JXB Advance Access originally published online on November 13, 2006
Journal of Experimental Botany 2006 57(15):4235-4243; doi:10.1093/jxb/erl201
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RESEARCH PAPER |
Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance
1Key Laboratory of Biotechnology and Crop Quality Improvement, Ministry of Agriculture, Biotechnology Research Center, Southwest University, Chongqing 400716, China
2Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA
To whom correspondence should be addressed. E-mail: peiyan3{at}swu.edu.cn
Received 26 April 2006; Accepted 13 September 2006
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Abstract |
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Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg2+) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg2+ transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg2+ deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 µM AlCl3 caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.
Key words: Aluminium toxicity, AtMGT1, magnesium, Nicotiana benthamiana
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Introduction |
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Aluminium (Al) is the most abundant metal in the earth's crust and comprises approximately 7% of its mass (Delhaize and Ryan, 1995). Al toxicity is the major factor limiting productivity of many crops grown in acid soils which cover about 40% of the world's arable land (Delhaize and Ryan, 1995; Kochian, 1995). In acid soils (pH <5.0), high Al concentrations in soil solution can result in the inhibition of root growth and disruption of cell division and the cytoskeleton, and hence reduce crop yield. A1 toxicity is also associated with decreased uptake and content of some cations, and leads to cation-deficiency symptoms (Foy, 1984; Rengel and Robinson, 1989; Rengel, 1990; Robinson and Rengel, 1991; Poschenrieder et al., 1995; Mariano and Keltjens, 2005). More recently, a fast change in cell patterning was observed in maize after exposure to Al for a short time (Doncheva et al., 2005). However, some plant species and cultivars showed tolerance to Al toxicity in acid soils (Kochian, 1995; Matsumoto, 2000; Barcelo and Poschenrieder, 2002; Kochian et al., 2005). Resistance to Al can be conferred via exclusion of Al from the root apex and/or via internal tolerance by sequestration of Al in the plant's symplasm. It has also been shown that applications of Mg2+ can reduce Al toxicity in several plant species (Tan et al., 1991, 1992; Keltjens and Tan, 1993; Matsumoto, 2000; Silva et al., 2001a, b, c; Mariano and Keltjens, 2005).
Magnesium (Mg2+) as an important part of chlorophyll is essential for photosynthesis in all green plants. Mg2+ also serves as a cofactor with ATP in a number of enzymatic reactions (e.g. ATPases and RNA polymerases). Despite these critical cellular functions, Mg2+ uptake, transport, and homeostasis in plants have only been elucidated recently. To date, the Mg2+ transport proteins characterized in plants include the Mg2+/H+ exchanger AtMHX (Shaul et al., 1999) and a family of Mrs2p homologues (Schock et al., 2000; Li et al., 2001). The AtMHX gene similar to the SLC8 family of Na+/Ca2+ exchanger genes in humans was observed to be expressed in a whole plant but higher in the vascular tissue (Shaul et al., 1999), suggesting that the physiological role of the AtMHX protein was to store Mg2+ in these tissues for later release as needed. Schock et al. (2000) identified the AtMRS2 family based on the similarity of the genes to the MRS2 gene of yeast (Saccharomyces cerevisiae), and established that the AtMRS2-1 gene could complement a 
mrs2 yeast mutant phenotype, indicating that the AtMRS2 family proteins mediated Mg2+ transport. In a more recent study, Li et al. (2001) reported a magnesium transport family (AtMGT) from Arabidopsis thaliana which was homologous to the MRS2 gene in yeast and to the CorA family in bacteria.
In previous studies, it had been established that overexpression of two yeast members of the CorA magnesium transport system, ALR1 and ALR2, could increase Al resistance on yeast (MacDiarmid and Gardner, 1998). Li et al. (2001) also reported that AtMGT1, a member of the AtMGT family, localized in the plasma membrane of Arabidopsis, functionally complemented an Mg2+ transport mutant of S. typhimurium, and AtMGT1 activity was inhibited by relatively low concentrations of Al3+. As far as is known, there is little information about whether magnesium transport proteins can improve Al tolerance in higher plants. In this study, to evaluate whether overexpression of magnesium transport genes can ameliorate Al toxicity in higher plants, the Arabidopsis magnesium transport gene, AtMGT1, under the control of the cauliflower mosaic virus (CaMV 35S) promoter, was expressed in N. benthamiana plants. The role of the AtMGT1 gene was investigated in mediating Al tolerance in transgenic plants.
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Materials and methods |
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Plasmid construction
The coding region of the A. thaliana AtMGT1 gene was amplified from cDNA with the primers AtMGT-1 5'-CGGATCCATGTCTGAACTCAAAGAGC-3' and AtMGT-2 5'-AGAGCTCTCACAGAGGCATGAGTCT-3'. BamHI and SacI restriction sites are underlined. A polymerase chain reaction (PCR) was performed for 35 cycles (denaturing at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 90 s). PCR products were applied to a DNA sequencer to confirm their sequences. The BamHI/SacI-amplified fragment was cloned downstream of the CaMV 35S promoter of binary vector pBI121 (Clonetech, San Francisco, CA, USA), followed by a nopaline synthase terminator. Subsequently, the 35S::AtMGT1 fragment was excised by HindIII/EcoRI and ligated to binary vector pCAMBIA1305.1 (Cambia, Canberra, Australia) which was digested with HindIII and EcoRI. The resulting plasmid was named pCAMGT (Fig. 1A).
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Plant transformation and growth conditions
The pCAMGT plasmid was transformed into N. benthamiana plants by Agrobacterium tumefaciens-mediated transformation, using the Agrobacterium strain LBA4404 (Horsch et al., 1985). Shoots of putative transformants were selected in MSB5 medium containing MS macro-salts and micro-salts (Murashige and Skoog, 1962), B5 vitamins, 1 mg l–1 benzylaminopurine, 0.1 mg l–1 naphthaleneacetic acid, and 100 mg l–1 kanamycin, and rooted in half-strength MSB5 medium with 100 mg l–1 kanamycin. Kanamycin-resistant transformants with well-developed roots were grown in HP Premier Pro-mix potting soil (Premier Horticulture Ltee, Rivere-du-Loup, Quebec, Canada) in a greenhouse under an 18 h light/6 h dark photoperiod at 25 °C.
Confirmation of transgenic plants
Total genomic DNA was isolated from young leaves of N. benthamiana using a modified CTAB (cetytriethylammonium) bromide procedure (Doyle and Doyle, 1990). PCR was used to identify 35S::AtMTG1 transgenic lines among the kanamycin-resistant lines obtained. The PCR primers used were as follows: the forward primer was directed against the 35S promoter with the sequence 5'-CCCAAGAAGGTTAAAGATGCAG-3', and the reverse primer was directed at the AtMTG1 gene with the sequence 5'-AGAGCTCTCACAGAGGCATGAGTCT-3'. PCR amplification was carried out under the following conditions: 1 cycle of 94 °C for 5 min; 30 cycles of 94 °C for 45 s, 50 °C for 10 min, 72 °C for 1 min; 72 °C for 10 min. The presence of the transgenes was further confirmed by the determination of ß-glucuronidase (GUS) activity in leaves using the histochemical assay (Jefferson et al., 1987).
Total RNA isolation
To isolate total RNA from N. benthamiana, leaf issues were homogenized in the extraction buffer consisting of 2% (w/v) CTAB, 2% (w/v) polyvinylpyrrolidone, 5 M sodium chloride, 2% (v/v) ß-mercaptoethanol, and then extracted with an equal volume of phenol:chloroform (1:1 v/v). Total RNA was recovered by centrifugation (5000 g, 3 min, 25 °C) and further purified at least twice using LiCl (final concentration 2 M) precipitation, and dissolved in sterile distilled water. The quality of the RNA was assessed by agarose gel electrophoresis and quantity was determined spectrophotometrically. Finally, the total RNA was precipitated with 2.5 volumes of 95% ethanol and stored at –80 °C until used for cDNA synthesis and northern blot hybridization.
Northern blot analysis and reverse transcription–polymerase chain reaction (RT-PCR)
Twenty micrograms of total RNA was separated on 1% (w/v) agarose gel containing 50% (v/v) formaldehyde and transferred to a nylon membrane (Amersham Pharmacia, Piscataway, NJ, USA) in 0.01 M NaOH transfer solution. A fragment of the AtMGT1 gene was used to probe for the RNA gel blots. Single-stranded probe was prepared according to the procedures oligolabelling kit (Amersham Pharmacia). Prehybridization and hybridization were performed at 65 °C for 8 h. The membrane was washed twice at 65 °C in a solution containing 1x SSC and 0.1% (w/v) SDS for 30 min. Hybridized membrane pieces were exposed to Kodak-X-OMAT AR film for 18 h with an intensifying screen.
For RT-PCR reactions, cDNA synthesis was performed with the ImProm-II TM reverse transcription system (Promega, Madison, WI, USA). The primers, A-1 (5'-CGGATCCATGTCTGAACTCAAAGAGC-3') and A-2 (5'-AGAGCTCTCACAGAGGCATGAGTCT-3'), were designed specifically to amplify the AtMGT1 cDNA. The primers, T-1 (5'-ATGCCCTCCCACATGCTATTC-3') and T-2 (5'-AACATGGTAGAGCCACCACTA-3') were used to amplify the TAC9 cDNA used for a control (Thangavelu et al., 1993; Wawrzynska et al., 2005). RT-PCR for AtMGT1 cDNA was conducted for 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 30 s, and finally 7 min at 72 °C. PCR for TAC9 expression was conducted under the above conditions but for only 25 cycles of amplification.
Mg treatments
To determine the tolerance of transgenic plants grown on low-Mg2+ and Mg2+-deficient media, wild-type and transgenic homozygous T2 seeds were germinated and grown on MSB5 medium for 2 weeks, and then transferred to MSB5 media containing 0 mM MgSO4 (MSB5-1) or 0.075 MgSO4 (MSB5-2) for 20 d, respectively. In order to maintain a constant anion/cation balance and to avoid sulphur deficiency, the MSB5-1 and MSB5-2 media were supplemented with 1.5 mM Na2SO4 and 1.425 mM Na2SO4, respectively.
Root elongation assay
Wild-type and transgenic lines (T2 homozygous) were tested for their sensitivity to Al using a modified procedure described by Murphy and Taiz (1995) and Ezaki et al. (2000). A plate assembly consisted of a 3-mm-thick glass plate, three squares of 1-mm-thick chromatography paper (3 MM CHR; Whatman, Maidstone, UK). The chromatography sheets were saturated with 1/6 MSB5 medium and then set on the glass plate. Sterilized seeds were germinated and 2-d-old seedlings were plated on the chromatography sheets. The plates were inclined at an angle of >80 °C in a sterilized plant growth rack containing 20 ml of 1/6 MSB5 medium (pH 4.3). Roots of the seedlings grown over the plate elongated in a straight line, enabling easy handling of the seedlings and accurate measurements of root lengths. After 5 d of growth, the young seedlings (with 1–1.5-cm-long roots) were transferred to a second plate, placed in a marked line on three new chromatography sheets. This new plate was inclined in the growth rack containing 1/6 MSB5 solution medium (pH 4.3) with or without Al. After exposure to the treatment solution for 2 d or 5 d, the final root lengths (between the root apex and marked line) of 10 plants, selected on the basis of seed availability, were measured. Root growth in each treatment was calculated relative to the control.
Elemental analysis
Homozygous T2 transgenic lines and wild-type plants were grown in HP Premier Pro-mix potting soil (Premier Horticulture Ltee) in a greenhouse under a 18 h light/6 h dark photoperiod at 25 °C. The 90-d-old plants were collected and the whole plants were dried overnight in an oven at 65 °C. Elemental analysis was carried out according to the method of Zarcinas et al. (1987). The concentrations of Mg, Mn, and Fe in plants were determined using atomic absorption spectroscopy in the Nutrient and Element Analysis Laboratory in the College of Resources and Environment at Southwest University (Chongqing, China).
Visualization of callose in roots
Five-day-old seedlings were exposed to 1/6 MSB5 medium containing 50 µM Al3+ on the chromatography sheets for 24 h, and transferred to the fixative containing 10% (v/v) formaldehyde, 5% (v/v) glacial acetic acid, and 45% (v/v) ethanol, and then vacuum infiltrated for 4 h. Fixed seedlings were stored in 0.1% (v/v) aniline blue (pH 9.0, 0.1 M K3PO4). Callose deposition and autofluorescence were visualized under a fluorescence microscope (Olympus, Japan). Levels of callose were qualitatively determined as described by Zhang et al. (1994).
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Results |
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Construction of transgenic plants overexpressing AtMGT1
To investigate effects of constitutive expression of AtMGT1 on Al tolerance in whole plants, the CaMV 35S promoter was used to drive expression of AtMGT1. The gene cassette 35S::AtMGT1 was inserted into a binary expression vector pCAMBIA, and the resulting construct was obtained and named p35S::AtMGT1 (Fig. 1A). Agrobacterium-mediated transformation of N. benthamiana with the 35S::AtMGT1 gene cassette resulted in 46 independent kanamycin-resistant lines. The primary transformants were screened by PCR and histochemical assay of GUS activity (data not shown). Twenty GUS- and PCR-positive lines were tested for their segregation ratios in T1 generation. Among these independent transgenic plants, five lines showed segregation ratios of 3:1 (GUS+:GUS–) indicating a single independent integration locus. The single locus homozygous lines were selected for detailed analysis.
AtMGT1 steady-state mRNA levels were examined in all five independent single locus homozygous lines by RT-PCR. The results showed that the 0.6 kb AtMGT1 DNA fragment was amplified in these 35S::AtMGT1 plants grown in a greenhouse (Fig. 1B). No amplification of cDNA was detected in non-transgenic plants. Northern blot analysis further confirmed the expression levels of AtMGT1 in the transgenic lines. The results for three lines (A12, A18, and A28) are shown in Fig. 1C. All three transgenic lines showed accumulation of AtMGT1 transcript. Lines A12 and A18 displayed high levels of AtMGT1 mRNA and line A28 accumulated lower but detectable levels. By contrast, the hybridization signal was not detected in non-transgenic plants.
Morphological effects of ectopic overexpression of AtMGT1 on N. benthamiana plants
Compared with wild-type N. benthamiana plants, 35S::AtMGT1 plants grown in a greenhouse at 25 °C, showed a wide range of phenotypic alterations, including a reduction in leaf and plant size (Fig. 2). Of the growth parameters observed, the average height of wild-type plants (78.1±3.3 cm) was greater than those of all transgenic lines (63.3±2.4 cm, 62.4±3.5 cm, 60.2±2.9 cm) (Table 1). Counting the numbers of internodes on the main stem revealed that no statistically significant differences in the number of nodes were observed (data not shown). Hence, the reduction in height growth of transgenic plants was primarily due to a decrease in internode lengths. Likewise, leaf development in transgenic lines was also affected. The leaves of 35S::AtMGT1 plants were shorter and narrower than wild-type plants (Fig. 2B; Table 1). In addition, there were slight differences in shoot and root biomass between transgenic and control plants when measured either on a fresh or a dry weight basis (Table 1). For instance, the shoot and root fresh weights in the A28 line were about 15% and 11% lower than in wild-type plants, and the dry weights were also reduced by 17% and 10%, respectively.
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AtMGT1 expression improves tolerance to Mg deficiency
In a previous study, it had been demonstrated that the AtMGT1 protein transports Mg2+ when expressed in Salmonella (Li et al., 2001). To determine whether AtMGT1 can function as an Mg2+ transporter in planta, AtMGT1 was overexpressed in N. benthamiana plants under control of the 35S promoter. Three independently transformed lines were selected for detailed study. One-week-old homozygous T2 seedlings growing on MSB5 medium were transferred to low-Mg2+ or Mg2+-deficient media. After additional cultivation of 20 d, the growth of both wild-type and transgenic plants was inhibited, but all transgenic plants grew much better than controls (Fig. 3). When grown on Mg2+-deficient medium, wild-type plants exhibited severe interveinal chlorosis on all fully expanded leaves of wild-type plants, whereas just mild chlorosis appeared on lower mature leaves of transgenic plants (Fig. 3). These results suggest that transgenic plants were tolerant of Mg deficiency.
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An additional experiment was carried out to test whether AtMGT1 overexpression affects the accumulation of magnesium (Mg), manganese (Mn), and iron (Fe) in N. benthamiana plants. The content of these metals in transgenic plants was evaluated by atomic absorption spectroscopy. As shown in Fig. 4A, Mg contents per unit dry weight in 35S::AtMGT1 lines were increased compared with wild-type plants. For instance, the Mg content per unit dry weight in the A28 line was increased by 30%. The accumulation of Mn and Fe per unit dry weight in transgenic plants increased markedly by contrast to non-transformed plants (Fig. 4B, C). Furthermore, the total contents of Mg, Mn, and Fe per plant were also increased compared with non-transformed plants. In the A28 line, the total contents of Mg, Mn, and Fe per plant were 10%, 40%, and 12% higher than in the control plants, respectively.
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Expression of AtMGT1 confers tolerance to Al
In order to study whether AtMGT1 overexpression could improve Al tolerance in plants, Al sensitivity of wild-type plants was tested using a root elongation assay. Results showed that root growth of seedlings was inhibited by increasing Al concentration. When treated with 100 µM AlCl3, root growth in wild-type plants was completely inhibited (data not shown). Subsequently, the effect of Al on root growth in 35S::AtMGT1 plants was evaluated. The results showed that the inhibition of Al-induced root elongation was significantly different between 35S::AtMGT1 and wild-type plants. Exposure to 50 µM AlCl3 for 5 d severely inhibited root elongation in wild-type plants but hardly inhibited it in transgenic 35S::AtMGT1 plants (Fig. 5A).
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To analyse root elongation quantitatively, wild-type, transgenic lines A12, A18, and A28 were grown in nutrient media supplemented with a range of Al concentrations (0 µM, 35 µM, 50 µM, and 100 µM) for 2 d. Treatment of non-transgenic plants with 35 µM AlCl3 resulted in a significant reduction in root growth rate by 76% compared with the untreated controls, whereas representative transgenic lines showed about 20% reduction in root growth rate under the same conditions. When exposed to 100 µM AlCl3, root growth in wild-type plants was inhibited completely, while roots of all three transgenic lines also showed some elongation (Fig. 5B), indicating that overexpression of AtMGT1 could confer tolerance to Al in N. benthamiana plants.
Callose accumulation
To confirm that expression of the 35S::AtMGT1 gene was correlated with Al tolerance in transgenic plants, callose deposition in the roots of transgenic and wild-type plants was examined after exposure to Al. Five-day-old seedlings were stained with 0.1% aniline blue (pH 9.0), and examined by fluorescence microscopy for callose accumulation in root tips. Wild-type root tips without Al treatment exhibited little background fluorescence, while intense fluorescence was observed in Al-treated wild-type root tips, indicating callose accumulation (Fig. 6A). By contrast, callose deposition in root tips of the 35S::AtMGT1 plants in the presence of Al was markedly reduced to levels slightly greater than that seen for the untreated controls (Fig. 6A). Qualitative analysis showed that the callose accumulation in the roots of the 35S::AtMGT1 line A28 was dramatically lower than in wild-type plants after Al treatment (Fig. 6B). These findings were consistent with the previous observation.
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Discussion |
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Al toxicity is the major factor limiting crop productivity on acid soils. In general, there are two strategies to deal with Al toxicity in plants: exclusion of Al from the root apex or development of Al tolerance once it enters the plant symplasm (Delhaize and Ryan, 1995; Kochian, 1995). The present results revealed that transgenic plants overexpressing AtMGT1 cDNA showed significantly higher relative root elongation and lower deposition of callose in the root tip region, indicating that Al tolerance in the transgenic plants was elevated significantly.
It has been reported previously that application of Mg can alleviate Al toxicity in a variety of plant species (Tan et al., 1991, 1992; Keltjens and Tan, 1993; Matsumoto, 2000; Silva et al., 2001a, b, c; Mariano and Keltjens, 2005). A possible explanation for this phenomenon may reside in a reduction in the negativity of the surface electrical potential of the plasma membrane (Kinraide et al., 1992, 2004; Kinraide, 1998). The differing negative electrical charges of the plasma membrane among plants are expected to differentially attract the positively charged Al ions (Kinraide et al., 1992) and may alter the phospholipid profile, thereby affecting the lipid-mediated signalling (Jones and Kochian, 1997). On the other hand, it was also proposed that Mg alleviation of Al toxicity may involve competition with Al for binding at sensitive sites (Kinraide and Parker, 1987) and a reduction in Al saturation in exchange sites of root cells (Grauer and Horst, 1992). Silva et al. (2001b, c) reported that alleviation of Al toxicity in soybean with micromolar additions of Mg could not be accounted for by the cation's effect on the surface electrical potential of the plasma membrane, but rather by involvement of Mg-induced production and efflux of citrate by roots.
In the present study, it was found that overexpression of the Mg transport gene AtMGT1 could also result in the amelioration of Al toxicity. Presumably, there are a number of reasons for this improvement. First, Mg2+ deficiency is alleviated by overexpression of AtMGT1 cDNA in transgenic plants. Direct uptake experiments had established that Al3+ inhibited Mg2+ uptake by intact Lolium multiflorum roots (Rengel and Robinson, 1989). Tan et al. (1991) reported that Al3+ induced Mg2+ deficiency in grasses and cereals. Moreover, Al3+ could displace Mg2+ at Mg-binding sites of enzymes and structural proteins due to the similar size of Al3+ and Mg2+ (Martin, 1992; Kochian, 1995; MacDiarmid and Gardner, 1996). Li et al. (2001) demonstrated that, based on the results of the tracer uptake assay, 10 µm Al3+ completely inhibited AtMGT1 activity in an Mg2+ transport mutant of S. typhimurium and proposed that Al3+ induced Mg2+ deficiency on account of inhibition of the AtMGT transport system. In the present experiments, the growth of 35S::AtMGT1 plants on low-Mg2+ medium is enhanced (Fig. 3). It is speculated that Mg2+ deficiency in plants is alleviated due to overexpression of AtMGT1, therefore leading to an improvement in Al tolerance in N. benthamiana.
Second, it is possible that overexpression of AtMGT1 increased organic acid production and exudation by roots. Chelation of Al by organic acids such as malate, oxalate, or citrate within root cells or in the rhizosphere has been proposed as a mechanism of Al tolerance (Taylor, 1991; Ryan et al., 1995). Kinetic and spectroscopic investigations of four key enzymes in the tricarboxylic acid cycle were carried out, and results showed that Mg2+ can activate these four key enzymes in vitro (Heaton, 1990). The presence of Mg2+ could perhaps stimulate the whole tricarboxylic acid cycle, leading to an increase in organic acid concentrations in the root tip. Meanwhile, Mg2+ could invigorate organic acid exudation by affecting the regulation of organic anion transporter. Silva et al. (2001c) demonstrated that application of Mg2+ elevated organic acid production and exudation, leading to amelioration of Al rhizotoxicity in soybean. In the present study, it was observed that the Mg content was higher in 35S::AtMGT1 plants than in wild-type plants (Fig. 4). It is speculated that, at higher Mg2+ concentration in transgenic plant cells, organic acid production and exudation are increased, resulting in improvement of Al tolerance in plants. But actually there is still no direct evidence to demonstrate it in the present study. Hence, additional metal element analysis and assays with organic acids could be helpful to confirm the validity of the role of the AtMGT1 gene in amelioration of Al toxicity.
In a previous work, radioactive Ni2+ tracer uptake analysis showed that, in bacteria, AtMGT1 was capable of transporting several divalent cations, including Mg2+, Ni2+, Co2+, Fe2+, Mn2+, and Cu2+, and had the highest affinity for Mg2+ (Li et al., 2001). Likewise, the present study showed that overexpression of AtMGT1 cDNA in transgenic plants increased the metal content of Fe, Mn, and Mg compared with wild-type plants (Fig. 4), consistent with the result that AtMGT1 protein could transport some divalent cations (Li et al., 2001). Moreover, in the Mg2+ deficiency assay, transgenic 35S::AtMGT1 plants displayed an enhanced ability to tolerate growth on low-Mg2+ and Mg2+-deficient media by contrast with wild-type plants (Fig. 5). These results further confirmed that AtMGT1 was involved in Mg acquisition from the environment and/or in Mg transport in plants.
Interestingly, in the present study it was found that the Al tolerance and morphological changes of different transgenic 35S::AtMGT1 plants were not proportional to the level of transgene expression. Based on the results from RT-PCR and northern blot analyses as shown in Fig. 1, the relative order of AtMGT1 expression in the three transgenic lines was A12 >A18 >A28, whereas, the order of Al tolerance was A12 >A28 >A18 and the morphological changes of transgenic line A28 were larger than lines A12 and A18. Moreover, the relative concentrations of Mg, Mn, and Fe varied in three transgenic lines (Fig. 4). Presumably, variability of transgene expression between different independent transgenic lines is attributed to transgene position effects in the chromosome (Dean, 1988; Peach and Velten, 1991; Gelvin, 1998). And it is also feasible that the expression level of AtMGT1 may not directly correlate with mediation of ion uptake and Al tolerance, because mRNA levels may not reflect protein levels. Previous studies revealed that expression of CorA, which is homologous with AtMGT1, did not coincide with protein levels (Chamnongpol and Groisman, 2002; Gardner, 2003).
Mg2+ deficiency in plants, which affects crop productivity, is a widespread problem worldwide (Bennett, 1997; Aitken et al., 1999; Mitchell et al., 1999). Mg2+ adsorbs less strongly to soil colloids than other cations due to a high hydrated radius, and therefore is highly prone to leaching, which is considered as the most important factor reducing Mg2+ availability for roots. Mg deficiency can also be induced by a failure of the roots to assimilate Mg2+, because of competitive inhibition of uptake by other cations such as Ca2+, K+, and NH4+ (Mengel and Kirkby, 1987). In the present study, AtMGT1-overexpressing transgenic plants showed enhanced tolerance to Mg2+ deficiency compared with wild-type plants (Fig. 3), indicating that AtMGT1 overexpression might be beneficial in the generation of plants which are capable of thriving on low-Mg2+ soils.
Callose deposition is an indicator of Al-induced stress because callose accumulates in the cell wall around plasmodesmata in response to the damage caused by Al stress in the roots of various plants (Wissemeier et al., 1987; Schreiner et al., 1994; Zhang et al., 1994). In the present study, after Al exposure, callose accumulation in the roots of 35S::AtMGT1 plants was reduced compared with the wild type (Fig. 6). This result further demonstrated that Al toxicity was ameliorated in transgenic plants overexpressing AtMGT1. As far as is known, this study presents the first report of overexpression of magnesium transport proteins for increased Al tolerance in higher plants.
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Acknowledgements |
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We would like to thank Dr Xiu Lan Zhao (College of Resources and Environment, Southwest University, Chongqing, China) for elemental analysis of plants. In China, this work was supported by the Special Funds for Major State Basic Research Program of China (973 Program) (No. 001CB108905). In the USA, the project was supported by USDA and CPBR/DOE to Yi Li.
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Footnotes |
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* These authors contributed equally to this research and are considered co-first authors.
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