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Published online 9 August 2005
Published in J Environ Qual 34:1508-1518 (2005)
DOI: 10.2134/jeq2005.0070
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Ecological Risk Assessment

Molecular Composition of Leaves and Stems of Genetically Modified Bt and Near-Isogenic Non-Bt Maize—Characterization of Lignin Patterns

Juergen Poerschmanna,*, Achim Gathmannb, Juergen Augustinc, Uwe Langera and Tadeusz Góreckid

a UFZ-Center for Environmental Research Leipzig-Halle Ltd., D-04318 Leipzig, Germany
b RWTH Aachen, Institute of Environmental Research (Biology V), Chair of Ecology, Ecotoxicology, Ecochemistry, D-52076 Aachen, Germany
c Leibniz-Center for Agricultural Landscape and Land Use Research, D-15374 Muncheberg, Germany
d Department of Chemistry, University of Waterloo, Waterloo, ON, N2L 3G1 Canada

* Corresponding author (juergen.poerschmann{at}ufz.de)

Received for publication February 25, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Transformation of crops, including maize (Zea mays L.), with the cry1Ab gene from Bacillus thuringiensis to combat lepidopteran pests results in pleiotropic effects regarding lignin biosynthesis. Lignin patterns in stems and leaves of two genetically modified Bt-maize varieties (Novelis T and Valmont T) were studied along with their non-Bt near-isolines (Nobilis and Prelude, respectively). Molecular-level based thermochemolysis using tetramethylammonium hydroxide (TMAH) in combination with gas chromatography–mass spectrometry (GC–MS) was used to quantitate the total lignin contents and to identify monomeric lignin subunits including p-hydroxyphenyl (P), guaiacyl (G), and syringyl (S) moieties. The results were supplemented and confirmed by cupric oxide oxidation. The stems of the transgenic lines had higher concentrations of total lignin than the respective isogenic lines: Valmont T/Prelude by 18% and Novelis T/Nobilis by 28%. In contrast, differences in the total lignin concentration of leaves between the transgenic and the respective near-isogenic lines were marginal. There were significant modifications in the ratio of p-hydroxyphenyl/guaiacyl/syringyl molecular marker units of stem lignin between transgenic and isogenic lines. The guaiacyl units (in particular the G18 marker) accounted chiefly for the higher total lignin contents in the transgenic lines. The leaf lignin patterns did not show significant differences in molecular markers between isogenic and transgenic lines. TMAH-induced thermochemolysis—conducted in both the on-line and off-line modes—provided detailed information on the molecular composition of lignin, thus proving superior to the established "wet chemistry" methods of lignin determination.

Abbreviations: amu, atomic mass unit • FFAP, free fatty acid phase • G, guaiacyl unit • GC–MS, gas chromatography–mass spectrometry • P, p-hydroxyphenyl unit • RSD, relative standard deviation • S, syringyl unit • TMAH, tetramethylammonium hydroxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
INSECT-RESISTANT transgenic plants, which express an array of insecticidal crystal proteins of the entomopathogen, Bacillus thuringiensis, to combat various insect pests, were among the first products of plant biotechnology to be approved for commercial use (Bruns and Abel, 2003; Wisniewski et al., 2002). The European corn borer, Ostrinia nubilalis (Lepidoptera, Crambidae), is regarded to be the most damaging insect pest of maize (Shelton, 2003; Traore et al., 2000).

There has been significant debate concerning the potential effects of transgenic crops on soil biological processes and functions in agroecosystems (Kremer and Motavalli, 2004; Bartsch and Schuphan, 2002). The risks include plant invasiveness or dispersal of the plant itself into the native ecosystem (Dunfield and Germida, 2004; Conner et al., 2003; Wandeler et al., 2002). The toxin (although having a molecular mass of about 66 kDa) is released in root exudates of Bt corn (Saxena and Stotzky, 2001a) or introduced into soil after harvest and plant decay, resulting in potential hazards for soil microbial communities.

Genetic transformation may also lead to pleiotropic effects, that is, it may alter the macroscopic plant characteristics as a result of the insertion of the protein (Saxena et al., 2002). There have been few reports on pleiotropic effects of Bt corn, which, to the best of our knowledge, have focused on lignin. Saxena and Stotzky (2001b) showed that stems of transgenic Bt plants had a higher lignin content (by 33 to 97%) than stems from the respective non-Bt isolines. By contrast, recent results by Jung and Sheaffer (2004) obtained with cry1 Ab transgenic corn hybrids and the corresponding isogenic lines (both lines grown in field environments) indicated that there was no trend for either increased or decreased lignin content in Bt hybrids compared with their respective non-Bt hybrids. Thus, the aim of this study was both to scrutinize these obviously contradictory findings, and to focus on more detailed, molecular-based information on lignin composition.

The phenolic heteropolymer lignin originates from the enzyme-mediated oxidative coupling of coumaryl-, coniferyl-, and sinapyl alcohols, resulting in hydroxyphenyl (P), guaiacyl (G), and syringyl (S) units, respectively (Whetten et al., 1998). In the framework of this paper, P-originating building blocks are considered to be lignin-derived, although, strictly speaking, P-type units are regarded to constitute non-core lignin ("tannin" polyphenols). Quantitative results obtained with fluorescence microscopy and the acetyl bromide method (Saxena and Stotzky, 2001b) or using wet chemistry (Jung and Sheaffer, 2004) regarded lignin to be a sum parameter, thus not considering the composition on the molecular level. The investigation of molecular-based lignin composition in transgenic and isogenic lines is not only of scientific interest, but it is mandatory when studying pleiotropic effects on digestibility, effect on herbivores, plant growth architecture, soil organic matter stabilization and/or decomposition processes after plant decay (e.g., higher lignin contents may lead to reduced decomposition in soil due to the high recalcitrance of lignin), etc. Similarly, the persistence of the toxin itself in soil, as well as its biological activity, are expected to be influenced by the protective mechanisms brought about by its sorption onto the sorbent lignin (Poerschmann and Kopinke, 2001; Stotzky, 2000; Saxena et al., 2002). When considering forage digestion in rumen, higher lignin proportions might be of concern due to the reduced availability of crop cell wall polysaccharides brought about by lignin. In fact, attempts have been made to down-regulate specific, rate limiting enzymes controlling carbon flux into the lignin biosynthesis pathway to reduce lignin content in crops (He et al., 2003). On the other hand, lignin confers strength, rigidity, and impermeability to water on plants. It also contributes to the defense against pathogens and insects in vascular plants.

An array of "wet chemistry" methods has been developed to quantify and structurally investigate lignin. Examples include acetyl bromide assays, thioacidolysis, and oxidation using permanganate or (mild) nitrobenzene (Fukushima and Hatfield, 2004). These methods share some common shortcomings: they are time-consuming, do not yield sufficient qualitative detail, and are prone to interference caused by other plant components. Thioacidolysis is not a simple technique to perform, and it requires careful optimization (Lu and Ralph, 1997) (e.g., to prevent losses of labile cinnamaldehydes). Cupric oxide oxidation (Hedges and Ertel, 1982) yielding simple VSC-phenols (VSC: vanillic acid + vanillin, syringic acid + syringaldehyde, and cinnamyl units including p-coumaric and ferulic acids), acetovanillone, acetosyringone, and others is an established method to characterize lignin patterns, yet it suffers from side chain reactions (Lehtonen et al., 2001), error-prone quantification, and poor yields (40–75%). Similarly, various derivatization protocols followed by reductive cleavage appear to be biased (Lu and Ralph, 1997), for example, by virtue of double bond saturation in p-coumarate units yielding phenylpropionates.

Degradative thermal methods combined with GC–MS proved to be very useful in both identifying monomeric lignin subunits and quantifying lignin without the necessity of its isolation (Filley et al., 2001). Tetramethylammonium hydroxide (TMAH)-induced thermochemolysis is an efficient method to study soft- and hardwood lignin (Challinor, 2001). It is also less time-consuming than the wet chemistry methods, especially in the on-line version (see below). The degradative, base-catalyzed thermochemolysis cleaves both the labile C–O bonds, including ester bonds (resulting in volatile methyl esters) and ether bonds (resulting in methoxy-compounds), thus protecting the structure of monomeric lignin building blocks. In the framework of this study, the nondiscriminating pyrolysis approach using a capacitive discharge in a Silcosteel tube ("one-step" on-line procedure) was used along with the off-line approach for reasons of comparison and method validation. The former technique was introduced into analytical practice recently by Górecki and Poerschmann (2001) and was detailed in Parsi et al. (2005). The basic reason to apply this kind of pyrolysis instead of using commercial pyrolyzers is that little or no discrimination occurs with this method over the entire range of retention times, which facilitates accurate quantification. To confirm the TMAH thermochemolysis results, CuO oxidation was applied as an independent method.

In the framework of this paper, lignin patterns of two transformation events (Novelis T and Valmont T), along with their respective non-Bt near-isolines (Nobilis and Prelude, respectively), were considered. In each case, the total lignin content and molecular lignin composition were studied in both leaves and stems. To the best of our knowledge, lignin analysis at the molecular level to differentiate between isogenic and transgenic plants has not been conducted previously. A future contribution will address fatty acid and sterol patterns of the corresponding transgenic and near-isogenic lines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Plant Growth
Two transgenic maize lines and their near-isogenic varieties were used: (i) Novelis (event MON-00810-6; Monsanto, St. Louis, MO) and its isogenic variety, Nobilis, and (ii) Valmont (event SYN-EV176-9; Syngenta, Basel, Switzerland) and its near-isogenic variety, Prelude. Maize was grown on a research field of the University of Aachen without additional fertilizer in 5- x 3-m plots. Herbicide treatment was done at the three leave stage. Infestation with European corn borer was not observed, because Aachen is outside the distribution range of the European corn borer. Plants grew under identical conditions; eight randomly selected plants of each type were harvested in the BBCH 75 growth stage (Meier, 1997). Harvesting maize plants at the same growth stage is essential, because lignin composition changes with development (e.g., increase of syringyl monomers) (Chen et al., 2002). After cutting the plants, the leaves and the stems were packed in moistened plastic bags and transported in an ice chest to the Centre for Environmental Research in Leipzig for sample preparation and analysis. Stem samples between the fourth and fifth internode, and leaves of node 5, were taken. After freeze-drying, samples from each plant compartment of a given line were combined and homogenized. Eight equal subsamples were used for the two TMAH-thermochemolysis approaches (off-line and on-line; four subsamples each), and two subsamples were used for the CuO treatment.

Thermochemolysis and Pyrolysis
On-Line Mode
The reaction was performed in 0.53-mm-i.d. deactivated stainless steel capillary tubing (Silcosteel; Restek, Bellefonte, PA) using about 150 µg of sample mass as described in Parsi et al. (2005). External quantification to obtain total lignin content in the samples was done using pure freeze-dried lignin isolated from maize by a fractionation scheme detailed in Augustin et al. (2002). Single response factors were used for all syringyl-derived analytes, guaiacyl-derived analytes, and p-hydroxyphenyl analytes, as described in Chefetz et al. (2000). For thermochemolysis, two 1-µL aliquots of the derivatizing reagent TMAH (25% in methanol) were injected into the pyrolysis tube containing the freeze-dried sample with a syringe, one through the top and one through the bottom of the tube, thus ensuring efficient "prewetting" of the sample. Thermochemolysis was conducted by capacitive discharge at 500°C. TMAH solutions were prepared fresh every day.

Off-Line Mode
Freeze-dried samples (about 2 mg) were placed into glass ampoules with 250 µL TMAH (25% in methanol). The ampoules were flushed with helium and sealed under vacuum and liquid nitrogen. Thermochemolysis occurred at a sub-pyrolysis temperature of 240°C for 2 h. After cooling to room temperature, the ampoules were cracked open, and the reaction mixtures were extracted as described by del Rio et al. (1998). Two deuterated standards, o-cresol-d7 (diagnostic ion m/z = 115 amu) and phenanthrene-d10 (diagnostic ion m/z = 188 amu), were used for calibration and quantification.

Cupric Oxide Oxidation
The method included lignin oxidation with CuO and 2 M NaOH at 170°C for 2 h (Hedges and Ertel, 1982). The oxidation was performed in Teflon vessels, which were flushed with helium just before sealing. After cooling, the suspension was centrifuged and the solid plant sample washed with water. The combined supernatants were spiked with internal standards (o-cresol-d7, and phenanthrene-d10), acidified with HCl to pH 2.5, and extracted with methylene chloride. The rotary-evaporated solvent containing the lignin-degradation products was analyzed by GC–MS using a polar free fatty acid phase (FFAP) stationary phase, on which analytes with free phenolic groups elute as symmetrical peaks. For better detection and identification of phenolic acids, the organic solvent was subjected to methylation using the well-known boron trifluoride/methanol procedure (70°C, 30 min).

Gas Chromatography–Mass Spectrometry Analysis
The GC–MS analysis was performed using an HP (Palo Alto, CA) 6890 GC and an HP 5973B mass spectrometric detector with data acquisition in full scan mode. The GC–MS analysis parameters were the following: 30 m x 0.25 mm, 0.25 µm DB-5 stationary phase, or 30 m x 0.25 mm, 1.2 µm DB-624 stationary phase (Agilent/Waldbronn). The thick film capillary was used to better resolve low boiling analytes. The initial temperature was 40°C (held for 2 min), followed by a linear increase of 8°C/min to a final temperature of 260°C with the medium polar cyanopropyl phase DB-624 and 300°C with the DB-5 phase. Splitless injection was applied in all cases. The GC column was protected against excess TMAH reagent (including its reaction products) by a 2-m noncoated, deactivated fused silica pre-column. Free phenols obtained on cupric oxidation were analyzed using an FFAP column (see above).

Statistical Analysis
Relative standard deviations (RSD) were calculated. The RSD is known to be a measure of how precise the mean value is (i.e., it indicates the similarity of replicate results). The RSD is expressed in percent and is obtained by multiplying the standard deviation (SD) by 100 and dividing this product by the mean (RSD = SD x 100/mean). Differences between mean values were evaluated by a one-way analysis of variance (ANOVA), followed by the least significant difference method (LSD) (SigmaStat 2.0; SPSS, 1997).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Identification and Assignment of TMAH Degradation Products
The structures of the monomeric building blocks are given in Fig. 1 (see also labels in Fig. 2) . Figure 2 presents a typical total ion chromatogram (TIC) obtained by thermochemolysis of the maize samples. The structural assignment of the labeled peaks (see Fig. 2) is given in Table 1. Peak identification should be done with care, so as not to confuse carbohydrate-originating analytes, including 1,2,4-trimethoxybenzene (1,2,4-TMB in Fig. 2), with lignin-related products (e.g., 1,2,3-TMB; shorthand designation in Fig. 2: S1). Other "misleading" candidates include 1,4-dimethoxybenzene (1,4-DMB) (Fabbri and Helleur, 1999) or methoxybenzene (the latter may originate from proteins). The monomeric building block pattern in Fig. 2 points to a typical grass-like lignin (wood samples do not contain any p-hydroxyphenyl units, with G-type prevailing). Figure 2 (along with Table 1) confirms that the mechanism of lignin thermochemolysis involves an efficient ß-O-4 bond cleavage in the presence of an aliphatic {alpha}-hydroxyl group on the aryl-aliphatic ether linkage, resulting in typical low molecular mass degradation products (Filley et al., 1999). In contrast to the pyrolysis–thermochemolysis of cross-linked humic organic matter, during which a solid carbonaceous residue accounting for more than 50% of the organic carbon was formed (Poerschmann et al., 2001), no significant solid residue was observed in the thermochemolysis of lignin. The formation of a solid residue derived from the organic carbon that contains the most important structural information reduces the diagnostic value of the thermoanalytical approach.



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  		Fig. 1. Structures of analytes released after tetramethylammonium hydroxide (TMAH)-induced thermochemolysis of lignin (P-type: R1 = H, R2 = H; G-type: R1 = OCH3, R2 = H, S-type: R1 = OCH3, R2 = OCH3); modified from del Rio et al. (1998).

 


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  		Fig. 2. Total ion chromatogram (TIC) of lignin breakdown products after tetramethylammonium hydroxide (TMAH) thermochemolysis. Sample: non-Bt maize stem (Prelude). Thermochemolysis: on-line, 500°C, 25% TMAH solution in methanol, capacitive discharge heating (see text). Stationary phase: DB-624 (GC conditions: see text). Peak labels: see Fig. 1 (molecular mass in Dalton in parentheses).

 

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  		Table 1. Major thermochemolysis products from maize stems and leaves with their diagnostic fragments and molecular weights.

 
Total Lignin Content
Table 2, presenting data obtained by off-line thermochemolysis, provides evidence that transgenic lines possessed higher lignin contents than the corresponding isogenic lines when considering stems. Differences in the total lignin contents levelled off, but remained significant when turning to leaves (transgenic to isogenic ratios of 1.04 and 1.06, RSD below 4%, see Table 2). The RSD values account for both the variability at the field location in Aachen, and the variability of the thermochemolysis process. The introduced gene is present both in stems and leaves. The difference observed between these compartments is possibly related to more intense lignin biosynthesis in stems compared with leaves, because lignin has to provide rigidity and stability to stems. Results obtained by on-line thermochemolysis were similar to the off-line results (Valmont TStem/PreludeStem = 1.10, Novelis TStem/NobilisStem = 1.20; with identical results for the pairs Valmont TLeaf/PreludeLeaf and Novelis TLeaf/NobilisLeaf), but the reproducibility was worse with the on-line method, probably due to the low TMAH reagent to sample ratio in this approach. However, the latter approach seems more appropriate for large sample series. These methodological peculiarities will be detailed in a forthcoming paper. The CuO oxidation approach was not considered in the determination of the total lignin content, because of the significant impact of reaction parameters (temperature, treatment duration, concentration of reagents) on lignin yields and lignin patterns, and side-chain reactions resulting in losses of glycerol-type building blocks. However, as shown below, CuO oxidation was useful in assigning lignin subunit markers.


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  		Table 2. Total lignin (in % referred to freeze-dried plant material) in different corn hybrids obtained by off-line thermochemolysis (RSD below 4.0%; n = 4).

 
The data presented here are in general agreement with those reported by Saxena and Stotzky (2001b), who used both fluorescence microscopy/staining and the acetyl bromide method. These authors reported lignin contents higher by 33 to 97% in the transgenic Bt varieties tested (Bt11, Bt176, MON810) compared to their near-isogenic varieties. Our results showed a higher lignin content in the transgenic plants, but the differences were significantly smaller than those found by Saxena and Stotzky (2001b). Nevertheless, both our results and the results of Saxena and Stotzky contradict the data published by Jung and Sheaffer (2004). These authors did not find any differences in lignin contents between six commercial transgenic/near-isogenic pairs. Jung and Shaeffer used both the acetyl bromide method for lignin solubilized in 25% acetyl bromide in glacial acetic acid, and the Klason method for the acid-insoluble residue solubilization–hydrolysis of cell wall polysaccharides with 72% sulfuric acid. Surprisingly, the total lignin content of a given sample as obtained by the two independent methods was also shown to be almost identical by Jung and Sheaffer (2004), which seems somewhat unusual. In contrast, a comparison of the acetyl bromide method and the Klason lignin method for the quantification of lignin, performed by Fukushima and Hatfield (2004), revealed significantly different results, which is in agreement with our experience. Differences between the total lignin contents obtained by the two methods appear unavoidable considering the possible underestimation of the Klason fraction due to the potential solubility of lignin in sulfuric acid and/or interference by compounds other than lignin when measuring absorption at 280 nm in the acetyl bromide procedure. In addition, Klason lignin might be obscured by aliphatic plant constituents, including cutin (Kögel-Knabner, 2002). The contradiction between our results and those of Saxena and Stotzky on the one hand, and those of Jung and Sheaffer on the other hand, prompted us to apply the classical Klason method, which was basically applied by Jung and Sheaffer. A protocol was used consisting of hot water (80°C, 10 min) pressurized liquid extraction to remove water-soluble analytes (including water-soluble lignin), methanol and chloroform (2:1, v/v) solvent extraction (80°C, 10 min) to extract lipids (which hamper phase separation), and a two-step hydrolysis of the residue with sulfuric acid (12 M at 30°C for 1 h; 1 M at 100°C for 3 h) (van Dam et al., 2004). Oven-dried stem samples used were similar to those used by both Saxena/Stotzky and Jung/Sheaffer. In general, the TMAH results were confirmed. The total lignin ratio found for the Novelis T/Nobilis pair was 1.26, and the corresponding ratio for Valmont T/Prelude was 1.16. To clarify the contradictory results published in the literature, a round-robin test could be organized, in the framework of which different lignin sum parameter determination methods would be compared using a well-homogenized freeze-dried isogenic sample along with its transgenic counterpart.

Lignin Patterns
The question arises which monomeric lignin building blocks accounted for the increased lignin contents in the transgenic lines. A comparison of the lignin patterns of the corresponding pairs clearly indicated that the G-type was overwhelmingly responsible for the increase, mainly due to the G18 subunit. P-type markers were also more abundant in stems of the transgenic lines, but to a lesser extent than the G-type. The pattern and the concentration of the syringyl units were almost identical in the respective transgenic and isogenic lines. Figure 3 illustrates the increased abundances of the lignin breakdown products, P18 and G18, in the transgenic lines.



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  		Fig. 3. Lignin markers P18 and G18 in stems of maize of the transgenic Bt line (Novelis T, top) and the near-isogenic non-Bt line (Nobilis, bottom) obtained by thermochemolysis. Stationary phase: DB-5ms. The chromatograms (shown in mirror representation, time in min) present the sum of extracted ions acquired in full scan mode: molecular ion m/z = 192 amu for P18, molecular ion m/z = 222 amu for G18 and molecular ion m/z = 188 amu for phenanthrene-d10 (internal standard, IST). Peak areas of the target analytes normalized to the peak areas of the internal standard for better comparison.

 
Similar results were obtained with the CuO oxidation approach, supporting the findings reported in the paper by Hatcher et al. (1995). Generally, patterns obtained after CuO oxidation were characterized by high abundances of G1, G3, S1, G4, G5, S4, and S5 units (listed in order of their retention times on FFAP stationary phase), as well as of S6, G18, and P18 (strictly speaking, their precursors are considered here). To analyze the carboxylic subunits (including S6, G18, and P18; see Fig. 1) by means of GC in a non-discriminative way, a derivatization step (e.g., formation of methyl esters; see Materials and Methods) is mandatory. Figure 4 compares the patterns of free phenol units G3, G4, S4, and S5 obtained on CuO oxidation of the stems of transgenic Novelis T and near-isogenic Nobilis. Both the patterns of the breakdown products shown in Fig. 4, and the quantitation results, were almost identical. Thus, it can be concluded that these units do not undergo substantial modifications on introduction of the Bt gene and do not account for the higher total lignin contents in the transgenic stems. These findings are in agreement with those obtained by the TMAH-induced thermochemolysis. However, when focusing on the G18 and P18 acids (Fig. 5 ; free acids are given along with their molecular masses), it becomes evident that it was chiefly the G18 unit that was responsible for the higher total lignin content. The good agreement with the findings obtained by TMAH-induced thermochemolysis is striking (Fig. 3, methyl esters are given along with their molecular masses).



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  		Fig. 4. Lignin markers obtained on CuO oxidation. Top: stem of Bt line Novelis T, bottom: stem of near-isogenic non-Bt line Nobilis. Data in parentheses: molecular mass of the free phenol (in Dalton). Peaks referred to identical internal standard (IST) (deuterated dimethylphenol; target molecular mass ion: m/z = 125 amu). GC–MS: free fatty acid phase (FFAP) stationary phase. Data presentation: Extracted ions (full scan data acquisition mode).

 


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  		Fig. 5. Lignin markers obtained by CuO oxidation; sample subjected to BF3/methanol methylation (see Materials and Methods). Top: Novelis T, Bt stem; bottom: Nobilis, non-Bt stem. Acids S6 and G18 (see Fig. 1) in methylated form. Presentation as superposition of three ion traces. GC–MS conditions: see caption to Fig. 4.

 
Table 3 along with Table 4 detail the composition of significant P-, G-, and S-type based lignin subunits. There were only slight differences between the S to G to P ratios in lignin patterns of leaves when the corresponding transgenic and near-isogenic pairs were studied (Table 4). The relative S to G ratios determined were characterized by RSD values below 6% in the on-line mode, and below 4% in the off-line approach using four replicates each. This finding agrees well with data given in Kuroda et al. (2002), who (using the off-line mode) observed an average RSD of 3.1% for a minimum of six repeated runs. The least significant difference (LSD 0.01) = 0.34581 of the calculated ANOVA revealed significant differences in the results between S/P-Novelis T (stem) and S/P-Nobilis (stem). A close comparison of data obtained by the on-line approach (explicit data not shown) with those obtained by the off-line method indicated that the former tended slightly, but significantly, to overemphasize the S-type units and discriminate against the P-type units. However, this finding is outside the scope of this paper, which focuses on the comparison of transgenic and isogenic lines. More important in this context is the fact that basic structure-related conclusions considering the transgenic–isogenic pairs were identical with those obtained with the off-line approach, thus supporting the usefulness of TMAH thermochemolysis in qualitative and quantitative studies of lignin.


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  		Table 3. Monomeric lignin subunits in Bt-transgenic and near-isogenic non-Bt lines in stems of maize as obtained by off-line thermochemolysis.

 

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  		Table 4. Monomeric lignin subunits in transgenic and isogenic lines in maize plant leaf as obtained by off-line thermochemolysis.

 
The susceptibility of lignin toward chemical and biological attack can be mirrored by both the 3,4-dimethoxybenzoate to 3,4-dimethoxybenzaldehyde (G6 to G4) ratio and the 3,4,5-trimethoxybenzoate to 3,4,5-trimethoxybenzaldehyde (S6 to S4) ratio (Vane et al., 2001). Generally, an enhanced acid to aldehyde ratio indicates a more pronounced oxidative cleavage at the C{alpha}–Cß bonds (del Rio et al., 1998). Data in Table 3 show that lignin in transgenic stems is expected to be slightly more susceptible to oxidation than in isogenic lines, whereas the corresponding leaf-related data are very similar to each other (see Table 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Our findings regarding the total lignin content, in agreement with those of Saxena and Stotzky (2001b), might be explained by the transgene being inserted accidentally into genes that control lignin biosynthesis. This pleiotropic effect could explain why (i) maize stems of transgenic maize are harder than those of the near-isogenic variety and (ii) the addition of biomass from Bt corn to soil results in a significantly lower gross metabolic activity (Stotzky, 2000). Lignin—synonymous with recalcitrance (e.g., much lower mineralization rate than carbohydrates)—serves both as connection between the cells along with reinforcing the cell walls of the xylem tissue, and as protection of the cell wall against microbial attack. Likewise, lignin is well-known for its capability to influence palatability and digestibility of plant material to herbivores and decomposers (Zwahlen et al., 2003a). Beyond that, higher lignin contents are expected to protect the associated carbohydrates. The lower gross metabolic activity, which in turn influences the stabilization processes of soil organic matter and carbon cycling, might also be caused by the toxin itself as a result of selecting toxin-resistant insects (both harmful and beneficial) and by influencing microbial communities in soils. As known (see Kögel-Knabner, 2002, and references cited therein), both insects and microorganisms are considered to be very important in the degradation of organic matter. Likewise, the impact of higher lignin contents along with the larger fraction of the G-type lignin on soil aggregation processes, as well as on the size and variability of different pools of soil organic matter (physically stabilized through microaggregation, associated with silt and clay particles, biochemically stabilized), need to be clarified yet. By contrast, it is rather unlikely that the randomly inserted Bt gene has a direct impact on the lignin biosynthetic pathways with different transformation events as used by Saxena and Stotzky (2001b) (MON810, Bt11, Bt176). Further work should explain if higher lignin content in transgenic crops is beneficial due to improved structure and (lignin-mediated) higher recalcitrance of soil, or harmful due to the lignin-mediated "preservation" of the toxin in soil. Other issues, such as the potential scavenging capabilities of free radicals and/or reactive oxygen species by higher quantities of lignin, might also be considered in the framework of the forthcoming research.

It is conventionally assumed that the G-type lignin is more resistant to chemical and biological breakdown (including oxidation and demethylation) than the S-type lignin (Filley et al., 2002). The G-type units have a 5-aromatic position available for strong C–C bonds (5–5 linkages), which makes them fairly resistant (this position is blocked by methoxy groups with the S-type). In this context, the lower S to G ratios obtained for stems of transgenic lines in comparison with their near-isogenic counterparts (see Table 3) might be counterproductive toward forage digestibility. In line with these considerations, efforts have been made to overexpress ferulate-5-hydroxylase and a specific 5-hydroxyferulic acid O-methyltransferase to enhance the S to G ratio (Boudet and Grima-Pettenati, 1996). The significance of this approach was confirmed by a better digestibility of transgenic alfalfa (Medicago sativa L.)—possessing higher S to G ratios—on fistulated steers as compared with isogenic lines (Guo et al., 2001). When considering the impact of both lignin content and lignin composition on digestibility, the lignin concentration of forage is more important than differences in S to G monomer ratios (Sewalt et al., 1997). However, there is no solid evidence in the literature that transgenic maize has lower food quality than non-transgenic maize. A substantial equivalence could be demonstrated for dairy cattle, growing bulls, sheep, pigs, or broilers (Barriere et al., 2001; Flachowsky and Aulrich, 2001; Donkin et al., 2003; Taylor et al., 2003). Similarly, no clear evidence has been presented that transgenic plants are associated with direct effects on soil insects (Escher et al., 2000), degradation of plant matter (Zwahlen et al., 2003a), or microbial communities (Blackwood and Buyer, 2004; Motavalli et al., 2004). These findings are in contrast to studies showing differences between Bt and non-Bt plants for adult earthworms in a 200-d feeding study (Zwahlen et al., 2003b), and for soil bacteria communities (Donegan et al., 1995). Thus, further integrated studies are necessary to fill the gaps in knowledge regarding these problems.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our results confirmed the occurrence of pleiotropic effects with regard to lignin biosynthesis in stems of Bt maize as described by Saxena and Stotzky (2001b), although to a lesser extent. Carboxylic groups bearing monomeric units including G18 and P18 accounted chiefly for the increase in the total lignin content of the transgenic lines. These effects were restricted to stems; they did not hold true for leaves. The results are expected to have significant implications on plant physiology (higher rigidity and stability of "transgenic" stems), stabilization of soil organic carbon (lignin is known to be refractory, in particular the guaiacyl subunits), and soil microbial communities. Investigations should be extended to roots, which were not under study here.

More research work is necessary to address the differences in composition of Bt maize in comparison with near-isogenic non-Bt lines for further compound classes, including lipids (non-saponifiable sterols, saponifiable fatty acids). A forthcoming joint research program managed at RWTH Aachen will address Bt-maize plant chemistry, soil biological processes, and functions in agroecosystems. Our results indicate that TMAH-induced thermochemolysis is a very effective method with which to study lignin on the molecular level.


   ACKNOWLEDGMENTS
 
Thanks are due to Evelyn Becker, Marion Hoyer, and Ursula Bachmann for excellent technical assistance. This study was supported by the German Research Council (DFG), priority program 1090. We would like to thank Dr. G. Stotzky for helpful comments. Thanks are due to Restek for donating the Silcosteel tubing used in the research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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