Journal of Experimental Botany, Vol. 54, No. 384, pp. 1023-1031,
March 1, 2003
© 2003 Oxford University Press
Expression of a putative high-affinity NO3– transporter and of an H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability
Received 30 July 2002; Accepted 27 November 2002
1 Department of Agricultural Biotechnology, University of Padua, Agripolis, Strada Romea 16, 35020 Legnaro (Padua), Italy
2 Department of Environmental Agronomy and Plant Production, University of Padua, Agripolis, Strada Romea 16, 35020 Legnaro (Padua), Italy
3 To whom correspondence should be addressed. Fax: +39 049 8272929. E-mail: silviaqu{at}unipd.it
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Abstract |
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Two maize genotypes differently responsive to nitrogen availability were characterized for their efficiency in nitrate accumulation via both the LATS (Low-Affinity Transport System) and HATS (High-Affinity Transport System) nitrate uptake systems. In addition, a full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.1) was isolated and its expression evaluated in both the roots and leaves of the two maize genotypes, together with the expression of a maize H+-ATPase isoform (Mha1). The data showed the importance of the iHATS (Inducible High-Affinity System) system efficiency as a physiological marker of adaptation to low input and suggested that the transcript accumulation of ZmNrt2.1 might be a key step for the regulation of iHATS. However, ZmNrt2.1 transcription cannot account for the differences found between the two hybrids in terms of the activity of their respective iHATS and, as a consequence, of their adaptation to low input. Therefore, the involvement of some other transporter(s) or of some post-transcriptional/post-translational mechanism of regulation affecting the efficiency of iHATS may be hypothesized. In addition, the data suggest that the transcription of the Mha1 gene may also be involved in the global efficiency of the iHATS system.
Key words: H+-ATPase, maize, nitrate accumulation, nitrate uptake system, nitrogen availability.
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Introduction |
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Nitrate is the major source of nitrogen for the vast majority of plants and its concentration in the soil solution can range from less than 1 mM to more than 10 mM depending on factors such as pH, oxygen availability, rainfall, and fertilizer supply (Reisenauer, 1966; Barber, 1984; Marschner, 1995). In the last three decades, nitrogen fertilization has been a powerful tool in increasing grain yield, especially for cereals such as maize and wheat, that have been principally selected for their adaptation to high fertilizer input (Castelberry et al., 1984). However, the optimization of the application of nitrogen fertilizers has become increasingly more important to avoid pollution by nitrate and, as a consequence, cereal cultivars that absorb and metabolize nitrogen in the most efficient way are being selected (Hirel et al., 2001; Ter Steege et al., 2001).
Intrinsic N-use efficiency of the whole plant is determined by the integrated biochemical processes in the plant, and improving crop N-use and yield with lower inputs and less pollution requires a better understanding of the whole system, from the level of genes, to metabolism, and finally to yield (Lawlor, 2002). In the last decades, the study of the regulation of the uptake and reduction of nitrate in higher plants has received considerable attention from scientists, leading to the unravelling of some important physiological and molecular events underlying these processes.
The first step in NO3– acquisition and use by plants is the active transport across the plasma membrane of root epidermal and cortical cells, and is coupled to the favourable H+ electrochemical gradient created by the plasma membrane H+-ATPases (Thibaud and Grignon, 1981; Ruiz-Cristin and Briskin, 1991; Meharg and Blatt, 1995; Miller and Smith, 1996). Physiological studies have demonstrated the existence of at least three different NO3– uptake systems in plants (Glass and Siddiqi, 1995; Forde and Clarkson, 1999). At high external NO3– concentrations, an essentially unregulated low-affinity transport system (LATS) operates and appears to be constitutively expressed. LATS is thought to contribute significantly to nitrate uptake at concentrations above 250 µM and to saturate at concentrations as high as 50 mM (Crawford and Glass, 1998), even though little information about its regulation in plants is available. At low external concentrations (0–0.5 mM), two high-affinity transport systems are active, one of these being constitutive (cHATS) (Km=6–20 µM), whereas the other is induced by nitrate (iHATS) (Km=20–100 µM) (Crawford and Glass, 1998). The cHATS has a higher affinity for NO3–, but iHATS has a much greater uptake capacity (Siddiqi et al., 1990; Aslam et al., 1992; Kronzucker et al., 1995).
Molecular data have established that multiple gene family members encoding putative transporters are present for both the high- and low-affinity systems (Glass et al., 2001). Genes that are thought to encode the HATS transporters, termed Nrt2 genes, have been isolated from various plant species and attributed to the NNP (nitrate–nitrite porter) family, belonging to the MFS (Major Facilitator Superfamily) superfamily of membrane transporters (Forde, 2000).
In all species, inducible high-affinity NO3– uptake is highly regulated, increasing upon initial nitrate supply, and decreasing in response to the accumulation of several endogenous nitrogen sources such as nitrate, ammonium and amino acids, that may be involved in its down-regulation, thus co-ordinating root uptake with shoot demand for nitrogen (Ismande and Touraine, 1994; Glass and Siddiqi, 1995; Crawford and Glass, 1998). Most of this regulation has been shown to take place at the steady-state mRNA levels of Nrt2 genes (Vidmar et al., 2000b). The high correlation reported between Nrt2 mRNA steady-state levels and NO3– uptake (Forde and Clarkson, 1999; Lejay et al., 1999; Zhuo et al., 1999) together with the nitrate uptake impaired phenotype of an Arabidopsis T-DNA mutant altered in NRT2.1 and NRT2.2 expression (Filleur et al., 2001) are strong arguments to date in favour of an involvement of NRT2 proteins in NO3– uptake and support the hypothesis that its regulation takes place preferentially at the transcriptional level. However, Fraisier et al. (2000), have shown in Nicotiana plumbaginifolia that at least the down-regulation of high-affinity nitrate transport is also controlled at the post-transcriptional level.
Despite the considerable progress made in the elucidation of the physiological and molecular aspects of nitrate uptake, also through the isolation of genes encoding putative nitrate transporters, the role played by nitrate uptake capacity and by its physiological and molecular regulation in defining the genetic variability for nitrogen use efficiency still remains largely uncharacterized.
To study the regulation of high-affinity nitrate uptake and its involvement in determining the plant response to nitrogen availability, two maize hybrids differently adapted to N input, named SL and TH, were compared for their responses to nitrate availability following a period of nitrate starvation. The two genotypes were chosen on the basis of their significantly different field performances, with SL having a higher yield in low nitrogen input compared to that of TH, and TH a greater yield in high nitrogen input compared to that of SL (KWS SAAT AG, personal communication). Their adaptation to nitrate availability was also evaluated in terms of shoot/root ratio, RGR and foliar pigment content, with SL seedlings having higher values in the presence of low nitrate concentration with respect to TH, and TH seedlings having higher values in the presence of high nitrate availability compared to SL (Schiavon, 2001).
In this study the tissue nitrate content and the efficiency of influx systems active at high and low nitrate concentrations were evaluated in seedlings of both hybrids. In addition, a cDNA clone encoding a putative high-affinity transporter, called ZmNrt2.1 (AY129953), was isolated and its expression characterized in both the roots and the leaves of the two genotypes. The expression of a gene encoding a maize H+-ATPase (Mha1, U09989, Jin and Bennetzen, 1994), putatively involved in the creation of a more favourable electrochemical gradient for nitrate active transport, was also investigated.
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Choice of the maize lines and plant growth conditions
The two maize genotypes used in this work are derived from two different lines, TH and SL, obtained after selection in high and low nitrogen input, respectively, and crossed with a tester line (KWS SAAT AG, personal comunication).
Seeds of two maize hybrids supplied by KWS SAAT AG (Germany) were surface-sterilized in 5% (v/v) sodium hypochlorite for 10 min, washed in distilled water and germinated on wet filter paper at 25 °C in the dark. After 3 d, seedlings were transferred to 35 l tanks containing a nitrogen-depleted aerated nutrient solution composed of 40 µM KH2PO4, 1 mM KCl, 200 µM MgSO4, and 10 µM FeNaEDTA. Unless otherwise indicated, all chemicals were purchased from Carlo Erba (Milano, Italia). Microelements were added as indicated by Hoagland and Arnon (1950). Nutrient solutions were changed every 2 d and the pH of the solutions ranged between 5.6 and 6.0. After 5 d the plants were transferred for an additional 2 d to a solution identical to that previously described, but supplemented with 1 mM KNO3 instead of KCl. The plants were grown in a controlled environment chamber with a day/night cycle of 14/10 h at 25/18 °C air temperature, 70/90% relative humidity, and 280 µmol m–2 s–1 photon flux density.
Plants for nitrate influx experiments and nitrate content were sampled for analysis at 0, 2, 6, 24, and 48 h after the transfer to the 1 mM NO3–-containing solution. Tissues used for gene expression analyses were collected at 0, 0.5, 2, 6, 24, and 48 h, immediately frozen in liquid nitrogen and kept at –80 °C for subsequent RNA extraction.
Nitrate influx
Nitrate influx was determined at external nitrate concentrations of 50 µM and 1 mM to evaluate the high- and low-affinity influx, respectively. Groups of five seedlings were harvested and transferred to a nutrient solution containing 15NO3– for 5 min, after a 3 min period of equilibration in an unlabelled solution identical to that used for the experiment. The plants were removed and their roots were immersed in ice-cold unlabelled nutrient solution for 2 min to eliminate the apoplastic fraction of the 15NO3–. The roots and leaves were harvested separately, weighed and ground in a ball mill. About 2 mg of the resulting powder was taken for total N/15N measurements. Samples were placed in tin cups and analysed with an elemental analyser connected in series with an isotope-ratio mass spectrometer (Roboprep CN and Tracermass, Europe Scientific, Crewe, UK). The abundance of 15N in the uptake solutions was determined after isotopic dilutions with a reference KNO3 solution so that each sample contained approximately 100 µg of N.
Nitrate content determination
Nitrate was extracted by grinding 1 g of frozen plant material in 10 ml of 10 mM HCl. The extract was filtered (0.22 µm) and the nitrate concentration was determined by ion chromatography using an Ion-Pac AS4A-SC separation column (Dionex, Sunnyvale, CA), with a solution of 1.8 mM K2CO3 and 1.7 mM KHCO3 as eluent, at a flux speed of 2 ml min–1. Nitrate quantification was obtained using a calibration curve.
RNA extraction and cDNA synthesis
Total RNA was isolated using the ‘Nucleon Phytopure’ kit (Amersham-Pharmacia, UK) following the protocol provided by the manufacturer. First-strand cDNA was synthesized from 5 µg of total RNA, after DNase treatment (Promega, Milano, Italy), using 200 U of MMLV Reverse Transcriptase (Promega, Milano, Italy) and oligodT as a primer, in 20 µl reactions, as described in Sambrook et al. (1989).
ZmNRT2.1 and Mha1 cDNA cloning and sequencing
The cloning of ZmNRT2.1 partial cDNA was performed by using degenerate primers designed on the basis of conserved sequence motifs of the high affinity (Nrt2) eukaryotic nitrate transporter proteins and using cDNA obtained from roots of seedlings grown in the presence of nitrate as template. The primers utilized for the ZmNRT2.1 cloning were: f-HANT-5'-CCATGGTCCTGTTCTC CATCKGNGCNCARGC-3'; r-HANT-5'-GAACATGGAGCCCC ACTGNGGRAARTG-3'.
Reactions were carried out with the Gene Amp PCR system 9700 (PE Biosystems, Branchburg, NJ, USA) using 0.025 U µl–1 Taq-polymerase (Amersham-Pharmacia, UK) under the following conditions: 5 min at 94 °C followed by 40 cycles of 30 s at 94 °C, 1 min at 55 °C, 30 s at 72 °C and 7 min of final extension at 72 °C. The obtained amplification products were subcloned into the pGEM-T Easy vector (Promega, Milano Italy) and plasmids from ten recombinant colonies were sequenced. Plasmids were prepared using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and sequenced, according to Sanger et al. (1977), using the ABIPRISM original Rhodamine Terminator kit (PE Biosystem, Branchburg, NJ, USA). Sequence comparisons were performed by using BLASTx and BLASTn computer programs (NCBI, National Center for Biotechnology Information).
The ZmNrt2.1 full-length clone was isolated by the RACE technique, as described by Schaefer (1995). The primers were: 5'-ATCTTCGGGGTCATCCCCTTTGTCT-3' for 3' RACE and 5'-CAGCGTGCACGCCATGATCAT-3' for 5'-RACE.
The amplification products obtained were subcloned and ten recombinants for both 3'-RACE and 5'-RACE were sequenced.
A Mha1 partial clone was obtained by using the following primers designed on the basis of the published sequence (accession number U09989, Jin and Bennetzen, 1994): 5'-GATGCTTGCCTCTGC GGTATAC-3', forward primer; 5'-TTCTTCTTGCTTGTGA ATGCGA-3', reverse primer. PCR conditions were as described above, but with an annealing temperature of 64 °C. The amplification product obtained was subcloned and sequenced.
ZmNrt2.1 and Mha1 expression analysis
RT-PCR experiments with specific primers were performed to evaluate the expression level of ZmNRT2.1 and Mha1 genes in roots and leaves of seedlings of the two maize hybrids at different times of exposure to nitrate (0, 0.5, 1, 2, 6, 24, and 48 h). For PCR, 1 µl of the cDNA obtained was used in 20 µl reactions, using 0.025 u µl–1 of Taq-polymerase (Amersham-Pharmacia-Biotech, Piscataway, NJ, USA). For each of these reactions a set of different numbers of cycles ranging between 15 and 25 was tested to choose those corresponding to the exponential phase for each gene. Each cycle was composed of a 30 s denaturation at 94 °C, 1 min of annealing at 65 °C and a 30 s extension at 72 °C; a 5 min denaturation at 94 °C period at the beginning of the reactions and a 7 min extension at 72 °C at the end were performed for all reactions. The specific primers used for ZmNRT2.1 were those used as specific primers in the 3' and 5'-RACE. Primers employed for Mha1 were those described for Mha1 cloning. A maize ubiquitin (U29162) and a maize actin (J0128) were used as constitutive internal standards and the primers used were: 5'-CCACTTGGTGCTGCGTCTTAG-3' (forward primer); 5'-CCTTCTGAATGTTGTAATCCGCA-3' (reverse primer) for ubiquitin and 5'-TGTTTCGCCTGAAGATCACCCTGTG-3' (forward); 5'-TGAACCTTTCTGACCCAATGGTGATGA-3' (reverse) for actin. PCR products obtained from expression analyses were sequenced to confirm the specificity of amplification of each gene. To confirm results, PCR reactions were performed on cDNAs obtained from two different RNA extractions performed on samples from two independent experiments and repeated at least three times for each cDNA. The PCR products were electrophoresed in 1–2% agarose gels, stained with ethidium bromide, transferred and fixed onto Geen-Screen membranes (NENTM Life Science, Boston, MA). The membranes were then hybridized with the cDNA probes corresponding to each gene and washed at high stringency as described in Sambrook et al. (1989) before exposure to X-ray films at –80 °C. Hybridization was performed at 65 °C in a solution containing 5x SSC, 5x Denhardt’s solution, 0.1% (w/v) SDS, 50 mM potassium phosphate, and 100 µg ml–1 sonicated herring sperm. Probes were prepared with the random priming method, using 32P-labelled dCTP and membranes were washed at 65 °C with 2x SSC, 0.1% (w/v) SDS, with 1x SSC, 0.1% (w/v) SDS, and with 0.1x SSC, 0.1% (w/v) SDS, for 20 min each.
Hybridization signals were quantified by using the LabImage 2.6 program (Kapelan).
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Nitrate accumulation
Since the tissue nitrate content may reflect nitrate acquisition and metabolism efficiency, it may be regarded as an important factor in nitrogen status sensing (Crawford and Glass, 1998). Therefore, the nitrate content of leaves and roots of the two maize hybrids was evaluated just before the transfer of seedlings to a nitrate-containing solution and at different times thereafter, as reported in Fig. 1A and B. TH and SL showed undetectable levels of nitrate at the beginning of the experiment and a subsequent continuous increase in terms of accumulation in response to longer times of exposure to 1 mM nitrate, in both roots and leaves. The level detected in roots (Fig. 1A) was markedly higher than that found in leaves (Fig. 1B) at all times in both genotypes.
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When the two hybrids are compared, roots of TH already showed a significantly higher nitrate content (4.7±0.3 µmol g–1 FW) than those of SL (3.3±0.2 µmol g–1 FW) after 2 h of nitrate exposure and a higher nitrate accumulation during the next 48 h, reaching values 50–60% greater then those measured for SL roots (Fig. 1A). Also in leaves, TH showed a higher nitrate accumulation throughout the whole experiment, with values 30–100% higher than those measured in SL leaves (Fig. 1B).
Influx at 1 mM
Nitrate influx measured in experiments conducted at a nitrate concentration of 1 mM, reflects the global activity of different transport systems active at both higher and lower nitrate concentrations ranging from 0 mM to 1 mM.
The nitrate influx rates measured at different times of exposure to 1 mM nitrate solution showed, both in SL and in TH, the existence of a constitutive activity, already present at the beginning of the experiment (time 0), of 37 and 40 µmol g–1 h–1 DW, respectively (Fig. 2). Inducible activities, that appeared not yet to have peaked throughout the experiment, were also observed in both TH and SL. In fact, nitrate influx increased immediately after nitrate supply and had already reached values 50–60% higher after 2 h. A steady increase in terms of influx was subsequently detected and, after 2 d of permanence in the nitrate solution, the influx values were four times higher than those performed by the constitutive system reaching 140 and 161 µmol g–1 h–1 DW in the SL and TH, respectively.
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Significant differences in terms of nitrate uptake between the two maize genotypes could already be detected 2 h after nitrate supply, with the TH performing the highest values of nitrate influx, indicating its major capability of nitrate transport in this range of concentration and supporting the tissue nitrate accumulation data.
Influx at 50 µM
Nitrate influx experiments carried out at 50 µM were aimed to evaluate the capability of the two maize genotypes to absorb nitrate from the solution by their high affinity systems known to be active at this concentration. For both hybrids, the activity of a constitutive high-affinity transport system could be detected during the first 2 h of exposure to nitrate and reached values not higher than 25 µmol NO3– g–1 h–1 DW and 60% lower than those found in the case of the constitutive system active at 1 mM influx experiments. No significant differences in terms of constitutive high-affinity influx could be detected between the two maize genotypes.
After 6 h of nitrate exposure, an increase in the high-affinity influx was detected in both SL and TH (Fig. 3). The SL hybrid always displayed higher values and already showed a 95% increase compared with its constitutive activity after 6 h, reaching a maximum of 51 µmol NO3– g–1 h–1 DW after 24 h, which was maintained up to 48 h.
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The activity detected for the TH hybrid reached a maximum of 39 µmol NO3– g–1 h–1 DW after 6 h of exposure (50% increase in comparison to its constitutive activity) and subsequently showed a slight progressive decrease 24 h and 48 h after the beginning of the experiment.
Cloning of ZmNRT2.1 and Mha1
Using specific primers designed on the sequence of a maize H+-ATPase (Mha1, Jin and Bennetzen, 1994; U09989) a partial cDNA clone of 500 bp showing 100% identity with the Mha1 sequence, was obtained.
As far as the ZmNrt2.1 cloning is concerned, using a degenerate PCR, a 264 bp cDNA fragment showing 87% identity with the nucleotide sequence of OsNrt2.1 (AB008519), was cloned. A full-length clone of 1789 bp, termed ZmNrt2.1 (AY129953), showing a deduced amino acid sequence 85.7% identical to that of OsNrt2.1 was isolated successively.
ZmNrt2.1 presents an open reading frame of 1575 bp encoding a protein of 524 amino acids characterized by 12 predicted trans-membrane domains and by the N-terminal and the C-terminal located at the cytosolic level. Moreover, an additional N-terminal sequence of 22 amino acids, typical of the NNP transporters belonging to the group IIIb (Forde, 2000) and several conserved recognition motifs of protein kinase C and casein-kinase II were found (Fig. 4). In addition, between the second and the third trans-membrane helix a highly conserved signature motif of the MFS superfamily was identified, together with two motifs located in the fifth trans-membrane domain typical of members of the NNP family.
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ZmNRT2.1 expression in roots and leaves
The analysis of the expression of ZmNrt2.1 in the roots of seedlings of the two maize genotypes that were without nitrate for 5 d and then supplied with this anion, evidenced the induction of the accumulation of transcripts corresponding to this gene in response to nitrate addition to the nutrient solution (Fig. 5A). Comparing both hybrids, SL showed the presence of a weak signal before nitrate supply to the solution (time 0) and a subsequent gradual increase in terms of ZmNrt2.1 transcript accumulation, after 30 min, which reached its maximum level after 48 h. The same experiment conducted on TH showed the appearance of a detectable signal only after 30 min, 10% lower than the corresponding level in SL, reaching a maximum, that was 32% higher than the corresponding value found in SL, 2 h after the transfer. The ZmNrt2.1 transcript level in TH subsequently underwent a slight decrease to a steady-state level maintained throughout the experiment, with a signal 20% lower than that found in SL after 48 h.
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The accumulation of transcripts corresponding to ZmNrt2.1 was also detected in leaves, although to a lesser extent than in root tissues (Fig. 5B). The expression analysis of ZmNrt2.1 in leaves showed an induction in terms of synthesis of this transcript in response to the presence of 1 mM nitrate, that peaked in both hybrids after 6 h and decreased successively after 24 h and 48 h. As reported for root tissues, SL showed a weak presence of the transcript before the supply of nitrate (T0). A marked decrease in terms of transcript accumulation to a basal level after 24 h and 48 h was observed for the SL genotype, in contrast to a more gradual down-regulation in the case of the TH genotype, which showed the presence of a transcript 51% and 31% higher than that of SL at, respectively, 24 h and 48 h.
Mha1 expression in roots and leaves
Because of the role played by H+-ATPases in nitrate uptake, the expression of a previously isolated gene encoding a H+-ATPase, named Mha1 (Jin and Bennetzen, 1994, U09989), was studied in the two maize genotypes. RT-PCR analysis of Mha1 transcript accumulation in roots and leaves of the SL hybrid indicated the presence of a constitutive signal showing no significant changes up to 48 h following the transfer to a nitrate-containing solution. By contrast, in the same conditions, in the roots and leaves of the TH hybrid, transcripts of Mha1 were almost undetectable (Fig. 6A, B).
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Discussion |
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In this study, two maize genotypes, obtained from a selection at high (TH) and low (SL) nitrogen input and significantly differing in their field performances (KWS SAAT AG, personal communication) and adaptation to nitrate availability (Schiavon, 2001), were used as a model for a physiological and molecular characterization of nitrate uptake efficiency. Different nitrate uptake systems characterized by high and low substrate affinity were demonstrated to exist in plants and thought to contribute differentially to the global efficiency of nitrate acquisition. Physiological studies have suggested that, at relatively high NO3– concentrations such as 1 mM, both high- and low-affinity systems may contribute significantly to overall uptake (Aslam et al., 1992; Glass et al., 1992; Peuke and Kaiser, 1996; Crawford and Glass, 1998; Vidmar et al., 2000a). Therefore, this concentration was chosen here to evaluate the physiological differences in terms of efficiency between the two maize cultivars in both their low- and high-affinity systems, paying special attention to the latter which may play an important role in the response of the plant to low nitrate availability.
Since the NO3– content of tissues may be a good marker to select maize genotypes with enhanced grain yield and grain nitrogen content (Hirel et al., 2001), this was evaluated in the two maize hybrids. In addition, this parameter can be one of the indicators of the plant’s nitrogen status (Lawlor, 2002) and a rather limited modification of the uptake of NO3– can significantly alter nitrate accumulation (Fraiser et al., 2000), which, in turn, can directly or indirectly down-regulate the NO3– high-affinity uptake systems (Forde and Clarkson, 1999; Vidmar et al., 2000b; Forde, 2002).
On the basis of these data, seedlings of TH had a greater nitrate content in both the roots and leaves throughout the experiment. This better ability of the TH to accumulate and to maintain a higher tissue content of NO3– than SL is consistent with its higher capability of nitrogen use efficiency at conditions of high external NO3– concentrations and correlates with the influx values measured at 1 mM NO3–. In fact, influx experiments conducted at this concentration revealed an uptake system significantly more efficient for TH than for SL. Even though nitrate uptake measured at 1 mM reflects the activity of both the high- and low-affinity systems, the contribution of the low-affinity system at a nitrate concentration higher then 250 µM is thought to be significant (Crawford and Glass, 1998). These data suggest that a direct connection between the low-affinity influx capability and the accumulation of nitrate in the tissue exists and, therefore, the differences among genotypes in their nitrogen use efficiency in the presence of unlimited availability may rely on different uptake capabilities, based principally on their low-affinity transport systems.
To characterize the high-affinity systems for NO3– uptake in the two maize hybrids, nitrate influx was measured at 50 µM NO3– after different times of induction of the transport system in the presence of 1 mM nitrate. On the basis of the data obtained, the cHATS of the two genotypes did not show any difference and had influx values several times lower than those performed by the iHATS, confirming the lower capability (Vmax) of the cHATS systems in maize as also reported in other species (Siddiqi et al., 1990, 1991; Aslam et al., 1992; Kronzucker et al., 1995; Min et al., 1998). By contrast, the two maize hybrids showed different behaviour in terms of iHATS in both absolute values and time-course, with SL showing the highest rate of induction and the lowest level of down-regulation compared with TH. The earlier and more consistent down-regulation shown in the case of TH correlates with its faster NO3– accumulation, and indeed with its better LATS performance. These results confirm the role of the accumulated NO3–, or of some products of its assimilation, in down-regulating the iHATS (King et al., 1993; Crawford and Glass, 1998; Forde and Clarkson, 1999; Vidmar et al., 2000b). In addition, the higher rate of influx induction and the less marked down-regulation observed in the case of the SL are consistent with its better field productivity performances at low nitrogen input conditions (KWS SAAT AG, personal communication) and its better adaptation to low nitrate availability (Schiavon, 2001), in which the HATS are supposed to play an important role (Glass et al., 2001).
To investigate the molecular events underlying the physiological differences between the two maize genotypes in terms of their iHATS systems, a clone, termed ZmNrt2.1, encoding a putative high affinity nitrate transporter, was isolated. The sequence of ZmNrt2.1 full-length cDNA encoded a deduced protein with an additional N-terminal of 22 amino acids highly conserved among the dicot members of the NRT2 family and thought to be dicot-specific. However, the same feature was found in a rice NRT2 protein (OsNrt2.1), suggesting that it is not, in fact, restricted to dicots. A number of highly conserved motifs and features of the NNP and MFS families supporting the putative role of ZmNrt2.1 in high-affinity nitrate transport were found. In addition, the presence of several casein-kinase II and protein-kinase C recognition motifs suggest that the activity of the protein may be subjected to post-translational regulation through phosphorylation events.
The analysis of the expression of ZmNrt2.1 in response to nitrate availability revealed an accumulation time-course similar to that of the induction of the high-affinity influx in roots of both TH and SL. In the case of SL, an increase of the transcript abundance throughout the experiment was observed. This correlated with the increase of the high-affinity influx, reaching its maximum after 24–48 h. In the case of TH, by contrast, the transcript accumulation had already reached its maximum after 2 h, decreasing to a steady-state level after 6 h in 1 mM NO3– solution, with a pattern similar to that shown by the time-course of the high-affinity influx. These data confirm the widely accepted hypothesis that the activity of the iHATS is mainly regulated at the transcriptional level, based on findings showing a correlation between transcript accumulation of specific putative high affinity transporters and nitrate influx (Lejay et al., 1999; Zhuo et al., 1999). In fact, while the time-course of transcript accumulation and iHATS activity showed a high degree of correlation, this can not account for differences in terms of absolute values of inducible high-affinity nitrate influx found when the two maize genotypes were compared. This may be explained by hypothesizing the existence of some other nitrate transporter that could be differentially expressed between the two genotypes and/or of some post-transcriptional mechanisms of regulation of the activity of ZmNrt2.1. The pH dependency of the activity of the nitrate plant plasma membrane transporters is well known, as well as the involvement of the H+-ATPase in the formation of a more favourable electrochemical gradient (Miller and Smith, 1996; Wang and Crawford, 1996). The expression analysis of a maize isoform of H+-ATPase (Mha1) in roots showed a significantly different presence of transcript between the two maize hybrids, with a constitutively higher level for SL, that could be related to its higher capacity of influx by iHATS.
This may lead to the supposition that the H+-ATPase might indirectly regulate the activity of high-affinity nitrate transporters at the post-transcriptional/post-translational level, by generating a more favourable apoplastic pH. The involvement of the post-transcriptional regulation in the control of the HATS-mediated influx has been also reported by Fraisier et al. (2000) in Nicotiana plumbaginifolia and a role for plasma membrane H+-ATPases in the regulation of nitrate uptake was also hypothesized by Santi et al. (1995), who showed an increase in the steady-state level of the enzyme in NO3–-induced maize roots. This is in contrast with these results showing that no changes in Mha1 gene expression occur in response to the presence of nitrate and it may be explained by either considering that enzyme isoforms other than Mha1 could be recognized by the polyclonal antibody used by Santi et al. or hypothesizing the existence of post-transcriptional/post-translational mechanisms of regulation of Mha1.
The analysis of ZmNrt2.1 expression also showed the presence of transcripts in leaf tissues, both in SL and TH, indicating a possible role of the protein encoded by this gene in nitrate loading from the xylem or in its compartmentation. This is consistent with the consideration of nitrate transport as an essential component not only for the uptake process in roots, but also for nitrate assimilation (Orsel et al., 2002). The ZmNrt2.1 transcript accumulation in leaves seems to be up-regulated by the presence of 1 mM nitrate and down-regulated faster than observed in the roots. Mha1 transcript accumulation in leaves confirmed data obtained in root tissues. Further studies will be necessary to elucidate the physiological role of ZmNrt2.1 and Mha1 in roots and leaves by determining their tissue and cellular localization.
In conclusion, the data point to a role for the regulation of nitrate uptake in determining responses to different N availability in maize. Based on these findings, a correlation exists between nitrate accumulation, the efficiency of the LATS system of nitrate uptake and field performances in the presence of high N input in a maize genotype (TH) selected to respond to high N fertilization. By contrast, the adaptation of SL to low nitrogen availability, selected to respond to low N input, seems to rely primarily on the higher efficiency of its iHATS system of nitrate uptake. As a consequence, the study of the regulation of this latter aspect is essential in order to select low-input crops and to reduce fertilization. In this study, it was shown that the time-course of iHATS activity highly correlates with the transcription of ZmNRT2.1 which may, therefore, play a role in the process. However, ZmNRT2.1 transcription alone cannot explain the global differences between two genotypes in their iHATS activities and in their adaptation to low N-input. Future work will be needed to assess the role played in determining the global efficiency of the iHATS system by the expression of other genes encoding additional nitrate transporters or by post-transcriptional regulatory mechanisms of ZmNrt2.1 expression, or by the creation of a more favourable pH for nitrate uptake through the activity of H+-ATPases, also considering the significantly higher level of Mha1 expression detected in the ‘low N-adapted’ SL genotype.
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Acknowledgements |
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We thank Dr D Carden for critical reading of the manuscript and Professor JC Davidian for allowing us to conduct the 15N analysis in his laboratory. Our appreciation to KWS SAAT AG for providing seeds of the two hybrids. Funds were provided by the MURST Giovani Ricercatori 2001 program (104).
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