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Dipartimento di Gestione dei Sistemi Agrari, e Forestali, Università degli Studi Mediterranea, Feo di Vito, 89060 Reggio Calabria, Italy
Dipartimento di Scienze e Tecnologie, Agroambientali, Università di Bologna, Via Fanin 40, 40127 Bologna, Italy
Dipartimento di Biochimica, Università di Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy
Dipartimento di Biotecnologie Agrarie, Facoltà di Agraria, Agripolis, Strada Romea 16–35020, Legnaro, Padova, Italy
* Corresponding author (amuscolo{at}unirc.it).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60%), are a key component of the terrestrial ecosystem, being responsible for many complex chemical reactions in soil (Stevenson, 1994). Humic substances are known to increase soil aggregation (Bremner, 1954; Whitehead, 1963), compactability (Soane, 1990), water-holding capacity (De Jong et al., 1983) and the cation exchange capacity (Stevenson, 1994). Directly, HS promote plant growth by acting on membrane permeability as protein carriers of ions, activating respiration, the Krebs cycle, photosynthesis, and the production of adenosine triphosphate and amino acids (Malcolm and Vaughan, 1978, 1979; Vaughan and Malcolm, 1985). Humic matter has a very complex biological activity, depending on its origin, molecular size, chemical characteristics, and concentration. It exhibits a range of different effects on plant metabolism in the diverse systems that have been tested (Maggioni et al., 1987; Albuzio et al., 1993; Nardi et al., 2002). During the last 10 yr, we have investigated the biological activity of HS derived from different soils (Dell'Agnola and Nardi, 1987; Muscolo et al., 1993; Nardi et al., 1994, 1998, 1999; Concheri et al., 1996; Muscolo et al., 1996, 1998, 1999a, 1999b, 2001a, 2001b; Muscolo and Nardi, 1997; Muscolo and Sidari, 1998; Panuccio et al., 2001), showing that HS induce morphogenetic and biological changes in leaf explants of Nicotiana plumbaginifolia, affecting the patterns of peroxidase and esterase, enzymes that are involved in organogenesis and may be indicators of somatic embryogenesis. These effects, peculiar to the humic fraction with a low relative molecular mass (<3500 Da), were similar to those produced by indole-3-acetic acid (IAA); the humic fraction with a high relative molecular mass (>3500 Da) had no effect on this vegetable system. A study on homogeneous carrot (Daucus carota L.) cell cultures compared the effects of the low-relative-molecular-mass humic fraction to different auxins. This humic fraction caused an increase in carrot cell growth similar to that induced by 2,4-dichlorophenoxyacetic acid (2,4-D) and promoted morphological changes similar to those induced by IAA. It is clear from this that low-molecular-weight components of HS are biologically active (Vaughan, 1967; Mato et al., 1972; Vaughan et al., 1974; Piccolo et al., 1992; Muscolo et al., 1998; Nardi et al., 2000), even if, in some experimental models, high-molecular-weight components appeared to be similarly active (Ladd and Butler, 1971; Malcolm and Vaughan, 1979).
The mechanism by which soil HS stimulate plant biological activity is not well clarified; this is in part due to their heterogeneity and to the difficulty of their characterization. Thus attempts to relate humus structure to biological activity have produced contrasting results. Although the molecular structure has not yet been extensively defined, according to the new view, HS are collections of diverse, relatively low molecular mass components forming dynamic associations stabilized by hydrophobic interactions and H bonds (Piccolo et al., 1996; Conte et al., 2003; Spaccini et al., 2006). These associations are capable of organizing, in suitable aqueous environments, into supramolecular structures stabilized by dispersive interactions and H bonding. Functional carboxylic and phenolic C groups in HS seem to have an important role in determining their biological activity (Mato et al., 1972; Malcolm and Vaughan, 1978; Pflug and Ziechmann, 1981), but the manner in which they act has still to be elucidated (Vaughan and Malcolm, 1985). Although it is very difficult to define a clear concept of their composition (Hayes, 1997), considerable progress has been made in the last few years in providing an awareness of some of the gross features of HS by using various spectroscopic procedures: infrared spectroscopy, electron spin resonance, Raman, ultraviolet-visible, fluorescence, and x-ray photoelectron spectroscopy. In recent years, considerable effort has been focused on 13C-NMR and 1H-NMR spectroscopy, considered the most powerful tools available to determine the structure of both organic and inorganic species.
To clarify if the biological activity of HS may be related to their chemical composition or molecular weight, 1H-NMR and DRIFT spectroscopy were used to obtain detailed chemical information that could be of use to elucidate the mechanism of action of HS. For this purpose, callus, a coherent and amorphous tissue, was used to study in vitro the effects of HS without environmental interference.
The use of tissue culture provides a rapid and inexpensive method to screen compounds and has been found to be representative of results obtained in whole-plant experiments studying different aspects (Souissi and Kremer, 1998; Ehsanpour and Fatahian, 2003; Vidal et al., 2004).
We compared the biological effects of two HS derived from an uncultivated couch grass field and a forest soil, both separated into high (>3500 Da) and low (<3500 Da) relative molecular mass fractions, on Pinus nigra callus growth. Biochemical parameters, such as sucrose, glucose, and fructose content, free amino acid pool, and the activity of the key enzymes of C and N metabolism were investigated.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Humic Substances Extraction
Humic substances were extracted from the air-dried samples with 0.1 M KOH (1:20 w/v) at room temperature for 16 h under an N2 atmosphere and were freed from the suspended material by centrifugation at 7000 x g for 20 min. Humic extracts were transferred into 18000 molecular weight cutoff dialysis tubings Visking (Medicell, London) and dialyzed against double-distilled water. The retained solution was desalted by ion exchange on Amberlite IR 120 (H+ form, Aldrich Chemical Co., Milwaukee, WI) and this fraction was treated with glacial acetic acid until pH 2.1 was attained. The acidified solution was dialyzed through 3500 molecular weight cutoff Spectra/Por 3 tubing (Spectrum, Gardena, CA) against distilled water. This procedure separated the total humic extract into high (>3500 Da) and low (<3500 Da) relative molecular mass (Mr) fractions. The high-molecular-weight humic fractions and low-molecular-weight humic fractions were collected inside and outside the dialysis tubing, respectively. These solutions were freed from unreacted acetic acid and reduced in volume to about 50 mL by vacuum distillation (Nardi et al., 1991).
Fourier-Transform Infrared Spectroscopy
For each analysis, 2 mg of dried sample was mixed with 148 mg of KBr (Fourier-transform infrared grade, Aldrich Chemical Co.), so that the mixture became homogeneous. After grinding, the sample mixture was heaped 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 Fourier-transform infrared spectrophotometer (Nicolet Instruments, Madison, WI) and fitted with an apparatus for diffuse reflectance (Spectra-Tech, Stamford, CT). Spectra were recorded with 200 scans collected at 4 cm–1 resolution. Spectra were collected and manipulated by using the Omnic (3.1) software supplied by Nicolet Instruments.
Hydrogen-1 Nuclear Magnetic Resonance Spectroscopy
Humic fractions (20 mg) were dissolved in 0.5 mL D2O (deuterium oxide). The spectra were recorded with a Bruker ACF 250 spectrometer (Bruker Optics, Bellerica, MA) using a 5-mm multinuclear probe. The 1H spectra were accumulated with 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 to 2000 scans were needed. Gated irradiation was applied between acquisitions to presaturate the residual water peak. A 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt, was added to the samples to provide a chemical shift standard (Francioso et al., 2000). The spectra were divided into three main regions: aromatic H from 6.0 to 8.0 ppm; H attached to O groups in C 
from 4.2 to 3.0 ppm (also defined as sugarlike), and aliphatic H from 3.0 to 0.5 ppm. In accordance with results obtained by Wilson et al. (1983), the sugarlike region was attributed to protons largely arising from polysaccharides. Wilson et al. (1983) and Simpson et al. (1997) showed that the aliphatic region might be affected by the presence of protons attached to aromatic rings in , ß, and 
positions.
Callus Growth
Excised leaves of P. nigra were grown in petri dishes, on MS medium (Murashige and Skoog, 1962) used as basal medium, supplemented with 3% sucrose, 0.5 mg L–1 2,4-D, and 0.25 mg L–1 6-benzylaminopurine (BAP) for 35 d to induce callusing. The pH was adjusted to 5.7 before adding 0.8% w/v Bacto agar (Oxoid 1), and the media were autoclaved for 20 min at 121°C. After 35 d, callus tissue was transferred for 28 d to the basal medium culture free (control) or with two humic fractions added that were derived from uncultivated couch grass—LMWG with low Mr (<3500 Da) or HMWG with high Mr (>3500 Da), or two forest humic fractions—LMWF with Mr < 3500 Da or HMWF with Mr > 3500 Da, or BAP (0.25 mg L–1), or IAA (2.0 mg L–1), or 2,4-D (0.5 mg L–1), or 2,4-D + BAP (0.5+0.25 mg L–1). Both filter-sterilized forest and grass humic fractions were used at a concentration of 1 mg C L–1, according to previous tests showing that, in the range of 0.1 to 5.0 mg C L–1, 1.0 mg C L–1 was the concentration of humic matter that most interfered with P. nigra callus (Muscolo et al. 2005). In addition, 1 mg C L–1 is the concentration of HS commonly present in forest soils (Cheng et al., 1996). All treatments were performed at 25°C in darkness. Samples of callus were taken at the end of the period (28 d) for all treatments for enzymatic analysis. The data are the means of five replicates.
Growth Parameters
Twenty-eight days after the beginning of the treatment, the callus growth was estimated as fresh weight. After weighing, the mean relative growth rate (RGR) was calculated as: RGR = ln (FW2 – FW1)/t2 – t1, where FW1 is the fresh weight at the beginning of the measurements, FW2 is the fresh weight at the end of the experiments (28 d), t1 is time at the beginning of the experiments, and t2 is time at the end of the experiments (Hunt, 1990).
Soluble Sugars Determination
Callus tissue was extracted three times with boiling 80% ethanol (v/v). Homogenates were centrifuged at 12000 x g and the ethanolic extract was evaporated under vacuum (Rotavapor RE 111, Büchi, Switzerland), resolubilized in distilled water, and subjected to enzymatic assay. Glucose, fructose, and sucrose were determined by using hexokinase, glucose-6-phosphate dehydrogenase, and phosphoglucose isomerase, respectively, and after enzymatic inversion to D-glucose and D-fructose by the enzyme ß-fructosidase (Boehring test combination 716260; Bergmeyer and Bernt, 1974). Absorbance was detected at 340 nm (UV-2100 spectrophotometer, Shimadzu, Japan).
Measurement of Free Amino Acids
The total free amino acid pool was determined by ninhydrin assay (Yemm and Cocking, 1955). Absorbance readings were converted to grams amino acid per kilogram fresh weight using a glycine standard curve.
Enzyme Extraction and Assay Conditions
Callus tissue was extracted according to the method of Zhifang et al. (1999), modified as follows: samples of callus tissues (1 g fresh weight) were homogenized in a chilled mortar with three volumes of chilled extraction buffer containing 100 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, and 1 mM dithiothreitol. The extract was filtered through two layers of muslin and clarified by centrifugation at 20000 x g for 15 min. The supernatant was used for enzymatic analysis. All steps were performed at 4°C.
Soluble acid invertase (SAI, ß-fructofuranosidase, ß-fructofuranoside fructohydrolase, EC 3.2.1.26) activity was assayed at 37°C by adding 50 µL of extract to 50 µL of 1 M NaOAc (pH 4.5). The enzyme reaction was started by the addition of 100 µL of a 120 mM sucrose solution. The reaction was stopped at 30 min by adding 30 µL of 2.5 M 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS, Trizma base) and boiling the mixture for 3 min. The concentration of glucose liberated was determined with a glucose test kit from Sigma-Aldrich (St. Louis, MO; Zhu et al., 1997).
Glucokinase (GK: D-glucose-6-phosphotransferase, EC 2.7.1.1) activity was measured by coupling hexose phosphate production with nicotinamide adenine dinucleotide (NAD+) reduction by glucose-6-phosphate dehydrogenase and monitoring at absorbance A340 (Huber and Akazawa, 1986).
Phosphoglucoisomerase (PGI, D-glucose-6-phosphate ketoisomerase, EC 5.3.1.9) assay contained 50 mM Hepes–NaOH (pH 7.2), 5 mM MgCl2, 5 mM fructose-6-phosphate, 1 mM NAD+, and 1 IU mL–1 glucose-6-phosphate dehydrogenase. The activity was measured spectrophotometrically at A340 (Tsai et al., 1970).
Aldolase (ALD, EC 4.1.2.13) was assayed spectrophotometrically at A340. The assay contained 50 mM Hepes–NaOH (pH 7.2), 5 mM MgCl2, 1 mM fructose-1,6-bis phosphate, 0.2 mM NADH, 1 IU mL–1 glycerol-3-phospho-dehydrogenase, 1 IU mL–1 triose phosphate isomerase, and 50 µL extract (Doehlert et al., 1988).
Pyruvate kinase (PK, EC 2.7.1.40) was assayed by adding to 450 µL 0.1 M triethanolamine (TEA), adjusted with 0.1 M NaOH to pH 7.75, 50 µL of 3 mM ß-NADH-Na2 in 0.1 M TEA (pH 7.75), 50 µL of 52 mM adenosine 5'-diphosphate-Na2 in 0.1 M TEA (pH 7.75), 50 µL of 0.15 M MgSO4·6H2O, 50 µL of 0.15 M KCl in 0.1 M TEA (pH 7.75), 50 µL of L-lactic dehydrogenase, and 50 µL of extract. The reaction was started after a lag time of 10 min at 30°C by adding 50 µL of 0.225 M 2-phosphoenolpyruvate–Na–H2O in 0.1 M TEA (pH 7.75) (Bergmeyer et al., 1986).
Glutamate synthase (GOGAT, EC 1.4.7.1) assay contained 25 mM HEPES-NaOH (pH 7.5), 2 mM L- glutamine, 1 mM -ketoglutaric acid, 0.1 mM NADH, 1 mM Na 2 EDTA, and 100 µL of enzyme extract. GOGAT was assayed spectrophotometrically by monitoring NADH oxidation at A340 (Avila et al., 1987).
To assay glutamine synthetase (GS, EC 6.3.1.2), the mixture for the transferase assay contained 90 mM imidazole-HCl (pH 7.0), 60 mM hydroxylamine (neutralized), 20 mM Na2HAsO4, 3 mM MnCl2, 0.4 mM adenosine diphosphate (ADP), 120 mM glutamine, and the appropriate amount of enzyme extract. The assay was performed in a final volume of 750 µL. The enzymatic reaction was developed for 15 min at 37°C. The -glutamyl hydroxamate was colorimetrically determined by addition of 250 µL of a mixture (1:1:1) of 10% (w/v) FeCl3·6H2O in 0.2 M HCl, 24% (w/v) trichloroacetic acid, and 50% (w/v) HCl. The optical density was recorded at A540 (Canovas et al., 1991).
Malate dehydrogenase (MDH, EC 1.1.1.37) was assayed at 25°C. The assay contained, in 3.17 mL, 94.6 mM phosphate buffer (pH 6.7), 0.2 mM NADH, 0.5 mM oxalacetic acid, and 1.67 mM MgCl2. The MDH was assayed spectrophotometrically by monitoring NADH oxidation at A340 (Bergmeyer et al., 1986).
The activity of NAD(H) glutamate dehydrogenase (GDH, EC 1.4.1.3) was measured at 340 nm at 30°C. The assay contained 150 mM tris-HCl at pH 8.0, 100 mM NH4Cl, 10 mM -ketoglutaric acid–KOH at pH 7.4, 0.3 mM NADH, and 100 µL of enzyme extract in a final volume adjusted to 1 mL with H2O (Refouvelet and Daguin, 1999).
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) enzymatic activity was spectrophotometrically measured by monitoring NADH oxidation at 340 nm for 5 min at 30°C. The assay medium (1 mL) contained 100 mM tris-HCl pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 0.2 mM NADH, 1.5 IU MDH, and 100 µL of enzyme extract (Pasqualini et al., 2001).
Statistical Analysis
The reported data represent mean values of five replicates. Comparisons between the means were made using the Student–Newman–Keuls test (Sokal and Rohlf, 1969).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-CH3 of acetoacetate and ether aliphatics, respectively (Pouchert and Behnke, 1993; Fan, 1996). On the contrary, the spectrum of LMWG was characterized only by a broad and unresolved region assigned to sugarlike components; however, in the spectrum we can see some resonances due to the presence of low-molecular-weight organic substances such as lactate (1.33 ppm) and acetoacetate (2.9 ppm) and a singlet at 1.47 ppm assigned to a proton in ß-CH3 of alanine. The aromatic region did not show resonances due to aromatic protons.
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-CH2, and at 3.24 ppm due to a proton in N-CH3 of glycinebetaine (Fan, 1996). Two quartets between 2.6 and 2.8 ppm were assigned to different forms of citrate (Fan, 1996). In the LMWF spectrum, the sugarlike region appeared less intense and characterized by different simple sugars. Glycinebetaine was also present, but with low relative intensity with respect to the HMWF fraction. Moreover, in this spectrum there appeared well-resolved signals corresponding to a singlet at 3.55 ppm of glycine, a singlet at 3.44 ppm of trimethyamine-N-oxide (Fan, 1996), a singlet at 2.39 ppm of succinate, a singlet at 2.37 of pyruvate, and finally, a singlet at 2.1 ppm of acetate (Fig. 2). The aliphatic region of HMWF (Fig. 3) was characterized by intense resonance of lactate (1.33 ppm), while the aliphatic protons were not well resolved. The LMWF spectrum (Fig. 3) showed a doublet at 1.47 ppm, assigned to alanine, and an intense resonance around 1.33 ppm, attributed to lactate complexed with cations. In this region, there were no signals due to protons of terminal methyl groups. No signal due to aromatic proton appeared in either spectra.
Fourier-Transform Infrared Spectroscopy
The DRIFT spectra are shown in Fig. 4
. The major spectra bands were assigned as follows: a broad band at around 3200 cm–1 was attributed to O–H stretching of carboxylic and alcoholic groups in different electrostatic environments (Bellamy, 1975); the band at around 2940 cm–1 was assigned to asymmetric C–H stretching of aliphatic groups. The band appearing at 1719 cm–1 is characteristic of undissociated carboxyl groups 
(C=O) vibrations (Rao, 1963; Niemeyer et al., 1992). The bands at 1660 and around 1514 cm–1 probaby correspond to carbonyl C=O stretching and both N–H deformation and C–N stretching vibrations. Moreover, these bands are due to C=C and C–C vibrations in aromatic rings, respectively. The region at around 1400 to 1300 cm–1 correspond to CH2 asymmetric bending and carboxylate symmetric stretching motions (Bellamy, 1975; Niemeyer et al., 1992; Stevenson, 1994); the band appearing at around 1230 cm–1 can be attributed to C–O stretch vibrations in alcohols, phenols, and carboxyl groups. The strong band appearing at around 1043 cm–1 was 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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The spectrum of humic fractions from forest showed different spectroscopic features assigned to different relative intensities due to COOH and COO– asymmetric stretching. The band at about 1400 cm–1 was identified as typical of monocarboxylic acids (Rao, 1963). The bands associated with acidic groups were identified in the 1H NMR spectrum (Fig. 2), such as acetate and citrate, while lactate was present in lower concentration with respect to the humic fractions from grass (LMWG). Other characteristic bands that appeared in the LMWF spectrum are due to the NH3 deformation at about 1540 cm–1 and the strong band at 1046 cm–1 assigned to C–C skeletal and C–N stretch vibrations. The CH stretching frequency at 3084 cm–1 might be linked to methyl groups connected with the N atom. In addition, the appearance of proton chemical shift at 3.24 ppm, corresponding to N–CH3 (Fig. 2), was assigned to glycinebetaine.
Callus Growth
Callus incubated on basal medium without growth regulators or humic fractions (control) had a slow growth after 28 d of subculture, reaching a weight of 2.41 g. A similar behavior was observed when IAA or BAP were added, but 2,4-D or 2,4-D plus BAP significantly increased the average mass of callus (Table 1). Exposure of P. nigra callus for 28 d to HMWF, LMWF, HMWF plus BAP, and LMWF plus BAP caused a significant decrease of callus growth compared with the other treatments (Table 1) and the callus tissue appeared totally brown and dying. A slight increase of callus growth was observed when HMWF plus IAA or LMWF plus IAA were used. The contemporaneous presence of HMWF or LMWF and 2,4-D or 2,4-D plus BAP significantly increased callus growth (Table 1), and produced callus with less visible brown areas than that grown with HMWF or LMWF.
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Carbohydrate Content
After 28 d of subculture, 2,4-D alone or in combination with BAP reduced the amount of hexoses compared with the control callus (Table 2). If treated with either forest humic fraction, a higher content of glucose and fructose in the callus tissue was observed compared with the other treatments. The combination of HMWF or LMWF and 2,4-D or 2,4-D plus BAP strongly decreased the amount of glucose and fructose in callus tissue with respect to that treated with humic fractions alone or in combination with BAP or IAA. In callus tissue grown with grass humic fractions alone or in combination with hormones, glucose and fructose contents were lower than that detected in the callus control or in callus grown with BAP or IAA, and was similar to that found in callus tissue treated with 2,4-D (Table 2).
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Enzyme Activities
In Table 3, the specific SAI activity of the different callus treatments is reported. Both forest humic fractions caused a significant increase of soluble acid invertase compared with control and hormone treatments. The contemporaneous presence of forest humic fractions and 2,4-D or 2,4-D plus BAP decreased the amount of SAI and the values were similar to those detected in callus grown with hormones alone. Instead, in callus tissue treated with forest humic fractions and BAP or IAA, specific SAI activity was similar to that observed in callus grown with HMWF or LMWF alone. In callus treated with grass humic fractions alone or in combination with hormones, the values of SAI were similar to those observed in callus tissue grown with plant growth regulators, except IAA.
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The highest levels of specific PGI activity were observed in callus treated with 2,4-D, BAP, or both, with values of 70.18, 75.85, and 71.85 mkat kg–1 protein, respectively (Table 3). Exposure of callus to HMWF or LMWF fractions caused a strong decrease of specific PGI activity up to values of 1.73 and 4.16 mkat kg–1 protein, respectively. The combination of HMWF or LMWF with 2,4-D or 2,4-D plus BAP increased the levels of this enzyme (Table 3). When the callus was supplemented with HMWF or LMWF plus BAP or IAA, the PGI activity was lower than the control or hormone treatments, but higher than forest HS alone. Exposure of callus tissue to grass humic fractions alone or in combination with hormones increased the PGI activity compared with the control (Table 3).
In callus grown with forest humic fractions, the specific ALD activity was low, with values of about 3 mkat kg–1 protein, showing an inhibition of about 70% with respect to hormones. When the callus tissue was treated with HMWF or LMWF and BAP, the specific ALD activity was low: 4.50 and 4.85 mkat kg–1 protein, respectively. Instead, when the callus was grown with forest humic fractions and 2,4-D or 2,4-D plus BAP or IAA, the ALD activity strongly increased, reaching values similar to those observed in callus grown with hormones (Table 3). In callus grown for 28 d on HMWG or LMWG alone or in combination with hormones, the ALD level increased compared with the control and it was similar to that detected in callus treated with hormones alone (Table 3).
The PK activity was lower in callus treated with HMWF or LMWF alone or in combination with BAP compared with the other treatments (Table 3). The callus grown with grass humic fractions alone did not show significant changes in PK activity with respect to control, 2,4-D, BAP, and IAA, but it was lower with respect to 2,4-D plus BAP (Table 3).
The GS activity was inhibited by the presence in the medium culture of HMWF or LMWF alone or in combination with BAP compared with the other treatments (Table 4). In callus grown in the presence of grass humic fractions alone or in combination with 2,4-D or 2,4-D plus BAP, the GS levels were similar to that detected in callus treated with 2,4-D plus BAP (Table 4).
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The GDH activity was lower in callus treated with forest humic fractions alone or with BAP, while it was increased by HMWF or LMWF and 2,4-D or 2,4-D plus BAP (Table 4). In the presence of grass humic fractions alone or in combination with 2,4-D, the GDH activity was similar to that detected in the control or in callus grown with 2,4-D, but it was lower than that found in callus treated with 2,4-D plus BAP alone or in combination with HMWG or LMWG (Table 4).
Both forest humic fractions alone or with BAP caused a decrease of MDH activity with respect to the other treatments (Table 4). Instead, in the presence of forest humic fractions and 2,4-D or 2,4-D plus BAP, it was possible to observe a strong increase in MDH activity compared with control (Table 4). When the grass humic fractions were added to medium culture, they strongly increased the MDH activity in callus compared with the control, IAA, or BAP, and the values were similar to that detected in callus treated with 2,4-D alone or in combination with grass humic fractions (Table 4).
In the control or in callus grown in the presence of IAA or BAP, the PEPC levels significantly decreased with respect to 2,4-D or 2,4-D plus BAP (Table 4). In callus grown with forest humic fractions alone or in combination with BAP or IAA, it was possible to observe a lack of this activity. On the contrary, in the presence of forest humic fractions and 2,4-D or 2,4-D plus BAP, the PEPC activity increased, reaching values similar to those observed in callus treated with 2,4-D plus BAP (Table 4). When the callus was supplemented with HMWG or LMWG alone or in combination with hormones, the PEPC levels were similar to that determined in callus treated with 2,4-D plus BAP (81.68 mkat kg–1 protein) and this value was higher than that determined in the control or in callus grown with IAA or BAP (Table 4).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One important level of competition between C and N metabolism is related to C chains that accept reduced N. The synthesis of amino acids interacts with the Krebs cycle, which provides a C skeleton for glutamate and aspartate (Alisdair et al., 2004). Different researchers have demonstrated an increase in amino acids with a simultaneous decrease in carbohydrate when NH4+ (Platt et al., 1977) or NO3– (Champigny and Foyer, 1992; Amancio et al., 1993) is supplied.
Thus, the drastic decrease of callus growth in the presence of forest HS is a consequence of the strong inhibition of PGI activity, which consequently causes a lower use of glucose and fructose in callus tissue, damage to C metabolism, and also an inhibition of PEPC, GDH, MDH, and GS, enzymes that provide a link between carbohydrate and amino acid metabolism (Stulen, 1986; Copeland et al., 1989; Robinson et al., 1992; Raab and Terry, 1995). These effects were reversed when the forest fractions were used concomitantly with 2,4-D or 2,4-D plus BAP, they were only partially reversed when used with IAA, but they were not reversed in the presence of BAP alone, suggesting that 2,4-D is the hormone that avoids, in the best way, the negative effect of these fractions on callus tissue. The findings that 2,4-D is better than IAA to reverse the negative effects of humic fractions from forest soil could be explained by the different roles that every auxin has on callus induction for various species (Tao et al., 2002).
An opposite behavior was observed when the callus was grown in the presence of grass humic fractions; in fact, they are capable of improving the growth of callus by increasing the levels of the activity of the key enzymes involved in C and N metabolism.
All the data support the view that the biological activity of HS utilized is independent of their molecular weight, since both fractions (high and low relative molecular mass) obtained from the same HS have a similar behavior on callus tissue.
Humic substances are considered as a sort of memory of microbial population and plant cover, where the active ingredients are not mineral nutrients, but organic acids and biologically active metabolites of various microbes; thus, as reported by Frankenberger and Arshad (1995), HS derived from soils with different plant cover may have a different chemical composition.
The results of 1H-NMR spectra and the DRIFT spectroscopy confirmed this, showing significant differences in the chemical structure between forest and grass HS. A great amount of aliphatic and H-sugarlike component and an intense chemical shift of the ß-CH3 region were observed in both grass humic fractions, while high contents of betaine, organic acids, and COOH groups were detected in both forest humic fractions.
In conclusion, this study shows that the parental organic materials may comprise compounds that serve as precursors or substrates for the synthesis of biologically active substances by the heterotrophic activity of soil biota, and, at the same time, gives evidence that the chemical structure of HS is well reflected in their biological activity.
As HS have very complex structures, it is very difficult to identify the relationship between the single compounds of HS and their biological activity. Therefore more research is necessary to explain the effects of humus, focusing attention on a much more detailed separation and analysis of these fractions, with a better chemical identification, and subsequent testing of pure compounds.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication February 9, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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