Introduction

Metal ions, such as Fe, Mn, Zn, and Cu are essential elements for plant growth and development (López-Millán et al. 2009). However, these ions (particularly Fe) have poor solubility in most types of soil (Guerinot and Yi 1994; Han et al. 2012), particularly in calcareous soil solution where the concentration of free Fe is far below 10−6 M, a required concentration for optimal plant growth (Han et al. 1998). Therefore, Fe deficiency is a worldwide problem for crop production. Iron deficiency chlorosis is a very common disease in apples, especially in North China (Gao et al. 2011), and largely limits the growth, yield, and quality of apples (Zhang et al. 2012).

To avoid such deficiencies, plants have developed sophisticated regulating mechanisms to acquire Fe from the soil, which have been classified into two strategies (strategies I and II) by Marschner and Romheld (1994). ‘Strategy I’ plants produce more ferric reductase-oxidase under Fe-deficiency stress, to reduce Fe(III) to Fe(II) and benefit Fe uptake (Zhang et al. 2009). The absorption and utilization of Fe in apple (Malus xiaojinensis included) follows the reduction mechanism of ‘Strategy I’ (Li et al. 2006a). Several changes have also been observed at the metabolic level in order to sustain the increased Fe uptake capacity of Fe-deficient plants.

In response to Fe deficiency, all non-graminaceous higher plants appear to adopt ‘Strategy I’, in which the role of the nicotianamine (NA) synthesis gene has not yet been determined. Additionally, NA can chelate Fe(II) for Fe transport through phloem (Stephan et al. 1994) and citric acid can chelate Fe(III) for its transport through xylem (Rellán-Álvarez et al. 2008) in plants. Recent study indicated that a transporter, FRD3, is necessary for efficient Fe translocation to the plant sap (Durrett et al. 2007). The Arabidopsis mutant, frd3, which performs Strategy I responses, showed constitutive expression regardless of the external Fe supply, therefore it has provided molecular evidence of the role of NA in iron transport (Rogers and Guerinot 2002; Green and Rogers 2004).

To identify Fe-efficient genotypes, we have collected more than 40 genus Malus samples of various species and ecotypes. Previous studies indicated that M. xiaojinensis is a Fe-efficient apple genotype (Han et al. 1994a, b). However, the role of NA synthesis gene in M. xiaojinensis and MxNas1 (a gene encoding putative NA synthase from M. xiaojinensis, Genbank accession no. DGB0458) is not very clear. The Nas1 gene plays a key role in synthesizing NA synthase, but the relationship between the Nas1 gene and iron transport or plant development remains unclear. In this study, we detected the expression of MxNas1 in different organs and investigated the relationship between the expression of MxNas1 and indoleacetic acid (IAA), abscisic acid (ABA) as well as Fe stress treatments. Importantly, overexpression of MxNas1 improved plant tolerance to Fe stress in transgenic tobacco, and resulted in late-flowering and abnormally shaped flowers.

Materials and Methods

Plant Material and Growth Conditions

M. xiaojinensis Cheng and Jiang test-tube seedlings are rapidly propagated on Murashige and Skoog medium (MS) + 0.5 mg L−1 6-BA + 0.5 mg L−1 indole-3-butytric acid (IBA) for 1 month, and then returned to MS + 1.0 mg L−1 IBA for one and a half months for rooting. Finally, the seedlings are transferred to Hoagland solution for 1 month for growth. When the plants have eight to nine mature leaves (fully expanded), plants were exposed to different iron concentrations Hoagland nutrient solutions (4, 40, and 160 μM). For IAA and ABA treatments, seedlings were respectively put in 0.1 mM IAA and 0.1 mM ABA Hoagland solution with normal Fe concentration (40 μM). All samples of control and treated plants were taken after treatments of respectively 0, 6, 12, 24, and 48 h, and were frozen immediately in liquid nitrogen, and stored at −80 °C for RNA extraction.

Real-Time PCR Analysis of MxNas1 Expression

Total RNA was extracted separately from root, phloem, xylem, young leaf (partly expanded), and mature leaf (fully expanded) using the CTAB method (Zhang et al. 2005). First-strand cDNA was synthesized with 1 μg total RNA and 1 μL superscript II enzyme (Invitrogen, USA) according to the manufacturer protocol. As a control, the Actin rRNA gene was amplified from M. xiaojinensis tissues using the following primers: Mx18SF, 5′-ACACGGGGAGGTAGTGACAA-3′ and Mx18SR, 5′-CCTCCAATGGATCCTCGTTA-3′.

The cDNA was diluted fivefold, and 2 μL of the dilution was used for quantitative RT-PCR. The iQ SYBR Green Supermix (Applied Biosystems, Foster City, CA, USA) was used for amplification on an Applied Biosystems 7500 RT-PCR system (Yang et al. 2011). The expression levels of each sample were normalized against glyceraldehyde-3-phosphate dehydrogenase mRNA expression levels. The methods for MxNas1 gene expression were designed for real-time polymerase chain reaction (real-time PCR) as follows. The primers for MxNas1 were designed for real-time PCR from partial sequences isolated in this work. The primers for MxNas1 (DGB0458) genes were designed from partial sequences published in the GenBank databases. The primer sets used are MF, 5′-GAGTCGACATGTGTTGCCAGGGA-3′ and MR, 5′-CAGGATGTTTTAAG*AAAGCTGCT-3′. The thermal cycling program was one initial cycle of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s (Jia et al. 2012). The relative gene expression data were analyzed using the 2-ΔΔCT method.

Subcellular Localization of the MxNas1 Protein

The MxNas1 ORF was cloned into the XalI and SamI sites of the pSAT6-GFP-N1 vector. This vector contains a modified redshifted green fluorescent protein (GFP) at XalI-SamI sites. The MxNas1–GFP construct was transformed into onion epidermal cells by particle bombardment as described earlier (Xu et al. 2011). The transient expression of the MxNas1–GFP fusion protein was observed under confocal microscopy.

Tobacco Transformation

To construct an expression vector for tobacco, the full-length MxNas1 cDNA was ligated into the vector pBI121 (Clotech) by replacing the gus gene. The MxNas1 gene driven under the cauliflower mosaic virus (CaMV) 35S promoter was introduced into tobacco plants by Agrobacterium-mediated GV3101 transformation (An et al. 1988). Nicotiana tabacum cv. Xanthi ecotype tobacco plants were transformed using the vacuum infiltration method. Transformants were selected on MS medium containing 50 μg mL−1 kanamycin. The primers for PCR detection are MF and MR, 18S rRNA gene was amplified from various tobacco tissues for Actin. T1 generation plants were used for further analysis.

Analysis of Fe Stress Tolerance of Transgenic Tobacco

The T1 generation plants of lines MxNas1-OE and wild type (WT) were used in the subsequent experiments. Twenty germinated seedlings from each line were carefully transferred to Hoagland solution supplemented with 4 μM (low Fe concentration), 40 μM (normal level), and 160 μM (high concentration) Fe. After 10 days of growth, the appearance was observed.

Detection of the Contents of Chlorophyll and NA

According to Aono et al. (1993), we measured the chlorophyll content of the T1 generation plants of lines MxNas1-OE (OE-2 and OE-9) and wild type.

Assays for the content of NA were performed by the method of high-performance liquid chromatography according to Higuchi et al. (2001). All experiments used pure NA (T. Hasegawa Co. Ltd, Japan) as an external standard. Experiments on the contents of chlorophyll and NA were conducted three times and the standard errors (±SE) were measured, respectively.

Determination of Metal Concentrations

According to Kojima and Iida (1986), new leaf and flower samples (including whole flower, petal, pistil, and stamen) weighing 100–200 mg were respectively placed with 2 mL of nitric acid in a sealed polytetrafluoroethylene vessel with a stainless steel jacket. The vessel was heated from room temperature to 150 °C in an oven for 90 min and then kept at 150 °C for 6 h. After cooling to room temperature, samples were filled to a constant volume and metal concentrations were determined using inductively coupled plasma emission spectrometry (SPS1200 VR; Seiko, Tokyo, Japan).

Results

Expression Analysis of MxNas1 in M. xiaojinensis

The expression level of the MxNas1 in these M. xiaojinensis tissues under normal Fe treatment was investigated using the real-time PCR assay. These M. xiaojinensis tissues were collected as described in “Materials and Methods”. Expression of MxNas1 was enriched in root and leaf, which was also detected in the phloem of stem, but was very low in the xylem (Fig. 1a). This expression pattern indicates that MxNas1 may play its role in active organs. To further analyze MxNas1 expression in M. xiaojinensis, we prepared total RNA from root (Fig. 1b) and mature leaf (Fig. 1c) of M. xiaojinensis in different treatments. The results show that the expression of MxNas1 increases in roots under a low Fe concentration (4 μM), IAA, and ABA treatments. Conversely, it decreases in roots under a high Fe concentration (160 μM; Fig. 1b). The expression of MxNas1 in mature leaf (Fig. 1c) was just the opposite to root under Fe stress. Treatments of IAA, ABA, and different Fe stresses affect the expression of MxNas1 in roots and leaves. IAA and ABA are considered as signals of Fe and other stresses in plants.

Fig. 1
figure 1

Time-course expression patterns of MxNas1 in M. xiaojinensis using real-time PCR. a Expression patterns of MxNas1 in young leaf (partly expanded of top) and mature leaf (fully expanded), root, xylem, and phloem in normal iron concentration (40 μM). b Expression patterns of MxNas1 in a low concentration of Fe (4 μM, −Fe), high concentration of Fe (160 μM, ++Fe), treated with 0.1 mM IAA (IAA) and 0.1 mM ABA (ABA) in roots at the following time points: 0, 6, 12, 24, and 48 h. The expression amounts were normalized to that of Mx18S. c Expression patterns of MxNas1 in mature leaf of M. xiaojinensis at the same conditions as above (root). Each data (mean ± SD, n = 3) represents the average of three independent plants; error bars indicate the standard deviation. Asterisks above the error bars indicate a significant difference between the treatment and control (0 h) using Student’s t test (p ≤ 0.05)

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Localization of MxNas1 in the Cell Membrane

The presence of a NA synthase, which commonly serves as a DNA-binding domain, suggests that MxNas1 is a functional gene. To examine subcellular localization of MxNas1 protein, the MxNas1–GFP fusion protein was introduced into onion epidermal cells by particle bombardment. As shown in Fig. 2, the MxNas1–GFP fusion protein was targeted into the cell membrane, whereas the control GFP alone was distributed throughout the cytoplasm. These results showed that the MxNas1 protein is a cell membrane localization protein.

Fig. 2
figure 2

Subcellular localization of MxNas1. Transient expression in onion epidermal cells of 35S–GFP and 35S–DgZFP–GFP translational product was visualized by fluorescence microscopy. The transient vector harboring 35S–GFP and 35S–MxNas1–GFP cassettes were transformed into onion epidermal cells by particle bombardment. The photos were taken in the bright light (left), in the dark for GFP images (right) after incubation for 20 h

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Overexpression of MxNas1 Confers Tolerance to Fe Stress in Transgenic Tobacco

In order to investigate the role of MxNas1 in response to Fe stresses in plant, we generated transgenic tobacco plants with overexpression of MxNas1 under control of the CaMV 35S promoter. Among 13 lines of transformants, six independent transgenic lines (OE-2, OE-4, OE-6, OE-7, OE-9, and OE-10) were confirmed by using RT-PCR analysis (Fig. 3).

Fig. 3
figure 3

Expression of MxNas1 in transgenic tobacco. The expression level of MxNas1 in wild-type (WT) and MxNas1-OE transgenic T0 lines. The results of semi-quantitative RT-PCR. Ethidium bromide staining of PCR products using MxNas1-specific primers with (top) and without (middle) prior reverse transcription, and the PT-PCR products with 18S rRNA gene primers (bottom) as Actin

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The T1 transgenic MxNas1-OE lines (OE-2 and OE-9) and wild-type tobacco seedlings were grown in soil for germination, then the seedlings were grown in Hoagland solution supplemented with 4 μM (low Fe stress), 40 μM (normal Fe level), and 160 μM (high Fe stress) Fe, respectively. As shown in Fig. 4, after 10 days of growth, the appearance was observed. In normal Fe concentration (40 μM) solution, tobaccos of both types grow well. The wild type has obvious chlorotic appearance, but transgenic tobacco has no obvious chlorotic appearance in Fe deficiency (4 μM) Hoagland solution. Transgenic plants of both lines have better appearance than wild type in high Fe concentration (160 μM).

Fig. 4
figure 4

Overexpression of MxNas1 in tobacco improved Fe stress tolerance in MxNas1 transgenic lines (OE-2 and OE-9). WT and MxNas1 were respectively shown the wild-type and transgenic tobacco seedlings phenotype grown in Hoagland solution supplied with 4 μM (low Fe concentration), 40 μM (normal level), and 160 μM (high concentration) Fe for 10 days

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As shown in Fig. 5, the transgenic tobaccos (lines OE-2 and OE-9; Fig. 5b and d) exhibited phenotypes of decreased growth and reduced leaf size (Fig. 5e right), compact plant shape and delayed flowering for about 15 days, compared to those of wild-type tobacco (Fig. 5a, c, and e; left). The transgenic tobaccos also have higher contents of chlorophyll (Fig. 5f) and NA (Fig. 5g) than wild type.

Fig. 5
figure 5

Overexpression of MxNas1 in tobacco. The phenotype of the MxNas1-OE T1 lines (b and d) and wild-type (a and c) tobacco at 25 and 90 days after transplanting in the soil. e Comparison of adaxial side of leaves in MxNas1-OE (right) and wild-type (left) tobacco. f–g Comparison of the contents of chlorophyll (f) and NA (g) in leaf of wild-type (WT) and MxNas1 transgenic lines (OE-2 and OE-9). All treatments are repeated at least three times. Scale bars 15 cm in (a–e). Significant differences between MxNas1-OE lines and wild-type (WT) were shown by the t test, *p ≤ 0.05; **p ≤ 0.01

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Overexpression of MxNas1 Confers Abnormally Shaped Flowers

In addition to changes of plant form, and contents of chlorophyll and NA, the MxNas1 tobacco (lines of OE-2 and OE-9) inflorescence developed marked morphological abnormalities (Fig. 6). The flowers of wild type had five petals, five stamens, and one pistil (Fig. 6a, i, and l). In contrast, MxNas1 tobacco produced four types of abnormally shaped flowers: (1) projected petals (Fig. 6b–f and h). (2) Chimeric flower organ. Petaloid filaments (Fig. 6f–h, o, r, s, and t) were observed. (3) Dehiscent flower. Dehiscent flowers were observed (Fig. 6 e, f, j, and k), with the corolla split open. (4) Abnormal number of flower organs. This type of flower showed supernumerary stamens and petals (Fig. 6b, c, e, f, h, j, k, n, o, and p) or a decreased number of petals and stamens (Fig. 6d and m).

Fig. 6
figure 6

Inflorescences of wild-type tobacco and MxNas1-OE tobacco (OE-2 and OE-9). a Wild-type flower, b–h MxNas1-OE tobacco flowers, b–d flower with projected petal, e–f staminoid petal, g–h petaloid stamen, i side view of the flower shown in (a), j side view of the flower shown in (e), k side view of the flower shown in (f), l pistil and stamen filaments of wild-type flower (a and i), m pistil and stamen filaments of (d), n pistil and stamen filaments of (b), o pistil and stamen filaments of (h), p pistils and stamen filaments of (k), q stamen of wild-type flower in (a and i), r–t petaloid stamen of the stamen filament shown in (g and h) that seems to be a petal. Scale bars 1 cm in a–t

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Overexpression of MxNas1 Increased Fe, Cu, Zn, and Mn Concentrations in Young Leaves and Flowers

Metal concentrations in the young leaves and flowers (including whole flower, petal, pistil, and stamen) of MxNas1-OE tobaccos (OE-2 and OE-9) were also analyzed (Fig. 7). In young leaves (Fig. 7a), whole flower (Fig. 7b), petal (Fig. 7c), pistil (Fig. 7d), and stamen (Fig. 7e) of the MxNas1 tobacco samples, the concentrations of Fe and Zn significantly increased as compared WT tobacco. The concentrations of Cu and Mn also increased but insignificantly. These results indicate that NA promoted the transport of metal ions, particularly Fe and Zn to young leaves and flowers.

Fig. 7
figure 7

Metal concentrations in young leaves and flowers. Metal concentrations in young leaves (a), whole flower (b), petal (c), pistil (d), and stamen (e) of MxNas1-OE tobacco (OE-2 and OE-9) and wild-type (WT) tobacco. All treatments are repeated at least three times. Significant differences between MxNas1-OE lines and wild type were shown by the t test, *p ≤ 0.05; **p ≤ 0.01

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Discussion

mRNA expression of MxNas1 was more enriched in root and leaf than that in phloem, while expression was nearly undetectable in xylem. This expression pattern indicates that MxNas1 may play its role in active organs. Treatments of IAA, ABA, and different Fe stresses affect the expression of MxNas1 in roots. Because IAA and ABA are considered as signals of Fe stress in plants (Schmidt et al. 2000; Schikora and Schmidt 2001), IAA and ABA treatments affect the expression of MxNas1, we believe that MxNas1 has probably participated in Fe transport. The results show that the expression of MxNas1 in M. xiaojinensis is upregulated in root under low Fe condition and down-regulated under high Fe stress. It is possible that MxNas1 plays a key role in regulating the responses of M. xiaojinensis to different treatments of Fe stress. When exposed to low Fe treatment, M. xiaojinensis increases the expression of MxNas1 to accelerate the synthesis of Nas and NA. Consequently, higher concentration of NA in plants will promote uptake of Fe from a poor Fe environment (Deinlein et al. 2012). In contrast, the expression of MxNas1 in roots was down-regulated in a rich Fe environment to reduce the synthesis of Nas and NA, so the uptake of Fe from the environment is decreased.

Overexpression of MxNas1 enhanced the tolerance to Fe stresses of high and low concentrations in transgenic tobacco and increased contents of chlorophyll and NA. It is possible that MxNas1 plays a crucial role in helping plants to survive Fe stress by regulating the synthesis of NA. Higher content of NA in MxNas1-OE tobacco helped to extract Fe from a poor Fe environment. Meanwhile, high concentration of NA is also helpful in chelating redundant Fe for detoxification when plants were exposed to a high Fe environment (Palmer and Guerinot 2009).

NA is essential for the transport of Fe, Mn, Cu, and Zn in veins (Haydon and Cobbett 2007). The concentrations of Zn and Fe were significantly higher in MxNas1-OE tobacco young leaves and flowers than in wild-type tobacco, which means that there must be some relationships between NA and Fe translocation in plants. It has been demonstrated that NA can chelate these four metal ions (Fe, Zn, Mn, and Cu) for their transport through phloem in plant (Schuler et al. 2012). Several researchers have reported an increase of NA content under Fe deficiency in phloem (Abadía et al. 2002). Metal ions are very important for plant growth, because they are important components of many critical proteins or enzymes. High content of metal ions can affect enzyme activity, which could depress the growth rate. Additionally, high concentration of NA, as a result of MxNas1 overexpression in transgenic tobacco, is also helpful in chelating redundant metal ions for detoxification when plants were exposed to heavy metal stress (Haydon et al. 2012).

NA is essential for reproductive growth. Normal flower development requires a specific NA concentration more strictly than that for normal leaf development (Takahashi et al. 2003). In this study, transgenic tobacco plants have higher contents of chlorophyll, NA, and metal ions and show improved tolerance to Fe stress. Leaves of transgenic tobacco grow well with increased content of chlorophyll caused by higher Fe concentration, but the shape of flower has changed and there are chimeric flower organs. The combination of NA with Fe(II) is essential for normal flower development. Other metal ions (particularly Zn and Cu) also participate in normal flower development (Conte and Walker 2011). NA, acting as a metal carrier, can help to transfer metal ions to organs such as leaves, developing pistils, and anthers. It also plays a role in the maturation of pollen and seeds. NA also could be involved in regulating functions of metal-requiring proteins (such as Zn finger proteins), so it may affect the number of flower organs, determine the shape of flower organs, and probably serve as a regulator of transcription factors.

Metal ions such as Fe, Cu, Zn, and Mn are very important for reproductive development of plants, because they are important components of many critical proteins during this stage (Kim and Guerinot 2007). It has been reported that Cu stress can cause male sterility (Dell 1981) and affect seed yield and quality (Bhakuni et al. 2009); Mn deficiency affects pollen productivity and viability (Dordas 2009). Zn stress would lead to decreased pollen fertility (Sharma et al. 1990) and seed quality (Chatterjee and Khurana 2007); Zn is also related to female fertility, because Zn finger Polycomb group proteins are necessary for proper female gametophyte and seed development (Grossniklaus et al. 1998; Brive et al. 2001). Zn plays an essential role in some key structural motifs of transcriptional regulatory proteins, including Zn finger, Zn cluster, and RING finger domains. Furthermore, transcriptional factors, including many Zn finger proteins, participate in flower development (Kapoor et al. 2002; Li et al. 2006b). In this study, the contents of metal ions (especially Zn) changed markedly, which probably affect the activity of critical proteins in reproductive development, and the function of transcriptional regulatory proteins, such as Zn finger, Zn cluster, and RING finger domains, and the abnormally shaped flowers of transgenic tobacco were produced as a result.

To our knowledge, this work is the first report regarding expression analysis by real-time PCR and function characterization of MxNas1 gene through transgenic tobacco. Clarifying the role of different domains of MxNas1 under metal stress response and that in abnormally shaped flowers will be helpful in breeding stress-resistance Malus by gene transformation. Further gene transfer experiments are required to identify the function of MxNas1 using knockout and RNAi technique, or through gene transformation into other Malus.