Plant Molecular Biology 28: 219-229, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 219

Promoter elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants

Thomas Kaiser 1, Karlheinz Emmler t, Thomas Kretsch 1, Bernd Weisshaar 2, Eberhard ScMfer t and Alfred Batschauer 1,, 1Biological Institute II, Albert-Ludwig-University, Schiinzlestrafie 1, 79104 Freiburg, Germany (*author

for correspondence)," 2 Max-Planck-Institut f~ir Z~ichtungsforschung, Carl-von-Linnd Weg 10, 50829 K61n, Germany

Received 14 October 1994; accepted in revised form 23 February 1995

Key words: Arabidopsis, chalcone synthase (expression), cis-acting elements (light, organ specifity), mustard (Sinapis alba L.), tobacco (Nicotiana tabacum), transgenic plants

Abstract

The expression of chalcone synthase (CHS) genes, which encode the first enzyme of the flavonoid pathway, is under developmental control as well as affected by external stimuli such as light. Varying fragments of the 1 kb upstream region of the CHS1 gene from white mustard (Sinapis alba L.) were fused to the GUS-coding region, and the light-regulated expression of these constructs was analysed in transgenic Arabidopsis and tobacco plants. Studies performed with Arabidopsis seedlings indicate the presence of two elements within the CHS1 promoter mediating light responses via different photore- ceptors. One element, located about 150 bp upstream of the transcription start site, is homologous to Unit 1 of the parsley CHS gene, the second, far more upstream element carries sequences similar to Unit 2 of the same gene. Detailed studies on Unit 1-driven expression indicate that this element transfers the expression characteristics of the original gene to both Arabidopsis and tobacco. Although the expression characteristics of Unit 1 are indistinguishable from those of the full-length promoter within the same species, we observed differences in mustard CHS promoter regulation between Arabidopsis and tobacco plants transgenic for the identical construct. The difference in photoreceptor usage by the same promoter element in different transgenic species (Unit 1 from mustard in Arabidopsis vs. tobacco) was also ob- served for different but homologous promoter elements in the same transgenic species (Unit 1 from mustard and parsley in tobacco). We therefore conclude that the same promoter and even the same promoter element (Unit 1) can mediate different spatial patterns of expression and modes of light regulation in different transgenic species.

Abbreviations: CAB, CAB protein typeI LHCII (Lhcbl); CHS, chalcone synthase; GUS, /%glucuronidase; ADH, alcohol dehydrogenase; CaMV, cauliflower mosaic virus

Introduction

Chalcone synthase is the first enzyme of the fla- vonoid pathway. The transcription rate of the

CHS genes is under strict developmental control. In addition, CHS gene transcription is affected by external stimuli such as light, fungal elicitors, and wounding (for review see [32]). In order to iden-

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tify cis-acting elements involved in light regulation and tissue-specific expression, promoters of sev- eral CHS genes have been investigated by tran- sient gene expression [3, 9, 15, 30, 31, 33, 35, 38, 44] as well as in stably transformed plants [13, 14, 25, 26, 37, 40, 42]. In vivo footprinting stud- ies with cultured parsley cells [39] demonstrated light-induced DNA-protein interactions in two regions of an element, called Unit 1 [44], of the parsley CHS promoter. In this case, one of the footprinted regions (Box II, G-box) is strongly bound in vitro by bZIP factors [1, 43]. The other binding activity (Box I-binding factors) has not yet been characterized in detail but there are in- dications that MYB-like proteins recognize Box I [36]. Recent analyses using transient gene ex- pression showed that Unit 1 from mustard ([35]; see [24] for Unit 1 sequences) and from parsley [43] CHS promoters, when fused to a heterolo- gous transcriptional start site, is capable of trans- ferring photoregulation to a chimeric gene.

We therefore wondered whether Unit 1, repre- senting 54 bp of the mustard CHS1 gene [2], could act as a light-responsive element in stably transformed Arabidopsis and tobacco plants and whether other regions of the mustard CHS1 pro- moter are also able to mediate photoregulation. We further investigated whether Unit 1 receives signals from different photoreceptors. In addi- tion, we analysed developmental and tissue- specific expression patterns confered by Unit 1 in our transgenic plants. These data were compared to those from plants carrying the full-length mus- tard CHSI promoter. To allow conclusive inter- pretation of the data we characterized the light responses of Arabidopsis to different light quali- ties. Here we present evidence strongly suggest- ing that Unit 1-mediated expression reflects the expression characteristics of the full-length CHS1 promoter fairly exactly. In addition, we demon- strate that Unit 1 is sufficient to mediate the dis- tinct expression patterns and modes of light ac- tion observed for CHS expression in different plant species.

Materials and methods

Plant transformation techniques and chimeric gene constructs

Arabidopsis thaliana ecotype Wassilewskija was transformed according to the root transformation protocol [41] with constructs carrying either the full-length promoter of the CHS1 gene from mus- tard [2] from position -907 to + 26, deletions thereof or the Unit 1 of the same gene represent- ing the sequence from position -146 to -92 rela- tive to the transcription start site. The full-length promoter and 5' deletions were cloned directly in front of the uidA open reading frame [23] in the binary vector pBI101 [22]. Unit 1 was cloned after dimerization and fusion to the minimal (-46) 35S promoter from CaMV [11] in vector pPCV812 [28]. In the case of 3' promoter dele- tions, transcription initiation signals were pro- vided by the Arabidopsis ADH gene promoter [ 7 ]. (For further details concerning the constructs see [24, 35].) Tobacco (Nicotiana tabacum cv. SR1) was transformed with the full-length CHS1 pro- moter construct (-907), the Unit 1 construct con- taining the dimerized mustard CHS1 Unit 1, and a construct containing the dimerized Unit 1 from the parsley CHS promoter [43] according to [20] using the Agrobacterium strain GV3101 [27]. In the Unit 1 constructs the 35S CaMV promoter region between -46 and + 8 [11] serves as a transcriptional initiation region.

Growth of pIants, light treatments and measurement of GUS activity

Seeds of transgenic F1 plants were sown on MS medium in Petri dishes, and kept at 4 °C over- night. The transgenic Arabidopsis lines used in this study were the same as described in the accom- panying paper [24]. Germination was induced by a red or white light treatment for 1 h. Afterwards, Petri dishes were transferred to darkness at 24 ° C for two (Arabidopsis) or four and eight days (to- bacco), followed by light treatment of the seed- lings for 24 h. Light sources used were as follows:

white light (W), Osram L40W/73+Phil ips TLD40W/18 lamps, fluence rate 10.9 W/m2; white light containing UV-B (W*), W + Philips TLD40W/12 lamps+ 310 nm cut-off filter, flu- ence rate 10.9 W/m2; red light (R), "~max = 660 nm, fluence rate 5 W/m2; far-red light (FR), "~max = 730 nm, fluence rate 3 W/m2; blue light (B), Philips TLD36W/18 lamps, plexiglass filter 627 (R6hm & Haas), /~max---= 436 nm, fluence rate 4.1 W/m2; UV-A light (UV), Osram L40W/73 lamps, ,~max = 350 nm, fluence rate 0.5 W/m 2. Light sources were described previously [2, 33]. Quan- titative measurements were performed by using equal numbers of seedlings from all lines trans- genic for a given construct. The material was pooled, protein extracted and used for determi- nation of GU S activity and protein concentration [24]. Fluorometric and histochemical determina- tion of GUS activity was performed as described [21].

RNA isolation and RNA-gel blot analysis

Total RNA was isolated from Arabidopsis or to- bacco seedlings at the age as indicated as de- scribed [34]. RNA isolated from Arabidopsis was separated on formaldehyde agarose gels, trans- ferred to Nitroplus (MSI, Westboro, MA) mem- branes, and probed with a 748 bp Hind III frag- ment of the genomic CHS clone SA-CHSI from white mustard [2] or a 2.1 kb Barn HI fragment of the mustard clone SA-CABI encoding Lhcbl [ 17]. Tobacco RNA was dotted on nitrocellulose filters as described [5], and probed with a 1.2 kb Hind III fragment of the genomic CHS clone CS2 from tobacco [8]. The DNA fragments represent- ing part of the coding regions were labelled with [~-32p]dATP (Amersham, Braunschweig, Ger- many; 110 TBq/mmol) using the Multiprime la- belling system (Amersham, Braunschweig, Ger- many) according to the manufacturer's manual. The extent of hybridization of tobacco RNA in each dot was measured by scintillation counting. Light treatments of plants were as described (see above).

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Results and discussion

CHS expression in Arabidopsis is regulated by dif- ferent photoreceptors

Although we have established recently a trans- formation procedure for white mustard [19], which would allow promoter studies in the ho- mologous system, we have choosen Arabidopsis and tobacco for transgenic studies for two rea- sons. First, the efficiency of mustard transforma- tion is still low making it difficult to analyse a great number of different constructs as it was necessary in the present study. Second, we wanted to address the question of how defined promoter elements behave in different back- grounds. Therefore it was required to study the expression of constructs (see [24]) in at least two different species. For this purpose we first char- acterized promoter elements involved in light regulation in seedlings of Arabidopsis which be- longs to the Brassicacea and thus is closely re- lated to Sinapis alba L. We then studied the ex- pression of functionally defined promoter elements in a more distantly related species (to- bacco).

In order to characterize the experimental sys- tem, light regulation of endogenous CHS expres- sion was analysed in Arabidopsis seedlings. Ara- bidopsis ecotype Wassilewskija was choosen since the same ecotype was used for generating trans- genic plants. Etiolated seedlings at the age of 6, 8 and 10 days after germination were treated with different light qualities for 24 h. Subsequently, total RNA was isolated and analysed for CHS and CAB transcripts (Fig. 1). CHS expression is induced by far-red light in 6-day-old seedlings, indicative for the high-irradiance response of phy- tochrome [ 16], whereas UV-A light is much more effective in 10-day-old seedlings. UV light induc- tion of CHS expression in Arabidopsis has been described before [12, 29]. In contrast, highest CAB expression was observed after red light treatment independently of the age of the seed- lings. These data suggest an developmental switch in the light regulation of CHS expression in Ara- bidopsis seedlings. Similar developmental changes

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Fig. 1. Light-induced CHS and CAB expression in Arabidop- sis seedlings. RNA-gel blot analyses of RNA isolated from seedlings which were grown for six, eight and ten days in darkness and light-treated afterwards for 24 h. D, dark con- trol; W, white light; R, red light; FR, far-red light; B, blue light; UV, UV-A light. For light sources see Materials and methods. Radio-labelled probes representing part of the coding regions of the mustard genes SA-CHS1 [2] and SA-CAB1 [17] were used for detection of transcripts encoding CHS and CAB, respectively. Ethidium bromide staining of gels indicated load- ing of equal amounts of RNA in each lane.

in the induction of CHS expression have been described for mustard and parsley [2, 15]. Since both phytochrome and blue/UV-A photorecep- tors seem to act in parallel on CHS expression in early stages as analysed for day six (Fig. 1) and day two (T. Kunkel, unpublished data), we de- cided to study the seedlings of transgenic lines at an age where effects of different photoreceptors on the expression of the introduced constructs are likely (see below).

Characterization of light-responsive elements in the mustard CHS 1 promoter

Transgenic Arabidopsis lines carrying CHS-pro- moter::uidA gene fusions (for constructs see [24]) were analysed for the effectiveness of different light qualities to induce expression from the vari- ous promoters. Specific GUS activity is inter- preted as a measure for the activity of the respec- tive promoter variant. Seedlings analysed were grown in complete darkness for two days and light-treated afterwards for 24 h.

Figure 2 shows the induced GUS expression driven by the full-length promoter (-907) and dif- ferent 5' promoter deletions (-655 to -129). The full-length promoter construct consisting of about 1 kb of the CHS1 promoter fused to the uidA open reading frame [23 ] showed induced expression by white, far-red, blue and UV-A light (Fig. 2A). Far-red light proved to be very effective, indicat- ing control by phytochrome A via a high- irradiance response (for review see [16]). The strong response to blue and UV-A light indicates that there is an additional involvement of blue and UV-A light photoreceptor systems on CHS expression. On the other hand, the lack of red light inducibility observed (Fig. 2) shows that phytochrome B (stable phytochrome; for review see [16]) is probably not involved in photoregu- lation of the chalcone synthase gene in Arabidop- sis seedlings at this developmental stage. As with the expression of the trans-gene, endogenous ex- pression of CHS in Arabidopsis seedlings shows strong induction by far-red and UV-A light but weak induction by red light (see Fig. 1). This mode of light regulation is different from that in mustard seedlings where CHS1 transcript levels are strongly increased both by far-red and red light [2, 10], indicating that the CHS1 gene in mustard probably responds to phytochrome B as well as phytochrome A.

The 5' deletions -655, -565, -334, and -237 showed a nearly identical photoregulation as the full-length promoter (Fig. 2A). Although the ex- pression of constructs -334 and -237 is weak, the data indicate that cis-acting elements down- stream of position -237 are able to mediate the

223

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Fig. 2. Light-induced GUS expression in transgenic Arabi- dopsis seedlings. Seedlings transgenic for the constructs indi- cated were grown for 2 days in complete darkness and then kept in darkness or treated for 24 h with different types of light2 D, dark control; W, white light; R, red light; FR, far-red light; B, blue light; UV, UV-A light. For light sources and growth conditions see Materials and methods. GUS activity was de- termined in total protein extracts [21]. The results represent an average of measurements from three independent experi- ments made on between six and eight (part A) or four and six (part B) independent transgenic lines. Error bars are 1 SD. A. Expression of 5' promoter deletion contructs (-907, -655, -565, -334 and -237) in seedlings grown on sugar-free MS medium. B. Expression of 5' promoter deletion constructs (-151 and -129) in seedlings kept on MS medium contain- ing 1% sucrose.

complete light regulation of the full-length pro- moter. In order to define more precisely the lo- cation of such c/s-acting elements, additional pro- moter deletions were analysed. For constructs

- 151 and - 129 no expression above background was observed in seedlings grown on sugar-free MS medium (data not shown). It is known that sugar can induce CHS expression (e.g. CHSA gene from Petunia [40]). Thus effects of light on

the expression of constructs - 151 and - 129 were reinvestigated with seedlings grown on MS me- dium containing 1~o sucrose. As shown in Fig. 2B, expression of construct -151 but not construct -129 is light-induced under such con- ditions. The G-box-like sequence of Unit 1 of the mustard CHS promoter [2, 24] is deleted in con- struct -129. These data indicate that in Arab# dopsis seedlings Unit 1 is probably involved in the light-induced expression driven by the mustard CHSI promoter and are in accordance with pre- vious findings from transient expression studies [3, 35, 43] which demonstrated that Unit 1 from the parsley as well as the mustard CHS promoters act as light-responsive elements.

To investigate whether Unit 1 of the mustard CHS1 gene is sufficient to mediate light-induced expression in Arabidopsis, a construct carrying the mustard Unit 1 as a dimer [24, 35] was trans- formed into Arabidopsis and the eleven resulting transgenic lines where analysed for light-induced GUS expression. The Unit 1 construct showed an almost identical photoregulation pattern with the exception of a somewhat higher dark expres- sion and therefore less pronounced light induc- tion (Fig. 3). We conclude that Unit 1 is sufficient for the light induction of the CHS1 promoter.

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Fig. 3. Full-length promoter and Unit 1-driven GUS expres- sion in transgenic Arabidopsis seedlings, Growth conditions and light treatments were as described in the legend of Fig. 2. Data are averages of four independent experiments on ten (Unit 1) and seven (full-length promoter, -907) independent transgenic lines. Error bars are 1 SD. The effectiveness of different lights is qualitatively the same for the full-length CHS1 promoter and the Unit 1 construct.

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In order to test whether the CHS1 promoter contains additional elements besides Unit 1 in- volved in light regulation, 3' promoter deletions were carried out and analysed in transgenic Ara- bidopsis seedlings (for constructs see [24]). The transcription initiation signals of the CHS1 gene were exchanged for those from the Arabidopsis alcohol dehydrogenase (ADH) gene [ 7 ]. The trun- cated ADH promoter (construct adh-GUS) by itself is unable to mediate light-induced expres- sion (data not shown and [7]). Construct -907/ -74 containing the complete Unit 1 is able to

mediate light-induced expression although about ten times reduced in comparison with the full- length promoter (Fig. 4). Surprisingly, red light was very effective, a result not seen for any other CHS1 promoter construct analysed in this study. The effectiveness of red light would indicate con- trol by phytochrome B [16]. Since neither the full-length promoter nor the Unit 1 construct showed induced expression by red light in Arab# dopsis seedlings, we assume that red light induc- tion seen for construct -907/ -74 might result from an unpredicted creation of an cis-acting el-

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Fig. 4. Expression of 3' promoter deletions in transgenic Ara- bidopsis seedlings. Transgenic Arabidopsis seedlings carrying constructs -907/-74, -907/-151, -907/-334 and -907/ -655 were grown and light-treated as described in legend of Fig. 2. In contrast to any other construct analysed, construct -907/-74 shows strong induction by red light. The shortest 3' deletion mediating light induction is construct -907/-655. For constructs see [24]. The results represent an average of four independent experiments. The number of transgenic lines analysed were as follows: five (-907/-74), five (-907/-151), five (-907/-334) and nine (-907/-655). Error bars are 1 SD.

ement due to the fusion of this particular pro- moter fragment with the truncated ADH pro- moter. A further explanation could be the destruction of an element responsible for the re- pression of the red light effect. If Unit 1 would be the only light-responsive element within the CHS1 promoter, one would predict that any 3' deletion removing Unit 1 would result in the loss of light induction. Since expression of construct -907/ -151 lacking Unit 1 is still induced by far-red and

blue light (Fig. 4), light-responsive element(s) must be present also upstream of position -151. Although the activity of construct -907 / - 151 is weak, expression after far-red and blue light treat- ments is significantly higher than in the dark con- trol. A further 3' deletion to position -334 (con- struct -907/-334) resulted in a complete loss of light induction (Fig. 4). Previous studies [24] have demonstrated that the region between -334 and -151 of the CHS1 promoter mediates expression

in leaves but not in cotyledons. Thus the inabil- ity of construct -907/-334 to react to light could be the result of either the deletion of a light- responsive element located between -334 and - 151 or the suppression of expression in cotyle- dons by sequences which might act independently of light-responsive elements. Note that the stud- ies were done with etiolated seedlings which lack true leaves. Since a further deletion (-907/-655, see below) is able to mediate light induction, plants containing construct -907/-334 are an ex- ample that correct spatial pattern of expression is a prerequisite for correct light induction. Interest- ingly, the photoregulation pattern mediated by the -907/-655 region is almost identical to that me-

diated by the full-length promoter or the Unit 1. Although all transgenic lines carrying the -907/ -655 construct (in total nine lines) mediate the same expression patterns in flowers [24] only five of them showed light-induced expression in seed- lings. Thus it seems likely that the region between -907 and -655 of the CHS1 promoter lacks el- ements necessary for strong expression in coty- ledons or other organs of the seedling but con- tains elements mediating light responses. A search for conserved sequences in the -907/-655 pro- moter region showed a striking similarity to Unit

2 of the parsley CHS promoter, an element which is involved in the light response of this gene [3, 38]. The regions in the mustard CHSI promoter with similarity to the footprinted regions (Box III and IV) of the parsley CHS gene [39] show iden- tity in four of eight positions (the ACGT core in Box III) and 11 of 16 positions (Box IV) as well as a nearly identical distance (13 bp in mustard CHS instead of 14 bp in parsley CHS; for se- quence comparison see also [24]). Although the conservation of a Unit 2-like element indicates an essential role in the mustard CHS1 gene, further studies are needed to determine the exact func- tion of this element.

Unit 1 mediates the same light regulation as the fulMength promoter

Data presented above together with results from transient expression studies [ 35, 43 ] demonstrate a major role of Unit 1 in light regulation of CHS expression. In order to analyse whether Unit 1 mediates light responses in other systems as well, we transformed tobacco with the Unit 1 and full- length promoter construct and studied their ex- pression. In 5-day-old transgenic tobacco seed- lings, the full-length promoter drives induced expression in white, red, far-red, blue, and UV-A light (Fig. 5, left). Again, the Unit 1 construct shows qualitatively the same photoregulation as the full-length promoter construct (Fig. 5, right).

The principal difference in CHSI promoter- driven GUS expression between Arabidopsis and tobacco is the pronounced induction by conti- nous red light seen in the latter. The effectiveness of red light indicates that Unit 1 may well respond to signals from phytochrome B in transgenic to- bacco seedlings as well as phytochrome A (see above). In contrast to Arabidopsis, measurements of endogenous CHS transcript levels in tobacco show major differences in light regulation to the expression of the chimeric CHS1 construct (see Fig. 6). For example, the induction of GUS ex- pression by red fight is not paralled by an increase in tobacco CHS m R N A (compare Fig. 5 and Fig. 6). This result indicates that the mode of light

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Fig. 5. GUS expression in transgenic tobacco seedlings driven by the full-length CHS1 promoter or the mustard Unit 1 hy- brid promoter construction. Transgenic tobacco seedlings car- rying mustard CHSI promoter constructs (full-length CHS1 promoter::GUS fusion, -907; Unit I::GUS construct, Unit 1) were grown in complete darkness for 4 days and then kept in darkness or treated for 24 h with different types of light. D, dark control; W, white light; R, red light; FR, far-red light; B, blue light; UV, UV-A light. For light sources and growth conditions see Materials and methods. The numbers of trans- genic lines analysed were as follows: seven (full-length pro- moter construct), nine (Unit 1 from mustard). Given are av- erages of three experiments and SD.

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Fig. 6. Light induction of CHS-mRNA levels in tobacco seed- lings. Tobacco seedlings were grown in complete darkness for 4 days and then kept in darkness or treated for 24 h with different fight qualities. Total RNA was then isolated and the relative amount of CHS-mRNA determined by dot blot hy- bridization as described in Materials and methods. Error bars are 1 SD. D, dark control; R, red fight; FR, far-red light; B, blue light; UV, UV-A light.

action on the expression of the trans-gene does not necessarily reflect the regulation of the corre- sponding endogenous gene. We therefore con-

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clude that the photoreceptor usage for the same promoter, and even for the same promoter ele- ment (Unit 1), may be different in different spe- cies used for transgenic analyses (Arabidopsis vs. tobacco) and may also be different from that of the corresponding endogenous genes. In accor- dance with this statement is our observation that conserved promoter elements from different genes (Unit 1 from the parsley and mustard CHS gene promoters) show significant differences in the patterns of light regulation in the same species (tobacco). For example, UV-B-containing white light is more efficient than far-red light in the in- duction of the parsley Unit 1-driven expression, whereas expression driven by Unit 1 from mus- tard CHS is induced to essentially the same ex- tent by far-red and UV-B-containing white light (Fig. 7). Similarily, differences in the in vitro bind- ing of Unit 1 sequences from mustard and pars- ley CHS have been described [35]. In conclusion, our results demonstrate that the system in which the trans-gene is expressed determines the expres- sion pattern and therefore, results obtained from studying one system should be interpreted with caution.

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Fig. 7. GUS expression in transgenic tobacco seedlings driven by Unit 1 from parsley and mustard CHS. For growth con- ditions and light treatments see the legend of Fig. 5. Nine (Unit 1 from mustard) or eight (Unit 1 from parsley) indepen- dent transgenic lines were analysed in three independent ex- periments. Given are means and SD. In contrast to mustard Unit 1, expression of parsley Unit 1-driven expression is stronger induced by UV-B containing white light (W*) than far-red and blue light. The data shown for mustard Unit 1 are the same as those presented in Fig. 5.

Unit I mediates the developmental switch in photo- receptor usage

It has previously been shown that the responsive- ness of CHS expression to far-red light decreases during development [2, 15]. The same phenom- enon is observed for CHS promoter activity in transgenic plants. The responsiveness to far-red and white light is nearly the same in 5-day-old tobacco seedlings (Fig. 5) whereas white light is clearly more than twice as effective in 9-day-old seedlings (Fig. 8). The relative decrease in the re- sponse to far-red light during development is seen for both the full-length promoter and the Unit 1 construct. The overall decrease in the light re- sponse during an extended dark period is again observed for the full-length promoter and for the Unit 1-driven GUS expression (compare Fig. 8 and Fig. 5).

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Fig. 8. Developmental changes in the responsiveness to dif- ferent photoreceptors for GUS expression driven by the full- length CHS1 promoter or the Unit 1.8-day-old etiolated to- bacco seedlings expressing GUS under the control of the full- length CHS1 promoter (-907, left-hand panel) or the Unit 1 (right-hand panel) were light-treated as described in Fig. 5 and Materials and methods. The results represent an average of measurements from three independent experiments with seven (-907) and nine (Unit 1) independent transgenic lines. Error bars are 1 SD. In contrast to 5-day-old tobacco seedlings (Fig. 5), white light is clearly more effective than far-red light, indicating a decrease in the effectiveness of far-red light dur- ing development.

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Unit 1 is sufficient for proper spatial expression patterns

The chalcone synthase promoter also directs tissue-specific expression in the transgenic plants. Full-length promoter-driven G U S expression was observed in cotyledons of dark-grown Arabidopsis seedlings, and the expression level is increased by light treatment (Fig. 9A). In contrast, the expres- sion of the same construct is strictly light- dependent in tobacco (Fig. 9B). We also observed differences in root expression between the two species. Whereas expression in this organ is not detectable in tobacco seedlings (Fig. 9B), there is a strong light-induced expression in Arabidopsis (Fig. 9A). Despite these differences in organ specificity between tobacco and Arabidopsis, the spatial expression pattern of the Unit 1 construct is indistinguishable from that for the full-length promoter in the same plant species (Fig. 9A and B).

Thus, we conclude from studies presented here and published previously [24, 35] that Unit 1 of the chalcone synthase promoter responds to ex- ternal and internal signals in the same way as the full-length promoter. This responsiveness is de- velopmentally controlled and species-specific. Other regions in this promoter have effects on modulating the expression levels and act indepen- dently when Unit 1 is deleted as shown in this study for the light regulation mediated by the -907/-655 region. Our results also do not ex- clude the action of additional elements mediating

Fig. 9. Spatial patterns of GUS expression in transgenic Ara- bidopsis (A) and tobacco (B) seedlings.Transgenic plants, car- rying the full-length CHS1 promoter:: G US construct (left-hand panels) or the Unit I::GUS construct (right-hand panels) were

grown in complete darkness for two (Arabidopsis) or four days (tobacco), respectively, followed either by a treatment with white light (W) or further darkness (D) for 24 h. In case of Arabidopsis, GUS activity is found in cotyledons Of etiolated and light-iLreated seedlings, whereas expression in roots and the upper part of the hypocotyl is light-dependent. In case of tobacco seedlings, GUS expression is strictly light-dependent and observed in the cotyledons and the upper part of the cotyledons but not in the roots. Although there are clear dif- ferences in the spatial patterns of GUS expression and their light dependence between Arabidopsis and tobacco seedlings, the expression patterns of the full-length promoter and the Unit 1 construct are very similar if not identical for the same species (Arabidopsis vs. tobacco).

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responses to stimuli such as wounding, not analy- sed in this study.

The observation that a short promoter element of a plant gene is sufficient for proper spatial and temporal expression as well as for regulated ex- pression by external light stimuli suggests a pos- sible major difference from gene regulation in ani- mals, where correct expression patterns in transfection experiments depend not only on pro- moter elements, but also on sequences (enhanc- ers) far distant from the promoter [4, 6, 18]. Fur- ther studies are needed to determine whether this difference holds more generally in plants.

Acknowledgements

The authors are very grateful to Dr W.R. Briggs for helpful comments and critical reading of the manuscript, and thank M. Rocholl for help in making the Unit 1 construct and M. Krenz for excellent technical assistance. Supported by DFG grants (to A.B. and E.S.) and a fellowship to T. Kaiser and K.E. from the Landesgraduierten- f~)rderung.

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