**1. Introduction**

230 Chromatography – The Most Versatile Method of Chemical Analysis

193-218.

dinitrophenylhydrazine. *J Org Chem* 18, 818-32.

capillary electrophoresis. *J Chromatogr* 717, 415-25.

[19] Reich H, Grane K F & Sanfilippo S J (1953) The reaction of steroid ketones with 2,4-

[20] Yang Q, Hartmann C, Verbeke J J & Massart D L (1995) Method development and validation for the determination of mineral elements in food and botanical materials by

[21] Hartmann C, Verbeke J J, Massart D L & McDowall R D (1998) Validation of pharmaceutical and biomedical chromatographic methods. *J Pharm and Biomed Anal* 17,

> The combination of chromatographic methods and mass spectrometry (MS) techniques is very useful in analysis of metabolites or components of complex biological samples. Chromatography has become the most important technique for separation and analyses, whereas MS is one of the most effective analytical methods used today for the determination of element concentrations, especially in the trace range; for the structural studies of organic and bioorganic compounds as well as isotopic analysis, due to its very high sensitivity, low detection limits and very small sample volumes needed[1-2]. Using chromatography and MS to separate and measure the concentration of amino acids has been well documented. Bengtsson[3] was the first to introduce a micro-method for the analysis of free amino acids in natural waters by gas chromatography (GC); the technique includes removal of interfering organic substances by chloroform extraction and purification of amino acids by cation exchange. Later, the procedure for analysis of amino acids from protein acid hydrolyzates as their *tert*-butyl dimethylsilyl derivatives by gas chromatography and mass spectrometry has been developed[4-9] and applied in biological matrix analysis[10-13]. Johansen et al.[12] and Rolin et al.[13] studied the nitrogen metabolism of external hyphae of the AM fungus using measurement of *tert*-butyldimethylsilyl(tBDMS)-derivatized amino acids levels by gas chromatography and mass spectrometry. Arbuscular mycorrhizal (AM) fungus is the oldest obligate symbiont,[14] benefiting the host plants by taking up N, P and other macronutrients, trace elements, and water from the soil. Among many components important for plant nutrition, nitrogen is often the most limiting but the AM fungi can improve the nitrogen (N) levels of their hosts.[15-17] The extraradical hyphae of the fungi effectively acquire nitrate (NO3- )[18-20] , ammonium (NH4+)[12,21-23], and amino acids[24-27] from the external medium. However, for a long time, it has been unclear in what form nitrogen is translocated along the

© 2012 HaiRu and Xiangyan, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 HaiRu and Xiangyan, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

hyphae (extraradical mycelium, ERM) of the fungus to the fungal structures within roots (intraradical mycelium, IRM), and how it is transferred across the mycorrhizal interface to the plant. To follow the uptake, assimilation and transfer of nitrogen in the arbuscular mycorrhizal symbiosis, we added isotopically labeled substrates to *in vitro* arbuscular mycorrhizal cultures of carrot (*Daucus carota* L.) roots colonized by *G. intraradices.*[28-30] When grown in divided Petri plates, this model mycorrhiza excludes other microorganisms and prevents diffusion of nonvolatile solutes between the compartments. This model system shows normal life cycle and development of fungal morphology.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 233

amino acids have displayed only negligible 15N labeling[30]. Thus, the Orn cycle seems to be an efficient path for Arg biosynthesis, nitrogen transfer to the host, and carbon recycling in

**Figure 1. A model of N uptake, translocation, degradation and transfer in the AM fungal symbiotic** 

In addition, the results of MS analysis of 15N-labeled amino acids isotopomers have indicated that germinating spores break down stored N (Arg or proteins) during presymbiotic growth.[32] HPLC analysis of amino acids derivatives using PITC[33] revealed that the germinating AM spores combine the C skeletons originating from the degradation of the internally stored lipids with the N released from the stored N compounds (Arg or proteins) for *de novo* biosynthesis of free amino acids, mostly producing serine and glycine. This is consistent with a large flux in the glyoxylate cycle and the utilization of lipids as a C source for carbohydrate and amino acid biosynthesis reported by Lammers at al.[34] Although exogenous N is not required for pre-symbiotic growth, it can be used for *de novo* biosynthesis of amino acids. MS analysis of 15N enrichment of amino acids after 15N labeling

assimilated, and incorporated into arginine (Arg) by the extraradical mycelium (ERM). The accumulated Arg as well as polyphosphate (PolyP) is then bi-directionally translocated along the coenocytic fungal hyphae from the ERM to the intraradical mycelium (IRM) or from the mycorrhizal compartment tissue to the ERM. Arg is catabolized through the catabolic arm of the urea cycle in the IRM, releasing NH3/NH4+ in the arbuscules. The NH4+ ion is deprotonated prior to its transport across the plant membrane by AMT protein and released in its uncharged NH3 form into the plant cytoplasm.

, NH4+, amino acids, peptides, and proteins) are taken up,

AM symbiosis.

**system.** Various forms of N sources (NO3-

Figure modified from Govindarajulul et al. (2005).

Following N uptake, its incorporation into amino acids via the glutamine synthetase/glutamine 2-oxoglutarate amidotransferase (GS/GOGAT) cycle has been observed in AM fungi. Using chromatographic separation and mass spectrometry analysis of amino acids of the hyphae, Johnsen et al.[12] have proved that *G. intraradices* grown in a medium containing 15NH4+ generated abundant free AAs in the ERM. Among these amino acids, 15N- labeled glutamate (Glu), glutamine (Gln), asparagine (Asn), aspartate, and alanine were predominant. Jin et al.[28] have confirmed the mechanism of N transport to the host plant via the AM fungi proposed by Bago et al.[31] (Fig.1). When mycorrhizae of *G. intraradices* and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes, containing 15NH4Cl synthetic medium in the fungal compartment, the measurement of amino acids N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) derivatives with GC-MS revealed that all free amino acids of the ERM were 15N-labeled to a high level. Among these amino acids, arginine (Arg) was the most abundant (over 90% of the total 15N in the free amino acids).This was confirmed by analysis of phenyl isothiocyanate (PITC)-derivatized amino acids with high performance liquid chromatography (HPLC). When the [U-13C] Arg (MTBSTFA derivative with an *m*/*z* of 448) was used as the labeled substrate in the fungal compartment, the isotopomer analysis with GC-MS confirmed that this amino acid was intact both in the mycorrhizal root tissue (*m*/*z* of 448) and in the ERM. This result suggests that Arg is either taken up or synthesized by the ERM and transported intact to the IRM; the conclusion is also consistent with the outcome of other [U-13C/U-15N] Arg labeling experiments.[30] This finding indicates that the AM fungi take up N and incorporate it into Arg, and this organic form of N is then transported to IRM (Fig.1).

When either [U-13C] or [U-15N/U-13C]-labeled Arg was added to the fungal compartment to follow up Arg catabolism in AM fungi (Fig. 1), GC-MS[30] analysis of Arg isotopomer found either [U-13C] or [U-15N/U-13C] ornithine(Orn) in the mycorrhizal root tissues. This result demonstrates that once Arg is translocated to the potential N-limited sites in the mycelium of AM fungi, it is degraded into Orn and urea. Although N released from Arg degradation is transferred to the host plant, it has been shown (using 13C1,2-acetate labeling in fungal compartment and subsequent mass spectrometry analysis of amino acids of mycorrhizal root) that the Arg-originated C is not incorporated into the host C pool and remains in the IRM[29]. Analysis of 15N-labeled Glu and Gln isotopomers using GC-MS has demonstrated that, following Arg degradation, Orn is recycled to Glu and Gln, which serve as C donors. Small amounts of 15N label have been found in Glu and Gln (in spite of their low levels) after [guanido-15N2] Arg translocation from the ERM to the mycorrhizal roots, whereas other amino acids have displayed only negligible 15N labeling[30]. Thus, the Orn cycle seems to be an efficient path for Arg biosynthesis, nitrogen transfer to the host, and carbon recycling in AM symbiosis.

232 Chromatography – The Most Versatile Method of Chemical Analysis

shows normal life cycle and development of fungal morphology.

and this organic form of N is then transported to IRM (Fig.1).

hyphae (extraradical mycelium, ERM) of the fungus to the fungal structures within roots (intraradical mycelium, IRM), and how it is transferred across the mycorrhizal interface to the plant. To follow the uptake, assimilation and transfer of nitrogen in the arbuscular mycorrhizal symbiosis, we added isotopically labeled substrates to *in vitro* arbuscular mycorrhizal cultures of carrot (*Daucus carota* L.) roots colonized by *G. intraradices.*[28-30] When grown in divided Petri plates, this model mycorrhiza excludes other microorganisms and prevents diffusion of nonvolatile solutes between the compartments. This model system

Following N uptake, its incorporation into amino acids via the glutamine synthetase/glutamine 2-oxoglutarate amidotransferase (GS/GOGAT) cycle has been observed in AM fungi. Using chromatographic separation and mass spectrometry analysis of amino acids of the hyphae, Johnsen et al.[12] have proved that *G. intraradices* grown in a medium containing 15NH4+ generated abundant free AAs in the ERM. Among these amino acids, 15N- labeled glutamate (Glu), glutamine (Gln), asparagine (Asn), aspartate, and alanine were predominant. Jin et al.[28] have confirmed the mechanism of N transport to the host plant via the AM fungi proposed by Bago et al.[31] (Fig.1). When mycorrhizae of *G. intraradices* and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes, containing 15NH4Cl synthetic medium in the fungal compartment, the measurement of amino acids N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) derivatives with GC-MS revealed that all free amino acids of the ERM were 15N-labeled to a high level. Among these amino acids, arginine (Arg) was the most abundant (over 90% of the total 15N in the free amino acids).This was confirmed by analysis of phenyl isothiocyanate (PITC)-derivatized amino acids with high performance liquid chromatography (HPLC). When the [U-13C] Arg (MTBSTFA derivative with an *m*/*z* of 448) was used as the labeled substrate in the fungal compartment, the isotopomer analysis with GC-MS confirmed that this amino acid was intact both in the mycorrhizal root tissue (*m*/*z* of 448) and in the ERM. This result suggests that Arg is either taken up or synthesized by the ERM and transported intact to the IRM; the conclusion is also consistent with the outcome of other [U-13C/U-15N] Arg labeling experiments.[30] This finding indicates that the AM fungi take up N and incorporate it into Arg,

When either [U-13C] or [U-15N/U-13C]-labeled Arg was added to the fungal compartment to follow up Arg catabolism in AM fungi (Fig. 1), GC-MS[30] analysis of Arg isotopomer found either [U-13C] or [U-15N/U-13C] ornithine(Orn) in the mycorrhizal root tissues. This result demonstrates that once Arg is translocated to the potential N-limited sites in the mycelium of AM fungi, it is degraded into Orn and urea. Although N released from Arg degradation is transferred to the host plant, it has been shown (using 13C1,2-acetate labeling in fungal compartment and subsequent mass spectrometry analysis of amino acids of mycorrhizal root) that the Arg-originated C is not incorporated into the host C pool and remains in the IRM[29]. Analysis of 15N-labeled Glu and Gln isotopomers using GC-MS has demonstrated that, following Arg degradation, Orn is recycled to Glu and Gln, which serve as C donors. Small amounts of 15N label have been found in Glu and Gln (in spite of their low levels) after [guanido-15N2] Arg translocation from the ERM to the mycorrhizal roots, whereas other

**Figure 1. A model of N uptake, translocation, degradation and transfer in the AM fungal symbiotic system.** Various forms of N sources (NO3- , NH4+, amino acids, peptides, and proteins) are taken up, assimilated, and incorporated into arginine (Arg) by the extraradical mycelium (ERM). The accumulated Arg as well as polyphosphate (PolyP) is then bi-directionally translocated along the coenocytic fungal hyphae from the ERM to the intraradical mycelium (IRM) or from the mycorrhizal compartment tissue to the ERM. Arg is catabolized through the catabolic arm of the urea cycle in the IRM, releasing NH3/NH4+ in the arbuscules. The NH4+ ion is deprotonated prior to its transport across the plant membrane by AMT protein and released in its uncharged NH3 form into the plant cytoplasm. Figure modified from Govindarajulul et al. (2005).

In addition, the results of MS analysis of 15N-labeled amino acids isotopomers have indicated that germinating spores break down stored N (Arg or proteins) during presymbiotic growth.[32] HPLC analysis of amino acids derivatives using PITC[33] revealed that the germinating AM spores combine the C skeletons originating from the degradation of the internally stored lipids with the N released from the stored N compounds (Arg or proteins) for *de novo* biosynthesis of free amino acids, mostly producing serine and glycine. This is consistent with a large flux in the glyoxylate cycle and the utilization of lipids as a C source for carbohydrate and amino acid biosynthesis reported by Lammers at al.[34] Although exogenous N is not required for pre-symbiotic growth, it can be used for *de novo* biosynthesis of amino acids. MS analysis of 15N enrichment of amino acids after 15N labeling experiment showed that NH4+ and urea are assimilated more rapidly than NO3 and exogenous amino acids. *De novo* biosynthesis of free amino acids in the AM spores was increased greatly after the uptake of exogenous NH4+, urea, and NO3- . In cases of a low C:N ratio (no exogenous glucose), the measurements of PITC-derivatized AAs with HPLC showed that Asn was the predominant amino acid in the AM spores. These results suggest that during spore germination, the main carbon source for amino acids biosynthesis is derived mostly from the degradation of stored lipids and the glyoxylate cycle. In contrast, HPLC analysis of PITC-amino acids derivatives has revealed that at a high C:N ratio (available exogenous glucose) Arg is the main amino acid produced and incorporated into the proteins of germinating AM spores[33]. This is consistent with the report of Tisserant et al [35] showing that the transcripts coding for the enzymes of Arg biosynthesis are highly expressed in germinating spores of AM fungus *G. intraradices*.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 235

Methylene chloride and water were added to the extraction solution to facilitate the separation of chloroform and the methanol–water phases. The methanol–water phase containing the amino acids was collected and evaporated in a rotary evaporator at 50°C Bengtsson & Odham[3] have pointed out that losses of amino acids during evaporation prior to derivatization are negligible, and, using a radioactive amino acid tracer, demonstrated low losses of amino acids co-precipitated with carbonates and hydroxides. Losses from nutrient rich samples were further reduced by acidifying the sample before evaporation. During evaporation, Maillard reaction can be avoided by keeping the temperature at 50°C and reducing evaporation pressure. A direct cation exchange has been shown to be inadequate in obtaining a sufficiently pure solution for derivative formation. However, it has been demonstrated that extraction of the aqueous sample with chloroform prior to ion exchange efficiently removes interfering organic substances without detectable losses of

Most amino acids are not soluble in nonpolar solvents and are soluble in water;[36] they display amphoteric properties (caused by COOH and NH2 groups), and many exist as

partly protonated whereas the ionization of the carboxyl group is very low. For the cation exchange, strong acidification is therefore necessary to convert the monoamino-acids completely to the univalent cation form. For example, at pH 2.5, 35% of Phe, 66% of Thr, and 100% of the diamino-acids are in the cationic form. In the micro-method procedure published by Bengtsson[3], the residues containing the amino acids were dissolved in 2 ml of 0.01 M HCl and loaded onto a cation exchange column, previously washed with 2 M NH4OH, deionized H2O and 2 M HCl, and followed by a wash with deionized H2O until the effluent was neutral. The neutral compounds, principally carbohydrates, were washed off the column with 5 ml of water, and the free amino acids (except cysteine(Cys) and methionine(Met), whose recoveries were low), were eluted with 2 ml of 1 M NH4OH. Sulfurcontaining amino acids are partly oxidized during the ion-exchange procedure or derivatization, therefore, this method is not suitable for recovery and purification of Cys and cystine. Nevertheless, Myung et al.[37] have developed a method employing SPME (solidphase micro-extraction) technique and GC–MS to determine homocysteine (Hcy), Cys and Met levels in aqueous samples. This method provides a new approach to the studies of S

**3. Determination of amino acid concentrations with high performance** 

Since amino acids are non-volatile compounds and most of them show low UV absorbance, they have been commonly analyzed by liquid chromatography (LC) methods with precolumn or post-column derivatization using UV chromophore or fluorophore reagents. The use of HPLC analysis is extremely common because this technique has no specific analyte volatility or thermal stability restrictions.[38-39] Derivatization can make the analysis more sensitive, gives a linear detection response and avoids specific interference. The common

. In acidic solutions, the amino groups are at least

amino acids.[12]

zwitterions in the form R-CH(NH3+)-COO-

uptake and transfer in AM symbiosis.

**liquid chromatography (HPLC)** 

To summarize, chromatographic separation and analysis of 15N/13C-labeled amino acids have determined in what form nitrogen is taken up and assimilated, and clarified the mechanisms of Arg transport and degradation in AM fungal symbiosis. In the following sections, we will discuss in some detail the application of chromatographic methods in the studies of nitrogen metabolism and transport in AM fungal system.
