Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload

Plant Physiology and Biochemistry 60 (2012) 180e189 Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal hom...

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Plant Physiology and Biochemistry 60 (2012) 180e189

Contents lists available at SciVerse ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload Sylvain Legay*, Cédric Guignard, Johanna Ziebel, Danièle Evers Centre de Recherche Public e Gabriel Lippmann, Department EVA, 41, rue du Brill, L-4422 Belvaux, Luxembourg

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2012 Accepted 13 August 2012 Available online 31 August 2012

Potato is one of the most important staple food in the world because it is a good source of vitamin C, vitamin B6 but also an interesting source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. The lack of iron constitutes the main form of micronutrient deficiency in the world, namely iron deficiency anemia, which strongly affects pregnant women and children from developing countries. Iron biofortification of major staple food such as potato is thus a crucial issue for populations from these countries. To better understand mechanisms leading to iron accumulation in potato, we followed in an in vitro culture experiment, by qPCR, in the cultivar Désirée, the influence of media iron content on the expression of genes related to iron uptake, transport and homeostasis. As expected, plantlets grown in a low iron medium (1 mg L1 FeNaEDTA) displayed a decreased iron content, a strong induction of iron deficiency-related genes and a decreased expression of ferritins. Inversely, plantlets grown in a high iron medium (120 mg L1 FeNaEDTA) strongly accumulated iron in roots; however, no significant change in the expression of our set of genes was observed compared to control (40 mg L1 FeNaEDTA). Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Potato Iron Homeostasis Uptake Storage In vitro culture Gene expression

1. Introduction Potato is the fourth most important crop in the world and is considered as an important staple food for developing countries. Potato is a good source of vitamin C, vitamin B6 but also a good source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. Iron content in unpeeled potato tuber may range between 30 and 45 mg g DW1 in Andean cultivars, which represents 3e5% of the recommended daily allowance [1]. Because of the strong occurrence of deficiencies for iron especially in developing countries, the valorization and the biofortification of the staple food potato is an important task. Iron is a crucial mineral in the living kingdom. In plants, it participates in a broad spectrum of processes including chlorophyll synthesis, respiration, energy transfer or nitrogen fixation and is present in many enzymes [2,3]. In agricultural land, the amount of iron available for the plant can be very variable. Additionally, the low solubility of iron in the soil at neutral or basic pH can lead to iron deficiency in the plant, which is mainly

* Corresponding author. Tel.: þ352 47 02 61 436; fax: þ352 47 02 64. E-mail addresses: [email protected] (S. Legay), [email protected] (C. Guignard), [email protected] (J. Ziebel), [email protected] (D. Evers). 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.08.003

observable by interveinal yellowing of the leaves [4]. Inversely, high free iron in cells can generate reactive oxygen species by the Fenton reaction [5]. Finally, due to the intrinsic chemistry of iron, it can precipitate at physiological state in the cytosol. Altogether, plants have to face challenges from the uptake, to the homeostasis and to storage management. Potato is considered as a crop that is sensitive to both excess and low iron conditions: non-optimal iron content in the substrate results in a consequent yield loss as well as a reduced tuber quality [6]. Iron uptake in potato belongs to the strategy I model, which consists in soil acidification, iron reduction from Fe3þ to Fe2þ and finally transport into the plant using an iron specific transporter called IRT1 [7]. Then, iron is complexed with molecules acting as metal chelators such as nicotianamine [8] and vehicles through the vessels in the plant. As previously mentioned, free iron is poorly soluble at physiological state and high accumulation can lead to oxidative stress. To avoid such cellular disorders, plants have several management strategies. On one hand, a broad spectrum of transporters has been characterized in model plants including the natural resistanceassociated macrophage (NRAMP) family protein, involved in iron homeostasis [9,10] or the vacuolar iron transporter (VIT1), involved in iron remobilization in Arabidopsis seeds during germination [4,]. On the other hand, a second strategy consists in sequestering iron

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in storage molecules such as ferritin increasing its solubility and avoiding cellular injuries. Ferritin is a widespread multimeric protein in the living kingdom constituted by 24 oligomers forming a sphere with a cavity in which 4500 iron atoms can be stored. In Arabidopsis thaliana, ferritins have been shown to be a major component of iron homeostasis in plants and oxidative stress prevention [11]. Despite an advanced knowledge of the mechanisms leading to iron uptake, homeostasis and storage in model plants, until now, few studies have been performed on potatoes, especially at the gene expression level. The present work describes iron dynamics in potato. To this end, an in vitro experiment was performed allowing an optimal control of the experimental growth conditions. The cultivar Désirée was grown in three different media: a control medium corresponding to the MS-based medium [12], and two other media with respectively 40 times less and 3 times more iron compared to the control medium. A detailed study on plant growth, mineral contents and gene expression of genes implicated in iron uptake, homeostasis and storage in different potato organs is presented.

Fig. 1. Shoot and root dry weight measured in plantlets growing in 1, 40 and 120 mg L1 FeNaEDTA media, 14, 21, 28 days after transfer. A: shoot dry weight B: root dry weight. Letters display statistically significant difference obtained using a multiple comparison analysis of mean of 3 biological replicates (10 plantlets per replicates) with a Tukey test (95% of limit of confidence).

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2. Results and discussion 2.1. Morphology Fourteen days after transfer, shoot dry weight displayed no significant difference between plantlets grown on the three different media, a slightly lower weight was however observed for

Fig. 2. Iron content (mg g DW1) measured in plantlets growing in 1, 40 and 120 mg L1 FeNaEDTA media, 14 and 28 days after transfer. A: leaves, B: stems, C: roots. Letters display a statistically significant difference obtained using a multiple comparison analysis of mean with a Tukey test (95% of limit of confidence).

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plantlets grown in the HIM (Fig. 1). This trend was confirmed 21 and 28 days after transfer: a significant decrease of shoot dry weight was observed in HIM plants compared to the CM. Low iron media did not affect shoot dry weight until 28 days after transfer when a significant decrease was observed compared to CM (Fig. 1). Concomitantly, a bleaching of the young leaves was observed in plantlets grown in the LIM. Twenty-one days after transfer, plantlets grown on the LIM had a lower root dry weight compared to the CM. This trend was also observed after 14 and 28 days but was not significant. Twenty-eight days after transfer, plantlets grown on the HIM had a higher root dry weight compared to the other treatments. 2.2. Total mineral content 2.2.1. Iron content Leaf iron content was different between treatments. 14 days after transfer, iron content increased concomitantly with the iron available in the media. After 28 days, no difference was observed between plantlets grown in control and high iron content media. Leaf iron content in LIM plantlets significantly decreased and remained significantly lower compared to the other treatments. Between 14 and 28 days after transfer, leaf iron content also decreased in the HIM and remained stable in the control medium (Fig. 2A). As observed in the leaves, the highest iron content in stems was observed in plantlets grown in the HIM. In all media, a decrease in iron content was observed between both time-points (Fig. 2B). This result might be explained by the increase of dry weight of the plantlets, which “dilutes” the iron content. After 28 days, iron content in stems seemed to be correlated with the iron available in the media. In roots (Fig. 2C), iron content did not significantly change in plantlets grown in low iron and control media at both time-points. However, a drastic iron accumulation was observed in plantlets from the HIM, especially 28 days after transfer, where the iron content reached 1699 mg/g DW. Overall, plantlets grown in the LIM generally displayed the lowest iron content in all plant parts (leaves, stems and roots) compared to the other treatments. This trend was clearly marked 28 days after transfer, when the iron content decreased even more. In the CM, the iron content remained nearly the same across the experiment in all organs. Plantlets grown in medium with 120 mg/L FeNaEDTA usually displayed the highest iron content especially in roots where the iron accumulation was five to thirty times higher than the one observed in the other conditions. It is also interesting

to note that this drastic accumulation was not observed in stems and leaves suggesting that plantlets might be able to limit the iron transport to the aerial parts and to store it in roots. 2.2.2. Manganese and zinc content Manganese and zinc shoot content were shown to increase in Arabidopsis shoot under iron deficiency condition [13], which was not observed, in our experiment, in potato shoots (see Supplementary data). Conversely, Mn, Zn and Co root contents increased in the wild type Arabidopsis plantlets grown in iron deficiency substrate without any changes in shoot [14]. In potato roots, some changes in Zn and especially Mn were observed. Fourteen days after transfer, Zn content displayed no significant difference between plantlets grown in the different media, albeit there was a slight trend of increase for those grown in LIM. After 28 days, we observed a decrease of the Zn content in plantlets grown in LIM and no significant changes in the other media (Fig. 3A). Comparatively, Mn content displayed a drastic increase in plantlets grown in LIM, 14 and 28 days after transfer whereas Mn content remained at a low level in the other media (Fig. 3B). These increases in Mn and Zn may be due to the enhanced expression of the iron uptake related genes, especially IRT1 which transports iron as well as Mn, Zn, Co and Cd (see below). 2.3. Gene expression Iron uptake, transport and storage have been well described in model plants such as A. thaliana or rice to name a few [3,7,15e17]. However in potato, few studies have been performed on the iron uptake, transport and storage mechanisms, especially at the gene expression level. 2.3.1. Iron uptake in potato In potato, iron uptake belongs to strategy I model [7]. According to this model, plants first acidify the soil using a proton-extruding HþeATPase and then Fe3þ is reduced to Fe2þ by a ferric chelate reductase (FRO). In our experiment, expression of the ferric chelate reductase was investigated in the three media described above (Fig. 4, Table 1). We designed qPCR primers from an EST (BQ045591.1), which is 94% homologous to the Solanum lycopersicum FRO1 (AAP46144.1) [18]. In roots, a drastic induction of this gene under iron deficiency condition was observed. Inversely, in the other media the ferric chelate reductase was only slightly expressed. In stems, nearly the same trend was observed whereas, in leaves, expression of FRO1 was higher in the low iron substrate compared to the other substrates at the beginning of the

Fig. 3. Zinc and manganese content (mg g DW1) measured in plantlets growing in 1, 40 and 120 mg L1 FeNaEDTA media, 14 and 28 days after transfer. A: Zn, B: Mn. Letters display a statistically significant difference obtained using a multiple comparison analysis of mean with a Tukey test (95% of limit of confidence).

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experiment (Fig. 4, Table 1). These data suggest that this gene is strongly regulated by the iron deficiency status of the plant, since it was only slightly repressed under excess of iron as previously described in S. lycopersicum [18]. The next step is the uptake of reduced Fe2þ by IRT (iron-regulated transporter), a member of the ZIP family protein involved in iron transport [2]. Two accessions homolog to LeIRT1 and LeIRT2 [19] were found; BQ514716.1 (called IRT1 in this study) was 100% homolog with LeIRT1. A second EST called CX161657 (IRTx in this study) was 87% homolog with both LeIRT1 and LeIRT2. It is difficult to assess that IRTx is the homologous gene of LeIRT2. But, in roots and stems, IRTx expression did not drastically change from one medium to the other and was even sometimes not observed in higher iron content media, supporting its weak role in iron deficiency responses. In Arabidopsis, IRT2 does not participate in iron uptake and is described as a secondary component of the iron homeostasis and might be involved in iron sequestration in vesicles [20]. However, we did not observe any induction of IRT2 in plantlets grown in HIM. Expression of IRT1 was low in the HIM and CM, in all the compartments of the plant (root, stem and leaves). However, in LIM, we observed a strong increase of IRT1 expression in roots (Fig. 4, Table 1) suggesting that IRT1 is involved in iron deficiency response as demonstrated [21]. Moreover, according to these authors, neither IRT2 nor FRO2 can complement the lack of irt1 gene expression. IRT1 is expressed in the root epidermis and is involved in iron homeostasis in plants. Additionally to iron, it can also transport other metals such as zinc, manganese and cadmium to name a few. This may explain the strong increase in Mn content observed in plantlets grown in LIM: enhanced expression of IRT1 under iron deficiency can trigger accumulation of other minerals such as Mn, Zn, Co and Cd [13,14]. 2.3.2. Iron homeostasis in cells After uptake by root cells, iron cannot remain in its free form due to its low solubility and its potential oxidative properties. Thus, iron can be chelated by nicotianamine (NA), a non proteinogenic amino acid, which helps to transport iron through the phloem [8]. In our study, the expression of nicotianamine synthase in roots and leaves was not different between the three media. However, in stems, a significantly increased expression was observed in plantlets grown in LIM. These data suggest that the expression of nicotianamine synthase is influenced by iron deficiency in the plant, although we could expect a lower synthesis of nicotianamine correlated with the lower amount of iron observed in stems from plantlets grown in LIM. Additionally, we designed qPCR primers targeting a FRD3-like pump, which belongs to multi-drug and toxin efflux (MATE) family and is considered to be involved in iron transport in the plants and to participate to iron deficiency signaling [22]. It is postulated that FRD3 transports citrateeFe complex into the xylem [23]. In roots, we observed a significantly increased expression of the FRD3 in LIM. The same trend was observed in the stems where the expression level was slightly lower (Table 1). In leaves, FRD3-like expression was not influenced by the iron status in the media. These results are in accordance with previous experiments performed in Arabidopsis, where FRD3 has been shown to be involved in iron deficiency responses, FRD3 being induced by iron starvation [24], but its main role in iron deficiency response is not yet clearly identified. We may speculate that, under iron deficiency, FRD3 expression is enhanced in order to remobilize iron from roots to leaves. NRAMP transporters (natural resistance-associated macrophage protein) are another broad family of membrane proteins, which are involved in metal transport including manganese, iron and cadmium. In order to find NRAMP potato mRNA sequences, six A. thaliana mRNA sequences (AF165125, AF141204, AF202539,

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AF202540, AJ292076, AJ291831) were used as input for BLAST search in the Solanum tuberosum EST database and 3 groups were found. NRAMP1 (CK640855) and NRAMP3 (CV492123) shared respectively 97% and 96% of homology with the S. lycopersicum NRAMP1 (AY196091) and NRAMP3 (AY196092) [25]. NRAMP2 shared 96% of homology with LeNRAMP2 (AAS6787.1) [26], 88% of homology with AtNRAMP2 mRNA [10], 100% homology with AtNRAMP4 and AtNRAMP5 mRNAs.

Fig. 4. Hierarchical clustering performed on the qPCR results with absolute correlation and centered data with complete linkage. Hierarchical clustering was performed independently in leaves, stems and roots. For better visualization, data from each gene of each organ were rescaled to a 0e1 range. In red: increased expression, in gray: low expression. For detailed results, refer to Table 1.

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Table 1 Normalized relative expression obtained on: ferritin 1, 2 3, NRAMP (natural resistance-associated macrophage protein) 1, 2, 3, IRT1 (iron regulated transporter 1), IRTx, FRO1 (ferric chelate reductase 1), FER (basic helix-loop-helix 29-like transcription factor), the VIT1 (vacuolar iron transporter 1), FDR3 and NicoS (nicotianamine synthase). Legend: nd: no data, NRE: mean value of normalized relative expression of three replicates, SD: standard deviation. HIM: high iron medium, CM: control medium, LIM: low iron medium. For each gene data were rescaled to 1 for the minimum observed value. Letters in the “Tukey” column display the result of a general linear mixed model with a Tukey Post-Hoc test performed on three biological replicates on each organ independently. Treatment

Ferritin 1

HIM

CM

LIM

Ferritin 2

HIM

CM

LIM

Ferritin 3

HIM

CM

LIM

NRAMP1

HIM

CM

LIM

NRAMP2

HIM

CM

LIM

NRAMP3

HIM

CM

LIM

IRT1

HIM

CM

LIM

Time-point (days)

14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28

Leaves

Stems

NRE

SD

152.98 143.30 270.78 78.10 49.30 167.73 263.53 181.44 38.78 9.78 11.75 12.53 4.69 3.56 7.57 5.20 2.48 1.08 35.99 30.69 44.91 23.97 23.39 48.00 11.14 14.95 16.62 nd nd nd nd nd nd nd nd nd 2.92 2.23 3.28 2.09 1.72 2.23 1.17 1.00 1.08 5.04 4.53 4.93 12.77 11.88 2.48 11.03 13.85 16.97 42.10 7.11 3.23 44.07 10.27 8.42 12.86 1.99 8.84

47.02 61.52 199.83 16.33 25.19 65.88 305.39 211.93 51.77 1.11 3.77 5.46 0.28 2.45 2.53 3.55 2.04 0.97 4.23 8.33 15.49 7.22 7.08 4.99 4.90 1.55 3.68 nd nd nd nd nd nd nd nd nd 0.75 0.29 1.12 0.12 0.46 0.28 0.22 0.26 0.22 0.22 1.62 0.32 3.83 4.71 0.23 4.66 7.05 4.20 6.80 7.09 2.93 28.00 7.23 5.16 14.68 1.75 8.49

Tukey a a a a a a a a a bc c c bc abc bc bc ab a cd bcd d bcd abcd d a ab abc

cd cd d bcd abc cd ab a a ab ab ab bc bc a bc bc c b ab a b ab ab ab a ab

Roots

NRE

SD

1069.13 637.47 1078.44 641.18 400.95 1246.03 65.36 79.37 79.37 36.27 32.13 49.93 24.35 20.03 41.41 6.30 7.69 4.59 26.86 19.79 34.41 16.77 19.28 38.71 7.39 9.74 9.34 nd nd nd nd nd nd 22.00 13.76 19.41 3.65 2.77 2.51 2.40 2.29 2.71 1.12 1.28 1.83 2.29 2.62 1.49 3.33 4.22 1.52 10.53 8.96 8.00 5.76 1.72 1.36 2.30 4.00 1.00 59.69 47.35 69.43

318.27 128.90 265.49 204.27 118.99 310.33 4.01 11.30 12.75 5.23 5.86 4.27 5.18 11.26 5.70 0.27 1.22 1.18 2.85 3.90 7.25 3.85 9.59 9.17 0.24 1.21 2.48 nd nd nd nd nd nd 13.12 7.38 26.21 0.07 0.12 0.18 0.07 0.47 0.20 0.21 0.23 0.11 0.16 0.49 0.20 1.08 1.60 0.23 0.80 3.36 1.89 5.37 0.87 0.83 0.82 2.92 0.62 8.92 19.97 86.67

Tukey cd bcd cd bc b d a a a bcd bcd d bc b cd a a a def cdef ef bcd bcde f a abc ab

a a a d cd bc bc bc cd a a b ab ab a b bc a d d cd a a a a a a b b b

NRE

SD

Tukey

143.27 330.50 1326.80 56.48 511.67 1934.74 1.00 3.83 27.83 11.73 24.43 30.03 11.64 26.75 40.19 1.00 2.43 5.27 8.01 14.33 24.79 8.25 17.33 28.91 1.00 2.32 5.16 1.33 3.18 5.62 1.00 4.69 9.79 102.65 164.90 177.86 2.34 2.60 2.48 1.76 2.75 3.28 1.20 1.42 2.01 1.13 1.38 1.00 1.85 1.36 1.25 6.66 4.29 3.09 5.81 8.56 3.32 3.96 13.88 20.42 728.17 566.58 563.83

137.18 46.80 250.50 19.97 444.80 407.27 0.10 2.27 14.04 3.04 5.75 11.11 1.57 10.53 2.88 0.14 0.37 1.28 2.13 4.83 2.99 0.88 5.06 2.97 0.16 0.07 0.97 0.49 0.28 0.43 0.19 2.51 4.52 16.30 9.18 9.76 0.32 0.13 0.43 0.13 0.41 0.22 0.23 0.11 0.24 0.05 0.19 0.07 0.26 0.12 0.21 1.18 0.93 0.68 1.99 2.93 1.28 1.23 9.93 14.24 236.74 182.31 46.49

bc cd de b cde e a a b d e e d e e a b c c de f cd ef f a b c ab bc cd a cd d e e e cde de cde bc de e a ab bcd a ab a b ab ab d c c ab ab a ab ab b c c c

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Table 1 (continued ) Treatment

IRTx

HIM

CM

LIM

FRO1

HIM

CM

LIM

FER

HIM

CM

LIM

VIT1

HIM

CM

LIM

FDR3

HIM

CM

LIM

NicoS

HIM

CM

LIM

Time-point (days)

14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28 14 21 28

Leaves

Stems

NRE

SD

nd 4.48 nd 16.75 8.93 7.27 3.01 1.07 8.80 137.23 277.21 1456.17 345.06 1568.38 1422.52 1227.05 1868.94 2045.77 1.50 1.32 6.98 2.47 4.85 5.56 9.54 8.37 5.91 2.14 2.06 2.93 2.35 2.50 2.11 1.49 1.44 1.68 2.08 2.84 11.00 4.50 7.74 9.61 6.58 5.75 3.61 2.42 1.23 4.70 1.14 1.00 3.33 5.49 3.59 3.57

nd 5.80 nd 9.78 5.94 2.68 2.58 0.20 8.24 32.32 141.45 700.44 52.31 411.52 407.28 276.82 887.99 114.10 0.50 0.52 5.19 0.57 1.71 2.16 5.01 3.83 1.17 0.20 0.32 0.85 0.43 0.31 0.42 0.43 0.40 0.16 0.51 1.09 6.91 1.29 4.01 9.28 1.46 3.27 1.32 0.39 0.39 5.18 0.37 0.61 1.98 4.74 1.61 0.28

NRAMP1 was mainly expressed in roots and slightly in stems, but its expression was not detectable in leaves (Table 1). NRAMP2 and NRAMP3 were expressed in all compartments. NRAMP1 and NRAMP3 displayed a higher expression under iron deficiency condition (LIM) (Fig. 4, Table 1). In roots, expression of NRAMP1 increased drastically (more than 20 fold increase) in all time-points in LIM compared to CM or HIM. In stems, expression of NRAMP1 was low in LIM and not detectable in the other media. As mentioned above, NRAMP3 was expressed in all compartments of the plantlets, but highest in leaves (Table 1). In roots and stems, NRAMP3 displayed an increased expression when plantlets were grown in LIM compared to the other media. Interestingly, in leaves, expression of NRAMP3 differed between the three media: in HIM, expression was stable across the experiment and remained at

Tukey ab b ab ab ab a ab a a b a b b b b b ab a bc abc abc bc c c bc ab ab b ab ab ab a a ab a a a a a a a a a a a a a a a a a a

Roots

NRE

SD

1.41 1.32 2.35 1.44 1.75 2.35 3.51 4.29 3.41 nd nd 58.33 33.90 130.85 66.29 1358.74 1206.56 622.58 3.13 1.00 2.30 1.73 2.71 1.59 26.82 22.09 28.26 2.80 2.10 1.56 2.36 2.10 1.40 1.65 1.35 1.62 1.55 1.72 1.44 1.00 2.10 1.44 5.01 5.05 4.13 6.45 3.92 4.27 3.15 3.57 2.80 8.18 8.64 8.76

0.10 0.06 0.48 0.59 0.08 0.44 1.24 1.74 0.29 nd nd 34.31 20.72 11.07 43.26 608.63 523.62 298.95 0.82 0.09 0.27 0.68 0.20 0.20 6.75 7.19 33.73 0.27 0.22 0.29 0.21 0.44 0.11 0.37 0.14 0.35 0.09 0.75 0.27 0.10 0.39 0.44 0.16 2.05 0.92 0.23 1.50 1.25 0.51 0.48 0.19 1.58 1.58 2.87

Tukey a a abc a ab abc bc c bc

ab a b ab c c c a a a a a a b b b c abc ab bc abc a ab a ab ab ab ab a bc ab d d d bc ab ab a ab a c c c

NRE

SD

Tukey

3.00 2.92 1.00 3.17 6.20 3.27 5.55 5.52 3.42 2.64 2.03 1.00 2.63 19.97 13.36 372.22 847.41 1616.49 64.05 74.22 74.41 31.82 45.04 57.89 237.97 184.79 149.97 1.42 1.04 1.43 1.07 1.09 1.39 1.00 1.10 1.36 5.00 4.06 7.33 3.12 4.14 8.36 12.16 11.49 13.29 7.68 5.89 4.67 5.35 5.36 3.75 9.36 8.28 5.82

0.43 1.58 0.57 0.84 4.72 2.86 0.48 0.79 0.64 0.92 1.14 0.87 0.64 17.77 13.07 158.93 470.09 495.85 8.97 25.05 5.67 5.23 11.54 15.20 26.25 16.38 12.98 0.17 0.03 0.28 0.18 0.06 0.05 0.05 0.15 0.12 1.05 0.40 0.53 0.68 0.98 3.32 2.44 1.78 2.23 1.74 1.39 2.14 0.63 2.06 0.88 4.64 2.13 1.05

ab ab a ab b ab b b ab ab a a ab b ab c c c b b b a ab ab c c c b ab b ab ab b a ab ab abc ab bcd a ab cd d d d a a a a a a a a a

a basal level whereas it tended to increase in plantlets grown in LIM. Interestingly, in the CM, expression of NRAMP3 strongly decreased after 28 days of the experiment (Table 1). In A. thaliana, NRAMP1 and NRAMP3 display the same expression pattern under iron deficiency condition [9,10], however they may act at different levels. On low iron substrate, AtNRAMP3 has been shown to be responsible for remobilization of iron from the vacuole during seed germination [9]. AtNRAMP1 is located in the plasma membrane, has a strong affinity for Mn2þ and can also transport Fe2þ [27]. Under low iron condition, AtNRAMP1 driven by the IRT1 promoter has been shown to partially complement the irt1-1 mutant, conferring a better ability to extract Fe2þ than the wild type plants and restoring the decrease in Mn2þ observed in the irt1-1 mutant [27]. This indicates that NRAMP1 might participate in the iron

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deficiency response. Moreover, the strong increase in Mn2þ in potato roots grown in LIM (Fig. 3B) might be due to both StIRT1 and StNRAMP1 enhanced expression. To summarize, under iron deficiency, NRAMP3 might release stored iron in the cytosol in order to counterbalance the lack of iron and NRAMP1 might assist IRT1 in the Fe2þ uptake. Compared to NRAMP1 and NRAMP3, NRAMP2 revealed a different gene expression pattern (Fig. 4, Table 1). In stems and leaves, NRAMP2 showed a lower expression in plantlets from the LIM and gene expression slightly increased in the HIM compared to the control media. In roots, NRAMP2 displayed a lower expression in plantlets grown in the LIM (Table 1). But, in both low iron and control substrate, expression increased during the experiment and became higher in the control medium than in the high iron one. This trend was also observed in A. thaliana where NRAMP2 displayed an inverse expression pattern compared to NRAMP1 [10]. As previously mentioned, in plant cells, high free iron can lead to production of reactive oxygen species and oxidative stress. Iron can be temporarily stored in the vacuole but ferritin proteins have been described as the main form for iron storage and support iron homeostasis in plants [11]. In order to get a clear overview of the expression of the ferritin gene, we distinguished several potato ferritin isoforms. A sequence alignment was performed from the ferritin mRNA of A. thaliana, Nicotiana tabacum and Glycine max, extracted from the NCBI UniGene database (Fig. 5). A short consensus amino acid sequence was then used as query in the S. tuberosum EST database to collect a high number of matching EST. Three distinct consensus sequences (Stu-Fer1, Stu-Fer2 and StuFer3) were found and specific primers were designed for sequencing validation (Fig. 5). qPCR primers were then designed to study the expression of these genes. Overall, ferritin 1 and 2 seemed to be mainly expressed in roots and stems whereas only a slight expression was observed in leaves (Table 1). Inversely, expression of ferritin 3 was expressed equally in all plant parts. In leaves, ferritin 2 and 3 displayed a slight increase of expression in the HIM but no significant difference was observed for the ferritin 1 isoform (Table 1). In stems, all ferritin isoforms were down-regulated in plantlets grown in iron deficiency condition (LIM). In roots, ferritin 1, 2 and 3 displayed the same expression pattern. The lowest expression was observed in plantlets grown in the LIM whereas in the CM and HIM, ferritin expression was significantly higher (Fig. 4). It is noticeable that ferritin expression was not considerably enhanced by the iron overload observed by ICP-MS in the roots of plantlets grown in the HIM, compared to control condition. Under high iron treatment, expression of AtFer1 and AtFer3 increases in both roots and leaves [28], this is in contradiction with our results. Investigation of ferritin

accumulation at the proteomic level might reveal a posttranscriptional regulation leading to a higher accumulation of ferritins in roots. There also might be other mechanisms of iron sequestration. In iron deficiency condition, the ferritin expression was quite well correlated with the iron content observed in plantlets. In addition, we designed a primer set targeting the vacuolar iron transporter 1 (VIT1) from the EST BG600564 based on the A. thaliana homolog (At2g01770) [4]. This gene is involved in iron homeostasis transporting iron from the cytosol to the vacuole [4]. In roots, StVIT1 was not or slightly differentially regulated in all treatments. In stems, expression of VIT1 remained low and stable along the experiment in the iron deficiency medium. Inversely, in the CM and HIM, transcript abundance of VIT1 was high and decreased during the experiment to reach the expression level observed in the LIM. Finally, in leaves, expression of VIT1 tended to increase during the experiment in the HIM, whereas it remained stable in the other media. But, the lowest expression level was observed in plantlets grown in the LIM. All these results suggest that VIT1 gene expression might be driven by the iron status of the plant. 2.3.3. Iron deficiency regulation via FER-like transcription factor We previously observed a strong induction of several genes such as IRT1 and NRAMP1 during iron deficiency, which could be driven by some common transcription factors, as described in plant models such as A. thaliana [3]. To assess this hypothesis, we designed qPCR primers from a S. tuberosum EST (BQ512373.1), which shared 90% identities with the S. lycopersicum FER transcription factor (AF437878) [29]. FER-like transcription factor was mainly expressed in roots; in leaves and stems, we only observed a low expression level, which slightly increased under iron deficiency (LIM) condition in stems (Table 1). In roots under control conditions, expression of the FERlike transcription factor remained at a low level as well as in the HIM, in which the expression was slightly higher. In LIM, a strong induction of this transcription factor was observed, suggesting that the expression of this gene was influenced by the iron status of the plantlets. Moreover, principal component analysis performed on qPCR results obtained in roots displayed a strong correlation between the IRT1, NRAMP1 and FER-like transcription factor genes and to a lesser extent with the NRAMP3 and FRO1 genes (Fig. 6A). Additionally, the results of the individual clustering reveal a strong difference in the expression pattern in plantlets grown in iron deficiency condition compared to the other treatments (Fig. 6B). Moreover, we observed a slight time-dependent distinction between results obtained after 2 and 3 weeks and those obtained 4

Fig. 5. Alignment of a partial sequence of the ferritin. Amino acid sequences were obtained from mRNA sequences coming from the NCBI UniGene database of Arabidopsis thaliana: Ath_Fer1, 2, 3 and 4; Glycine max: Gma_Fer1, 2, 3 and 4; Nicotiana tabacum: Nta_Fer1 and 2; Solanum tuberosum: Stu_Fer1 and x (undefined name). The three S. tuberosum sequenced ESTs obtained from the in silico analysis are also added: Stu_Fer1_seq, Stu_Fer2_seq and Stu_Fer3_seq.

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Fig. 6. Principal component analysis performed on qPCR results obtained from roots. A: gene expression correlation. B: sample clustering obtained from normalized expression value of all genes. Labeling of the data-points: days after transfer e medium type (1-LIM, 40-CM, 120-HIM) e replicate number.

weeks after transfer, suggesting that overall gene expression was influenced by the exposure time to the several treatments. No clear difference was observed between the high iron treatment and the control condition. Altogether, we observed a positive correlation between the expression of FER-like transcription factor and the NRAMP1 and IRT1 genes. Inversely, FER-like transcription factor might not influence the expression of ferritin (Fig. 6). These results might indicate that the FER-like transcription factor might be involved in the iron deficiency response and also impact the expression of several genes, as mentioned in previous studies in A. thaliana and S. lycopersicum [29e31]. All plants belonging to iron uptake strategy 1 may possess a main regulator of iron efficiency response [31], which is homolog to the LeFER gene. LeFR01 is driven by the FER transcription factor [18], which also regulates LeIRT1 and

LeNRAMP1 under iron starvation [25,29]. Inversely, expression of LeFER transcription factor was suppressed by high iron and thus expression of the genes mentioned above would be reduced [30]. Further studies have to be performed in order to confirm that the FER-like transcription factor is a key regulator of the iron deficiency response in potato. 3. Conclusion The present work provides a first report of the expression of genes involved in iron uptake, homeostasis and storage in potato. Under iron deficiency condition, our results were in agreement with the literature displaying the important role of IRT1, NRAMP family proteins or ferritins. However, the qPCR results obtained with plantlets grown in HIM did not allow to give clear answers on

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the mechanisms leading to the high iron content observed in the roots by ICP-MS. Further experiments should be performed at the protein level in order to elucidate this point. If the present in vitro experiment allowed an optimal control of the iron level in the substrate, further steps will include investigations on potato cultivated ex vitro, in a soil substrate, allowing to understand the mechanisms of iron accumulation in the tuber.

purity and concentration were assessed measuring the absorbance at 230 nm, 260 nm and 280 nm using a Nanodrop ND1000 spectrophotometer (Thermo scientific, Villebon-sur-Yvette, France). 4.6. Reverse transcription and qPCR

The S. tuberosum cultivar Désirée was used for the experiment. In vitro plantlets were obtained from a home collection multiplied each month on an MS½ medium. Before the experiment, 3 multiplication cycles were performed on MS medium with 4 weeks of growth period per cycle and 10 plantlets per box (10 cm (L)  10 cm (H)  5 cm (W)). At day 0, 1.5 cm of the plantlets apexes were transferred into the final experimental media. Plantlets were grown in a climatized room at 20  C with 16 h-photoperiod. Light was provided by GROLUX T8 30W tubes (Sylvania, Antwerpen, Belgium) with 40 mmol m2 s1 of irradiance.

Primers were designed with the Primer3 software (http://frodo. wi.mit.edu/) with the following criteria: primer size between 18 and 25 base pairs, GC content between 30 and 70%, amplicon size from 50 to 200 base pairs, Tm of primers in the 59e61  C range. Matching primer sets were validated using NetPrimer (http:// www.premierbiosoft.com/netprimer/index.html) for unexpected secondary structures (DG should be below 5 kcal/mol with default settings). In order to test the specificity of primers, an in silico BLAST engine was used. Reverse transcription was performed using the Superscript II reverse transcriptase (Invitrogen, Carlsbad, NM, USA) from 1 mg RNA following the manufacturer guidelines in a 20 mL final volume. PCR was performed using Mesa Green Low Rox Real-time PCR Kits (Eurogentec, Liège, Belgium) with the following final concentrations in 25 mL final volume: 1 MasterMix, 100 nM forward and reverse primers, 0.4 ng mL1 cDNA on a 7500 Fast system (Applied Biosystems). Thermal cycling conditions were: initial 5 min denaturation at 95  C, followed by 50 cycles of 15 s at 95  C and 1 min at 60  C, and a final dissociation step. Primer specificity was controlled by the presence of a single peak in the melting curve and PCR efficiency was assessed using decreasing five-fold dilution (from 25 ng to 0.04 ng and no cDNA). Relative expression was calculated taking into account multiple reference genes and gene-specific PCR efficiency [32,33]. The two best reference genes among the set previously described [34] were selected using the Normfinder tool. Sub-groups corresponding to plant organs and treatments were assigned for the analysis. For this experiment, the two most stable housekeeping genes were the cytoplasmic ribosomal protein L2 and the adenine phosphoribosyl transferase (APRT). For details see Supplementary data.

4.3. Sampling

4.7. ICP-MS analysis

Three biological replicates were sampled (10 plantlets ¼ 1 box constitute one biological replicate). Sampling for gene expression was performed 14, 21 and 28 days after the beginning of the experiment and after 14 and 28 days for ICP-MS analysis. Leaves, stems and roots were sampled into individual RNase free Eppendorf tubes, flash-freezed and stored at 80  C. Prior to ICP-MS analysis, plantlets were washed two times each in 1 L of 10 mM Na2EDTA and 1 L of ultrapure water.

Samples, 250 mg of dry weight, were digested in 7 mL HNO3 (Plasma Pure 67e70%, SCP Science) (Courtaboeuf, France) and 3 mL H2O2 (30% in weight for metal trace analysis, Fisher Scientific) (Tournai, Belgium). Acid digestion was performed in Teflon tubes in a microwave oven (Anton Paar Multiwave 3000) (Graz, Austria) by increasing temperature and pressure until 200  C and 30 bars. At the end of the protocol, samples were diluted with ultrapure water up to 25 mL and kept at 4  C until analysis. Blank and certified reference material (spinach, NCS ZC 73013, LGC Standards) (Molsheim, France) were included at each mineralization cycle for quality control. Samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer Elan DRC-e) (Waltham, MA, USA).

4. Material and methods 4.1. Culture media In the MS medium [12], iron is mainly supplied as FeNaEDTA. Thus, experimental media were prepared based on MS medium with three different concentrations of FeNaEDTA: 40.4 mg L1 (control medium: CM), 120 mg L1 (high iron medium: HIM) and 1 mg L1 (low iron medium: LIM) FeNaEDTA. EDTA concentration was adjusted in all media with Na2EDTA to the maximum EDTA concentration, obtained in the 120 mg L1 FeNaEDTA medium. All media were supplemented with 30 g L1 sucrose (Beghin Say) and 8 g L1 agar (Kalys AgarÔ HP 696, Kalys, France); pH was adjusted to 5.8 using a KOH 0.1 M solution. All media were autoclaved before use. 4.2. Plant material and culture conditions

4.4. Determination of dry weight For each biological replicate, roots and shoots were collected from one box corresponding to 10 plantlets. Samples were dried at 60  C during 48 h in an oven. Dry weight was measured using a Mettler Toledo AG204 balance. 4.5. RNA extraction and quality control Total RNA was extracted from 100 mg of frozen leaves using the RNeasy Plant Mini Kit (Qiagen, Leusden, The Netherlands) including DNase treatment (following the manufacturer’s instructions). Quality control was performed with the RNA Nano 6000 assay (Agilent Technologies, Diegem, Belgium) using a 2100 Bioanalyzer with quality parameters adapted to plant RNA profiles (Agilent Technologies). RNA samples with a RIN (RNA integrity number) lower than 7 were excluded from the experiment. RNA

4.8. Statistics For all experimental datasets, 3 biological replicates (consisting each of a pool of 10 plantlets) were used. Normal distribution was assessed and a general linear mixed model with a Tukey Post-Hoc test was applied using the SPSS 16 software. For dry weight measurement, ICP-MS measurement and relative expression, data corresponding to each organ (shoot and root for dry weight measurement or root, stem and leaves for ICP-MS and gene expression analysis) were analyzed independently. Results of the Post-Hoc test are displayed on the charts using letters, different letters indicating significantly different datapoints.

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