Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome M.W. Elmlinger and H. Mohr*
BiologischesInstitut II der Universitfit,Sch/inzlestrasse 1, W-7800 Freiburg i.Br., Federal Republicof Germany Received 15 April; accepted 22 May 1992 Abstract. The appearance of glutamine synthetase (GS.
EC 188.8.131.52) in response to light and nitrogen (NOj, NH2) was studied in the organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine (Pinus sylvestris L.) seedling. Although GS activity was found to be mainly (> 80%) located in the whorl where it increased strongly in response to light, a significant GS synthesis was also detected in dark-grown seedlings. Anion-exchange chromatography was used to resolve two GS isoforms which appeared to be regulated differentially in the cotyledonary whorls. The isoform (presumably plastidic GS2) which eluted from the column at 90 mM KC1 increased drastically in response to light. The other isoform (presumably cytosolic GS1), which eluted at 200 mM KC1, was not stimulated by light but tended to disappear during the experimental period (4 to 12 d after sowing). Immunoblotting of pine extract yielded a prominent band with a molecular weight of 43 kDa. The linear correlation between GS activity and immunodetectable GS protein could be extrapolated through zero, showing that any increase of GS2 activity is to be attributed to the de-novo synthesis of GS protein. Gelfiltration chromatography yielded a molecular mass for the GS holoenzyme of 340 kDa, a value which supports an octameric quarternary structure as previously suggested for angiosperms. While supplying seedlings with 10 mM NO~- stimulated GS synthesis in the whorl by 12 %, 10 mM NH2 caused an incipient ammonium toxicity. Experiments using dischromatic light (simultaneous treatment with two light beams to vary the level of the physiologically active form of phytochrome, Pfr, in blue
Abbreviations and symbols: B=blue light; D=darkness; FdGOGAT = ferredoxin-dependentglutamate synthase (EC 184.108.40.206); GS = glutaminesynthetase(EC 220.127.116.11); R = red light; RG9 = longwavelength far-red light defined by the properties of Schott glass filter RG9; ~%=Pfr/Ptot=far-red absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochromesystem * To whom correspondence should be addressed; FAX: 49 (761) 203 2712
light) revealed that synthesis of GS2 was controlled by light in the same way as previously shown for ferredoxindependent glutamate synthase (Fd-GOGAT; EC 18.104.22.168). Up to 10 d after sowing the strong light effect could be attributed to phytochrome action whereas between 10 and 12 d after sowing phytochrome control of GS-synthesis failed if no blue/ultraviolet-A light was provided. The data show that blue light is required to maintain responsiveness of GS2 synthesis to phytochrome. Both enzymes, GS2 as well as Fd-GOGAT, appear to be regulated coordinately to meet the demands of ammonium assimilation. Key words: Ammonium assimilation -
Coaction of photoreceptors - Glutamine synthetase - Pinus
Assimilation of ammonium in plant cells is catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The reactions involved are referred to as the GS/GOGAT cycle by which glutamine and glutamate are synthesized from ammonia and 2-oxoglutarate (Miflin and Lea 1980). Both GS and GOGAT are known to exist as distinct isoforms in different subcellular compartments. In the case of GS one isoform, GS1, is located in the cytosol and the other, GS2, is in the plastid (Hirel and Gadal 1980). In the case of GOGAT, an NAD(P)Hdependent isoform (EC 22.214.171.124) could be localized in the cytosol while the ferredoxin-dependent isoform (FdGOGAT) is located in the plastid (Hecht et al. 1988). As far as regulation of gene expression is concerned, a coordinate regulation of GS2 and Fd-GOGAT was postulated to be essential to meet the demands of ammonium assimilation in the light (McGrath and Coruzzi 1991). We have previously studied the control of the appearance of Fd- and NADH-GOGATs in the major organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine
M.W. Elmlinger and H. Mohr: Glutamine synthetase in Scots pine seedlings
(Pinus sylvestris L.) seedling (Elmlinger and M o h r 1991). It was f o u n d that cytosolic N A D H - G O G A T d r o p p e d to a low level during the experimental period (from 4 to 12 d after sowing) and was n o t affected by light. O n the other hand, the level o f plastidic F d - G O G A T increased strongly in response to light. The kind o f light control was intriguing: red light, (R) operating via p h y t o c h r o m e , could fully replace white light b u t only up to 10 d after sowing. Thereafter, there was an absolute requirement for blue light (B) for a further increase in the enzyme level. D i c h r o m a t i c experiments (simultaneous t r e a t m e n t o f the seedlings with two light beams to vary the level o f the far-red a b s o r b i n g f o r m o f p h y t o c h r o m e , Pfr, in blue light) showed that B does not affect enzyme appearance if the Pfr level is low. It was concluded that B is required to m a i n t a i n responsiveness o f F d - G O G A T synthesis to Pfr b e y o n d 10 d after sowing. A m o d e l for this kind o f coaction between B / U V - A and light a b s o r b e d by phyt o c h r o m e was a d v a n c e d previously ( M o h r 1986). In the present study using the Scots pine seedling we have addressed the following questions: (i) T o w h a t extent is the appearance o f GS in the m a j o r organs o f the Scots pine seedling controlled by light, nitrate a n d amm o n i u m ? (ii) Is the a p p e a r a n c e o f G S activity to be attributed to synthesis de n o v o o f the e n z y m e p r o t e i n ? (iii) C a n isoforms be resolved by c h r o m a t o g r a p h y ? (iv) D o e s the sophisticated light c o n t r o l o f e n z y m e appearance as described in the case o f F d - G O G A T also apply in the case o f G S 2 ? As pointed out a b o v e it is tempting to speculate that b o t h gene expressions are co-ordinately regulated to meet p h o t o r e s p i r a t o r y d e m a n d s ( M c G r a t h and Coruzzi 1991). O n l y little is k o w n so far a b o u t the enzymes o f nitrate/ a m m o n i u m assimilation in conifers and their control, even t h o u g h it has b e c o m e clear in recent years that a m m o n i u m m e t a b o l i s m in forest trees poses a considerable practical p r o b l e m (see Flaig a n d M o h r 1992; H o c h stein and H i l d e b r a n d 1992, for reviews).
Material and methods
Growth conditions and light treatments. Seeds of Pinus sylvestris L. were obtained from Staatsklenge (Nagold, FRG). Seedlings were cultivated and subjected to different light treatments (light-pulse treatment, dichromatic irradiation and continuous light) as described previously (Elmlinger and Mohr 1991). After 4 d in weak R (0.68 W - m -2) to synchronize germination the experimental treatment was started (onset of experimental period). Nutrient solutions or water were given from sowing onwards. Enzyme assay. Samples of 15 organs from pine seedlings (cotyledonary whorl, hypocotyl, roots) were homogenized at 4~ C with a total 9 volume of 6 ml of extraction buffer (500 mM Tris-HC1, pH 8.5; 0.5 mM EDTA; 1% (v/v) Tween 80; 20 mM dithiothreitol). From the supernatant of the centrifuged (39000 99, 25 min) homogenate 100 ~tl aliquots were removed for the synthase assay (Shapiro and Stadtman 1970, with modifications as indicated). The reaction buffer (300 mM imidazole, pH 6.8; 20 mM EDTA) contained 500 mM glutamate, 40 mM of the ammonium-analogous substrate hydroxylamine, 50 mM ATP, and 100 mM Mg 2§ as a cofactor. The reaction was terminated by adding 500 ~tl of stopping solution (400 mM trichloracetic acid, 2 M HC1, 740 mM FeC13) and the synthesized 4-glutamylhydroxamate measured colorimetrically at
540nm. Samples containing 100 ~1 of heat-denatured extract (5 min, 95~ C) served as controls.
Anion-exchange chromatography. The GS present in the cotyledonary whorls of pine was characterized by anion-exchange chromatography (fast protein liquid chromatography, Mono Q HR 5/5. Pharmacia, Uppsala, Sweden) as follows. Twenty whorls were extracted with a final volume of 6 rnl of extraction buffer which additionally contained 0.3 % albumin and 3 % betaine to prevent protein aggregation. According to Schmidt and Mohr (1989), the homogenate was centrifuged, submitted to a gel filtration on Sephadex G-25 to remove low-molecular-weight substances, micro-filtered and applied to a Mono Q HR 5/5 column previously equilibrated with buffer (15 mM triethanolamine, pH 8.0; 10 mM Mg/+; 3% betaine). Proteins were eluted with a linear gradient from of 0 to 400 mM KC1, fractions of 0.5 ml collected and assayed for GS activity as described above.
Gel-filtration chromatography. For estimation of the apparent molecular mass of the native GS protein, gel-filtration chromatography on a Superose 6 column was performed according to Pharmacia instructions. The column was calibrated by determining Vo (void volume) and vt (total volume) and then elution volumes (ve) of molecular-weight standard proteins (Pharmacia, Uppsala, Sweden). Values for the eluation coefficient (kay) were calculated after the equation Kav=(vo-ve)/(vt-Vo) Two milliliters of extract from 20 whorls grown for 8 d in B were concentrated eight fold, desalted and partially purified with centrifugal micro-concentrators (Centricon 100; Amicon, Beverly, Mass., USA). A 150-111sample of the concentrate was applied to the column previously equilibrated with 5 volumes of elution buffer (50 mM Tris-HC1, pH 8.0; 10 mM Mg2+; 0.5 mM EDTA; 0.1% (v/v) mercaptoethanol). Samples of 0.4 ml were collected and assayed for GS activity. The elution volume of the GS activity peak was used to determine the Kavvalue and thus the molecular mass of the GS holoenzyme.
Western blotting. From each sample the proteins of 10 lal of heatdenatured (5 min, 95~ C) extract were separated in the presence of sodium dodecyl sulphate on a 10% polyaerylamide gel (Laemmli 1970) and then electro-blotted semi-dry (BioRad, Miinchen, FRG) to a nitrocellulose membrane (Schleicher & Schuell, Dassel, FRG). The membrane was incubated with polyclonal antibodies raised in rabbit against GS2 protein from seedlings of white mustard (Sinapis alba L.; kindly provided by Professor A. Wild, Institut ffir Allgemeine Botanik, Mainz, FRG). Thereafter a commercial peroxidase-conjugated anti-rabbit IgG antibody (Sigma, Deisenhofen, FRG) was added. Chemiluminescence produced by the peroxidase-reaction (ECL Western-detection system; Amersham, Braunschweig, FRG) at the sites where the antibody had bound was fixed on ECL-films after an exposure time of 10-20 s. Quantification of immunoresponse. Different volumes of extract (1, 2, 5, 10 lxl) from whorls grown in darkness (D) or B (10 W" m -z) for 8 d after onset of the experimental period resulted in GS bands of different intensities which were cut out of the film. Silver grains were solubilized in 1 ml 1 N NaOH and detected in a photometer (Uvikon 1860; Kontron, Ziirich, Switzerland) at 500 nm according to Suissa (1983). Results
Immunodetection of GS polypeptides. Figure 1 shows a strong i m m u n o l o g i c a l cross-reaction o f d e n a t u r e d G S polypeptides separated by s o d i u m dodecyl sulfatep o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) with polyclonal antibodies against purified m u s t a r d GS2. Western blotting revealed a p r o m i n e n t b a n d with a m o lecular weight o f 43 k D a and a m i n o r b a n d (48 k D a )
M.W. Elmlinger and H. Mohr: Glutamine synthetase in Scots pine seedlings