Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana

Plant Physiology and Biochemistry 114 (2017) 111e118

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Research article

Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana Florinda Gama a, *, Teresa Saavedra a, Susana Dandlen a, Amarilis de Varennes b, Pedro J. Correia a, Maribela Pestana a, Gustavo Nolasco a a

MeditBio e Center for Mediterranean Bioresources and Food, University of Algarve, FCT, Ed8, Campus of Gambelas, 8005-139 Faro, Portugal LEAF e Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisbon, Portugal b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 November 2016 Received in revised form 2 March 2017 Accepted 3 March 2017 Available online 6 March 2017

To evaluate the dynamic role of the ferric-chelate reductase enzyme (FCR) and to identify possible pathways of regulation of its activity in different plant organs an investigation was conducted by virusinduced gene silencing (VIGS) using tobacco rattle virus (TRV) to silence the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. Half of Nicotiana benthamiana plants received the VIGS vector and the rest remained as control. Four treatments were imposed: two levels of Fe in the nutrient solution (0 or 2.5 mM of Fe), each one with silenced or non-silenced (VIGS-0; VIGS-2.5) plants. Plants grown without iron (0; VIGS-0) developed typical symptoms of iron deficiency in the youngest leaves. To prove that FRO1 silencing had occurred, resupply of Fe (R) was done by adding 2.5 mM of Fe to the nutrient solution in a subset of chlorotic plants (0-R; VIGS-R). Twelve days after resupply, 0-R plants had recovered from Fe deficiency while plants containing the VIGS vector (VIGS-R) remained chlorotic and both FRO1 gene expression and FCR activity were considerably reduced, consequently preventing Fe uptake. With the VIGS technique we were able to silence the FRO1 gene in N. benthamiana and point out its importance in chlorophyll synthesis and Fe partition. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Ferric-chelate reductase Ferric reductase oxidase gene Iron deficiency Tobacco rattle virus Virus-induced gene silencing

1. Introduction Iron (Fe) deficiency (chlorosis) is an abiotic stress of worldwide importance affecting several crops, especially when grown in calcareous soils. Fe is an essential micronutrient and its deficiency reduces plant growth and fruit quality (Walker and Connolly, 2008). It participates in numerous key processes such as photosynthesis, respiration and chlorophyll (Chl) biosynthesis and is a component of heme, the Fe-sulphur cluster and other Fe-binding sites (Kobayashi and Nishizawa, 2012). Therefore, it is essential to understand how plants maintain Fe homeostasis. In Fe limiting conditions, plants induce a set of responses which can be divided

Abbreviations: BPDS, Fe(II)-bathophenantrolinedisulfonate; Chl, chlorophyll; DW, dry weight; EC, electrical conductivity; EDDHA, ethylenediamine-N-N'bis(ohydroxyphenylacetic) acid; EDTA, ethylenediamine tetraacetic acid; FCR, ferricchelate reductase; Fe, iron; FRO, ferric reductase oxidase; FW, fresh weight; MES, 2(N-morpholino)ethanesulfonic acid; SPAD, soil and plant analyser development; TRV, tobacco rattle virus; VIGS, virus-induced gene silencing. * Corresponding author. E-mail address: [email protected] (F. Gama). http://dx.doi.org/10.1016/j.plaphy.2017.03.004 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved.

into two groups: Strategy I and Strategy II. In Strategy I, typical of non-gramineous plants, Fe reduction by the root ferric-chelate reductase (FCR), located in the plasma membrane, is a necessary step for Fe uptake. The absorption of Fe in these plants include mainly three steps localized at the plasma membrane of root cells: a proton pump Hþ-ATPase (HA) that acidifies the rhizosphere and is thought to help solubilize Fe(III); the FCR and iron regulated transporters (IRT) that move Fe(II) and also other metals across the plasma membrane and into cells (Walker and Connolly, 2008; Kobayashi and Nishizawa, 2012; Barberon et al., 2014). Strategy II is confined to grasses which rely on the secretion of phytosiderophores into the rhizosphere together with the induction of a highaffinity system for Fe (III)-phytosiderophore uptake (Hindt and Guerinot, 2012). The reduction of Fe is an enzymatic reaction executed by the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. In Arabidopsis thaliana FRO2 and IRT1 genes are regulated at the transcriptional level and upregulated under Fe deficiency conditions by the central basic helix-loop-helix (bHLH) transcription factor FER-LIKE Fe deficiency-induced transcription factor (FIT) (Brumbarova et al., 2015). In regards to Fe deficiency signalling

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pathways, nitric oxide (Graziano and Lamattina, 2007) and plant phytohormones such as auxin (Chen et al., 2010) and ethylene (Lucena et al., 2015) are described to positively modulate the uptake of Fe under deficiency conditions. The FRO1 exhibits different expression levels depending on the plant tissue and location within cells being the vacuole, the chloroplast and the mitochondria the main compartments responsible for Fe homeostasis. In Arabidopsis, eight members of the FRO family are known and these play different roles in Fe acquisition and uptake throughout the plant. For example, FRO2, FRO3 and FRO5 are mainly expressed in roots, while FRO6, FRO7 and FRO8 are shoot dependent. FRO1 and FRO4 genes have been shown to be expressed in both roots and leaves but with relatively low expression (Mukherjee et al., 2006; Jeong and Guerinot, 2009; Hindt and Guerinot, 2012; Ivanov et al., 2012; Jain et al., 2014). The tissue specific expression of the FRO1 gene in tomato has been described in all organs but the gene is only highly induced by Fe deficiency in roots (Li et al., 2004). In pea the FRO1 expression was seen in roots and leaves and highly induced under conditions of Fe deficiency (Waters et al., 2002). Virus-induced gene silencing (VIGS) is a reliable technique which provides several advantages in the study of tolerance to plant abiotic stresses. This method exploits one of the natural defence mechanisms that plants use against viral attack and results in degradation of viral mRNAs. If a host gene is cloned and inserted in the viral genome, then the mRNAs derived from the plant's copy of the gene will also be degraded through the silencing mechanism called Post-Transcriptional Gene Silencing (PTGS) and the effect of its inactivation on the phenotype of the plant can be studied (Senthil-Kumar and Mysore, 2011; Agüero et al., 2014). There are several viral vectors developed for VIGS with tobacco rattle virus (TRV) as one of the most frequently used (Liu et al., 2002; Valentine et al., 2004). TRV has two particles containing the RNA viral genome and required for infection. The pTRV-RNA1 contains the genes required for viral replication and movement, and the pTRV-RNA2 encodes the proteins necessary for virion formation. The target gene's partial sequence can be cloned into a multiple cloning site of pTRV-RNA2 which replaces viral proteins not required for VIGS (Senthil-Kumar and Mysore, 2011, 2014; Sahu et al., 2012). The VIGS vector is usually introduced into the plant through agro-infiltration with strains of Agrobacterium tumefaciens containing T-DNA constructs harbouring the modified viral genome. N. benthamiana is broadly used as an experimental host for VIGS assays due to a high susceptibility to a large number of plant viruses and fast manifest of the deprived phenotype. Another important aspect is that the expressed sequence tag (EST) of this species is commonly homologous to several solanaceous crops such as tomato, potato and pepper (Goodin et al., 2008; Senthil-Kumar and Mysore, 2011).

Lycopersicon esculentum ferric-chelate reductase (LeFRO1) gene (AY224079.1 - bases 1094e1396) was selected for this work. 29 bp attB recombination sequences were added to this sequence (purchased from Life Technologies®). The fragment was PCR amplified using Pfu DNA polymerase with designed primers 50 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-30 and 50 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3’. The resulting PCR product was gel purified with the Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corp.) and inserted through Gateway (Invitrogen) recombination into a pDONR-Zeo vector containing the attP1 plus attP2 recombination sites using the BP Clonase enzyme. The entry clones were then transformed into E. coli Mach1 chemical competent cells and selected on LB (Luria-Bertani) agar medium plates containing Zeocin (50 mg mL1). Plasmid DNA from recombinant E. coli cultures was purified using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). Total DNA was quantified using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) at a 260 nm. The ratio of absorbance at 260 nm and 280 nm, used to assess the purity of the DNA sample, was greater than 1.80. The identity of the construct was confirmed by DNA sequencing. The expression clone, TRV2FRO1, was generated through LR recombination by mobilizing the entry clone containing attL recombination sites to the TRV RNA2 vector (known as pYL279) containing attR recombination sites by using the LR Clonase enzyme. This mixture was transformed into E. coli Mach1 chemical competent cells and selected on LB plates containing Kanamycin (50 mg mL1). Plasmid DNA from recombinant E.coli cultures was purified using GeneJET Plasmid Miniprep Kit (Thermo Scientific) and total DNA was quantified and the final construct was confirmed by PCR.

1.1. Aim and hypothesis

N. benthamiana seeds were germinated in a mixture of vermiculite and peat in a 1:2 proportion. Four week-old plants were then transferred to pots containing peat and were grown at 25  C in a growth chamber under 16/8 h light/dark cycle. Once they had developed 4 to 5 leaves, at least 8 plants were selected for agroinfiltration with the VIGS vector (TRV-FRO1) and 7 plants as control, i.e., without agro-infiltration. After an acclimatization period of 15 days, plants were transferred to individual containers filled with aerated half strength Hoagland's nutrient solution with the following composition (in mM): 2.5 Ca(NO3)2$4H2O, 2.5 KNO3, 0.5 KH2PO4, 1 MgSO4$7H2O, and (in mM): 23 H3BO3, 0.4 ZnSO4$7H2O, 0.2 CuSO4$5H2O, 4.5 MnCl2$4H2O and 0.01 (NH4)6Mo7O27$H2O. Two Fe levels were imposed: 0 and 2.5 mM of Fe as Fe-EDDHA, resulting in the final following four treatments: i) plants without VIGS vector grown without Fe (0) or ii) with 2.5 mM of Fe as Fe-

The aim of this work was to analyse the effect of silencing the FRO1 gene using the VIGS technique in N. benthamiana plants. It was expected that silenced plants (VIGS treatments) would not be able to produce the enzyme and therefore, Fe would not be available for metabolic pathways (e.g. Chl synthesis). It is expected to demonstrate that the VIGS technique is a useful tool for studying FCR activity in plants grown with different Fe availability. 2. Material and methods 2.1. Plasmid construction TRV2-FRO1 A 361 bp synthetic sequence corresponding to part of the

2.2. VIGS assay For the VIGS assay, pTRV1 or pTRV2-FRO1 were introduced into chemically competent Agrobacterium tumefaciens strain C58C1 by electroporation, selected on LB plates containing Kanamicin, Gentamicin and Rifampicin, each at 50 mg mL1. Cultures of pTRV1 and pTRV2-FRO1 were grown separately overnight at 28  C in LB medium containing 10 mM MES (2-(N-morpholino) ethanesulfonic acid), 20 mM acetosyringone and the appropriate antibiotics. The next day, Agrobacterium cultures containing pTRV1 or pTRV2-FRO1 reached an optical density at 600 nm (OD600) of 0.6. They were then centrifuged and re-suspended by mixing the two cultures in an buffer (1:1 ratio) containing 10 mM MES, 10 mM magnesium chloride (MgCl2) and 200 mM acetosyringone (pH 5.6). This suspension was left at room temperature for approximately 3 h. All leaves of plants with 6 weeks-old were infiltrated using a 1 mL needleless syringe on the underside part. 2.3. Hydroponic experiment

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EDDHA (2.5); and iii) plants with VIGS vector grown without Fe (VIGS-0) or i) with 2.5 mM of Fe as Fe-EDDHA (VIGS-2.5). After 37 days, control and infiltrated plants growing in 2.5 mM Fe remained green but plants growing without Fe were chlorotic. At this time, chlorotic plants grown without Fe (0 and VIGS-0) were divided into two groups, which were then subject to Fe treatments: 0 and at 2.5 mM of Fe in the nutrient solution, 0-R and VIGS-R treatments, respectively.

2.4. Total leaf chlorophyll and plant growth measurements The degree of chlorosis was determined in young (apical) leaves and in mature (basal) leaves using a SPAD-502 apparatus (Minolta Corp., Osaka, Japan). A calibration curve was previously created by correlating SPAD values and Chl concentration. Leaf discs of 1 cm diameter were taken from leaves exhibiting different degrees of chlorosis using a calibrated cork borer and the respective SPAD values were registered. Each disc was then extracted with 100% acetone and sodium ascorbate as described by Abadía and Abadía (1993). Chlorophylls were estimated according to the equations by Lichtenthaler (1987), and a linear regression model (Fig. 1) was adjusted between SPAD (X) values and total Chl concentration (Y, mmol m2). During the experiment, stem height (in centimetres) and total number of leaves were also measured.

2.5. Fe content in shoots Plants from each treatment were harvested at the end of the experiment and divided into stems, mature leaves and young leaves. Samples were washed with a non-ionic detergent (0.1%) to remove surface contamination, then with tap water and finally rinsed three times with deionised water. Dry weight (DW) was determined after oven dried at 65  C, at least during 48 h or until constant weight. Fe concentration was determined by atomic absorption spectrometry (AAS) in dried stems, mature leaves, young leaves and flowers after they were ashed at 450  C and digested in HCl 1 M following standardized procedures (A.O.A.C, 1990). Fe content was calculated by multiplying the DW of each plant organ by Fe concentration. Due to the small amount of plant material for each organ of each treatment, a composite sample consisting of multiple samples taken from different repetitions were combined together for analysis. Fe partition (%) was calculated in relation to total leaf content.

Chl (μmol m-2)

800 Chl = 21.56 * SPAD + 10.24 r² = 0.97; n = 23; P < 0.001

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2.6. Ferric-chelate reductase activity The activity of FCR (EC 1.16.1.17) was measured by the formation of the Fe(II)-bathophenantrolinedisulfonate (BPDS) complex from Fe(III)-EDTA (Bienfait et al., 1983). Measurements were performed before resupply of Fe and at the end of the experiment. Determinations were conducted in root tips, young and mature leaves using at least three replicates from each plant. Root tips with 2 cm were excised with a razor blade and leaf discs of 0.13 cm2 were taken with a calibrated cork borer. Root tips and leaf discs were incubated in the dark for one hour and two hours, respectively, in an Eppendorf tube containing 900 ml of micronutrient-free half strength Hoagland's nutrient solution, 300 mM BPDS, 500 mM Fe(III)-EDTA and 5 mM MES, pH 6.0. An extinction coefficient of 22.14 mM cm1 was used. Blank controls without plant material were also used to correct for any unspecific Fe reduction.

2.7. RNA isolation and qRT-PCR analysis RNA was extracted from root tips, young leaves and mature leaves at the end of the experiment. Three biological replicates for each Fe treatment were assessed. Total RNA was extracted using approximately 100 mg of plant FW by using TRIzol® Reagent (Ambion). RNA preparations were cleaned up with Turbo DNA-free Kit (Applied Biosystems) according to manufacturer instructions. Each RNA sample was quantified using the NanoDrop 2000c Spectrophotometer at a 260 nm and the ratio of absorbance (260 nm/280 nm) that determines RNA purity was assessed in all samples. Quantification of mRNA expression was performed by real time reverse transcriptional polymerase chain reaction (qRT-PCR) in an iCycler IQ (Biorad) using the iScript One-Step RT-PCR Kit with SYBR Green (Biorad). The amplification mixtures were prepared in a total volume of 15 ml containing 7.3 ml of 2 SYBR Green master mix, 0.3 ml of each amplification primer, 0.3 ml of iScript reverse transcriptase and 1 ml of the RNA template. In regard to the primers, 200 nM of each of the FRO1 forward and reverse primers used annealed outside the region targeted for silencing so that endogenous gene could be quantified. RT-FRO1 Fwd: 50 -TTG AGA TTC TTA CAG TCA CGA -30 and RT-FRO1 Rev: 50 -CTT AAA CCT TTA GTC TTG GAG -3’. The thermo cycling parameters were: reverse transcription for 10 min at 50  C, denaturation for 5 min at 95 , followed by 40 cycles of 10 s denaturation at 95  C, 30 s for annealing at 55  C and 30 s at 72  C for extension. The relative expression level of FRO1 mRNA was determined in roots, mature leaves and young leaves. To quantify the gene expression a calibration curve was generated using a seven point serial dilution and a PCR efficiency of 91.3% was obtained for the transcript (Pfaffl, 2001). For each sample the relative expression was calculated based on the real time PCR efficiency and the difference of cycle threshold between a control (in this case a control 2.5 plant) and the sample. FRO1 relative expression was normalized for each sample by divining by the amount of secondary roots formed by each treatment. The relative expression ratio of a target gene was computed, based on its real-time PCR efficiencies (E) or a static efficiency of 2, and the crossing point (CP) difference (D) of one unknown sample (treatment) versus one control (DCP control - treatment).

2.8. Statistical analyses

0 0

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SPAD Fig. 1. Relationship between total leaf chlorophyll (Chl) concentration (mmol m2) and SPAD values in N. benthamiana plants.

The effects of Fe treatments were evaluated by analysis of variance (ANOVA; F test) and the means compared using the Duncan Multiple Range Test (DMRT) at P < 0.05 (IBM SPSS® software version 20).

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had significantly lower Chl values compared to 2.5 plants (VIGS0 ¼ 329 ± 32 mmol m-2; 2.5 ¼ 489 ± 88 mmol m-2). At the end of the experiment, 0-R plants reached Chl values of 2.5 plants but VIGS-R plants were unable to attain these values. At day 37, plants grown without Fe (0 and VIGS-0) had fewer leaves than 2.5 plants; however, after resupply 0-R plants developed leaves and reach the same amount as control plants (Fig. 4). On the contrary, VIGS-R had fewer leaves than 2.5 plants at the end of the experiment. Plants grown with Fe had higher stems compared to plants grown without Fe in the nutrient solution (Fig. 4). The resupply treatments (0-R and VIGS-R) had shorter stems and neither reached the values of Fe sufficient treatments. In roots, plants had the highest FRO1 expression (Table 1). In young leaves, 0 plants had greater expression values compared to 0-R, 2.5 and VIGS-2.5 plants. The gene expression was similar in mature leaves of all treatments. In general, greater FCR activity was registered in root tips

3. Results The variation of chlorophyll concentration during the experiment is presented in Fig. 2. Measurements started after the plants were transferred to nutrient solution (Day 1). Chl values declined in young leaves of Fe-deprived plants. Typical Fe chlorosis symptoms appeared in VIGS-0 plants 5 days after growth without Fe (Chl ¼ 265 ± 47 mmol m2) and symptoms in 0 plants (Chl ¼ 234 ± 32 mmol m2) occurred after 10 days of growth in the absence of Fe. Fe-sufficient plants: 2.5 and VIGS-2.5, maintained Chl values above 440 mmol m2 in young leaves and no significant differences were obtained between these treatments throughout the experiment. At the end of the experiment (twelve days after the beginning of the resupply; Figs. 2 and 3) 0-R plants had completely recovered from Fe chlorosis (Chl ¼ 620 ± 24 mmol m2 in young leaves) while VIGS-R plants remained chlorotic (Chl ¼ 107 ± 49 mmol m2). Twenty days after Fe deprivation, mature leaves of VIGS-0 plants

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Fig. 2. Chlorophyll values (Chl; mmol m2) of young (A) and mature (B) leaves during the experimental period. Treatments: 0 e plants without Fe in the nutrient solution; 2.5 e plants with 2.5 mM of Fe in the nutrient solution; VIGS-0 - plants inoculated with the VIGS vector and without Fe in the nutrient solution; VIGS-2.5 plants inoculated with the VIGS vector and with 2.5 mM of Fe in the nutrient solution; 0-R e control plants grown without Fe followed by a resupply of 2.5 mM of Fe; VIGS-R - plants containing VIGS vector grown without Fe followed a resupply of 2.5 mM of Fe. For each date, averages with different letters are statistically different at P < 0.05 (Duncan's multiple range test).

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Fig. 3. General aspect of N. benthamiana at the end of the experiment. Treatments: 0 e plants without Fe in the nutrient solution; 2.5 e plants with 2.5 mM of Fe in the nutrient solution; VIGS-0 - plants inoculated with the VIGS vector and without Fe in the nutrient solution; VIGS-2.5 plants inoculated with the VIGS vector and with 2.5 mM of Fe in the nutrient solution; 0-R e plants grown without Fe followed by a resupply of 2.5 mM of Fe; VIGS-R - plants containing VIGS vector grown without Fe followed a resupply of 2.5 mM of Fe.

compared to leaves (Table 1). At the end of the experiment control 0 plants had significantly higher root FCR activity (69 ± 19 nmol Fe (II) min1 g1 FW) and no significant differences were obtained for the other treatments. Recovered plants (0-R) lowered the root FCR activity and reached values of plants growing always in Fe sufficient conditions (2.5 plants). Plants containing the VIGS vector (VIGS-0; VIGS-R and VIGS-2.5) maintained low root activity, irrespective of Fe concentration in nutrient solution. In regards to mature leaves control 0 plants had higher activity and VIGS-0 registered the lowest activity. Concerning young leaves, VIGS-2.5 plants had significant higher FCR activity. Fe accumulated mainly in mature leaves of all treatments as observed by the Fe partition between theses organs (Table 1). Total leaf Fe content in plants containing the VIGS vector was almost half compared to control plants. Whereas in mature leaves the VIGS0 had the lowest Fe content, in young leaves control 0 plants had the least Fe quantity. After Fe resupply, Fe was mainly transported to mature leaves in both treatments (0-R and VIGS-R) although 0-R doubled the amount of Fe compared to VIGS-R. The amount of Fe in young leaves was similar in those treatments. VIGS-0 and 0 plants showed an increase in the formation of secondary roots but in a different manner: control 0 plants revealed a swelling of the root tip and smaller secondary roots, while VIGS0 plants had less swelling and thinner root tips with fine and lengthened secondary roots (Fig. 5).

4. Discussion In deficient conditions, gene expression may be induced or repressed to guarantee Fe homeostasis in plants (Mukherjee et al., 2006; Vigani et al., 2013). Lucena et al. (2006) proposed an integrative model to explain the upregulation of Fe acquisition genes as a response to Fe chlorosis in plants based on a cross-talk between leaves and roots induced by hormone signals (ethylene, nitric oxide) or by Fe, itself, in phloem. In our work, FRO1 gene expression was detected in several organs of N. benthamiana plants under Fe deficiency such as roots, young and mature leaves. These results are in accordance with those previously observed in tomato (Li et al., 2004), pea (Waters et al., 2002) and cucumber (Waters et al., 2007) plants. In this study, FRO1 gene of N. benthamiana was silenced using VIGS, a technique with much advantages over transgenic gene silencing (Senthil-Kumar and Mysore, 2011). To our knowledge the functional characterization of FRO1 gene in N. benthamiana has not been studied yet. Several studies demonstrate changes in FRO gene expression but more advances in functional characterization are required (Mukherjee et al., 2006; Enomoto et al., 2007; Sperotto et al., 2010; Gonzalo et al., 2011; Murgia et al., 2011; Santos et al., 2013; Vigani et al., 2013). As expected, 0-plants showed chlorosis symptoms in young leaves and a decline in overall growth. As a response to Fe deficiency an increase in root FCR activity and root FRO1 gene

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Fig. 4. Number of leaves and plant height (in cm) for the treatments during the experimental period. Treatments: 0 e plants without Fe in the nutrient solution; 2.5 e plants with 2.5 mM of Fe in the nutrient solution; VIGS-0 - plants inoculated with the VIGS vector and without Fe in the nutrient solution; VIGS-2.5 plants inoculated with the VIGS vector and with 2.5 mM of Fe in the nutrient solution; 0-R e control plants grown without Fe followed by a resupply of 2.5 mM of Fe; VIGS-R - plants containing VIGS vector grown without Fe followed a resupply of 2.5 mM of Fe. For each date, averages with different letters are statistically different at P < 0.05 (Duncan's multiple range test).

Table 1 Ferric-chelate reductase activity (nmol Fe (II) min1 g1 FW; FCR; n ¼ 9) and FRO1 relative expression (n ¼ 3) for the different treatments at the end of the experiment. For each parameter, averages with different letters indicate significant differences at P < 0.05 (Duncan's multiple range test). Fe values were obtained in one composite sample per each type of organ. Fe partition (%) was calculated in relation to total leaf content. Treatments: 0 e plants without Fe in the nutrient solution; 2.5 e plants with 2.5 mM of Fe in the nutrient solution; VIGS-0 - plants inoculated with the VIGS vector and without Fe in the nutrient solution; VIGS-2.5 plants inoculated with the VIGS vector and with 2.5 mM of Fe in the nutrient solution; 0-R e control plants grown without Fe followed by a resupply of 2.5 mM of Fe; VIGS-R - plants containing VIGS vector grown without Fe followed a resupply of 2.5 mM of Fe. Data are averages ±standard error.

Roots FRO1 FCR Mature Leaves FRO1 FCR Fe (mg) Fe (%) Young Leaves FRO1 FCR Fe (mg) Fe (%)

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35.3 ± 22.9 a 69.3 ± 19.8 a

4.6 ± 2.8 b 11.7 ± 1.6 b

1.8 ± 1.2 b 7.4 ± 1.8 b

0.8 ± 0.3 b 14.2 ± 5.0 b

0.4 ± 0.1 b 2.2 ± 0.3 b

3.3 ± 2.0 b 7.2 ± 1.8 b

0.6 ± 0.3 a 5.3 ± 2.3 a 399 79

0.1 ± 0.0 a 1.9 ± 0.5 ab 1211 93

0.5 ± 0.3 a 3.4 ± 0.2 ab 2030 87

0.1 ± 0.0 a 1.1 ± 0.1 b 139 47

0.7 ± 0.4 a 4.4 ± 1.0 ab 653 89

0.3 ± 0.2 a 2.2 ± 0.6 ab 1091 72

21.7 ± 1.6 a 1.4 ± 0.4 b 105 21

0.4 ± 0.2 b 2.2 ± 0.5 b 98 7

0.9 ± 0.1 b 1.1 ± 0.1 b 301 13

9.4 ± 3.9 ab 1.1 ± 0.0 b 154 53

11.7 ± 5.6 ab 1.1 ± 0.3 b 80 11

0.9 ± 0.0 b 5.6 ± 2.1 a 418 28

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Fig. 5. General aspect of root tips (2 cm in length) from plants subjected to different treatments after testing the FCR activity FCR at the end of the experiment. The rose colouration of root tips indicates localization of FCR activity. Treatments: 0 e plants without Fe in the nutrient solution; 2.5 e plants with 2.5 mM of Fe in the nutrient solution; VIGS-0 - plants inoculated with the VIGS vector and without Fe in the nutrient solution; VIGS-2.5 plants inoculated with the VIGS vector and with 2.5 mM of Fe in the nutrient solution; 0-R e control plants grown without Fe followed by a resupply of 2.5 mM of Fe; VIGS-R - plants containing VIGS vector grown without Fe followed a resupply of 2.5 mM of Fe.

expression occurred. The symptoms described are observed in plants of strategy I (dicots and non-grasses) already defined by several authors in other crops as peach (El-Jendoubi et al., 2014),
pez-Milla
n et al., 2001a,b) citrus (Pestana et al., 2005) sugar beet (Lo and strawberry plants (Gama et al., 2016), among others. This reduction-based method was also associated with the induction of root morphological changes (Pestana et al., 2001; McCluskey et al., 2004; Long et al., 2010; Pestana et al., 2012). However, in VIGS0 plants these root morphological changes are less evident and associated with a repressed expression of root FRO1 gene. The decrease observed in young leaf Chl content was slightly anticipated in VIGS-0 plants since two simultaneous stresses were imposed: absence of Fe and gene silencing. Since FRO1 gene is silenced, FCR activity of roots was not boosted and consequently, a marked decrease of total leaf Fe content was recorded. These results are in accordance with the importance of the FRO1 gene in encoding the FCR enzyme to circulate Fe(II) throughout plant, specially Fe translocation between mature and young leaves (Legay et al., 2012). The FRO1 gene is strongly regulated by Fe deficiency due to an increment observed in gene expression in roots and in young leaves of 0-plants of N. benthamiana. The induction of the FRO1 expression as a response to Fe deficiency has been reported in other plant species namely in Prunus (Gonzalo et al., 2011), potato (Legay et al., 2012) and in pea (Waters et al., 2002). In mature leaves of N. benthamiana the FRO1 expression was low and independent of Fe availability probably due to the high Fe content in these leaves and low mobility of this nutrient to other plant parts. After Fe resupply to the nutrient solution, the recovery of leaf symptoms was only observed in 0-R plants (chlorotic non-silenced plants) which attained greater Chl values than 2.5 plants, that always grew in non-limiting Fe concentration. This suggests that Fe stress did not cause permanent damages in the photosynthetic apparatus due to the quick access of Fe via xylem to young leaves as
pez-Milla
n et al., 2001a,b), strawberry observed in sugar beet (Lo
rio et al., 2014) and peach (Ferna
ndez et al., 2008). Root FCR (Oso activity and root FRO1 gene expression was reduced to values similar to 2.5 plants combined with a normalized Fe partition between young and mature leaves. This further demonstrates a recovery from Fe chlorosis and a deactivation of the mechanisms involved in Strategy I Fe acquisition. In VIGS-R plants the resupply of Fe to nutrient solution did not lead to any visible recovery and young leaves remained chlorotic, root FCR activity remained suppressed and the relative root FRO1

gene expression remained low. Considering this global response, the Fe uptake in silenced plants was insufficient to mobilize Fe to new developing leaves. Other FRO genes may have been involved in regulating Fe homeostasis at shoot level as observed by the pattern of the Fe partition between mature leaves and young leaves. 5. Conclusion In N. benthamiana roots FCR activity and FRO1 gene expression decreased in plants containing the VIGS vector indicating the effect of FRO1 gene silencing. When resupplied with Fe, silenced plants did not recover from the deficiency further demonstrating the strongly repressed function of the FRO1 gene, as these plants were unable to resume the metabolism of Chl synthesis, growth and root FCR activity. These results demonstrate how VIGS was effective to analyse the FRO1 gene function associated to Fe deficiency. Contributions F. Gama wrote the manuscript and executed the experiments. T. Saavedra assisted in hydroponic experiments. G. Nolasco and S. Dandlen supervised the VIGS assay. A. de Varennes was responsible for mineral composition analysis. P.J. Correia and M. Pestana contributed to the layout experiment and discussion of the results. Acknowledgements This study was funded by the National Project (PTDC/AGR-PRO/ ~o para a Cie ^ncia e a Tecnologia (FCT) 3861/2012) from the Fundaça and Florinda Gama is thankful to FCT for the PhD Grant SFRH/BD/ 89521/2012. References A.O.A.C, 1990. Association of Official Agricultural Chemists. Official Methods of Analysis, Washington, D.C. Abadía, J., Abadía, A., 1993. Iron and pigments. In: Barton, L.L., Hemming, B.C. (Eds.), Iron Chelation in Plants and Soil Microorganisms. Academic Press Inc, San Diego, CA, USA, pp. 327e343.
zquez, K., Pina, J.A., Navarro, L., Moreno, P., Agüero, J., del Carmen Vives, M., Vela Guerri, J., 2014. Effectiveness of gene silencing induced by viral vectors based on Citrus leaf blotch virus is different in Nicotiana benthamiana and citrus plants. Virology 460, 154e164. Barberon, M., Dubeaux, G., Kolb, C., Isono, E., Zelazny, E., Vert, G., 2014. Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc. Natl. Acad. Sci. 111 (22), 8293e8298. Bienfait, H.F., Bino, R.J., Van der Blick, A.M., Duivenvoorden, J.F., Fontaine, J.M., 1983.

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