Research NoteFerredoxin glutamate synthase (ferredoxin glutamine: oxoglutarate aminotransferase, or Fd-GOGAT, EC 1.4.7.1) activity was detected in grapevine (Vitis vinifera L.). Highest Fd-GOGAT activity was found in lamina tissue. Moderate levels of activity were found in petiole, tendril, rachis, and flower tissues. No activity was detected in extracts from pedicel tissue. Western blotting with rice Fd-GOGAT antibodies resulted in a positive reaction with single grape protein band of 73 kDa in all tissue types tested

Breen, P. J.

Creasy, G. L.

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JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 36 (4), 207-208 (1997) Research Note Evidence for ferredoxin glutamate synthase activity in grape shoot tissues Summary : Ferredoxin glutamate synthase (ferredoxin glutamine: oxoglutarate aminotransferase, or Fd-GOGAT, EC 1.4. 7 .I) activity was detected in grapevine ( Vitis vinifera L. ). Highest Fd-GOGAT activity was found in Iamina tissue. Moder-ate Ievels of activity were found in petiole, tendril, rachis, and flower tissues. No activity was detected in extracts from pedicel tissue. Western blotting with rice Fd-GOGAT antibodies resulted in a positive reaction with single grape proteinband of73 kDa in all tissue types tested. K e y wo r d s : Fd-glutamate synthase, ammonium assimi-lation. Introduction: Glutamate synthase (GOGAT) has been characterized in many herbaceous plants, but little research in woody perennials has been reported. Additionally, to avoid high Ievels of phenolics prevalent in these plant spe-cies, material used in earlier studies were either from seed-lings or dark-grown suspension cultures. In grape, RouBELAKIS-ANGELAKIS and KLrEWER (1983) were unable to detect GOGAT activity in Chenin blanc root or leaf extracts, but JoRDAN et al. (1992) reported NADH-GOGAT activity in crude grape leaf extracts from Cabemet Sauvignon. Gu et al. ( 1994 ), working with Pinot noir shoot cuttings, used an inhibitor of GOGAT to infer that certain grape tissues had less GOGAT activity than others. Here, we report the presence and activity of the most important form of GOGAT in photosynthetic tissues: that which utilizes Fd as the reductant. Materials and methods: Grapevine tissue was obtained from both greenhouse and field-grown Pinot noir vines. Flower clusters were separated into flowers, pedicels, and rachis prior to freezing. Separation was done over ice in a cold room ( 4 °C) using fine-tipped scissors to clip first the flowers, then the pedicels from the inflorescence. Tissues were then frozen in liquid nitrogen and stored at -80 °C. Frozen tissue for the westem blots was ground in a mortar with 25% (w/w) polyvinylpolypyrrolidone (PVPP), then mixed with 5 volumes buffer (50 mM phosphate, pH 7.6, with 5 mM ethylenediaminetetraacetic acid (EDTA), 1 % (v/v) Triton X -100, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1) National Wine and Grape lndustry Centre, Charles Sturt University, P.O.Box 588, Wagga Wagga NSW 2678, Australia. Fax +61-2-6933 2107. E-mail: gcreasy@csu.edu.au 2 ) Department of Horticulture, Oregon State University, 4017 Agriculture & Life Seiences Bldg., Corvallis OR 97331-7304 USA and 14 mM ß-mercapto-ethanol, ß-ME). After centrifuga-tion, proteins were precipitated with 5 volumes of 0.1 M ammoniumacetatein methanol at -20 oc ovemight, washed twice with fresh 0.1 M ammoniumacetatein methanol, then twice with -20 oc acetone. The proteins were resuspended in sample buffer, their protein concentration determined (BRADFORD 1976), then loaded onto SDS mini gels (Mini-PROTEAN II apparatus, Bio-Rad Laboratories, Richmond, CA). Proteins were trans-ferred to a nitrocellulose membrane using a Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell. Membranes were probed with lgG anti-rice Fd-GOGAT (SuzuKr et al. 1982) and developed using a Bio-Rad Immun-Blot Assay Kit (AP goat anti-rabbit IgG). For the enzyme assay, frozen tissues were ground with 75% (w/w) PVPP, then added to 12 volumes 200 mM phos-phate buffer, pH 7 .6, containing 2.5 mM EDTA, 75 mM borax decahydrate, 28 mM ß-ME, 5 mM dithiothreitol (DTT), 0.2 mM PMSF, and 75 % (w/w) PVPP. Ammoniumsulfate precipitate between 25 % and 85 % saturation was resus-pended in 2.0 ml buffer (10 mM phosphate, pH 7.6, with 2.5 mM DTT) and dialyzed (Spectra!Por MWCO: 6-8000 tub-ing, Spectrum Medical Industries, lnc., Los Angeles, CA) ovemight at 4 °C. The protein concentration ofthe resulting solutionwas measured and then used for the assays, which were carried out at 30 oc for 2 min. The 1.2 ml reaction vol-ume contained buffer (1 0 mM phosphate, pH 7 .6, with 0.5 mM a-keto glutarate, 1.0 mM glutamine, 0.48 mM DTT, 20 mM KCl, and 0.4 mM EDTA), 0.05 mg ferredoxin, and 150 111 pro-tein solution. The reaction was started with the addition of sodium hydrosulfite and stopped by immersion in boiling water. Glutamate in the reaction solutionwas separated and quantified using the method of MATOH et al. ( 1980). Results: Distinct positive reactions occurred on the ni-trocellulose membrane in lanes containing each grape tis-sue tested, though response was weaker for grape Iamina and rachis. The major band for rice leaf corresponded to a molecular weight of 177 kDa, while that for grape tissues was approximately 73 kDa. Analysis of free amino acid content of enzyme assay eluates showed glutamate as the only significant free amino acid present. Addition of 1 mM azaserine to the assay mix-ture effectively blocked enzyme activity, while addition of methionine sulfoximine (an inhibitor of glutamine synthetase) had no effect on glutamate production (CREASY 1996). Fd-GOGAT activity was detected in all but one tissues tested (Table ). Extracts from laminas showed the greatest activity, but no activity was found in the extract from pedicel tissue. Other tissues exhibited moderate, and similar, Ievels of activity. Discussion: Western blots using anti-Fd-GOGAT IgG from rice leaf showed a positive reaction in grape tissues corresponding to a protein of approximately 73 kDa. The size of subunit F d-GOGAT proteinthat has been reported in other plant species ranges from 115 kDa in rice (SuzuKI et al. 1982) to 165 kDa in pine seedling cotyledons (GARCiA-GunERREZ et al. 1995). Fd-GOGAT from a variety ofplant 208 G. L. CREASY and P. J. BREEN Tab I e GOGAT activity data (± standard error) from a variety of shoot tissues from Pinot noir grapevine. Assay conditions were as described in the text. The unit of enzyme activity (U) is defined as I J.Lmol glutamate equivalent produced per minute ofreaction time Activity Tissue Source U·mg·1 protein ug·l fresh weight Lamina Greenhause 0.431 ±0.093 0.921 ±0.180 Petiole 1 Field 0.177±0.026 0.105±0.015 Petiole2 Field 0.165±0.054 0.053 ± 0.017 Flower I Field 0.152 ± 0.053 0.180 ± 0.036 Flower2 Field 0.129±0.025 0.125±0.143 Pedicel Field n.d. n.d. Rachis Greenhouse 0.119±0.009 0.161 ±0.012 Tendril Field 0.172 ± 0.029 0.132 ± 0.022 n.d.: not detectable. species have been sequenced, and are fairly highly con-served ( ZEHNACKERet a/. 1992, GARCiA-GUTU':RREZ et a/. 1995). Fd-GOGAT antibody from one species has successfully been used to probe other species, including monocotyledon to dicotyledon probes (GARCiA-GUTIERREZ et al. 1995), so it is likely that the positive reaction reported here indicates a protein related to Fd-GOGAT in grape. Grape Iamina and rachis tissue had a lesser response in reaction to the antibody than other tissues, possibly be-cause the enzyme may be expressed preferentially orto vary-ing extents in tissues of different ages or types. In tobacco, high amounts ofFd-GOGAT protein were found in Ieaves, lesser amounts in pistils and anthers, and nonein the corol-las or stems ( ZEHNACKERet al. 1992). Gelfiltration ofthe partially purified enzyme extract from grape Ieaves suggested that the native form Fd-GOGAT is a dimer or a trimer of the 73 kDa protein identified with the polyclonal antibody (CREASY 1996). Leaftissue had a Ievel ofFd-GOGAT activity similar to published values, e.g. 0.195 Umg· 1 in rice (SuZUKI and GADAL 1982) and 2.64 Umg·1 in tobacco ( ZEHNACKERet al. 1992). This is a preliminary study into the action ofFd-GOGAT in grape tissues. The recent isolation of DNA sequences encoding for Fd-GOGAT in grape (K. A. RoUBELA KIS-ANGELAKIS, personal communication) should do much to help elucidate ammonium cycling within grapevines. The authors wish to thank Dr. AKIRA SuzuKI for the gift of lgG anti-rice Fd-GOGAT and Dr. D. ÄRP, Dr. S. PRICE, and Dr. M. KELLER for help in the preparation of this manuscript. Financial support provided in part by the Oregon Wine Advisory board is gratefully acknowledged. BRADFORD, M. M.; 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. CR EASY, G. L.; 1996: 1nflorescence necrosis, ammonium, and evi-dence for ferredoxin-glutamate synthase activity in grape ( Vitis vinifera L.). Ph. D. Thesis. Oregon State Univ., Corvallis, Or-egon, USA. GARCiA-GUTIERREZ, A.; CANTÖN, F. R.; GAI.LARDO, F.; SANCHEZ-JIMENEZ, F.; CANOVAS, F. M.; 1995: Expression of ferredoxin-dependent glut-amate synthase in dark-grown pine seedlings. PI. Molec. Bio!. 27, 115-128. Gu, S.; LOMBARD, P. 8. ; PRICE, S. F.; 1994: Inflorescence necrosis induced from ammonium incubation and deterred by a-keto glutarate and ammonium assimilation in Pinot noir grapevines. Am er. J. Enol. Viticult. 45, 155-160. JoRDAN, 0.; PETERSON, C.; PEAKE, J.; CLARK, C.; 1992: Glutamate synthase activity in grape tissue. In: Proc. 4th Intern. Symp. Grapevine Physiology. San Micheie and Torino, May 11-15, 1992, 469-472. MATOH, T.; IDA, S.; TAKAIIASIII, E.; 1980: A rapid and sensitive assay for ferredoxin-glutamate synthase. Bull . Res. lnst. Food Sei., Kyoto Univ. 43, 1-6. RouBELAKIS-ANGELAKIS, K. A.; KLIEWER, W. M.; 1983: Ammonia assimi-lation in Vitis vinifera L.: II. Leaf and root glutamine synthetase. Vitis 22, 299-305. SuzuKI, A.; GADAL, P. ; 1982: Glutamate synthase from rice leaves. PI. Physiol. 69, 848-852. - -; ViDAL, J.; GADAL, P.; 1982: Glutamate synthase isoforms in rice. Immunological studies of enzymes in green leaf, etiolated leaf, and root tissues. PI. Physiol. 70, 827-832. ZEHNACKER, C.; BECKER, T. W.; SUZUKI, A.; CARRAYOL, E.; CABOCHE, M.; HlREL, 8.; 1992: Purification and properlies of tobacco ferredoxin-dependent glutamate synthase, and isolation of corresponding cDNA clones. Planta 187, 266-274. 

Evidence for ferredoxin glutamate synthase activity in grape shoot tissues

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Evidence for ferredoxin glutamate synthase activity in grape shoot tissues

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