JXB Advance Access originally published online on March 1, 2006
Journal of Experimental Botany 2006 57(5):1129-1135; doi:10.1093/jxb/erj133
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RESEARCH PAPER |
Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stress-protectant glycine betaine
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
2Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N 0W9
* To whom correspondence should be addressed. E-mail: ray.wu{at}cornell.edu
Received 25 August 2005; Accepted 24 January 2006
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Abstract |
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Glycine betaine (GB) is a compatible solute that is also capable of stabilizing the structure and function of macromolecules. Several GB-producing transgenic rice lines were generated in which the Arthrobacter pascens choline oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI) gene promoter that is considered to be constitutive. This comparison led to interesting observations that suggest complex regulation with respect to GB synthesis and plant growth response under stress. In spite of the use of the well-studied stress-inducible promoter, the highest level of GB accumulation (up to 2.60 µmol g–1 DW) in the SIP lines grown under saline conditions was not as high as in the UBI lines (up to 3.12 µmol g–1 DW). Therefore, the use of an ABA-inducible promoter was not more beneficial for de novo production of GB. Interestingly, saline growth conditions enhanced GB accumulation by up to 89% in the SIP lines, whereas up to 44% increase was seen in a UBI line. In all these cases the GB levels were many-fold below the range reported for plant species that produce GB naturally. In spite of lower GB concentrations, statistically greater levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress protection observed in SIP plants cannot be totally explained by the increase in the GB content.
Key words: Choline oxidase, glycine betaine, salt stress, transgenic rice
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Salt stress can directly or indirectly affect the physiological status of plants by altering the metabolism, growth, and development (Garg et al., 2002
). One of the most extensively studied compounds related to the tolerance of salt stress is glycine betaine (GB) (Jain and Selvaraj, 1997; Sakamoto and Murata, 2001; Sulpice et al., 2003; Kumar et al., 2004). Under salt-stress, GB contributes to the osmotic potential in the cytoplasm to maintain an appropriate water content (Robinson and Jones, 1986; Gabbay-Azaria et al., 1988), and may also protect the intactness of macromolecules including proteins (Incharoensakdi et al., 1986; Rontein et al., 2002). In photosynthetic organisms, GB may contribute to the stability of oxygen-evolving photosystem II complex at high concentrations of NaCl (Papageorgiou and Murata, 1995).
The pathways for the synthesis of GB that start with choline proceed through reactions that involve one or two enzymes for the oxidation of choline to GB (Rontein et al., 2002). The reaction in the soil bacteria Arthrobacter pascens and A. globiformis is catalysed by a soluble flavoprotein choline oxidase, COX (EC 1.1.3.17
[EC]
), which is capable of converting choline to GB via betaine aldehyde as the intermediate. In the chloroplasts of higher plants, the first step is catalysed by a ferredoxin-dependent choline mono-oxygenase (CMO) and the second step is catalysed by an NAD+-dependent betaine aldehyde dehydrogenase (BADH). It has been reported that transgenic expression of choline oxidase-encoding genes in transgenic plants confers variable levels of stress tolerance against salt (Huang et al., 2000; Mohanty et al., 2002; Kumar et al., 2004), low temperature (Hayashi et al., 1997), and high temperature (Alia et al., 1998). Constitutive expression of a choline oxidase transgene in rice has been reported to result in the biosynthesis of GB and tolerance to salt and/or cold and/or drought (Sakamoto et al., 1998; Mohanty et al., 2002; Sawahel, 2003). In the present study, both a stress-inducible promoter and a constitutive promoter were used to drive COX gene expression in rice in order to compare the effectiveness of the two promoters in producing GB and in tolerating salt stress. The results suggest that the system is complex, and the observed effect cannot be explained by GB production alone: in transgenic rice grown in soil under salt stress, the extent of biomass production was significantly higher (P <0.01) in plants harbouring the COX transgene driven by a stress-inducible promoter as compared to those plants harbouring a constitutive-promoter-driven COX gene that resulted in at least as much GB as in the former.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Construction of COX gene-containing plasmids
It has been shown that GB is synthesized in the chloroplasts of higher plants (Rontein et al., 2002
). In order to produce a chloroplast-targeting transit peptide (TP)–COX fusion protein, a TP coding sequence from Pisum sativum Rubisco small subunit (Guerineau et al., 1988) fused in frame with A. pascens COX coding sequence (Rozwadowski et al., 1991) was isolated from pHS990 (R Hirji and G Selvaraj, unpublished construct) by EcoRI-XbaI digestion and blunted with Klenow DNA polymerase. To construct a stress-inducible expression plasmid pSIP-COX (Fig. 1), the TP-COX fragment was cloned into the SmaI site of a binary vector pHW36 containing a stress-inducible promoter complex which has been proved to drive inducible expression of transgenes under salt or drought stress (Su et al., 1998). The 2.1 kb TP-COX fragment was inserted at a blunted BamHI-digested pHW37 vector to create the constitutive expression plasmid pUBI-COX (Fig. 1). Both pSIP-COX and pUBI-COX contain the hygromycin phosphotransferase gene, which served as the selection marker in the transformation of rice. In addition, two copies of a tobacco matrix attachment region (MAR) sequence were included in each plasmid to enhance COX transgene expression (Cheng et al., 2001).
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Rice transformation
Agrobacterium-mediated transformation was carried out to generate COX-transgenic rice plants as described by Roy and Wu (2001)
. Mature seed scutellum-derived embryogenic calli of rice, Oryza sativa cv. TNG67 (Japonica), were used. Three-week-old calli were co-cultivated with Agrobacterium strain LBA4404 harbouring either pSIP-COX or pUBI-COX. After transformation, rice calli were selected in MS medium supplemented with hygromycin (50 mg l–1) as selective agent for 4 weeks (subcultured every 2 weeks). The resistant calli were then transferred to MS regeneration medium containing 50 mg l–1 hygromycin to regenerate into plants. Regenerated plants were transplanted into sterilized soil and grown in the greenhouse (30/22 °C day/night) with supplemental light for 10 h. Plants germinated from the seeds of regenerated plants were named the R1 generation and R2 seeds were produced from the R1 rice plants. R2 plants were produced from the R2 seeds.
DNA blot hybridization analysis of transgenic rice plants
Genomic DNA from rice plants was extracted as previously described (Cao et al., 1992). DNA (6 µg) was digested with EcoRI and separated in a 0.8% agarose gel. A DIG-labelled 1.9 kb COX coding sequence was used as a probe. Gel preparation, hybridization, and washing were performed according to the standard protocol (Sambrook et al., 1989) and the instruction manual supplied with the DIG-labelling and hybridization kit (Roche Co.).
Growth and salt treatment of plants in soil to test the growth performance under salt stress
R2 seeds were germinated on half-strength MS medium containing 50 mg l–1 hygromycin for 8 d. Non-transgenic (NT) plants were grown in half-strength MS medium without hygromycin. No significant difference in growth performance was found between the transgenic plants in hygromycin-containing MS medium and NT plants in hygromycin-free MS medium (data not shown). The 8-d-old seedlings were transplanted into MetroMix soil in small pots (18x18 cm) with holes in the bottom (4–6 plants per pots). The pots were kept in flat-bottomed trays containing water. The seedlings were grown for an additional 16 d. Two plants with similar heights and weights from each pot were selected for salt treatment. The 24-d-old plants were salinized daily with 150 mM NaCl in half-strength Yoshida nutrient solution (Yoshida et al., 1976) by irrigating through trays underneath the pots. In order to determine the NaCl concentration in the soil, 5 g of NaCl-treated soil were mixed well with 20 ml water. Then the extracted salty soil solution was used to measure the electrical conductivity (EC) with a conductivity meter (PM6304, CONTROL Co.). The final NaCl concentration in the salinity-treated soil was determined according to the EC readings as compared with EC readings from a standard NaCl solution mixed with untreated soil. At 8 h after adding the NaCl solution, the NaCl concentration in MetroMix soil in the pots measured about 90 mM. On the second day, at 8 h after adding fresh salt solution into the trays, the NaCl concentration in the pots reached 150 mM. After 6 d of salt treatment, stressed rice plants were resupplied with fresh water every day, by adding water directly to the pots, for 6 more days to allow plants to recover. On the third day during the recovery period, the plants were watered with 200 ppm fertilizer solution (15-5-15, Cal-Mag, Scotts Co.) for 2 d. The composition of the fertilizer solution is N:P:K:Ca:Mg with the following ratio by weight of 100:15:83:33:13. The 200 ppm fertilizer solution contains approximately 7 mM of salt. On the first day of recovery, the soil in the pots still contained 40 mM NaCl. After the second day of recovery, the salt was no longer detectable. These plants were then subjected to a longer (12 d) salt treatment with 150 mM NaCl in half-strength Yoshida nutrient solution as before, followed by watering without salt for 10 d for plants to recover. On the fifth day the stressed plants were fertilized again with 200 ppm of the fertilized solution. Five more days were then allowed for the plants to recover.
COX enzymatic activity assay
Third generation rice plants (R2 plants) were germinated on hygromycin-containing MS medium for 10 d to select transgenic plants. Germinated R2 seedlings were transplanted into MetroMix 360 (SUN GRO Co., 35–45% Canadian Sphagnum peat moss, Vermiculite, Bark ash, Pine Bark, Dolomitic limestone) soil in the pots (18x18 cm) and grown in the greenhouse for 30 d. The rice plants were fertilized weekly with 200 ppm (15-5-15 Cal-Mag, Scotts Co.) of a fertilizer solution. For assaying COX enzyme activity, leaf samples from 42-d-old R2 plants were collected after treating with 150 mM NaCl for 2 d. The 42-d-old R2 plants were watered directly and the trays holding the rice plants were also filled fully with 150 mM NaCl solution. On the first day, 8 h after adding 150 mM NaCl solution, the NaCl concentration in the soil measured 90 mM. On the second day, 8 h after applying the fresh salt solution, the NaCl concentration in the soil reached 150 mM. As controls, leaf samples from non-stressed, 42-d-old R2 plants were collected. COX enzyme isolation and activity assay were carried out as described by Huang et al. (2000). Approximately 5 g fresh leaves from NaCl-stressed or non-stressed plants were collected and homogenized in 10 ml of buffer A containing 50 mM HEPES-KOH (pH 8.0), 10 mM EDTA, 25 mM DTT, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 mM ß-mercaptoethanol, and 5% (w/v) insoluble polyvinylpyrrolidone. The homogenates were centrifuged at 10 000 g for 10 min at 4 °C. Solid (NH4)2SO4 was added to the supernatant, and the fraction between 40% and 80% (NH4)2SO4 saturation was collected by centrifugation. The (NH4)2SO4 pellet, containing the COX protein, was dissolved in 2.5 ml of buffer B [50 mM HEPES-KOH (pH 8.0), 10 mM EDTA, and 10% (v/v) glycerol], desalted with Sephadex G-25 columns PD-10 (Pharmacia Biotech), and eluted with 3.5 ml of buffer B. Protein concentration was determined according to the method of Bradford (Bradford, 1976) using a dye concentrate from Bio-Rad. COX activity was measured spectrophotometrically by a BADH-coupled enzymatic reduction of NAD+ at 22 °C. BADH activity was assayed independently by betaine aldehyde-specific reduction of NAD+ at 22 °C (Weretilnyk and Hanson, 1989). The reactions were carried out in a final volume of 0.5 ml containing 50 mM HEPES-KOH (pH 8.0), 10 mM EDTA, 1 mM NAD+, 65 units of E. coli BADH (Boyd et al., 1991), 20 mM choline, and protein extract.
Determination of glycine betaine in R2 transgenic rice plants
R2 plants were germinated on half-strength MS medium containing 50 mg l–1 hygromycin to select the transgenic plants. To determine the GB level under salt-stress, 6-week-old R2 plants were treated daily with 150 mM NaCl, for 3 d. The rice plants in pots were watered directly with 150 mM NaCl solution, and the trays holding the pots were also fully filled with 150 mM NaCl solution. On the first day, 8 h after adding NaCl solution, the NaCl concentration in the soil measured 90 mM and it reached 150 mM NaCl at 8 h after applying fresh 150 mM NaCl solution (please see above for the determination of the salt concentration in the soil). The rice leaves from plants with or without stress were collected for measuring the GB level. GB was extracted as previously described (Rhodes et al., 1989; Huang et al., 2000). Oven-dried R2 leaf material (30–45 mg) was ground in methanol:chloroform:water (12:5:1 by vol.) with d9-betaine (various defined levels, after preliminary analyses) as an internal standard added prior to grinding with white quartz sand. The homogenate was extracted with chloroform:water (1:1.5 v:v). The aqueous phase was separated by centrifugation at 10 000 g for 10 min and dried under a stream of N2 at 45 °C. The sample was dissolved in 1 ml of water and then passed through Dowex-1-OH– and Dowex-50-OH+ as described in Selvaraj et al. (1995). The GB fraction was eluted with 6 M NH4OH, dried under a stream of N2 at 45 °C, and dissolved in 200 µl of 0.7% acetic acid. A hydrophilic interaction chromatography column (HILIC, 2.1x50 mm, Waters) was used for liquid chromatographic separation followed by Electrospray Ionization Tandem Mass Spectrometry to generate m/z values for GB analyses; this was done by the Mass Spectrometry Unit at the Plant Biotechnology Institute, National Research Council of Canada.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plasmid construction
Transgenic production of GB under the control of a stress-inducible promoter has not been examined. Therefore, two plasmids (Fig. 1) were constructed to address this. The pSIP-COX contains an ABA-inducible promoter complex AIPC (Su et al., 1998
), and the pUBI-COX contains a constitutive maize ubiquitin promoter, and the latter served as the control. The stress-inducible expression of AIPC-directed transgene expression was verified by both GUS activity assay (Su et al., 1998) and proline synthesis (Su and Wu, 2004). The AIPC used in the expression plasmid pSIP-COX contains four copies of ABRC (ABA response complex), the rice Act1 minimal promoter, and the Hva22 intron (Su et al., 1998). In addition, two copies of a tobacco matrix attachment region (MAR) sequence were also included in each plasmid to enhance COX transgene expression (Cheng et al., 2001).
Production of transgenic rice plants
The two plasmids, pSIP-COX and pUBI-COX, were separately introduced into the rice genome by Agrobacterium-mediated transformation. A total of 16 independent first generation (R0) rice plants harbouring either the SIP-COX or the UBI-COX transgene were generated, and verified by Southern blot hybridization analysis (data not shown). For further analysis, three SIP-COX R1 lines were chosen, each harbouring a single copy of the transgene. For comparison, two UBI-COX R1 lines with a single copy and one line with two copies of the transgene were also chosen. Southern blot hybridization results shown in Fig. 2 confirm that SIP-COX lines 3, 7, and 21, and UBI-COX lines 5 and 16, each contain a single copy of the transgene. UBI-COX line 7 harbours two copies of the transgene.
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Transgenic plants produce active choline oxidase
The COX assay originally described for bacterial extracts gave unreliable results with plant extracts. Therefore an assay was used, based on the spectrophotometric assay for betaine aldehyde-dependent reduction of NAD+ by BADH (Weretilnyk and Hanson, 1989
). However, an exogenous supply of betaine aldehyde was not provided, as outlined in Huang et al. (2000). Escherichia coli BADH was added to couple COX-generated betaine aldehyde from choline in order to reduce NAD+. As shown in Fig. 3, NADH production was undetectable in the extracts from non-transgenic (NT) control plants either before or after 150 mM NaCl treatment. However, various levels of COX enzyme activity as measured by NADH production were observed in all R2 transgenic plants under either salt stress or non-stress conditions. The very low COX activity in SIP3 and UBI5 is probably due to a rearrangement or position effect of the input plasmid in transgenic plants. It is important to note the COX activity of SIP-COX plants after NaCl treatment was increased by 1.6–1.8-fold compared with that of non-stress (0 mM NaCl) plants. By contrast, in two UBI-COX plants (UBI7 and UBI16), the levels of COX activity remained high under stress and non-stress conditions due to constitutive expression of the COX gene.
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Glycine betaine accumulation in R2 COX-transgenic rice plants
To test whether COX enzyme accumulation can increase GB synthesis in COX-transgenic plants, the GB levels in R2 transgenic plants were measured next under salt-stress or non-stress conditions. As shown in Table 1, COX transgene expression has resulted in significant amounts of GB accumulation in the transgenic rice over the control. For example, the UBI7 plant harbouring the plasmid with a constitutive promoter accumulated 3.4 µmol g–1 DW of GB when stressed with NaCl treatment in contrast to a similarly treated non-transgenic control that showed 0.3 µmol g–1 DW. The best SIP-COX line accumulated as much as 2.9 µmol g–1 DW under stress conditions compared with 2 µmol g–1 DW under no such stress. As shown in Table 1, there were variations among individual lines in spite of the use of MAR regions. When individual lines were examined, it is clear that the relative increase in GB under stress is neither a general feature of the SIP lines nor an attribute that is restricted to these plants. The highest GB content observed is still much less than the nearly 100 µmol g–1 DW seen in natural GB producers (Rhodes and Hanson, 1993
). This reinforces the view that, without engineering, the upstream steps for enhanced choline supply to chloroplasts, the natural high levels of GB are not attainable (Nuccio et al., 2000). Furthermore, there are also other fundamental issues to be addressed. For example, plants that produce GB do so naturally in their chloroplasts, whereas cytosolic installation of the pathway in engineered plants has produced relatively more GB than in those plants where the pathway was compartmentalized to the chloroplasts (Rontein et al., 2002). There is no evidence for active choline uptake in engineered chloroplasts even if choline production in the cytosol were to be enhanced, nor is there any clear indication of GB being exported to the cytosol. Thus, stress inducibility alone is not a factor in terms of GB production in these transgenic rice plants.
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Biomass production in SIP-COX and UBI-COX plants under NaCl stress
Rice, a salt-sensitive cereal crop, cannot produce GB as a consequence of the apparent absence of the activities of the two enzymes required for GB synthesis (Rathinasabapathi et al., 1993
). However, these results showed the presence of GB at 0.3 µmol g–1 DW. This low value for this particular rice cultivar is probably real in view of the sensitivity of the method employed. By engineering expression of a bacterial COX gene in transgenic rice plants, measurable amounts of GB have been synthesized. It should be emphasized that, while numerically the amounts are significant in comparison to non-transgenic controls, physiologically the amounts are not sufficient to afford osmotic balance. This has been the case with all efforts thus far to generate transgenics capable of producing high levels of GB. To determine whether the GB accumulation can confer salt-tolerance to the transgenic plants, in keeping with the observations that have been made with rice producing GB constitutively (Sakamoto et al., 1998), R2 plants were grown in soil for a growth performance test. The goal of this research was to measure the biomass production of transgenic rice plants in which the COX expression was driven by a stress-inducible promoter and to compare it to an identical coding sequence expressed in rice under the control of a constitutive promoter. To create high soil salinity, 150 mM NaCl in half-strength Yoshida solution was added to the trays in which the pots (with holes in bottom) were placed. After 6 d of salt stress, the control plants showed slow growth rate, whereas the COX-transgenic plants still showed relatively healthy growth. After watering and allowing plants to recover for 6 d, a second period of salt stress was applied by watering plants with 150 mM NaCl. Twelve days later, the control plants started to wilt and the leaves turned yellow. UBI-COX plants also showed some salt-stress symptoms such as slow growth rate and some yellowish leaves. On the contrary, the SIP-COX plants still remained green and the growth rate was relatively fast. The average dry root and shoot weights are shown in Table 2. These values would suggest that GB accumulation confers a measure of stress tolerance to transgenic rice plants, because both UBI-COX plants and SIP-COX plants grew significantly larger (P <0.01 except P <0.05 for UBI16::sNeT root) under salt-stress condition compared with control plants (sNeT and sNT). More importantly, the SIP-COX plants with inducible synthesis of GB showed significantly (P <0.01) higher growth rate than UBI-COX plants with constitutive synthesis of GB. In addition, the biomass values of a non-expressing transgenic line SIP22 (NeT) and the transgenic plants without NaCl treatment were measured during the entire period in the salt-stress experiments. No significant differences between NeT control plants and the transgenic plants were found under non-stress condition (data not shown). The results of NeT and sNeT plants indicated that 150 mM NaCl solution severely inhibits rice plant growth as reflected by the dry weights (root: sNeT/NeT=32/550=6%, a 94% inhibition; shoot: sNeT/Net=435/2481=18%, an 82% inhibition).
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In summary, transgenic rice plants were successfully generated in which the Arthrobacter pascens choline oxidase gene was driven by a stress-inducible promoter. The transgenic plants contained COX enzyme and also produced GB de novo without supplementation with the substrate choline. The underlying objective of this experiment was to determine if this would afford a better strategy for GB production than using a constitutive promoter. The results show that the use of a stress-inducible promoter did not result in enhanced GB accumulation over a line in which UBI, a strong constitutive promoter, was used. Paradoxically, the SIP line performed better than the UBI line in biomass production under stress.
GB stabilizes enzymes and other cellular entities (Sakamoto and Murata, 2001; Rontein et al., 2002). Thus the apparent effects of GB biosynthesis on stress tolerance may be attributed to protective effects other than osmotic balance due to GB, for which much greater levels of GB would be required. Indeed the amount of GB synthesized in any transgenic plant system to date is insufficient to raise cellular osmotic pressure in the face of the osmotic stress to which the plants have been subjected. While various other explanations, such as protection against oxidative stress, and in vitro studies on protection against protein denaturation, have been advanced, it is not yet clear how GB mediates the effects in vivo. The observations that SIP lines with less GB fared better in terms of plant growth under salt stress than the UBI lines with more GB, suggests that the mechanisms are likely to be very complex. Huang et al. (2000) have also noted that small protective effects seen with GB production are not consistent across three different species or across various stress conditions within one species. It should also be noted that all of the stress conditions employed are under laboratory settings and are largely arbitrary. To date, none of the GB transgenics have been tested under field conditions of salinity. However, from this study, SIP transgenics are a step closer to producing GB under stress conditions and are thus likely to be more useful for studying the effect of GB accumulation than those producing GB constitutively.
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Acknowledgements |
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Jin Su was supported in part by a biotechnology career fellowship from the Rockefeller Foundation. The authors are grateful to Steve Ambrose and Richard Hughes for mass spectrometry.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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