Journal of Experimental Botany, Vol. 52, No. 358, pp. 1071-1081,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations
1 Centro de Investigación sobre Fijación de Nitrógeno, UNAM, Apartado Postal 565-A, Cuernavaca, Mor. México
2 Photosynthesis Research Unit of USDA/ARS and Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA
3 Facultad de Química, UNAM, México
Received 4 January 2001; Accepted 23 January 2001
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Abstract |
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Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40–50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution.
Key words: GS1, tobacco, Nicotiana tabacum, transgenic, ammonia assimilation.
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Introduction |
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The United Nations Environment Programme recently reported that worldwide nitrogen pollution is one of the main threats to human survival and the environment, along with water shortage and global warming (UNEP, 1999
). Human activities now result in greater fixation of N2 than all natural processes combined (Vitousek et al., 1997). Sixty per cent of this anthropogenic nitrogen is in the form of inorganic crop fertilizer, the global use of which has increased 10-fold in the last half century (UNEP, 1999). Plants typically take up less than half of the N fertilizer applied, with the majority lost to the atmosphere or dissolved in surface or groundwater (Vitousek et al., 1997). In the last 100 years the nitrate concentration in some US rivers has increased 10-fold, primarily from agricultural run-off, so that drinking water requires expensive additional water purification to safeguard consumers (Carpenter et al., 1998).
The development of new crop varieties with a low N requirement may provide a solution. For that reason manipulation of the enzymes involved in N uptake and metabolism, by molecular and genetic techniques, is necessary to increase the efficiency of nitrogen use in crops (Foyer and Ferrario, 1994). In non-leguminous plants, the majority of primary nitrogen enters through the roots as nitrate. Nitrate reductase and nitrite reductase sequentially reduce the N to ammonium. Glutamine synthetase (GS, EC 6.3.1.2) catalyses the ATP-dependent condensation of ammonia with glutamate to produce glutamine. Subsequently, glutamate synthase (GOGAT) transfers the amide group of glutamine to 
-ketoglutarate producing two molecules of glutamate. Glutamine and glutamate are the primary source of organic N for proteins, nucleic acids and chlorophyll (Lea, 1997). It has been suggested that ‘although plants do not appear to be limited by their ability to take up or reduce nitrate, the ability of some crop plants to incorporate nitrogen into proteins does appear to be limited’ (Lam et al., 1995).
In plants, GS is an octameric protein with a native molecular weight of 350–400 kDa (Lea, 1997). Two types of GS isozymes, located in the cytosol (GS1) or in the chloroplast (GS2), have been identified in plants. The GS isozymes are coded by a gene family in all plant species examined to date. A thorough characterization of the GS family, in several plant species, revealed a single nuclear gene for chloroplastic GS2 and multiple genes for cytosolic GS1 (Lam et al., 1996).
The GS2 gene is expressed primarily in leaf mesophyll cells. The physiological role of chloroplast GS2 is the assimilation of ammonia generated by both photorespiration and nitrite reduction. GS2 genes are tightly regulated by light, at least in part mediated by phytochrome activation (Lam et al., 1996).
Little is known about the regulation of GS1 gene expression in plants or the significance of multiple GS1 family members. Expression of some of these members is localized to the phloem elements (Brugière et al., 2000), the N2-fixing root nodules of legumes (Edwards et al., 1990), cotyledons (Cantón et al., 1999) and senescing leaves (Bernhard and Matile, 1994; Brugière et al., 2000; Masclaux et al., 2000), suggesting a role in N uptake, translocation and mobilization. With the aim of improving the understanding of GS1 gene regulation, several research groups have attempted to manipulate GS1 levels by either over-expressing or down-regulating their expression (Temple and Sengupta-Gopalan, 1997). These studies have been performed in both non-legume and legume plants which can assimilate ammonia in their root nodules when this is provided by their microsymbiont (Rhizobium and related species) from fixed atmospheric N2.
The soybean GS1 gene fused to the CaMV 35S promoter and expressed in tobacco induces a concomitant expression of an otherwise silent native cytosolic GS1 gene in the leaf mesophyll cells (Hirel et al., 1992). These plants show little change in growth or in leaf protein content under optimal growth conditions (Hirel et al., 1997). Over-expression in Lotus corniculatus results in changes in ammonium assimilation and accelerated plant development, leading to early senescence and premature flowering when plants are grown on ammonia-rich medium (Vincent et al., 1997). During the symbiosis with Rhizobium loti these transgenic plants form more nodules and show an increase in shoot and root biomass, which may be due to a more efficient ammonium assimilation in the transgenic nodules, as indicated by an increase in amino acids and decrease in carbohydrate content (Hirel et al. 1997). Under low N conditions rice expressing a bacterial GS gene ‘grew better’ than controls (Su et al., 1995), and potted poplar trees expressing a conifer GS gene were taller than untransformed controls after 6 months of growth without fertilizer (Gallardo et al., 1999). These intriguing observations suggest that this approach to increasing crop nitrogen use efficiency is feasible.
In this study tobacco plants were transformed to induce constitutive expression of alfalfa GS1, and it is demonstrated that elevated cytosolic GS activity can increase photosynthetic productivity under N deprivation.
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Materials and methods |
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Vectors construction
Plasmid pGS100 (DasSarma et al., 1986
), kindly provided by Dr HM Goodman (Department of Molecular Biology and Genetics, Harvard Medical School, MA, USA), contains the whole cDNA coding region of alfalfa (Medicago sativa) constitutive class of GS1, cloned from a cell culture line. The pIC20H vector (Marsh et al., 1984) was used to clone, by blunt end ligation, a 1.56 kb AccI-EcoRI fragment from pGS100 containing the whole GS1 coding sequence, between the CaMV 35S promoter from cauliflower mosaic virus (35Sp) and the nopaline synthase terminator (NOSt), thus generating the intermediate plasmid pGH219. A 2.3 kb HindIII-HindIII fragment from pGH219 containing the 35Sp-GS1-NOSt chimeric gene was subcloned into the HindIII site of the binary vector pBI121 (Jefferson et al., 1987), between gus and nptII marker genes, thus generating plasmid pGH114 (Fig. 1).
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Plant transformation and growth
Agrobacterium tumefaciens LBA4404 strains bearing the binary vectors pGH114 or pBI121 (Jefferson et al., 1987) were used to transform Nicotiana tabacum L. cv. Xanthi plants by the leaf disc transformation procedure (Horsch et al., 1985). Shoots of putative transformants were selected in MS medium (Murashige and Skoog, 1962) containing 0.3 mg l-1 indoleacetic-3-acid (IAA), 10 mg l-1 dimethylaminopurine and 100 mg l-1 kanamycin (Km) and were rooted in MS medium containing 0.2 mg l-1 IAA and 100 mg l-1 Km. Putative, Km-resistant transformants with well-developed roots were transferred to pots containing vermiculite and grown under greenhouse conditions (day/night air temperature 22±3 °C; maximum photosynthetically active photon flux density [PPFD] c. 1100 µmol m-2 s-1; 12 h photoperiod; vapour pressure deficit [VPD] c. 0.9 kPa). The plants were watered with half-strength MS nutrient solution every week. The presence and expression of the transgenes in primary transformants (T0), was verified by PCR analysis and by the determination of GUS activity in leaves, using the histochemical assay (Jefferson et al., 1987). The primary transformants were allowed to self-fertilize and seeds from the T1 generation were used. The presence of the transgenes in the T1 transformants analysed was verified by Southern blot analysis. The T1 transformants were grown in either of the following N regimes. In the high nitrogen regime (N+), plants were watered with Jensen nutrient solution (Vincent, 1970), containing MS micronutrients and 12.5 mM ammonium sulphate as the N source for approximately 16 weeks. In the N deprivation regime (N-), plants were grown for 8 weeks in the high nitrogen regime, then washed and transferred to new pots with sterile vermiculite and watered with Jensen nutrient solution plus MS micronutrients without an N source for another 8 weeks. For the photosynthesis measurements the conditions were identical, except that T1 plants were grown in a growth chamber (22 °C; 0.8 kPa VPD; 800 µmol m-2 s-1 PPFD). For both treatments, at least three independent experiments were performed and similar results were obtained each time.
RNA isolation and Northern blot hybridization
Total RNA was isolated from fully expanded leaves using TRIzolTM (Gibco BRL, Life Technologies, Inc., Grand Island, NY) reagent as described by the manufacturer. RNA samples (30 µg per lane) were separated by electrophoresis on formaldehyde gels and transferred to positively charged nylon membranes by capillary action. Membranes were prehybridized for 2 h at 60 °C in a buffer containing 7% SDS, 250 mM sodium phosphate, 1% BSA and 1 mM Na2-EDTA. Hybridization was performed overnight with a PCR amplified 0.8 kb fragment containing from nucleotide 94 to nucleotide 993 of the coding GS1 sequence (DasSarma et al., 1986). A DNA probe coding for maize 18S and 28S rRNA was used to standardize RNA loading. The probes were labelled with [32P] dCTP (Amersham Life Science, Little Chalfont, UK) using the Random Prime labelling kit (Boehringer-Mannheim, Germany) following the manufacturer's instructions. The filters were washed as described previously (Beltran-Pena et al., 1995). The specific activity of the cDNA probes was routinely 107 cpm µg-1.
GS extraction and assay
Fully expanded leaves were homogenized, at 4 °C, with 2 vols (w/v) extraction buffer (50 mM Trizma [pH 8.3], 10 mM magnesium acetate, 1 mM Na2-EDTA, 10% [v/v] glycerol, and 1 mM PMSF), and 10% of polyvinylpolypyrrolidone. The homogenates were centrifuged twice at 12 000 g for 15 min. The clear supernatant was recovered to determine the GS activity. For initial assays of GS activity in seven transformed GS lines and two control lines, the transferase assay (Ferguson and Sims, 1971) was used. For the assays of leaf GS activity in T1 transformants of the two GS over-expressing and two control lines, grown on different nitrogen regimes, both the transferase and the synthetase assays (Ferguson and Sims, 1971) were used.
Protein extraction and Western immunoblotting
Preparation of protein extracts from fully expanded leaves was carried out at 4 °C. Plant material was homogenized in a mortar and pestle with 3 vols (w/v) of extraction buffer containing: 50 mM HEPES, 1 mM EDTA, 20% glycerol, 5% ethylene glycol, and protease inhibitor cocktail tablets (completeTM Mini, Boehringer-Mannheim, Germany). The homogenates were centrifuged at 10 000 g for 15 min. Supernatants were fractionated with ammonium sulphate, the 30–75% fraction was recovered, desalted and resuspended in 50 mM Tris-HCl buffer [pH 7]. Protein content was determined with a microassay based on the Bradford procedures (Bradford, 1976). Protein samples (50 µg) were separated by discontinuous SDS-PAGE electrophoresis (Laemmli, 1970) (15% resolving gel and 5% stacking gel), blotted onto nitrocellulose and incubated at room temperature with the primary antibody directed against bean nodule GS1, kindly provided by Dr Miguel Lara (CIFN-UNAM, México). An alkaline phosphatase conjugated secondary antibody was used and revealed with 5-bromo-4-chloro-indolyl phosphate and nitroblue tetrazolium salt as substrates (Sigma Chemical Co., St Louis, MO, USA).
Chlorophyll and free amino acid content
Chlorophyll was extracted from freshly harvested discs (7.7 cm2) taken from a fully expanded leaf (the 4th to 6th leaf, numbered from the youngest fully expanded leaf) using 80% acetone. Total chlorophyll content was calculated as described earlier (Graan and Ort, 1984). Soluble amino acids were extracted from samples (0.2–0.3 g fresh weight) of fully expanded leaves (4th to 6th leaf) as reported previously (Raab and Terry, 1995). After centrifugation, the samples were lyophilized. Amino acids were determined by HPLC reverse phase analysis using a pre-column derivation technique with 9-fluoromethyl chloroformate and a Nova-Pack C18 column (Waters International, Hertfordshire, UK).
Photosynthesis
To examine the effects on photosynthesis of N+ and N- treatments in GS1 transformants and control plants, leaf gas-exchange (Li-6400, Li-Cor Inc., Lincoln, NE, USA) was used to measure the relationship between light-saturated net CO2 assimilation rate (A) and the intercellular CO2 concentration (ci), as described previously (Nogués et al., 1998). These measurements were undertaken with leaf temperature, VPD and PPFD maintained at 25 °C, <1.4 kP and 1600 µmol m-2 s-1, respectively. From this A/ci relationship in vivo estimates of the maximum carboxylation velocity of Rubisco (Vc,max), the maximum rate of electron transport contributing to RuBP regeneration (Jmax) (McMurtrie and Wang, 1993) and light-saturated A at ambient CO2 (Asat) were calculated as described previously (Allen et al., 1997). The linear initial slope of the A/PPFD response was used to determine the maximum quantum efficiency of CO2 assimilation (
CO2) on an absorbed light basis. Leaf PPFD absorptance was measured using a Taylor integrating sphere (Li-1800-12, Li-Cor Inc., Lincoln, NE, USA).
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Selection of transgenic tobacco plants with the GS1 chimeric gene
Transgenic tobacco plants containing the nptII and gus marker genes plus the 35Sp-GS1-NOSt chimeric gene were obtained after transformation with pGH114 (Fig. 1
). Also, transgenic tobacco plants containing only those marker genes from pBI121 were obtained, and used as control plants. pGH114 transformed lines were designated GS lines, control transgenic lines were designated as C lines. The presence and expression of the transgenes in Km-resistant, primary transformants (T0) of independent GS and C lines was confirmed by PCR analysis and by positive GUS activity in leaves.
GS activity was determined in leaves of three T0 plants from seven independent GS lines and from two C lines. All of the GS lines (GS-2, GS-3, GS-4, GS-5, GS-6, GS-7, and GS-8) showed higher GS activity, ranging from 4–13-fold, as compared to the C lines (Fig. 2). The plants with the lowest increase in GS activity were discarded – the GS-6 and GS-7 lines, since transgenic tobacco plants with a small increase in GS activity and showing similar phenotypes to control plants had already been reported (Hirel et al., 1992; Temple et al., 1993; Temple and Sengupta-Gopalan, 1997). Four other lines: GS-2, GS-3, GS-4, and GS-5, showed a similar 8-fold increase in GS activity. The GS-8 line showed the highest GS activity, which was 13-fold higher than the control. Therefore two transgenic lines with high GS activity: GS-5 and GS-8, and two control transgenic lines: C-1 and C-2, were chosen for further analysis. Southern analysis of GS-5 and GS-8 transgenic lines revealed that a single copy of the 35Sp-GS1-NOSt gene was stably incorporated (data not shown).
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Over-expression of GS1 in tobacco GS lines
Molecular analysis of GS-5 and GS-8 plants, grown under high N conditions, was performed to determine the over-expression of the GS1 transgene. Northern blot analysis revealed an over-expression of GS1 transcript in leaves (Fig. 3A) with a molecular weight corresponding to that reported for GS1 RNA. The very faint hybridization signal in the control plants (Fig. 3A) was due solely to the cross-hybridization of the alfalfa GS1 probe with the native tobacco leaf GS RNA. The hybridization signal in the GS-5 and GS-8 plants, however, was substantially higher because the GS1 probe hybridized not only with the native tobacco leaf GS RNA, but also with the over-expressed GS1 RNA (Fig. 3A).
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The over-expression of GS observed at the RNA level (Fig. 3A
) correlated with elevated GS activity (Fig. 4). The GS activity from leaves of GS-5, GS-8 and control lines was determined by the rapid and commonly used transferase assay (Ferguson and Sims, 1971), which allows detection of high rates of ‘apparent’ activity (Lea et al., 1990) (Fig. 4A). However, the synthetase assay (Ferguson and Sims, 1971), which measures GS in a biosynthetic direction, was also used to confirm the increase in GS activity in the transgenic lines (Fig. 4B). Both enzyme assays gave identical results: the GS specific activity from leaves of GS-5 and GS-8 lines, expressed on a leaf area basis, was 6–7-fold higher than the activity present in leaves from control plants at high N (Fig. 4A, B).
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Western blot analysis of crude leaf extracts revealed that C-1, C-2, GS-5, and GS-8 lines contained a similar amount of the GS2 (chloroplastic) polypeptide (44 kDa). In addition the GS-5 and GS-8 lines contained a GS1 polypeptide (39 kDa), which co-migrated with alfalfa cytosolic GS1 (Fig. 5
). Since there was no change in the amount of GS2 protein in the GS-5 and GS-8 lines (Fig. 5), the increase in foliar GS activity (Fig. 4A) can be attributed to the additional cytosolic alfalfa GS1 protein expressed. A similar response was observed when the N supply was ammonium nitrate, rather than ammonium sulphate (data not shown).
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) (white bars). Values are the mean ±standard error from fully expanded leaves of 4–6 control, GS-5 and GS-8 plants.Effect of GS1 over-expression under high N
Plants from the GS-5 and GS-8 lines grown in the N+ regime were fertile and showed a similar phenotype to the control plants, with no difference in shoot and root dry weight (Fig. 6A
, B), leaf area (Fig. 7A) or chlorophyll content (Fig. 8A). The foliar free amino acid composition and content was similar in each line, except that the GS-5 and GS-8 lines had twice as much glutamine, than the controls (Fig. 9A), which may be the result of their higher GS activity (Fig. 4A, B). There was no difference in light-saturated photosynthesis (Asat) between control and GS1 over-expressing plants, due to similar maximum Rubisco activities (Vc,max) and RuBP regeneration rates (Jmax) (Fig. 10). There was also no difference in light-limited photosynthesis between control and GS1 over-expressing plants at high N, as the maximum quantum yield of CO2 assimilation (CO2) was unchanged (Fig. 11).
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Effects of GS1 over-expression under low N
Plants over-expressing GS1 showed an improved phenotype when compared with the control plants under low N, i.e. greener and bigger leaves (Fig. 12). Under such N- conditions, Northern blot analysis revealed an increased GS RNA content in leaves of GS-5 and GS-8 plants when compared to the controls (Fig. 3B). Leaf GS activity was reduced by 20–50% in all lines at low N (Fig. 4). However, GS activity, determined both by the transferase and the synthetase assays, in the GS-5 and GS-8 lines remained substantially higher (6–7-fold) than the controls in low N (Fig. 4C, D).
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After 8 weeks of N deprivation, control plants had 80% less shoot dry weight (Fig. 6C
) and 90% less root dry weight (Fig. 6D) than plants maintained at high N (Fig. 6A, B). Also, the leaf area of N-deprived control plants was 50% less than that of plants under high N (Fig. 7). The dry weight and leaf area of GS1 over-expressing plants under N deprivation was not decreased as much as in N- control plants, with GS-5 and GS-8 having c. 100% more shoot dry weight, c. 70% more root dry weight (Fig. 6) and c. 50% more leaf area (Fig. 7) than C-1 and C-2 at low N. Identical trends were observed in the chlorophyll content of these plants, with GS1 over-expressing plants having a higher content under N starvation, than control plants (Fig. 8B). Under N deprivation in control plants there was a 50% loss of total foliar free amino acids, on a leaf area basis (Fig. 9). Combined with the decline in leaf area (Fig. 7), it is clear that whole plant foliar free amino acid content declines substantially under N deprivation. However, GS1 over-expression entirely ameliorated this loss of total free foliar amino acids, on a leaf area basis (Fig. 9). The most abundant free foliar amino acid changed from glutamine to glutamate in all lines under N deprivation (Fig. 9B). Although reduced from N+ conditions, free foliar glutamine concentrations under N deprivation remained substantially higher (c. 4-fold) in plants with elevated GS activity (Fig. 9).
Nitrogen deprivation in control plants reduced light-saturated photosynthesis by c. 40%, due to similar declines in Rubisco activity and RuBP regeneration rate (Fig. 10). There was also a substantial decline in the light-limited maximum quantum yield of CO2 fixation (Fig. 11). Remarkably, in GS1 over-expressing plants, the low N treatment had no effect on light-saturated or light-limited photosynthesis, with Rubisco activity, RuBP regeneration rate (Fig. 10) and the maximum quantum yield of CO2 assimilation (Fig. 11) maintained close to N+ values.
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Discussion |
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In this paper, the first description is provided of an increase in photosynthetic productivity under N deficiency achieved by the over-expression of cytoplasmic GS1. This supports previous observations where the over-expression of GS1 under low N conditions produced plants which ‘grew better’ (rice; Su et al., 1995
) or were taller (poplar; Gallardo et al., 1999) than controls. When growing tobacco plants for two months under N-deprivation conditions, the transgenic lines with elevated GS expression and activity (Figs 3, 4) had an improved phenotype when compared to the N- control plants (Fig. 12). This included an increase in shoot (70%) and root (100%) dry weight as well as larger leaf area (50%) (Figs 6, 7). This faster growth is likely to be a consequence of both higher light-limited (Fig. 11) and light-saturated (Fig. 10) photosynthetic rates. It is remarkable that in the GS over-expressing plants, N deprivation had no significant effect on photosynthesis, while the control plants exhibited a 30–50% depression (Figs 10, 11). No GS1 effect on photosynthetic productivity was apparent under normal high N growth conditions (Figs 10, 11). The only previous study that observed a similar substantial over-expression of GS1, driven by the CaMV 35S promoter, in tobacco was conducted under optimum N conditions (Eckes et al., 1989). Other attempts to over-express GS1 in tobacco using the CaMV 35S promoter only reported small increases in GS activity (Hirel et al., 1992; Temple et al., 1993; Temple and Sengupta-Gopalan, 1997). Some transgenic plants were also obtained with minor increases in GS activity (Fig. 2). The differences in the degree of GS1 over-expression in transgenic tobacco bearing similar chimeric genes may be related to differences in the transgene, the positional effect or growth conditions.
It has been argued that transferase assay of GS activity may not reflect the physiological activity (Lea et al., 1990). In this work, it was confirmed that both the GS transferase activity and the GS synthetase activity were increased to a similar degree (6-fold) in GS-5 and GS-8 lines (Fig. 4). The synthetase assay measures GS in a biosynthetic direction and there is evidence that the kinetic constants with hydroxylamine are similar to those with ammonia (Stewart et al., 1980).
It was found that over-expressing GS1 (with the CaMV 35S promoter) in the legume Lotus corniculatus, had no effect on shoot or root biomass after 3 weeks of hydroponic growth with 4 or 12 mM 
as the sole N source (Vincent et al., 1997). The N deprivation reported here is likely to be much more severe than the low N used by Vincent et al., as after the tobacco plants were fertilized with 25 mM for 8 weeks, they received no further N supply for another 8 weeks. Therefore, there will be little primary N available for assimilation, and N for growth will come from reassimilation of photorespiratory ammonia and recycling of other endogenous nitrogenous compounds.
Nitrogen cycling through the photorespiratory cycle is approximately 10 times the normal rate of primary N uptake by C3 plants (Keys et al., 1978). GS1 does not normally contribute to the reassimilation of photorespiratory ammonia, as demonstrated by the inability of GS1 activity to rescue chloroplastic GS2-deficient barley mutants from the drain in organic N under photorespiratory conditions (Wallsgrove et al., 1987). This can be explained by the localization of GS1 in vascular tissue and not in mesophyll cells where photorespiration occurs (Forde et al., 1989; Edwards et al., 1990). However, the over-expression of cytosolic GS1 driven by the CaMV 35S promoter in this study was likely to result in GS1 accumulation in all cells, including mesophyll cells. Unlike the controls, significant GS1 protein was observed in leaf tissue (Fig. 5). At low N, GS1 over-expressing plants had a higher maximum Rubisco activity than the control plants (Fig. 10). As this was not accompanied by any change in ci (data not shown), the absolute rate of photorespiration and N input into this pathway is likely to be higher than in the controls, under N deprivation. It is not known whether the increased cytosolic GS1 contributed to the reassimilation of photorespiratory N, to support greater growth and maintain a larger free amino acid pool (Figs 6–9), despite this extra drain on plant N resources.
In addition to photorespiration, other cellular processes result in the recycling of N through GS. An increase in GS activity could therefore potentially accelerate and improve the efficiency of this recycling, maintaining photosynthetic enzymes and growth. As mature 4-month-old vegetative plants were used in this study, there was some senescence of the oldest leaves by the time of analysis. Between 50% and 90% of protein in senescing, leaf tissue is remobilized to other parts of the plant (Matile, 1982; Bernhard and Matile, 1994). Cytosolic GS1 is one of the relatively few genes up-regulated (relative to total RNA) during leaf senescence (Kawakami and Watanabe, 1988; Kamachi et al., 1992; Bernhard and Matile, 1994; Buchanan-Wollaston and Ainsworth, 1997). Moreover, of the nitrogen assimilation enzymes in senescing tobacco leaves, the expression of nitrate reductase, GS2and Fd-GOGAT are down-regulated while GS1 and glutamate dehydrogenase are concomitantly up-regulated, suggesting a contribution of these two enzymes in the reassimilation of ammonia in older leaves (Masclaux et al., 2000). The increased expression of GS1 was immunolocalized to the cytosol, suggesting that ammonia assimilation is shifted to the mesophyll cytoplasm during leaf senescence (Brugiére et al., 2000). It is possible that the over-expression of GS1 during leaf senescence increased the N available for translocation to the rest of the plant.
Glutamine and asparagine are common catabolites of leaf proteins during senescence, and they are subsequently transported to other parts of the plant (Lea and Miflin, 1980). To estimate the fate of remobilized amino acids, 14C- and 15N-labelled asparagine was fed to maturing Lupinus albus seeds (Atkins et al., 1975). There was a rapid appearance of 15, and a range of 15N-labelled amino acids, whereas the 14C becomes primarily incorporated in non-amino compounds (Atkins et al., 1975). This separation of the N and C of the transported asparagine means that a significant proportion of the N recycled from senescing leaves is likely to be utilized in de novo synthesis of amino acids through and the GS-GOGAT pathway. Translocation of N from a source, such as a senescing leaf, is proposed to be regulated by the sink strength elsewhere in the plant (Simpson, 1986; Staswick, 1994). Therefore, it could be envisioned that an over-expression of GS1 increased the scavenging of in developing leaves and other tissues, enhancing the sink strength and N remobilization from senescing leaves.
Degradation of protein during senescence is only a small proportion of the proteolysis occurring in plants, as there is rapid recycling of proteins in all tissue. Under low N stress, plants increase the rate of protein turnover and divert a higher proportion of N into proteins essential for continued photosynthetic productivity (Hatfield and Vierstra, 1997). Over-expression of GS1 may facilitate a more efficient scavenging of liberated during protein and amino acid catabolism, for incorporation into new proteins. This over-expression may also allow greater recycling of ammonia released during lignin, phenylpropanoid, methionine, and isoleucine synthesis (Lea, 1993; Lam et al., 1996). The over-expression of GS1 using tissue and ontogenetic specific promoters, instead of constitutive CaMV 35S, could help determine the mechanism behind this response.
Why do these crops not have sufficient GS activity to maximize productivity under N deprivation, when GS is an enzyme present in many isoforms and locations in all plants? Cultivar selection is typically undertaken under optimum growth conditions, and so there is unlikely to have been significant selective pressure for adaptations only apparent under extreme N deprivation. The authors are not aware of any studies that compare GS activity under low N conditions, in species or cultivars adapted to different N environments. However, the loss of an ability to up-regulate GS1 activity in response to low N is unlikely to be the cause, as leaves of both control and transgenic tobacco (Fig. 3) and soybean, pea and bean roots (Hoelzle et al., 1992), actually decrease GS activity at low N. Furthermore, a decrease in GS1 mRNA is observed at reduced ammonia fertilization rates in soybean (Hirel et al., 1987) and in two out of three isoforms of maize (Sukanya et al., 1994).
Field experiments are required to confirm whether these exciting preliminary findings translate to an increase in crop yield under low N conditions. In particular, it will be important to determine whether this protection of photosynthetic productivity at low N is at the expense of a loss of other important characters, particularly secondary metabolites including those involved in disease and pest resistance and, of course, nicotine production. For these results to be extended to other crops like maize, wheat, rice, and soybean, careful examination of any additional GS1 over-expression effects on seed production and quality is of particular importance.
In summary, it has been demonstrated for the first time that constitutive over-expression of cytosolic GS1 increases photosynthetic productivity under N deficiency. These results suggest that using transgenic technology, or other methods, to elevate GS activity in crops offers a promising approach to reducing agricultural N fertilization and the concurrent deleterious impacts on the environment.
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Acknowledgments |
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This work was partially supported by grants 4822-N9406 from CONACYT and IN205595 from DGAPA-UNAM. We are indebted to Donald R Ort (USDA/ARS and University of Illinois, Urbana, IL, USA) for discussions and the use of equipment, in particular photosynthesis measuring equipment and growth chambers. We acknowledge the technical assistance of Sandra Contreras and Yolanda Mora in the amino acids determinations, of Jesús Arellano in the Western blot and of Ramón Suárez in the Southern blot analyses. We are grateful to Drs Howard Goodman (Harvard Medical School, Cambridge, MA, USA) and Miguel Lara (CIFN-UNAM, Cuernavaca, México) for providing a plasmid and an antiserum used in this work.
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Notes |
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4 Present address: Joslin Diabetes Center, Harvard Medical School, MA02215, USA.
5 To whom correspondence should be addressed. Fax: +527 311 6710. E-mail: gina{at}cifn.unam.mx
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Abbreviations |
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CO2, maximum quantum efficiency of CO2 assimilation on an absorbed light basis; Asat, light-saturated net CO2 assimilation rate; CaMV, cauliflower mosaic virus; ci, intercellular CO2 concentration; GS1, cytosolic glutamine synthetase; GS2, chloroplastic glutamine synthetase; Jmax, maximum potential rate of electron transport contributing to ribulose 1,5-bisphosphate regeneration; PPFD, photosynthetically-active photon flux density; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate; carboxylase/oxygenase; Vc,max, maximum carboxylation velocity of Rubisco; VPD, vapour pressure deficit. |
References |
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