regulation of glutamine synthetase expression in sunflower cells exposed to salt and osmotic stress

Scientia Horticulturae 103 (2004) 101–111

Regulation of glutamine synthetase expression insunflower cells exposed to salt and osmotic stress

Conceição Santosa,∗, Anabela Pereiraa,Susana Pereirab, Jorge Teixeirab

a Department of Biology, University of Aveiro, Portugalb Department of Botany and IBMC, University of Porto, Portugal

Accepted 16 April 2004

Abstract

Sunflower (Helianthus annuusL. cv. SH222) plants and calluses were exposed to 100 mM NaCl andto osmotic stress induced by polyethylene glycol 6000 (PEG 6000; 50 g/l). Salt and osmotic stressesincreased cytosolic glutamine synthetase (GS1) mRNA and the corresponding polypeptides in plants.In calluses, GS1 mRNA and polypeptide contents increased in response to salt stress. Determinationof glutamine synthetase isoenzyme activities showed increases of GS1 activity in NaCl-stressed cells(increases of 3.5; 6.2 and 1.3 times, respectively in leaves, roots and calluses) and in PEG-stressedplants (increases of 4.0 and 6.9 times in leaves and roots, respectively), while plastidial glutaminesynthetase (GS2) activity decreased in NaCl-stressed leaves. This increase of GS1 expression wasaccompanied by an increase of ammonium levels in stressed leaves. This report shows that NaCland osmotic stresses induced GS1 expression by increasing GS1 mRNA and polypeptide leading toincreased enzymatic GS1 activity. These data confirm previous suggestions about the increase of GS1expression in senescent cells and support the role of this isoenzyme on nitrogen mobilization in plantcells.© 2004 Elsevier B.V. All rights reserved.

Keywords:Ammonium; Glutamine synthetase;Helianthus annuus; Osmotic stress; Salt stress

1. Introduction

Salt and water stresses severely affect plant growth and nutrient accumulation (e.g.Joneset al., 1980; Santos and Caldeira, 1999) and induce cell senescence (Lutts et al., 1996; Linand Kao, 1998; Santos and Caldeira, 1999). In particular, they affect nitrogen absorption

Abbreviations:GA3, gibberellic acid; GS1, cytosolic glutamine synthetase; GS2, plastidic glutamine syn-thetase; NAA, naphthalene acetic acid; BA, benzylaminopurine; PEG, polyethyleneglycol

∗ Corresponding author. Tel.:+351-234370780; fax:+351-234426408.E-mail address:[email protected] (C. Santos).

0304-4238/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.scienta.2004.04.010

102 C. Santos et al. / Scientia Horticulturae 103 (2004) 101–111

and metabolism (Grattan and Grieve, 1994; Santos et al., 2002) leading to ammoniumaccumulation that is toxic to the cell (Lin and Kao, 1998). It was suggested a correlationbetween ammonium accumulation and senescence induction (Lin and Kao, 1998). Onepossible process to eliminate ammonium released during senescence (e.g. from proteinbreakdown) is the synthesis of Gln by glutamine synthetase (GS) an enzyme that uses Gluand ammonium as substrates.

Most plant leaf cells contain two GS isoforms, one cytosolic (GS1) and one locatedin the chloroplast stroma (GS2). GS isoenzymes are located in specific organs and cellssuggesting non-overlapping functions in plant metabolism (Pereira et al., 1992). It waspostulated that GS2 plays a role in the assimilation of ammonium produced by nitratereduction or photorespiration, while in shoots a cytosolic isoform is located in phloemcompanion cells (Carvalho et al., 1992; Pereira et al., 1992) and is probably involved inthe synthesis of amides during senescence (Bauer et al., 1997; Finnemann and Schjoerring,1999).

GS expression is affected by biotic and abiotic factors such as drought (Bauer et al., 1997),Na2SO4 stress (Santos et al., 2002), nitrogen deficiency (Finnemann and Schjoerring, 1998),Rhizobium-induced nodulation (Robertson et al., 1975) and microbe infection (Perez-Garciaet al., 1998). On the other hand, the influence of N source on GS isoforms expressionalthough largely studied (Finnemann and Schjoerring, 1998, 1999; Haba et al., 1992) isstill controversial (Finnemann and Schjoerring, 1998). Total GS activity increased in leavesand decreased in roots of NaCl-stressed soybean (Glycine maxL.) plants grown in thepresence of NO3− but was not affected when stressed plants were growing in the presence ofNO3

−/NH4+ (Bourgeais-Chaillou et al., 1992). These authors, however, did not differentiate

cytosolic from plastidial activities.Recent works support the idea that GS1 is particularly important in nitrogen remobiliza-

tion during natural or induced senescence (Bauer et al., 1997; Finnemann and Schjoerring,1999; Santos et al., 2002). For example, cytosolic glutamine synthetase gene expressionincreased in water-stressed tomato leaves while the plastidial isoform remained unchanged(Bauer et al., 1997). GS1 polypeptides or their corresponding mRNA also increased withaging and natural senescence in tomato (Perez-Rodriguez and Valpuesta, 1996) and riceplants (Kamachi et al., 1992) while GS2 polypeptides decreased (Kamachi et al., 1991,1992; Perez-Rodriguez and Valpuesta, 1996). The aim of this work was to study the influ-ence of salt and osmotic stress on GS isoforms, namely on GS1 mRNA and polypeptidequantities and GS1 enzyme activity in sunflower plants and calluses cells.

2. Materials and methods

2.1. Biological material

Helianthus annuusL. cv. SH222 seeds were supplied by Sclepal, Portugal. Seedlings weregrown in a greenhouse in hydroponic culture with Long Ashton medium (Meidner, 1984)under the conditions previously described bySantos and Caldeira (1999). Four-day-oldplants (n = 15 for each treatment) were exposed for 3 weeks to 0 and 100 mM NaCl or to50 g/l polyethylene glycol (PEG) 6000.

C. Santos et al. / Scientia Horticulturae 103 (2004) 101–111 103

Calluses were induced from sunflower (H. annuusL. cv. SH222) explants derived fromeight-day-old seedlings growing under aseptic conditions described bySantos and Caldeira(1999). These explants and calluses were grown on a modified MS (Murashige and Skoog,1962) medium containing 2.6�M NAA, 2.2 �M BA and 2.8�M GA3 and incubated ina growth chamber under the light and temperature conditions described bySantos et al.(2001). One-month-old calluses (n = 15 for each treatment) were transferred to the samemedium containing 0 mM NaCl, 100 mM NaCl and 50 g/l PEG 6000. Calluses were exposedto salt and osmotic stress for 3 weeks.

2.2. Antibodies

Polyclonal antibodies raised against GS fromPhaseolus vulgarisroot nodules, that alsorecognise other plant GS proteins (Cullimore and Mifflin, 1984), were a generous gift ofJ.V. Cullimore. Peroxidase conjugated goat anti-rabbit IgG was obtained by Vector, UK.Goat anti-rabbit Ig conjugated with 15 nm colloidal gold was obtained from Amersham,UK.

2.3. Total RNA isolation and dot blot

Leaves andcalluses(1 ± 0.001 g) were homogenised in liquid N2. RNA was extractedusing the RNAgents Total RNA Isolation System Kit (Promega, USA) according to theprotocol recommended by the manufacture.

Total RNA samples (0.5, 5 and 50�g) were loaded onto a nylon membrane (Stratagene)using a dot-blot apparatus (Schleicher and Schuell, Germany). The GS transcripts presenton the membrane were hybridised with a cDNA probe for the cytosolic GS. This cDNAprobe had been previously obtained from a cDNA library fromSolanum tuberosumtuber(Teixeira et al., 1996) and constructed in the� ZAPII vector (Lothar Willmitzer, Max PlankInstitute fur Molekulare Pflanzenphysiologie). The probe was labelled using multi-primeDNA labelling systems RPN.1601Z (Amersham, UK), then denaturised for 3 min and addedto the hybridization solution.

Hybridization took place for 16 h at 42◦C. The membrane was washed with 2× SSC (1×SSC= 150 mM NaCl, 15 mM Na citrate) with 1% (w/v) sodium dodecyl sulphate (SDS) at42◦C. Finally, the membrane was washed in 0.2× SSC with 0.1% SDS (w/v) at 60◦C for15 min and exposed to X-ray film (Agfa) at−70◦C. The negative and positive controls were� DNA and the same DNA used as probe. Band dimension/intensity was determined usinga Quantity One Program Software (BR) and a Mitsubishi video with a Hantarex Cameraand Image Analysis Program.

2.4. Protein extraction and immunoblotting

Soluble proteins were extracted according toLaemmli (1970). SDS-polyacrylamide gelelectrophoresis was carried out on 12% (w/v) SDS polyacrylamide gels with equal amountsof proteins (20�g) in all lanes. After separation, denaturised proteins were transferred ontoa nitro-cellulose membrane (Stratagene, Hoeffer Scientific Instruments). The blots weredeveloped with 4-chloro-1-naphtol and H2O2 according toHarlow and Lane (1988). Aspositive controls, protein extracts fromS. tuberosumleaves were used (Pereira et al., 1992).


Band dimension/intensity was determined by using a Quantity One Program Software (BR)and a Mitsubishi video with a Hantarex Camera and Image Analysis Program.

2.5. Protein isolation and isoenzyme activities

For GS1 and GS2 activity determination, proteins were extracted from leaves, roots andcalluses according toBhatia et al. (1994)but with the addition of polyvinylpyrrolidone(PVP) 4% (w/v) to the extraction buffer. Protein was quantified by theBradford (1976)method using bovine serum albumin (BSA, Sigma) as standard. Separation of GS1 andGS2 was performed by chromatography according toBhatia et al. (1994)but with theaddition of PVP 4% (w/v) to the elution buffer. Activities of GS1 and GS2 were measuredusing Mg2+ dependent biosynthetic assay based on the�-glutamylhydroxamate formationaccording toBhatia et al. (1994). One unit of enzyme activity (U) represents the amount ofenzyme required to catalyse the formation of 1�mol of �-glutamylhydroxamate per minunder optimal assay conditions (Bhatia et al., 1994).

2.6. Ammonium quantification

For ammonium determination, leaf segments and calluses (n = 3) were homogenised insulphosalicylic acid at pH 3.5 and the content of free ammonium determined according toDüball and Wild (1988).

2.7. Fisher and t-tests

RNA expression, protein amount and activity and ammonium quantification were per-formed in triplicate (n = 3). Variance analysis was performed by Fisher test and the meanswere statistically tested using a two-sidedt-test. Statistical significance is assumed atP <

0.05.

3. Results

3.1. Influence of NaCl and PEG on cytosolic GS mRNA

Sunflower calluses contain more cytosolic glutamine synthetase mRNA than leavesand roots of cytosolic GS1 present in calluses (Fig. 1). The amounts of GS1 mRNA insalt-stressed leaves, calluses and roots were two to six times higher than in controls (Fig. 1).Osmotic stress increased the amount of GS1 transcript only in roots (approximately fourtimes) and leaves (approximately seven times) while no significant changes were observedin calluses.

3.2. Influence of NaCl and PEG on cytosolic GS polypeptide and enzyme activity

The antibody used in this work recognised both GS1 (44 kDa) and GS2 (40 kDa) polypep-tides with high efficiency. In control cells and for equal amounts of protein, calluses had


Fig. 1. “Dot-blot” showing changes of translatable mRNA for GS1 in leaves, roots andcallusesof sunflower, afterexposure to 0, 100 mM NaCl and to PEG 50 g/l; 0.5, 5 and 50�g correspond to the amount of RNA loaded in eachwell.

more GS1 polypeptide than leaves and roots (Fig. 2a), supporting the finding that calluseshad more GS1 mRNA. With this technique, GS2 was only detected, up to the moment, inleaves, while both roots and calluses showed only cytosolic GS (40 kDa). The concentrationof cytosolic GS increased in leaves (1.3 times), roots (1.4 times) and calluses (1.2) exposedto NaCl 100 mM. PEG stress also increased the concentration of GS1 polypeptides in leaves(1.7 times higher) and roots (1.6 times), but did not affect this isoenzyme concentration instressed calluses (Fig. 2a and b).

Once it was shown that NaCl and osmotic stress increased GS1 polypeptide quantityand its transcript, the next step was to evaluate if there was a corresponding increase ofGS1 activity. Enzymatic activity of control leaves showed higher activity of GS2 than GS1(Fig. 3a). However, salt stress increased GS1 activity 3.5 times, while GS2 activity decreased(Fig. 3b). Total GS activity decreased in NaCl-stressed leaves mainly due to the observeddecrease of GS2 activity while in PEG-stressed leaves GS2 was not affected (Fig. 3c). Also,GS1 activity increased 4.0 times in PEG-stressed leaves.

It was only found an active peak of GS activity in roots corresponding to GS1 (approx-imately at 126 mM NaCl, similar to the value found for GS1 in leaves) (Fig. 3d). GS1


Fig. 2. Images from one single blot (each well was loaded with 20 mg/protein) showing changes in polypeptides ofGS1 and GS2 in control, 100 mM NaCl and PEG-stressed leaves, roots andcalluses. (a) Crude extracts from leavescorresponding to the same amount of protein were electrophoresed and blotted. (b) Corresponding GS1 intensitydetermination: values in leaves, roots and calluses are expressed in percentage on a GS1 control leaf (100%)basis, control roots (100%) basis and control calluses (100%), respectively. Dimension/intensity was calculatedfrom three independent assays (always the same amount of protein loaded in each well). Vertical bars: Standarddeviation; S: statistically different; NS: statistically not different means (P < 0.05).

activity increased in salt (6.2 times) and PEG-stressed roots (6.9 times), respectively, tocontrol roots (Fig. 3e and f). In control calluses, GS1 and GS2 separation showed onlyone active peak (at 126 mM NaCl) corresponding to GS1 (Fig. 3g). Calluses exposed toNaCl had an activity of cytosolic GS slightly higher (1.26 times) than control calluses(Fig. 3h).

The addition of PVP 4% to the extraction and the elution buffers was necessary forthe successful separation of GS isoenzymes in PEG-stressed cells (Fig. 3c, f and i). Inplants, PEG induced higher increases of GS1 activity than NaCl. However, PEG stress didnot affect GS1 activity in calluses or GS2 activity in leaves. Therefore, total GS activityincreased in PEG-stressed leaves at the expenses of GS1 activity increase, while it decreasedin NaCl-stressed leaves mainly due to the decrease of GS2 activity.

3.3. Influence of NaCl and PEG stress on ammonium content

Ammonium levels increased in leaves and roots exposed to NaCl and PEG and inNaCl-stressed calluses, but were not affected in calluses exposed to PEG (Table 1).


Fig. 3. Activities of GS isoforms (20�g of total soluble proteins) in sunflower leaves: control (a), NaCl-stressed(b) and PEG-stressed (c). Activities of GS isoforms in roots: control (d), NaCl-stressed (e) and PEG-stressed (f)of sunflower. Activities of GS isoforms in sunflower calluses: control (g), NaCl-stressed (h) and PEG-stressed (i).Isoenzymes were separated by ion-exchange chromatography. (—) GS activity; (- - -) NaCl gradient.

Table 1Changes in free ammonium content (expressed in relation to gram of fresh weight) in sunflower leaves, roots andcalluses exposed to 0 and 100 mM NaCl and to PEG 50 g/l

Leaves (�mol g−1 FW) Roots (�mol g−1 FW) Calluses (�mol g−1 FW)

Unstressed 3.8± 0.3 aa 3.1± 1.2 a 7.8± 2.1 aPEG-stressed 8.2± 1.1 b 9.3± 1.7 b 8.3± 2.4 aNaCl-stressed 12.7± 1.0 c 11.8± 1.9 b 16.3± 3.6 b

a Values are the mean± S.E. of three independent determinations. Different letters mean statistically differentmeans (P < 0.05) from the control.


4. Discussion

Previous studies have shown that salt and water stress decreased chlorophyll and proteincontents and induced membrane degradation and nutritional imbalance in calluses of thiscultivar (Santos and Caldeira, 1999; Santos et al., 2002) leading to cell senescence. Inparticular, the effect of salt stress on N metabolism has been largely studied. For example,osmotic stress and salt stress reduced nitrate content in sunflower while ammonium levelsincreased (Santos et al., 2002).

The increase of ammonium levels found in salt-stressed sunflower cells, together withthe increase of free amino acid levels, previously found in salt-stressed cells (Santos andCaldeira, 1999; Santos et al., 2002) may result, at least in part, from an increase of proteolysis(Santos and Caldeira, 1999). This protein degradation in regions under senescence maysupply some of the carbon and nitrogen necessary for the development of other regions inthe plant (Bauer et al., 1997).

As most of the nitrogen transport in plants is done in the form of Asn or Gln, aminoacids released during proteolysis may have a role in both osmotic adjustment and in thesynthesis of other amino acids (e.g. Gln), requiring the activation of glutamine synthetase(Bauer et al., 1997). The ammonium accumulation reported here together with the increaseof free amino acid levels that was previously reported for these salt-stressed cells (Santosand Caldeira, 1999) seem to be associated with an induction of GS1 expression in sun-flower. These facts support the idea that GS1 is involved in regulating ammonium releasedduring proteolysis induced by salt stress. In fact, under stress conditions, that acceleratesenescence symptoms, an increase of GS1 activity can be expected increasing Gln levels.This isoenzyme will therefore, and by the same reaction, stimulate N translocation throughthe plant (due to increases of Gln that may be translocated through the phloem) and controlammonium levels that increase under salt stress conditions. Similar postulations were pro-posed for other species under different stress conditions.Perez-Garcia et al. (1998)found anovel GS1 isoform expressed in photosynthetic cells of infected tomato leaves, suggestingthat this novel isoform may have an important role in N remobilization from rubisco andother chloroplastic protein degradation. Also,Bauer et al. (1997)found a significant de-crease of rubisco content in water-stressed tomato leaves, together with an increase of GS1expression. These findings strongly support the proposed role for GS1 in the assimilation ofammonium released during cell senescence (Bauer et al., 1997). Other authors also found acorrelation between senescence and the increase of GS1 expression (Kamachi et al., 1991,1992; Wollaston, 1997).

To our knowledge, this is the first report to GS1 expression (GS1 mRNA, GS1 polypep-tide and enzymatic activity) in in vitro cells exposed to NaCl. Calluses had a higher GS1expression than plants. This fact may be explained by the high concentration of nitrogen(NO3

− and NH4+) in the MS medium obligating callus cells to metabolise the ammonium

probably using GS1, but this subject should be further clarified in the future. The presence ofnitrate in the medium was also found to stimulate GS1 activity in other species (Cordovillaet al., 1996), and a stimulation of GS1 activity was also reported in sunflower cotyledonsgrowing in the presence of nitrate (Haba et al., 1992).

Only PEG-stressed calluses did not show a significant change in GS1 mRNA and polypep-tide contents. Contrarily, PEG- and NaCl-stressed plants had an increase of GS1 polypeptide


contents. Similar results were obtained in water-stressed tomato plants (Bauer et al., 1997),in Picea abies(Manderscheid and Jager, 1993) and inMorus albaplants (Ramanjulu andSudhakar, 1997) under osmotic stress.Santos and Caldeira (1999)reported that sunflowercalluses are more tolerant to 50 g/l PEG than plants. This higher sensitivity exhibited byplants may explain the higher increases of ammonium levels observed in plants under PEGstress when compared with calluses, together with lower levels of soluble protein contents(Santos and Caldeira, 1999) that may be involved in a higher induction of cytosolic GSactivity.

This report shows that salt stress decreases total GS activity in sunflower leaves (althoughincreasing GS1 activity). The decrease of GS activity under salt stress seems therefore tobe due to a decrease of plastidial GS, corroborating other authors that report a decrease ofGS2 activity under senescence conditions (Kamachi et al., 1991, 1992; Perez-Rodriguezand Valpuesta, 1996). Contrarily, osmotic stress increases total GS activity by increasingGS1 activity and having no effect on GS2 activity. Osmotic and salt stress effects on GSactivity are still controversial:Bhullar et al. (1996)found an increase of GS2 activity withosmotic stress andBourgeais-Chaillou et al. (1992)found a reduction in total GS activity inroots exposed to NaCl stress but an increase in leaves. However, these authors did not searchfor isoenzyme variations, and therefore the influence of NaCl on GS1 and GS2 expressionwas not followed.

Other stress conditions were reported to decrease total GS activity: KNO3 stress(Cordovilla et al., 1996), senescence induced by cuttings (Jacoby et al., 1994) or by in-fection (Perez-Garcia et al., 1995). These last authors showed, however, that this decreaseof total GS activity masked an increase of GS1 activity. Similarly to their results, this reportshows that NaCl and PEG stress may induce a decrease of total GS activity but increaseGS1 activity and GS1 transcript in sunflower cells. We propose that the induction of GS1by salt/osmotic stress is related to senescence mechanisms in stressed cells—where highlevels of ammonium are accumulated (most probably due to increased proteolysis), andthat it is essential for N remobilization and ammonium levels control in the cell. Besidesthis proposed role for GS1, the correlation of this isoenzyme and the proline biosynthesisunder stress is not clear yet. In their study with plants and calluses of this genotype ex-posed to NaCl,Santos and Caldeira (1999)reported that NaCl increased proline level whileglutamate decreased. This fact may indicate a substantial flux from Glu to proline and thepossible link between GS1 activity and this “proline pool” under stress conditions must bestudied in more detail.

Acknowledgements

This work was supported by FCT project PNAT/1999/AGR/15011/C.

References

Bauer, D., Biehler, K., Fock, H., Carrayol, E., Hirel, B., Migg, A., Becker, T., 1997. A role for cytosolic glutaminesynthetase in the remobilisation of leaf nitrogen during water stress in tomato. Physiol. Plant. 99, 241–248.


Bhatia, P., Mahajan, M., Vaidyanathan, C., 1994. Chloroplastic glutamine synthetase from normal andwater-stressed saflower (Carthamus tinctoriusL.) leaves. Plant Sci. 95, 153–164.

Bhullar, S., Desamsetty, N., Behura, S., Singh, P., 1996. Differential changes in glutamine synthetase isoforms incotyledons of germinating chickpea (Cicer arietinumL.) seeds in response to osmotic stress. J. Plant. Physiol.149, 201–204.

Bourgeais-Chaillou, P., Perez-Alfocea, F., Guerrier, G., 1992. Comparative effects of N-sources on growth andphysiological responses of soybean exposed to NaCl stress. J. Exp. Bot. 43 (254), 1225–1233.

Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizingthe principle of protein binding. Anal. Biochem. 72, 248–254.

Carvalho, H., Pereira, S., Sunkel, C., Salema, R., 1992. Detection of a cytosolic glutamine synthetase in leaves ofNicotiana tabacumby immunocytochemical methods. Plant Physiol. 100, 1591–1594.

Cordovilla, M.P., Ligero, F., Luch, C., 1996. Growth and nitrogen assimilation in nodules in response to nitratelevels inVicia fabaunder salt stress. J. Exp. Bot. 47, 203–210.

Cullimore, J.V., Mifflin, B., 1984. Immunological studies on glutamine synthetase using antisera raised againsttwo plant forms of enzyme fromPhaseolusroot nodules. J. Exp. Bot. 35, 581–587.

Düball, S., Wild, A., 1988. Investigation on the nitrogen metabolism of spruce needles in relation to the occurrenceof novel forest decline. J. Plant. Physiol. 132, 491–498.

Finnemann, J., Schjoerring, J., 1998. Ammonium and soluble amide-bound nitrogen in leaves ofBrassica napusas related to glutamine synthetase activity and external N supply. Plant Physiol. Biochem. 36 (5), 339–346.

Finnemann, J., Schjoerring, J., 1999. Translocation of NH4+ in oilseed rape plants in relation to glutamine

synthetase isogene expression and activity. Physiol. Plant. 105, 469–477.Grattan, S., Grieve, C., 1994. Mineral element acquisition and response of plants grown in saline environments.

In: Pessaraki, A. (Ed.), Handbook of Plant and Crop Stress. Marcel Dekker, NY, pp. 203–227. ISBN0-8247-8987-3.

Haba, P., Cabello, P., Maldonado, J., 1992. Glutamine-synthetase isoforms appearing in sunflower cotyledonsduring germination. Planta 186, 577–581.

Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Springer Verlag, NYJacoby, A., Katinakis, P., Werner, D., 1994. Artificially induced senescence of soybean root nodules affects different

polypeptides and nodulins in the symbiosome membrane compared to physiological ageing. J. Plant. Physiol.144, 533–540.

Jones, M., Osmond, C., Turner, N., 1980. Accumulation of solutes in leaves of sorghum and sunflower in responseto water deficit. Aust. J. Plant. Physiol. 7, 193–205.

Kamachi, K., Yamaya, T., Mae, T., Ojima, K., 1991. A role for glutamine synthetase in the remobilization of leafnitrogen during natural senescence in rice leaves. Plant Physiol. 96, 411–417.

Kamachi, K., Yamaya, T., Mae, T., Ojima, K., 1992. Changes in cytosolic glutamine synthetase polypeptide andits mRNA in a leaf blade of rice plants during natural senescence. Plant Physiol. 98, 1323–1329.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriofage T4. Nature227, 680–685.

Lin, J., Kao, C., 1998. Water stress, ammonium, and leaf senescence in detached rice leaves. Plant Growth Regul.26, 165–169.

Lutts, S., Kinet, J., Bouharmont, J., 1996. NaCl induced senescence in leaves of rice (Oryza sativa) cultivarsdiffering in salinity resistance. Ann. Bot. 78, 389–398.

Manderscheid, R., Jager, H., 1993. Seasonal changes in nitrogen metabolism of spruce needles (Picea abiesL.Karst) as affected by water stress and ambient air pollutants. J. Plant. Physiol. 141, 497–501.

Meidner, H., 1984. Class Experiments in Plant Physiology. G. Allen & Unwin, London.Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures.

Physiol. Plant. 15, 473–497.Pereira, S., Carvalho, H., Sunkel, C., Salema, R., 1992. Immunocytolocalization of glutamine synthetase in

mesophyll and phloem of leaves ofSolanum tuberosumL. Protoplasma 167, 66–73.Perez-Garcia, A., Cánovas, F., Gallardo, F., Hirel, B., Vicente, A., 1995. Differential expression of glutamine

synthetase isoforms in tomato detached leaflets infected withPsudomonas syringaepv. Tomato. MPMI 8,96–103.

Perez-Garcia, A., Pereira, S., Pissara, J., Gutierrez, A., Cazorla, F., Salema, R., Vicente, A., Cánovas, F., 1998.Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response tobacterial infection or phosphinothricin treatment. Planta 206, 426–434.


Perez-Rodriguez, J., Valpuesta, V., 1996. Expression of glutamine synthetase genes during natural senescence oftomato leaves. Physiol. Plant. 97, 576–582.

Ramanjulu, S., Sudhakar, C., 1997. Drought tolerance is partly related to amino acid accumulation and ammoniaassimilation: a comparative study in two mulberry genotypes differing in drought sensitivity. J. Plant. Physiol.150, 345–350.

Robertson, J., Farnden, K., Warburton, M., Banks, J., 1975. Induction of glutamine synthetase during noduledevelopment in lupin. Aust. J. Plant. Physiol. 2, 265–272.

Santos, C., Caldeira, G., 1999. Comparative responses ofHelianthus annuusplants andcallusesexposed to NaCl.I. Growth rate and osmotic regulation in intact plants andcalluses. J. Plant. Physiol. 155, 769–777.

Santos, C., Campos, A., Azevedo, H., Caldeira, G., 2001. Nutritional imbalance and senescence induced in plantsand calluses exposed to KCl. J. Exp. Bot. 52, 351–360.

Santos, C., Pinto, G., Loureiro, J., Oliveira, H., Costa, A., 2002. Response of sunflower cells under Na2SO4. I.Osmotic adjustment and nutrient responses and proline metabolism in sunflower cells under Na2SO4 stress.J. Plant Nut. Soil Sci. 165 (3), 366–372.

Teixeira, A., Pereira, S., Sunkel, C., Salema, R., 1996. Isolation and characterization of glutamine synthetase-relatedcDNA sequences fromSolanum tuberosumL. In: Proceedings of the XXXI Annual Meeting of the PortugueseSociety for Electron Microscopy and Cell Biology, SPMEBC-Cell and Molecular Biology’96, Oeiras, Portugal.

Wollaston, V.B., 1997. The molecular biology of leaf senescence. J. Exp. Bot. 48 (307), 181–199.

regulation of glutamine synthetase expression in sunflower cells exposed to salt and osmotic stress

Documents