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and other nitrogen compounds within the plant. In: Stumpf PK,. Conn EE (eds) The biochemistry of plants, vol 5. Academic. Press, New York, pp 569±607.
Planta (1998) 206: 426±434

Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment A. PeÂrez-GarcõÂ a1, S. Pereira3, J. Pissarra3, A. GarcõÂ a GutieÂrrez2, F.M. Cazorla1, R. Salema3, A. de Vicente1 F.M. CaÂnovas2 1 Departamento de MicrobiologõÂ a, Facultad de Ciencias-Instituto Andaluz de BiotecnologõÂ a, Universidad de MaÂlaga, E-29071 MaÂlaga, Spain 2 Laboratorio de BioquõÂ mica y BiologõÂ a Molecular, Facultad de Ciencias-Instituto Andaluz de BiotecnologõÂ a, Universidad de MaÂlaga, E-29071 MaÂlaga, Spain 3 Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto 4150, Portugal

Received: 16 January 1998 / Accepted 21 April 1998

Abstract. In tomato (Lycopersicon esculentum Mill.) leaves, the predominant glutamine synthetase (GS; EC 6.3.1.2) is chloroplastic (GS2; 45 kDa) whereas the cytosolic isoform (GS1; 39 kDa) is represented as a minor enzyme. Following either infection by Pseudomonas syringae pv. tomato (Pst) or treatment with phosphinothricin (PPT), a GS inhibitor, GS1 accumulated in the leaves. In contrast to healthy control leaves, where GS1 was restricted to the veins, in infected and PPT-treated leaves the GS1 polypeptide was also detected in the leaf blade; moreover, it was more abundant than GS2. Di€erent immunological approaches were therefore used to investigate whether or not the GS1 polypeptide expressed in Pst-infected and PPTtreated tomato leaves was distributed among di€erent tissues and subcellular compartments in the same way as the constitutive GS1 expressed in healthy leaves. By tissue-printing analysis, a similar GS immunostaining was observed in epidermis, mesophyll and phloem of lea¯et midrib cross-sections of control, infected and PPT-treated leaves. Immunocytochemical localization revealed that GS protein was present in the chloroplast of mesophyll cells and the cytoplasm of phloem cells in healthy leaves; however, in Pst-infected or PPT-treated leaves, a strong labelling was observed in the cytoplasm of mesophyll cells. Two-dimensional analysis of GS polypeptides showed that, in addition to the constitutive GS1, a GS1 polypeptide di€erent in charge was present in tomato lea¯ets after microbial infection or herbicide treatment. All these results indicate that a novel cytosolic GS is induced in mesophyll cells of Pst-infected or PPT-treated leaves. A possible role for this new cytosolic GS in the remobilization of leaf nitrogen during infection is proposed.

Abbreviations: GS1 ˆ cytosolic glutamine synthetase; GS2 ˆ plastidic glutamine synthetase; MS ˆ Murashige and Skoog medium; Pst ˆ Pseudomonas syringae pv. tomato; PPT ˆ phosphinothricin Correspondence to: A. de Vicente; E-mail: [email protected]; Fax: 34 (95) 2132000

Key words: Glutamine synthetase ± Lycopersicon ± Nitrogen remobilization ± Phosphinothricin ± Plant defense ± Pseudomonas

Introduction The enzyme responsible for ammonium assimilation in higher plants is glutamine synthetase (GS, EC 6.3.1.2), which catalyzes the ®rst reaction in the main pathway for ammonium assimilation: the glutamine synthetase and glutamate synthase cycle (Lam et al. 1996). The products of this cycle, glutamine and glutamate, serve as nitrogen donors in the biosynthesis of essentially all amino acids, nucleotides and other nitrogen-containing compounds such as chlorophylls (Lea et al. 1990). In angiosperms, the ocurrence of two glutamine synthetases, a cytosolic isoform (GS1) and a chloroplastlocated isoenzyme (GS2) is well established (McNally and Hirel 1983). Molecular studies have shown that GS isoforms are encoded by a family of homologous but di€erent nuclear genes. All angiosperm plants examined to date appear to possess a single nuclear gene for chloroplastic GS2 and several genes for cytosolic GS1 (Cullimore et al. 1984; Peterman and Goodman 1991; Lam et al. 1996). In leaves, GS isoforms are expressed in di€erent cell types, suggesting that they play nonoverlapping physiological roles in plant metabolism. The isoform GS2 is localized in photosynthetic cells, where it could be involved in the assimilation of ammonium derived from nitrate reduction and photorespiration, generating glutamine for amino acid biosynthesis inside the chloroplast (Edwards et al. 1990). The isoform GS1 is associated with phloem cells, where it could be involved in the generation of glutamine for nitrogen transport (Carvalho et al. 1992; Kamachi et al. 1992). Tomato plants, like other solanaceaous species with very active photosynthetic metabolism, present high levels of GS2 in their leaves. In our laboratory, the predominant GS2 isoform of tomato leaves has been

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

Fig. 1. Immunoblot analysis of GS polypeptides in tomato leaves. Relative abundance of GS polypeptides on western blots of leaf blade (L) and leaf midrib (M ) extracts prepared from control leaves, infected with P. syringae pv. tomato (Pst) or treated with phosphinothricin (PPT ). In these experiments, detached leaves were maintained for 8, 10 and 5 d in MS medium, respectively. The size of major cross-reacting bands is indicated on the left. The GS polypeptides are marked on the right. Faster-migrating bands also cross-reacted with the GS antibodies in Pst and PPT treatments, but they were interpreted as degradation products of the intact GS polypeptides. The same amount of protein (25 lg) was loaded per lane

puri®ed, its molecular properties studied and its speci®c localization in the chloroplast determined by electron microscopy (CaÂnovas et al. 1984; Botella et al. 1988). Pseudomonas syringae pv. tomato (Pst) causes bacterial speck of tomato, and it has been suggested that the

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accumulation of ammonium may contribute to the development of the disease symptoms (Bashan et al. 1986). Phosphinothricin (PPT) is a GS inhibitor that has been widely used as a herbicide; its application induces a rapid accumulation of ammonium ions (Wild et al. 1987). In many di€erent plants, drastic metabolic changes in response to microbial infection have been extensively reported, basically the pathogenesis-related (PR) induction of proteins (Linthorst 1991). In previous work we have reported that during Pst-infection or PPTtreatment of tomato leaves, there is a change in the GS isoform pattern; GS2 is down-regulated and GS1 appears to be the predominant isoform in leaves, and its expression is regulated at the transcriptional level in a light-dependent fashion (PeÂrez-Garcõ a et al. 1995, 1998). The aim of this study was to determine the speci®c tissue in which this protein is accumulated and whether it is the same isoprotein as the constitutive cytosolic GS. The ®ndings reported here strongly suggest that a di€erent cytosolic GS is induced in tomato leaves in response to bacterial infection or herbicide treatment. Materials and methods Bacterial strain and culture conditions. Pseudomonas syringae pv. tomato strain HM-1 was isolated from infected tomato plants and identi®ed by standard biochemical tests, and by hypersensitivity and pathogenicity tests towards tobacco and tomato, respectively. This strain was routinely cultured on King's medium B at 27 °C (PeÂrez-Garcõ a et al. 1995).

Fig. 2A±D. Tissue-print localization of GS in tomato leaves. A Leaf cross-section stained with azure II/methylene blue to show the anatomy. B±D Tissue prints of leaf cross-sections on nitrocellulose from control (B; 8 d), infected (C; 10 d) and PPTtreated (D; 5 d) leaves. Prints were incubated with anti-GS antibodies and labelling revealed with peroxidase-conjugated antirabbit IgG. Immunolabelling was localized in the leaf blade, in the internal and external phloem, and in the epidermis. b, leaf blade; e, epidermis; ep, external phloem; ip, internal phloem; x, xylem. Bars ˆ 500 lm

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A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

Fig. 3A±D. Immunolocalization of GS by transmission electron microscopy in sections of mesophyll and phloem cells from healthy tomato leaves after immunogold staining. A Mesophyll cell showing appreciable labelling limited to the chloroplast. B Phloem cell showing abundant labelling in the cytoplasm. C, D When preimmune rabbit antiserum was used instead of primary antibody in control sections of mesophyll (C) and phloem cells (D), the nonspeci®c labelling was very low. ch, chloroplast; cy, cytoplasm; m, mitochondrion. Bars ˆ 1 lm Plant material and experimental design. Lycopersicon esculentum Mill cv. Hellfrucht FruÈhstamm (seeds were obtained from the Experimental Station La Mayora, CSIC, MaÂlaga, Spain) was grown from seed in a greenhouse under natural light. All experiments were performed using detached lea¯ets from 6 to 8week-old tomato plants. Prior to the di€erent experiments, tomato leaves were detached, disinfected and placed in Murashige and Skoog (MS) medium. Bacterial inoculations were carried out as described elsewhere (PeÂrez-Garcõ a et al. 1995). The PPT-treatment was performed using MS medium supplemented with 25 lM phosphinothricin (glufosinate) as a commercial formulation, Finale (Hoechts IbeÂrica, Barcelona, Spain. PeÂrez-Garcõ a et al. 1998). Detached leaves were incubated under a 16-h photoperiod (30 lmol photons m)2 s)1). Four to six tomato plants were harvested for each experimental time point of treatment. All experiments were repeated at least three times. Untreated and noninfected detached lea¯ets maintained in MS medium were included in all experiments as controls. Antibodies. Polyclonal antibodies raised against Scots pine (Pinus sylvestris L.) GS recombinantly expressed in Escherichia coli (CantoÂn et al. 1996) were used. Peroxidase-conjugated goat antirabbit IgG was obtained from Vector Lab. Inc. (Burlingame, Calif., USA). Goat anti-rabbit IgG conjugated with 15-nm colloidal gold was obtained from Amersham (Buckinghamshire, UK). Protein extraction, electrophoresis and western blot analysis. Extraction procedures, total protein determination in crude extracts and SDS-PAGE were carried out as described earlier (PeÂrez-Garcõ a et al. 1998). Two-dimensional electrophoresis was performed as described by Bollag and Edelstein (1991). After denaturing separation, proteins were electroblotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) according to CaÂnovas et al. (1991). The GS antibodies were used at 1:2500

dilution. Immunocomplexes were detected using peroxidase-conjugated antibodies. Peroxidase activity was viewed by incubation in H2O2 and 4-chloro-1-naphthol in phosphate-bu€ered saline. Tissue-print and immunoblot localization. Tissue prints were obtained by pressing freshly cut sections of lea¯ets, similar to those used for leaf extracts, onto a nitrocellulose membrane (Schleicher and Schuell). Antibody staining for GS on the tissue print was performed as described for western blots of SDS-PAGE gels, except that endogenous peroxidases were ®rst blocked by treatment of tissue prints with 1% periodic acid for 30 min (Pereira et al. 1992). Antigen-antibody complexes on the tissue prints were located with a Nikon microscope (Tokyo, Japan). For observation of lea¯et anatomy, cross-sections were ®xed as described below for immunogold cytolocalization. Semithin sections of the blocks were cut on an ultramicrotome (LKB, Uppsala, Sweden) and collected on glass slides. Sections were stained with 1% azure II, 1% methylene blue in 1% sodium borate and photographed under a Nikon microscope. Subcellular immunocytolocalization. Small lea¯et pieces were ®xed in 3% paraformaldehyde and 0.5% glutaraldehyde, 2% sucrose, 0.05% CaCl2 in 1.25% Pipes bu€er (pH 7.2) for 2 h. Following ®xation, leaf pieces were washed several times in 2% Pipes bu€er (pH 7.2) with 1 mM glycylglycine, dehydrated in a graded series of ethanol and embedded in LR White resin (London Resin Co., London, UK). Polymerization took place at 50 °C for 22 h. Ultrathin sections were cut on an LKB (Uppsala, Sweden) ultramicrotome and collected on uncoated 400-mesh grids. Immunogold labelling of GS was performed as described by Pereira et al. (1992) with minor modi®cations. Grids were ¯oated on a blocking solution of 1% globulin-free BSA, 0.2% Tween-20 and 3% NaCl made in PBS and incubated in primary antibody (anti-GS) diluted 1:100 in the same blocking solution. After several washes in

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

Fig. 4A±D. Immunolocalization of GS by transmission electron microscopy in sections of mesophyll and phloem cells from tomato leaves infected with P. syringae pv. tomato after immunogold staining. A±C Mesophyll cells showing substantial labelling in the cytoplasm and less in the chloroplast. D Phloem cell also showing substantial labelling in the cytoplasm. ch, chloroplast; cy, cytoplasm; m, mitochondrion; cw, cell wall. Bars ˆ 1 lm (A, C±D) and 0.5 lm (B) blocking solution, grids were incubated with the secondary antibody (anti-rabbit IgG conjugated with gold) diluted 1:25 in the blocking solution. The grids were then washed in the same solution and double distilled water, stained with uranyl acetate and lead citrate, and viewed in a Zeiss (Oberkochen, Germany) electron microscope. Control sections were prepared as experimental sections except for the fact that primary antibody was replaced with rabbit preimmune serum. Determination of amino acid content. Total amino acids were extracted from tomato lea¯ets in a mixture of 40 mM Li2CO3/ methanol (1/4, v/v) at pH 9.5. Extracts were clari®ed by centrifugation at 15 000 g and the supernatants concentrated by evaporation at 80 °C. Amino acids were derivatized using dansyl chloride and separated by HPLC on a 5-mm Ultrasphere ODS column

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(4.6 mm i.d. ´ 250 mm; Beckman, Palo Alto, Calif. USA) as previously described (Gallardo et al. 1993). Quantitative determinations were carried out at 254 nm.

Results Immunoblot analysis of GS polypeptides in blade and midrib extracts. First of all, GS polypeptides were analyzed by western blotting in separated extracts of blades and midribs from control, Pst-infected and PPTtreated tomato leaves, using antibodies, raised against Pinus sylvestris GS, that recognized GS1 and GS2 polypeptides on the western blots (Fig. 1). In blade extracts of control leaves, a GS polypeptide of 45 kDa (GS2) was exclusively detected, whereas in midrib extracts two GS polypeptides of 45 and 39 kDa (GS1) were observed, the molecular species of 45 kDa being predominant. In infected and herbicide-treated leaves the 39-kDa polypeptide was the most abundant isoprotein both in blade and midrib extracts. Figure 1 also shows (compare lanes L) that the GS1 polypeptide was

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Fig. 5A±D. Immunolocalization of GS by transmission electron microscopy in sections of mesophyll and phloem cells from PPTtreated tomato leaves after immunogold staining. A±C Mesophyll cells showing substantial labelling in the cytoplasm and only slight labelling in the chloroplast. D Phloem cell also showing abundant labelling in the cytoplasm. ch, chloroplast; cy, cytoplasm. Bars ˆ 1 lm (A, D) and 0.5 lm (B, C)

absent in blades of healthy plants, but was clearly detected in blades of Pst-infected lea¯ets, and especially in PPT-treated leaves. Immunolocalization of GS in lea¯et cross-sections. Tissue printing was used to further characterize GS tissue distribution in tomato lea¯ets. Similar patterns of scattered immunostaining were found throughout the leaf blade and leaf midrib in control, Pst-infected and PPT-treated leaves, but staining was most prominent in the leaf blade, internal and external phloem, and epidermis in all studied leaves (Fig. 2). Immunocytolocalization of GS in mesophyll and phloem cells. Transmission electron microscopy was used to determine the subcellular distribution of GS polypeptides in tomato leaves. Ultrathin sections of mesophyll and vascular tissues from control leaves maintained for

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

8 d in MS medium, from bacterially infected leaves 10 d after inoculation and from PPT-treated leaves after 5 d of treatment were examined after immunogold labelling. In mesophyll of untreated leaves this analysis revealed highly vacuolated mesophyll cells with chloroplasts containing well-di€erentiated grana and stroma lamellae with appreciable labelling con®ned to the chloroplast (Fig. 3A). In the vascular system of the same leaves, substantial labelling occurred in the phloem, almost exclusively distributed throughout the cytosol of companion cells (Fig. 3B). In Pst-infected and PPT-treated leaves a few gold grains were observed in the chloroplast of mesophyll cells, but substantial staining was evident in the cytosol of the same cells (Figs. 4A±C, 5A±C). In the vascular tissue of bacterially infected and herbicidetreated leaves, substantial labelling was also observed in the cytosol of the companion cells (Figs. 4D, 5D). Characterization of GS polypeptides by two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis was used to distinguish the di€erent GS polypeptides according to their relative charge and to study if the GS induced by infection or herbicide treatment was the same as or a di€erent isoprotein from the constitutive cytosolic GS polypeptide (Fig. 6). When separated by

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

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tides (Fig. 6A), in agreement with the data described by Lara et al. (1984) for Phaseolus vulgaris. In control midrib extracts, two isoforms of GS of 45 and 39 kDa were identi®ed, presumably corresponding to the chloroplastic GS (GS2) of photosynthetic cells and cytosolic GS (GS1) of phloem cells, respectively (Fig. 6B). In infected and herbicide-treated leaves the predominant GS polypeptide was similar in size to the cytosolic GS1 polypeptide (39 kDa) found in control midribs, but distinct in charge (Fig. 6C and D, arrowheads). This was con®rmed when extracts of Pst-infected or PPT-treated leaves were co-electrophoresed with control midrib extract (Fig. 6E,F). In addition to GS1, leaves under stress conditions contained low levels of the 45-kDa GS2 polypeptide.

Fig. 6A±F. Two-dimensional gel analysis of GS polypeptides from control tomato leaves and midribs, and from infected and PPT-treated leaves. Soluble proteins were extracted, separated by isoelectric focusing (IEF, pH 4±6.5) and SDS-PAGE (SDS), electroblotted onto nitrocellulose, and GS polypeptides identi®ed by binding to anti-GS antibody. Protein extracts analyzed were obtained from: A control healthy and untreated leaves; B midribs of healthy leaves; C leaves infected with P. syringae pv. tomato; D leaves treated with phosphinothricin; E control midribs plus Pst-infected leaves; F control midribs plus PPT-treated leaves. The positions of the constitutive GS polypeptides are marked (arrows) as are those of the novel GS polypeptide induced by Pst and PPT (arrowheads)

size and charge, several GS polypeptides were detected in tomato leaves at di€erent relative abundances under the studied experimental conditions. In control leaves, the predominant isoform of GS was the 45-kDa chloroplast GS2, comprised of at least three polypepTable 1. Changes in amino acid contents [nmol á (g FW))1] in tomato leaves infected by P. syringae pv. tomato or treated with PPT. The data are means (‹ SD) of two determinations from three di€erent experiments

Amino acid contents in tomato leaves. To study the metabolic impact of GS changes under stress conditions, the levels of several amino acids were examined following infection and herbicide treatment. Total amino acids were extracted from non-detached tomato leaves, from control leaves maintained for 10 d in MS medium, from infected leaves 10 d after inoculation and from PPTtreated leaves after 5 d of treatment, and the amino acid contents were determined by HPLC (Table 1). In comparison with the healthy leaves maintained in MS medium, infected leaves showed a very high increase in the amount of asparagine and a noticeable reduction in glutamate. In contrast, a general decrease in the amounts of all amino acids examined was observed in PPT-treated leaves, especially in glutamine levels. Discussion It is well documented that microbial infection induces in plants a wide range of metabolic changes. In a previous work we have described a di€erential expression of tomato GS in response to Pst infection, and suggested that GS1 could be considered as a pathogenesis-related protein (PeÂrez-Garcõ a et al. 1995). We have characterized GS subunit polypeptides on the basis of their molecular size and charge, and determined their distribution in tomato leaves infected by Pst or treated with PPT. The aim of this experimental approach was to obtain insight into the physiological role of the cytosolic GS isoprotein induced in response to bacterial infection

Amino acid

In plantaa

Asparagine Serine Glutamine Aspartate Glutamate Glycine Alanine

25.7 290.3 1353.3 1726.4 2506.3 1502.4 151.3

a

‹ ‹ ‹ ‹ ‹ ‹ ‹

28.1 159.3 442.3 314.4 729.1 285.7 65.8

Control 10b

Pst 10c

1150.2 1196.1 628.2 1187.5 5659.3 1118.5 513.8

19843.4 538.1 1010.4 3112.4 799.1 525.6 222.8

‹ ‹ ‹ ‹ ‹ ‹ ‹

11.1 33.8 125.0 588.8 134.4 101.1 12.0

PPT 5d ‹ ‹ ‹ ‹ ‹ ‹ ‹

2654.5 258.2 204.9 436.2 77.1 75.9 36.9

Non-detached leaves Detached leaves maintained for 10 d in MS medium c Detached leaves 10 d after inoculation with P. syringae pv. tomato d Detached leaves after 5 d of treatment with PPT b

152.8 355.5 56.3 1092.4 479.6 859.6 218.9

‹ ‹ ‹ ‹ ‹ ‹ ‹

4.7 41.9 4.1 21.6 132.3 281.4 15.7

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or herbicide treatment. A similar accumulation of GS1 isoform has been described during senescence of leaves and cotyledons (Kawakami and Watanabe 1988; Kamachi et al. 1991; PeÂrez-RodrõÂ guez and Valpuesta 1996), and during water stress (Bauer et al. 1997), but there are no data about its tissue and cellular localization. In healthy tomato leaves the presence of GS1 was restricted to the vascular tissue whereas GS2 was the unique GS species detectable in photosynthetic cells, in agreement with data reported for other plant species (Edwards et al. 1990; Carvalho et al. 1992; Kamachi et al. 1992). However, under stress conditions the accumulation of a GS polypeptide of 39 kDa was observed both in blade and midrib protein extracts (Fig. 1), suggesting the possibility of GS1 expression in mesophyll cells. As the presence of cytosolic GS in this cell type has not been demonstrated yet, additional experiments were done to examine the cellular and subcellular distribution of GS protein in tomato leaves. Immunoblots of tissue prints of lea¯et cross-sections revealed similar patterns of GS distribution in healthy, infected and PPT-treated leaves (Fig. 2). Label was observed in the phloem, where the GS isoform was assumed to be GS1, epidermis and leaf blade. These results are similar to those reported for tobacco and potato leaves (Carvalho et al. 1992; Pereira et al. 1992) and indicate no alteration in GS expression in the vascular tissue of the studied tomato leaves. High-resolution localization by immunocytochemical techniques showed that in healthy leaves GS is con®ned to the chloroplasts of mesophyll cells and the cytosol of phloem companion cells, in accordance with earlier observations in tomato and other solanaceaous species (Botella et al. 1988; Carvalho et al. 1992; Pereira et al. 1992). However, in infected and herbicide-treated leaves,

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

labelling was also noticeable in the cytosol of mesophyll cells, where fewer gold particles were found in the chloroplasts, compared to control leaves. Detection of GS-speci®c staining in the cytosol of mesophyll cells can be explained by the expression of GS1 in this cell type, in addition to phloem cells. Alternatively, the accumulation of a new GS isoform under stress could also be considered. The characterization of GS polypeptides by two-dimensional gel electrophoresis and western blotting showed that the cytosolic GS isoform induced during infection or herbicide treatment is a polypeptide similar in size (39 kDa) to the constitutive GS1 isoform present in the vascular tissue, but distinct in charge. Our results suggest that during infection or PPT-treatment a novel cytosolic GS, which is di€erent from the constitutive GS1 associated with the vascular tissue, is expressed in the photosynthetic cells. However, we cannot conclude whether the new GS corresponds to a modi®ed form of the pre-existing GS1, or is the product of a separate GS1 gene. In parallel, there is a reduction in the expression of the GS2 gene (PeÂrez-Garcõ a et al. 1995, 1998) and consequently a minor presence of GS2 in the same cells. What could be the function of this novel GS? During infection, ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco) and other chloroplast polypeptides are degraded (PeÂrez-Garcõ a et al. 1995). Carbon skeletons released can be used as precursors for the biosynthesis of molecules involved in plant defense or oxidized in the tricarboxylic acid cycle supplying energy, while ammonium is reassimilated by GS enhancing the formation of glutamine. Glutamate can be provided by the action of aminotransferases or glutamate dehydrogenase. Expression of a cytosolic GS in mesophyll cells during pathogenesis can assure the reassimilation of

Fig. 7A,B. Schematic representation of the proposed physiological roles for GS isoforms in tomato leaves. A Currently accepted function of GS2 and GS1 in mesophyll and phloem cells of healthy leaf tissue. GS2 would be involved in the assimilation of ammonium derived from nitrate reduction and photorespiration in photosynthetic cells (mesophyll). GS1, however, would be involved in the generation of glutamine (Gln) for nitrogen transport in vascular tissues. B Proposed role for cytosolic GS in mesophyll and phloem cells of leaves infected with P. syringae pv. tomato. The cytosolic isoform (GS1) present in mesophyll cells of infected leaves would be implied in the reassimilation of ammonium released in the catabolism of chloroplast components. Glutamine (Gln) would be converted into asparagine (Asn) for the transport of nitrogen to the healthy parts of the plant. In the phloem cells, constitutive GS1 collaborates in the intercellular transport of nitrogen

A. PeÂrez-GarcõÂ a et al.: A novel glutamine synthetase in tomato leaves

nitrogen released from chloroplast disassembly. Then, glutamine can be a vehicle for nitrogen transport (Urquhart and Joy 1981) from mesophyll to vascular tissue, and from here to healthy parts of the plant where it can be used for the biosynthesis of defense-related proteins. Nevertheless, the amino acid levels detected in infected leaves indicate that it is not glutamine but possibly asparagine the amino acid mainly involved in the transport of nitrogen produced in infected tissues from protein catabolism. Actually, asparagine levels increased 20-fold in infected leaves when compared with control detached leaves (Table 1). Asparagine has a high nitrogen:carbon ratio compared with glutamine, and therefore is a major transport and transient-storage nitrogenous compound in conditions of limiting carbohydrate (Lea and Mi¯in 1980; Urquhart and Joy 1981; Lam et al. 1996). Although several routes of asparagine synthesis in higher plants have been proposed (Sieciechowicz et al. 1988), the conversion of aspartate to asparagine by glutamine-dependent asparagine synthetase is the most important (Eason et al. 1996). Thus GS could be responsible for the biosynthesis of glutamine as substrate for asparagine biosynthesis, and it would represent a route for transferring ammonium released from protein catabolism into asparagine. This hypothesis is summarized in Fig. 7, and it is consistent with the low levels of these amino acids detected in PPT-treated leaves, where the total inhibition of GS activity (PeÂrezGarcõÂ a et al. 1998) would prevent the synthesis of glutamine and consequently asparagine biosynthesis. The accumulation of asparagine has also been reported in senescent plant tissues and under sugar starvation (Brouquisse et al. 1992; Eason et al. 1996); and, asparagine is the major amino acid involved in the remobilization of leaf nitrogen during natural senescence (Lea and Mi¯in 1980). The authors thank Mrs. Eugenia Rivera and Mr. Francisco Olea, Universidad de MaÂlaga, Spain, and Mrs. Andrea Costa, Universidade do Porto, Portugal for assistance with electrophoresis and photography. This study was supported by grants from DireccioÂn General CientõÂ ®ca y TeÂcnica (PB95-0482), ComisioÂn Interministerial de Ciencia y TecnologõÂ a (AGF 95-0962-CO3-02) and Junta de AndalucõÂ a (Grupos AGR-0169 and CVI-0114). A. P.-G. thanks the Universidad de MaÂlaga for a fellowship covering his stay in the Universidade do Porto.

References Bashan Y, Okon Y, Henis Y (1986) A possible role for proteases and deaminases in the symptoms of bacterial speck disease in tomato caused by Pseudomonas syringae pv. tomato. Physiol Mol Plant Pathol 28: 15±31 Bauer D, Biehler K, Fock H, Carrayol E, Hirel B, Migge A, Becker TW (1997) A role for cytosolic glutamine synthetase in the remobilization of leaf nitrogen during water stress in tomato. Physiol Plant 99: 241±248 Bollag DM, Edelstein SJ (1991) Isoelectric focusing and two dimensional gel electrophoresis. In: Bollag DM, Edelstein SJ (eds) Protein methods. Wiley-Liss, New York, pp 161±179 Botella JR, Verbelen JP, Valpuesta V (1988) Immunocytolocalization of glutamine synthetase in green leaves and cotyledons of Lycopersicon esculentum. Plant Physiol 88: 943±946

433 Brouquisse R, James F, Pradet A, Raymond P (1992) Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips. Planta 188: 384±395 CaÂnovas F, Valpuesta V, NuÂnÄez de Castro I (1984) Characterization of glutamine synthetase from tomato leaves. Plant Sci Lett 37: 79±85 CaÂnovas FM, CantoÂn FR, Gallardo F, Garcõ a-GutieÂrrez A, de Vicente A (1991) Accumulation of glutamine synthetase during the early development of maritime pine (Pinus pinaster) seedlings. Planta 185: 372±378 CantoÂn FR, Garcõ a-GutieÂrrez A, Crespillo R, CaÂnovas FM (1996) High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinat protein and expression studies in pine seedlings. FEBS Lett 393: 205±210 Carvalho H, Pereira S, Sunkel C, Salema R (1992) Detection of cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods. Plant Physiol 100: 1591±1594 Cullimore JV, Gebhardt C, Saarelainen R, Mi¯in BJ, Idler KB, Barker RF (1984) Glutamine synthetase of Phaseolus vulgaris L.: organ-speci®c expression of a multigene family. J Mol Appl Genet 2: 589±599 Eason JR, O'Donoghue EM, King GA (1996) Asparagine synthesis and localization of transcripts for asparagine synthetase in tips of harvested asparagus spears. J Plant Physiol 149: 251±256 Edwards JW, Walker EL, Coruzzi GM (1990) Cell-speci®c expression in transgenic plants reveals nonoverlapping roles for chloroplastic and cytosolic glutamine synthetase. Proc Natl Acad Sci USA 87: 3459±3463 Gallardo F, CantoÂn FR, Garcõ a-GutieÂrrez A, CaÂnovas FM (1993) Change in photorespiratory enzymes and glutamate synthases in ripening tomatoes. Plant Physiol Biochem 31: 189±196 Kamachi K, Yamaya T, Mae T, Ojima K (1991) A role for glutamine synthetase in the remobilization of leaf nitrogen during natural senescence in rice leaves. Plant Physiol 96: 411± 417 Kamachi K, Yamaya T, Hayakawa T, Mae T, Ojima K (1992) Vascular bundle-speci®c localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol 99: 1481±1486 Kawakami N, Watanabe A (1988) Senescence-speci®c increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons. Plant Physiol 88: 1430±1434 Lam HM, Coschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM (1996) The molecular genetics of nitrogen assimilation into amino acids in higher plants. Annu Rev Plant Physiol Plant Mol Biol 47: 569±593 Lara M, Porta H, Padilla J, Folch J, SaÂnchez F (1984) Heterogeneity of glutamine synthetase polypeptides in Phaseolus vulgaris L. Plant Physiol 76: 1019±1023 Lea PJ, Mi¯in BJ (1980) Transport and metabolism of asparagine and other nitrogen compounds within the plant. In: Stumpf PK, Conn EE (eds) The biochemistry of plants, vol 5. Academic Press, New York, pp 569±607 Lea PJ, Robinson SA, Stewart GR (1990) The enzymology and metabolism of glutamine, glutamate and asparagine. In: Mi¯in BJ, Lea PJ (eds) The biochemistry of plants, vol 16. Academic Press, New York, pp. 121±159 Linthorst HJM (1991) Pathogenesis-related proteins of plants. Crit Rev Plant Sci 10: 123±150 McNally SF, Hirel B (1983) Glutamine synthetase isoforms in higher plants. Physiol VeÁg 21: 761±774 Pereira S, Carvalho H, Sukel C, Salema R (1992) Immunocytolocalization of glutamine synthetase in mesophyll cells and phloem of leaves of Solanum tuberosum L. Protoplasma 167: 66±73 PeÂrez-Garcõ a A, CaÂnovas FM, Gallardo F, Hirel B, de Vicente A (1995) Di€erential expression of glutamine synthetase isoforms in tomato detached lea¯ets infected with Pseudomonas syringae pv. tomato. Mol Plant-Microbe Interac 8: 96±103 PeÂrez-Garcõ a A, de Vicente A, CantoÂn FR, Cazorla FM, Codina JC, Garcõ a-GutieÂrrez A, CaÂnovas FM (1998) Light-dependent changes of tomato glutamine synthetase in response to

434 Pseudomonas syringae infection or phosphinothricin treatment. Physiol Plant 102: 377±384 PeÂrez-Rodrõ guez J, Valpuesta V (1996) Expression of glutamine synthetase genes during natural senescence of tomato leaves. Physiol Plant 97: 576±582 Peterman TK, Goodman HM (1991) The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and di€erential expression in leaves, roots, and seeds. Mol Gen Genet 230: 145±154

A. PeÂrez-Garcõ a et al.: A novel glutamine synthetase in tomato leaves Sieciechowicz KA, Joy KW, Ireland RJ (1988) The metabolism of asparagine in plants. Phytochemistry 27: 663±671 Urquhart AA, Joy KW (1981) Use of the phloem exudate technique in the study of amino acid transport in pea plants. Plant Physiol 68: 750±754 Wild A, Sauer H, Ruhle W (1987) The e€ect of phosphinothricin (glufosinate) on photosynthesis. I. Inhibition of photosynthesis and accumulation of ammonia. Z Naturforsch 42c: 263±269