Plant Molecular Biology Reporter pages 37-46 Volume 10(1) 1992

Protocol

Non-Destructive Assay Systems for Detection of ~-Glucuronidase Activity in Higher Plants Thomas Martin, Renate Schmidt, Thomas Altmann, and

Wolf B. Frommer Institut fiir Genbiologische Forschung, Abt. Willmitzer, Ihnestr. 63, D-1000

Berlin 33, Germany, (RS, present address: PSR Cambridge Laboratory, John Innes Center for Plant Science Research, Colney Lane, Norwich NR4 7UH,

UK)

Abstract:. 13-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incuba- tions in X-gluc or by spraying 4-MUG.

T he bacterial gene, uidA, which encodes ]3-glucuronidase and is commonly referred to as the "GUS gene," has become the major reporter used to s tudy gene expression in higher plants (Walden

and Schell, 1990). As plants usually contain only low endogenous activities of GUS under commonly used assay conditions, very sensitive detection systems can be used in order to obtain both quantitative measurements (i.e., level of expression) and qualitative measurements (i.e., specificity of expression in tissues and organs) of gene expression in transgenic plants. GUS is used for a wide range of applications ranging f rom the deve lopment of transformation protocols to the analysis of p romoter activity (Martin et al., 1991). A further improvement of the system was the introduction of a portable intron into the GUS gene, which leads to nearly complete repression of its expression in Agrobac- teria and thus allows one to visualize transformation events of single cells at early stages after cocultivation (Vancanneyt et al., 1990).

Abbreviations and gene names. X-gluc, 5-bromo-4-chloro-3-indolyl-~-glucu- ronic acid; 4-MUG, 4-methylumberlli ferylq3-glucuronide; G US, ~-glucuronidase; uidA, gene encoding 13-glucuronidase.

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38 Martin et al.

The commonly used histochemical protocols are destructive, as they involve fixation, vacuum infiltration, and prolonged incubations of the plant material in solutions containing X-gluc, detergents, and organic solvents. As blue staining is difficult to evaluate in green tissues, the material subsequently has to be destained in ethanol. This is seen as the major disadvantage of the GUS system, in comparison to other reporters, e.g., the luciferase system (Walden and Schell, 1990). The spectrum of use could be further broadened if GUS assays could be performed in a non- destructive manner, such that plants would survive the staining and be able to set seeds.

Gould and Smith (1989) circumvented the problem by applying 4- MUG to spent media after transfer of the plant material to new media. They also showed that callus material for example is not severely ha rmed by short-term incubations in 4-MUG. These assay systems are useful during early stages of regeneration, but are less optimal to test offspring, as the plants have to be wounded in order to test the leaking fluid for GUS activity (Gould and Smith, 1989). Also such a system is not suitable for screening large populations.

We have therefore developed fast and simple protocols that allow the analysis of large populations in vivo without irreversibly harming or destroying the plants. This opens up new areas such as the screening for mutants involved in signal transduction chains and makes possible the analysis of offspring at early stages.

Mater ia l s Solutions required.

MS medium (Murashige and Skoog, 1962), supplemented with 2% sucrose.

X-gluc solution: 2 mM X-gluc (Biosynth AG, Switzerland, predissolved to 0.5 % in N,N'-dimethylformamide) in 50 mM phosphate buffer pH 7-8, 0.5 % Triton X-100. The solution is sterile-filtered before use.

4 -MUG (Sigma, Heid el berg, FRG): d issoI ved in phosphate buffer pH 7.5, 0.01% Sapogenat Tl10 (detergent, Hoechst AG, Frankfurt).

DNA constructs. B33G is a class I patatin promoter from Solanum tuberosum fused to tha J3-glucuronidase reporter gene (Rocha-Sosa et al., 1989). In transgenic Arabidopsis, it gives sucrose-dependent expression in roots (W. Frommer, manuscript in preparation). D4E is a construct consisting of a truncated B33 patatin promoter (-195/+18) fused to the CaMV 35S enhancer (-373/-32) driving the ~-glucuronidase gene. Simi-

Non-Destructive G US Assay 39

lar constructs are described in Liu et al. (1990). In transgenic Arabidopsis, it leads to strong constitutive expression in all tissues. The pattern of expression is indistinguishable from the pattern obtained by the CaMV 35S promoter itself (W. Frommer, manuscript in preparation).

Transformation of Arabidopsis thaliana C24 was performed as de- scribed by Schmidt and Willmitzer (1989).

P r o c e d u r e s

Standard staining protocol. The standard protocol for assaying b- glucuronidase activity has been described in detail by Jefferson (1987) and Martin et al. (1992). In short, this involves fixation of the plant material in paraformaldehyde, followed by vacuum infiltration and incubation in a buffered X-gluc solution containing 0.1% Triton X 100. Green tissues are then washed several times in ethanol to remove chlorophyll in order to allow detection of the blue stain. This procedure is obviously lethal when applied to whole plants.

Nevertheless modifications of the protocol allow non-destructive GUS detection. Depending on the expression profile of the gene of interest, roots, above ground parts of the plant or whole plants can be analyzed for GUS expression in vivo.

Non-destructive assay using X-gluc for root staining. As 5-bromo-4- chloro-indigo, the blue end-product of X-gluc staining, is less mobile in the plant than the fluorescent 4-MU, a staining procedure for roots using X-gluc was developed. As in the case of 4-MUG, X-gluc and its products do not seem tobe lethal to the plan t when exposed only for short periods. Addition of X-gluc solution directly onto the agar around the plantlets grown in tissue culture allows detection of the blue staining in the roots (Fig. 1). Transfer to new medium can thus be used to rescue the plants after staining.

�9 Germinate sterile seeds on 15 mL of solid MS-medium (e.g., in 15- cm petri dishes) for approximate ly 10 days (cotyledon to primary-leaf stage).

�9 Add 2 mL 2 mM X-gluc solution. �9 Incubate overnight under normal growth chamber conditions

(22~ �9 Evaluate root staining. �9 Rescue by transfer to fresh MS medium. �9 Separate plantlets into tubes for seed setting or transfer to soil.

40 Martin et al.

Under such conditions, more than 90% of Arabidopsis seedlings sur- vive. The staining in roots is still tissue specific as can be taken from the absence of staining in root tips of B33G transformants (Fig. 1a/b) com- pared to the complete staining in case of D4E transformants. The same pat tem is observed (data not shown) when single plants were stained by the standard protocol. Relatively strong promoters are necessary to detect activity, as the staining conditions are suboptimal, e.g., the acidic pH of the medium, the low concentrations of X-gluc around roots and the low temperature. If no staining can be detected, it is possible either to increase the pH of the medium to 7, to increase the amount and concen- tration of X-gluc, or to prolong the incubations to several days and to perform the assay at 37~ These treatments however result in reduced survival rates.

We have observed that the addition of an oxidation catalyst (0.5 to 2 mM ferrocyanide) strongly enhances the staining due to oxidation of the reaction intermediate 5-bromo-4-chloro-indol to the blue product 5- bromo-4-chloro-indigo (Jefferson, 1987, and references therein).

Care should be taken when transferring the plantlets to fresh medium as the seedlings are very sensitive at this stage. As many plant species contain endogenous G US activity with an acidic pH opt imum (Heberle- Bors, pers. comm.; Martin et al., 1992), untransformed control plants should be treated the same way. Endogenous GUS activity can be suppressed by performing the assay in a p H 7.5 MS medium. The roots can be better observed if the plates are incubated vertically. Depending on the promoter, staining can be observed even at younger developmen- tal stages. As certain promoters are regulated by environmental conditions (e.g. light), the staining patterns should be controlled by comparison to plants stained with the usual procedure (Jefferson, 1987; Martin et al., 1992).

Non-destructive assay using X-gluc in liquid media for whole plant analysis. Arabidopsis can be cultured for short times in liquid media, which allows analysis ofuidA expression in all tissues of intact plants:

�9 Germinate seeds in petri dishes on solid MS until they reach the four-leaf stage (cotyledons plus primary leaves).

�9 Transfer seedlings to liquid MS medium in petri dishes or for large populations into flasks.

�9 Add X-gluc to 1 mM final concentration. �9 Incubate over night under normal growth chamber conditions

(22~

Non-Destructive G US Assay 41

Fig. 1. Non-destructive X-gluc staining of Ara- bidopsis thaliana roots. Arabidopsis thaliana plants (C24) were trans- formed with a construct ofuidA fused toa patatin class I (B33G) promoter, and grown in sterile cul- ture on 2MS medium. Roots of these plants were stained according to the standard protocol, Non-destructive assay us- ing X-glucfor root staining. (a) B33G, overview of s ta ined roots pho to - graphed from the bottom of the plate (staining is absent from root tips); (b) B33G, a more detailed view of the roots from a single transformant; (c) D4E, complete staining of roots.

42 Martin et al.

�9 Evaluate staining. �9 Rescue plants by transfer to solid medium.

For promoters active in large areas of the plant, survival rates are low. In case of strong promoters, incubation time and X-gluc concentration can be reduced in order to decrease damage. As the staining in green tissues is difficult to evaluate, this system is most suitable for root expression. The expression in leaves can be better studied in plants that are grown in the dark.

Non-destructive assay by spraying 4-MUG. Due to difficulty in detec- tion of the blue color derived from X-gluc staining in green parts of the plant, a fluorescent assay was developed that is suitable for detecting GUS in above-ground parts of the plant, i.e., leaves and stems, based on sprayingof4-MUG. TransgenicArabidopsis plants sprayed with 4-MUG solution show strong staining in leaves (Fig. 2).

�9 Grow plants in sterile culture. �9 Spray 2 mM 4-MUG. �9 Incubate plants at 22~ to 37~ �9 Evaluate fluorescence under UV light after different periods of

time. �9 Document on polaroid film with Wratten 2E filter (Kodak).

Control of both age of the plants and growth conditions is essential to obtain a uniform staining due to improved accessibility of the substrate. Plantlets at the four-leaf stage after cultivation in glass containers gave the best results. Background fluorescence due to leakage of the enzyme can be reduced by covering the agar with sterile sand before putting out the seeds. In contrast, plants grown in soil at a stage shortly before flowering gave patchy staining both with X-gluc and 4-MU (Fig. 3). Higher concentrations of detergent (Sapogenat T110) led to faster stain- ing with 0.5% detergent within three hours for D4E-GUS (W. Frommer, unpublished result), but also to a decrease of the survival rates. Pro- longed irradiations should be avoided as fluorescence fades, survival is reduced, and probably mutations are induced. W- l i gh t with an emis- sion maximum at 365 nm should be less harmful than light of 305 nm and is, moreover, closer to the absorption opt imum of 4-MU.

D i s c u s s i o n

For most applications, such as promoter analysis (Liu et al., 1990; Keil et al., 1989, Stockhaus et al., 1989) or the dissection of gene families

Non-Destructive G US Assay 43

Fig. 2. Non destructive 4-MUG staining of Arabidopsis thaliana. Arabidopsis thaliana plants (C 24) transformed with a truncated patatin promoter (D4E) fused to the CaMV 35S enhancer and uidA were grown together with wild-type C24 plants on MS in sterile culture. Plants at the two- to six-leaf stage were sprayed with 4-MUG as described in the section, Non-destructive assay by spraying 4-MUG. (a) Arabidopsis thaliana transformants (right) and wild-type (left) plants under white light; (b) same plants as in (a) under UV-light.

44 Martin et al.

(Rocha-Sosa et al., 1989; K6ster-T6pfer et al., 1989), the GUS-reporter system has proven to be well suited. In transgenic Arabidopsis plants the pattern of GUS activities can be analyzed in large numbers of transfor- mants at the same time by staining whole plantlets with X-gluc in small tubes (Frommer et al., 1991). The commonly used assays for GUS can be used to establish or improve transformation procedures by analyzing early transformation events, but in many cases the stained cells and their progeny are required to survive. Therefore, non-destructive methods using4-MUG as a substrate have been developed for the analysis of early transformation events (Gould and Smith, 1989). Also in all cases where an early analysis of seedlings is necessary, e.g., to test crosses or to follow the excision of transposable elements, a non-destructive assay system is essential. Certainly one way to do so is to analyze single leaves or parts of the plants as has been described for the screening of starch metabolism mutants (Caspar et al., 1986).

The assay systems described above were mainly developed in order to dissect signal-transduction pathways of individual promoters. For this purpose, transgenic seeds containing the promoter of interest (e.g., a light-regulated promoter) fused to a reporter gene, such as uidA, are mutagenized by conventional methods. The M2 generation is then screened for mutants that show an altered phenotype in respect to the

Fig. 3. Patchy fluorescence pattern of mature transgenicArabidopsis thaliana grown in the greenhouse. Patchy staining pattern when sprayed with 4-MUG may be due to a differential accessability to substrate. The same result was obtained when assays were performed according to the standard X-gluc assay (data not shown).

Non-Destructive G US Assay 45

express ion of the r epor t e r gene. This should lead to the isolat ion of m u t a n t s in the signal t r ansduc t ion of the respect ive gene. Arabidopsis thaliana was chosen as a target due to its un ique features that h a v e been referenced extens ive ly e l sewhere (Meyerowi tz , 1989; D a m m et al., 1991). As bo th qual i ta t ive and quant i ta t ive changes can be mon i to red , uidA seems to be the p re fe r red choice as a marke r . Non-des t ruc t ive assay s y s t e m s were necesqary in order to p r e v e n t sacrificing pu t a t i ve mutan t s . O u r m a i n focus was therefore to develop in-vivo s ta ining pro tocols for GUS that are sui table for screening large popu la t ions of Arabidopsis plants . By c o m b i n i n g the non-des t ruc t ive assay sy s t em for roots us ing X-gluc and the sp ray ing of 4-MUG onto the leaves, it is n o w possible to detect and fol low bo th quant i ta t ive ly and qual i ta t ive ly the express ion of ch imer ic GUS genes in mos t t issues of the p lant w i thou t des t roy ing the plants . Both assay sys t ems h a v e been s h o w n to be sui table to detect bo th u n t r a n s f o r m e d p lan ts a m o n g a major i ty of GUS-pos i t ive p lan t s and vice versa. Both sy s t ems should also be sui table to ana lyze seedl ings of m a n y o ther species, 'u t ue re tested on ly with Arabidopsis.

Acknowledgments: We are grateful to Lothar Willmitzer for ideas, discussions and support. We also thank Erwin H eberle-Bors for data prior to publication, and Peter Morris and Burkhard Schulz for critical reading of the manuscript. This work was supported by Bundesministerium hlir Forschung und Technologie (BCT 389, Molekular- und Zellbiolgie von h6heren Pflanzen und Pilzen).

R e f e r e n c e s .

Caspar, T., S.C. Huber and C. SomerviUe. 1986. Alterations in growth, photosynthesis and respiration in a starchless mutant of Arabidopsis thaliana (L.) Heynh. deficient in chloroplast phosphoglucomutase activity. Plant Physiol. 79:1-7.

Damm, B., U. Halfter, T. Altmann and L. Wi[lmitzer. 1991. Transgenic Arabidopsis. In: Transgenic Plants, S.D. Kung and R.W. Butterworths, eds. In press.

Frommer, W.B., T. Martin, R. Schmidt, S. Hummel and L. Willmitzer. 199I. Patatin promoters as a tool to identify sink related signal transduction pathways. In: Recent Advances in Phloem Transport and Assimilate Compartmentation, J.C. Bonnemain, S. Delrot, J. Dainty and W.J. Lucas, eds. Ouest Editions, Paris pp 254-257.

Gould, J.H. and R.H. Smith. 1989. A non-destructive assay for GUS in the media of plant tissue cul~tres. Plant Mol. Biol. Rep. 7:209-216.

Jefferson, R.A. 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5:387-405.

Keil, M., J.J. Sanchez-Serrano and L. Willmitzer. 1989. Both wound-inducible and tuber- specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family. EMBO J. 8:1323-1330.

K6ster-T6pfer, M., W. Frommer, M. Rocha-Sosa, S. Rosahl, J. Schell and L. WiUmitzer. 1989. A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants. Mol. Gen. Genet. 219:390-396.

IAu, X.J., S. Prat, L. Willmitzer and W.B. Frommer. 1990. Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/

46 Martin et al.

GUS-gene fusionMol. Gen. Genet. 223:401-406. Martin, T., R.V. W6hner, S. Hummel, L. Willmitzer and W.B. Frommer. 1992. The GUS

reporter system as a tool to study plant gene expression. In: GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, S. GaUagher, ed. Academic Press, in press.

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tobacco tissue cultures. Physiol. Plant. 15:473-497. Rocha-Sosa, M., U. Sonnewald, W.B. Frommer, M. Stratmann, J. Schell and L. Willmitzer.

1989. Both developmental and metabolic signals activate the promoter c'~a class I patatin gene. EMBO J. 8:23-29.

Schmidt, R., WiIlmitzer, L. (1988) High efficiency Agrobacterium tumefaciens mediated transformation ofArabidopsis thaliana leaf and cotelydon explants. Plant Cell Rep. 7:583- 586.

Stockhaus, J., J. Schell and L. Willmitzer. 1989. Correlation of the expression of the photosynthetic gene ST-LS1 with the presence of chloroplasts. EMBO J. 8:2445-2451

Vancanneyt, G., R. Schmiclt, A. O'Connor-Sanchez, L. Willmitzer and M. Rocha-Sosa. 1990. Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transfor- mation. Mol. Gen. Genet. 220:245-250.

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