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​We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of f-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Key words: chimaeric genes/plant transformation/reporter gene/Agrobacterium

Introduction

Control of gene activity can be manifested at many levels, including the initiation of transcription or translation and the processing, transport or degradation of mRNA or protein. The use of precise gene fusions can simplify analysis of these complex processes and delineate the contribution of transcriptional control by eliminating the specific signals for post-transcriptional controls and replacing them with sequences from a readily assayed reporter gene. In addition, members of multi-gene families whose products are very similar can be regulated differentially during development. By using gene fusions to individual members of such families and introducing these fusions into the germline one can study the expression of individual genes separate from the background of the other members of the gene family. Analysis of mutationally altered genes in organisms accessible to transformation techniques is greatly facilitated by the use of sensitive reporter enzymes. By using a reporter gene that encodes an enzyme activity not found in the organism being studied, the sensitivity with which chimaeric gene activity can be measured is limited only by the properties of the reporter enzyme and the quality of the available assays for the enzyme.

To date, at least six reporter genes have been used in studies of gene expression in higher plants. Gene fusions using the Escherichia coli f-galactosidase (Helmer et al., 1984) proved difficult to assay because of high endogenous 3-galactosidase activity in plants. Use of the Agrobacterium tumefaciens Ti-plasmid-encoded genes nopaline synthase (Depicker et al., 1982; Bevan et al., 1983a) and octopine synthase (DeGreve et al., 1982) promised to overcome problems associated with endogenous activity because the opines produced by these genes are not found in normal plant cells. However, these reporter genes are not widely used because the assays are cumbersome and difficult to quantitate, they cannot be used to demonstrate enzyme localization (Otten and Schilperoort, 1978), and octopine synthase cannot tolerate amino-terminal fusions (Jones et al., 1985). The two most useful reporter genes to date have been the bacterial genes chloramphenicol acetyl transferase (CAT) and neomycin phosphotransferase (NPTfl) which encode enzymes with specificities not normally found in plant tissues (Bevan et al., 1983b; Fraley et al., 1983; Herrera-Estrella et al., 1983a,b). In addition, NPTfl can tolerate amino-terminal fusions and remain enzymatically active, making it useful for studying organelle transport in plants (van den Broeck et al., 1985). However, both CAT and NPTII are relatively difficult, tedious and expensive to assay (Gorman et al., 1982; Reiss et al., 1984). Competing reactions catalyzed by endogenous esterases, phosphatases, transferases and other enzymes also limit sensitivity and make quantitation of CAT or NPTII by enzyme kinetics very difficult. Recently, the firefly luciferase gene has been used as a marker in transgenic plants (Ow et al., 1986), but the enzyme is labile and difficult to assay with accuracy (DeLuca and McElroy, 1978). The reaction is complex and there is little, if any, potential for routine histochemical analysis or fusion genetics.

We believe that future advances in the study of plant gene expression require the development of new gene fusion systems that are easy to quantitate and highly sensitive, thus allowing analysis of genes whose products are of moderate and low abundance. This is contingent on a complete absence of any intrinsic reporter enzyme activity in plants. Activity of the reporter enzyme should be maintained when fused to other proteins at its amino terminus to allow the study of translation and the processing events involved in protein transport. The reporter enzyme should be detectable with sensitive histochemical assays to localize gene activity in particular cell types. Finally, the reaction catalyzed by the reporter enzyme should be sufficiently specific to minimize interference with normal cellular metabolism and general enough to allow the use of a variety of novel substrates to maximize the potential for fusion genetics and in vivo analysis.

To meet these criteria, we have developed the E. coli beta-glucuronidase gene as a reporter gene system for transformation of plants. ,B-Glucuronidase (GUS, EC 3.2.1.31), encoded by the uidA locus (Novel and Novel, 1973), is a hydrolase that catalyses the cleavage of a wide variety of f-glucuronides (Stoeber, 1961), many of which are available commercially as spectrophotometric, fluorometric and histochemical substrates. The beta-glucuronidase gene has been cloned and sequenced, and encodes a stable enzyme that has desirable properties for the construction and analysis of gene fusions (Jefferson, 1985; Jefferson et al., 1986; Jefferson et al., 1987). In this paper we describe several useful features of GUS which make it a superior reporter gene system for plant studies. Many plants assayed to date lack detectable glucuronidase activity, providing a null background in which to assay chimaeric gene expression. We show that glucuronidase is easily, sensitively and cheaply assayed in vitro and can also be assayed histochemically to localize GUS activity in cells and tissues.

Results

Many higher plants contain no detectable beta-glucuronidase activity. Roots, stems and leaves from wheat, tobacco, tomato, potato, Brassica napus and Arabidopsis thaliana, potato tubers, and seed from wheat and tobacco were homogenized with GUS extraction buffer containing a variety of protease inhibitors such as PMSF and leupeptin. The plant extracts were incubated in a standard assay at 37°C for 4 to 16 h, and the fluorescence of 4-methylumbelliferone (MU) was measured. Endogenous activity was below the limits of detection. Extremely lengthy assays occasionally gave low levels of MU fluorescence, but the kinetics of MU accumulation were consistent with a slow conversion of the glucuronide into another form, possibly a glucoside, that was subsequently cleaved by intrinsic glycosidases. 3-Galactosidase assays performed under similar conditions on tobacco and potato extracts were off-scale (at least 10 000 times higher than the minimal detectable signal) within 30 min. Reconstruction experiments were performed with purified GUS added to tobacco and potato extracts to demonstrate the ability of these extracts to support beta-glucuronidase activity.

Construction of plasmids for transformation ofplants with GUS fusions. A general purpose vector for constructing gene fusions was made by ligating the coding region of GUS (Jefferson et al., 1986) 5′ of the nopaline synthase polyadenylation site (Bevan et al., 1983a) in the polylinker site of pBIN19 (Bevan, 1984). This vector, pBI101 (Figure 1), contains unique restriction sites for HindIII, Sall, XbaI, BamHI and SmaI upstream of the AUG initiator codon of GUS, to which promoter DNA fragments can be conveniently ligated. The cauliflower mosaic virus (CaMV) 35S promoter (Odell et al., 1985) as described in the expression vector pROK1 (Baulcombe et al., 1986) was ligated into the Hindlll and BamHI sites to create pBI121. Similarly, the promoter from a tobacco gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) Ntss23 (Mazur and Chui, 1985) deleted of rbcS coding sequences, was fused to pBI101 to make pBI131.

Chimaeric GUS genes are expressed in transformed plants Nicotiana tabacum var. Samsun plants were transformed with Agrobacterium binary vectors (Bevan, 1984) containing transcriptional fusions of either the CaMV 35S promoter or the tobacco rbcS promoter with the coding region of GUS as shown in Figure 1. Several kanamycin resistant plants were regenerated from each transformation. Two rbcS -GUS transformants and two CaMV -GUS transformants were chosen for further study. We first assayed various organs of one plant from each transformation, axenically cultured in 3000 lux white light, 18 h day, 6 h night. The results of this analysis are shown in Figure 2, and tabulated in Table I using either of two normalization methods (see Discussion). The plant containing a rbcS-GUS fusion (rbcS -GUS 2) exhibited a pattern of gene expression consistent with earlier studies using heterologous rbcS gene fusions (e.g. Simpson et al., 1986a). The highest sp. act., using either protein or DNA as a denominator, was found in older leaves (-. 8 cm long), with progressively less activity in very young leaves ( < 5 mm), stems and roots. The other rbcS -GUS fusion plant showed a similar pattern. 

The two plants transformed with the CaMV 35S -GUS fusion displayed a pattern of gene expression distinct from that of the rbcS -GUS fusion plants. The highest levels of activity were found in roots, with similar levels in stems. GUS activity was also high in leaves, consistent with previous observations that the CaMV 35S promoter is expressed in all plant organs (Odell et al., 1985).

To verify that no significant rearangements of the transforming DNA had occurred, a Southern blot analysis was conducted as shown in Figure 3. Digestion of DNA extracted from all of the tranformants with HindlII and EcoRI released a single internal fragment of T-DNA consisting of the nopaline synthase polyadenylation site, the GUS coding region and the promoter (CaMV 35S or rbcS). RbcS-GUS transformants contained three copies (rbcS -GUS 2, Figure 3, lane 6) and about seven copies (rbcS -GUS 5, lane 8) of the predicted 3.1 kb HindIH -EcoRI fragment. Digestion with EcoRI revealed multiple border fragments (Figure 3, lanes 5 and 7), confirming the copy number estimates deduced from the double digestions. Similarly, CaMV35S -GUS plants had multiple insertions as shown in Figure 3, lanes 1-4. CaMV-GUS 21 had three copies of the predicted 2.9 kb fragment, while CaMV -GUS 29 had two copies. No hybridization of the labelled GUS coding region to untransformed plant tissue was observed (lanes 9 and 10).

GUS activity in plants can be visualized using histochemical methods. To determine whether we would be able to use histochemistry to investigate single-cell or tissue-specific expression of GUS gene fusions in plants, preliminary experiments were carried out on sections of stems of several independently transformed rbcS-GUS and CaMV-GUS plants. Typical results are shown in Figure 4. Stem sections were chosen both for their ease of manipulation and because most of the cell types of a mature plant are represented in stem. To illustrate the light-regulated nature of the rbsC -GUS fusion, the plants were illuminated from one side only for 1 week before sectioning. Sections from both plants stained intensely with the substrate while non-transformed tissue did not stain (Figure 4c). Stem sections of CaMV -GUS plants always show highest levels of activity in phloem tissues along the inside and outside of the vascular ring, most prominently in a punctate pattern that overlies the internal phloem and in the rays of the phloem parenchyma which join the internal and external phloem (Esau, 1977). There is also variable lighter staining throughout the parenchymal cells in the cortex and in the pith, and also in epidermal cells, including the trichomes (Figure 4a).

RbcS -GUS stem sections rarely if ever show intense staining in the trichomes, epidermis, vascular cells or pith, but tend to stain most intensely over the cortical parenchyma cells containing chloroplasts (chlorenchyma), with faint and variable staining in the pith. Although we most often see the strongest stainingin a symmetrical ring around the vascular tissue just inside the epidermis, we sometimes observe an asymmetric distribution of staining in the cortical stem cells. Suspecting that this pattern was due to uneven lighting, we illuminated a plant from one side for 1 week before sectioning, and found that the staining was asymmetric, with intense staining in the chloroplast-containingcells proximal to the light source (Figure 4b). The staining patterns we observe for both the CaMV 35S GUS and the rbcS -GUS transformants are consistent between several independent transformants. Untransformed plants never show staining with 5-bromo4-chloro-3-indoyl f-D-glucuronide (X-Gluc), even after extended assays of several days (Figure 4c).

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