JXB Advance Access originally published online on March 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 803-813, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.)
Received 23 June 2003; Accepted 2 December 2003
1 Dipartimento di Biotecnologie Agrarie, Agripolis, Università di Padova, Viale dell’Università 16, I-35020 Legnaro (Padova), Italy
2 Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via delle Science 208, I-33100 Udine, Italy
3 Dipartimento di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum, Università di Bologna,Viale Fanin 40, I-40127 Bologna, Italy
4 Dipartimento di Biochimica, Università di Bologna, Via Belmeloro 8/2, I-40127 Bologna, Italy
* To whom correspondence should be addressed. Fax: +39 49 8272929. E-mail: serenella.nardi{at}unipd.it
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Abstract |
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In this study, a detailed characterization of earthworm low molecular size humic substances (LMS) was performed and these substances were used to study their effect on the nitrate influx in roots, tissue nitrate content, and expression of maize genes putatively involved in nitrate uptake in maize (Zea mays L.). The results show that the humic fraction with low molecular size used in this study is endowed with the characteristic structural network described for most humic substances so far isolated and confirm the presence of IAA in this fraction. The results also show that the LMS fraction of humic substances stimulates the uptake of nitrate by roots and the accumulation of the anion at the leaf level. Moreover, the analysis of the expression of genes encoding two putative maize nitrate transporters (ZmNrt2.1 and ZmNrt1.1) and of two maize H+-ATPase isoforms (Mha1 and Mha2) show that these substances may exert direct effects on gene transcription in roots, as shown for the Mha2 gene, and long-distance effects in shoots, as observed for the ZmNrt2.1 gene.
Key words: Gene transciption, humic substances, low molecular size (LMS), maize, nitrate content, nitrate influx, roots.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The environmental impact of crops and cropping systems, the low cost of production and, as a consequence, the necessity of reducing chemical applications to the soil, have become important objectives in agriculture, previously dominated essentially by productivity (Gastal and Lemaire, 2002). To avoid nitrate pollution and to preserve their economic margin, farmers must optimize the application of nitrogen fertilization (Hirel et al., 2001), that has been a powerful tool in increasing grain yield in the last three decades. This is especially so for cereal crops, such as maize and wheat, that have been principally selected for their adaptation to high fertilizer input (Castelberry et al., 1984). In addition, fallow periods are often reduced in shifting cultivation leading to irreversible soil degradation, a decrease of the cation exchange capacity (CEC) and to a lowered efficiency of applied mineral fertilizers, especially when the loss of mobile nutrients such as NO3– or K+ from the topsoil is enhanced by rainfall (Cahn et al., 1993).
For these reasons, studying the improvement in efficiency of nutrient uptake by plants, either by selecting low-input crops or by reducing the amount of soil mineral losses, has received considerable attention from scientists in the last decades. The application of manure or biomass with a different level of humification has frequently been shown to increase soil fertility (Glaser et al., 2002). As a consequence, the unravelling of the physiological and molecular events underlying the effects of humic substances (HS) on plant metabolism, and, in particular, on plant nutrient use efficiency, has become a primary goal to understand and improve the mechanisms involved in the plant response to nutritional stress.
Many papers have shown that HS may improve physical and chemical soil properties, favour a higher concentration of ions in soil solution, and act as source and sink for nutrients such as P, N, and K (Vaughan et al., 1985). In the rhizosphere, an interaction between the root system and humic matter is possible when humic molecules present in the soil solution are small enough to flow into the apoplast and reach the plasma membrane. Many authors have demonstrated the capacity of the low molecular size (LMS) humic fraction to accumulate in the apoplast and to reach, at least in part, the plasma membrane (Vaughan, 1986; Muscolo and Nardi, 1999). This event occurs in the vicinity of the root surface, where the simultaneous release of protons and organic acids by both the root and microbes enables the dissociation of the humic macrostructures and the subsequent release of the otherwise unavailable bioactive fractions (Dell’Agnola and Nardi, 1987; Piccolo et al., 1992). These LMS may enter the plant and affect plant metabolism by either inducing or repressing protein synthesis (Vaughan and MacDonald, 1971; Dell’Agnola et al., 1981), by enzyme activation or inhibition (Butler and Ladd, 1969), and by inducing morpho-functional changes in root architecture (Nardi et al., 1996; Canellas et al., 2002).
The effects of humic substances on ion uptake (nitrate, sulphate, and phosphate) have been discussed by many authors (Vaughan et al., 1985; Chen and Aviad, 1990; Varanini and Pinton, 2001; Clapp et al., 2001) and appear to be selective and quantitatively related to the concentration of HS and to the pH of the medium. Such effects are far from being fully understood, owing to the complex and still unknown nature of the humic substances, and may be related to a syndrome of physiological effects on plant metabolism involving, directly or indirectly, a modulation of ion uptake.
It has been suggested that humic fractions exhibit high hormonal activity and, in particular, auxin-like activity (for a review, see Nardi et al., 2002). Nardi et al. (1994), by using two inhibitors of auxin (TIBA=2,3,5-tri-iodobenzoic acid and PCIB=4-chlorophenoxy-isobutyric acid), demonstrated in Nicotiana plumbaginifolia that the LMS component of humic matter is the fraction endowed with auxin-like activity, although the pathways followed by IAA and the LMS fraction in inducing their effects may be somewhat different.
Pinton et al. (1999), showed that the low molecular weight water extractable humic fraction (WEHS) affects nitrate uptake and plasma membrane (PM) H+-ATPase activity in maize roots. The authors demonstrated that maize seedlings exposed to the WEHS have a higher capacity to absorb NO3– and a faster induction of nitrate uptake. The same pattern was also observed for the activity of PM H+-ATPase, leading the authors to suppose a possible role of WEHS in the modulation of nitrate uptake via an interaction with the PM H+-ATPase. More recently, Canellas et al. (2002) showed that humic acids, isolated from earthworm compost, induced maize H+-ATPase activity by enhancing the content of the enzyme. Despite the considerable amount of physiological and biochemical data and the progress made in the isolation of genes involved in the regulation of nitrate transport no information is available at the molecular level on the effects of humic substances on the expression of these genes.
The active transport of nitrate across the plasma membrane is a key step of nitrate metabolism, requiring specific membrane transporters and is powered by the favourable proton electrochemical difference generated by the PM H+-ATPases (Thibaud and Grignon, 1981; Ruiz-Cristin and Briskin, 1991; Meharg and Blatt, 1995; Miller and Smith, 1996; Wang and Crawford, 1996). Physiological studies have demonstrated the existence of at least three different NO3– uptake systems in plants, with an essentially unregulated low-affinity transport system (LATS) active at high external NO3– concentrations and two high-affinity transport systems, one of these being constitutive (cHATS) whereas the other is induced by nitrate (iHATS), which are active at low external concentrations (Glass and Siddiqi, 1995; Forde and Clarkson, 1999). Molecular data have established that multiple gene family members encoding putative nitrate transporters are present for both the high- and low-affinity systems (Glass et al., 2001). Those encoding the HATS transporters, termed Nrt2 genes, have been attributed to the NNP (nitrate-nitrite porter) family, whereas those encoding the LATS transporters, termed Nrt1 genes, to the PTR (peptide transporter) family, both belonging to the MFS (Major Facilitator Superfamily) superfamily of membrane transporters (Forde, 2000).
The goal of this research was to characterize the chemical composition and structure of the LMS fraction of humic substances used in this study and to verify if LMS has an effect on nitrate uptake and accumulation in maize. Therefore, the NO32– influx by roots, and the nitrate accumulation in roots and leaves were determined and the expression level of a cDNA, termed ZmNrt2.1, encoding a putative high-affinity nitrate transporter (Quaggiotti et al., 2003; accession number AY129953 [GenBank] ) was evaluated in both roots and shoots of maize seedlings in response to the treatment with LMS. In addition, a maize partial cDNA, highly homologous to the members of the PTR gene family, was isolated (ZmNrt1.1, AY187878 [GenBank] ) and its transcript accumulation was measured in both roots and shoots, together with the expression level of two maize genes encoding two H+-ATPase isoforms, Mha1 (Jin and Bennetzen, 1994; accession number U09989 [GenBank] ) and Mha2 (Frias et al., 1996; accession number X85805 [GenBank] ), to clarify the role of H+-ATPases in the response of this species to the LMS treatment.
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Materials and methods |
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Earthworm culture conditions
The faeces of Nicodrilus (=Allolobophora (Eisen)=Aporrectodea (Oerley)) caliginosus (Savigny) and Allolobophora rosea (Savigny) were collected from the surface of the uncultivated couchgrass (Agropyron repens L.) fields at the College of Agriculture farm (University of Padua) as already described in Muscolo et al. (1999). The soils were all classified as Calcaric Cambisol (CMc, FAO classification).
Extraction of the LMS humic fraction
Humic compounds were extracted from the faeces of Nicodrilus (=Allolobophora (Eisen)=Aporrectodea (Oerley) caliginosus (Savigny) and Allolobophora rosea (Savigny) using 0.1 N KOH as already described in Muscolo et al. (1999). The extract was desalted using 14 kDa cut-off dialysis Visking (Medicell, UK) tubing against distilled water. Subsequently the extract was desalted on ion exchange Amberlite IR-120 (H+ form) (Stevenson, 1994). This fraction was then acidified to pH 2.1 with glacial acetic acid and no precipitate was formed, in contrast to when inorganic acids were used for acidification. The acidified fraction was dialysed using Spectra/Por® 3 tubing (MW 3500 cut-off) against distilled water. The eluted LMS fraction was purified by vacuum distillation and ion exchange chromatography (Nardi et al., 1991; Muscolo et al., 1999) and then freeze-dried. The humic carbon content was measured by means of the oxidimetric method (Stevenson, 1994).
Diffuse Reflectance Infrared Fourier Transforms (DRIFT)
The LMS fraction was homogenized with KBr (FT-IR grade, Aldrich Chemical Co. Milwaukee, WI). After grinding, the sample mixture was placed over the top of the micro sample cup. Any excess material was removed with a straight-edged tool. For the background a micro sample cup of pure KBr was prepared. The spectra were recorded with a Nicolet Impact 400 FT-IR Spectrophotometer (Madison, WI) and fitted with an apparatus for diffuse reflectance (Spectra-Tech. Inc., Stamford, CT). DRIFT spectra were recorded with 200 scans collected at 4 cm–1 resolution.
1H NMR spectroscopy
The LMS fraction (20 mg) was dissolved in 0.5 ml D2O. The spectra were recorded with a Bruker ACF 250 spectrometer using a 5 mm multinuclear probe. 1H spectra were accumulated with a 16 K data point, one pulse sequence, 40° pulse angle, 3 s relaxation delay, and a sweep width of 2.5 kHz. To obtain a satisfactory signal-to-noise ratio 1000–2000 scans were needed. Gated irradiation was applied between acquisitions to presaturate the residual water peak. Sodium 3-trimethylsilyl-propionate-2,2,3,3-d4 (TSP) was added to the samples to provide a chemical shift standard. The spectra were divided into three main regions: aromatic hydrogens (Hr) from 6.0–8.0 ppm; H attached to oxygen groups in carbon (Hc-o) from 4.2–3.0 ppm, and aliphatic H (Ha) from 3.0–0.5 ppm. In accordance with results obtained by Wilson et al. (1983) the Hc-o region was attributed to protons largely arising from polysaccharides. Wilson et al. (1983), Gil-Sotres et al. (1994), and Simpson et al. (1997) showed that the Hal region might be affected by the presence of protons attached to aromatic rings in 
, ß, and 
positions.
13C NMR spectroscopy
The relative intensity observed for a given chemical shift value, expressed as a fraction of the total signal intensity acquired, represents the proportion of that type of C present in the sample (Baldock and Preston, 1995). The spectrum was divided into chemical shift regions assigned to chemical group classes alkyl C (0–50 ppm), O-alkyl C (50–100 ppm), aromatic C (110–160 ppm), and carboxyl C (160–185 ppm), and carbonyl C (185–220 ppm), respectively (Mathers et al., 2000; Knicker et al., 2000). By integrating the signal intensity contained within each chemical shift region, the proportion of a given type of C was calculated.
Indoleacetic acid content determination
The quantitative determination of IAA in the LMS fraction was made by an enzyme linked immuno-sorbent assay (ELISA) (Phytodetek-IAA, Sigma, St Louis, MO). Tracer and standard solutions were prepared following the manufacturer’s instructions. To each well 100 µl of standard or LMS fraction and 100 µl of diluted tracer were added. After an incubation at 4 °C for 3 h, the wells were decanted and washed by adding 200 µl of wash solution and then 200 µl of substrate solution. After 60 min at 37 °C, 50 µl of stop reagent was added to each well and the colour absorbance was than read at 405 nm using a 450 Biorad microplater reader (Biorad, Hercules, CA).
Plant material
Maize seeds (Zea mays L. var. Mitos, Pioneer) were soaked for one night in running water and germinated in the dark at 27 °C on a filter paper wetted with 1 mM CaSO4 for 96 h (Nardi et al., 2000). Seedlings were grown in 400 ml of Hoagland no. 2 solution (Hoagland and Arnon, 1950) for 14 d as follows: 16 h of light at 25 °C and 60% relative humidity, 8 h of dark at 18 °C and 80% relative humidity. The seedlings were then incubated in solutions containing different concentrations of LMS fraction (0.25, 0.5, 0.75, 1 mg l–1 C), or 1 mM CaSO4 (control) for an additional 48 h (Nardi et al., 2000). The LMS fraction concentrations were chosen according to Nardi et al. (2000) and previous experiments (data not published) which indicated that the concentration of 0.75 mg l–1 C expressed maximal activity. At the end of this period, the plants were collected.
NO32– influx and NO32– content determination
After 48 h of incubation with LMS the seedlings were transferred to a solution containing 1.5 mM KNO3 for 5 h. Successively, groups of five seedlings were transferred to a nutrient solution containing 0.5 mM 15NO3– for 5 min, after a 3 min period of equilibration in a unlabelled solution identical to that used for the experiment. Successively, 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–. Roots and leaves were harvested separately, weighted, and ground in a ball mill. Samples were analysed with a continuous flow isotope ratio mass spectrometry (FINIGAN MAT Mod. Delta Plus) and 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 was extracted by grinding 1 g of frozen plant material in 10 ml of 10 mM HCl and the extract was filtered (0.22 µm). 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.
Cloning and sequencing of ZmNrt1.1
The ZmNRT1.1 partial cDNA cloning was performed by use of degenerate primers to conserved sequence motifs of the low affinity (Nrt1) eukaryotic nitrate transporter genes and using cDNA obtained from roots of seedlings grown in the presence of nitrate as a template. The primers used were:
f-LANT-5'-CCGTGACGCAGGTCGARGANBTNAA-3' and
r-LANT-5'-GCACAGGACGCCCATCADCCARWARAA-3'.
Reactions were carried out with the Gene Amp PCR system 9700 (Applied Biosystems, Branchburg, NJ) using 0.025 U µl–1 Taq-polymerase (Amersham Biosciences, Piscataway, NJ) under the following conditions: 5 min at 94 °C followed by 40 cycles of 30 s at 94 °C, 1 min at 58 °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, Italia) 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 (Applied Biosystem, Branchburg, NJ). Sequence comparisons were performed by using BlastX and BlastN computer programs (NCBI, National Center for Biotechnology Information).
The ZmNrt1.1 5'-end clone was isolated by the 5'-RACE technique as described by Schaefer (1995). The primer used for the 5'-RACE was 5'-GACCGTGTTGAGGTACGACCC-3'. The obtained amplification products were subcloned and ten recombinants were sequenced.
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, Italia), using 200 U of MMLV Reverse Transcriptase (Promega, Milano, Italia) and oligo dT as a primer, in 20 µl reactions, as described in Sambrook et al. (1989).
ZmNrt2.1, ZmNrt1.1, Mha1, and Mha2 expression analysis
RT-PCR experiments with specific primers were performed to evaluate the expression level of ZmNRT2.1, ZmNrt1.1, Mha1, and Mha2 genes in roots and leaves of seedlings treated and untreated with LMS for 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). For each of these reactions a set of a different number of cycles ranging between 10 and 25 was tested to choose those corresponding to the exponential phase for each gene. Each cycle was composed by a 30 s denaturation at 94 °C, 1 min of anealing at 65 °C and a 30 s extension at 72 °C; a 5 min denaturation period at 94 °C at the beginning of the reactions and a 7 min extension at 72 °C at the end were performed for all reactions. Specific primers for ZmNrt2.1, Mha1, and Mha2 were designed on the basis of sequences corresponding to those genes available in the databases and in the case of ZmNrt1.1 of sequence of partial cDNA isolated as described above (Table 1).
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A maize Ubiquitin (U29162 [GenBank] ) and a maize Actin (J0128) were used as constitutive internal standards and the primers used were: 5'-CCACTTGGTGCTGCGTCTTAG-3' (forward primer); 5'-CCTTC TGAATGTTGTAATCCGCA-3' (reverse primer) for Ubiquitin and 5'-TGTTTCGCCTGAAGATCACCCTGTG-3' (forward); 5'-TGA ACCTTTCTGACCCAATGGTGATGA-3' (reverse) for Actin.
PCR products obtained from expression analyses were sequenced to confirm the specificity of amplification of each gene. To confirm the 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). 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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Results |
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Characterization of LMS fraction
DRIFT spectroscopy: The interpretation of the DRIFT spectrum (Fig. 1) was according to spectra published by Baes and Bloom (1989), Niemeyer et al. (1992), Stevenson (1994), Johnston et al. (1994), and Francioso et al. (2001, 2002). The spectrum was dominated by a broad band at around 3200 cm–1 attributed to
(O-H) vibration of carboxylic and alcoholic groups in different electrostatic environments (Bellamy, 1975); the band at around 2960 cm–1 was assigned to asymmetric (C-H) motions of aliphatic groups. The strong and sharp band appearing at 1719 cm–1 is characteristic of undissociated carboxyl groups (C=O) vibrations (Rao, 1963; Niemeyer et al., 1992; Francioso et al., 1998, 2002). In addition, the appearance of a broad band at 2626 cm–1 was attributed to (O-H) vibration of carboxyl groups (Rao, 1963). This band is undoubtedly due to the formation of intermolecular hydrogen bonding between OH groups in oxygenated compounds (Rao, 1963). The bands at 1612 and 1514 cm–1 probably correspond to (C=O) vibration in amide I and both 
(N-H) and (C-N) vibrations in amide II. Moreover, these bands are due to C=C and C-C vibrations in aromatic rings, respectively. The band at 1413 cm–1 corresponds to (CH2) and s(COO–) symmetric stretching motions (Bellamy, 1975; Niemeyer et al., 1992; Stevenson, 1994). The band appearing at 1220 cm–1 can be attributed to (C-O) vibrations of alcohol, phenol, and carboxyl groups. The band appearing at 1083 cm–1 is attributed to C-O stretching of carbohydrates and alcohols (Bellamy, 1975; Stevenson, 1994), as well as to C-C stretching motions of aliphatic groups, and in-plane C-H bending of aromatic rings.
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1H NMR spectroscopy: The aromatic proton region, poorly resolved in the spectrum (Fig. 2), suggested that the LMS fraction was at an early stage of humification. This observation was confirmed by the intense and broad region (3.0–5.0 ppm) attributed to sugar-like components, polyether materials or methoxy groups (Wilson et al., 1983; Wershaw, 1985) lignin derivatives, which can survive the early stages of humification. Inspecting the aliphatic region revealed a finer proton structure with the appearance of a very well resolved signal which may correspond to low molecular weight organic acids (aliphatic) and other components such as ethers (Francioso et al., 2000). An intense doublet at 1.43 ppm is identified as the lactate groups (Wilson et al., 1988; Fan, 1996; Francioso et al., 2000). Moreover, other broad and intense resonances at 0.83 ppm and 1.3 ppm are due to the protons of terminal methyl groups of methylene chains, respectively (Malcolm, 1990). In particular, the more intense resonances appeared at 1.9 ppm (acetate), 2.70 ppm (tentatively acetoacetate), and 3.70 ppm (indolacetic) (Aldrich Library, 1993; Fan, 1996).
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Furthermore, the presence of low molecular weight organic acids was also corroborated by the bands at 2626, 1719, and 1220 cm–1 in the DRIFT spectrum (Fig. 1) which were attributed to vibrations of COOH groups in acid dimers.
13C NMR spectroscopy: The NMR spectrum (figure not showed) showed an intense sharp singlet around C –20, characteristic of CH3 groups in alkyl chains. In particular, the peaks appearing at 20.76 (ßCH3) and 183.40 (COO–) ppm confirmed the presence of lactate as revealed by the 1H NMR spectrum. According to Canellas et al. (2002) the broad signal observed in the region between C –44 to –57 suggested the low level of humification of the LMS fraction isolated from the earthworm faeces, because of an increase in C bonded to mono and di-O.
The peak at C –60 is assigned to OCH3-groups. The region between C –60 to –70 can be assigned to C of C-O in primary alcohols and polysaccharides. In particular, the shoulder around C –70 indicated the presence of C atoms bound to N in amino acids (Canellas et al., 2002). The low signal of aromatic C suggested the early stage in formation of this humic like fraction. The peaks between C –160 to –190 indicated the presence of differently substituted carbonyl C atoms.
Quantitatively, the spectra revealed 22.5% carboxyl, 4.4% phenolic, 13.8% aromatic, 32.7% peptide and carbohydrate, and 26.6% other aliphatic carbons (Table 2).
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IAA content: The IAA content in the LMS fraction determined by an immunoassay method revealed an IAA concentration of 37 nmol mg–1 C, corresponding to a final concentration of 27.75 nM in the 0.75 mg C l–1 solution (Table 2).
NO32– influx and tissue content
Nitrate influx experiments were carried out with the aim of evaluating the activity of both constitutive and inducible high affinity nitrate uptake systems and low affinity uptake systems globally active at 0.5 mM NO32–. Maize seedlings pretreated with LMS for 48 h and then transferred to a solution containing 1.5 mM of nitrate showed an influx value 70% higher than control plants (Fig. 3).
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As far as the tissues nitrate content was concerned, roots of treated and untreated plants didn’t show any significant difference, whereas the nitrate content of leaves showed a value 50% higher in seedlings treated with LMS than in leaves of control plants (Fig. 3).
Cloning of ZmNrt1.1 and analysis of expression of ZmNRT1.1 and ZmNRT2.1 in roots and shoots
To investigate the role of transcriptional regulation of maize nitrate transporters on the induction of the nitrate uptake observed after the treatment with LMS, the expression of two genes encoding putative maize nitrate transporters was evaluated in both treated and untreated seedlings. To this end, a partial cDNA clone of 1570 bp (accession number AY187878
[GenBank]
), termed ZmNRT1.1, encoding a putative low-affinity nitrate transporter, was isolated by use of degenerate PCR and 5'-RACE. Its expression was evaluated together with that of a previously characterized putative high-affinity nitrate transporter termed ZmNRT2.1 (accession number AY129953
[GenBank]
, Quaggiotti et al., 2003). The deduced amino acid sequence of ZmNRT1.1 showed a conserved domain of about 400 amino acids typical of the PTR family of low-affinity nitrate transporters and a 65% sequence similarity with a rice nitrate transporter.
The analyses of the expression of ZmNrt2.1, a putative high-affinity nitrate transporter, in roots of maize seedlings that had been grown for 48 h in the presence of LMS, did not show any induction in terms of accumulation of transcripts corresponding to this gene, showing the presence of a similar constitutive signal in both control and LMS treated seedlings (Fig. 4).
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As far as the shoots are concerned (Fig. 4), ZmNrt2.1 transcripts could not be detected in control seedlings while, on the contrary, in seedlings treated with LMS humic substances for 48 h, the presence of a clear signal was detected. This indicates an induction of ZmNrt2.1 transcript accumulation by LMS treatment in the shoot.
The expression analysis of ZmNrt1.1, encoding a putative low-affinity nitrate transporter in roots and shoots (Fig. 4) of maize seedlings, showed the presence of a constitutive signal in both control and LMS-treated seedlings, with no significant differences between the organs and treatments considered.
Expression of Mha1 and Mha2
Because of the role played by H+-ATPases in nitrate uptake (Thibaud and Grignon, 1981; Ruiz-Cristin and Briskin, 1991; Meharg and Blatt, 1995; Miller and Smith, 1996; Hirsch and Sussman, 1999), the expression of two previously isolated genes, encoding two maize H+-ATPases, named Mha1 (Jin and Bennetzen, 1994, accession number U09989
[GenBank]
) and Mha2 (Frias et al., 1996; accession number X85805
[GenBank]
), was studied in control and in LMS-treated seedlings.
RT-PCR analysis of Mha1 transcript accumulation in roots showed the presence of a constitutive signal in both treated and untreated plants, with no significant changes in terms of transcript accumulation for up to 48 h of treatment with low molecular weight humic substances (Fig. 5). In shoots of untreated seedlings, Mha1 transcripts were found to accumulate to a higher extent than in roots while, in contrast, they remained almost undetectable in the shoots of LMS treated seedlings (Fig. 5), thus suggesting a down-regulation of the Mha1 expression in maize shoot tissues by treatment with LMS.
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As far as Mha2 transcript accumulation was concerned, a weak signal was detected in roots of untreated seedlings while a significant increase (8 times) was found in roots of seedlings that were grown in the presence of LMS humic substances for 48 h (Fig. 5). These results suggest an up-regulation of the transcription of the Mha2 gene in response to the supply of humic acids. By contrast with the roots, a very weak presence of Mha2 transcripts was detected in the shoots, with no significant differences between treated and untreated seedlings (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, the investigation of some physiological and molecular aspects of the effect of humic substances on nitrate uptake in maize seedlings is reported through the evaluation of nitrate influx in roots, together with the nitrate tissue content and the analysis of the expression of specific genes putatively involved in the uptake process. Maize was used because of its worldwide economic relevance (Mann, 1999) and because of its importance as one of the best model plants to combine physiological and agronomic studies (Hirel et al., 2001).
Previous reports from the authors and other groups have shown the stimulatory effects of humic substances on physiological processes related to growth and productivity in maize, such as nutrient uptake and reduction ability (Vaughan et al., 1985; Chen and Aviad, 1990; Nardi et al., 2000; Varanini and Pinton, 2001; Clapp et al., 2001). Despite the considerable amount of physiological and biochemical data, no information is available at the molecular level on the effects of HS on the expression of specific genes, even though an action on the synthesis of proteins by LMS has been reported (Vaughan and MacDonald, 1971; Dell’Agnola et al., 1981; Nardi et al., 2000). In addition, progress in research on HS has been considerably hampered by the lack of characterization of the humic fraction being used. To this end a detailed characterization of earthworm HS by DRIFT (Diffuse Reflectance Infrared Fourier Transforms), 1H- and 13C-NMR spectroscopy has been undertaken, and these substances were used to study their effect on nitrate uptake and on the expression of maize genes putatively involved in nitrate uptake.
The LMS humic fraction used in this research showed, in comparison with typical values reported for an average soil HA (Schnitzer, 1991), a high content of carboxyl, peptide, and carbohydrate C and a low content of aliphatic and phenolic C. These findings point out that the LMS humic fraction used in this study was at an early stage of humification, as that described by Canellas et al. (2002), with which it shares common features. On the other hand, these data also show that this fraction is endowed with the characteristic structural network described for most humic substances isolated from different sources of organic matter (Clapp and Hayes, 1999) and, together with the data reported by Canellas et al. (2002), confirm that common pathways lead to the formation in different environments of LMS fractions with similar characteristics. In addition, this study’s results confirmed the presence of IAA in the LMS fraction, as already found by Muscolo et al. (1998) and by Nardi et al. (2002), through an immunological approach, and by Canellas et al. (2002), by means of gas chromatography-mass spectrometry.
To study the specific effects of these substances on nitrate uptake, nitrate influx in roots and nitrate tissue content were measured and the characterization of the accumulation of transcripts of some genes implicated in nitrate transport was also performed to evaluate the involvement of their transcriptional regulation in determining the maize response to the treatment with LMS.
In these experiments both control and LMS-treated seedlings were transferred for a 5 h period in a solution containing 1.5 mM nitrate to measure the induced nitrate influx. The results showed a nitrate influx 70% higher in LMS-treated seedlings than in the control, confirming the results of other groups.
Tissue nitrate content only showed significant differences in leaves, with a higher amount of nitrate in LMS-treated plants than in control seedlings, suggesting that LMS may stimulate nitrate accumulation in leaves, possibly as a consequence of a more efficient NO32– uptake and translocation to shoots.
In roots, no increase in terms of transcript accumulation of a gene encoding a novel putative maize low-affinity nitrate transporter, termed ZmNrt1.1, and of a previously characterized putative high-affinity nitrate transporter, ZmNrt2.1 (Quaggiotti et al., 2003), was found in response to treatment with LMS for 48 h. Even though the involvement of other nitrate transporters in the root response to humic acids cannot be ruled out, the absence of stimulation of the transcription of these two genes, and especially of that of ZmNRT2.1, which was shown to be transcriptionally up-regulated in root tissues by nitrate availability with a high degree of correlation with the induction of the iHATS system for nitrate uptake (Quaggiotti et al., 2003), suggest that the increase of nitrate uptake measured after 48 h of permanence of maize seedlings in a solution containing LMS may involve post-transcriptional/post-translational mechanisms of regulation of ZmNrt2.1 and/or an indirect modulation of the uptake process. This latter effect may rely on the action of H+-ATPases responsible for generating the proton electrochemical difference across the plasma membrane essential for nutrient uptake (Serrano, 1989), also considering that nitrate uptake by plants is thought to be a secondary transport driven by the proton electrochemical difference generated by the proton pump (Thibaud and Grignon, 1981; Ruiz-Cristin and Briskin, 1991; Meharg and Blatt, 1995; Miller and Smith, 1996; Wang and Crawford, 1996). These results, showing no changes in transcript accumulation of the Mha1 gene (Jin and Bennetzen, 1994) in roots of seedlings grown in the presence of LMS, suggest that the transcription of this gene is not involved in the regulation of nitrate uptake in response to this treatment. The analysis of the expression of Mha2, a major maize isoform of H+-ATPase preferentially expressed in guard cells, phloem, and root epidermal cells (Frias et al., 1996), showed a strong induction in terms of accumulation of its transcripts in roots of maize seedlings grown in the presence of LMS for 48 h. This is in agreement with data recently reported by Canellas et al. (2002), showing, in roots of maize plants exposed to earthworm compost for 7 d, an increase of PM H+-ATPase content measured by western blot analysis using antibodies raised against the PMA2 isoform from Nicotiana plumbaginifolia. The authors hypothesized that this could be due to an increase of the Mha2 isoform through an effect on Mha2 transcription and, considering that Frias et al. (1996) demonstrated as the most significant regulatory feature of the Mha2 gene a 3-fold increase in its steady-state mRNA level in response to auxin, concluded that the action of the LMS on PM H+-ATPase may rely on the auxin-dependent activation of the Mha2 gene. The present results, showing a stimulation of Mha2 mRNA synthesis by LMS exclusively at the root level already after 48 h of treatment, may further support this hypothesis and suggest a putative role for the IAA present in LMS in the mechanism of regulation of the plant’s response to humic substances through an effect on the expression of specific genes. In addition, these data taken together with those on ZmNrt1.1 and ZmNrt2.1 expression, point out that the increase of nitrate uptake measured in maize after treatment with LMS more likely relies on the stimulation of Mha2 gene expression and, as a consequence, of the efficiency of the nitrate symport system through the generation of a more favourable proton electrochemical difference rather than on the up-regulation of genes encoding specific transporters.
As far as the shoots are concerned, even though the effects of HS on shoot physiology are mediated by root–shoot signalling events yet to be characterized, the induction of the synthesis of transcripts of ZmNrt2.1, observed in seedlings exposed to LMS, together with the absence of significant changes in ZmNrt1.1 and Mha2 transcription and a remarkable down-regulation of Mha1 expression, point out that LMS treatment also affects gene expression in shoots with striking differences compared with roots. ZmNrt2.1 transcription was also shown to be up-regulated in maize leaves, as well as in root tissues, in response to nitrate availability, and therefore also claimed to be probably involved in nitrate translocation and compartmentation at the whole plant level (Quaggiotti et al., 2003). For this reason, it is possible to speculate that the LMS stimulation of the ZmNrt2.1 transcript accumulation in shoots, that is consistent with a higher nitrate content in leaves of LMS-treated plants, may implicate a more efficient translocation of nitrate to its metabolic sinks and reflect a better nitrogen use efficiency, also believed to be connected to a more efficient nitrogen remobilization (Hirel et al., 2001). Since the specific role of Mha1 H+-ATPase still remains partially unknown, the exact physiological meaning of these results showing the marked down-regulation of its gene expression will require future studies aimed at the characterization of the role of additional genes encoding H+-ATPase isoforms and a better understanding of the factors involved in the root–shoot signalling events evoked in response to LMS treatment.
In conclusion, these data taken together suggest that the LMS fraction of humic substances stimulates nitrate uptake in maize, possibly through the up-regulation of mRNA synthesis of the major maize H+-ATPase form Mha2. The results also suggest that the LMS fraction, besides exerting direct effects on gene transcription in roots, may also induce long-distance effects on shoot gene expression as observed for the ZmNrt2.1 and Mha1 genes. Further studies should be aimed at elucidating the features of auxin contained in LMS and its contribution in regulating these processes and the global plant responses to humic substances.
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Acknowledgements |
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We thank Professor Angelo Vianello, University of Udine, and Dr David Carden, University of Padua, for critical reading of the manuscript. Funds were provided by the MURST Giovani Ricercatori Program 2000.
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