Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Plant Science, 87 (1992) 115-122 Elsevier Scientific Publishers Ireland Ltd.

115

Detection, expression and specific elimination of endogenous /3-glucuronidase activity in transgenic and non-transgenic plants L e n e H o d a l a, A n j a B o c h a r d t a, J o h n E. Nielsen b, Ole M a t t s s o n a a n d F i n n T. O k k e l s b alnstitute of Plant Physiology, University of Copenhagen, 2A Oster Farimagsgade, DK-1353 and/'Maribo Seed Biotechnology, Danisco .4/S. P.O. Box 17. DK-IO01 Copenhagen K (Denmark/ (Received June 9th, 1992; revision received August 7th, 1992: accepted August 10th, 1992)

With the aim of making a system using the GUS gene as a selection gene, the effects of two toxin glucuronides were tested on transgenic tobacco cells (Nicotiana tabacum L.) containing a/3-glucuronidase (GUS) gene from Eseherichia coli and on non-transgenic cells. No significant difference in toxicity was observed between transgenic and non-transgenic cells. We found that this most probably was due to the activity of an endogenous GUS enzyme, which could be detected in all plant species tested, e.g. tobacco, sugar beet (Beta vulgaris L.), oilseed rape (Brassica napus L.), pea ( Pisum sativum L.), wheat ( Triticum sativum L.) and rhubarb (Rheum rhaponticum L.). This indicates that GUS may be ubiquitous to plants contrary to earlier assumptions. The endogenous enzyme is active at pH 4-5 and the activity is eliminated without reducing the introduced GUS activity when pH is elevated. In addition the endogenous GUS can be selectively inhibited at high temperatures. Modifications according to these findings can be employed in standard GUS assays to avoid misinterpretations when the expression of tissue specific promoters is tested.

Key words." endogenous B-glucuronidase: toxin glucuronides: D-saccharic acid 1,4-1actone: tobacco (Nieotiana tabacum L.I

Introduction

The widespread use of the 13-glucuronidase (GUS) gene from E. coli as a reporter gene has been based on the assumption that no endogenous GUS activity was present in plants [1]. Furthermore, it has been proposed that the GUS gene can be used as a positive or negative selection gene in tissue cultures [2] and the use of pollen or anther specific expression of GUS genes to activate toxin glucuronides sprayed on the plants to induce male sterility has been suggested [3]. Correspondence to: Anja Bochardt, Institute of Plant Physiology, University of Copenhagen, 2A Oster Farimagsgade, DK-1353 Copenhagen K, Denmark. Abbreviations." CAP, chloramphenicol; CAPG, chloramphenicol glucuronide; 2,4-D, dichlorophenoxy acetic acid: GUS, ~glucuronidase; MS-medium, Murashige and Skoog minimal organics medium; PNP, p-nitrophenol: PNPG, p-nitrophenyl glucuronide; SL, D-saccharic acid 1,4-1actone; X-gluc, 5bromo-4-chloro-3-indolyl-/3-D-glucuronide.

Introduction of foreign GUS genes should allow hydrolysis of various/3-glucuronides and thereby activation of the aglycons, e.g. a toxin. Using two toxin glucuronides, chloramphenicol glucuronide (CAPG) and p-nitrophenyl glucuronide (PNPG), we have been trying to establish tissue culture systems in which transgenic cells containing inactive GUS genes may be selected. We found that one of the prerequisites for the use of the GUS gene as a selection gene is not fulfilled. The glucuronides are not stable in plant cells not expressing the introduced GUS gene because these cells contain endogenous GUS. Lately, putative endogenous activity has been reported in certain tissues in various plant species [4-9] when assayed according to the standard conditions (pH 7.0, 37°C) described by Jefferson et al. [21. Here we present data showing that plants in general contain a native GUS enzyme which is responsible for the endogenous activity appearing

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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in the assays and this native enzyme is expressed in most tissues. The endogenous GUS activity differs from the introduced GUS activity with respect to optimal pH and heat tolerance. Assay conditions, which selectively reduce or eliminate endogenous GUS and enhance activity of the introduced GUS are described. Materials and Methods

Plant material and growth conditions Plants regenerated from leaf discs of primary transgenic plants of tobacco (Nicotiana tabacum L. cv. Wisconsin 38 and Petit Havana) transformed with the Ti-plasmid pMON9749 [10] (plant) or pBI 121 [2] (suspension cells) were used. The TDNA of both contain the uidA gene from E. coli (encoding GUS) [111 driven by the CaMV 35S promoter and other genes of no interest to this study. The young plants were cultured on hormone free Murashige and Skoog minimal organics medium (MS-medium) [12] (pH 5.7), supplemented with 0.7% (w/v) agarose (Sigma type I) and 600 mg/l carbenicillin (disodium salt). Mature plants were grown in the greenhouse in soil. Material from the following species was used: young (3-4 cm tall) transgenic and non-transgenic tobacco plants cultured in vitro, non-transgenic mature (at flowering stage) tobacco plants, rhubarb petioles (Rheum rhaponticum L.), bought locally, in vitro grown shoot cultures of sugar beet (Beta vulgaris L.), oilseed rape (Brassica napus L.), wheat (Triticum sativum L.) and pea (Pisum sativum L.). Sugar beet, oilseed rape and pea were grown on MS-medium (pH 5.7), supplemented with 0.25 mg/l BA and 0.7% (w/v) agarose. Tissues of wood-sorrel (Oxalis acetosella L.) and duckweed (Lemna minor L.) plants, collected locally, were also used. Suspension cultures of tobacco were established from callus induced on leaf discs. The discs were placed on MS-medium (pH 5.7), supplemented with 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4D), 0.1 mg/l BA and 0.7% (w/v) agarose. Cultures were grown in liquid MS-medium (pH 5.7) containing 1 mg/l 2,4-D and 0.1 mg/l BA at 25°C under continuous light (20 #mol m -2 s-~), on a shaker set to 80 rev./min. Cultures were sub-

cultivated every seventh day to a settled cell volume of 30%.

Histochemical GUS assays Tissue and suspension cells were assayed for GUS activity with the indigogenic substrate, 5-bromo-4-chloro-3-indolyl-/3-D-glucuronide (Xgluc), according to the procedure described by Jefferson et al. [2] with some modifications. The testing solution contained 1 mg/ml X-gluc, cyclohexylammonium salt (Sigma) in 0.1 M sodium phosphate buffer, 10 mM Na~EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 and 3% (w/v) sorbitol. Leaf discs, without major veins, 5 mm in diameter were punched out. Transverse sections of stems, petioles and roots were cut by hand. The reaction was stopped by placing the samples on ice. Chlorophylls were cleared by soaking the tissues in 96% ethanol before data were taken. The results are based on a minimum of three replications. Samples of suspension cultures were filtered through a 100-/zm mesh and 50 mg of cells was incubated with 200 #1 of testing solution. The stained cells were counted under the microscope. The pH was adjusted from 3.0 to 9.0 as indicated with NaOH or HCI. Experiments with preincubation of tissues were performed in a testing solution without X-gluc at pH 5.0-9.0 for 5 h at room temperature, followed by 8 h of incubation at 37°C in testing solution with a similar pH. When the effect of temperature of incubation was tested, the plant material was incubated for 8 h in preheated testing solution at 37, 50, 55, 60 or 65°C. As a control, 10 mM D-saccharic acid 1,4lactone (SL) was added to the incubation medium. According to Levvy [13] SL is a specific inhibitor of GUS. Test of toxin glucuronides on suspension cells Suspension cells, transgenic and non-transgenic, diluted to 10% (w/v) in MS-medium supplemented with 1 mg/1 2.4-D and 0.1 mg/l BA, were homogenized (Ultra-Turrax, Janke & Kunkel, Germany) for 10 s. at 1000 rev./min to separate the cells from each other. A volume of 1.5 ml was transferred to 20 ml MS-medium solidified with 7% (w/v) agarose (Sigma, type I) and sup-

117

plemented with 1 mg/l 2,4-D, 0.1 mg/l BA and P N P G or p-nitrophenol (PNP), in concentrations from 3.6 mM to 9.0 mM. The petri dishes (diameter 9 cm) were sealed with polyethylene film and placed in the dark at 25°C. Growth was measured after 3 weeks as number of colonies formed (diameter > 2 mm). Of these colonies 210 were tested for GUS activity in the X-gluc assay. To test if the introduced GUS was able to hydrolyse PNPG, the spectrophotometric GUS assay described by Jefferson [1] was employed. Cells were lysed by a French Press at 68 atm, incubated for one hour with P N P G and the amount of PNP liberated was monitored on a spectrophotometer. The effect of addition of SL (10 mM) stabilized at pH 5.7 to this assay was examined. The effect of SL without any toxin glucuronides was also tested on transgenic and non-transgenic cells, using the plating procedure described above. Number of colonies was scored after three weeks. Extracts of transgenic cells containing the E. coli GUS enzyme were added to solid medium with or without PNPG. The change in colour was observed visually after 20 h at 25°C. The plating procedure described above was used to test the effect of CAPG or chloramphenicol (CAP) on suspension cells. To examine if the effect of C A P G on nontransgenic cells was caused by activity of endogenous GUS, non-transgenic cells were exposed to combinations of CAPG and SL. The plating procedure was as previously described and colony formation was scored after 6 weeks.

Table I. Effect of CAPG and CAP on growth of transgenic and non-transgenic suspension cells of N. tabacum. Number of colonies was scored after exposure to 5 ~tM CAPG or 5 #M CAP for 4 weeks. GUS, transgenic; CON, non-transgenic. (n = 3, coefficient of variation: S.D. /~ × 100 in brackets)

Codename

GUS CON

No. of colonies in percent of control Control

CAPG

CAP

100 (28) 100 (26)

62 (29) 53 (30)

34 (17) 23 (28)

Table lI. Effect of CAPG in combination with SL on growth of control suspension cells of N. tabacum. Number of colonies in percent of control (no CAPG) after exposure to CAPG in combination with SL for 6 weeks. (n = 8, coefficient of variation: S.D./~ x 100 in brackets)

SL (mM)

0 3.75

CAPG (aM) 0

12.5

25

100 (14) 100(23)

15 (30) 25 (31)

5 137) 15 (43)

Results

The effect of toxin glucuronides Growth of both transgenic and non-transgenic suspension cells of tobacco was inhibited after exposure to 5/~M CAPG for 4 weeks (Table I). The non-transgenic cells were slightly more affected by both CAP and CAPG than the transgenic cells. The inhibitory effect of CAPG (5/~M) was approx. 60% of the effect of CAP (5 #M) in both cultures. At 50 /~M of C A P G or CAP no colonies was observed after 4 weeks. After 8 weeks a few colonies of both transgenic and non-transgenic cells appeared on both media. All colonies of transgenic cells reacted positive in X-gluc assay at pH 7. When non-transgenic suspension cells were grown on CAPG medium supplemented with SL at a concentration of 3.75 mM, the inhibitory effect of CAPG could be reduced (Table II). When SL was tested alone at concentrations ranging from 10 to 30 mM it showed pronounced inhibitory effects (Table III). The growth of non-

Table IlL Effect of SL on growth on transgenic and nontransgenic suspension cells of N. tabacum. Number of colonies of suspension cells after exposure to SL for 3 weeks in percentage of the control. GUS, transgenic; CON, non-transgenic. (n = 4, coefficient of variation: S.D./~ × 100 in brackets)

Codename

GUS CON

SL (mM) 0

10

30

100 (23) 100 (29)

85 (27) 46 (33)

18 (36) 15 (31)

118 transgenic cells was reduced more than the growth of transgenic cells at low SL concentration (10 mM). When the concentration was increased to 30 mM, the growth was strongly reduced for both the transgenic and the non-transgenic cells (Table III). PNP completely inhibits growth of both transgenic and non-transgenic cells at the lowest concentration tested (3.6 raM). At that concentration PNPG had no effect. Growth was inhibited up to 19% for the transgenic cells and up to 27% for nontransgenic cells by 9.0 mM PNPG. When the colonies of transgenic cells grown on PNPG containing medium were examined for GUS activity in X-gluc assay (pH 7) all showed a positive reaction. No reaction was observed (at pH 7) when the colonies of non-transgenic cells were tested. Extracts of the GUS enzyme from the transgenic suspension cells were able to liberate PNP both when P N P G was found in liquid (PNPG assay) and in solid medium. The latter was detected as a shift in colour in the solid P N P G containing medium from colourless to the same yellow colour as observed for the medium containing PNP.

The effect of p H of the testing solution on the G US activity At 37°C the transgenic suspension cells of tobacco reacted positive in X-gluc assay at pH 5 and pH 7 (Table IV). Non-transgenic cells reacted at pH 5, while no GUS activity was detected at pH 7 (Table IV). Addition of SL (10 mM) eliminated

Table IV. The effect of elevation of the incubation temperature on the GUS activity in transgenic and non-transgenic tobacco suspension cells. Samples of suspension cultures were filtred and tested in X-gluc assays performed at pH 5 and 7. The temperature of incubation was 37, 45, 50. 55 or 65°C.

Temp(°C)

37 45 50 55 65

GUS expressingcells (%) Non-transgenic cells

Transgeniccells

pH 7

pH 5

pH 7

pH 5

0.0 a 0.0 0.0 0.0 0.0

99.3 97.9 16.9 3.0 0.0

97.2 95.6 96.6 97.0 96.0

100.0 99.6 99.9 99.9 79.3

a Values are means (n = 3, each of 2250 ceils).

Table V. G U S activity in non-transgenic plants. X-gluc assays were performed on plant tissues at pH 3 - 7 and at 37°C for 8 h. Assays on rhubarb and pea were performed on petioles and hypocotyls, respectively, all others were performed on leaves or suspension cells (tobacco). Species

pH 7

6

5

4

3

Duckweed

+a

nd b

nd

nd

nd

Rhubarb Wood-sorrel Pea Oilseed rape Sugar beet Tobacco Tobacco cells Wheat

+ +

+ nd

+ nd

nd nd

nd nd

0

0

+

+

0

(+) (+) 0 0

+ + 0 0

+ + + +

+ + + +

+ + 0 0

0

+

+

+

0

aRating scale; 0, no reaction; +, positive reaction; (+), occasional positive reaction. b nd, not determined.

measurable GUS activity in transgenic (pH 5 and pH 7) and in non-transgenic cells (pH 5) (not shown). When non-transgenic plants of different species were tested in X-gluc assays at standard conditions (pH 7 and 37°C), intense GUS activity was observed in duckweed, rhubarb and wood-sorrel (Table V). At this pH, no GUS activity was detected in tobacco, pea and wheat. Similarly no activity was observed in oilseed rape and sugar beet, except for occasional reactions in epidermis, stomata, trichomes and in xylem parenchyma. In contrast, intense GUS activity was observed in all species when the pH of the testing solution was adjusted to 5 (Table V). Addition of 10 mM SL to the Xgluc assay eliminated detectable GUS activity. As SL is a specific inhibitor of all GUSs tested [14], the inhibition by SL found in the present study indicate that the endogenous reaction is caused by an endogenous GUS. In accordance with this glucuronic acid and selected glucuronides inhibited the endogenous activity, while gluconolactone, a specific inhibitor of glucosidases [15] and galactose and EDTA, inhibitors of UDPglucuronid transferases [16], had no effects (data not shown). Control experiments without plant tissue show-

119 Table VI. G U S activity in mature and young non-transgenic tobacco plants. X-gluc assays were performed at pH 5 or 7 at 37°C, 8 h incubation. Leaves (from mature: 140 m m in length, young: 50 m m in length) were dissected from the shoot apex. Structure/tissue

Stem Pith Pith rays External phloem Internal phloem Trichomes Epidermis Cortex

Mature plant

Young plant

pH 7

pH 5

pH 7

pH 5

0a 0 0 0 (+) 0 0

+ 0 ++ + +++ 0 0

0 0 0 0 0 0 0

0 +++ +++ +++ ++ (+) 0

Leaf Mesophyll Vascular bundles Trichomes Stomata

0 0 0 0

++ +++ +++ +++

0 0 0 0

+++ +++ +++ +++

Root Root hairs Cortex Vascular bundles

0 0 0

0 ++ +++

0 0 0

0 +++ +++

aRating scale of intensity: 0. no; (+), occasionally low; +, low; ++, medium; +++, high reaction.

ed no detectable spontaneous hydrolysis of X-gluc in the range pH 3-8, nor did leaves incubated in testing solution without X-gluc react. Analysis of fourteen different tissues/structures, representing stem, leaf and root, of either young or mature tobacco plants were performed (Table VI). At pH 7, no GUS activity was observed, except occasionally in the trichomes on stems of mature plants. At pH 5, GUS activity was observed in ten of the fourteen tissues/structures analyzed. In leaves and roots, GUS activity was observed in a similar pattern in the mature and the young plant, but in general a higher intensity of the reaction was seen in the young plant compared to the mature plant. In rhubarb the endogenous GUS activity detected at standard conditions in the X-gluc assay could be eliminated if the tissue was preincubated and tested at elevated pH (pH 9) (Table VII). When pH of the testing solution was measured after rhubarb had been incubated the pH was

Table VII. Effect of elevation of the pH in the testing solution on G U S activity of rhubarb and tobacco plants. Segments of petiole from rhubarb and stems of tobacco were preincubated in buffer at pH 5, 7, 8 or 9 for 5 h, followed by incubation in X-gluc testing solution at similar pH at 37°C for 8 h. pH

Non-transgenic rhubarb

Non-transgenic tobacco

Transgenic tobacco

5

+++a

+++

+++

7

+++

0

8

++

0

9

0

0

+++ +++ +++

aRating scale: 0, no reaction; +++, high intensity of reaction.

decreased from 9.0 to 6.1. In non-transgenic tobacco plants the GUS activity was eliminated if pH was above 5 in the assay, whereas in transgenic tobacco plants GUS activity was not reduced when raising the pH up to 9. (Table VII). This indicates that the X-gluc assay is functioning at elevated pH and the elimination of the reaction at high pH is due to specific inactivation of an endogenous enzyme.

The effect of the &cubation temperature on the GUS activity In non-transgenic tobacco plants, elevation of the incubation temperature at pH 5 reduced the

Slructure Tissue

pH5

Stem

pH 7

'!

Pith

.t ]

i!,i)i", i't

Pith rays External phloem Internal phloem Trlchomes Eplderrnls Cortex Leaf

Mesophyll Vascular bundles Trlchomes Stomata 37 50 55 60 65

37 50 55 60 65 °C

i

Reaction in transgenlc and non-transgenlc plants

[~

Reachon only in transgemc plants

Fig. 1. The effect of elevation of the incubation temperature on the GUS activity in tissues of young, non-transgenic and transgenic tobacco plants. X-gluc assays were performed at pH 5 and pH 7.

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number of GUS expressing structures and the intensity of the reaction (Fig. 1). At 60°C, no GUS activity was detectable in any structure. In transgenic plants, no reduction in the number of GUS expressing structures was observed in leaves as temperature was increased; on the contrary the intensity of the reactions was somewhat enhanced. The activity in the stem was gradually reduced and at 65°C no GUS activity was observed. A similar pattern was seen in the transgenic plants when the assay was performed at pH 7 (Fig. 1). An effect of raising the temperature could also be demonstrated on suspension cells of tobacco (Table IV). At 37°C practically all non-transgenic cells expressed GUS activity when assayed at pH 5. When temperature was raised the number of GUS expressing cells was gradually diminished and at 65°C no activity could be detected. In the transgenic culture, only a weak reduction in the number of GUS expressing cells was observed at pH 5 when temperature was increased up to 65°C. When the X-gluc assay were performed at pH 7 there were no effect of raising the temperature up to 65°C. Discussion

In the present study none of the tested toxin glucuronides has selective inhibitory effect on the transgenic cells (Table I). The toxic effect was more pronounced for the non-transgenic cells in both instances. To test whether this toxicity was caused by hydrolysis of the toxin glucuronides by endogenous GUS, non-transgenic cells were exposed to CAPG in combination with SL. In this case the growth inhibiting effect of CAPG was decreased (Table II). This indicates that the effect of CAPG on non-transgenic cells was caused by an endogenous GUS enzyme hydrolyzing the glucuronide, rather than the toxin glucuronide being toxic before hydrolysis. Since SL also inhibits the introduced GUS enzyme it cannot be used for selective elimination of the effect of the native GUS. Our results indicate that suspension cells of tobacco have a limited uptake capacity of glucuronides. First, the inhibitory effect of both the toxin glucuronides was smaller than that of the

toxins and at the same time the transgenic cells were not more affected by the toxin glucuronides than the non-transgenic cells even though the transgenic cells expressed the introduced GUS gene. Secondly, CAP is much more toxic to plant cells than PNP, as is CAPG compared to PNPG, but the difference in toxicity between CAP and CAPG is not very pronounced, while PNP exhibits much higher toxicity than PNPG. This may simply be due to differences in the uptake of CAPG and PNPG, or that the uptake of small amounts of glucuronides (CAPG) is possible while uptake of larger amounts of glucuronides (PNPG) cannot happen. Such limited uptake capacity of glucuronides of weak toxins will not result in detectable toxic symptoms. As indicated by the effect of SL in combination with CAPG on non-transgenic cells of tobacco, the activity of an endogenous GUS could be detected in the X-gluc assay. This enzyme was not active at standard conditions (pH 7), but could be detected if pH in X-gluc assay was decreased (Table V). At pH 5 endogenous GUS activity was seen in a broad range of plants (Table V). In some species (duckweed, rhubarb, wood-sorrel) endogenous activity could be detected at pH 7. These species have an acidic vacuolar sap and are rich in oxalate [17-19] and the lower pH of the tissues decreases the initial pH of the testing solution and thus might cause a more favourable condition for activity of the endogenous GUS. In agreement with this, it was possible to eliminate the endogenous GUS activity when raising the pH in the tissues of rhubarb by preincubation followed by incubation at pH 9 (Table VII). Plegt and Bino [7] observed endogenous GUS activity in certain floral organs of non-transgenic tobacco plants when the histochemical assay was performed at pH 6, whereas Hauffe et al. [20] found no activity at pH 7. This discrepancy may be explained by the differences in pH during the assays. Only the lower pH makes detection of the endogenous activity possible. The occasional observations of GUS activity at pH 7 in the trichomes of mature tobacco and in the stomata, trichomes, epidermis and xylem parenchyma of sugar beet in the present study, may be due to an acidic environment in these cells, which

121

favours activity of the endogenous GUS. Idioblasts with large amounts of oxalic acid are common in certain tissues in a number of plants species [17,21]. They occur in trichomes, epidermis and in cells connected to the vascular system [17,21] and endogenous GUS activity is occasionally observed in these tissues at pH 7. This activity may be due to the occurrence of more oxalic acid than the buffer in the standard testing solution can neutralize. Hu et al. [4], who performed GUS assays at pH 7 in various tissues of different plants, interpreted the observed endogenous GUS activity as tissue specific expression of native GUS gene(s) in plants. Since we detected GUS activity at pH 5 in most of the analyzed tissues, including those of beet and tobacco where Hu et al. found no activity, we suggest an alternative interpretation. Rather than a tissue specific gene expression the GUS activity in certain tissues at pH 7 may reflect a tissue specific occurrence of acid containing cells. This also explains, why the authors detected binding of antibodies to GUS in extracts of tissues, which did not express GUS in assays at pH 7. The enzyme is present, but not active at the assay conditions used. Other reports on organ or tissue specific GUS activity at pH 7 in various tissues and plants species [5-9] may be explained in a similar way. With the knowledge of the pH range for activity of the endogenous GUS, it is possible to analyze the true expression pattern of this enzyme. As shown in Table VI, the endogenous enzyme is found to be expressed in most tissues or cell types, while in a few, e.g. root hairs there is no detectable activity. GUS activity in root hairs of transgenic tobacco plants containing the GUS gene from E. coli (data not shown) indicates that differences in detected activity are not due to selective functioning of the assay, e.g. differences in uptake of X-gluc in the different cell types. Besides the difference in pH range between endogenous GUS and the E. coli derived GUS it was found that it was possible to eliminate selectively the endogenous GUS activity by increasing the incubation temperature to 60°C. The E. coliderived GUS is rather resistant to thermal inactivation with a half-life at 55°C of about 2 h [1]

and apparently an increased activity caused by the increased temperature more than compensate for a possible shortening of its half-life. The introduced GUS enzyme is also active in the assay at pH 5, indicated by the observation that all leaf tissues examined still show GUS activity above 55°C in the transgenic plants, although endogenous activity is eliminated at that temperature in the non-transgenic plants. This is in agreement with the GUS from E. coli having maximal activity between pH 5.0 and 7.5 [ll]. Thus the present observed GUS reaction at pH 5 and 37°C in transgenic plants is the total effect of the activities of the introduced and the endogenous GUS. The general function of GUS in plants is not known. Treatment with SL leads to inhibition of growth, the non-transgenic cells being the most sensitive (Table III). This difference could be due to an expected higher GUS activity in transgenic cells and indicates that GUS activity is somehow important in plant cells. Alternatively, SL could be toxic in general, but this does not explain why the non-transgenic cells are the more sensitive. In conclusion, it has been found that plants in general contain an endogenous GUS enzyme. This enzyme has a pH and temperature range different from the E. coli enzyme which makes it possible to eliminate the activity selectively when the E. coli gene is used as a reporter gene. The endogenous GUS enzyme complicate the use of glucuronides for selective purposes. Although, the glucuronides seem to be stable and inactive in plant cells when inhibitors of GUS are added, endogenous GUS activity in most tissues hydrolyse the glucuronides, irrespectively of the expression of an introduced GUS gene. Additionally, toxicity of especially weak toxin glucuronides is limited by uptake rather than by the level of GUS present in the cells. This last point introduces the possibility that introduction of the gene encoding glucuronide permease [22] expressed in a tissue specific manner could be used to obtain tissue specific effects of glucuronides which are not readily taken up by plant cells, e.g. PNPG. On the other hand, toxin glucuronides, e.g. CAPG, which are taken up in toxic concentrations by plant cells may be used to select for plants or cells with blocked or altered ex-

122

pression of the endogenous GUS gene. Such plants could be used to investigate the function of this enzyme.

9

Acknowledgement Monsanto Company, USA, is acknowledged for supplying us with Agrobacterium MON9749. Jette Rasmussen (MSB), Klaus K. Nielsen (MSB) and Sven Bode Andersen (Agricultural University, Copenhagen) are acknowledged for their kind donations of some of the in vitro cultured plant species. Morten Joersboe (MSB) and especially Gorm Palmgren (University of Copenhagen) are thanked for helpful discussions during this work.

I0

References

13

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