Annals of Botany 77 : 429–432, 1996

Tissue-Specific Distribution of Glutamine Synthetase in Potato Tubers

SUSANA PEREIRA*, J. PISSARRA, C. SUNKEL and R. SALEMA

Centro de Citologia Experimental and Instituto de BotaW nica, Uni�ersidade do Porto, Rua do Campo Alegre, 823,

4150 Porto, Portugal

Received: 30 June 1995 Accepted: 27 October 1995

Cytosolic isoforms of the enzyme glutamine synthetase (GS) located in the phloem have been implicated in themobilization of nitrogen for intracellular transport in higher plants. The potato tuber represents an importantreservoir of nitrogen and an approach was made to the characterization of GS in this organ, particularly at the stagesof sprouting and of new tuber formation. By immunoblotting after SDS-PAGE, and by immunological tissueprinting, it was possible to conclude that a cytosolic GS is present in tubers and sprouts, and that it is mainly expressedin the internal phloem, in a very precise tissue-specific pattern of distribution. These data provide additional clues tothe interpretation of the functional role of GS in the mobilization of nitrogen and its utilization in growing parts ofthe plant. The importance of morphological data and localization studies in complementing molecular andbiochemical work is emphasized. The proposed functional importance of the internal phloem in Solanum tuberosumorgans is also reinforced. # 1996 Annals of Botany Company

Key words : Solanum tuberosum L. cv. Desire! e, potato, tubers, plant glutamine synthetase, tissue-specific distribution,phloem, nitrogen mobilization, in situ localization, tissue printing.

INTRODUCTION

In plants, glutamine synthetase (GS) is the key enzyme inthe assimilation of ammonium into glutamine, whichprovides nitrogen for other organic compounds (Sechley,Yamaya and Oaks, 1992). GS isoforms are encoded by asmall family of genes which are differentially expressed inspecific organs and cell types of plants (Forde and Cullimore,1989). Promoters for cytosolic and chloroplastic GSisoforms direct a tissue-specific expression of reporter geneGUS (β-glucuronidase) in transgenic plants (Forde et al.,1989; Edwards, Walker and Coruzzi, 1990; Cock, Hemonand Cullimore, 1992).

Edwards et al. (1990) demonstrated that the promoter ofone of the cytosolic GS genes of pea directed the expressionof reporter gene GUS in the phloem of transgenic tobaccowhich suggested a possible involvement of this isozyme ingenerating glutamine for intercellular nitrogen transport.Cytosolic GS had already been implicated in the mobil-ization of nitrogen for export by Kawakami and Watanabe(1988) and the subsequent localization of GS in the vasculartissues gave further meaning to those studies.

This paper is one of a sequence in which immunogoldelectron microscopy was used to detect the actual GSprotein (and not the reporter gene GUS) in Solanumtuberosum and Nicotiana tabacum leaves (Carvalho et al.,1992; Pereira et al., 1992). Cytosolic GS was previouslyconsidered to be non-existent in Solanaceae leaves (McNallyet al., 1983), but a striking abundance of GS protein has

* For correspondence at : Centro de Citologia Experimental,Universidade do Porto, Rua do Campo Alegre, 823, 4150 Porto,Portugal.

now been observed in the cytosol of phloem companioncells, thus identifying the precise cell type in which it occurs.Moreover, the presence of GS was most evident in theinternal phloem, restricted to some dicotyledoneous familiesincluding Solanaceae, which could have a physiological rolein GS metabolism (Carvalho et al., 1992; Pereira et al.,1992). These data provided further support for the proposedrole of cytosolic GS in mobilizing nitrogen for export(Kawakami and Watanabe, 1988; Edwards et al., 1990).

The cell specificity presented by GS isoforms, and itsphysiological implications, can be evaluated only by in situtechniques. One of these techniques is immunological tissueprinting, in which imprints of in �i�o tissue sections on anitrocellulose membrane are immunostained allowing thevisualization of cell-specific expression patterns of proteins(Cassab and Varner, 1987). The antigen is accessible toantibodies in the nitrocellulose membrane and in its nativeform and location.

In the important crop plant S. tuberosum (potato), tubersrepresent an important reservoir of nitrogen both in theform of proteins and of a free aminoacid pool whichconsists mainly of glutamine and asparagine (Burton, 1989).In this communication we report the localization of GS intubers and sprouts by means of tissue printing techniques.

MATERIALS AND METHODS

Sprouting seed tubers’ aerial stemsand leaves anddevelopingtubers, of Solanum tuberosum L. cv. Desire! e were obtainedfrom plants grown in vermiculite in a growth chamber.

Total soluble proteins (20 µg) were separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane.The blots were incubated with primary anti-GS antibody

0305-7364}96}050429­04 $18.00}0 # 1996 Annals of Botany Company

430 Pereira et al.—Distribution of Glutamine Synthetase in Potato Tubers

produced against GS from Phaseolus �ulgaris root nodules(Cullimore and Miflin, 1984). Secondary peroxidase-conjugated goat anti-rabbit antibodies were used and theblots were further developed with 4-chloro-1-naphtol.

Hand cut sections of plant tissue made with a sharp razorblade were pressed on nitrocellulose membranes and theprints were stained for GS as described for immunoblots ofSDS gels, except that tissue prints were first treated with 1%periodic acid for 30 min to block endogenous peroxidases.Tissue prints were examined under a stereomicroscope anda Nikon photomicroscope.

RESULTS AND DISCUSSION

On immunoblots of tuber and sprout extracts analysed bySDS-PAGE, a GS polypeptide of about 42 kDa, cor-responding to the cytosolic form could be detected (Fig. 1).The GS isoform content of the leaf is also shown forcomparison.

The immunostaining of tuber and sprout tissue printsgave an overall picture of GS distribution over large areasof tissue sections but also revealed expression confined tocertain cells, with a good level of resolution. It should bestressed that one of the main advantages of the tissueprinting technique is that the actual protein is detected in itsnative form in its original location, since there are noartifacts induced by fixation, dehydration or embeddingprocedures.

In young developing tubers (Fig. 2) immunostaining wasmost evident in the vascular tissue of a longitudinal section,near the heel or stem end of the tuber. The periphery of thetuber was also stained.

In cross-sections of mature tubers (Fig. 3) labellingappeared as well defined spots, sometimes strands, in theperimedullary zone inside the vascular ring, in a very clearpattern of distribution superimposable on that of theinternal phloem. Strands of internal phloem differentiate ingroups in the parenchyma in the perimedullary region andanastomosis can occur between them. The localization ofthe label in relation to the xylem is most evident at highermagnifications (Fig. 3C, detail). In the periphery of thetuber, well defined areas in the periderm (Fig. 3B), wereparticularly stained. Immunostaining was also very intensein the ‘eye’ region (Fig. 3D) and in the basal portion of thesprouts (Fig. 3E). In cross-sections of sprouts (Fig. 4) theinternal phloem was also evidently labelled.

Potato tubers represent both a nitrogen source and sink indifferent stages of plant development. Both the mobilizationof nitrogen from the source organs and the degradation oftransport products in sink tissues generate ammoniumwhich is probably reassimilated by GS (Sechley et al., 1992).Developing tubers represent a sink for carbon and nitrogensupplied by the leaves (Burton, 1989). The internal phloem,which is a complex tissue characteristic of some dicotyle-doneous families including Solanaceae, has been describedas the pathway for transport of carbohydrates from theleaves into developing tubers (Burton, 1989). The suggestionthat utilization of nitrogenous transport products could alsotake place in this tissue is supported by the presence of GS.

A B C

GS2

GS1

F. 1. GS detection on immunoblots of sprout (B) and tuber extracts(C) of Solanum tuberosum analysed by SDS-PAGE. GS content of leaf-extracts (A) is also shown for comparison. Chloroplastic GS (GS2) isthe predominant form in the green leaf whereas only one bandcorresponding to the cytosolic form (GS1) appears to be present in the

tubers and sprouts.

v

F. 2. Tissue print of a longitudinal section of a young developingtuber. The heel or stem end is visible (arrow); labelling is localized inthe vascular region (v) and also at the periphery of the tuber. ¬2±7.

As the tuber approaches maturity, meristematic activitybecomes progressively more localized in the ‘eye’ regionsand bud ends (Reeve, Hautala and Weaver, 1969). In seedtubers, dormancy is considered to be broken when thetransport of amino acids is directed toward the externalbud-containing tissues, so that the protein synthesis canbegin in these tissues (Hemberg, 1985). At the onset ofsprouting, the high levels of GS detected by immunoblottingcould be important in the mobilization of seed tuberreserves and production of glutamine for transport forgrowing parts of the plant. The abundance of this enzyme inthe vascular tissue gives further support to this hypothesis.GS localization in primary meristematic areas such as

Pereira et al.—Distribution of Glutamine Synthetase in Potato Tubers 431

A

v

pm

c

p

B

v

c

C

x

D E

pdpd

F. 3. Localization of GS in tissue prints of cross sections of mature sprouting tubers. General aspect (A, ¬2±2) showing staining localized inthe inner portion of the vascular ring (v) and also scattered in the perimedullary zone (pm). Labelling sometimes appears in strands. Well definedareas (arrow) in the periderm (pd) are also immunostained (B, ¬6). Staining is precisely confined to certain cells whose localization in the innerportion of the vascular ring (C, ¬7) is evident from the position in relation to the xylem cells (x) which are easily identified in the detail (¬48).

An intense label is shown in an ‘eye’ region (D, ¬5) and in the basal portion (arrow) of the sprouts (E, ¬7). c, Cortex; p, pith.

sprout apices suggests an involvement of this enzyme in theutilization of nitrogen in these areas for protein synthesis.

Labelling in some areas of the periderm both in developingand mature tubers, could be related to other processes suchas suberization of the outer layers of periderm or even othertissue response to wounding or pathogen attack. Duringthese events, the flux of ammonium generated by thephenylpropanoid metabolism can be very large (Sechley etal., 1992). GS in tubers may be involved in the assimilationof ammonium derived from different biochemical pathways.The identification of the precise cell-types in which these

metabolic reactions occur can be achieved only by studies atelectron microscopy level.

A cytosolic GS has recently been implicated in thesynthesis of glutamine for export from source organs(Kawakami and Watanabe, 1988). Localization of theexpression of cytosolic GS in vascular tissues by differentmethods and techniques (Edwards et al., 1990; Carvalho etal., 1992; Kamachi et al., 1992; Pereira et al., 1992) givessupport to this hypothesis.

The present results, from an economically-importantorgan, confirm and extend those previous studies. The

432 Pereira et al.—Distribution of Glutamine Synthetase in Potato Tubers

A

B

x

x

F. 4. Localization of GS in tissue prints of cross sections of sprouts.The labelling (arrow) is confined to the internal phloem (A, ¬16; B).The localization in relation to the xylem (x) is more evident in higher

magnifications (B, ¬44).

presence of a cytosolic GS in the internal phloem of bothdeveloping (sink) and mature sprouting tubers (source)gives new clues to guide further investigations.

ACKNOWLEDGEMENTS

We are very grateful to Dr Julie V. Cullimore (INRA-CNRS, Castanet-Tolosan) for the antibodies against puri-fied GS from Phaseolus nodules. The skillful photographicassistance of Mrs Andrea Costa is gratefully acknowledged.

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