Transgenic Research 2, 33-47 (1993)

Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants

K E I T H L I N D S E Y * , W E N B I N W E I , M I C H A E L C. C L A R K E , H A Y L E Y F. M c A R D L E , L E O N I E M. R O O K E and J E N N I F E R F . T O P P I N G

Department of Botany, University of Leicester, University Road, Leicester LE1 7RH, UK (Fax: +44 533 551219)

Received 17 June 1992; revised 24 August 1992; accepted 14 September 1992

As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterless uidA (gusA) gene, encoding the [3-glucuronidase (GUS) reporter, was introduced into populations of tobacco, Arabidopsis and potato by Agrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of the gusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns of gusA activation in diverse organs and cell types in those populations, gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency of gusA activation was differentially biased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T 2 and T 3 progeny. Developmentally-regulated and environmentally-inducible expression of gusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.

Keywords: plant gene expression; gene tagging; promoter trap; Arabidopsis thaliana; Nicotiana tabacum; Solanum tuberosum

Introduction

Working towards an understanding of the organization of coordinate gene expression during cytodifferentiation and organogenesis is a central aim of plant developmental biology. It is clear that discrete populations of mRNAs are transcribed in spatially and temporally distinct patterns during the life cycles of higher plants and, furthermore, specific genes are activated by diverse environmental and biochemical cues, reflecting the characteristic plasticity of plant growth and development (Kuhlemeier et al., 1987; Goldberg, 1988; Edwards and Coruzzi, 1990).

A number of plant genes which exhibit tissue-specific or developmentally- or environmentally-regulated expres- sion have been identified. The majority of studies have been carried out on a relatively small number of develop- mental or environmentally-regulated processes, such as seed development (e.g. Kreis et al., 1986; Goldberg et al.,

*To whom correspondence should be addressed.

0962-8819 �9 1993 Chapman & Hall

1989), aspects of flower development (e.g. Koltunow et al., 1990; Coen and Meyerowitz, 1991; Twell et al., 1991), light-regulated gene expression (Gilmartin et al., 1990) and response to pathogen attack (Dixon et al., 1986; Lamb et al., 1989). The most common approaches to the isolation of novel genes have been the differential screening of cDNA libraries made from mRNAs isolated from different organs, and the synthesis of oligonucleo- tides from protein sequence data, following protein purifi- cation or in vitro translation of isolated mRNAs. A corollary of these approaches is that the genes encoding relatively abundant transcripts or proteins are isolated preferentially. Other developmentally interesting genes, which may not necessarily encode high abundance tran- scripts or proteins, have been identified by transposon tagging (e.g. Sheridan, 1988) and the relatively new technique of T-DNA insertional mutagenesis (Feldmann, 1991; Walden etal., 1991).

A more recent strategy, which has been used to study

34

gene expression in Drosophila, mouse, Caenorhabditis elegans and, to a lesser extent, plants, relies on the activation of reporter genes, which possess either a minimal promoter or no promoter, by native gene regu- latory sequences following transformation and random integration (Teeri et al., 1986; Allen et al., 1988; Bellen et al., 1989; Koncz et al., 1989; Hope, 1991; Kertbundit, 1991). We have described such activatable reporter sequences as 'interposons' (Topping et al., 1991). The rationale of the technique is that promoterless interposons may be transcriptionally activated by integration down- stream of a native gene promoter, to create either tran- scriptional or translational fusions ('promoter trapping'); while weak promoter interposons may be activated by nearby native enhancer sequences, and will function in the absence of gene disruption ('enhancer trapping'). In both cases it is expected that the pattern of reporter expression would reflect the pattern of expression of the native gene, and indeed this has been formally demonstrated in both Drosophila (Wilson et al., 1989) and recently in Arabi- dopsis (Kertbundit et al., 1991).

There is currently very little information available concerning either the mechanisms determining the frequency of interposon activation, the types of genes into which insertion may occur (is integration of foreign DNA into genes random?), or the range of plant developmental processes to which this type of approach can be applied. In this paper we provide a detailed description of the range of patterns found following activation of a promo- terless uidA (gusA) reporter gene, encoding the enzyme [5-glucuronidase (GUS; Jefferson et al., 1987), in three plant species of significantly different genome sizes, amounts of repetitive DNA and ploidy levels: Nicotiana tabacum (tobacco; amphidiploid, 2C -- 7.8 pg), Arabi- dopsis thaliana (diploid, 2C -- 0.15 pg) and Solanum tuberosum (potato; tetraploid, 2C --- 4.2pg). We demonstrate that activation occurs in a wide range of cell types in all organs studied, but is differentially biased in different species; and developmentally regulated gene expression patterns can be revealed. The value of this approach to generate cell type-specific markers of deve- lopment, and in characterizing genomic sequences that direct specific patterns of gene transcription during development, is discussed.

Materials and methods

PLANT TRANSFORMATION

Arabidopsis thaliana ecotype C24 was transformed by Agrobacterium tumefaciens strain LBA4404 (Ooms et al., 1982) according to the method of Clarke et al. (1992). Transgenic tobacco plants (Nicotiana tabacum cv. Petit Havana SR1) were generated essentially as according to Horsch et al. (1985), using A. tumefaciens LBA4404 as described by Topping et al. (1991). Transgenic potato

Lindsey et al.

plants (Solanum tuberosum cv. Desiree) were produced by inoculating either tuber explants (Sheerman and Bevan, 1988), or leaf or stem explants, with A. tume- faciens LBA4404. Leaf and stem explants were derived from aseptic shoot cultures maintained on Murashige and Skoog (1962) medium (Sigma, Poole, UK) containing 30 g 1-1 sucrose (MS30). For potato regeneration from leaf and stem, inoculated explants were cultured initially on a callus-inducing medium (MS30 supplemented with 0.9 mg 1-1 thiamine-HC1 [MS301], 0.19 mg 1-1 napthal- eneacetic acid (NAA), 2.25 mg 1-1 6-benzylamino purine (BAP), 10 mg 1-1 gibberellic acid (GA). After 2-3 weeks, explants were transferred to shoot-inducing medium (MS301 containing 1.75mg1-1 zeatin riboside and 0.87 mg 1-1 indole-3-acetic acid (IAA)-aspartic acid conjugate). Shoots were rooted on growth substance-free MS301. The following selective conditions were used: 100 mg 1-1 kanamycin sulfate (Sigma, Poole, UK) for tobacco; 50 mg 1-1 kanamycin sulfate for potato and 35 mg 1-1 kanamycin sulfate for A. thaliana. To eliminate Agrobacterium media were supplemented also with either 200 mg 1-1 cefotaxime (Claforan, Roussel, Dublin, Eire) for tobacco and potato or 850 mg 1-1 vancomycin-HC1 (Sigma, Poole, UK) for Arabidopsis (Clarke et al., 1992). Segregation analysis for kanamycin resistance was deter- mined by plating out ca 200 seeds, surface sterilized with 5% v/v sodium hypochlorite solution for 20 min, on half- strength Murashige and Skoog (1962) medium supple- mented with 10 g 1-1 sucrose and 100 mg 1-1 (for tobacco) or 25 mg 1-1 (for A. thaliana) kanamycin sulfate, and counting the proportion of resistant seedlings at 14 days post germination.

G E N E C O N S T R U C T I O N

The binary vector pAgusBinl9 (Topping et al., 1991) comprises the gusA coding region plus nos termination sequence inserted as a 2.2 kb Hin dlII-Eco RI fragment into the multiple cloning site of pBin19 (Bevan, 1984). The 5' end of the gusA sequence is adjacent to the T-DNA left border.

G U S E N Z Y M E A C T I V I T Y A N A L Y S I S

Fluorimetry

For quantification of GUS enzyme activity, crude extracts of aseptic tissues were made according to Jefferson et al. (1987). Absence of viable A. turnefaciens cells was confirmed by routinely incubating tissue homogenates on solidified Luria-Bertani medium (Maniatis et al., 1982) at 30 ~ C. GUS activities were determined using 4-methyl- umbelliferone glucuronide (MUG, Sigma, Poole, UK) as substrate, and the fluorescent product 4-methylumbelli- ferone (MU) was assayed using a Perkin Elmer LS-50 Luminescence Spectrometer, using an excitation wave- length of 365 nm and an emission wavelength of 455 nm.

Protein concentrations in plant extracts were

Promoter trapping in plants

determined using the Bradford reagent (BioRad Laboratories, Hemel Hempstead, UK), according to the manufacturer's instructions. Specific GUS enzyme activities were expressed as pmol MU produced per mg protein per min of reaction.

H istochernistry

Tissue localization of GUS enzyme activity was deter- mined by staining for up to 12 h at room temperature in 1 mM 5-bromo-4-chloro-3-indolyl [5-D-glucuronic acid (X-glu, Biosynth G, Staad, Switzerland) according to the method of Jefferson et al. (1987) as modified by the use of buffer comprising 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.1% v/v Triton X-100 (Stomp, 1990) and 1 mM potassium ferricyanide to inhibit diffusion of the reaction intermediate. Stained tissues were cleared of chlorophyll by soaking in 70% ethanol. No background staining was observed in any tissue of untransformed plants studied. Photographs were taken on Ektachrome 160 tungsten-balanced film, using Nikon Optiphot and Zeiss Stemi SV8 microscopes.

PLANT DNA EXTRACTION AND ANALYSIS

Leaf tissue (ca 250 rag) was ground to a powder in liquid nitrogen and DNA was extracted according to the method described by Lindsey and Jones (1989). Purified DNA (10 gg for tobacco and potato, 0.5 ~tg for A. thaliana) was digested with 100 units of restriction enzymes and separated in 1% (w/v) agarose gels (SeaKem, FMC Bioproducts, Rockland, USA) and transferred to Gene- Screen Plus nylon membranes (DuPont, Boston, USA) by capillary transfer according to the manufacturer's instruc- tions. DNA fragments for use as hybridization probes were purified from agarose gels by electroelution (Mani- atis et al., 1982) and radioactively labelled by the oligo- labelling method (Feinberg and Vogelstein, 1984) to a specific activity of ca 2 X 109 disintegrations per rain per ~tg DNA. Membrane-bound DNA was prehybridized and hybridized according to the membrane manufacturer's instructions.

INVERSE PCR AMPLIFICATION OF T-DNA FLANKING SEQUENCES

Purified genomic DNA from transgenic lines of A. thaliana was analysed by Southern hybridization following digestion with a range of restriction enzymes designed to generate T-DNA/plant DNA border fragments, containing up to 2 kb of plant flanking DNA. Digested DNA fragments (2 gg in 40 ~tl) were ligated under conditions designed to generate monomeric circles (Collins and Weissman, 1984) in 200 ~tl 5 • ligation buffer (250 mM Tris-HC1 pH 7.4; 50 mM MgCIe; 50 mM dithiothreitol; 5 mM ATP) plus 14 btl T4 ligase (1 unit ~tl-1; BRL/Gibco, Middlesex, UK), made up to a final volume of 1 ml with sterile distilled water. With phenol/

35

chloroform, 500 ~tl ligated DNA was extracted, precipi- tated in ethanol and resuspended to a concentration of 20 ng ~xl -~. For the inverse polymerase chain reaction (IPCR) amplification, 50 ng DNA was used as substrate in the following reaction mixture: 2.5 ~tl 10 • PCR buffer (Promega, Southampton, UK); 2.5 ~tl 2 mM dNTP; 1 ~tl each primer (100 ng ml-l); 2.5 pl MgC12; 0.4 ~tl Taq polymerase (0.5 units per 0.1 ~tl); made up to 17 ~tl with sterile distilled water. PCR amplification was carried out in a Techne PHC-3 thermal cycler over 30 cycles using the following conditions: denaturation at 95* C, 4 min; primer annealing at 55 ~ C, 2 min; extension by Taq polymerase at 72* C, 10 min; denaturation at 95* C, 1 rain; and final extension at 72 ~ C, 10 min. The reaction products were separated in a 1% w/v agarose gel (SeaKem, FMC Bio- products, Rockland, USA) and stained with ethidium bromide. The following oligonucleotides were used as PCR reaction primers: Seq 1: 5' CTGAATGGCGAA- TGGCGC 3'; nested 2: 5' CAGGACGTAACATA- A G G G 3'; nested 3: 5' G A C T G G C A T G A A C T r C G 3'; and npt-ll: 5' GTCATAGCCGAATAGCCTC 3' (Fig. 1).

(a) RE 1 RE 2 RE 1 RE 2

~ ,usA m

seq 1 nested 2

(b) [ gusA [ l a c Z 3 ' [ L B ]

nested 2 seq 1

Plant 5'

RE 1 nested 3

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IPCR Product (5' plant fragment)

(c) RE 2 npt-ll

I .p,.. ] no~s'l RB [ Plan, 3' gusA [

nested 2

IPCR Product (3' plant fragment)

Fig. 1. Schematic representation of the positions of oligonucleotide primers (arrows) for IPCR amplification of T-DNA flanking sequences (Plant 5', Plant 3'; (a) and of the expected PCR products (b,c). RE = restriction enzyme site; LB = T-DNA left border repeat; RB = T-DNA right border repeat. The relative positions of gusA, nptII, nos 5' and 3' and lacZ sequences are shown. Digestion of plant DNA with RE 1 allows amplification of left border genomic sequences (b); digestion with RE 2 allows amplification of right border genomic sequences (c).

36

Resul t s

Frequencies of promoterless gusA activation are different in homologous organs of different species The binary vector pAgusBinl9 (Topping et al., 1991 and Fig. 1) contains in its T-DNA the promoterless gusA gene, which is adjacent to the left border repeat, and a selectable nptlI gene near the right border. This plasmid was introduced into populations of tobacco, Arabidopsis and potato by Agrobacterium tumefaciens-mediated transformation, and kanamycin-resistant regenerants were grown up for analysis of GUS enzyme activity in a range of organs and tissues.

In order to quantify the frequency of gusA activation following transformation, fluorimetric GUS enzyme assays and histochemical localization studies were carried out on leaves, roots and floral organs of individual T a (primary transformant) lines of tobacco and Arabidopsis, and leaf, root, stem and tuber extracts of potato; the results are presented in Table 1. Some of the data for tobacco leaf and root have been summarized previously (Topping et al., 1991). Activation was found to occur at a high frequency in all three species. Approximately 20-30% of the transformants of each species exhibited gusA activ- ation in leaf, but differential frequencies of activation were observed in other organs. For example, a relatively high frequency of activation was identified in the roots of tobacco (up to 75% of all transformants), while the expression frequency in root was lower for Arabidopsis (30%) and potato (9%), as determined by quantitative fluorimetric enzyme assay. Histochemical localization of enzyme activity in the flowers of transgenic Arabidopsis revealed that 22% were GUS-positive, but for tobacco

Table 1. Frequency of activation of pAgusBin19 in transgenic populations of tobacco, Arabidopsis and potato as determined by fluorimetric GUS analysis of tissue extracts or histochemistry*

Organ Frequency

Tobacco Leaf 52/234 (22%) Root 175/234 (75%) Flower 22/24 (92%)

A rab idopsis Leaf 45/162 (28%) Root 28/94 (30%) Silique 70/430 (16%) *Flower 17/76 (22%)

Potato Leaf 11/48 (23%) Root 4/42 (9%) Stem 12/48 (25%) Tuber 5/48 (10%)

Lindsey et al.

this value was 92%. Histochemical staining was found to be very sensitive: for example, three lines of Arabidopsis exhibiting GUS activities of 55.8, 56.2 and 56.5 pmol MU mg protein -1 min -1 respectively in leaf extracts, each stained positively in X-glu. Untransformed plants showed no staining reaction in any organ.

Expression levels in vegetative tissues GUS activities were determined by fluorimetry in leaf and root of expressing individuals in populations of transgenic tobacco, Arabidopsis and potato. The data are in Fig. 2. The range of activities in leaf extracts of Arabidopsis transformed with the gusA gene under the transcriptional control of a cauliflower mosaic virus (CaMV)35S promoter (pBI121), has been published elsewhere (Clarke et al., 1992). Although different sizes of populations of the three species were analysed, and the frequencies of gusA activation were different, the ranges of scatter of GUS activities were broadly similar, with a clustering of activi- ties at relatively low levels ( < 400 pmol MU mg protein -1 min-~). On a population basis, the levels of expression of the activated promoterless gusA gene were several-fold lower than the levels obtained when the CaMV35s promoter was used. The GUS-positive CaMV35S-gus Arabidopsis plants had a mean activity in leaf of 2508 pmol MU mg protein -~ mill -1 in leaf extracts (n ---- 64; 7/71 lines showed no detectable expression). Previously published data for tobacco plants transformed with the same (CaMV35S-gus) gene construction are in broad agreement with these levels of activity (e.g. Jefferson et al., 1987; Topping etal., 1991). The mean activities for GUS- positive promoterless gusA plants are presented in Table 2. The relatively low levels of expression in Agus plants were correlated with spatially-restricted expression patterns. For example, of the lines of Arabidopsis which

Table 2. Mean GUS activities in diverse organs of plants transformed with pAgusBinl9

GUS activity Organ (pmol MU mg protein -1 min -1)

Tobacco Leaf 58 (n = 52) Root 115 (n = 175)

Arabidopsis Leaf 210 (n = 45) Root 235 (n = 28) Silique 165 (n = 70)

Potato Leaf 383 (n = 11) Root 416 (n = 4) Stem 400 (n = 12) Tuber 640 (n = 5)

Promoter trapping in plants

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Fig. 2. GUS enzyme activity in leaf and root tissue extracts of populations of GUS-positive tobacco, Arabidopsis and potato plants transformed with pAgusBin19. Each dot represents the activity measured in one transgenic individual, and each figure represents a scatter of activities throughout the respective populations. The frequency of GUS-positive individuals is indicated in the boxes.

expressed in leaf or root, up to 40% showed GUS activity localized to the vascular tissue only in these organs. Of the transformants studied for this expression pattern, 4% of the kanamycin-resistant plants (11 /260 lines) exhibited GUS activity in the leaf or stem vascular tissue, while 12% (10 /87) expressed in the root vascular tissue.

Expression levels in floral organs The relatively large size of tobacco flowers allowed a

detailed quantitative analysis of gusA activation in different floral organs. Eighteen tobacco lines which exhibited GUS activity in leaf and/or root were analysed for expression in fully expanded flowers, by fluorimetric assay, and the activities are given in Table 3. It is apparent that the levels of GUS activity within a given organ show a scatter within the population, and the patterns of activity are diverse. Indeed, 12 different patterns of expression were observed within the population of 18 lines, as

38

Table 3. GUS enzyme activities in leaf, root and flowers of 18 tobacco T1 plants transformed with pAgusBinl9. GUS activity is measured in pmol MU mg protein -1 min -1. Background GUS activities in control (untransformed) plants were calculated as the mean activities plus 3 SD in 33 (for leaf and root) or in seven (for floral organs) independent plants. (ND = activities below this background level)

Plant Leaf Root Sepal Petal Stamen Pistil

Control 27.9 38.9 30.4 31.7 15.5 16.9 WWl8 ND 149.1 ND 36.4 19.7 ND WW31 37.9 183.3 42.2 ND 22.4 26.5 WW40 35.3 222.5 ND ND 79.5 ND WW44 29.2 309.3 35.2 33.0 43.0 ND WW50 82.1 324.7 43.5 ND 52.6 ND WW53 52.8 275.2 ND ND 35.1 ND WW59 ND 81.3 ND ND 37.3 ND WW79 77.4 283.1 ND ND ND ND WWl04 70.8 41 ND ND ND 19.5 W W l l l ND 645 ND ND ND 19.6 WW124 33.6 352 90.4 ND 24.6 ND WW125 ND 39.9 ND ND ND ND WW139 ND 207.6 ND ND ND 20.1 WW171 37.5 ND ND ND 23.8 ND WW183 ND 66.0 ND ND 16.8 ND WW203 59.4 60.0 ND ND 72.9 ND WW225 ND 374.2 36.1 ND ND ND WW228 ND 369 ND ND 41.3 18.0

Table 4. Patterns of GUS expression in 18 T1 tobacco plants transformed with pAgusBinl9 which express GUS activity in root and/or leaf

Pattern Number of transformants

Root only Root and leaf Root, leaf, calyx, corolla and stamen Root, leaf, calyx and stamen Root, leaf, calyx, stamen and pistil Root, leaf and stamen Root, leaf and pistil Root and calyx Root, corolla and stamen Root and stamen Root, stamen and pistil Leaf and stamen

Lindsey et al.

Table 5. GUS enzyme activities in flowers of six tobacco T 1 plants transformed with pAgusBin19 which express no GUS activity in either root and leaf. GUS activity is measured in pmol MU mg protein -1 min-l( ND = activities below background levels (see Table 3))

Plant Sepal Petal Stamen Pistil

WW25 ND ND 55.3 24.3 WW88 ND ND 24.7 21.5 WW96 ND ND 27.9 ND W W l l 8 ND ND ND 23.0 WW229 ND ND ND 18.4 WW232 ND ND ND 20.1

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Fig. 3. GUS enzyme activities in diverse organs of three tobacco lines (wwl l l , 124, 225) transformed with pAgusBinl9. Each value represents the mean of at least three enzyme assays. For details of histochemical localization of GUS activity, see text.

Fig. 4. Localization of GUS enzyme activity in diverse organs in Arabidopsis lines transformed with pAgusBin19, by X-glu staining. (a) Magnification • AtST-1 stigma; (b) magnification • AtVS-1 stem; (c) magnification • AtTAP-1 anthers; (d) magnification • AtVT-1 roots; (e) magnification x96, AtVT-1 anthers: arrow indicates activity in tapetum; (f) magnification • AtPS-1 anthers.

Promoter trapping in plants 39

40 Lindsey et al.

summarized in Table 4. Of the lines, 89% (16/18 lines) exhibited GUS activity in at least one floral organ: 12 lines expressed in the stamen, five expressed in the calyx, five expressed in the pistil, and two expressed in the corolla.

Six transgenic lines of tobacco which had no detectable GUS activity in either root or leaf were also analysed for expression in mature floral organs (Table 5). All six lines showed gusA activation in at least one organ. Of these lines, five exhibited expression in the pistil, three expressed in the stamen, and none expressed in either the calyx or corolla.

These results demonstrate clearly that, within a popul- ation of transformed plants, activation of a promoterless gusA gene occurs at high frequency in a wide range of organs.

Frequencies of single locus T-DNA insertion events In order to exploit usefully this experimental system to study patterns of gene expression during plant develop- ment, it was considered important to characterize lines transformed with the interposon integrated at a single locus. This would simplify the interpretation of expression patterns, which might be complex should transcription be initiated from two or more promoters; and would facilitate cloning of a genomic sequence directing an expression pattern of interest.

The number of loci of T-DNA sequences in a number of transgenic tobacco and Arabidopsis lines was deter- mined by segregation of the kanamycin resistance pheno- type. Of the tobacco lines studied, 33% (15/46) exhibited segregation ratios of approximately 3 kant: 1 kan S (indica- tive of a single locus insertion; Heberle-Bors et al. 1988), and for Arabidopsis this frequency was 52% (49/95). For specific lines of interest, the number of T-DNA copies was determined by Southern blot/hybridization analysis.

Expression patterns in single versus multiple locus transf ormants To investigate the effect of T-DNA insert number on gusA expression pattern, three independent lines of tobacco (ww62, ww124, ww225), and two lines of Arabidopsis (AtTAP-1 and AtPS-1), which were found to have single T-DNA inserts, and one tobacco line (ww111) and two Arabidopsis lines (AtST-1, AtVT-2) containing two (ww111) or more than two (AtST-1, AtVT-2) inserts, were analysed for GUS activity in seedling leaf and root and in mature floral organs. For tobacco line ww62, a more detailed developmental study was carried out.

GUS activities in the three tobacco lines were analysed by fluorimetry and histochemistry (Fig. 3). All three lines exhibited highest levels of GUS activity in roots, although each in a different cell type. Line ww124 showed expres- sion in root vascular system (370 pmol MU mg protein -~ min-t), leaf (81 pmol MU mg protein -~ min-l), stamen (25 pmol MU mg protein -1 min -~) and sepal (90 pmol

MU mg protein -1 min-1). Line ww25 showed gusA expression in the root cortex (374 pmo! MU mg protein -1 min -1) and low levels of activity in the sepal (36 pmol MU mg protein -1 min-~). Line ww111 exhibited gusA expres- sion in the root elongation zone (645 pmol MU mg -1 rain-) and low levels of expression in the pistil (20 pmol MU mg protein -1 min-1). Although w w l l l had two T-DNA copies, it had the most restricted expression pattern of the three tobacco lines studied.

The two Arabidopsis lines that contained at least 3 T-DNA copies (i.e. which exhibited kanamycin resistance segregation ratios of > 15 kanr: 1 kan s) also exhibited very restricted expression patterns. AtST_ 1 showed expression specifically in the stigma surface (Fig. 4a); and AtVS_ 1 (Fig. 4b) showed expression in the vascular tissue only. Of two lines that contained single T-DNA copies, AtTAP_a showed expression specifically in anthers (tapetum; Fig. 4c); AtVT_I showed expression in the vascular tissue and tapetum (Fig. 4d,e); and AtPS_~ showed expression only in mature pollen (post-microspore mitosis; Fig. 4f).

Developmentally-regulated and inducible expression One example of developmentally-regulated expression can be described in some detail. The tobacco line ww62 was found to contain a single copy of the gusA gene, as determined by DNA restriction digest and gel blot analysis; analysis of four other lines (ww25, 68, 75 and 121) is presented for comparison, three of which contain one and one of which contains two hybridizing gusA sequences (Fig. 5a). Line ww62 is phenotypically normal in the heterozygous and homozygous conditions. Activa- tion of the gusA gene was analysed by fluorimetry and histochemistry during seed germination, seedling growth and in the mature plant. No GUS activity was detectable in the mature embryo, but during seed germination, gusA expression occurred in the testa at the site of radicle emergence (Fig. 5b). In the emerging seedling, GUS activity was localized to the hypocotyl (Fig. 5c). In the 7 day-old seedling, expression was found in the elongation zone of the root and, less strongly, in the cotyledons. By 14 days after germination, expression was observed in the lamina and vascular tissue of older leaves (Fig. 5d), but not in emerging young leaves. In flowering plants (100 days old), expression was localized to the leaf: relatively strongly in the vascular tissue (notably in the xylem, Fig. 5e), faintly in the mesophyll; and also in the root vascular tissue (Fig. 5f). In the mature floral organs, expression was detected by fluorimetry in the calyx, corolla, androecium (notably the pollen), and gynoecium; untransformed plants exhibited extremely low levels of activity in the respective organs (Fig. 5g). Histochemical analysis revealed expression in these organs was restricted to the ovary placental tissue and ovules, and the nectaries. A low level of GUS activity was detectable in the corolla by fluorimetry (up to around 70 pmol MU mg protein -1

Promoter trapping in plants

Fig. 5a. DNA gel blot analysis of five tobacco lines (ww25, 62, 68, 75, 121) transformed with pAgusBinl9. The 10 gg genomic DNA was digested either with (1) both Hin dill and Eco RI, to excise a 2.2 kb gusA internal fragment from the T-DNA or (2) Hin dill, to generate T-DNA border fragments, expected to be of different sizes for each transgenic line, according to the site of insertion. The DNA was separated and blotted, and probed with the purified and oligolabelled 1.8kb Sac I/Xba I gusA coding region fragment. Lines ww25, 62, 68 and 121 each contain one gusA insert, while ww75 contains two.

min -1) but not by histochemistry. In plants hemizygous for the gusA gene, 50% of the pollen showed GUS activity, indicating gametophytic expression of a single active gusA gene (Fig. 5h), while homozygotes showed 100% pollen staining (Fig. 5i). No GUS activity was detectable in the anther filaments or the root or shoot meristems. This expression pattern was found to be stably transmitted through meiosis to the progeny as a single dominant Mendelian trait.

When leaves of ww62 were mechanically wounded with forceps, low levels of GUS activity were induced at the wound sites and subsequently throughout the wounded leaf; the strongest expression was detectable in the wounded petiole (Fig. 5j). When the third leaf was wounded, for example, a doubling of GUS activity, from 266 + 11 pmol MU mg protein -1 min -1 to 531 +_ 14 pmol MU mg protein -1 min -1 (n --- 3) was observed within 6 h post-wounding. Plants which were transformed with the CaMV35S-gus gene and wounded in the same way did not show this pattern of GUS activation (activity of 5086 + 379 pmol MU mg protein -1 rain -1 at time 0; 4551 ___892 pmol MU mg protein -1 min -1 at 6 h post- wounding). The genomic DNA which flanks the gusA insert in this line is currently being characterized, and the results will be presented elsewhere.

Aberrant phenotypes In view of the relatively high frequency of gusA activation in a wide range of tissues, presumably due to T-DNA

41

integration into transcription units, it might be expected that diverse aberrant phenotypes would be generated in homozygotes of Arabidopsis (which is naturally predomi- nantly diploid) carrying recessive mutations created by insertional mutagenesis. Of 10 transformed lines in which the karyotypes were examined, all 10 were confirmed to be diploid, indicating that polyploidization due to trans- formation or regeneration had only occurred, if at all, at a low frequency, and that mutants would not commonly be masked by gene redundancy.

Within the transgenic Arabidopsis population (cur- rently we have produced over 2100 independent trans- formed lines), a number of putative mutants have been identified, although only about 300 of these lines have been screened to date. All screening has been carried out on T2 or T 3 generations. Aberrant phenotypes observed include: transparent testa (Fig. 6a); male sterile (Fig. 6b); dwarf (Fig. 6c); curly root (Fig. 6d); short root; agravi- tropic root; bushy shoot; embryo-lethal; albino seedling (Fig. 6e); triple cotyledon; cotyledonless seedling (Fig. 6f); and rootless seedling. A wild-type seedling is given in Fig. 6g for comparison. Segregation of some of these phenotypes in the T 2 and T 3 generations has been demon- strated to occur (transparent testa, albino, dwarf, triple cotyledon, cotyledonless, male sterile). For example, the male-sterile phenotype of line AtTAP-1 (Fig. 6b) segre- gates as about 25% (10/48 plants) of the T 2 progeny; interestingly GUS activity is restricted to the tapetum (Fig. 4c). Efforts are currently underway to determine whether these phenotypes are linked to the T-DNA inserts, by co- segregation analysis, which, if the case, would greatly facilitate the isolation of the mutated genes.

Isolation of T-DNA flanking sequences In order to isolate genomic sequences flanking the T-DNA inserts in single copy transformants, DNA restric- tion analysis and Southern blot/hybridization analysis was carried out for several lines. Using oligonucleotide primers corresponding to T-DNA sequences, flanking sequences were amplified from five transformed lines of A. thaliana by the inverse polymerase chain reaction, and the results are summarized in Table 6. The predicted minimum sizes of the plant genomic sequences, as well as the total sizes of the respective IPCR products, are presented.

In four cases (AtVT-1, AtVS-1, AtVR-1, AtPS-1) the cloned fragments were amplified from the 5' flank of the gusA coding region (Fig. lb), and are expected to contain at least some of the elements which direct the observed patterns of gusA activation in the transformants. For the fifth transgenic line, AtTr--1, the nrpH primer (Fig. lc) was used to amplify putative coding region DNA at the right T-DNA border. In all cases studied the PCR products retained the characteristic restriction sites, following amplification of the T-DNA multiple cloning site between the respective primer site and plant DNA.

42

Table 6. T-DNA flanking sequences generated from transgenic lines of A. thaliana by IPCR. For AtVT-1, AtVS-1, AtVR-1 and AtPS-1, the primers nested 2 and nested 3 were used initially, followed by reamplification with seq 1 and nested 3 for confirmation (Fig. lb). For AtTT-1, the primers nested 2 and nptII were used (Fig. lc). The first number presented represents the expected size of the plant genomic sequence, and the numbers in parentheses represent the actual sizes, in kb, of IPCR products generated, including T-DNA sequences. The sizes of the genomic fragments described are probably underestimates, since some truncation of the T-DNA sequences is possible

Phenotype Size of genomic Line (mature piano fragment (kb)

AtVT-1 GUS expression in vascular tissue and tapetum 0.5 (1.2)

AtVS-1 GUS expression throughout vascular tissue 0.5 (1.2)

AtVR-1 GUS expression in vascular tissue of root 0.8 (1.5)

AtPS-1 GUS expression in mature pollen 0.3 (1.0) AtTr-1 Transparent testa, no GUS activity 1.0 (1.7)

These results indicate the feasibility of IPCR as a strategy to isolate T-DNA flanking sequences with relative ease. The 5' flanking sequences have been introduced into pUC- and pBinl9-based gusA expression vectors for functional analysis in both transient expression and stable retransformation studies, and the results will be published elsewhere.

Discussion

The expression of specific genes has been demonstrated to be under tight tissue-specific and developmental stage- specific regulation during the life cycle of plants (reviewed by Edwards and Coruzzi, 1990). Furthermore, the devel- opment of a given organ system is characterized by the coordinate up-regulation and down-regulation of different sets of transcripts (e.g. Goldberg et al., 1989; Koltunow et al., 1990). The results we present in this paper demonstrate that the approach of promoter trapping provides a valuable means of analysing the regulatory

Lindsey et al.

properties of diverse genomic sequences in vivo, and of facilitated isolation of those sequences by virtue of linkage to the foreign DNA.

The frequencies of promoterless gusA activation described here for tobacco, Arabidopsis and potato are broadly similar to those described recently by other workers (Koncz et al., 1989; Kertbundit et al., 1991), although different reporter genes and/or T-DNA contexts of those reporters were used. Koncz et al. (1989) found that, on average, 30% of T-DNA inserts carrying a promoterless aph(3')II gene at the right border formed functional gene fusions in Nicotiana and Arabidopsis. Kertbundit et al. (1991) found that a promoterless gusA gene, also located at the right T-DNA border, generated functional gene fusions in Arabidopsis at a frequency of 1.6% and 54%, after introducing vectors designed to create translational and transcriptional fusions respect- ively. Our transformation vector pAgusBin19 carries the promoterless gusA sequence at the left, rather than the right, T-DNA border. Truncation of the left border region may, however, occur during T-DNA transfer/integration (Topping et al. 1991), which may eliminate the stop codons and could result in the generation of translational fusions. We are currently analysing T-DNA-plant DNA junctions to determine features of T-DNA integration in specific lines of interest, and the results will be presented elsewhere.

The high frequences of gusA activation in tobacco, potato and Arabidopsis, which have widely different genome sizes and amounts of repetitive DNA (Bennett and Smith, 1976; Zimmerman and Goldberg, 1977), further supports the hypothesis that T-DNA integration is primarily into regions of chromatin which are transcrip- tionally active or potentially active. This suggests that this approach may be more efficient than an alternative method of detecting promoter or enhancer activity, namely in which random genomic fragments are spliced upstream of reporter genes in plasmid libraries, for subsequent analysis following transformation (Ott and Chua, 1990; Claes et al., 1991). Furthermore, since gene insertion is detectable in the absence of a mutant pheno- type, the technique allows gene detection in polyploid species which exhibit gene redundancy.

Our results do suggest, however, that while integration may result in gusA expression in a wide range of cell types, there is a bias of gene fusion expression patterns, which are different in different species. For example, while

5b-j. Localization of GUS enzyme activity in diverse organs of tobacco line ww62 transformed with pAgusBinl9. (b) Magnification • seed immediately prior to seedling emergence; (c) magnification • seedling < 1 day post-germination; (d) magnification • leaf, 14 days post-germination; (e) magnification • 146, leaf showing xylem vessel (XV), 100 days post-germination; (f) magnification • root tip, 100 days post-germination; (g) fluorimetric assay of GUS enzyme activity in floral organs: S = sepals, P = petals, A = androecium, G - gynoecium, R = root; solid bars represent activities in untransformed control plants; (h,i) magnification x146, mature pollen in heterozygotes (h) and homozygotes (i); (j) magnification • 7.3, mechanically wounded leaf, 24 h post-wounding before staining: wound sites indicated by arrows; note extensive blue staining throughout the lamina and at the cut petiole.

Promoter trapping in plants 43

44

22% of the transgenic Arabidopsis plants were found to express the Agus interposon in floral organs, 92% of the tobacco plants were found to have GUS-positive flowers. Of these, 66% showed expression in stamens, while only 8% expressed in the corolla, 21% in the calyx and 42% in the pistil. We previously described a high frequency of Agus expression in roots of tobacco, with 75% of trans- formants expressing in that organ; here, in contrast, we found that only 30% of Arabidopsis and 9% of potato Agus plants expressed in roots. This result excludes the possibility that T-DNA sequences in pAgusBinl9 direct root-enhanced expression, and points to the possibility that patterns of T-DNA integration and/or subsequent expression are qualitatively different in different species. There exists the further possibility, discussed previously (Topping et al., 1991), that the chromatin structure within the inoculated cells may determine the activation pattern of promoterless interposons, and may be variable between, but characteristic of, a particular species. It is now generally considered that T-DNA insertion is not strictly random (Walden et al., 1991), but the differential spatial activation of interposons in different species has not been described previously.

Although the observed expression pattern is biased, a wide range of tissue-specific or enhanced markers were nevertheless identified in Arabidopsis and tobacco, and correlated with steady state gusA mRNA levels (data not shown). The range of expression patterns was not corre- lated with T-DNA copy number: for example, tobacco line wwl 11 had two T-DNA copies, but a more restricted pattern of GUS activity than did lines ww62, 124 and 225 which had single copies of the gusA gene. Levels of GUS activity in Agus plants were generally low compared with transformants containing a CaMV35S-GUS gene, and there are a number of possible explanations. It may be that the majority of the transcriptional activation sequences tagged have relatively low promoter activities: it should be noted that genes identified by more conventional tech- niques such as cDNA library screening represent those which are the more transcriptionally active, producing the more abundant or stable mRNAs. However, we also observed that high GUS activities can occur in very restricted cell types, but this activity is diluted by adjacent non-expressing cells, giving a low total extractable enzyme activity. It may also be that, in some cases, the trans- latability or stability of fusion transcripts is, in some as yet uncharacterized way, reduced; this possibility requires further investigation.

The transcription promoting properties of several tagged sequences, isolated by IPCR, are currently being

L i n d s e y et al.

defined in both transient expression and stable retrans- formation studies. It may be possible that at least some of the fusion gene expression patterns are the consequence of activation by 'pseudo-promoters', that is, sequences which may have fortuitous transcription-promotion activity but may not be associated with genes. While this possibility cannot be excluded at present, it is clear from work with Drosophila (Wilson et al., 1989) that one can expect that at least some of the reporter fusions will be with native genes. In view of the lack of extensive dispersed repeats (approximately 1%) and so the high density of gene organization in the Arabidopsis genome (Meyerowitz, 1987), it might be expected that insertion into genes would occur at high frequency. In any case, the possible identification of 'pseudo-promoters' by this approach might provide valuable information on the evolution of gene structure and regulation.

With the assumption that the expression patterns described in this paper are due to regulatory sequences associated with active genes, what can we infer of their function? Arabidopsis line AtST-1 expresses predomin- antly in the stigma surface and (to a lesser extent) in the stylar transmitting tissue, and is similar in this respect to reported S-locus gene expression in Brassica (Nasrallah et al., 1988; Sato et al., 1991). Anther-specific expression, as observed in Arabidopsis line AtTAP-1, has also been described by e.g. Koltunow et al. (1990) in tobacco, Scott et al. (1991) in Brassica napus, Smith et al. (1990) in tomato and van Tunen et al. (1990) for the Petunia chi genes. Arabidopsis AtPS-1, which expresses gene fusion activity in late stage pollen, has similarities in expression pattern to late pollen genes of tomato (Twell et al. 1990, 1991). The flanking sequence in this and other pollen- expressing lines are being isolated for structural and functional comparisons. Expression in tobacco ww62 was followed throughout development and exhibited certain features characteristic of genes encoding enzymes in phenylpropanoid metabolism: in the xylem parenchyma, pollen and in the wounded testa during seedling emer- gence. It is also inducible in leaves by wounding. This is similar to reported expression patterns of, for example, the parsley gene encoding 4-coumarate ligase in transgenic tobacco (Hauffe et al., 1991), except that the ww62 gene is expressed at only low levels in petals; and to genes encoding phenylalanine ammonia-lyase from Arabidopsis (PAL-1-GUS fusions; Ohl et al., 1990)and bean (Bevan et al., 1989; Liang et al., 1989). The observation that ww62 does not express in shoot and roo t meristems is similar to that for the Arabidopsis, but not the bean, PAL genes. Other potential elicitors of the ww62 expression

Fig. 6. Aberrant phenotypes of Arabidopsis lines transformed with pAgusBin19. (a) Transparent testa (tt) and wild type (wt) seed; (b) two male sterile plants (MS), which lack fully-developed siliques, and one wild-type individual (WT) of line AtTAP- 1; (r dwarf (arrowed) and wild-type; (d) curly root; (e) albino; (D cotyledonless; (g) wild-type.

Promoter trapping in plants 45

46 Lindsey et al.

pattern, such as ultra-violet light and fungi, are yet to be investigated.

A number of the observed phenotypic mutants of Arabidopsis fall into the classes of T-DNA insertion mutants described recently by Feldmann (1991). While a number of the phenotypes have been found to segregate, rigorous genetic studies must be completed to determine linkage to T-DNA sequences. Nevertheless, the potential advantage of combining promoter trapping and T-DNA insertion mutagenesis is clear, and the approach may be of particular value for the identification of genes expressed in embryogenesis which may be lethal in homozygotes, but detectable in heterozygotes by GUS activation. The generation of large numbers of visual markers of diverse aspects of development also provides a means of investi- gating transacting factors which may regulate specific patterns of transcription, through further mutagenic studies on the transformants followed by screening for loss of G~JS activity. We believe that this general technique provides a valuable means of dissecting the developmental genetics of plants, and is a powerful complement to standard T-DNA and transposon mutagenesis.

Acknowledgements

We are grateful for financial support from AFRC (M.C. Clarke, W. Wei and H.F. McArdle), and CEC (J.F. Topping). L.M. Rooke is supported by a SERC CASE studentship in collaboration with Dalgety plc (Cambridge, UK). We also wish to thank Ila Pandya, Paul Muskett, Melanie Mackay and Philippe Lessard for assistance with enzyme assays and DNA isolation. This research was carried out under MAFF Plant Health licence number PHF 1277/32(110).

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