Medicago truncatula ecotypes A17 and R108 differed in their response to iron deficiency

Journal of Plant Physiology 171 (2014) 639–647

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Physiology

Medicago truncatula ecotypes A17 and R108 differed in their response to iron deficiency Gen Li a,1 , Baolan Wang a,1 , Qiuying Tian a , Tianzuo Wang a , Wen-Hao Zhang a,b,∗ a b

State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China Research Network of Global Change Biology, Beijing Institutes of Life Sciences, Chinese Academy of Sciences, Beijing, China

a r t i c l e

i n f o

Article history: Received 17 August 2013 Received in revised form 19 December 2013 Accepted 20 December 2013 Available online 21 March 2014 Keywords: Medicago truncatula Ecotype A17 Ecotype R108 Fe deficiency Ferric chelate reductase activity

s u m m a r y Medicago truncatula Gaertn is a model legume species with a wide genetic diversity. To evaluate the responses of the two M. truncatula ecotypes, the effect of Fe deficiency on ecotype A17 and ecotype R108, which have been widely used in physiological and molecular studies, was investigated. A greater reduction in shoot Fe concentration of R108 plants than that of A17 plants was observed under Fedeficient conditions. Exposure to Fe-deficient medium led to a greater increase in ferric chelate reductase (FCR) activity in roots of A17 than those of R108 plants, while expression of genes encoding FCR in roots of A17 and R108 plants was similarly up-regulated by Fe deficiency. Exposure of A17 plants to Fe-deficient medium evoked an ethylene evolution from roots, while the same treatment had no effect on ethylene evolution from R108 roots. There was a significant increase in expression of MtIRT encoding a Fe transporter in A17, but not in R108 plants, upon exposure to Fe-deficient medium. Transcripts of MtFRD3 that is responsible for loading of iron chelator citrate into xylem were up-regulated by Fe deficiency in A17, but not in R108 plants. These results suggest that M. truncatula ecotypes A17 and R108 differed in their response and adaptation to Fe deficiency, and that ethylene may play an important role in regulation of greater tolerance of A17 plant to Fe deficiency. These findings provide important clues for further elucidation of molecular mechanism by which legume plants respond and adapt to low soil Fe availability. © 2014 Elsevier GmbH. All rights reserved.

Introduction Iron (Fe) is an essential nutrient for plant growth and development. Although Fe is the fourth most abundant element in the earth’s crust, it usually exists in the form of Fe (III)-oxides with low availability for plants, especially in calcareous soils (Guerinot and Yi, 1994). Therefore, Fe deficiency often limits crop production and nutritional quality. In addition, more than 2 billion people have been reported to be at risk of Fe-deficiency-induced anemia worldwide (WHO, 2007). Given that plants are a primary Fe source for humans, understanding mechanisms by which plants efficiently acquire Fe from soil, particularly under Fe-deficient conditions, would allow for breeding crop cultivars with high Fe nutritional quality, thus alleviating Fe malnutrition of humans. Two strategies have been developed by plants to mobilize and acquire Fe from soil (Römheld and Marschner, 1981). These strategies are defined as Strategy I in non-graminaceous monocots and

∗ Corresponding author at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China. Tel.: +86 10 6283 6697; fax: +86 10 6259 2430. E-mail address: [email protected] (W.-H. Zhang). 1 These authors contributed equally to this work. 0176-1617/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.12.018

dicots, and Strategy II in graminaceous monocots (Römheld and Marschner, 1981; Kobayashi and Nishizawa, 2012). In Strategy I plants, numerous changes in morphological and physiological processes are initiated under Fe-deficient conditions. These include sub-apical swelling with proliferation of root hairs, development of transfer cells, increases in ferric chelate reductase (FCR) activity, acidification of the rhizosphere, and up-regulation of Fe (II) transporters (Curie and Briat, 2003; Hell and Stephan, 2003; Romera and Alcántara, 2004; Morrissey and Guerinot, 2009; Conte and Walker, 2011). In contrast, the Strategy II plants are characterized by enhanced release of phytosiderophores and highly specific uptake system for Fe (III)-phytosiderophores in response to Fe deficiency (Römheld and Marschner, 1994; Inoue et al., 2003; Nozoye et al., 2011). Fe (III) has to be reduced to Fe (II) by a plasma membranebound FCR prior to uptake by roots in Strategy I plants (Romera and Alcántara, 2004; Kobayashi and Nishizawa, 2012). FRO2, which was first isolated from Arabidopsis, encodes a plasma membrane Fe (III) chelate reductase and is involved in mediation of Fe (II) acquisition by Strategy I plants (Robinson et al., 1999a,b). Once reduced, uptake of Fe (II) into roots is mediated by a divalent transporter of Iron-Regulated Transport 1 (IRT1) (Vert et al., 2002). The Arabidopsis knockout mutant of IRT1 showed chlorosis and was unable to take up Fe and other divalent cations in low Fe medium,

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indicating that the IRT1 is a major transporter responsible for highaffinity metal uptake under Fe-deficient conditions (Vert et al., 2002). AtIRT1 belongs to ZIP (ZRT/IRT-related protein) family, and an orthologue of ZIP has been also identified in other plant species, including Lycopersicum esculentum (Eckhardt et al., 2000), Medicago truncatula (López-Millán et al., 2004) and Arachis hypogaea (Ding et al., 2010). After taken up by root cells, Fe has to be loaded into the xylem and transported to the shoot via transpiration stream. FRD3, a protein belonging to the multidrug and toxin efflux (MATE) family, is involved in loading of citrate, which is an iron chelator necessary for distribution of Fe throughout the plant, into the xylem (Green and Rogers, 2004; Durrett et al., 2007; Rellan-Alvarez et al., 2009; Roschzttardtz et al., 2011). FRD3 plays a key role in the regulation of Fe translocation from roots to shoots (Durrett et al., 2007). In addition, FRD3 is expressed at detectable levels in roots, as well as in seeds and flowers in Arabidopsis, but its function appears to be root-specific (Rogers and Guerinot, 2002; Roschzttardtz et al., 2011). Several hormones are involved in the regulation of Fe-deficiency responses, including ethylene (Lucena et al., 2006; Waters et al., 2007), cytokinins (Séguéla et al., 2008), and brassinosteroids (Wang et al., 2012). Among the phytohormones, ethylene is one of the best characterized phytohormones associated with Fe-deficiency response in strategy I plants. For instance, exposure of cucumber seedlings to Fe-deficient medium up-regulates the expression of CsACS2 and CsACO2 that encode ACC synthase and ACC oxidase, two enzymes responsible for ethylene biosynthesis (Garcia et al., 2011). The Fe deficiency-induced ethylene has been shown to act as an enhancer to regulate FRC activity and IRT in response to Strategy I plants to Fe deficiency (Romera and Alcántara, 1994, 2004). M. truncatula has been used as a model plant to study genomics of legume plants because of its small diploid genome size and relatively easy transformation (Cook, 1999; Wang et al., 2011). There are different ecotypes (accession lines) of M. truncatula with wide genetic variations (Ellwood et al., 2006). Among the M. truncatula ecotypes, M. truncatula Jemalong A17 (A17) has been used for the whole-genome sequencing project (Choi et al., 2004; Young et al., 2005), while the ecotype R108 is often used for gene transformation, due to its superior in vitro regeneration (Hoffmann et al., 1997). There are reports demonstrating that the A17 differs from R108 in their phenotypes (Schnurr et al., 2007; Bolingue et al., 2010) and in their responses to abiotic stress (de Lorenzo et al., 2007) and biotic stress (Salzer et al., 2004; Gaige et al., 2012). However, there have been few studies to investigate whether the two ecotypes differ in their response to deficiency in mineral nutrients. Identification of key physiological processes underlying tolerance of legume plants to nutrient deficiency is of importance for molecular improvement of plants grown in nutrient-poor soils. In the present study, we compared the effect of Fe deficiency on the two ecotypes of legume model species of M. truncatula at physiological, morphological and molecular levels.

Materials and methods

MgSO4 ·7H2 O, 0.125 mM CaCl2 , 1.25 mM KNO3 , 0.5 mM NH4 NO3 , 15 ␮M H3 BO3 , 2.5 ␮M MnSO4 ·H2 O, 0.5 ␮M ZnSO4 ·7H2 O, 0.5 ␮M CuSO4 ·5H2 O, 0.35 ␮M NaMoO4 ·2H2 O, and 50 ␮M Fe (III) EDTA with a pH of 6.0. Seedlings were firstly cultured in half-strength nutrient solution for 5 days and then transferred to full strength nutrient solution for another 10 days. Thereafter, the 15-day-old seedlings were exposed to the Fe-deficient nutrient solution (−Fe) in which Fe (III) EDTA was removed, while the nutrient solution containing 50 ␮M Fe (III) EDTA is referred to as Fe-sufficient medium (+Fe). Nutrient solution was replaced every 5 days. Plants were grown in an environment-controlled chamber with a photosynthetic photon flux density of 350 ␮mol m−2 s−2 photo-synthetically active radiation, 80% relative humidity, and at a 16 h 26 ◦ C/8 h 19 ◦ C day/night regime. Determination of chlorophyll and root morphology Plants exposed to Fe-deficient medium for 10 days were used to measure chlorophyll (Chl) concentration and root morphology. Secondary root density is defined as the ratio of secondary root number to primary root length. Newly formed leaves were excised and weighed, then extracted in aqueous acetone (80%, v/v) as described previously (Wang et al., 2012). The extract solutions were measured in 663 nm and 645 nm with a spectrophotometer (Rio-Rad, USA). Total Chl concentration (mg L−1 ) was calculated as 8.02A663 + 20.21A645, and Chl concentration was expressed as mg Chl g−1 fresh weight. Excised roots were washed with deionized water and the length of primary root and numbers of secondary roots were determined. Determination of Fe, Mn and Zn concentrations The concentrations of Fe, Zn and Mn in roots and shoots of plants grown in Fe-sufficient and Fe-deficient medium were measured using an inductively couple plasma-atomic emission spectrometry (ICP-OES) with a detection limit of 0.01 ppm for Fe as described previously (Wang et al., 2012). Samples of shoots and roots of both ecotypes cultured in Fe-sufficient (+Fe) and Fe-deficient (−Fe) medium for 10 days were harvested and dried at 75 ◦ C and digested with the mixture of nitric acid and hydrogen peroxide using a microwave system (CEM, USA). The digested samples were analyzed by iCAP6300 (Thermo Electron, USA). Measurements of Fe (III) chelate reductase activity Activities of FCR in roots grown in Fe-sufficient and Fe-deficient medium were determined spectrophotometrically following the protocols described by Yi and Guerinot (1996). Briefly, intact root systems were submerged in the reductase assay solution containing 0.1 mM Fe (III) EDTA and 0.3 mM ferrozine (pH 5.0). Samples were incubated in a culture chamber for 1 h, and then the absorbance of the assay solution was read with a spectrophotometer (Rio-Rad, USA) at 562 nm. The concentration of Fe (III)-ferrozine was calculated with the molar extinction coefficient of 36.485 mM−1 cm−1 and the results were expressed in nmol Fe (II) per gram fresh weight per hour.

Plant growth Detection of ethylene production Two Medicago truncatula Gaertn ecotypes (Jemalong) A17 and R108 were used in this study. Seeds of both ecotypes were dipped in sulfuric acid for 10 min to degrade the seed coat, and rinsed thoroughly with sterilized water. The seeds were then sown on 0.8% agar to germinate at 25 ◦ C until the radicals were about 2 cm. The seedlings were planted in plastic buckets (12 seedlings per bucket) filled with 2.5 L aerated nutrient solution. The composition of full-strength nutrient solution is: 0.25 mM KH2 PO4 , 0.5 mM

Ethylene production from roots was detected with a gas chromatograph as described previously (Sun et al., 2007; Li et al., 2009). Briefly, excised roots of the two ecotypes grown in Fe-sufficient and Fe-deficient medium for 10 days were rinsed with distilled water and placed into a sealed glass jar (10 cm3 ) for 1 h at room temperature. To minimize a wounding effect, the excised roots were incubated in aerated distilled water for 1 h in the glass jar

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prior to sealing up. Ethylene was sampled with 1 cm3 syringes from the headspace of the sealed container and the concentration was determined with a gas chromatograph (GC-7A, Shimadzu, Japan) fitted with a flame ionization detector and an activated glass column filled with GDX502 (Shimadzu, Japan). The detection limit was 0.013 cm3 m−3 . Ethylene production was calculated based on fresh weight (FW) of root samples.

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Statistical analysis Analysis of variance was conducted between the different treatments. Significant differences between the two ecotypes under Fe-sufficient and Fe-deficient conditions were evaluated by LSD multiple range tests (P ≤ 0.05) using the SAS statistical software. Results

Xylem sap collection and determination of Fe and citrate concentration Xylem sap was collected with a pressure chamber (PMS, Instruments, Corvallis, OR) as described previously (Li et al., 2009). Briefly, the excised roots from the two ecotypes grown in Fe-sufficient and Fe-deficient medium for 10 days were put into the chamber. To minimize the diurnal fluctuations in the concentration of xylem sap contents, xylem sap was collected in the morning between 10:00 and 12:00 am, and the collection was made from A17 and R108 plants alternatively. A pressure of approximately 1.1 MPa was applied for 15 min to allow for collection of the xylem sap. The collected xylem sap was used for measuring Fe concentrations using an ICP after the first few drops of xylem sap from excised roots were discarded. Citrate concentration in the xylem sap was measured by a reverse-phase HPLC system following the protocols described by Wang et al. (2006). Separation was conducted on a 250 mm × 4.6 mm reverse-phase column (Alltima C18, 5 Micron; Alltech Associates, Inc., Deerfield, IL, USA). The mobile phase was 25 mM KH2 PO4 (pH 2.5) with a flow rate of 1 mL min−1 at 28 ◦ C, and detection of organic anions was carried out at 214 nm.

RNA isolation and quantitative real-time PCR RNA isolation was carried out as described preciously by Sun et al. (2010). Briefly, roots and young leaves of plants exposed to Fedeficient medium for 10 days were sampled. MtActin gene was used as a reference to quantify the relative transcript level. The RT-qPCR primers designed using Primer Premier5.0 software were as follows: MtFRO2 (MTR 7g038180) (5 -ATGGAACCGTCCAATGCTTGT3 and 5 -TTGCCGTACTCTGCCGCTGT-3 ), MtFRD3 (MTR 3g029510) (5 -AGTGACATTCTGCGTGACCTT-3 and 5 -ACCATCAGCGAGAAGG GA-3 ), MtIRT (MTR 4g083570) (5 -AGTGCTCGTCCAAATATGAAGG TG-3 and 5 -TGCTGGGATCGAAGTTGTGAAA-3 ), MtACO1 (MTR 3g083370) (5 -CCAAAGGGCTAGAGGCTGTTC-3 and 5 -GGTAGGT GACGCAAATGGA AA-3 ), MtACO2 (MTR 2g025120) (5 -GTTAGTAA CTACCCTCCTTGTCCT AAGC-3 and 5 -AAGGATGATGCCACCAGCAT3 ), MtACS2 (AY062022) (5 -TGCCTACACCTTACTATCCAG-3 and 5 -TCTGTCCATAACTGCCTAA-3 ), MtACS3 (MTR 8g101820) (5 GTCTACCAGGTTTCAGAGTTG-3 and 5 -CTCTTCTTCAATCTTTCCC TAT-3 ), MtActin (MTR 7g026230) (5 -ACGAGCGTTTCA GATG-3 and 5 -ACCTCCGATCCAGACA-3 ). All primers for the genes were selected on the basis of available A17 sequence data. To verify that the primers are identical to the genes in R108 plants, we cloned and sequenced these genes in R108 plants. The sequences in R108 plants were perfectly matched for A17 plants (Data not shown). Real-time PCR was performed using SYBR GreenERTM qPCR SuperMix Universal (Invitrogen) in 96-well reaction plates (Applied Biosystems) on a Step One Plus Real-Time PCR System (Applied Biosystems). The thermal cycle used was 95 ◦ C for 2 min, 40 cycles of 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 30 s. Each sample was carried out by three independent experiments and the relative expression level was analyzed by the comparative CT method as described by Livak and Schmittgen (2001).

General effect of Fe deficiency on A17 and R108 Distinct symptoms associated with Fe deficiency such as chlorosis and suppressed shoot growth were observed in the R108 seedlings after growth in Fe-deficient medium (0 ␮M Fe) for 10 days (Fig. 1A). By contrast, no such symptoms appeared in A17 seedlings when they were exposed to the identical Fe-deficient medium (Fig. 1A). A reduction in chlorophyll content is one of the responses of plants to Fe deficiency (Graziano et al., 2002). No significant difference in chlorophyll concentration was observed in A17 plants after grown in Fe-deficient medium for 10 days (Fig. 1B). In contrast, there was a marked reduction in chlorophyll concentration in R108 plants when exposed to the same Fe-deficient medium for 10 days (Fig. 1B). Chlorophyll concentration in A17 and R108 plants was comparable under Fe-sufficient conditions (P = 0.512), but it became lower in R108 than that in A17 plants under Fedeficient conditions (P = 0.002). These phenotypes suggest that A17 plants are more tolerant to low Fe availability in growth medium than R108 plants. Exposure of the two ecotypes to Fe deficiency led to significant decreases in shoot biomass of both ecotypes (Table 1). There was a significant decrease in root biomass of R108 plants, but not of A17 plants when transferring them to Fe-deficient medium for 10 days (Table 1). In addition to root biomass, we also investigated the effect of Fe deficiency on root morphology of the two ecotypes. Exposure of R108 seedlings to Fe-deficient medium led to a significant decrease in their primary root length and number of secondary root (Table 1). However, the secondary root density of R108 plants remained unchanged due to a reduction in primary root length when challenged by Fe deficiency (Table 1). In contrast to R108 plants, no changes in primary root length, secondary root number, and secondary root density were found in A17 plants when challenged by the identical Fe-deficient regimes. Note that the secondary root number of A17 plants was significantly higher (P = 0.001) than that of R108 plants under Fe-sufficient conditions (Table 1), and that exposure to Fe-deficient medium led to the secondary root number in R108 plants being much less than that in A17 plants (Table 1). Effect of Fe deficiency on Fe concentration Fe concentration in shoots of A17 was significantly higher (P = 0.014) than that in R108 plans under Fe sufficient conditions (Fig. 2A). There was a marked decrease in Fe concentrations of A17 and R108 plants upon exposure of these plants to Fe-deficient medium, and the decrease in shoot Fe concentration was greater in R108 than in A17 plants (Fig. 2A). For example, Fe concentration in A17 shoots was reduced by 43% in response to treatment with Fe deficiency, while shoot Fe concentration in R108 plants was reduced by 68% by the same Fe-deficient treatment, leading to the shoot Fe content in A17 plants being 2.1-fold higher than in R108 plants under Fe-deficient conditions. A similar decease in Fe concentrations in roots of both A17 and R108 plants was also observed when these plants were grown in Fe-deficient medium (Fig. 2B). However, unlike Fe concentration in shoots, a decrease in root Fe concentration was comparable between A17 and R108 plants,

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leading to a similar Fe concentration in roots of A17 and R108 plants (Fig. 2B). In Fe-sufficient seedlings, Fe concentration in the xylem of A17 plants was not significantly different (P = 0.142) from that of R108 plants (Fig. 2C). Fe concentration in the xylem of both A17 and R108 plants was reduced by exposure to Fedeficient medium, and Fe concentration in the xylem of R108 plants was not significantly different (P = 0.071) from that of A17 plants (Fig. 2D). Similarly, citrate concentration in the xylem was significantly higher (P = 0.045) in A17 plants than in R108 plants when grown in Fe-deficient medium, while citrate concentration in the xylem of A17 and R108 plants was comparable when grown under Fe-sufficient conditions (Fig. 2E).

Effect of Fe deficiency on FCR activity and expression of MtFRO2 Ferric chelate reductase (FCR) activity in roots of A17 and R108 plants was not significantly different (P = 0.172) when grown in Fe-sufficient medium (Fig. 3A). A marked increase in FCR activity was observed in roots of A17 and R108 plants upon exposure of A17 and R108 to Fe-deficient medium for 10 days, leading to no significant difference in FCR activity between A17 and R108 plants under Fe-deficient conditions (P = 0.663) (Fig. 3A). For example, FCR activity in A17 plants was increased from 47.82 ± 10.85 to 140.50 ± 28.71 nmol Fe(II) g−1 WT−1 h−1 (n = 4) after 10 days of transferring from Fe-sufficient medium to Fedeficient medium The same treatment led to an increase FCR activity of R108 plants from 78.96 ± 16.93 to 128.40 ± 8.76 nmol Fe(II) g−1 WT−1 h−1 (n = 4). Similar to FCR activity, exposure of A17 and R108 plants led to significant increases in expression level of MtFRO2 that encodes ferric chelate reductase (Fig. 3B).

Effect of Fe deficiency on concentration of Zn and Mn In addition to Fe, Fe transporters can also mediate transport of other divalent metals such as Zn and Mn in plants (Vert et al., 2002). We thus investigated the effect of Fe deficiency on Zn and Mn concentrations in shoots and roots of the two ecotypes. There were increases in Zn concentration in shoots and roots of both A17 and R108 plants when grown in Fe-deficient medium compared to those grown in Fe-sufficient medium (Table 2). The increases in Zn concentration in both roots and shoots of R108 plants were greater than those of A17 plants (Table 2). Under Fe-sufficient conditions, R108 plants had higher (P = 0.0001) Zn concentration in their roots than A17 plants (Table 2). Similar increases in Mn concentrations in roots and shoots of A17 and R108 plants were also observed when these plants were grown in Fe-deficient medium (Table 2). Unlike Zn concentrations, A17 and R108 plants had similar (P = 0.432) Mn concentrations in their roots under Fe-sufficient conditions, while Mn concentrations in R108 shoots were significantly lower (P = 0.006; P = 0.012) than in A17 leaves under Fe-sufficient and Fedeficient conditions (Table 2).

Fig. 1. Chlorophyll concentrations in newly formed leaves of M. truncatula A17 and R108 seedlings grown in Fe-sufficient (+Fe) and Fe-deficient medium (−Fe). Fifteenday-old seedlings of A17 and R108 plants pre-cultured in Fe-sufficient medium were transferred to either Fe-deficient or Fe-sufficient medium and young leaves were harvested for determination of chlorophyll concentrations after growth in either Fe-sufficient and Fe-deficient medium for 10 days. Data are means ± SE with four replicates. *Significant differences between Fe-sufficient (+Fe) and Fe-deficient (−Fe) for each ecotype at the level of P ≤ 0.05.

Table 1 Growth and root morphological characteristics of M. truncatula A17 and R108 plants grown in Fe-sufficient (+Fe) and Fe-deficiency (−Fe) medium. Fifteen-day-old seedlings that were exposed to Fe-deficient medium (−Fe) for 10 days were used to determine biomass (dry weight, DW) and morphological parameters. Data are means ± SE with four replicates, and significant differences between +Fe and −Fe treatments within each ecotype were shown as * (P ≤ 0.05), respectively. Ecotypes

A17

R108 −Fe

+Fe Shoot DW (mg) Root DW (mg) Primary root length (cm) Secondary root number Secondary root density

21.58 5.41 34.63 67.67 1.9 6

± ± ± ± ±

0.97 0.33 0.43 1.91 0.06

14.94 4.95 35.98 69.50 1.93

−Fe

+Fe ± ± ± ± ±

0.96* 0.26 0.68 1.73 0.04

20.27 6.1 8 39.48 45.20 1.14

± ± ± ± ±

0.59 0.48 0.53 5.00 0.12

15.18 4.40 31.98 37.83 1.18

± ± ± ± ±

0.48* 0.24* 1.30* 2.52* 0.07

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Fig. 2. Effect of Fe deficiency on Fe concentration in shoot (A), root (B and C), Fe concentration in the xylem (C) and citrate concentration in the xylem (D) of M. truncatula ecotypes A17 and R108. Fifteen-day-old seedlings pre-cultured in Fe-sufficient medium were transferred to Fe-deficient medium for 10 days, and concentrations of Fe and citrate in shoots, roots and xylem sap were determined. Data are means ± SE with four replicates. *Significant differences between Fe-sufficient (+Fe) and Fe-deficient (−Fe) for each ecotype at the level of P ≤ 0.05.

Table 2 Concentrations of Zn and Mn in leaves and roots of A17 and R108 plants grown in Fe-sufficient (+Fe) and Fe-deficient (−Fe) medium. Zn and Mn concentrations in leaves and roots of A17 and R108 plants were measured after they were grown in Fe-deficient medium for 10 days. Data are means ± SE with four replicates, and significant differences between +Fe and −Fe treatments within each ecotype were shown as * (P ≤ 0.05), respectively. Concentration (mg g−1 DW)

R108

A17 −Fe

+Fe Leaf Zn Root Zn Leaf Mn Root Mn

0.02 0.05 0.22 0.52

± ± ± ±

0.00 0.01 0.01 0.09

0.10 0.64 0.42 2.62

−Fe

+Fe ± ± ± ±

0.00* 0.02* 0.02* 0.07*

0.03 0.14 0.16 0.61

± ± ± ±

0.01 0.01 0.00 0.07

0.19 0.76 0.37 4.26

± ± ± ±

0.02* 0.01* 0.05* 0.24*

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Fig. 3. Ferric chelate reductase (FCR) activity for root of A17 and R108 plants grown in Fe-sufficient and Fe-deficient medium. Fifteen-day-old seedlings of A17 and R108 plants pre-cultured in Fe-sufficient medium were exposed to Fe-deficient medium for 10 days, and were used to determine FCR activity. Data are means ± SE with four replicates. *Significant differences between Fe-sufficient (+Fe) and Fe-deficient (−Fe) for each ecotype at the level of P ≤ 0.05.

Effects of Fe deficiency on expression of MtIRT and MtFRD3

Fig. 4. Effect of Fe deficiency on expression patterns of genes encoding IRT (MtIRT) (A) and FRD3 (MtFRD3) (B). Roots of A17 and R108 seedlings pre-culture in Fesufficient medium for 15 days were sampled at 10 days after exposure of A17 and R108 seedlings to Fe-deficient medium were used to extract total RNA for detection of relative gene expression by real-time PCR. Data are means ± SE with three biological replicates. *Significant differences between Fe-sufficient (+Fe) and Fe-deficient (−Fe) for each ecotype at the level of P ≤ 0.05.

To further verify the different responses of A17 and R108 plants to Fe deficiency, two genes (MtIRT and MtFRD3) involved in Fe uptake and transport were studied at the transcript level. Expression of MtIRT that encodes IRT was increased in A17 plants 2-fold by Fe deficiency (Fig. 4A). In contrast to A17 plants, expression of MtIRT in R108 plants was insensitive to Fe deficiency (Fig. 4A). Under Fesufficient conditions, abundance of MtFRD3 transcript in A17 plants was comparable with that in R108 plants, and expression of MtFRD3 in A17 plants was enhanced by Fe deficiency, while MtFRD3 transcripts in R108 plants remained relatively constant in response to Fe deficiency (Fig. 4B). Effect of Fe deficiency on ethylene production and expression of ACS and ACO Ethylene has been shown to be a key regulator to mediate response of plants to Fe deficiency (Lucena et al., 2006). To evaluate whether Fe deficiency-induced ethylene production is involved in the difference in Fe mobilization and transport in A17 and R108 plants, the effect of Fe deficiency on ethylene production in roots of the two plants was studied. Ethylene level in roots of A17 and R108 roots was comparable (P = 0.091) when they were grown in Fe-sufficient medium, and a marked increase in ethylene production from A17 roots was observed when exposed to Fe-deficient medium (Fig. 5). In contrast, exposure of R018 plants to the same

Fig. 5. Effect of Fe deficiency on ethylene production from roots of A17 and R108 plants. Fifteen-day-old seedlings of A17 and R108 pre-cultured in Fe-sufficient medium were exposed to Fe-deficient medium for 10 days, and ethylene evolution rates were determined. Data are mean ± SE with three replicates. *Significant differences between Fe-sufficient (+Fe) and Fe-deficient (−Fe) for each ecotype at the level of P ≤ 0.05.

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Fig. 6. Effect of Fe deficiency on expression of MtACS2 (A), MtACS3 (B), MtACO1 (C) and MtACO2 (D) of A17 and R108 plants. Fifteen-day-old A17 and R108 seedlings were exposed to Fe-deficient medium for 10 days, and total RNA was extracted for monitoring transcript levels by real-time PCR. MtActin was used as an internal control. Data are mean ± SE with three biological replicates.

Fe-deficient medium did not evoke ethylene production in roots of R108 plants (Fig. 5). Ethylene production in plants is catalyzed by two enzymes, 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylicacid oxidase (ACO) (Kende, 1993). To further study the differential response of ethylene production in the two ecotypes to Fe deficiency, effect of Fe deficiency on ethylene biosynthesis in the two ecotypes was studied at the transcriptional level. The expression levels of MtACS2, MtACS3 that encode ACS in A17 and R108 plants were comparable when grown in Fe-sufficient medium (Fig. 6A). Exposure to Fe-deficient medium led to a significant up-regulation of MtACS2 in A17 plants, but not in R108 plants (Fig. 6A). Expression of MtACS3 was significantly up-regulated by Fe deficiency in both A17 and R108 plants with the increase being greater in A17 than in R108 plants (Fig. 6B). In addition to ACS, expression patterns of MtACO1 and MtACO2 that encode ACO in A17 and R108 plants also differed in their response to Fe deficiency. For example, up-regulation of MtACO1 expression in R108, but not in A17 plants, was observed when challenged by Fe deficiency (Fig. 6C), while exposure to Fe-deficient medium significantly enhanced expression of MtACO2 in both A17 and R108 plants (Fig. 6D). Discussion The two widely used ecotypes of legume model plant A17 and R108 differ in their tolerance to salt stress (de Lorenzo et al., 2007), morphology (Schnurr et al., 2007), dormancy behavior of

seeds (Bolingue et al., 2010) and susceptibility to fungal pathogen Macrophomina phaseolina (Gaige et al., 2012). However, there has been no study to compare the responses of the two ecotypes to deficiencies in mineral nutrients. In the present study, we demonstrate that the M. truncatula ecotype A17 and ecotype R108 differed in their response to Fe deficiency, such that there were less change in leaf chlorophyll concentration, root growth and morphology, and shoot Fe concentrations in A17 than in R108 plants in response to exposure to Fe-deficient medium. The physiological mechanisms underlying the differential responses of the two ecotypes to Fe deficiency were explored. An increase in activity of root FCR has been reported in Strategy I plants when exposed to Fe-deficient medium (Schmidt, 2003). The enhanced activity of FCR is an adaptive response to Fe deficiency by facilitating reduction of rhizosphere Fe (III), thus allowing plants for more efficient acquisition of Fe. We found an increase in root FCR activity in A17 and R108 roots, but the magnitude of Fe deficiency-induced increase in FCR activity was higher in A17 than in R108 plants (Fig. 3). A similar Fe deficiency-induced increase in FCR activity of M. truncatula A17 plants has been reported (Andaluz et al., 2009). At the transcriptional level, we observed a greater up-regulation of MtFRO2 in A108 than in A17 plants under Fe-deficient conditions (Fig. 3), suggesting that the difference in FCR activity between the two ecotypes is likely to be accounted for post-transcriptionally and/or other unknown mechanisms. The greater increase in FCR activity of A17 roots than R108 plants under Fe-deficient conditions may allow A17 plants to be more efficient reduction of Fe (III) to Fe (II), thus leading to higher Fe

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concentrations in leaves of A17 than R108 plants. A similar argument has been used to explain the differential tolerance of pea genotypes to Fe deficiency (Kabir et al., 2012). The Fe concentrations in roots of both ecotypes (approx. 4500 ppm) under Fe-sufficient conditions appeared higher than those reported in the literature for other dicot plants, but these values were comparable to Fe concentration in roots of Arabidopsis grown in Fe-sufficient conditions (Barberon et al., 2011). Fe is taken up by roots after reduction from Fe (III) to Fe (II) by a membrane-bound FCR in the rhizosphere. In Strategy I plants, Fe transport from the rhizosphere to root cells has been suggested to be mediated by an iron-regulated transporter belonging to ZIP family, referred to as IRT1 (Vert et al., 2002). A recent study showed that MTR 4g083570 is the most likely ortholog of AtIRT1 (Rodriguez-Celma et al., 2013). In the present study, we found that Fe deficiency up-regulated expression of MtIRT in A17 plants, while the expression of MtIRT in R108 plants was insensitive to Fe deficiency (Fig. 4A). The difference in MtIRT activity between A17 and R108 plants may explain the higher Fe concentrations in shoot of A17 than R018 under Fe-deficient conditions (Fig. 2B). This result also suggests that the IRT-mediated transport may be a major determinant for the observed difference in tolerance of the two ecotypes to Fe deficiency. In addition to mediation of Fe transport, IRT can also mediate transport of other divalent cations such as Zn and Mn into roots (Vert et al., 2002). Less sensitivity of FCR activity in R108 than in A17 plants may result in the lower amount of Fe (II) for acquisition by plants, thus leading to greater accumulation of Zn and Mn by R108 plants under Fe-deficient conditions. Another interesting observation is that there was a higher Mn concentration in R108 roots than in A17 plants when grown in Fe-deficient medium (Table 2). Expression of MtIRT1 in R108 was lower than in A17 plants in Fe-deficient medium (Fig. 4A), implying that other mechanisms may exist in R108 plants which underlie the observed greater accumulation of Mn in R108 plants. Fe concentration in leaves of A17 plants was comparable with R108 plants under Fe-sufficient conditions, while Fe concentration in A17 leaves was higher than in R108 leaves under Fe-deficient conditions (Fig. 2). Fe concentration in roots of the two plants was comparable regardless of Fe supply (Fig. 2). These results imply that A17 plant is more efficient than R108 in terms of Fe acquisition under Fe-deficient conditions. In addition, the less inhibitory effect of Fe deficiency on Fe concentrations in shoots of A17 than R108 plants may also be explained by more efficient translocation of Fe from roots to shoots in A17 plants than R108 plants. FRD3 is a key player in controlling Fe translocation from roots to shoots by mediating loading of Fe (III) chelator citrate into the xylem (Durrett et al., 2007). In the present study, we found that the expression level of MtFRD3 was significantly up-regulated by Fe-deficiency in A17, while expression of MtFRD3 in R108 plants was insensitive to Fe deficiency (Fig. 4B). These results suggest that A17 plants can up-regulate loading of citrate into the xylem in response to Fe deficiency, thus conferring efficient Fe translocation of Fe from roots to shoots. Moreover, the more abundance of FRD3 transcripts in A17 than in R108 plants under Fe-deficient conditions (Fig. 4B) may also be responsible for the higher concentrations of citrate and Fe in the xylem of A17 plants than that of R108 plants (Fig. 2C and D). Therefore, our results highlight an important role of FRD3 played in regulation of Fe translocation, thus determining the tolerance capacity of M. truncatula to Fe deficiency. In a recent study, Kabir et al. (2012) reported that citrate concentrations in roots of pea genotype tolerant to Fe deficiency are substantially increased in response to Fe-deficient treatment, while the same treatment leads to a marginal decrease in citrate concentrations in roots of pea genotype less tolerant to Fe deficiency. Ethylene has been shown to be a key regulator to mediate response of plants to Fe deficiency. For instance, it has been

suggested that Fe deficiency-induced ethylene production acts as a signal to regulate expression of transcription factor FER and FCR activity (Romera and Alcántara, 2004; Lucena et al., 2006). A marked increase in ethylene evolution from A17 roots, but not R108 roots, was found upon exposure to Fe-deficient medium (Fig. 5). A similar difference in Fe deficiency-induced in ethylene evolution between pea genotypes differing in tolerance to Fe deficiency has been observed (Kabir et al., 2012). We further demonstrated that the expression of MtACS2, MtACS3, and MtACO2, which encode ACS and ACO, in A17 differed from R108 plants in response to Fe deficiency (Fig. 6). ACS activity is a limiting enzyme in ethylene biosynthesis (Kende, 1993). Therefore, the greater up-regulation of MtACS2 and MtACS3 induced by Fe deficiency in A17 plants than in R108 plants may account for the Fe deficiency-induce ethylene production in roots of A17 plants (Fig. 5). Given that A17 plants had higher Fe concentration in leaves and the xylem and ethylene production than R108 plants under Fe-deficient conditions, it is conceivable that ethylene may be an important regulator to modulate Fe translocation by targeting FRD-dependent physiological processes, thus conferring A17 plants more efficient utilization of Fe under Fedeficient conditions. Further studies by screening large numbers of M. truncatula ecotypes can test the relationship between ethylene production and Fe utilization efficiency. In summary, we demonstrate that M. truncatula ecotype A17 was more tolerant to Fe deficiency than ecotype R108. The high use efficiency of Fe in M. truncatula A17 plants than in R108 plants may result from the more effective acquisition of Fe due to higher FCR activity and greater up-regulation of FRD3. Our findings also demonstrate that A17 plants can evolve ethylene in response to Fe deficiency, and that the evolved ethylene may act as a signal to enhance mobilization and translocation of Fe under Fe-deficient conditions, thus conferring M. truncatula ecotype A17 more tolerant to Fe deficiency than ecotype R108.

Acknowledgements This study was supported by the National Natural Science Foundation of China (31101594 and 31272234) and State Key Laboratory of Vegetation and Environmental Change. We thank the two anonymous reviewers for their constructive suggestions on the previous version of the manuscript.

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