Journal of Experimental Botany, Vol. 52, No. 364, pp. 2079-2087,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Transgenic tobacco plants that overexpress alfalfa NADH-glutamate synthase have higher carbon and nitrogen content
1 Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Mor, México
2 Department of Agronomy and Plant Genetics, 411 Borlaug Hall, 1991 Upper Buford Circle, University of Minnesota and USDA, Agricultural Research Service, Plant Science Research Unit, St Paul MN, 55108 USA
Received 21 December 2000; Accepted 12 June 2001
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Abstract |
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This work reports the characterization of transgenic tobacco (Nicotiana tabacum L.) plants that constitutively overexpress NADH-GOGAT. Three independent transformants, designated GOS10, GOS13 and GOS19 (for GOGAT sense), with stable integration of the chimeric alfalfa NADH-GOGAT gene fused to the CaMV 35S promoter were studied. The transgene was stably integrated and inherited by the progeny. In these GOS lines, the expression of NADH-GOGAT mRNA and protein was detected at low levels in roots and leaves, while the expression of the host tobacco NADH-GOGAT gene was nearly undetectable. The roots of GOS lines showed an elevated (15–40%) enzyme activity as compared to control plants. When GOS plants were grown under greenhouse conditions and fed with either nitrate or ammonium as the sole nitrogen source, they showed higher total carbon and nitrogen content in shoots and increased shoot dry weight when plants were entering into the flowering stage, as compared to control plants. The observed phenotype of GOS plants was interpreted as reflecting a higher capacity to assimilate nitrogen due to a higher NADH-GOGAT activity.
Key words: Nitrogen assimilation, NADH-GOGAT, transgenic tobacco.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Nitrogen is the major limiting nutrient in most plant species. Irrespective of the nitrogen source the reduced form of nitrogen ultimately available to higher plants for direct assimilation is ammonium. In most plants ammonium is assimilated into amino acids through the co-operative activity of two enzymes: glutamine synthetase (GS) and glutamate synthase (GOGAT) (Lea and Miflin, 1974
). GS catalyses the incorporation of ammonium into glutamate, producing glutamine. GOGAT catalyses the transfer of the amide group of glutamine to 2-oxoglutarate, resulting in the formation of two molecules of glutamate. Glutamine and glutamate serve as nitrogen donors for the biosynthesis of many other compounds (Lea and Ireland, 1999; Temple et al., 1998).
In plants, GOGAT occurs in two distinct forms that use NADH (NADH-GOGAT) (EC 1.4.1.14) or Fd (Fd-GOGAT) (EC 1.4.7.1) as electron carriers. These enzymes differ in molecular mass, kinetics and location within the plant and are the products of distinct genes (Vance et al., 1995; Lam et al., 1996). Fd-GOGAT is more abundant in photosynthetic tissue, and its major role is the reassimilation of ammonium released from photorespiration (Lea, 1997).
NADH-GOGAT is found primarily in non-green tissues such as roots and legume root nodules and it is located in the plastids (Hayakawa et al., 1999; Trepp et al., 1999a, b). NADH-GOGAT activity, enzyme protein and mRNA increase markedly (Egli et al., 1989; Gregerson et al., 1993) and are regulated temporally and spatially (Trepp et al., 1999a, b) during the development of effective alfalfa root nodules. NADH-GOGAT activity and mRNA is maintained at low or undetectable levels in other tissues of alfalfa (Vance et al., 1995), but is detectable in the flowers (Schoenbeck et al., 2000). Such studies confirm that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed nitrogen in legume nodules and during pollen development. Rice NADH-GOGAT protein accumulates in specific cell types of the vascular bundles of developing young leaves and spikelets, suggesting that NADH-GOGAT is important in the synthesis of glutamate from the glutamine that is transported through the vascular system from senescing organs and roots in rice plants (Hayakawa et al., 1994). In rice, the mRNA and protein for NADH-GOGAT accumulated markedly in two cell layers of the root surface within a few hours of supplying low concentrations of ammonium (Yamaya et al., 1995; Hirose et al., 1997; Ishiyama et al., 1998). Thus, the expression of the gene in rice plants is regulated in an age-, cell type- and nitrogen-responsive manner (Hayakawa et al., 1999; Hirose and Yamaya, 1999).
The NADH-GOGAT cDNA and genes from alfalfa (Gregerson et al., 1993; Vance et al., 1995) and rice (Goto et al., 1998) have been isolated. The alfalfa NADH-GOGAT cDNA (7.2 kb) contains a single long open reading frame coding for a 240 kDa protein, with the mature processed protein being 229 kDa (Gregerson et al., 1993).
Research on nitrogen assimilation into amino acids in plants has been complicated by the fact that the reactions are catalysed by multiple isozymes located in distinct cellular compartments. Using traditional biochemical approaches it has been difficult to define the precise function and regulation of each isozyme in plant nitrogen metabolism. Transgenic and mutational approaches provide an alternative way to define the in vivo function of a particular isozyme through the analysis of plant mutants and/or transgenic plants. The analysis of mutants and transgenic plants with altered expression of the cytosolic GS1 and plastidic GS2 forms of GS (Eckes et al., 1989; Hirel et al., 1992; Temple et al., 1993; Kozaki and Takeba, 1996; Wallsgrove et al., 1987; Harrison et al., 2000; Migge and Becker, 2000), and with reduced expression of Fd-GOGAT and NADH-GOGAT (Somerville and Ogren, 1980; Kendall et al., 1986; Avila et al., 1993; Ferrario-Méry et al., 2000; Schoenbeck et al., 2000) have contributed to defining the specific roles of some isozymes of the ammonium assimilation cycle.
This paper reports on the constitutive expression of alfalfa NADH-GOGAT in transgenic tobacco. As compared to control plants, the transgenic tobacco plants, had elevated NADH-GOGAT specific activity and when entering the flowering stage they had increased shoot weight and shoot total nitrogen and carbon content when supplemented with different inorganic nitrogen sources, such as nitrate and ammonium.
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Materials and methods |
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Vector construction
Plasmid pBIGOS was constructed in two steps. In the first step, the BamHI-SacI fragment from pBI121 (Jefferson et al., 1987
) containing the gus gene was excised and replaced by the BamHI-SacI fragment of the multiple cloning site from pSL1190 (Brosius, 1989), thus generating the intermediate vector pBI90. In the second step, the 7.2 kb Asp718-SacI fragment from pG7.2 (Gregerson et al., 1993) containing the NADH-GOGAT full length cDNA from alfalfa nodules, was cloned into the Asp718-SacI sites of the multiple cloning site of pBI90, thus generating plasmid pBIGOS (Fig. 1).
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Plant transformation and growth
Agrobacterium tumefaciens EHA105 strains with the binary vectors pBIGOS or pBIN19 were used to transform tobacco (Nicotiana tabacum L. cv. Xanthi) by a modification of the leaf disc method (Horsch et al., 1985). Regenerated kanamycin resistant T0 transgenic plants were propagated in vitro and transferred to pots with vermiculite for maintenance in growth chambers at 24 °C with a 168 h photoperiod provided by cold fluorescent lights or in the greenhouse. The plants were watered with half-strength Jensen solution (Vincent, 1970) supplemented with 7.5 mM KNO3 or 3.75 mM (NH4)2SO4, as sole nitrogen source. The primary transformants were allowed to self-fertilize and seeds from the T1 generation were used for further experiments. All measurements and analyses were performed on plants at the vegetative stage of growth before transition to flowering.
DNA isolation, PCR and Southern blot analysis
Tobacco genomic DNA isolation was carried out using the PUREGENE (GENTRA Systems, Minneapolis, MN) plant tissue DNA isolation kit. PCR was performed, using AmpliTaq polymerase for the amplification of PCR1 and PCR2 fragments or rTth polymerase for amplification of the large PCR3 fragment, both from Applied Biosystems/Roche Molecular Systems (Branchburg, NJ) (Fig. 1
). The primer pair used for the amplification of a 0.8 kb fragment from the nptII gene was reported previously (Blake et al., 1991). The primer pairs 5-p+3-bG (5'-CCC ACA GAT GGT TAG AGA GG -3'+5'-TGC CCA ACC CTG TGT TAT CC-3') and 5-eG+3-t (5'-GGT TGT GGT CAT TGG CGG GGG-3'+5'-CAA GAC CGG CAA CAG GAT TCA-3') were used to amplify PCR1, PCR2, and PCR3 fragments (Fig. 1). For Southern blot analysis, 15 µg of digested plant DNA was electrophoresed and transferred to Hybond-N+ membrane (Amersham Life Sciences, UK). Hybridization was performed at 65 °C using 32P-labelled S1 or S2 internal fragments from NADH-GOGAT cDNA gene (Fig. 1) as probes.
RNA isolation and reverse transcriptase (RT)-PCR analysis
Total RNA was isolated from freshly collected tissues using TRIzol reagent (GIBCO BRL Life Technologies, Inc., Grand Island, NY), treated with DNAseI (Boehringer Mannheim, Germany), and used as template for RT-PCR following the manufacturer's instructions for the RNA PCR kit (Applied Biosystems/Roche Molecular Systems, Branchburg, NJ). The primer pair 5-bG (5'-AAG CCA AAA CTC GAA AAC GC-3') and 3-bG (see above) was used for RT-PCR to amplify the RT internal fragment of alfalfa NADH-GOGAT (Fig. 1). To monitor the absence of DNA contamination in RNA samples, control reverse-transcriptase free RT-PCR reactions were carried out.
Western blot analysis
Plant material was homogenized in a mortar and pestle with 1 vol. (w/v) of extraction buffer containing: 100 mM MES, 100 mM sucrose, 2% 2-mercaptoethanol, 15% ethylene glycol (pH 6.8), and protease inhibitor cocktail tablets (completeTM Mini, Boehringer Mannheim, Germany). After centrifugation the supernatants were precipitated with acetone. The pellets were resuspended in 100 mM Tris-HCl [pH 6.8]. Protein samples (150 µg) were separated in 7% SDS polyacrylamide gel and blotted onto nitrocellulose. The membranes were incubated with the primary antibody directed against NADH-GOGAT from alfalfa nodules (Anderson et al., 1989). After treatment with alkaline phosphatase conjugated secondary antibody, these were revealed with 5-bromo-4-chloro-indolyl phosphate and nitro blue tetrazolium salt (Sigma Chemical Co., St Louis, MO).
Determination of NADH-GOGAT activity
For the assay of root NADH-GOGAT activity, T1 plants were grown in vitro in Magenta boxes with liquid modified MS medium, containing 25 mM KNO3 or 20 mM (NH4)2SO4 as the sole nitrogen source. These growth conditions allowed a more homogeneous population with regard to values of enzyme activity in different plants from each line. Plant material was ground in the extraction buffer mentioned above with the addition of 2 mM phenylmethylsulphonyl fluoride. Supernatants were recovered to determine NADH-GOGAT enzyme activity by a spectrophotometric assay described earlier (Groat and Vance, 1981). The data were analysed by unpaired t-test.
Determination of total nitrogen and carbon content
Tobacco seeds were germinated and grown for 4 weeks on MS medium with kanamycin and then transferred into pots. Plants were grown in a greenhouse with natural light and temperature ranging from 30 °C to 22 °C between day and night. After 6 and 8 weeks of growth four or five plants from each line were harvested, and their shoot dry weight and carbon and nitrogen content were determined. A LECO CN-2000 carbon and nitrogen analyser (LECO, St Joseph, MI) was used for the determination of carbon and nitrogen content in 0.2 g of finely ground plant sample. The data were analysed by the statistical analysis of variance (ANOVA) and the mean values were compared by the Newman–Keuls statistical method.
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Results |
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Selection of transgenic tobacco plants with the alfalfa NADH-GOGAT chimeric gene
The alfalfa NADH-GOGAT cDNA fused to the 35S CaMV promoter was introduced to tobacco Nicotiana tabacum cv. Xanthi. Also, control transgenic tobacco plants containing only the nptII marker gene from pBIN19 were obtained. The presence of transgenes in regenerated plants selected on kanamycin media was first confirmed by PCR analysis. The nptII PCR fragment was observed in all of the 16 primary independent plants transformed with pBIGOS and all 12 plants transformed with pBIN19, whereas the alfalfa NADH-GOGAT PCR1 fragment (Fig. 1
) was only observed in 11 (71%) out of the 16 plants transformed with the pBIGOS plasmid. pBIGOS transformed lines were designated as GOS (GOGAT sense) lines, control transgenic lines were designated as C (control) lines. From these primary transformants three independent transformed plants with the NADH-GOGAT transgene (GOS10, GOS13 and GOS19) and two independent control plants (C2 and C5) were selected for further study after initial analysis of NADH-GOGAT activity in roots. These plants were self-pollinated and their kanamycin-resistant T1 progeny were used in all of the following experiments. Seed germination in selective media allowed the elimination of non-transformed kanamycin-sensitive plants and analysis of the kanamycin-resistant plants only.
The presence of the alfalfa NADH-GOGAT transgene was confirmed in every kanamycin-resistant T1 plant analysed by PCR amplification. Transgenic plants from the GOS10, GOS13 and GOS 19 lines showed the three expected PCR fragments, while the control plants did not show any, indicating that the full length chimeric NADH-GOGAT transgene was present in these GOS lines (Fig. 2A). Stable integration of the NADH-GOGAT transgene in all three GOS lines was confirmed by Southern hybridization. Using the S1 fragment as probe (Fig. 1), hybridization bands of the expected sizes were obtained both in the EcoR1 and the HindIII digested total DNA (Fig. 2B). To assess copy number of the inserted NADH-GOGAT gene, DNA digested with BamHI or HindIII was hybridized with the S2 fragment (Fig. 1). The hybridization patterns indicated that GOS13 and GOS19 had one insertion, while GOS10 could have two insertions (Fig. 2C). No hybridization band was revealed for DNA from control lines indicating that, under the stringent hybridization conditions used, the host tobacco NADH-GOGAT gene did not hybridize with the alfalfa NADH-GOGAT gene fragments used as probes (Fig. 2B, C).
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Expression of alfalfa NADH-GOGAT transgene in tobacco GOS lines
The expression of the transgene was analysed in both leaves and roots of the GOS10, GOS13 and GOS19 lines. RT-PCR analysis was used to assess the mRNA expression of the transgene (Fig. 3). The 35S promoter used in the chimeric construct directed the transcription of alfalfa NADH-GOGAT in every plant organ analysed from all three GOS lines. The RT-PCR fragment corresponding to alfalfa NADH-GOGAT was not detected in the control plants.
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The expression of alfalfa NADH-GOGAT polypeptide was analysed by Western blotting (Fig. 4
). In both root and leaf extracts from plants of the GOS lines, a band that co-migrated with the band corresponding to NADH-GOGAT from alfalfa nodules was observed. NADH-GOGAT polypeptide was observed in both tissues from plants grown either in nitrate (Fig. 4A) or in ammonium (Fig. 4B). A band with extremely low intensity could be seen for the control plants.
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The NADH-GOGAT specific activity was determined in roots of 15–20 plants from each GOS and control line grown in vitro in either of the nitrogen sources (Fig. 5
). The enzyme activity present in GOS10, GOS13 and GOS19 lines was significantly greater than that of control lines C2 and C5 (P=0.05), grown in either nitrate or ammonium (Fig. 5). In nitrate, GOS lines had from 15–40% (Fig. 5A) and in ammonium from 25–35% (Fig. 5B) higher enzyme activity than control lines.
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Shoot dry weight and shoot total nitrogen and carbon content of the GOS lines
Plantlets from GOS and C lines were germinated in vitro on kanamycin supplemented medium and transferred to pots. After 6 and 8 weeks of growth on nitrate or ammonium the plants were harvested and the shoot dry weight (Fig. 6) and total nitrogen and carbon content in shoots (Fig. 7) was determined. The data were statistically compared by means of three-way ANOVA analysis which involved genotype, nitrogen source, and either shoot dry weight or shoot total carbon and nitrogen content. ANOVA for these two characteristics, dry weight and nitrogen and carbon content were performed global or separately for 6 and 8 weeks. At both time points and for both characteristics there were significant differences (P<0.005) between genotypes, indicating that either dry weight or the nitrogen and carbon content of the GOS lines was higher than that of the control lines. The analysis showed that the contents of carbon and nitrogen were related and that there was no significant difference between the treatments with either nitrogen source (nitrate or ammonium). The comparison of the mean values for both characteristics by Newman–Keuls test showed that C2 and C5 lines were equal and different from either GOS10, GOS 13 and GOS19. As for the GOS lines, GOS10 is equal to GOS13 and GOS19 is different from those two lines.
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It is suggested that the constitutive expression of alfalfa NADH-GOGAT in transgenic tobacco enhanced its capacity to assimilate inorganic nitrogen provided either as nitrate or as ammonium, which resulted in an increased weight of plant shoots at the start of the flowering stage, as compared to control plants.
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Discussion |
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Current understanding of the precise roles and regulation of the enzymes involved in ammonium assimilation in plants has improved through the isolation and study of both defective mutants and transgenic plants which overexpress or down-regulate a particular enzyme.
For the two enzymes which participate in the ammonium assimilation cycle in plants, much more biochemical, genetic and metabolic information exists about GS than about GOGAT. With regard to GOGAT, although plant mutants specifically lacking Fd-GOGAT have been well characterized (Somerville and Ogren, 1980; Kendall et al., 1986; Avila et al., 1993), no mutants defective in NADH-GOGAT have been reported. Only recently a transgenic alfalfa line with a down-regulation of NADH- GOGAT expression was reported (Schoenbeck et al., 2000).
As far as is known, this is the first report on the overexpression of NADH-GOGAT in transgenic plants. The expression in tobacco of an alfalfa NADH-GOGAT cDNA (Gregerson et al., 1993) driven by the CaMV 35S promoter was achieved. The transgene was stably integrated and inherited.
Assimilation of inorganic nitrogen sources such as nitrate or ammonium varies according to plant organ. In most plants, nitrogen from ammonium is processed entirely in the roots, while nitrate may be reduced to ammonium in the roots or transferred to the shoot where it can be processed (Oaks, 1992). Therefore, the transgenic tobacco plants with the alfalfa NADH-GOGAT chimeric gene were analysed when grown either in nitrate or in ammonium to determine if the transgene was expressed in different organs and if this could result in similar or in different phenotypes related to different sites of nitrogen assimilation and/or regulation of enzyme activities.
While the alfalfa NADH-GOGAT transgene was expressed in both leaves and roots of the transgenic plants, only low levels of RNA accumulated. This was evidenced by RT-PCR analysis (Fig. 3) since the transgene RNA could only be detected by Northern blot hybridization when a high amount (2 µg) of poly (A)+ RNA was used (data not shown). Such results were unexpected since the expression of the alfalfa NADH-GOGAT transgene was driven by the strong 35S CaMV constitutive promoter. The low levels of alfalfa NADH-GOGAT mRNA in the transgenic tobacco plants could be due to inherent properties such as its length. The alfalfa NADH-GOGAT cDNA is quite large (7.2 kb); no other reports on the expression of coding sequences of this size in transgenic plants were found. The excessive length of this transgene could possibly negatively influence some process(es) after transcriptional initiation and/or the mRNA stability. Various examples of differential mRNA expression or stability for transgenes driven by the 35S CaMV promoter have been reported (Williams et al., 1992; Friedrich et al., 1995). It is also known that NADH-GOGAT is expressed at high levels only in nitrogen-fixing nodules, therefore, for high expression levels the enzyme might need some stabilizing factor absent in other plant organs.
In accordance with the low level of the transgene mRNA, a moderate expression of alfalfa NADH-GOGAT at the level of protein and enzyme activity was also detected (Figs 4, 5), while in the nodule extracts NADH-GOGAT is readily detected in a 5 µg sample of total protein. In the roots of control plants the enzyme activity of native NADH-GOGAT was detected (Fig. 5), but only a very faint polypetide band corresponding to NADH-GOGAT could be observed (Fig. 4), probably due to a low affinity of the antibody used for the native tobacco enzyme. Nevertheless, NADH-GOGAT enzyme activity present in the roots of GOS plants was higher (from 15–40%) than in roots of control plants (Fig. 5), as a result of the expression of the alfalfa NADH-GOGAT transgene. The assay used to determine root NADH-GOGAT activity is generally used to determine enzyme activity from root nodules (Groat and Vance, 1981; Gregerson et al., 1993; Schoenbeck et al., 2000). Unfortunately this method could not be used for determining the activity from leaf extracts because of the presence of interfering compounds; in addition, the instability of the enzyme did not allow the desalting or purifying of the extracts prior to the enzyme assay. Although the leaf NADH-GOGAT enzyme activity was not determined, the alfalfa NADH-GOGAT polypeptide was observed in leaves of GOS plants (Fig. 4). The alfalfa NADH-GOGAT seems to be active in the leaves of GOS plants and it may contribute to a higher total enzyme activity.
It has been assumed that in most plants nitrogen is reduced more efficiently in leaves than in roots because of the ready availability of photosynthate (carbon, reductant, and energy) in leaves (Oaks, 1992). With nitrate as the nitrogen source, the GOS plants showed higher shoot dry weight than control plants after 6 and 8 weeks of growth (Fig. 6). In ammonium-fed GOS plants, after 6 weeks of growth shoot dry weight was similar, and after 8 weeks it was higher for the plants of GOS lines than for the plants of C lines (Fig. 6). The demand for photosynthate by the roots of ammonium-fed GOS plants does not seem to be adequately satisfied at the early stages of plant development when smaller young leaves have less photosynthetic capacity. However, when fed either nitrate of ammonium, the plants of GOS lines had higher shoot dry weight and total nitrogen and carbon content in shoots than control plants at the time of switching to generative development. Keeping in mind that shoot production is more important than root production not only for tobacco but also for most other plant species important for agriculture, the detailed analysis of shoot biomass was performed. Preliminary data on root biomass suggest that the increase in shoot dry weight is a result of the increase of the total plant biomass rather than a different shoot/root partitioning. The root biomass of the line GOS 19 which showed the higher shoot dry weight, is indeed higher than the root biomass of control plants.
Since nitrogen is the major limiting nutrient for crop species, developing plants with improved capacities to assimilate soil nitrogen could be important in agricultural systems. In this work transgenic tobacco plants have been obtained with constitutively increased NADH-GOGAT activity that have higher shoot weight and shoot total carbon and nitrogen content at the start of flowering when fed with two different inorganic nitrogen sources in controlled greenhouse conditions. It remains to be tested if the reported effects of tobacco plants with higher NADH-GOGAT can occur under field conditions, and if these can be obtained in other engineered plant species of agricultural interest.
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Acknowledgments |
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We thank the participation of Stanislav Svoboda (Czech Republic) in the initial stage of this work, Miguel Lara for helpful discussion and Michael Dunn for critical review of the manuscript. This work was partially supported by grants IN205595 from DGAPA-UNAM and 4822-N9406 from CONACYT, México.
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Notes |
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3 To whom correspondence should be addressed. Fax: +527 311 6710. E-mail: gina{at}cifn.unam.mx
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