Agricultural Chemistry, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.01.009

apparatus, the supply of a minimum amount of Fe to chloroplasts is mandatory for a strong tolerance to Fe deficiency because both photosystems require large amounts of Fe to function. Efficient Fe supply to chloroplasts cannot be explained only by the acquisition of a large amount of Fe from soil, because the Fe concentration in Fe-deficient barley leaves is as low as that of rice, which is susceptible to Fe deficiency [9]. Thus, barley should have a better preferential Fe transport to the chloroplasts under Fe-deficient conditions than rice. In this study, we show that barley preferentially allocates Fe to thylakoid membranes under Fe-deficient conditions. In sorghum, which is susceptible to Fe deficiency and whose photosynthesis is severely damaged under Fe-deficient conditions, the proportion of thylakoid Fe among leaf tissues was not altered by Fe deficiency. The relationship between the protection of photosynthesis and the light-dependent FCR activity on plasma membranes and chloroplast envelopes is also discussed. 2. Materials and methods 2.1. Plant materials Barley (Hordeum vulgare L. “Ehimehadaka No. 1”) and sorghum (Sorghum bicolor L. Moench “Keller” [sweet sorghum]) seeds were germinated on moist filter paper; the seedlings were grown hydroponically in a growth chamber before the Fe deficiency treatment as described previously [10]. Barley was grown at 24
C day/20
C night and sorghum at 28
C day/25
C night; both

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were kept under a light intensity of 200 mmol photon m2 s1 photosynthetic photon flux density (PPFD) and under a 14/10 h light/dark cycle. The seedlings were grown in half-strength standard nutrient solution as described in Maruyama et al. [9], with 30 mM Fe(III)-ethylenediamine-N,N,N0 ,N0 -tetraacetic acid (EDTA). When the second leaf of barley or the third leaf of sorghum appeared, plants were transferred into a newly prepared hydroponic solution containing 0.3 (Fe-deficient condition) or 30 (control) mM Fe(III)-EDTA. 2.2. Photosynthetic rates and PAM chlorophyll fluorescence The photosynthetic rates of fully expanded leaves with sufficient area were measured. Leaf gas-exchange measurements were coupled with measurements of pulse amplitude modulation (PAM) chlorophyll fluorescence, using an open gas-exchange system (LI6400XT; LICOR Inc., Lincoln, NE) with an integrated fluorescence chamber head (LI-6400-40 leaf chamber fluorometer; LICOR Inc.). The CO2 assimilation rate (AN) (mmol CO2 m2 s1) was measured by using the light-curve mode of the stored program of the fluorescence chamber head. Details of other measured parameters are described in the Materials and methods section of our previous report [8]. The CO2 assimilation rate of C4 plant sorghum was corrected following the manufacturer’s instructions. 2.3. Isolation of protoplasts, chloroplasts, and thylakoid membranes Protoplasts were isolated as described by Gardestrom and Wigge [11] and González-Vallejo et al. [12]. Chloroplasts were isolated as described by Smith et al. [13], Gardestrom and Wigge [11], and Jenkins and Boag [14]. Thylakoid membranes were isolated as described by Smith et al. [13] and Bughio et al. [15]. The details of these procedures are described in the Supplementary materials section. In brief, to release protoplasts, fresh leaves were finely chopped with a sharp blade, and cell walls were digested with cellulase and pectinase. Protoplasts were disrupted using a 20-mm mesh to release chloroplasts. Chloroplasts were purified with 15e80% Percoll gradient centrifugation. Purified chloroplasts were disrupted by osmotic shock to obtain thylakoid membranes. The integrity and numbers of protoplasts and chloroplasts were assessed by light microscopy (Suppl. Fig. 1). Before the measurement of FCR activity, the intactness of protoplasts was confirmed using Evans Blue dye [16]. The total protein contents of thylakoid membranes were measured using the bicinchoninic acid (BCA) method [17]. 2.4. Determination of Fe

Vallejo et al. [12] and Oki et al. [19]. The concentration of protoplasts was adjusted to 2.0  105/400 mL. One milliliter reaction buffer (0.5 M sorbitol, 2.25 mM KHCO3, 750 mM bathophenanthroline disulfonic acid [BPDS], and a 0.05 volume of 10  MES; see Supplementary materials), 400 mL protoplast suspension, and 100 mL 7.5 mM Fe(III)-EDTA were mixed and incubated at 20
C under LED lighting (white color: 430e680 nm, 100 mmol photon m2 s1) or in the dark for 30 min. The reaction mixtures were gently resuspended twice with a pipette during incubation. After 30 min, the reaction mixtures were immediately centrifuged with 100g for 1 min at 4
C. The absorbance of the supernatants at 535 nm was measured within 15 min. An FeSO4 standard was used for calibration. The measurement of chloroplast FCR activity was performed as described by Jeong et al. [5]. BPDS (450 mM) in HS buffer (see Supplementary materials) was used as a reaction buffer in addition to Fe(III)-EDTA (1.5 mM) in HS buffer. The temperature, light, and reaction time were the same as described above. The reaction mixtures were immediately centrifuged at 2000g for 5 min at 4
C. 2.7. Cloning of HvFRO genes and RT-PCR Total RNA from barley and sorghum leaves was isolated using the RNeasy Plant Mini Kit (QIAGEN, Tokyo, Japan). cDNA was synthesized using the PrimeScript 1st-strand cDNA synthesis kit with the supplied oligo-dT primer (Takara Bio Inc., Ohtsu, Japan). First, the partial PCR products from barley were obtained; FROlike genes were amplified with the FRO1 (TaFRO1-F þ TaFRO1-R) and FRO2 primer sets (TaFRO2-F2 þ OsFRO2-R) (Table 1) using Ehimehadaka No. 1 cDNA as the template. We then amplified the 50 and 30 flanking regions of HvFRO1 and HvFRO2 fragments by using inverse PCR with the following primer sets: HvFRO1-InvN1 þ HvFRO1-InvC1, HvFRO1-InvN2 þ HvFRO1-InvC2, and HvFRO2-InvN1 þ HvFRO2InvC1. The open reading frames (ORFs) of HvFRO1 and HvFRO2 were amplified with the following primer sets: HvFRO1-N-XbaI þ HvFRO1C and HvFRO2-N-BamHI þ HvFRO2-C, using Ehimehadaka No.1 cDNA as a template. We sequenced several PCR products for confirmation and registered these sequences as DDBJ accessions AB564557 and AB564558. We used KOD FX DNA polymerase (Toyobo, Osaka, Japan) for amplification. We performed RT-PCR with KOD FX DNA polymerase. The FRO1 (TaFRO1-F þ TaFRO1-R) and FRO2 (TaFRO2-F2 þ OsFRO2-R) primer sets were used at annealing temperatures of 56
C and 60
C, respectively. To calibrate the quantities of the cDNA templates, an amplified cDNA fragment of EF1a was used.

Dried leaves and thylakoid membranes were digested in a solution of concentrated HNO3 at 150
C and dissolved in 1% HNO3. Fe contents were subsequently measured with an atomic absorption spectrometer (AA-6300; Shimadzu Co. Ltd., Japan).

3. Results

2.5. Chlorophyll measurement

We used barley and sorghum grown in a nutrient solution with 0.3 (Fe-deficient condition) or 30 (control) mM Fe for 7 days (barley) or 5 days (sorghum). The Fe and chlorophyll concentrations of young fully developed leaves in Fe-deficient plants decreased to similar levels when compared to the corresponding control plants (Fig. 1AeD). The following were also measured: AN and PAM chlorophyll fluorescence of the leaves (to measure photosynthetic rates), the Fv/Fm ratio (to measure the maximum quantum efficiency of PSII’s primary photochemistry), the electron transport rate (ETR), and nonphotochemical quenching (NPQ) (Table 2). Fe deficiency in barley resulted in a decreased Fv/Fm ratio and ETR, and markedly increased NPQ; however, CO2 assimilation did not stop completely as we reported previously [8]. Meanwhile, Fv/Fm and

Chlorophyll was extracted with 80% acetone from liquid nitrogeneground leaves, protoplasts, chloroplasts, and thylakoid membranes; the absorbance of the supernatants was measured spectrophotometrically at 663.6, 646.6, and 750 nm (UV1600; Shimadzu Co., Ltd., Japan), using the method described by Porra et al. [18]. 2.6. FCR activity All steps described below were performed in a dimly lit room. Protoplast FCR activities were measured as described by González-

3.1. Differences in the adaptation of barley and sorghum photosynthetic apparatus to Fe deficiency

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515

Table 1 A list of polymerase chain reaction primers used in this study. Primer name

Primer sequence

TaFRO1-F TaFRO1-R TaFRO2-F2 OsFRO2-R EF1aF EF1aR HvIRT1-F HvIRT1-R HvFRO1-InvN1 HvFRO1-InvC1 HvFRO1-InvN2 HvFRO1-InvC2 HvFRO2-InvN1 HvFRO2-InvC1 HvFRO1-N-XbaI HvFRO1-C HvFRO2-N-BamHI HvFRO2-C HvFRO1N-attB1

50 -GACATTCCATTTGAGCATGCTAC-30 50 -GTCGAAGCTGTGGCTGTTG-30 50 -TGCGTCTCGCGGCTCCAATG-30 50 -CCTCCTCCGCTTGTTCCACA-30 50 -ACCACTGGTGGTTTTGAGGC-30 50 -ATCATCATGAACCACCCTGG-30 50 -CCAGATGTTTGAGGGGATGG-30 50 -GATAGACACAAGACACACCC-30 50 -AGCACAAGCCGTGCAGCGTA-30 50 -AGTCCATCAGCTCATCCGTC-30 50 -GCCATTGTGAGATGCCCCAA-30 50 -GCATCTGGACATCCAAGCCT-30 50 -CCACCCGCCTCTGTTCTTGA-30 50 -GCCAGAACGATGCTGAACCT-30 50 -AGAGAGTCTAGATGACCCAAGAACGGGAGCCGCT-30 50 -CTAAAGGTCGAAGCTGTGGCTGT-30 50 -TCTCTCGGATCCTTCTGATAGTGAAACATGCAG-30 50 -CTAAACAAAACCAGATGTACGTCT-30 50 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGACCCAAGAACGGGAGC-30

HvFRO1C-attB2

50 -GGGGACCACTTTGTACAAGAAAGCTGGGTCAAGGTCGAAGCTGTGGCTGT-30

HvFRO2N-attB1

50 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGCAGCTCAGCTCCCTGCA-30

HvFRO2C-attB2

50 -GGGGACCACTTTGTACAAGAAAGCTGGGTCAACAAAACCAGATGTACGTCTGAAG-30

The underlined nucleotides represent XbaI (TCTAGA) BamHI (GGATCC) restriction sites. The double-underlined nucleotides are parts of FRO genes. HvIRT1-F and HvIRT1-R are described in Pedas et al., 2008 [23].

ETR markedly decreased; NPQ increased by a lesser extent, and CO2 assimilation was not detectable in sorghum under the 14-day Fedeficient condition (Table 2). 3.2. Comparison of Fe contents of thylakoid membranes between barley and sorghum under Fe-deficient conditions Although the total Fe concentration of young fully developed leaves was similar in barley and sorghum (Fig. 1C, D), the photosynthetic parameters were more affected in sorghum than in barley (Table 2). Therefore, we focused on the localization of Fe. We isolated thylakoid membranes, which contain protein complexes with high Fe content, and determined the Fe concentration on a chlorophyll and protein basis (Fig. 1EeH). We were unable to isolate thylakoid membranes from sorghum grown for 14 days under the Fe-deficient conditions. Thylakoid Fe on a chlorophyll basis did not decrease as a result of Fe deficiency in barley, but it exhibited a nonsignificant decrease in sorghum. Thylakoid Fe on a protein basis decreased as result of Fe deficiency in both barley and sorghum to 76% and 60% in the short term, respectively. Thylakoid Fe in barley did not decrease after 14 days Fe deficiency. Similar to the total Fe concentration of leaves, the levels of Fe concentration in the thylakoid membranes were almost similar in Fe-deficient barley and sorghum. However, the percentages of thylakoid Fe to leaf Fe ([thylakoid Fe ng/mg chlorophyll]/[leaf Fe ng/mg chlorophyll]) were increased by Fe deficiency in barley but were not altered in sorghum (Fig. 1I, J). These results suggest that barley can efficiently allocate Fe within leaf tissues, in contrast to sorghum.

and numbers of protoplasts and chloroplasts were assessed by light microscopy (Suppl. Fig. 1). The number of chloroplasts per protoplast seemed to have decreased as a result of Fe deficiency. All FCR activities were light inducible (Fig. 2). Blanks without Fe(III)EDTA or protoplasts/chloroplasts exhibited no activity (data not shown). No reducing agents were added to the reaction mixtures; thus, each FCR activity is dependent on the reducing agents elicited by light within protoplasts or chloroplasts. FCR activities per protoplast in barley and sorghum decreased to 51% and 35% of their normal levels, respectively, as a result of Fe deficiency (Fig. 2A, B). Fe deficiency resulted in a slight increase and a decrease in the protoplast FCR activities, when analyzed on a chlorophyll basis, in barley and sorghum, respectively (data not shown). The average diameter of barley protoplasts decreased significantly from 41.6 to 36.0 mm with Fe deficiency (Student’s ttest, P < 0.01), while that of sorghum protoplasts was not altered (data not shown). Therefore, the FCR activities in barley protoplasts are less damaged as a result of Fe deficiency than those in sorghum protoplasts. The FCR activity per chloroplast did not decrease in sorghum, although it slightly increased as a result of Fe deficiency in barley (Fig. 2C, D). Fe deficiency increased the chloroplast FCR activities, when analyzed on a chlorophyll basis, in both barley and sorghum (data not shown). The average diameters of barley and sorghum chloroplasts were not significantly altered by Fe deficiency (data not shown). Thus, the FCR activities in both barley and sorghum chloroplasts are not severely damaged by Fe deficiency.

3.4. Cloning and expression patterns of HvFRO genes 3.3. Comparison of FCR activities between barley and sorghum under Fe-deficient conditions Bughio et al. suggested the involvement of FCR in the process of Fe influx into barley chloroplasts [20]. FCR could also contribute to the allocation of Fe to mesophyll cells. Therefore, we determined the FCR activities in intact protoplasts and chloroplasts isolated from barley and sorghum grown with 0.3 or 30 mM Fe. The integrity

Our previous microarray analysis revealed that Fe deficiency induces the gene responsible for FRO2-like proteins in barley leaves [8]. Two FRO-like fragments were found in the barley database; rice also has 2 FRO genes [21]. Since we only found 2 types of FRO-like fragments from graminaceous plants in the databases, we designed consensus PCR primer sets for graminaceous FRO1 (TaFRO1F þ TaFRO1-R) and FRO2 (TaFRO2-F2 þ OsFRO2-R) (Table 1). We

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Chl a+b (µg·g-1 FW)

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**

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Fe in thylakoid membranes (ng·µg-1 Chl) Fe in thylakoid membranes (ng·µg-1 Protein)

**

150

0

**

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200

Thylakoid Fe/Leaf Fe (%)

Sorghum 2000

7d

0

ΔFe

+Fe 5d

Fig. 1. Chlorophyll and Fe concentrations of young fully developed leaves of Fe-sufficient and Fe-deficient plants. Barley and sorghum were grown in hydroponic solutions containing 30 mM (þFe) or 0.3 mM (DFe) EDTA-Fe for 7 days (barley), 5 days (sorghum), or 14 days (barley and sorghum). Values represent the means (SE) of 4 independent experiments. *P < 0.05 and **P < 0.01 indicate significant differences (according to Student’s t-test) between values connected by a line. (A, B) Chlorophyll a þ b concentration. (C, D) Total Fe concentrations in leaves. Total Fe concentration of isolated thylakoid membranes normalized to chlorophyll (E, F) and protein (G, H) amounts. Total Fe concentration of isolated thylakoid membranes normalized to protein amounts. (I, J) The percentage of Fe in thylakoid membranes to Fe in leaves. Yields of thylakoid membranes were calculated based on chlorophyll content. We could not isolate thylakoid membranes from sorghum grown for 14 days under the Fe-deficient conditions.

subsequently obtained partial cDNA sequences of HvFRO1 and HvFRO2, their genomic clones by inverted PCR, and ORF sequences from barley cDNA (AB564557, AB564558). The protein sequences of HvFRO1 and HvFRO2 were compared with those of rice and Arabidopsis thaliana FROs (Fig. 3). HvFRO1 was similar to OsFRO1, AtFRO6, and AtFRO7; HvFRO2 was similar to OsFRO2. We confirmed the expression of HvFRO1 and HvFRO2 in barley leaves (Fig. 4). Fe deficiency slightly increased HvFRO1 expression.

These genes were not expressed in roots. An FRO1-like gene was also expressed in sorghum leaves. Since we could not find a FRO2like maize gene in the database, this suggests that members of the Andropogoneae family may only have 1 FRO gene. Furthermore, Fe(II) should be efficiently transported by some transporter; on the root surfaces of Strategy I plants, Fe(II) is transported by IRT1 [22]. Since a previous study reports that HvIRT1 is expressed in barley roots [23], we examined HvIRT1 expression in barley leaves (Fig. 4).

Y. Mikami et al. / Plant Physiology and Biochemistry 49 (2011) 513e519 Table 2 Photosynthetic rates and PAM chlorophyll fluorescence. Fv/Fm, the maximum quantum efficiency of PSII’s primary photochemistry; ETR, electron transport rate (mmol quanta m2 s1); NPQ, nonphotochemical quenching; AN, photosynthetic rate (mmol CO2 m2 s1). Chlorophyll fluorescence values and photosynthetic rates were measured at 500 mmol photon m2 s1 containing 10% blue light. Barley and sorghum were grown in hydroponic solution containing 30 mM (þFe) or 0.3 mM (DFe) Fe(III)-EDTA for 7 days (barley) and 5 days (sorghum). Data were obtained from young fully developed leaves. Data represent the mean (SE) of at least 3 replicates. ND means “not detectable.” *P < 0.05 and **P < 0.01 indicate significant differences (according to Student’s t-test) between þ Fe and DFe.

DFe

þFe Barley

7d

14 d

Sorghum

5d

14 d

DFe/þFe (%)

Fv/Fm ETR NPQ AN Fv/Fm ETR NPQ AN

0.80 114 0.74 32.2 0.78 123 0.74 30.7

(0.002) (0.99) (0.02) (0.71) (0.002) (1.40) (0.02) (0.52)

0.77 82.8 1.68 14.7 0.67 34.9 1.68 3.23

(0.01) (0.40) (0.27) (2.77) (0.02) (2.21) (0.27) (0.43)

96.9 72.6* 227* 45.7** 84.7** 28.4** 414** 10.5**

Fv/Fm ETR NPQ AN Fv/Fm ETR NPQ AN

0.77 73.2 1.12 29.6 0.78 86.3 0.76 37.7

(0.001) (1.37) (0.06) (1.93) (0.004) (1.68) (0.09) (3.00)

0.67 19.8 2.32 4.58 0.50 8.31 1.63 ND

(0.01) (3.75) (0.14) (0.55) (0.003) (6.21) (0.22)

86.2** 27.0** 206** 15.5** 64.6** 9.63** 214* 0

We found that HvIRT1 is expressed in barley leaves and is not affected by Fe deficiency. 4. Discussion

µmol Fe (ll) µmol Fe (ll) 108 chloroplasts–1·min–1 105 protoplasts–1·min–1

We previously demonstrated that barley chloroplasts are resistant to damage caused by Fe deficiency, although Fe and chlorophyll concentrations were as low as those in rice [8]. Likewise, the photosynthetic functions of Fe-deficient barley leaves were less damaged than those of Fe-deficient sorghum leaves at the same Fe and chlorophyll concentrations (Table 2, Fig. 1AeD). Therefore, the total Fe concentration in whole leaves is not correlated with

the maintenance of photosynthetic functions in barley, although the photosynthetic apparatus requires a large amount of Fe. We found that the relative amount of leaf Fe allocated to thylakoids in barley leaves increased as a result of Fe deficiency, whereas thylakoid Fe concentration on a chlorophyll or protein basis was similar between Fe-deficient barley and sorghum (Fig. 1EeJ). These results suggest that barley may allocate Fe more efficiently to its thylakoids under Fe-deficient conditions than sorghum. At least one of the following three Fe transportation routes should be improved under Fe-deficient conditions for an efficient Fe allocation: from apoplasts to mesophyll cells across plasma membrane, from the cytoplasm to chloroplasts across the chloroplast envelope, or from labile-Fe pools into thylakoid proteins within chloroplasts. Fe is likely to be in insoluble forms in plant tissues [24e26]. Besides chelators and transporters, the activities of ferric reductases are also important for the efficient distribution of Fe in shoots [12]. We show that the FCR activities on the surfaces of barley and sorghum protoplasts and chloroplasts are light-dependent (Fig. 2). In addition, we confirmed the expression of HvIRT1 [23] in barley leaves; thus, Fe(III) can be reduced to Fe(II) and can therefore be subsequently transported via IRT1 in barley leaves. Several molecules can reduce Fe(III) [27]. In our experiment, blanks without Fe(III)-EDTA or protoplasts/chloroplasts exhibited no activity and no reducing agent was added to the reaction mixture. Thus, each FCR activity depended on the reducing agents elicited by light in protoplasts or chloroplasts, probably from photosynthetic apparatus. Iron deficiency did not decrease FCR activities per chloroplast in barley or sorghum (Fig. 2C, D). These results may be explained by the methods used for chloroplast isolation; in particular, fully developed and functional chloroplasts with suitable density were purified by Percoll gradient centrifugation and used to determine FCR activity. In reality, the amount of fresh Fe-deficient leaves required to obtain enough chloroplasts was larger than that required for Fe-sufficient leaves. Reduced photosynthetic rates as a result of Fe deficiency (Table 2) may be partially explained by the decrease of the number of functional chloroplasts. On the other hand, FCR activities per protoplast decreased as a result of Fe deficiency in both barley and sorghum

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Fig. 2. Ferric chelate reductase activities of leaves. Barley and sorghum were grown in hydroponic solutions containing 30 mM (þFe) or 0.3 mM (DFe) EDTA-Fe for 7 days (barley) and 5 days (sorghum). Values represent the means (SE) of 3 independent experiments. 0.1, *P < 0.05 and **P < 0.01 indicate significant differences (according to Student’s t-test) between values connected by a line. Ferric chelate reductase activities of isolated protoplasts (A, B) and chloroplasts (C, D) were determined under light and dark conditions.

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Fig. 3. Phylogenic tree of FRO proteins constructed with Clustal W. Protein sequences from barley, rice, and A. thaliana include HvFRO1 (AB564557), HvFRO2 (AB564558), OsFRO1 (AB126084), OsFRO2 (AB126085), AtFRO1 (Y09581), AtFRO2 (Y09581), AtFRO3 (NM_102150), AtFRO4 (NM_122303), AtFRO5 (NM_122304), AtFRO6 (NM_124351), AtFRO7 (NM_124352), and AtFRO8 (NM_124395).

(Fig. 2A, B). Because the FCR activities of protoplasts were also lightdependent, they seem to be supported by the reducing agents arising from photosynthetic apparatus. Therefore, the decreases in FCR activities per protoplast as a result of Fe deficiency may be due to a decrease in the number of functional chloroplasts. However, the decrease of the activity of barley protoplasts was smaller than that of sorghum protoplasts. The protection of barley photosynthetic apparatus against damage caused by Fe deficiency may contribute to the efficient reduction of Fe(III). The differences between C3 and C4 plants also should be considered, because the bundle sheath cells of C4 plants have increased PSI content [28]. PSI is the primary system that is affected by Fe deficiency, probably because of its high iron concentration (12 Fe atoms per PSI unit) [2]. Thus, the photosynthetic apparatus of sorghum could be more susceptible to Fe deficiency than that of barley. However, we did not distinguish between bundle sheath and mesophyll cells in this study. Two-dimensional electrophoresis and subsequent western blot analysis of sorghum thylakoid proteins from different cell types should be performed to verify this hypothesis. Another possible factor affecting the efficient reduction of Fe(III) is the disturbance of cellular redox status caused by imbalances of

Fe and other metals. We previously reported that Fe-deficient maize suffer from Zn excess stress possibly due to the difference of the substrate specificities of transporters of metal-mugineic acid complexes [29]. Sorghum and maize belong to the Andropogoneae family, and older leaves of Fe-deficient sorghum exhibit necrosis spots the same as maize (data not shown). Thus, the system for Fe(III) reduction in sorghum leaves could be more susceptible to the various adverse effects of Fe deficiency compared to that in barley leaves. In plant tissues, proteins from the FRO family are known to reduce Fe(III) [30]. AtFRO6 and AtFRO7 are localized on the plasma membrane of mesophyll cells and on the chloroplasts of Arabidopsis, respectively [5]. We confirmed the expression of FRO genes in barley and sorghum leaves (Fig. 4). We cloned 2 FRO genes from barley cDNA and found that deduced amino acid sequence of HvFRO1 is similar to those of AtFRO6 and AtFRO7 (Fig. 3). However, GFP-fusion proteins, HvFRO1:GFP and HvFRO2:GFP, were localized in both the cytoplasm and nuclei in a similar manner to GFP alone in onion epidermal or Physcomitrella patens cells (data not shown). Known plant FROs are localized in membranes or secretory pathways, and none of them exhibit a diffuse cellular distribution [30,31]. Yeast FRE7:GFP-fusion proteins are reported to exhibit a variable and diffuse cellular distribution [32]. However, there are no existing reports on the localization and FCR activities of graminaceous FROs. In contrast to Arabidopsis, the rice genome only has two FRO genes; maize may only have one, and barley only two. Thus, it is also possible that graminaceous FRO catalyze some redox reactions other than those catalyzed by FCR. Arabidopsis cytochrome b561 is reported to reduce Fe(III)-chelate in tonoplasts [33]. Future studies should search for other molecules that exhibit FCR activity on the membranes of graminaceous mesophyll cells. In this study, we show that barley allocates Fe to thylakoid membranes more efficiently than sorghum. Protection of the photosynthetic apparatus may support the production of reducing agents. Reducing Fe(III) may promote efficient Fe trafficking in mesophyll cells, which would allow thylakoid membranes to preferentially receive Fe. The allocation of Fe to thylakoid membranes is essential for maintaining photosynthetic apparatus. A synergistic relationship between the protection of photosynthetic apparatus and the acquisition of Fe within barley mesophyll cells may exist. Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.plaphy.2011.01.009. References

Fig. 4. Semiquantitative RT-PCR analysis of FRO genes in barley and sorghum and HvIRT1 in barley. Barley and sorghum were grown in hydroponic solution containing 30 mM (þFe) or 0.3 mM (DFe) EDTA-Fe for 7 days (barley) and 5 days (sorghum). EF1a was amplified as a loading control. The numbers of PCR cycles were 24, 28, 30, 35, 20, and 23 for FRO1, FRO2, root HvIRT1, leaf HvIRT1, upper panel EF1-a, and lower panel EF1-a, respectively.

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