Introduction

Heavy metal (HM) is commonly defined as those having a specific density of more than 5 g cm−3. The main threats to human health from heavy metal are associated with the exposure to lead, cadmium, mercury, and arsenic (Jarup 2003), and thus, remediation of the environment assumes priority. Chemical strategies for the remediation of metal pollutants have their share of associated consequential environmental problems. Use of plants for alleviating biotic and abiotic stress, i.e., phytoremediation is fast gaining scientific acceptance and various mechanisms of phytoremediation have been reported (Cobbett and Goldsbrough 2000). Root exudation may alter plant response to heavy metal stress as these can either increase the mobilization and uptake or inhibit the uptake of toxic elements in the rhizosphere (Marschner 1995). Graminaceous species are known to synthesize and secrete Fe-chelating substances known as phytosiderophore (PS), the non-proteinaceous amino acids belonging to the mugineic acid (MAs) family, that dissolve sparingly soluble Fe compounds in the rhizosphere (Ma and Nomoto 1996) and even facilitate the transport of Fe as FeIII-PS complex to the shoot even grain (Curie et al. 2001; Grillet et al. 2013). However, phytosiderophore also posses the capacity to chelate other metal besides iron (Romheld et al. 1996; Pratibha 2007). The above approach holds promise as plant PS can solubilize and mobilize variety of metal nutrients with no harmful effect to the environment vis-a-vis the synthetic chelators (Kudo et al. 2007).

In general, the amount of PS secreted correlates positively with the ability of the plants to tolerate Fe deficiency (Ma et al. 1999). Information pertaining to regulation of PS release under metal stresses other than Fe is limited and inconsistent. Enhanced release of PS and consequent uptake of Zn-PS under Zn deficiency (Tolay et al. 2001; Singh et al. 2002) were reported to be an indirect response caused by Zn deficiency-induced Fe deficiency (Walter et al. 1994) rather than a direct effect of Zn deficiency (Suzuki et al. 2006). Effect of arsenic on phytosiderophores and mineral nutrition of barley seedlings grown in iron-depleted medium has been studied by Shaibur et al. (2009)) where increasing the As concentration in rhizosphere lowered the release of PS. Enhanced release of phytosiderophores has also been reported for maize plants that were grown in the presence of Cd (Hill et al. 2002), and inhibition of Cd uptake via phytosiderophore chelation has been suggested as a mechanism that protects grain crops from Cd toxicity (Pratibha 2007); however, phytosiderophore-mediated Cd mobilization would depend on the experimental conditions (Awad and Romheld 2000; Shenker et al. 2001). Thus, understanding the regulatory relationship between HM and PS biosynthesis and release is important to assign the role of phytosiderophore in heavy metal sequestration. Meda et al. (2007) postulated the role for PS in heavy metal tolerance of graminaceous plants; however, this shall depend upon favorable regulation of genes involved in PS biosynthesis, i.e., nicotianamine synthase (NAS) which results in the production of nicotianamine, that aids in retranslocation of metal within the plant, and nicotianamine amino transferase (NAAT) which guides the production of PS from nicotianamine. To achieve this objective, it is important to decipher the regulation of phytosiderophore biosynthesis as affected by heavy metal.

Fe deficiency induced the expression of NAS and NAAT genes, i.e., HvNAS1, HvNAAT-A, and HvNAAT-B in barley (Hordeum vulgare L.); OsNAS1, OsNAS2, and OsNAAT1 rice (Oryza sativa L.); and AtNAS1, AtNAS2, and AtNAS4 in arabidopsis (Higuchi et al. 1999, 2001; Bashir et al. 2006; Satoshi et al. 2007). These results reveal that a favorable regulation of NAS and NAAT gene expression is imperative for optimal plant Fe nutrition. Further, Suzuki et al. (2006) showed that DMA synthesis in shoot of barley is also induced by Zn deficiency. However, there is dearth of information on regulation of NAS and NAAT genes under heavy metal stress. The present study was, thus, taken up to ascertain physiological and molecular basis of the regulation of PS biosynthesis and PS release in wheat grown on Fe-depleted nutrient medium in the presence and absence of different concentrations of heavy metals such as Cd, Pb, and Ni.

Materials and methods

Experimental materials

The investigations were carried out with bread wheat (Triticum aestivum cv. PBW-343) procured from the Division of Genetics and Plant Breeding, IARI, New Delhi. Grains were rinsed for 3 min in 70% ethanol followed by 10 min in 15% hydrogen peroxide solution and finally in distilled water and sown on autoclaved sand in plastic trays. Trays were kept in a germinator in dark at 25 °C for proper germination. During germination, the trays were watered as and when necessary. After 3 days into germination, the emerging seedlings were moved to light to prevent etiolation. Five-day-old seedlings were removed from trays gently while preventing any damage to the root system. The roots were washed of any sand particles with deionized water and the seedlings were transferred to nutrient solution in glass tanks (10 l capacity) with darkened sides and bases. Plants were raised in iron-deficient and iron-sufficient nutrient solution (Singh et al. 2006) under controlled climatic conditions (light/dark regimes of 16/8 h, temp 18/22 °C) in the Nuclear Research Laboratory, IARI. Composition of the nutrient solution (in mM concentration, AR grade chemicals) was CaNO3 (2.0), KH2PO4 (0.25), MgSO4 (1.0), KCl (0.1), H3BO3 (1.0), MnSO4 (0.5), CuSO4 (0.2), (NH4)2Mo7O24 (0.002), and ZnSO4 (0.001). Iron was supplied as Fe-EDTA at concentration of 0.0 μM to iron-deficient plants and 100 μM to iron-sufficient plants (Khobra et al. 2014). The nutrient solution was changed every 3 days and was continuously aerated. Ten-day-old seedlings of wheat grown in Fe-deficient condition were transferred into 250-ml volumetric flask containing nutrient solution plus different concentrations of heavy metal (Cd, Pb, and Ni at 2.5, 5.0, and 10 mM). Seedlings were allowed to grow in nutrient solution containing heavy metal for 72 h, and plant tissue and root exudates, i.e., PS were collected at 2 h after the onset of the light period from 8 am till 2 pm (Singh et al. 2006) after 24, 48, and 72 h of the heavy metal treatment. Continuous aeration was provided for uniform circulation of the nutrients in the growing solution and to avoid anoxia.

Estimation of phytosiderophore

Phytosiderophore release by roots of wheat seedlings was estimated following the method of Romheld (1991) modified from Takagi (1976). Working solution of Fe was prepared by adding 1 N NaOH into 4 mM FeCl3 (pH 6.0) until precipitation of Fe(OH)3 occurred. Eight milliliters of the PS was added to 2 ml of the working Fe solution (Fe(OH)3) and 500 μl of 0.5 M sodium acetate buffer (pH 5.6) was added after shaking for 30 min. Contents were filtered (Whatman no. 1) and 0.2 ml of H2SO4 (3 M) was added for acidification of the solution. Five hundred microliters of hydroxyl amino chloride was then added and the resultant mixture was heated for 20 min at 55 °C. This was followed by the addition of 0.2 ml of ferrozin (10 mM) along with 1 ml of sodium acetate buffer (0.5 M, pH 4.6). Intensity of the violet color developed due to the formation of Fe(II)-ferrozin complex was measured at 562 nm using UV-visible spectrophotometer (EC GS5701V, ECIL, Hyderabad, India).

Amount of PS production per unit time was calculated by the following formula:

$$ \mathrm{P}\mathrm{S}=\frac{A562\times \mathrm{total}\ \mathrm{volume}\ \mathrm{of}\ \mathrm{collected}\ \mathrm{root}\ \mathrm{exudate}}{2.15\times \mathrm{volume}\ \mathrm{of}\ \mathrm{PS}\ \mathrm{used}\ \mathrm{in}\ \mathrm{assay}} $$

Amount of PS was expressed as micromole Fe equivalent per plant per unit of time (μmol Fe equivalent plant−1 h−1).

Estimation of lead, cadmium, nickel, and iron

Heavy metal concentration of root and shoot tissues was analyzed according to Singh et al. (2002) and Pratibha (2007) using atomic absorption spectrophotometer (AAS; ECIL, Hyderabad, India) All samples were analyzed in triplicate and HM concentration was read against the standards of Fe, Pb, Cd, and Ni procured from Merck, Germany.

Expression studies of NAS and NAAT transcripts using reverse transcription polymerase chain reaction

Nucleotide sequences for the candidate genes NAS and NAAT were obtained from the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/) was used to identify the homologs of these genes. For the reverse transcription polymerase chain reaction (RT-PCR) expression analysis, the following oligonucleotide primers were designed manually (Table 1), using Oligoanalyzer 3.0 tool (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/,IntegratedDNA Technologies, Coralville, IA 52241, USA).

Table 1 List of gene primers used in the present experiment
Full size table

Gene expression studies were carried out in 10-day-old seedlings subjected to different HM treatments up to 72 h. Gene expression of NAS and NAAT was studied in root tissue after 24, 48, and 72 h of HM treatment. Root samples were harvested from control and treated seedlings and total RNA was extracted using TRI REAGENT® RT reagent (Molecular Research Center, Inc., USA) according to the manufacturer’s standard protocol. One microgram of total RNA was reverse transcribed using gene-specific primers and Qiagen one step RT-PCR kit. PCR conditions were standardized using gene-specific primers for ACTIN. Amplification carried out in BioRad Thermal Block under the following conditions: initial PCR activation step 15 min at 95 °C, reverse transcription 30 min at 50 °C, denaturation 1 min at 94 °C, annealing 1 min at 59 °C (NAS and NAAT) and 60 °C (ACTIN), extension 2 min at 72 °C, and final extension 10 min at 72 °C. The amplification products were separated by electrophoresis on 0.8% agarose gel at 5 V/cm in TAE buffer (Tris Cl, KCI, (NH4)2SO4, 12.5 mM MgCI2, DTT; pH 8.7 at 20 °C) using 1-kb DNA ladders. RT-PCR reactions were repeated many times to standardize the time and temperature of annealing. Reported gel photographs were selected from the best of three PCR reactions after staining with ethidium bromide and as visualized on Uvi Pro Gel Documentation system (Alpha Digidoc, Alpha Innotech Corporation, USA).

The purified complementary DNA (cDNA) for each gene were sequenced and multiple sequence alignment was done using CLUSTAL 2.0.12.

Statistical analysis

The data collected were analyzed statistically using factorial completely randomized design (CRD) and mean value of three replicates is presented along with standard error (SE).

Results

Iron and heavy metal accumulation by wheat seedlings

In general, iron-sufficient wheat seedlings showed better growth and measured a root and shoot iron concentration of 350 and 542 μg/g DW as compared to the Fe-deficient wheat seedlings which possessed a much lower Fe at 8.3 and 20.2 μg/g DW, respectively (Fig. 1), i.e., an average reduction of 97.7 and 96.3% in Fe− over Fe+ treatment, respectively, for root and shoot tissues, on the day of latter’s transfer to the respective heavy metal treatment. Irrespective of the Fe treatment, the shoot Fe was higher than the root Fe concentration. A comparison of the Fe concentration in the root, after 3 days of heavy metal exposure (i.e., at 13-day stage) between the HM-treated and the Fe-deficient seedlings of wheat showed a significant inhibition in root Fe with all the metal. The decline in root Fe accumulation in Cd-treated seedlings was concentration dependant. Lowest Cd concentration in fact had no effect on the Fe content of root. Pb and Ni treatments although lowered the root Fe levels, the response was independent of the metal concentration (Fig. 2). Shoot Fe of Cd-treated plants was in general, reduced while those of Pb- and Ni-treated seedlings were increased in comparison to Fe-deficient control. The effects of heavy metal on the shoot Fe were independent of treatment concentration of Cd, Pb, and Ni. Respective heavy metal concentration in the treated wheat seedlings, irrespective of the concentrations supplied, was higher than those of Fe-deficient control seedlings.

Fig. 1
figure 1

Iron accumulation in shoot and root of wheat plants raised on iron sufficient (+Fe) and deficient (−Fe) nutrient solution

Full size image
Fig. 2
figure 2

Effect of heavy metal treatments on iron accumulation in shoot and root of wheat plants raised on Fe-deficient (−Fe) nutrient solution

Full size image

Cd accumulation in the wheat seedlings increased with an increase in the concentration of the exogenously applied Cd, i.e., from 2.5 to 10 mM, i.e., an increase of 84.6%. Plant Pb concentration increased only up to 5 mM Pb external concentration (+39.3%) and then decreased at 10 mM (−43.6%) when compared with 2.5 mM Pb treatment. Plant Ni concentration, on the other hand, was although higher than that of the Fe-deficient control seedlings, the response was independent of the Ni supply (Fig. 3).

Fig. 3
figure 3

Accumulation of respective heavy metals, viz Fe, Cd, Pb, and Ni in Fe-deficient wheat plants in the presence of varied heavy metal concentration in the nutrient solution

Full size image

Phytosiderophore release in the presence of heavy metal

The capacity of Fe deficient wheat seedlings to release phytosiderophore in the presence of varying concentration of different heavy metals was measured and is presented in Fig. 4. One thing which came out very clearly from the results was that the PS release capacity was not inhibited by the presence of Cd (+35.8%), Pb (+47.1%), and Ni (+30.8%) in the root zone when compared with the PS release under Fe-deficiency treatment. While comparing different heavy metals, irrespective of their concentration, in general, the release of PS was lower in response to Ni than that under Cd and Pb treatments. Release of PS increased with an increase in rhizospheric Cd. In the case of lead supplementation, the PS release increased till 5.0 mM concentration but decreased thereafter marginally and was still higher than the PS release by Fe-deficient control seedlings. Nickel treatment showed a trend similar to that of lead; however, the variation between the concentration treatments was relatively much lower than the Pb. While comparing different heavy metals, irrespective of their concentration, in general, the release of PS was lower in response to Ni than that under Cd and Pb treatments.

Fig. 4
figure 4

Phytosiderophore release capacity of iron-deficient wheat plants raised in nutrient solution in presence of varied concentration of heavy metals

Full size image

Influence of heavy metal on transcript level expression of NAS and NAAT

RT-PCR was carried out to determine the effect of different heavy metals (Cd, Pb, and Ni) on the transcript expression of NAS and NAAT genes (Fig. 5) at 24, 48, and 72 h of exposure to 2.5, 5, and 10 mM of the metal concentration. Expectedly, the expression of both NAS and NAAT genes was observed under Fe-deficient but not under Fe-sufficient condition. Further, in the presence of different heavy metals, in general, the Fe deficiency-induced expression of the phytosidrophore biosynthesis genes was not completely shut down or inhibited.

Fig. 5
figure 5

Phytosiderophore biosynthesis pathway in graminae crops. (SAM S-adenosyl-L-methionine, NAS nicotianamine synthase, NAAT nicotianamine amino transferase, DMAs 2′-deoxymugineic acid synthase)

Full size image

Nicotianamine synthase expression

Cadmium treatment, irrespective of the concentration, did not alter the nicotianamine synthase (NAS) expression at 24 h when compared with NAS expression under Fe-deficient condition. It was, however, upregulated at 48 h at 2.5, 5.0, and 10.0 mM Cd but downregulated at 72 h particularly at concentration above 25 mM (Figs. 6, 7, and 8). However, compared to NAS expression at 24 h stage, the expression was still higher at 72 h in response to Cd treatment.

Fig. 6
figure 6

Expression pattern of nicotianamine synthase (NAS) as affected by Cd

Full size image
Fig. 7
figure 7

Expression pattern of nicotianamine synthase (NAS) as affected by Pb

Full size image
Fig. 8
figure 8

Expression pattern of nicotianamine synthase (NAS) as affected by Ni

Full size image

Lead treatment also showed a similar effect as Cd at 24 h stage in which the NAS expression activity was more or less similar to the NAS expression under Fe deficiency and was upregulated at 48 h irrespective of Pb concentration (2.5, 5.0, or 10.0 mM). In clear contrast to Cd, NAS expression was switched off at 72 h of Pb treatment at all concentrations. Nickel treatment caused an increase in the expression of NAS over the Fe-deficient control which appeared to be upregulated with time and concentration.

Nicotianamine amino transferase expression

Cadmium treatment to the Fe-deficient plants, irrespective of its concentration, did not inhibit the nicotianamine amino transferase (NAAT) expression at 24 h stage and it was upregulated at 48 and 72 h compared to the expression activity at 24 h. However, between the 48 and 72 h, the NAAT transcript levels decreased at the latter stage under all Cd levels but it was still higher than that of the Fe-deficient control plants (Figs. 9, 10, and 11). Cadmium-mediated changes in NAAT gene expression were independent of the metal concentration.

Fig. 9
figure 9

Expression pattern of nicotianamine amino transferase (NAAT) as affected by Cd

Full size image
Fig. 10
figure 10

Expression pattern of nicotianamine amino transferase (NAAT) as affected by Pb

Full size image
Fig. 11
figure 11

Expression pattern of nicotianamine amino transferase (NAAT) as affected by Ni

Full size image

Lead exposure caused a decline in NAAT expression at 24 h when compared with the expression profile of the Fe-deficient control. However at 48 h, NAAT expression was upregulated in a manner similar to the Cd-induced stimulation of NAAT. However, unlike NAS, Pb treatment did not switch off the NAAT activity at 72 h stage irrespective of the Pb concentration. The NAAT expression at 72 h however was relatively lower than that observed at the 24 and 48 h stage and that of the Fe-deficient control. The NAAT expression profile in response to nickel treatment showed a marginal downregulation of NAAT expression at 24 h which was however upregulated at 48 h and beyond and was higher than the NAAT expression of Fe-deficient control. Expression of NAAT was regulated independently of the Ni metal concentration.

Sequencing of NAS gene and sequence alignment with HvNAS

The purified PCR products of NAS were sequenced which yielded a 998-bp sequence conforming NAS (Fig. 12). Multiple sequence alignment of wheat NAS using CLUSTAL 2.0.12 revealed 92% sequence similarity with barley (H. vulgare) NAS cDNA (GenBank Id: AK248255; Supplementary Fig. 1).

Fig. 12
figure 12

Total cDNA sequence of NAS gene from Triticum aestivum

Full size image

Discussion

The present study was aimed at determining the effect of heavy metals like Cd, Pb, and Ni on the Fe deficiency-induced release of PS and latter’s role in the mobilization and uptake of heavy metal using wheat as a monocot model system. It has been shown that these Fe chelators released by roots of Fe-deficient grasses (Romheld 1991; Marschner 1995) could mobilize from Fe-deficient soils not only Fe but also other metals such as Zn, Cu, or Cd (Romheld and Awad 2000; Chaignon et al. 2002). Since phytosiderophores are prominently released under Fe deficiency, we used Fe-deficient wheat seedlings as a control for different heavy metal treatments. We postulate that Fe deficiency along with an enhanced PS release can lead to increased mobilization and acquisition of toxic heavy metal by plants, a strategy termed as phytoremediation. Fe-deficient plants in this case possessed a significantly lower Fe and a chlorotic appearance than the Fe-sufficient plants. Roots, in general, accumulated lesser Fe than the shoot. The shoot Fe concentration of the heavy metal-treated seedlings was higher than the Fe-deficient control and could be related to an enhanced translocation of the root Fe to the shoot or to the heavy metal response on the formation and uptake of the Fe-PS complex, which may be related to the differential affinities of Cd, Pb, and Ni for the PS (Pratibha 2007). Uptake of Fe appears to be more directly affected by the presence of Cd and it will be worthwhile to examine if the interaction between Cd and Fe has anything to do with the Cd influence on the Fe deficiency-induced PS release. Further, a concentration-dependant increase in the seedling Cd was recorded in this study. It is apparent from the study that the Cd supplied Fe-deficient wheat seedlings took up more Cd chiefly at the cost of Fe, while it was not the case for the Pb- and Ni-treated seedlings which showed a much more increase in Fe than the respective metal at different concentration treatments. Role of phytosiderophore in improving Fe availability and uptake in Graminaceous crop species has been reported by several workers (Takagi 1976; Alam et al. 2000 and 2005; von Wiren et al. 1996, 2000 and 2005; Cassin et al. 2009; Zheng et al. 2010; Khobra et al. 2014). For their involvement in HM dynamics in soil/plant system, Pratibha (2007) found that Cd-PS interaction operates at the level of immobilization and that the complex of Cd and PS may not be taken up by plants owing to their large molecular weight. However, no evidence to this effect is available in the literature. Further, these results also hint towards a relatively higher affinity of PS for Cd than Ni and Pb in the same order.

Phytosiderophrore release as observed by Fe-deficient plants was uninhibited by heavy metal used in this study. In fact, PS release was induced under HM treatments particularly up to 5 mM. A decline in PS release at 10 mM particularly in Pb and Ni treatments might be related to a greater metal toxicity response of roots, which was not so prominent in case of Cd due to a greater stability of the PS-Cd moiety (Pratibha 2007). A higher chelation strength of Cd-PS may result in a reduced in plant metal toxicity. An increase in PS release in response to heavy metal is well in accordance with earlier studies (Chaignon et al. 2002; Shenker et al. 2001). However, Kudo et al. 2007 on the contrary found a decline in PS release in the presence of Cd and concluded that Fe-deficient condition led to an increased influx of Cd in the root of barley. It is suggested that PS in order to play a role in the chelator-assisted phytoextraction of Cd might be involved in the mobilization of Cd, resulting in the enhancement of Cd concentration in the rhizosphere. Some reports suggest a relationship between expression of the gene encoding Fe2+ transporter and Cd uptake (Eide et al. 1996; Rogers et al. 2000; Nakanishi et al. 2006). However, to exploit phytosiderophore as a phytoremediation strategy (Raskin et al. 1994; Salt et al. 1998) would necessitate that the remediated target element or environment does not adversely impact the phytosiderophore production.

Increased levels of the HvNAS1, HvNAAT-A, HvNAAT-B, HvIDS2, and HvIDS3 transcripts in Zn-deficient roots have been shown by Suzuki and co-workers (2008). Transgenic rice plants expressing OsNAS2 and OsNAAT1 fused to the synthetic green fluorescent protein (sGFP) under the control of their own promoters were used to prove that OsNAS2 is localized in the RER-derived vesicles and that it is involved in the DMA production in rice roots (Tomoko 2010). Several studies involving manipulation of genomic content of cereal crops like rice and barley with genes of phytosiderophore biosynthetic pathway have shown variable results for the interaction of these transgenic lines with Fe and other related metals. Kobayashi et al. (2008) produced transgenic rice plants that were high not only on the phytosiderophore production but had also acquired tolerance to low iron availability in the field condition. Increased Fe and Zn concentrations in transgenic rice lines possessing barley genome fragments containing genes for MAs synthesis (i.e., HvNAS1, HvNAS1, HvNAAT-A, and HvNAAT-B or IDS3) have been reported by Masuda et al. (2008). However, PS synthesis and release need not always correlate with each other (Singh et al. 2002). Transcript expression analysis of NAS and NAAT in the present study suggests no limitation in PS biosynthesis in the presence of heavy metal and suggests that molecular manipulation of the physiderophore biosynthesis through overexpression of NAS and NAAT could boost the phytosiderophore-mediated heavy metal remediation capacity of graminaceous plants. Non-food chain and high biomass producing grasses could serve as suitable experimental material for developing phytosiderophore overproducing transgenic events.

Conclusion

Finding simple biosynthetic molecule(s) with selective chelating ability that a plant can produce and secrete into the rhizosphere at adequate concentrations and simultaneously creating a selective transport protein that facilitates the metal-chelate translocation is desirable to develop a unique HM phytoremediation strategy. Our study suggests that natural metal chelators like phytosiderophore could favor the removal of heavy metals as the phytosiderophore biosynthesis and release are not hindered in the presence of different heavy metals in the rhizosphere. There are indications for the involvement of PS in altering the interaction of Fe and the heavy metal and consequently their uptake and the remediation process. In future, transgenic plants, with high constitutive expression of NAS and NAAT, can be developed that produce large quantities of metal selective ligands into the rhizosphere in order to solubilize elements of phytoremediation interest.