Purification and Characterization of Glutamine Synthetase Isoenzymes from Leaves and Roots of Brassica napus (L.)

]. Plant PhysioL Vol. 147. pp. 1-8 (1995)

Purification and Characterization of Glutamine Synthetase Isoenzymes from Leaves and Roots of Brassica napus (L.) GUNTHER OCHS, GERALD SCHOCK,

and ALOYSIUS WILD*

Institut fUr Allgemeine Botanik, Johannes Gutenberg-Universitlit, 55099 Mainz, Germany Received September 8,1994 . Accepted May 22,1995

Summary

The glutamine synthetase enzymes from leaves (GS2) and roots (GSR) of Brassica napus L. have been purified to homogeneity by the application of a three-stage isolation procedure comprising anion-exchange chromatography, adsorption by hydroxylapatite and gel filtration on Sephacryl S-300 HR. The isoforms of the enzyme show a differential distribution in leaf and root tissues. Elution profiles of hydroxylapatite chromatography showed a distinct behaviour for GS proteins found in leaves and roots. Denaturing SDS-PAGE and Western blot experiments revealed molecular masses of approximately 43.5 and 40.5 kD for GS2 and GSRsubunits, respectively. Moreover, kinetic properties determined using crude extracts confirmed different physiological roles for both GS enzymes, especially pH-optima and apparent Km-values. The affinity for glutamate was observed to be six times higher for GSR compared with GS2 • Northern analysis of leaf and root RNA revealed two distinct GS mRNAs: a 1.6 kb GS transcript was present in leaves, while a 1.4 kb GS mRNA was detected in roots. These results provide evidence for the predominant existence of a plastidic glutamine synthetase in leaves, whereas the roots of Brassica napus contain mainly cytosolic GS isoenzymes.

Key words: Brassicaceae; Brassica napus; allopolyploid; ammonia assimilation; glutamine synthetase; isoenzymes; protein purification. Abbreviatiom: GOGAT .. glutamine-oxoglutarate aminotransferase; GS .. glutamine synthetase; GS 1 "" cytosolic GS; GS2 .. plastidic GS; GSR .. root GS; IgG .. immunoglobulin G; TBS .. Tris-buffered saline; TBBS .. TBS + 0.5 % (w/v) BSA; SSC .. standard saline citrate. IntroductIon

In higher plants the GS/GOGAT cycle represents the main pathway for assimilation of ammonia (Lea, 1993). In this respect glutamine synthetase (GS; EC 6.3.1.2) occupies the same key position in nitrogen metabolism as ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) does in carbon metabolism. Ammonium is produced by a diversity of primary and secondary processes, such as seed germination, photorespiration, nitrite reduction, dinitrogen fixation, phenylpropanoid metabolism, and primary ammonia assimilation from the 1

Correspondence.

© 1995 by Gustav Fischer Verlag, Stuttgan

soil (Miflin and Lea, 1980; Wallsgrove and Lea, 1985). Glutamine synthetase therefore occurs in multiple isoenzymic forms that are associated with different organs and cell compartments (Stewart et al., 1980; McNally et al., 1983; Cullimore et al., 1984). In the case of leaves and green tissues two distinct GS proteins are commonly found. These are localized in the chloroplast (GS2) and in the cytosol (GS 1). The plastidic GS isoenzyme is usually much more abundant than the cytosolic GS in leaves of most C3 plants (McNally et al., 1983). Some C 3 species, including spinach, tobacco and mustard, exhibit only GS2 activity in green leaves (Hirel et al., 1984; Ericson, 1985; Hopfner et al., 1988; Becker et al., 1992). The isolation of barley mutants lacking GS2 activity confirmed the sugges-

2

GONTHEll OCHS, GEIlALD ScHOCK, and ALOYSIUS WILD

tion that ammonia released during photorespiration in C 3 plants is reassimilated by the chloroplast GS (Blackwell et al., 1987; Wallsgrove et al., 1987; Edwards and Coruzzi, 1989). Roots generally contain cytosolic GS isoenzymes (GSR), which are almost indistinguishable from the leaf cytosolic enzyme (GS 1). The kinetic and regulatory properties of the root and leaf eytosolic GS were identical in barley (Mann et al., 1980) and in rice (Hirel and Gadal, 1980). Further investigations concerning the protein structures of GS 1 and GSR in bean (Lara et al., 1984), pea (Tingey et al., 1987) and soybean (Hirel et al., 1987) indicate that both holoenzymes possess similar arrangements of subunits. Determination of the subunit molecular weights of plastidic and eytosolic gluti\mine synthetases based on SDS-PAGE reveal that the chloroplast subunit is slightly larger (43-45kD) than the cytosolic subunit (37 -43 kD) (Tingey and Coruzzi, 1987; Forde and Cullimore, 1989). In the past, extensive analysis of the expression of GS genes in legumes has been reported (Donn et al., 1984; Tingey et al., 1987; Lightfoot et al., 1988; Tingey et al., 1988; Forde and Cullimore, 1989; Walker and Coruzzi, 1989; Edwards et al., 1990; Cock et al., 1992). Previous studies pertaining to GS isoenzyme patterns and GS gene expression in non-legume species have been carried out (Sakamoto et al., 1989; Hopfner et al., 1991; Peterman and Goodman, 1991; Becker et al., 1992; Sakakibara et al., 1992). In this study we report the purification and characterization of GS polypeptides from another non-legume plant (Brassica napus) a field crop with increasing importance.

Materials and Methods

Plant Material and Growth Conditions Seeds of winter oilseed rape (Brassica napus L. cv. Arabella) were germinated in burnt clay (Lecaton) with Hoagland solution for 5 to 6days as described previously (Wild and Manderscheid, 1984). Seedlings were then placed in special constructions, which allowed a hydroponic culture. Plants were illuminated with light of approximately 6OWm- 2 (HQI 2000W, Osram) for a 16h day. During plant growth the temperature was kept constant at 21°C with an approximate relative humidity of 75 %.

!'reparation ofCrude Extracts Green leaves and roots were harvested from 21-day-old plants and homogenized as described earlier (Hopfner et al., 1988). In the case of leaves, approximately 60 g wet weight material was extracted with two volumes of TMME-buffer (50 mmol/L Tris-HCI, pH 8.3, 1mmoVL MgS04, 0.5 mmoVL EDTA, 30 mmol/L 2-mercaptoethanol). Homogenization of roots (approximately loog) was per· formed with one volume TMME-buffer. DEAE·Seph~el

Chromatography

The crude extracts were applied to a DEAE-Sephacel column (6x3cm), previously equilibrated with TMME-buffer, pH 7.8. Unbound proteins were washed off by rinsing the chromatographic matrix with several volumes of equilibration buffer. In the case of GS purification from root tissue, the washing buffer was supple-

mented with 80mmol/L. Linear KCI-gradients (O-400mmol/L and 80-4oommol/L) in TMME-buffer were used to elute leaf or root proteins, respectively. The flow rate was adjusted to 48 mL h- I and fractions (8 mL) were collected.

Hydro;rylapatite Chromatography Fractions containing the majority of GS activity were pooled and layered on a hydroxyl-apatite column (11 x 2 cm) equilibrated with TMME-buffer, pH 7.8. After loading with partially purified leaf extract, the column was rinsed with TMME-buffer, followed by elution with a linear gradient of 100 to 350 mmol/L K2HPO•. In the case of the partially purified root extract, the washing buffer included 60 mmol/L K2HP0 4 and the proteins were eluted using a 60 to 200 mmol/L K2HP0 4 gradient in washing buffer. Fractions (7 mL) were collected at a flow rate of 24 mL h -1.

Gel Filtration After hydroxylapatite chromatography, the pooled fractions were concentrated approximately 3D-fold by ultraft.ltration (Ornegacell/OlO, Filtron). Concentration was carried out on ice and the pressure regulated to 200 kPa. Finally, a volume of 1mL was loaded on a Sephacryl $0300 HR column previously equilibrated with TMME-buffer, pH 7.8. Elution was performed using the above buffer at a flow rate of 10 mL h -1 and fractions (1 mL) were collected.

Assay Methods GS activity was determined as described by Hopfner et al. (1988) using the synthetase reaction (Stewart et al., 1980). One unit of activity (U) represents the formation of J'-glutamylhydroxamate in limol min -I. The protein content was determined colorimetrically by the dye binding method of Bradford (1976) using ovalbumin as a standard.

Denaturing Polyacrylamide Gel Electrophoresis Molecular weight determinations of GS subunits were performed using an SD$oPAGE system according to Laemmli (1970). This system employed a 10 to 20 % (w/v) analyzing gradient gel and a 2.4 % (w/v) stacking gel. Electrophoresis (180V, 20 rnA) was carried out at 4°C. For molecular weight estimations, the following protein standards were used: phosphorylase b (92.5 kD), BSA (67 kD), ovalbumin (45kD), carboanhydrase (29kD), soybean trypsin inhibitor (21 kD), cytochrome c (12.5 kD), and bovine lung trypsin inhibitor (6.5kD). After separation, the proteins were either stained with silver using a method based on that of Meccil et al. (1981) or the gels were subjected to Western blot analysis.

Western Blot Analysis Following electrophoresis, proteins were transferred to a sheet of nitrocellulose according to the method of Bjerrum and SchaferNielsen (1986). After transfer, the nitrocellulose blot was blocked by immersion in 3 % (w/v) BSA dissolved in Tris-saline buffer (20 mmoVL TrisHCI, pH 7.5, with 150 mmol/L NaCl) for 1h. After five washing steps (each 10 min) in TB$obuffer containing 0.5% (w/v) BSA, the filters were incubated overnight at 37°C with a polyclonal antibody raised against purified mustard plastidic GS (Hopfner et al., 1990). This primary antibody was diluted 1: 500 in TBB$obuffer. The blots were then washed five times for 10 min each in TBB$obuffer prior to incubation (1 h, 37°C) with horseradish peroxidase con-

Isoenzymes of Glutamine synthetase in rape

3

jugated to goat antibodies raised against rabbit IgG. Finally, the filters were washed with TBS alone and peroxidase activity was visualized by incubation with TBS-buffer containing 0.05% (w/v) 3,3'diaminobenzidine and 0.003 % (v/v) H 20 2•

Kinetic Studies In order to investigate the kinetic properties of GS isoenzymes from leaf and root tissue, freshly prepared crude extracts were used. The homogenization buffer contained 20 mmol/L DTE instead of 2-mercaptoethanol, because preliminary examinations revealed that glutamate saturation could not be obtained by using 2-mercaptoethanol. The apparent Km-values for glutamate, ATP and NH 20H were determined by assaying GS activity at different substrate concentrations. For each Km-determination only one substrate concentration was varied while the others were kept at saturation. LineweaverBurk double reciprocal plots were used to evaluate the achieved results and to calculate the Km-values. The pH-optima curves were recorded by employing a buffer combination composed of 0.2 mol/L sodium acetate (pH 5.6 - 6.4) and 0.2 mol/L imidazole-HCI (pH 6.4-7.4).

Preparation a/Total RNA Frozen leaf or root material was ground in liquid nitrogen using a mortar and pestle. The fine powder was mixed with 10 mL RNAbuffer (50mmol/L Tris-HCI, pH 7.6, 2.5mmol/L MgCh, 1 % [w/v] NaCI) and 10 mL phenol/chloroform/isoamylalcohol (25: 24: 1; v/v/v). The mixture was swirled vigorously for 15 min and centrifuged at 5000 x g for 20 min. The aqueous phase was collected and the nucleic acids were precipitated with one volume isopropanol. DNA and small RNA species were removed by CsCI density-gradient centrifugation (17 h, 15°C, 220,000 x g). The pellets of total RNA were dissolved in RNA-TE (10mmol/L Tris-HCI, pH 7.6, 1mmol/L EDTA), precipitated with 2.5 volumes of ethanol and stored at -70°C.

67kD

67kD

45kD

45kD

29kD

29kD

21 kD

21 kD

1 25kD

6,SkD

12,5 kD

6,5kO

Fig. 1: Denaturing polyacrylamide gel electrophoresis of leaf and root extracts at different stages of the purification of GS. Protein concentrations: crude extracts (leaf, 8.5 J.Lg; root, 5.1 J.Lg), DEAESephacel (leaf, 8.0 J.Lg; root, 1.2 J.Lg), hydroxylapatite (leaf, 0.1 J.Lg; root, 1.6 J.Lg), Sephacryl S-3oo HR (leaf, 0.08 J.Lg; root, 0.3 }lg). Protein was visualised by silver staining. Positions of the molecularweight markers are indicated.

Results

Purification and Chromatographic Properties Northern Blot Analysis Total RNA (10 }lg/lane) was denatured with formaldehyde and separated on 1.2 % agarose gels containing 0.66 mol/L formaldehyde as described in Davis et al. (1986). The RNA patterns were transferred to nylon membranes (Duralon-UV, Stratagene) by vacuum blotting using 20 x SSC (1 x SSC contains 150 mmol/L NaCl and 15mmol/L NaJ-citrate, pH 7.0) and were immobilized by irradiation with short-wave UV light for 2 min. Prior to hybridization (42°C, 22h), the blots were prehybridized at 42°C for at least 2 h in the hybridization buffer (50 % formamide, 5xSSC, 5 x Denhardt's solution, 1% SDS and O.lgL -I denatured herring sperm DNA). The probes were labeled according to Feinberg and Vogelstein (1983) using a 32 P-dATP and the Megaprime DNA labelling system (Amersham, Braunschweig). The blots were washed with 1 x SSC, 0.1 % SDS at room temperature for 10 min followed by three washes with 0.1 x SSC, 0.1 % SDS at 55°C for 15 min and exposed to X-ray film (X-OMAT AR, Kodak) at -70°C for 22 h. Two full-length GS cDNAs (BnGS18, BnGSR1) were used as hybridization probes. BnGS 18 encodes the chloroplastic GS subunit and was isolated from a leaf cDNA library (Ochs et al., 1993). BnGSRl encodes a smaller eytosolic GS subunit and was found in a root specific cDNA library (Schock et al., 1994).

Both isolations delivered nearly pure GS enzyme preparations (Fig. 1). In the case of leaf enzyme, GS2 , the enrichment was 42.5-fold, whereas the isolation procedure yielded a 145-fold purification of the root specific GSR. Moreover, small aliquots of each purification step were analyzed by SDS-PAGE and silver staining, demonstrating homogeneity of both preparations (Fig. 1). The elution patterns from a DEAE-Sephacel column and a hydroxylapatite column invariably showed a single peak of GS activity (Fig. 2) and thus support the evidence that not only green leaves, but also roots of Brassica napus contain one predominant isoenzyme of GS. Both enzymes eluted from the anion-exchange column at similar ionic strengths, approximately 200 mmoVL and 180 mmoVL KCI, for GS2 and GSR, respectively (Fig. 2 A + C). Contrary to this, elution properties during hydroxylapatite chromatography revealed evident differences. The leaf specific enzyme eluted at approximately 260 mmol/L K2 HPO., whereas the root GS lett the column at a lower salt concentration of approximately 110mmoVL (Fig. 2 B+D). This behaviour supported the assumption that leaves and roots of Brassica napus express two different GS isoenzymes,

4

GONTHER OCHS, GERALD SCHOCll, and ALOYSIUS WILD

Table 1: Purification of glutamine synthetase from rape leaves and roots. Recovery

Total protein [mg]

Total GS activity [units]

Specific activity [U/mg protein]

[%]

Leaf enzyme (GS2) Crude extract DEAE-Sephacel Hydroxylapatite Sephacryl 5-300 HR

1070.0 606.0 9.0 3.2

390 248 133 49

0.36 0.41 14.80 15.30

100.0 63.6 34.1 12.6

1.0 1.1 41.1 42.5

Root enzyme (GSR) Crude extract DEAE-Sephacel Hydroxylapatite Sephacryl 5-300 HR

936.0 55.0 7.4 0.3

243 116 23 11

0.26 2.11 3.11 37.70

100.0 47.7 9.5 4.7

1.0 8.1 12.0 145.0

Purification step

Hydroxylapatite

DEAE-Sephacel

m

Q)

KCI[M]

GS activity [Ulmq

5

A

4

0.4

2

0

0.6

10

20

30

40

4

0.2

2

0

0

0.4 0.2 0 10

0

50

20

30

40

2

C

4

0

0.8

0.8

1.5

3 2

0

0.8

6

0

~

B

0.6

5

-

K 2 HPO.[M]

8 0.8

3

0

Purification factor [fold]

0.6

0.6

0.4

0.4

0.2 0.5

0.2

0 0

10

20

30

40

50

fractions

0

0 0

10

20

30

40

fractions

Fig. 2: Elution of DEAE- phacel column chromatography (A + C) and hydroxyl-apatite column chromatography (B + D) from leaf (A + B) and root extracts (C+ D). - - protein (reI. A2IO) G5-activity A--A salt gradient.

corresponding to the distribution of GS isoforms found in other higher plants (Forde and Cullimore, 1989).

Molecular Weight Determination ofGS Subunits The root preparation yielded a single polypeptide with an apparent molecular mass of approximately 40.5 kD (Fig. 1), confirming that native root GS (GSR) consists of identical subunits. In contrast, the isolation of leaf GS revealed two protein bands with slight differences in size by SDS-PAGE

(43.8 and 43.3 kD; Fig. 1). Such a doublet has also been observed during purification of plastidic GS enzymes from other plants, for example Pisum sativum (Tingey et al., 1987), Phaseolus vulgaris (Lightfoot et al., 1988), or Lycopersicon esculentum (Valpuesta et al., 1989; Becker et al., 1992). Assuming that GS 2 and GSR are composed of eight identical subunits the holoenzymes would have molecular masses of approximately 350 and 320 kD, respectively. These estimations are in good agreement with values obtained by native PAGE (data not shown).

Isoenzymes of Glutamine synthetase in rape

Immunoblotting To confirm the GS specific character of the purified proteins, SDS-polyacrylamide gels (Fig. 1) were subjected to Western blot analysis. The results presented in Fig. 3 show that antibodies raised against plastidic GS from Sinapis alba recognize the isolated polypeptides. In addition, it is notable that both bands of the GS2 doublet were detected. The faint bands also observed may be explained by un· specific cross reactivity of the polyclonal serum. For exam· pIe the diffuse band at about 50 kD can be explained by an artifactual reaction of the primary antibody with the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase, which is highly expressed in green tissues (Fig. 3, left panel). Nevertheless, very small amounts of plastidic GS subunit in root extract and cytosolic GS subunit in crude extract from leaf tissue were also detected.

5

Table 2: Functional properties of GS isoenzymes in leaves and roots of Brassica napus. Property

leaf enzmye

root enzyme (GSR)

(GS z)

pH optimum

K m (mM) Glu

NHzOH ATP

6.8

6.4

33 3 1.2

5 0.6 0.8

GS activity [%] 100

80

llGS aetlvtty [11%]

B

80

Kinetic Properties

40

Various substrate affinities of leaf and root localized GS enzymes are summarized in Table II. Comparison of apparent Km values for glutamate and hydroxylamine revealed striking differences, while ATP affinities for both enzymes appeared nearly identical. The greater than 6-fold glutamate affinity of GSR (Fig. 4), compared with that for GS2 , supports the existence of distinct GS isoforms in leaves and roots of Brassica napus.

20 0.2

0..

0.6

0.8

1.0

1/g1utamate [l/mM)

O+-----,-----,-----,r----,----c-----l

o

50

100

150

200

250

glutamate [mM]

Fig.4: Glutamate saturation curves (A) of leaf ( . - - . ) and root (0--0) crude extracts. The small inset (B) illustrates the double reciprocal presentation according to Lineweaver-Burk.

The pH-optima for GS 2 and GSR were found to be similar. . However, in comparison to GSR, it was obvious that the activity of GS2 appeared to be shifted towards a more alkaline pH (not shown).

Northern blot analysis 117kD

-ll7kD

UIID -

-UIID

2t1lD -

-2tkD

21110

-

12,8 lID CI,8kD

21 lID

-

12,8110

-

1,5110

Fig. 3: Western blot analysis of leaf and root extracts at different stages of the purification of GS. Extracts were subjected to SDS-PAGE (Fig. 1) and transferred to nitrocellulose. Filters were probed with mustard anti-GSrantibodies. Protein markers are indicated on the right and left margins of the blots.

In order to confirm the results obtained by conventional biochemical methods, Northern hybridization experiments were also performed to analyse the abundance of the corresponding GS mRNAs. When the cDNA for plastidic GS (BnGS 18; Dchs et al., 1993) was hybridized to total RNA from mature leaves, a large amount of a 1.6 kb transcript was detected. In contrast, the hybridization with a cDNA encoding a eytosolic GS subunit (BnGSRl; Schock et al., 1994) yielded low or undetectable levels of a 1.4 kb transcript (Fig. 5). In the case of total RNA extracted from root tissue, a vice versa hybridization pattern resulted (Fig. 5). Discussion

Early biochemical studies demonstrated that many higher plants contain distinct GS isoenzymes, resolvable by ion-exchange chromatography, which are associated with different

6

GONTHER OCHS, GERALD SCHOCK, and ALOYSIUS WILD

thaliana (Peterman and Goodman, 1991), and many other

1.&kb _

-1.4kb

BnGS18

BnGSR1

Fig. 5: Northern blot showing the expression of plastidic GS (BnGS18) and cytosolic GS (BnGSR1) in leaf and root tissue of Brassica napus. Each lane contained 10 I1g of total RNA. The blots were washed at high stringency and exposed to X-ray film for 22 h.

organs or subcellular compartments (McNally and Hirel, 1983; McNally et al., 1983). In contrast, Garda-Fernandez et al. (1994) recently suggested that the separation of two GS activities by ion-exchange chromatography does not conclusively mean the occurrence of two enzymic forms. From this, it may be concluded that the differentiation of GS isoenzymes by anion-exchange chromatography is an uncertain method that should be complemented by other experimental approaches. In view of these contradictory results we have purified the GS activities of leaves and roots from Brassica napus to apparent homogeneity utilising different chromatographic methods. The elution profiles obtained with DEAE-Sephacel revealed that conventional anion-exchange column chromatography is not suitable for the separation of cytosolic and plastidic GS isoenzymes from Brassica napus. Similar observations have been previously reported for other plant species of the Brassicaceae family, such as Raphanus sativus (Kawakami and Watanabe, 1988) and Sinapis alba (Schmidt and Mohr, 1989; Hopfner et al., 1990, 1991). In contrast, the hydroxylapatite chromatography of GS containing DEAE-Sephacel fractions revealed distinct elution properties. In common, both elution profiles showed a single GS activity peak suggesting the existence of a predominant GS enzyme in roots as well as in leaves. On the other hand, the peaks eluted at markedly different salt concentrations, indicating the existence of distinct GS isoforms. These data are further supported by SDS-PAGE, Western blot analysis, and kinetic studies. Denaturing polyacrylamide gel electrophoresis and immunoblotting revealed that Brassica napus. contains distinct size classes of GS polypeptides. In leaves mainly a 43.5 kD GS polypeptide was found while in roots a predominant 40.5 kD GS subunit was detected. Distinct GS polypeptides have also been resolved by Western blot analysis in Phaseolus vulgaris (Bennett and Cullimore, 1989), Pisum sativum (Tingey et al., 1987), Arabidopsis

plants. In all cases the larger (42-44kD) GS polypeptide is located in the chloroplast while the small polypeptides (37 40 kD) are cytosolic. By analogy it is likely that the leaf-specific polypeptide of Brassica napus is chloroplastic while the root-specific polypeptide is located in the cytosol. Moreover, our Western blot results implied the existence of two plastidic GS polypeptides with slightly different molecular weights. Two size variants of the GS2 subunit from Phaseolus vulgaris have already been discussed. Since similar variants were observed after in vitro import of a single precursor polypeptide into isolated chloroplasts, they are thought to be derived from processing at two different cleavage sites (Lightfoot et al., 1988). Alternatives for subunit heterogeneity in the chloroplast enzyme could be limited proteolysis, other post-translational modifications such as glycosylation and phosphorylation, the allopolyploid genome structure, or some artefacts arising during enzyme extraction (Nato et al., 1984; Valpuesta et al., 1989; Becker et al., 1992). Northern blot experiments indicate that leaves and roots of Brassica napus possess at least two distinct size variants of GS mRNA species. In total RNA isolated from rape leaves a predominant 1.6 kb transcript was found, whereas a large amount of a 1.4 kb mRNA was present in total RNA from root tissue. These results are consistent with the derived data of enzyme purifications, Western blot analysis and kinetic studies at the protein level. In conclusion, the work described in this paper has shown that the leaf and root tissues of Brassica napus contain two distinct GS enzymes, and for the first time the purification and characterization of a cytosolic GS isoform from roots of a Brassicaceae species has been reported. Further investigations are now in progress to study the expression and regulation of the Brassica napus GS gene family. Acknowledgements

We are grateful to Dr. S. Page and M. Page for their critical reading of the manuscript.

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Isoenzymes of Glutamine synthetase in rape

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