β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background

Plant Science 113 (1996) 209-219

P-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background B. Thomasset*a, M. MCnarda, H. Boetti”, L.A. Denmata, D. Inzkb, D. Thomasa aLaboratoire de Technologie Enzymatique. URA No. 1442 du CNRS, UniversitP de Technologie de Compi&gne. B.P. 529, 60205 Compiegne Cedex, France bLuboratoire Assock! INRA,

Laboratorium voor Genetika, Lea’eganckstraat 35, B-%WOGent, Belgium

Received 24 July 1995; revision received 16 October 1995; accepted 6 November 1995

Abstract Bacterial fl-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in trans-

genie plants could lead to an underestimation of GUS. In this paper, a decrease of the V,,, values and a greater affinity (K,,,) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure @-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene. Keywords: E. coli &glucuronidase;

Endogenous activity; Nicotiana tabacum; Plant inhibitors; Plant gene expression;

GUS assay protocol

1. Introduction Reporter genes are important tools both for monitoring expression patterns controlled by specific gene regulatory domains and for establishing Abbreviations: MU, 4-methylumhelliferone; MUG, 4methylumhelliferyl /3-o-glucuronide; p-NP, Cnitrophenol; pNPG, pnitrophenyl B-D-glucuronide; GUS, fl-glucuronidase; SL, saccharic acid l-4 lactone; commercial GUS enzyme, Boehringer GUS enzyme; E. coli GUS enzyme, enzyme from uidA acne. * Corresponding author.

and optimizing transformation methods in plants. A series of reporter genes such as chloramphenicol acetyl transferase (cur), o&opine and nopaline synthases (OCS,nos), /3-galactosidase (fucz) or luciferase (kc) genes have been used in higher plants [I]. However, the bacterial fl-glucuronidase gene @idA) has become the most commonly and routinely used reporter gene system for several reasons [2-41: l The enzyme is very stable under different physiological conditions. l GUS specifically catalyses the hydrolysis of a

0168-9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04286-7

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wide range of substrates and the GUS activity can be easily and sensitively measured either by spectrophotometric of fluorometric methods. l The GUS activity can also be detected in situ by histochemical analysis. For some time, this system was known to have a low background of endogenous /3-glucuronidase activity in many plants [3]. Nevertheless, more and more investigators were troubled by so-called ‘false positive’ results, ‘background’, or ‘endogenous’ activities when GUS assays were performed. These intrinsic GUS activities in higher plants were reported in several organs such as seeds, pollen grains, fruits, embryos, stems, leaves and roots [5-lo] and in numerous plant species according to the extensive study by Hu et al. [ 111.In some cases, false results were found to be due to endophytic microorganisms which express fl-glucuronidase [ 12- 141. Such a background activity can hide the activity originating from the introduced GUS gene resulting in insufficient information on GUS gene expression especially when the expression level is low or when inducible expression systems are tested [ 15,161. Moreover, some crude extracts of plant tissues contain endogenous phenolic compounds or endogenous inhibitors which either mimic or abolish the enzyme activity, respectively, leading to an over or underestimation of GUS v71.

Several attempts were made to minimize the endogenous GUS or GUS-like reaction [7-9, 18-201. Histochemical artifacts can arise through the diffusion of the soluble indolyl reagent into GUS negative neighbouring cells. In this case, addition of a mixture of ferrilferrocyanide can prevent intracellular diffusion of the reaction intermediate [7,21]. Kosugi et al. [18] have also developed a technique using organic solvents added to the reaction mixture that inhibit endogenous activity both in the histochemical and fluorometric assays. However, this technique has been tested by other groups and it seems that the observed inhibition is not specific of endogenous GUS activity [20,22] but can be also explained by the effect methanol has on the cell membrane permeability [20]. Other results supported that the GUS-like activity was due to a plant derived enzyme analytically distinguishable from GUS en-

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coded by the uidA gene from E. coli. Appropriate experimental conditions will be searched to separate the two enzyme activities [8,9]. In this study, we have tested the effect of crude or purified protein extracts from untransformed tobacco cell suspension, on the properties (I’,, K,,,) of the commercial GUS enzyme and conditions were established to minimize the endogenous GUS inhibition. We have developed a simple and rapid protocol that allows the quantitative GUS analysis of large number of samples and prevents artifacts. Furthermore, we have tested the protocol on untransformed coffee, tobacco and carrot cells and on transgenic cells to determine the endogenous GUS activity or the GUS activity encoded by the E. coli uidA gene. 2. Materials and methods 2.1. Plant cell materials Nicotiana tabacum var. Petit havana SRI callus or suspension cultures derived from leaf protoplasts were kept in a growth chamber at 24°C under periodic light (16th day, 33 pm01 rns2 s-’ irradiance). The cells were cultured on a Linsmaier and Skoog medium (LS medium) with 2 mg 1-l NAA, 0.3 mg 1-l kinetin, 30 g 1-l sucrose and 9 g 1-l Difco agar, pH 5.7 [23]. Suspension cultures of Coffea arabica L. were established from nonembryogenic roots via callus cultures (a gift of FRANCERECO, France) and cultured on a modified Gamborg B5 medium with 1 mg 1-l 2,4-D, 0.06 mg 1-l kinetin 20 g 1-l sucrose at pH 5.8 [24]. Daucus carota cell suspensions were established from hypocotyls via callus cultures and cultured on a MS medium [25] with 1 mg 1-l 2,4-D, 0.5 mg 1-l kinetin and 30 g 1-l sucrose at pH 5.8. Cultures of Coffea arabica and Daucus carota were continuously shaken (110 rev./mm ) at 24°C.

2.2. Plant cell transformation GUS transformed SRI tobacco cell lines were obtained via protoplast transformation as described by Van Lysebettens et al. [26]. Clonal callus lines were produced by co-cultivation of protoplasts with Agrobacterium C58ClRR if (pGV

B. Thornasser et al. /Plant

2260) [27] harboring a binary T-DNA vector containing a CaMV 35s promoter fl-glucuronidase @i&t) marker gene fusion associated with a neomycine phosphotransferase selection gene under the control of the nos promoter (ptDE4 plasmid) kindly provided by Dr Bottennan (Plant Genetic System NV, Gent, Belgium). The development of an inducible gene expression system was achieved by introducing a T-DNA containing the uidA gene under the control of the inducible promoter of the Rub&o SSU from Arabidopsis thaliana (Plant Genetic System NV, Gent, Belgium) into tobacco cells by using the method described by Van Lysebettens et al. [26]. The uidA gene is turned on after a light stimulus. Such an induction step was performed with lo-day-old plant cells after a light exposure (3.4 W m-*) during 16 h, at 24°C and under continuous shaking (110 rev./mm). Thereafter, the GUS activity was recorded by using fluorometric method [28]. 2.3. Protein and GUS assays A total of 0.4 g of lo-day-old cells was ground in a mortar in 1.5 ml of extraction buffer containing 50 mM Na3P04 buffer pH 7, 1 mM EDTA, 0.1% Triton X-100 and 10 mM P-mercaptoethanol. The plant tissue extract was centrifuged for 10 min at 4°C at 13 000 rev./min. The supernatant was collected and for some experiments it was purified by passing through Sephadex G-25 spin columns (Boehringer-Mannheim) prior to the assay. Protein concentration in the plant cell extract was determined by the method of Bradford [29] using the Biorad reagent and bovine serum albumin as standard. GUS activity was measured in microtiter plates (Nunc 2/6920) using p-nitrophenol-15-D glucuronide as substrate (1 mM; p-NPG; Sigma N-1627) and following the method described by Jefferson [28]. The hydrolysis of p-NPG was performed at 37°C and the formed 4-para-nitrophenol (p-NP) was measured spectrophotometrically at 410 nm by a Dynatech MR 5000 plate reader. In such a condition, 1 OD unit represents 52.9 nmol of pNP. The fluorometric reaction was carried with 3 mM rlmethylumbelliferyl &&glucuronic acid (4 MUG) in extraction buffer. The reaction was in-

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cubated at 37°C and aliquots were removed at different time points (0, 15, 30, 45, 60, 90 and 120 min). The reaction was terminated with the addition of 0.2 M Na2C03 in the dark. Fluorescence of 4-methylumbelliferone (MU) was measured at 455 nm after excitation at 365 nm on a PerkinElmer LS 5 spectrofluorometer. For each experiment, the fluorometer was calibrated with freshly prepared MU standards (l-50 nM) in the same buffer. In experiments performed with the pure enzyme, commercial &glucuronidase enzyme (E. cofi. Boehringer N 172501) was diluted into the extraction buffer before tested by using spectrophotometric or spectrofluorometric method as described above. For heat treatments, the protein extracts or commercial /I-glucuronidase enzyme were preincubated at 65°C for 5, 10 or 30 min or at 100°C for 5 min prior to addition of MUG and assay at 37°C. Analysis of pH influence on GUS specific activity was performed as above with the following modifications: the reaction buffers were citrate 100 mM for pH 4-6, phosphate 100 mM for pH 6-7.5 and Tris 100 mM for pH 7-8. Substrate (MUG) was solubilized in reaction buffer at the same pH and added. DSaccharic acid, l-4 lactone (SL; l-5 mM) solutions were freshly prepared for each use. All assays were performed in triplicate with each experiment. 3. Results and discussion 3. I. Properties of the commercial P-glucuronidase The E. coli uidA gene, encoding P-glucuronidase, is frequently used as a reporter gene for studying the activity of constitutive or inducible promoters in transgenic tobacco cells [16]. It is known that plant extracts can interfere with GUS assays [8,9,11], we have initiated a study of how plant extracts affect GUS activity and the GUS enzyme kinetic parameters (V,,,, K,). To this end, paranitrophenol @-glucuronide (p-NPG) and 4methylumbelliferyl /3-D-glucuronide (CMUG) were used as standard spectrophotometric and fluorogenic substrates of GUS activity in vitro. The commercial &glucuronidase was tested direct-

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ly in the extraction buffer with different concentrations of substrate. The initial rate data have been used to evaluate the kinetic parameters (I’,,,, K,,,). The kinetic model can be described by a rectangular hyperbolic function similar to the Michaelis-Menten equation:

vnlp r=

K,,,+P

where r is the p-nitrophenol (p-NP) or methylumbelliferone (MU) production rate, V,,, the maximum p-NP (MU) production rate and K,,, the p-NPG (4-MUG) concentration for which the p-NP (MU) production rate is half maximal. In this study, the initial rates were plotted as a function of the p-NPG @l-MUG) concentrations and

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the curves were fitted by a weighted least square non-linear regression. For the pure GUS enzyme, the K,,, values were estimated to be 0.1 * 0.01 mM for p-NPG (Table 1) and 0.33 * 0.03 mM for 4-MUG whatever the concentration of enzyme. The last result is in accordance with those (0.57 mM) of Gallagher [30], calculated from a Lineweaver Burk double reciprocal (l/V, l/[a) plot of GUS activity. Due to these low Km values, the two substrates (p-NPG, CMUG) will be commonly used as standards in our experiments. To ensure that the enzyme activity is kept maximal during the length of the assay, the concentration of substrate in each of the following experiments was fixed approximately 10 times the K,,,. The substrate concentrations used were 1 mM and 3 mM for p-NPG and CMUG,

Table 1 Influence of callus extracts on kinetic parameters (K,,,, V,,,) of commercial GUS enzyme

Enzyme (jog ml-‘)

0.1937

Protein Crude extract concentration in the V,,, callus extract (run01 pNp h-‘) (Pg ml-‘) 0 10.5 32.0 66.6

0.3875

87 f 3 85 zt 3 (NS) 71 t4 (P < 0.001) 55

l

2

Purified extract K,,, (mW

V, (nmol pNP h-t)

K, (mW

0.10 l 0.02 0.10 f 0.02 (NS) 0.09 f 0.02 W)

87 zt 3 84*6 (NS) 88 f 4 (NS)

0.10 f 0.02 0.10 f 0.02 (NS) 0.10 f 0.02 (NS)

0.040

l

0.009

(P < 0.001)

(P < 0.001)

165 f 7 121 l 5 (P < 0.001) 100 l 5

0.11 l 0.02 0.10 * 0.02 (NS) 0.08 zt 0.02

(P < 0.001) 81 zt 3 (P < 0.001)

(P < 0.001) 0.04 * 0.01 (P < 0.001)

73.7

ND

ND

*180.3

ND

ND

0 10.5 32.0 66.6

91 *4

0.09 f 0.02

(NS) 165 f 7 168 zt 8 (NS) 171 f 8

(NS) 0.11 f 0.02 0.11 * 0.02 (NS) 0.11 f 0.02

(NS) 172 f 8

(W 0.10

(W

(W

168 zk 6 (NS) 161 f 8 (NS)

0.12 f 0.01 (NS) 0.120 f 0.006 (P < 0.05)

l

0.02

Data were estimated from eight assays for each concentration of callus extracts, except for 180.3 pg ml-’ where only seven assays were performed. Statistical variability was determined for 11 independent experiments (Student t-test). Each V, or K,,, values have been compared with those of the pure enzyme. NS, non-significantly different. P < 0.05 = significantly different at 95%. P < 0.001 = significantly different at 99%. The callus extracts were used as crude extract or as purified extract after passing through a spin column Sephadex G-25 prior to assays.

B. Thomassel et al. /Plant Science 113 (19%)

respectively, excess.

without

inhibition

by

substrate

3.2. Influence of tobacco callus extracts on GUS properties To obtain quantitative information on GUS activity in transgenic plant cells, it is necessary to establish the relationship between the concentration of GUS enzyme and the activity and to identify compounds that inhibit the GUS activity. The experiments described below are performed with a solution of commercial GUS diluted into the extraction buffer (0.1937 or 0.3875 pg ml-‘) containing fixed amounts of extracts from non-transformed callus (0, 10.5, 32, 66.6, 73.7 or 180.3 pg ml-’ protein for each commercial enzyme concentration). In spectrophotometric assays, the amount of hydrolyzed substrate increased linearly over a large range of concentrations for the pure enzyme (Fig. 1). The addition of non-transformed callus extracts (3 1 or 62 rg ml-‘) to commercial GUS enzyme clearly shows an inhibition of GUS activity with p-NPG as substrate (Fig. 1). Indeed, more than 30% and 45% inhibition of commercial GUS enzyme activity was observed when 31 c(g ml-’ or 62 pg ml-’ of non transformed callus respectively were added in the reaction media (Fig. 1). Such an inhibition occurs when more than 10 Fg of crude extract were used in the assay with p-NPG (Table 1). Rao and Flynn [ 171 have also recommended that a total of 4 pg tobacco leaf proteins not be exceeded with 4-

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MUG as substrate. According to these results, it is necessary to determine the optimal amounts of proteins to use in the assays. The changes observed in the properties of the pure enzyme may be related to the presence of endogenous inhibitors in the crude protein extract. By using the previous kinetic model relating p-NP production rate to the substrate concentration (Eq. a), the apparent kinetic parameter (V,,,, K,,,) of the system has been calculated and summarized in Table 1 (crude extract). The significance of the difference between the kinetic parameters of the pure enzyme in the presence or the absence of the crude extracts was established using the Student ttest (P or NS, Table 1). A significant decrease of both the specific GUS activity (I’,,,; P < 0.001) and the &-values (P < 0.001) has been observed when more than 10 pg ml-’ of crude protein extract was added to the pure enzyme (Table 1). The observed inhibition is not competitive (Fig. 2). In this case, the inhibitors do not recognize the catalytic site but it binds to the enzyme at another site to form an active complex ES1 (Fig. 3) [3 11.Indeed, the synthesis of p-NP is highly reduced with increasing concentrations of inhibitor since the constants Ki and KS have been affected by a factor CYand K2 by a factor /3 (Figs. 2, 3). Fig. 2 indicates that the straight lines converge at a unique point for a definite enzyme concentration and different concentrations of plant inhibitor. In our case, cx

,011 ,

: =. z .f = = , ,

Fig. 1. Influence of plant extract on the bacterial GUS activity. The GUS activity was expressed as a function of the concentration of the bacterial enzyme (H) and in the presence of crude extracts from non-transformed callus (31 cg ml-’ (0) and 62 cg ml-’ (0)).

Fig. 2. Lineweaver Burk double reciprocal plot of bacterial GUS activity. Influence of plant extracts. Influence of crude extracts of non-transformed callus (9.2 cg ml-’ (A, A) and 18.4 bg ml-’ (0, 0)) on 0.1875 cg ml-’ (A, 0) and 0.375 pg ml-’ ( A, 0) or bacterial enzyme. Commercial enzyme: 0. I875 pg ml-’ (@ and 0.375 pg ml-’ (U).

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weight compounds (< 5000). However, it is highly probable that, depending on the tissue, the concentration of the inhibitors will change and the optimal amounts of protein should be verified for each plant tissue or species used. 3.3. Properties of E. coli GUS enzyme in transgenic plant cells Fig. 3. Global inhibition model. The model assumes that the fixation of the inhibitor (I) modify the constants KS and Ki by a factor Q. The reaction rate will be also modified by a factor j3 in the presence of the compound I. The inhibition occurs when j3 c 1. We can deduce that a must be inferior to I and a c fi [3l]. (E, enzyme; S, substrate; P, product.) For such a model, the equation of the reaction can be written as:

and fl are less than 1 whereas cr > /3 as observed in Fig. 2 [30]. Moreover, the endogenous inhibitor increases the affinity of the enzyme for its substrate (Table 1). In order to quantitate GUS activity in transgenic plant callus it is necessary to eliminate this endogenous inhibitor. The inhibitory effect is partially removed when the non-transformed callus extract is purified by passing through a Sephadex G-25 spin column prior to the assays (Table 1; purified extract) [12,17,32]. Such a treatment eliminates almost all the polyphenolics, pigments and low molecular weight contaminants from the extract. Whatever the concentration of the commercial GUS enzyme and for concentrations of callus extract less than 180 pg ml-‘, the specific activity is not significantly different from those of the pure enzyme (Table 1; purified extract). However, the &-values for 180 c(g ml-‘, differ from each other at a confidence level higher than 95% (Table 1). With the purified callus extracts, no inhibition of GUS activity is observed with 75 pg ml-’ of tobacco protein. The optimal amount of proteins to use in the assays should not exceed 180 pg ml-’ in order to measure GUS activity in transgenic callus. The nature of the inhibitors were not further investigated but they were probably low molecular

The bacterial uidA coding sequence has been used in gene fusion to different regulatory sequences allowing either constitutive or inducible GUS expression in callus cultures or suspension cultures. Such experiments are used both to verify the expression level of the introduced gene or to have a time-dependent expression of the foreign gene in the plant cells. In our experiments, the TDNA containing the uidA gene under the control of either the constitutive 35s cauliflower mosaic virus promoter or the light inducible promoter of an Arabidopsis gene encoding the small subunit of Rubisco was introduced via Agrobacterium tumefaciens into tobacco protoplasts following the method described by Van Lysebettens et al. [26]. Then, for each transformation (constitutive or inducible), the transgenic cells were embedded in agarose. In this way, each transformed cell resulting from a single transformation event can be collected separately. Numerous cell lines have been obtained for each constitutive or inducible system. Furthermore, each cell line was quantitatively analyzed for GUS expression. These experiments were performed by spectrophotometric or fluorometric assays on the transgenic cell lines having integrated a constitutive or an inducible uidA gene, respectively. As previously described [16] the expression levels of the introduced gene differed considerably in different transformants and for each construct we chose the cell line with the highest GUS activity to determine the kinetic parameters of the GUS enzyme. In the transgenic cells, whatever the integrated constructs (35s GUS, SSU GUS), the K,,, values greatly decreased when increasing amounts of protein from a crude extract of transgenic cells were tested (Table 2: crude extract). On the other hand, the E. coli GUS enzyme showed the typical behaviour of the commercial GUS enzyme when the plant extracts were purified on a G-25 Sephadex spin column (Table

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215

Table 2 The behaviour of E. coli GUS enzyme in transformed tobacco cell suspensions Protein concentration in cell suspension extract (Pg ml-‘) 35s GUS

2.66 8.30

SSU GUS

Crude extract V, (nmol p-NP h-‘)

1.8 l 0.02 8~1

33.4

28 f 1

2.4

9.8 * 0.2

Purified extract K, (mW

0.1 f 0.02 (NS) 0.087 zt 0.003 (P < 0.05) 0.07 l 0.03 0.32 ZL0.02

V, (nmol p-NP h-‘)

2.2 f 0.04 9.66 f 0.06 35 f 2

K, (mM)

0.10 f 0.02 (NS) 0.10 f 0.01 (NS) 0.09 f 0.02

ND

ND

24.5 + 0.8

0.38 zt 0.04 (NS) 0.35 f 0.04 (NS) 0.35 f 0.05

(P < O.OOOl)

4.8

12.2 f 0.2

0.23 f 0.10 (P < 0.001)

9.8 12.1

27.8 + 0.4

0.088 f 0.008

49 * 2

ND

ND

70 f 3

The 35s GUS calli were obtained after introducing the gene coding for the fl-glucuronidase under the control of the constitutive 35s promoter of cauliflower mosaic virus via Agrobacterium tumefaciens. The results were expressed in nmol p-NP h-l. Data were estimated from 10 assays for each concentration of plant extracts, statistical variability was determined following Student t-test and each K,,,-values have been compared with those of the first protein concentration (2.66 pg ml-‘). The SSU GUS calli were obtained by introducing the GUS coding sequence under the control of inducible promoter of the rub&o SSU gene from Arabidopsis. The induction was performed after an overnight light exposure of the plant cells at 24°C under continuously shaking (I 10 rev./mm). The results are expressed in nmol MU h-l. Data were estimated from six assays for each concentration of plant extracts and each K,,,values have been compared with those of the first protein concentration (2.4 pg ml-‘). The plant extracts were used as crude extracts or as purified extracts after passing through a Sephadex G-25 spin column prior to assays. NS, non-significantly different. P < 0.05 = significantly different at 95%. P < 0.001 = significantly different at 99%. ND, non-determined.

2: purified extract). In such experiments, not only the GUS activity was restored (Table 2). Indeed, the V,-values were increased by a factor of around 1.2 or 1.9 in the assays with purified extracts of the 3% GUS or SSU GUS lines, respectively (Table 2: compared 2.2 to 1.8 nmol p-NPG h-’ for 3% GUS and 24.5 to 12.2 nmol MU h-’ for inducible GUS). The presence of crude plant extract decreases the GUS activity which was completely restored when purified extract was added to the enzyme (Table 1). In fact, such a treatment allows the elimination of the small compounds that inhibit the GUS activity [17,32]. 3.4. GUS-like activity Many plants have various levels of endogenous GUS or GUS-like activity which can interfere with

the activity originating from the introduced GUS gene, especially if this level is low [8,9,11,16]. In our experiments, the GUS-like activity was determined for various non-transgenic plant species. Leaves, calli or suspension cultured cells of tobacco, coffee and carrot were homogenized in the extraction buffer. Initial GUS activities were determined in these extracts by the method described by Jefferson et al. [3] using 3 mM 4MUG as a substrate. Prior to the assays, substrate was incubated in the absence of plant extract to determine the background rate of hydrolysis. No hydrolysis of 4-MUG has been observed in these control experiments. Fluorometric analysis of GUS activity in nontransformed tobacco tissue indicate the presence of endogenous activity capable of converting 4MUG to the fluorescent product (MU; Table 3).

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Table 3 Intrinsic GUS activity Crude extract (pmol MU h-’ pg-’ protein)

Purified extract (pmol MU h-’ rg-’ protein)

Reaction media without substrate tobacco callus or cell suspensions

1.32 Z’Z0.01

ND

Tobacco Callus Cell suspensions Leaves

1.05 f 0.22 0.75 f 0.08 1.4 * 0.4

NS

0.6 f 0.2 0.20 f 0.04 1.0 f 0.6

Coffee Callus Cell suspensions

5*3 1.3 * 0.4

NS NS

5 f 0.5 0.9 f 0.3

Carrot Callus Cell suspensions

5.7 l 0.4 1.20 f 0.05

P < 0.001

3.5 f 2 I.1 f 0.3

P < 0.001 P < 0.001

NS

Calli, suspensions and leaves (400 mg fresh weight) were homogenized in I .5 ml of extraction buffer as described in Section 2, Materials and methods. Extracts containing 10 to 75 pg of protein were used for GUS assays. The values are the means of IO experimental data and were calculated from the integrated curve of GUS activity represented as a function of the protein amounts. NS, nonsignificantly different. P < 0.001 = significantly different at 99%. ND, not detected. In these experiments the value 1.32 pmol MU h-’ cg-’ protein obtained with tobacco callus or cell suspension in the absence of substrate has been subtracted from the tobacco callus or cell suspension values.

The intrinsic GUS activity of callus of N. tabacum was around l.Cfold higher than for suspension cells (1.05 f 0.2 to 0.75 f 0.08 pmol MU h-’ kg-’ protein; Table 3). According to the results of Kosugi et al. [18], obtained on various plant species, the levels of endogenous GUS activity differed among cell types in the same plant. In calli, the GUS activities were always higher than those of suspension cultured cells whatever the plant species (Table 3). Numerous studies have shown that changes in the pattern of intrinsic GUS-like activities can be modified during seed germination, stages of development, tissue source, age and treatment [9,11,18]. Moreover, the intrinsic GUS like activity seems to be strongly genotype dependent since high activity has been recorded in Nicotiana tabacum var. Petit Havana SRl (1.05 pmol MU h-’ rg-’ protein; Table 3) compared with those obtained for callus of Nicotiana tabacum Bright Yellow (0.56 pmol MU h-’ pg-’ protein) [18]. Such a clear genotype dependency has also been recently reported by HHnsch et al. [ 101on different genotypes of barley. We have noticed that the incubation of crude ex-

tract in the reaction media and in the absence of substrate has always produced a significant background [12]. The background level was measured at around 1.32 f 0.01 pmol MU h-’ rg-’ protein in tobacco crude extract and was completely eliminated after passing through a spin column of Sephadex G-25 prior to the assays (Table 3). After such a treatment, the intrinsic GUS activity recorded from tobacco callus or cell suspension decreases significantly (Table 3). On the contrary, the intrinsic GUS activity is not modified after purification of extracts from coffee or carrot cells (Table 3). Indeed, it is highly probable that depending on the tissue tested the nature and the concentration of inhibitors or endogenous enzymes will change. These interfering unspecific GUS activities are not present in all tissues and plants [11,19]. Although the E. coli GUS enzyme is very sensitive to low or high pH (< 5 or > 7.5) no change in the GUS activity has been detected at pH-values lower than 7.5 with the crude or the purified tobacco callus extract in our experiments contrary to those obtained by Alwen et al. [8] and Wozniak

B. Thomasset et al. /Plant Science 113 (19%) 209-219 Table 4 Effect of the saccharic acid-l$lactone

217

on the GUS activity SL concentration (mM) 0

Bacterial enzyme Bacterial enzyme + crude extract from non-transformed cells Bacterial enzyme + purified extract from non-transformed cells Tobacco crude extract from calli Tobacco purifiedextractfrom calli

I

2.5

5

(5.1 l 0.2) x 106 (2.9 f 0.1) x IO6

(0.75 f 0.01) x I06 (0.9 l 0.2) x 106

-

(0.025 f 0.006) x IO6 (0.32 zt 0.07) x IO6

(5.01 zt 0.06) x IO6

(0.84 f 0.15) x IO6

-

(0.27 f 0.04) x IO6

1.4 l 0.2

0.6 A 0.2

-

I.0 * 0.3

0.8 f 0.2

-

0.7 f 0.1

0.59l 0.02

The inhibitor was added at the indicated concentrations to the reaction mixtures containing bacteria1 GUS enzyme or nontransformed tobacco protein extracts. Extracts containing IO to 75 pg of protein were used for GUS assays. Data (pmol MU h-’ cg-’ protein) were estimated from five independent assays.

and Owens [9]. Moreover, the GUS-like enzyme present in the tobacco callus extracts is more resistant to heat treatment than the commercial GUS enzyme. Only around 50% of inhibition has been observed with crude or purified callus extracts after 10 min at 6YC or 5 min at 100°C compared with more than 98% of inhibition with the commercial GUS enzyme after similar treatments (data not shown). Several papers have underlined the tolerance of the intrinsic GUS-like enzyme to D-saccharic acid l-4 lactone (SL) when tested in the crude protein extracts [9,18]. We have tested the effect of such an inhibitor on the behaviour of the commercial /3glucuronidase in the presence or absence of nontransformed protein extract (Table 4). According to the Lewy’s results [33] the commercial @glucuronidase is very sensitive to the specific SL inhibitor (Table 4: inhibition superior than 85% and 99.5% for 1 and 5 mM SL, respectively). When the commercial GUS was tested in the presence of purified non-transformed extract, total GUS activity decreased about 83% and 95% with 1 mM and 5 mM inhibitor, respectively when compared to untreated samples (Table 4; compare 0.84 and 0.27 rmol p-NP h-i pg-’ protein). Such a behaviour is similar to that of the commercial enzyme. On the contrary, the addition of crude extracts induced a decrease of the GUS activity (Table 4: compare 2.9 to 5.1 pmol p-NP h-’ rg-’ protein) due to the complex formed between the intrinsic plant inhibi-

tor and the pure enzyme (Table 1). Such a complex prevents the specific effect of the SL inhibitor since only 70 and 89% inhibition have been recorded with 1 and 5 mM SL, respectively (Table 4: compare 0.9 and 0.32 to 2.9 pmol p-NP h-’ pg-’ protein). Such a behaviour can explain the difference in the level of the inhibition of the intrinsic GUS enzyme in crude extracts as observed previously [8,9,18], since there might be competition between the intrinsic plant inhibitor and the SL inhibitor. In our case, no inhibition was observed when SL was incubated in the presence of the purified extract of non-transformed callus (Table 4). After elimination of low molecular-weight compounds in the protein extracts, SL does not inhibit the endogenous GUS activity. As such, the intrinsic GUS-like activity probably depends on the presence of other enzymes. 4. Conclusions The use of uidA as a reporter gene is often limited by the presence of high levels of GUS-like activities masking the activity of the introduced gene. Our results indicate that the filtration of the plant protein extracts can both minimize the interfering unspecific activity and prevent them from the inhibitory effect of small compounds. However, the resulting intrinsic GUS activity in plant extracts may or may not depend on plant &lucuronidase. We have confirmed that SL, a specific inhibitor of the commercial GUS enzyme [32] does not inhibit

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this endogenous GUS enzyme. Moreover, the endogenous enzyme seems to be less sensitive to heat treatment than the commercial GUS and seems to be independent of the pH of the reaction. However, after purification of the protein extracts on G-25 spin columns, the intrinsic GUS activity always remains very low compared with the E. coli GUS expression systems. The major point of this study is to be aware of the fact that GUS activity in transgenic tissues will depend on the possible effect of both inhibitory compounds and endogenous GUS-like activity. After purification of the extracts, transgenic cell lines can be unequivocally distinguished from non-transgenic plant cells and the expression of the uid4 gene can be accurately quantified in each transgenic cell line. Acknowledgements

This work was financially supported by the Biopole VCgetal de Picardie. We thank Valerie Devillers and Laurence Chevalier for technical assistance. We are grateful to Dr Botterman (Plant Genetic Systems NV, Gent, Belgium) for providing the ptDE4 and the pSSU plasmids. References

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