BIOLOGIA PLANTARUM 35 (1): 151-155, 1993 Quantitative [3-glucuonidase assay in tansgenic plants S. VITHA, K. BENE~, M. MICHALOV~, M. OND17~EJ Institute of Plant Molecula Biology, Academy of Science of the Czech Republic, BanigovskA 31, ~eskd Buddjovice, Czech Republic Abstact Seveal factos influencing eliability of the quantitative fluoimetic ~-glucuonidase (GUS) assay in tlansgenic plant tissue have been investigated. We obtained linea dependence of fluoescence on both the duation of hydolysis and the extact concentation. The stability of the enzyme in the homogenate was faily high, the same as the stability of the substate solution and of the final eaction poduct. The modification of the extactionincubation buffe was poposed, esulting in seveal times highe activity in compaison with oiginal pocedue. Intoduction The tansfomation of plant cells by Agobacteium tumefaciens caying the 13-glucuonidase (GUS) gene fom Escheichia coli and the poof of its expession fequently have been studied. Relatively little attention, howeve, has been payed to quantitative assay of GUS activity in tansfomed cells. The aim of the pesent communication was to fill this gap. Mateial and methods The expeiments wee done using leaf tissue of tobacco (Nicotiana tabacum L. cv. Samsun) tansfomed two yeas ago by Agobacteium tumefaciens stain LBA 444 with pbi 121.1 plasmid caying chimeic GUS and NPT II (neomycin phosphotansfease II, i.e. kanamycin esistance) genes (Jeffeson 1988). Afte egeneation on Muashige and Skoog (MS) medium with gowth egulatos and antibiotics and the initial cultivation on plain MS media only with antibiotics, the plants wee kept on MS media and subcultued evey month (see Ond~ej et al. 1991). The leaves of coesponding ontogenetic stage wee assayed. Received 28 Novembe 1991, accepted 14 Febuay 1992. Contibution pesented to the 5th Czechoslovak Semina "Plant Gene Engineeing" oganized bythe Institute of Plant Molecula Biology in ~esk6 Bud~jovice, 2-13 Septembe 1991. 151
S. VITHA et al. We modified the fluoescence method with 4-methyl umbellifeyl I~-Dglucuonide of Jeffeson (1987). 25 mg of leaf tissue was homogenized in 1 cm 3 of extactionincubation (eli) buffe (containing 245 cm 3 of H2, 85 cm 3 of 1-1 M NaH2PO 4, 165 cm 3 of 1-1 M Na2HPO 4, 185 mg Na 2 EDTA, 345 mm3 [~-mecaptoethanol, 5 cm 3 1 % Titon X-1) in glass homogenize of Potte-Elvehjem type. 3 mm 3 of the homogenate was added to 45 mm3 of incubation medium [2 mg 4- methylumbellifeyl ls-d-glucuonide (Sigma) dissolved in 5 cm 3 of ei buffe]. Afte 2 min at oom tempeatue 4 mm 3 of this eaction mixtue was tansfeed into 3.5 cm 3 of stop-solution (.2 M Na2CO3). In ode to evaluate spontaneous decomposition of the substate (blank) 3 mm 3 of ei buffe was used instead of the homogenate. The fluoescence was assayed using Tune 111 fluoomete, 3.5 cm 3 cuvette, excitation by adiation of wavelength 365 nm, emission measued at 455 nm (gay absoption filte - 1% ange, Kodak-Wanen 96 was used to attenuate the signal). The esults ae expessed as woking units diectly measued o ecounted. In the pesent expeimental system the fluoescence of 1-7 M 4-metylumbellifeone (Clontech) dissolved in the fluid obtained by mixing eli buffe and stop solution as given above was 2.33 (sx=l.85). Results and discussion Using the above mentioned pocedue we obtained linea dependence of fluoescence on both the duation of the hydolysis (Fig. 1) and the sample quantity (extact concentation, Fig. 2). "~ 25 >.,e 2.,e 15o Zl~ 1 w e 5 2 15 1 5 I I I I i I 25 5 75 1 125 15 INCUBATION TIME [mini Fig. 1. The dependence of activity on the incubation time. Fig.2. The dependence of activity on the concentation of the extact. I I I 5 1OO 15 ~2 EXTRACT CONCENTRATION [ag cm -3] Futhe we tested the dependence of activity on the composition of incubation medium (Fig. 3). The highest activity was found in the medium containing Titon X- 1, Na2EDTA and [~-mecaptoethanol. That is why we chose this medium to be used in ou standad pocedue, omitting sodium lauoyl sacosine (sakosyl;.1% 152
GLUCURONIDASE ASSAY IN TRNSGENIC PLANTS vv). Consideable spontaneous decomposition of substate in plain buffe must be emphasized in compaison with eli buffe (about ten times highe). Although the incubation medium made of citate buffe povides somewhat highe activity than that with phosphate buffe, we pefe to use phosphate in the standad pocedue (Fig.4) which allows the compaison with othe authos' esults. 6 E 6 F - V '-,to ~, 7, < <4, tlj o 2 < v 2 ~ ", o "~[--7],,, ~ o ) 2 3 4 s 6 7 8 9 ~o i z 3 4 Fig.3. The enzyme activity in media with diffeent composition (phosphate buffe, the same amounts as in the standad medium; sakosyl.1% vv, 1 - sakosyl, 2 - Titon, 3 - EDTA, 4 - mecaptoethanol, 5 - EDTA+l~-mecaptoethanol, 6 - Titon+EDTA, 7 - Titon+l}-mecaptoethanol, 8 - Titon+EDTA+l~-mecaptoethanol, 9 - Titon+EDTA+[~-mecaptoethanol+sakosyl, 1 - plain buffe. Fig. 4. The activity in incubation media based on diffeent buffes (ph 6.5; 1 - acetate buffe, 2 - phosphate buffe, 3 - tis-maleate buffe, 4 - citate buffe). 4O 4 >" 3.a z 3 2 3 2 1 5.6 6.2 6.7 7.2 7.7 8.1 I i I i i 5 1 15 2 25 ph TIME OF EXTRACT PRESERVATION [h] Fig. 5. The activity in the standad incubation media at diffeent ph. Fig. 6. The stability of the extact (ei buffe, activity afte the given time of pesevation). Fo evaluation the stability of homogenate the activity was measued using fesh homogenate and then afte 3, 6 and 24 h stoage in a efigeato (8 ~ in dakness. The homogenate was faily stable (Fig. 6). We also tested the stability of the incubation medium, assaying the activity of the same homogenate using incubation medium of diffeent age (Fig. 7). It follows that the incubation media can be used 153
S. VITHA et al, even afte one month stoage in a efigeato in dakness. The fluoescence of final solution (afte mixing with stop-solution) does not change duing 24 h when kept fee in the laboatoy (Fig. 8). The eliability of fluoescence measuements was investigated by gadual dilution of the given sample by the stop-solution containing coesponding amount of ei buffe (Fig. 9). "~ 4O >, 7---- 4 "~ 3 m 2 "7.i,.t,,i "I, j,;-,,~ :. 2 1 2 3 36 3 6 24 AGE OF SUBSTRATE SOLUTION [d] TIME AFTER STOPPING REACTION [hi Fig. 7. The stability of the incubation medium (hatched columns - samples, empty columns - blanks). Fig. 8. The stability of the final eaction poduct (the same sample measued afte the given time). 75 6o ~ 45 ~ 3o ; i i i.2.4.6 G.;, '~.,2' SAMPLE DILUTION Fig. 9. "l~e poof of the eliability of the fluoescence measuements (gadual dilutions of the sample). The accuacy and eliability of the assay was veified in 9 paalel assays of the same mateial. The final fluoescence was measued epeatedly thee times immediately afte stopping the eaction and thee times 1 h late (Table 1). Othe data in this espect can be obtained fom Fig. 6 and Fig. 7. Fig. 6 shows seveal independent assays using the same incubation medium and the same extact eithe fesh o stoed fo 3 and 6 h, as given peviously. Fig. 7 epesents paallel assays using the same extact and diffeent incubation media. The vaiation of paticula measuements is within the ange + 1 % of the mean. When compaing method of Jeffeson (Jeffeson 1987, Jeffeson et al. 1986, 1987) and its modifications (Bustos et al. 1989, Hu et al. 199, Kosugi et al. 199, 154
GLUCURONIDASE ASSAY IN TRNSGENIC PLANTS Putteil and Gadne 1989, Spolein et al. 1991) ou wok is a contibution to effots to find the most convenient and eliable pocedue fo quantitative GUS assays. Table 1. The accuacy of the pocedue tested by paallel assays and epeated measuements Sample Fluoescence measuement 1 2 3 4 5 6 1 49 5 5 51 53 51 2 53 53 54 56 56 56 3 54 53 56 56 58 57 4 54 53 56 57 58 57 5 52 52 55 56 56 56 6 53 55 57 58 58 58 7 53 54 56 57 56 57 8 54 54 56 57 57 57 9 54 53 55 57 56 56 Refeences Bustos, M.M., Guiltian, M.J., Begum, D., Kalkan, F.A., Hall, T.C.: Regulation of ~-glucuonidase expession in tansgenic tobacco plants by an AT ich, cis acting sequence found upsteam of a Fench bean ~phaseolin gene. - Plant Cell 1: 839-853, 1989. Hu,.C., Chee, P.P. Cbesney, R.H., Zhou, J.H., Mille, P.D., O'Bien, W.T.: Intinsic GUS-like activities in seed plants. - Plant Cell Rep. 9: 1-5, 199. Jeffeson, R.A.: Assaying chimeic genes in plants: The Gus gene fusion system. - Plant tool. Biol. Rep. 5: 387-45, 1987. Jeffeson, R.A., Bugess, S.M., I-Iisch, D.: [$-glucuonidase fom Escheichia coil as a gene-fusion make. - Poc. nat. Acad. Sci. USA 83: 8447-8451, 1986. Jeffeson, R.A., Kavanagh, T.A., Bevan, M.W.: GUS fusions: ~glucuonidase as a sensitive and vesatile gene fusion make in highe plants. - EMBO J. 6: 391-397, 1987. Kosugi, S., Ohashi, Y., Nakajima, K., Aai, Y.: An impoved assay fo ~glucuohidase in tansfomed ceils: methanol almost completely suppesses a putative endogenous glucuonidase activity. - Plant Sci. 7: 133-14, 199. Ondl'ej, M., Bavina, T.V., Dudko, N., I-Iouda, M., Keknie, J., Lozhnikova, V.N., Mach~kov~, I., Seidlov~t, F., Vlasfl J.: Tansgenic tobacco plants with T-DNA phytohomone synthesis genes. - Biol. Plant. 33: 4-48, 1991. Putteil, J.J., Gadne, R.C.: Initiation of tanslation of the I$-glucuonidase epote gene at intenal AUG codons in plant cells. - Plant Sci. 62: 199-25, 1989. Spolein, B., Maye, A., Dahlfeld, G., Koop, H.U.: A micoasay fo quantitative detemination of ~- glucuonidase epote gene activities in individnally selected single highe plant cells. - Plant Sci. 78. ~ 73-8, 1991. 155