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RESEARCH PAPER |
Effect of the expression of cyanamide hydratase on metabolites in cyanamide-treated soybean plants kept in the light or dark
University of Illinois, Department of Crop Sciences, Edward R. Madigan Laboratory, 1201 W. Gregory, Urbana, IL 61801, USA
* To whom correspondence should be addressed. E-mail: widholm{at}uiuc.edu
Received 25 July 2007; Revised 31 October 2007 Accepted 1 November 2007
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Abstract |
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Metabolite profiling of untransformed and cyanamide hydratase- (Cah) transformed (denoted 1C) soybean (Glycine max [L.] Merrill) leaves revealed only small differences in plants grown in the greenhouse or in the dark for 24 h, indicating that the Cah enzyme that converts cyanamide to urea has no substrates in soybean leaves and does not affect metabolism. Untransformed leaves sprayed with 0.5% cyanamide developed necrotic lesions within 2 h in the light but not in the dark. The sprayed 1C leaves showed little visible damage and accumulated high concentrations of urea, amino acids, and some sugars, but sucrose decreased over a 24 h period. The untransformed necrotic leaves also accumulated some urea and amino acids apparently due to cyanamide degradation, while sucrose and some organic acids decreased. Sprayed 1C leaves in the dark for 24 h contained very little urea and lower sugar levels. The untransformed sprayed leaves accumulated some organic acids, some sugars including sucrose, and urea and total amino acids. Unsprayed plants of both lines placed in the dark for 24 h showed increases in some amino acids and phosphate, and decreases in other amino acids, sugars, and organic acids. Thus the Cah enzyme can detoxify cyanamide by conversion to urea that is converted to amino acids. Other metabolic changes associated with leaf necrosis and darkness are also described. Principal component analysis confirmed the similarities and differences observed. Comparison of the GC-MS metabolic profiling analysis of amino acids with a dedicated system shows large differences, indicating a limitation of the former system.
Key words: Cyanamide, cyanamide hydratase, metabolic profiling, plant dark response, plant necrotic response, soybean, urea
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Introduction |
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Cyanamide is a nitrogen-rich compound that has been used as a nitrogen fertilizer, defoliant, and herbicide (Miller and Hall, 1963), and to break fruit flower bud dormancy (Nir et al., 1986). Due to its toxicity, cyanamide has also been used as a selection agent for plant transformation using a gene for the enzyme cyanamide hydratase (Cah) isolated from the soil fungus Myrothecium verrucaria (Maier-Greiner et al., 1991b). This enzyme converts cyanamide to the common metabolite urea. The Cah gene and cyanamide selection has been used to select transformants of wheat (Weeks et al., 2000) and Arabidopsis, potato, rice, and tomato (Damm, 1998). Expression of the Cah gene in tobacco also imparted cyanamide resistance (Maier-Greiner et al., 1991a). The cause of the cyanamide toxicity is not known, but the breaking of flower bud dormancy in grapevine by cyanamide was associated with a decrease in catalase activity (Nir et al., 1986). Barr and Crane (1980) reported that disodium cyanamide inhibited the high pH site of photosystem II of spinach chloroplasts, and cyanamide has been shown to inhibit rat liver aldehyde dehydrogenase (Svanas and Weiner, 1985).
Cyanamide is usually not considered to be a naturally occurring compound in plants, but Kamo et al. (2006a) found that Vicia villosa (hairy vetch) and Vicia cracca both contained cyanamide while the compound was undetectable in five other Vica species and 94 other species analysed. Kamo et al. (2003) showed that the cyanamide in V. villosa could act as an allelochemical and was synthesized by the plant (Kamo et al., 2000b).
The Cah gene has been introduced into soybean (Glycine max [L.] Merrill) using hygromycin phosphotransferase as the selectable marker gene and hygromycin as the selection agent (Zhang et al., 2005). Plants and tissue cultures that expressed the Cah gene were more resistant to cyanamide than the untransformed control plants and cultures. While no abnormalities were noted with any of the plants expressing the Cah gene, it is possible that the enzyme might affect metabolism in some way. Thus out metabolic profiling analyses were carried to determine if any differences could be found in any metabolite measured. Leaf samples were also analysed after spraying with cyanamide to document the changes that occur in both the damaged untransformed control and the Cah-expressing plants that show little visible damage. The effect of light on the toxicity and metabolites profile was also studied.
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Materials and methods |
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Plant material
Seeds of the untransformed control Jack and line 1 (denoted 1C here) from Zhang et al. (2005) that was transformed with the Cah gene driven by the Arabidopsis thaliana actin-2 promoter with the actin-2 terminator were used. Line 1C was homozygous for the transgenes. Seeds were planted in December 2006 in steamed soil mix (1:1:1; soil:perlite:sand; v:v:v) in 15 cm diameter plastic pots and grown in the greenhouse with supplemental lighting (Philips Metal Halide 1000 W bulbs were set to turn on at 6 am and off at 8 pm). The mid-day light intensity was
1000 µmol photons m–2 s–1. The average temperature in the greenhouse was 23.6 °C. The plants were sprayed 24 d after planting when the first trifoliolate leaf was fully expanded with 0.5% cyanamide in water until wet. Plants were divided into two groups after spraying with 0.5% cyanamide: the first group was placed in the dark and sampled after 8 h and 24 h; the second group was grown under normal light conditions (14 h day–1) and sampled 0.5, 4, 8, and 24 h after spraying. Each variant consisted of three independent plants, and the first trifoliolate leaves were analysed. During the experiments plants were not watered.
Cyanamide hydratase assay
The Cah activity assay that measures cyanamide disappearance was performed according to Zhang et al. (2005). About 100 mg of soybean leaf tissue from the untransformed Jack and transgenic 1C plants were ground in liquid N2 and extracted with 5 mM sodium phosphate buffer (pH 8.0) containing 1x protease inhibitor cocktail (Sigma, St Louis, MO, USA). The extracts were centrifuged at 4 °C for 10 min at 9400 g. On ice, 3 µl of 1 M cyanamide was added to 150 µl of supernatant. The mixture was incubated at 37 °C for 0, 4, and 6 h. At each time point an aliquot of 40 µl of the solution was mixed with 27 µl of 0.1 N sodium carbonate buffer (pH 10.4) and 3.5 µl of freshly made colour reagent [4% (w/v) sodium amminepentacyanoferrate (II); Sigma-Aldrich] that had been filtered through a Whatman No. 1 filter disc and kept in the dark. The reaction was incubated at room temperature for 10 min in the dark (Zhang et al., 2005). Absorbance at 530 nm was recorded.
Extracts were prepared from leaves of five individual untransformed and transgenic plants. Extraction buffer without plant extract was used as the blank control. The A530 changes were calculated by subtracting the readings at 4 h and 6 h from their respective readings at 0 h (Zhang et al., 2005).
The total soluble protein concentration of leaf samples was measured with the Bio-Rad protein assay kit (Bradford, 1976) using bovine serum albumin as the standard. The relative Cah enzyme activity was defined as µmol of cyanamide converted mg–1 protein min–1 using the data from the reactions at 4 h. A standard curve was created using cyanamide concentrations of 0, 10, 20, 40, 60, 80, 100, 150, 200, 400, 600, and 800 µM, and 1, 2, 5, 10, and 20 mM (Zhang et al., 2005).
Metabolic profiling GC-MS analysis
A 10–15 mg aliquot of dried soybean leaves was extracted and derivatized as described (Fiehn et al., 2000Roessner et al., 2000). A sample volume of 2 µl was injected with a split ratio of 6:5:1. The GC-MS system consisted of a HP5890 gas chromatograph, a HP5973 mass selective detector, and a HP 7673A autosampler (Agilent Inc., Palo Alto, CA, USA).
Gas chromatography was performed on a 30 m SPB-50 column with 0.25 mm ID and 0.25 µm film thickness (Supelco, Belfonte, CA, USA) with an injection temperature of 230 °C, the interface set to 250 °C, and the ion source adjusted to 200 °C. The helium carrier gas was set at a constant flow rate of 1 ml min–1. The temperature program was 5 min isothermal heating at 70 °C, followed by an oven temperature increase of 5 °C min–1 to 310 °C and a final 10 min at 310 °C. Mass spectra were recorded in the m/z 50–600 scanning range. Spectra evaluation was performed as described (Lozovaya et al., 2006).
Analysis of free amino acids
A 50 mg aliquot of the freeze-dried material was extracted and derivatized as described (Silva et al., 2003; Inaba et al., 2007). The samples were analysed with an Agilent 6890N gas chromatograph equipped with a Agilent 7683B autosampler, 5973 series mass selective detector (Agilent Inc., Palo Alto, CA, USA) and an SPB-1701 (30 mx0.25 mm ID and 0.25 µm film thickness) capillary column (Supelco, Belfonte, CA, USA). Both injector and detector were set at 250 °C. The helium carrier gas flow rate was 1 ml min–1. Temperature program was: 100 °C for 1 min (solvent delay 3 min) to 280 °C at 20 °C min–1 and 280 °C for 5 min. Mass spectra were recorded in the m/z 50–300 scanning range. Spectra were evaluated according to Inaba et al. (2007).
Validation of extraction, derivatization, and GC-MS analysis
The precision of the analytical methods was evaluated by calculating the SD for each standard compound in seven repeated injections: a standard mixture of 104 compounds was divided onto seven identical aliquots, derivatized and analysed by GC-MS. The SD was 5% for most of the compounds and, in the case of the metabolite profiling, no value was >10%. In the case of the amino acid analysis where five standard solutions of 24 compounds were derivatized and processed by GC-MS, again most values were 5% and all were <10%, except histidine which had an SD of 12.2%.
Statistical analysis
Data sets were statistically analysed by t-test and one-way analysis of variance (ANOVA) using the algorithm incorporated into Microsoft Excel 2002 (Microsoft Corporation, Seattle, WA, USA). Differences were determined to be statistically significant at P <0.05. Multivariate statistical analysis was performed using PCA (principal component analysis) implemented in XLSTAT V.2007.4. (Addinsoft, New York, NY, USA) to distinguish metabolite and group correlations to Cah gene functions and cyanamide treatment. PCA is a technique used to reduce multidimensional data sets of related variables to lower dimension sets of uncorrelated variables (PCs) forming clusters (Joliffe, 1986; Roessner et al., 2001). The first PC accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible. Each PC corresponds to one dimension (axis) such that each object is characterized by its proximity to a particular axis (Taylor et al., 2002).
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Results |
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Cah enzyme activity measurement
Since the soybean line 1C is transformed with the Cah gene and previously has been shown to express the gene and have Cah enzyme activity in leaf and embryogenic tissue culture tissues by both enzyme assays and enzyme-linked immunosorbemt assay (ELISA) (Zhang et al., 2005), enzyme assays were carried out on leaf tissue, and activity levels of 103.1±7.6 µmol cyanamide mg–1 protein min–1 for 1C and 0.04±0.0 µmol cyanamide mg–1 protein min–1 for the untransformed line Jack were found. This is similar to the values reported by Zhang et al. (2005) for the same lines.
Metabolic profiles and free amino acid analysis of 1C and Jack leaves
Analysis by GC-MS of extracts from soybean leaves revealed that 1670 peaks and 260 spectra were unique. A total of 116 compounds could be identified that are listed in Table 1. Of these the quantitation of 63 compounds was possible using known standards (Tables 2 and 3). The amino acid concentrations determined both by this metabolic profiling methodology (Tables 2 and 3) and by the methods of Silva et al. (2003) (Table 4) that were developed for plant materials have been included for comparison. The latter method was shown by Silva et al. (2003) to give a high percentage recovery from samples spiked with known amounts of amino acid standards. Neither this nor the metabolic profiling method measure Arg which has been shown to be converted to Orn during the derivitization procedures used (Halket et al., 2005). Comparison of the amino acid concentrations determined by both methods shows that levels of most amino acids measured by the metabolic profiling methods are lower, and His, homoserine, Ile, Lys, Met, and pipecolic acid were not detected. However, in the case of Asn, Asp, Gln, and Gly, the concentrations measured by the metabolic profiling method were higher in some samples. Overall the method developed for amino acids detects more amino acids and should provide a more accurate measurement; therefore, the results obtained with this method (Table 4) were used.
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When the concentrations of the 63 compounds and seven additional free amino acids were measured in greenhouse-grown 1C and Jack trifoliolate leaves over a 24 h period, there was not much variation over time (Tables 2, 3, and 4). There were also only small differences in the compound levels between 1C and Jack that were not statistically significant, except that Jack had about double the galactinol and Gly concentrations, and 1C had about double the F-6-P, ribose, and mannitol concentrations. Homoserine and Hyp were detected at low levels in all 1C samples but not in Jack. When PCA was applied, the unsprayed Jack and 1C data form a tight cluster (Fig. 1), confirming the similarities. Additional evidence is presented below showing the similarity of the metabolite profiles of unsprayed 1C and Jack plants kept in the dark. These results indicate that expression of the Cah gene did not affect metabolism. This is what one would expect since cyanamide is the substrate for the Cah enzyme and should not normally be present.
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Effect of cyanamide on 1C and Jack plants in the light and dark
When 1C and Jack plants were sprayed with 0.5% cyanamide, the 1C plants showed only slight leaf damage even after 24 h, while the Jack leaves began to exhibit necrosis by 2 h that seriously damaged the whole trifoliolate leaf by 24 h (Fig. 2A, B). The visible damage was light dependent since if the sprayed Jack plants were kept in the dark for 24 h no damage was seen (Fig. 2C), but symptoms appeared within 2 h when they then placed in the light (data not shown).
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Effect of cyanamide spraying on metabolite profiles of soybean leaves
When 1C plant leaves were analysed over a 24 h period after spraying with 0.5% cyanamide in the greenhouse, many changes were noted (Tables 2 and 4). To simplify the analysis of the data, Table 5 lists the compounds where increases of >100% and decreases of >50% were seen in 1C leaves during the 24 h period in comparison with unsprayed leaves. In all cases these changes were statistically significant at the P <0.05 level. There were increases in some sugars, glycerol, quinate, and shikimate, but the greatest increases were in urea (Fig. 3) and almost all of the amino acids (Tables 4 and 5) that were apparently produced from the high concentration of urea. These results are expected since urea is the product of the Cah enzyme reaction with cyanamide as substrate.
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The only compounds that decreased greatly in 1C leaves sprayed with cyanamide were ascorbate, threonic acid, and F-6-P (Table 5). Sucrose (Suc), the most abundant sugar, also decreased by almost 50% (Table 2).
However, when Jack leaves were sprayed with cyanamide, the tissue damage noted was associated with increases in some sugars, glycerol, phosphate, many amino acids, and urea (Tables 3, 4, and 5; Fig. 3). However, the cyanamide-sprayed Jack leaves also show decreases in several acids, Suc, and some amino acids. PCA of this data show that Jack and 1C form a large cluster including a small subcluster of both at the 0.5 h time point and two sublusters of sprayed Jack and sprayed 1C showing the differential response to cyanamide treatment in normal light conditions.
Effect of darkness on cyanamide-sprayed and unsprayed leaf metabolite profiles
The compound profile that was increased in cyanamide-sprayed 1C leaves kept in the dark when measured at 8 h and 24 h (Tables 2, 4, and 6) was very similar to that of sprayed leaves in the light (Table 5) except the acids citrate, p-hydroxybenzoate, and threonic acid were increased, and melibiose and sorbose were the only sugars that increased. There were also very few amino acids that increased, and urea did not increase at all (Fig. 3). Malate, 3-PGA, 
KG, many sugars and alcohols, and the total sugar content decreased in the dark, indicating the general lack of energy caused by the lack of photosynthesis.
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The sprayed Jack leaves kept in the dark showed increases in many acids, some sugars including Suc, alcohols, and amino acids (Tables 3, 4, and 6). Urea also increased greatly when compared with the unsprayed control (Table 3; Fig. 3). Over 50% decreases were found in N-acetylglucosamine, Glc, isomaltose, sorbose, rhamnose, R-5-P, Gln, His, Leu, Lys, Phe, Pro, Tyr, and Val. When these data were subjected to PCA, Jack and 1C were in a large cluster but clearly separated from each other (Fig. 1), confirming the differential response to cyanamide spraying.
When unsprayed 1C and Jack plants were placed in the dark and analysed at 8 h and 24 h, the changes in compound concentrations in comparison with plants kept under normal greenhouse conditions were very similar in leaves of both lines (Table 7), and PCA of the data placed unsprayed 1C and Jack in the dark in a tight cluster (Fig.1), confirming the similarities. Increases of >100% in quinate, shikimate, syringic acid, glycerol, Asp, β-ala, GABA, homoserine, Hyp, Met, pyroGlu, Ser, and Tyr were seen in both lines. More than 50% decreases in aconitate, citrate, fumarate, 3-PGA, KG, total sugars, Suc, Fru, Gal, F-6-P, R-5-P, maltose, myo-inositol, galactinol, Ala, Gln, Glu, His, Leu, Orn, Trp, Val, and ethanolamine were also found in both lines. The sorbose concentration was increased after 8 h in the dark and then decreased by 24 h in both lines in comparison with plants kept under greenhouse conditions. The only differences found between 1C and Jack was that Ile, p-hydroxybenzoate, ribose, trehalose, and melezitose increased by >100% in Jack leaves, while a clear but lesser increase was noted in 1C (Table 4). Also Ser increased by >100% in 1C while a smaller but clear increase was found in Jack leaves (Tables 2 and 3). Both lines showed >40% increases in phosphate. Thus both 1C and Jack responded similarly to darkness, with only slight differences. The largest changes noted in both lines in the dark are decreases in organic acids and sugars that would be associated with the lack of energy available in the dark. Total amino acids were also increased by about one-third in both lines (Table 4).
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Discussion |
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These studies show that the expression of the Cah enzyme in soybean leaves does not greatly affect the concentrations of any of the metabolites measured in unsprayed 1C and Jack plants grown under normal conditions or when placed in the dark for 24 h. This might be expected since the enzyme is apparently very specific for cyanamide and showed no activity with a number of chemically related compounds including formylcyanamide, acetylcyanamide, acetonitrile, cyanate, cyanide, dicyandiamide, cyanourea, formamide, urea, or creatine (Maier-Greiner et al., 1991b). Thus this marker gene could be used in soybean without any apparent modification of plant metabolism.
However, in leaves sprayed with cyanamide, the Cah-expressing plants showed no visible damage while the untransformed control showed necrotic lesions within 2 h in the light. The very rapid formation of symptoms caused by cyanamide in the light is similar to that caused by the photosystem I inhibitors paraquat and diquat. However, the leaf symptom caused by these herbicides in the light is water soaking (Hess, 2000) that appears to be different from the cyanamide symptoms (Fig. 2). The toxicity of tree of heaven (Ailanthus altissima L.) extracts on alfalfa seedlings was also enhanced by light (Tsao et al., 2002).
The Cah-expressing plants produced large amounts of urea that was then apparently converted to ammonia by urease (Gerendas et al., 1999). The formation of ammonia has also been documented when urea was sprayed on potato leaves (Witte et al., 2003). The Cah-expressing leaves then apparently utilized the ammonia to produce large amounts of amino acids.
The untransformed control leaves sprayed with cyanamide also contained more urea than unsprayed leaves. This could be due to cyanamide degradation to urea, as was reported by Miller and Hall (1963) who used 14C-labelled cyanamide applied to cotton leaves or injected into bolls. In the case of soybean reported here, the cyanamide degradation that occurred in Jack leaves was apparently not due to endogenous cyanamide hydratase activity since none was detected here or by Zhang et al. (2005). It is also possible that urea could accumulate in damaged leaf tissue.
The 1C plants accumulate >4-fold the normal level of free amino acids after 24 h in the greenhouse following spraying, and this coincides with a decline in total sugars (Table 4) that may be used for amino acid biosynthesis. In contrast, sprayed Jack leaves accumulate total sugars and have a doubling of total free amino acids (Table 4), probably due to tissue death. When sprayed Jack plants are kept in the dark, total sugars also increase but total amino acids (mostly Glu, Gly, and Ile) and urea also increase (Tables 3 and 4; Fig. 3). However, sprayed 1C plants kept in the dark show decreased total sugars and increased total amino acids, but almost no urea (Tables 2 and 4, Fig. 3). This indicates that 1C plants are not damaged by cyanamide spraying when in the dark, while Jack plants may be since sugars accumulate, but the nitrogen from urea, apparently formed from cyanamide, can be utilized to form amino acids. The lack of accumulation of urea in the sprayed 1C plants in the dark may indicate that amino acid synthesis occurs rapidly enough to use all the urea that is apparently formed by the Cah enzyme.
Both quinate and shikimate, compounds related to the shikimate pathway, showed large increases in leaves of both lines both sprayed and unsprayed when kept in the dark and in sprayed 1C leaves in the greenhouse, all in comparison with unsprayed leaves kept in the greenhouse (Tables 2–6
). Sprayed Jack leaves showed decreases in both compounds when under greenhouse conditions. The quinate and shikimate concentrations usually correlate with changes in total amino acids (Tables 4–6) including sprayed 1C leaves kept in the dark when total amino acids increased greatly over time when compared with unsprayed leaves, as was seen in sprayed leaves kept in the greenhouse (Table 2). The exception was the Jack sprayed leaves where quinate and shikimate concentrations decreased while total amino acids increased. Kolbe et al. (2006) showed that shikimate increased in Arabidopsis plants treated with dithiothreitol (DTT) and proposed that the increased thioredoxin formed as a result activated the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-P synthase, leading to an increase in shikimate and Tyr. A number of amino acids also increased in the DTT-treated plants.
When the overall changes in total amino acids and sugars are examined, the results show that these are relatively stable over the 24 h period when measured at 0.5, 4, 8, and 24 h in unsprayed plants kept in the greenhouse. When both unsprayed 1C and Jack plants are placed in the dark for this 24 h period the total sugar concentrations decline by 60%, organic acids by 30%, and the total amino acid concentrations increase by about one-third (Tables 2, 3, and 4). This is probably due to the lack of photosynthesis that would decrease sugar formation and amino acid utilization.
When PCA was applied to all the data obtained, clusters were formed that confirmed the observations made (Fig. 1): unsprayed 1C and Jack are very similar metabolically under normal light and dark conditions but when sprayed with cyanamide show differences as expected.
Studies with spinach, pea, wheat, and barley leaves and protoplasts showed that the sugar concentration declined and the phosphate concentration increased in the dark (Stitt et al., 1985) as was found here. In addition, the present results with dark-grown plants were very similar to those found with Arabidopsis plants placed in the dark for 
3 d where the sugar and organic acid levels decreased greatly and amino acids including pyroGlu (pyroglutamate, 5-oxoproline) increased (Ishizaki et al., 2005). The increase in amino acids was proposed to be due to protein breakdown. PyroGlu can be formed by the post-translational cyclization of peptide N-terminal glutaminyl residues by the enzyme glutamine cyclotransferase that has been studied in Carica papaya (Dahl et al., 2000) so could be increased if proteins containing this N-terminal residue are degraded. It was found that the pyroGlu was only detected in leaves kept in the dark in the case of both sprayed and unsprayed leaves of both 1C and Jack lines (Tables 2, 3, and 4).
A microarray study of Arabidopsis plants grown in the light or dark (3 d) showed that the expression of a number of genes was up- or down-regulated by light (Ma et al., 2001). When classified into functional groups, those up-regulated by light that would seem relevant to the present results include photosynthesis genes, sucrose, amino acid, cell wall, and phenylpropanoid synthesis genes, and glycolysis and tricarboxylic acid cycle genes. Those down-regulated by light were sulphur and nitrogen assimilation genes and glyoxylate cycle genes.
While the amino acid concentrations determined by the GC-MS metabolite profiling methods used here give good comparisons of the relative concentrations between the two lines and different conditions, it was found previously that comparison of the concentrations found by the methods used here and the methods of Silva et al. (2003) that were developed especially for all free amino acids except arginine showed some differences (Inaba et al., 2007). In most cases, the concentrations measured by the methods of Silva were higher and more amino acids were detectable, indicating that the metabolite profiling derivatization reaction was not efficient with all amino acids. Thus it was felt that the dedicated amino acid analysis method is the most accurate method for amino acid measurement, and the data obtained by this method were used here.
A preliminary cyanamide spraying experiment was carried out earlier (July) and the plant injury responses and metabolite changes measured by the metabolic profiling method were similar to those presented here (data not shown). However, comparison of the amino acid analysis data presented here for unsprayed Jack plants grown in the greenhouse in December with the results presented by Inaba et al. (2007), where older leaves were sampled in July, shows that the concentrations of most amino acids found here are much lower. The greenhouse temperature is controlled, but sunlight and day length are quite variable. Others have reported variability between experiments that could be caused by plant age and season (Gebrehiwot et al., 2002), and that might also be affected by circadian clocks (Wijnen and Young, 2006).
The results presented here document the changes in metabolites that occur in leaves of an important crop plant, soybean, when treated with a toxic N-containing compound that can be detoxified by expression of a transgene where a large increase in N-containing compounds occurs. The Cah expression does not affect the metabolism of soybean plants under normal conditions. In the case of the untransformed plant, the toxicity results in a series of metabolic changes that have not been documented previously. The changes that occur under 24 h darkness are similar to those shown before with Arabidopsis.
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Acknowledgements |
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This research was supported in part by the United Soybean Board, by soybean checkoff funding from the Illinois Soybean Association, and the Illinois Agricultural Experiment Station.
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Abbreviations |
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KG, -ketoglutarate; F-6-P, fructose-6-phosphate; G-6-P, glucose-6-phosphate; GABA, 
-aminobenzoate; PCA, principal component analysis; pyroGlu, pyroglutamate; R-5-P, ribose-5-phosphate. |
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