Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2683-2686, April 1991 Botany

Deletion analysis of a phytochrome-regulated monocot rbcS promoter in a transient assay system (Lemna gibba/particle bombardment/ribulose 1,5-bisphosphate carboxylase/oxygenase)

STEPHEN A. ROLFEt

AND

ELAINE M. TOBIN*

Department of Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024-1606

Communicated by Bernard 0. Phinney, January 3, 1991 (received for review November 28, 1990)

ABSTRACT We have developed a transient gene expression assay system in the aquatic monocot Lemna gibba in which DNA was introduced into intact tissue by particle bombardment. Constructs based on the Lemna rbcS gene SSU5B, which is positively regulated by phytochrome in vivo, also showed phytochrome regulation in the transient assay system. Reporter gene expression increased 12-fold over dark levels in response to a single treatment with red light. This increase was not observed if far-red light was immediately followed by the red light. A 5' deletion analysis of the promoter defined a region from position -205 to position -83 relative to the start of transcription as necessary to observe the phytochrome response. This region contains the binding site for the lightinduced binding activity (LRF-1) found in Lemna nuclear extracts. Upstream of position -205, we found evidence for the presence of at least two upstream activating sequences and a silencer. The photoreceptor phytochrome is involved in the regulation of expression of many higher plant genes. Phytochrome exists in two photochemically interconvertible forms. In dark grown plants, phytochrome is synthesized as the inactive form Pr, which is converted to the active form Pfr by light; this conversion can be reversed by far-red light (FR) (1). The transcription of a number of nuclear genes has been shown to be regulated either positively or negatively by phytochrome action (2, 3) and sequences upstream of some of these genes have been demonstrated to be important in mediating such regulation (e.g., ref. 4 and references therein). In several species, the expression of genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcS genes) is positively regulated by phytochrome. In the aquatic monocot Lemna gibba, the rbcS gene family has 12-14 members (5), 6 of which have been isolated as genomic clones and a seventh as a cDNA clone (6, 7); the expression of these individual family members can be regulated by the action of phytochrome (6). Transcription of the family as a whole, as measured by in vitro transcription experiments, is stimulated by 1 min of red light (R), and this effect can be reversed if immediately followed by 10 min of FR (8). Of the available Lemna rbcS genomic clones, SSU5B is the most abundantly expressed in response to R illumination of dark-treated plants (6). Furthermore, a protein factor, designated LRF-1, that binds a specific sequence upstream of SSU5B has been identified in nuclear extracts of Lemna gibba (9). A 2-fold to 3-fold increase in this binding activity is seen when dark-treated plants are exposed to white light or 2 min of R, suggesting that this binding activity is itself phytochrome regulated. The region to which this activity binds overlaps a region (box X) that is highly conserved among all rbcS genes. Two additional regions of the SSU5B The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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promoter (boxes Y and Z) have high nucleotide identity with two other Lemna rbcS genes (9). In the work reported here, we have developed and used a transient expression assay to test which regions of this promoter function in vivo to confer sensitivity to regulation by phytochrome. Numerous studies have analyzed light-regulated promoters by using reporter gene fusions introduced into dicots (e.g., refs. 10-14). Five rbcS binding factors have been identified in nuclear extracts from dicots, GT-1, GBF, GA-1, AT-1, and 3AF1 (15-18), but their binding is not affected by light. Although promoters from monocot chlorophyll a/b apoprotein genes of Lemna (19) and wheat (20) can direct phytochrome-regulated expression of reporter genes in tobacco (a dicot), a homologous expression system is preferable for promoter analysis because it is likely to give a more faithful picture ofthe normal signal transduction process. The analysis of constructs in monocots has only recently become possible with the successful transformation and regeneration of fertile maize (21) and rice (22). No transformation system is yet available for Lemna. Recently, transient assay systems have been used to examine gene expression in monocot tissues and protoplasts. Such systems offer several advantages. Gene expression can be studied more rapidly compared to generating stably transformed dicots. Furthermore, the particle bombardment system that has been used to study gene expression in several cereals offers the possibility of studying the expression of introduced monocot genes in the homologous system. Constructs encoding components of the anthocyanin biosynthesis pathway have been introduced into intact maize tissue and restored anthocyanin synthesis in mutant lines (23, 24). The promoter from the oat phytochrome gene was introduced into rice seedlings and shown to be negatively regulated by phytochrome (25). Deletions within the promoter of this gene identified three positive elements, but the element(s) responsible for the strong repression of expression by the active form Pfr was not identified (26). We report here the development of a transient assay system in Lemna gibba using a particle acceleration device. We have used the assay to localize a region in the SSU5B promoter that is essential for the regulation of transcription

by phytochrome. MATERIALS AND METHODS Growth of Plants. Etiolated Lemna gibba L. G-3 were cultured aseptically in liquid E medium supplemented with 3 ,uM kinetin. Plants were grown in darkness and exposed to 2. min of R every 8 hr (27). Two days prior to bombardment, fronds were transferred to E medium solidified with 1% Abbreviations: CAT, chloramphenicol acetyltransferase; FR, farred light; R, red light. tPresent address: Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN England. tTo whom reprint requests should be addressed.

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phytagar and arranged as a monolayer 4 cm in diameter. All manipulations were performed under a dim green safelight. The plants were irradiated with 10 min of FR and then placed in total darkness for 2 days. Construction of SSUSB Promoter Fusions. The construct pSBCATNOS contains SSU5B sequence from position -%5 to position +197 joined as a translational fusion to the chloramphenicol acetyltransferase (CAT) gene with a nopaline synthase (nos) 3' untranslated region. SSU5B sequence was isolated from the phage A genomic clone ASSU5B (5) as two subclones: (i) plasmid pXB985 contained the region from position -965 to position +20 as an Xba I-BamHI fragment in the vector pUC19 and (ii) plasmid pSSU500 contained the region from position -256 to position +197 as a Sal I fragment in pBR322. The SSU5B sequences were joined as a translational fusion to the CAT gene because it has been reported that the transit peptide from the Arabidopsis rbcS gene increased expression in transgenic plants (28). To obtain the translational fusion, two fragments were ligated into the vector pBluescript SK(-) (Stratagene) digested with BamHI and EcoRI. The first fragment, containing SSUSB sequence from position +20 to position + 197, was isolated from pSSU500 by digestion with Sal I, trimming with mung bean nuclease to create a blunt end, and further digestion with BamHI. The second fragment containing the 5' coding region of the CAT gene was isolated from pBR328 (29) by digestion with Taq I, trimming with mung bean nuclease, and digestion with EcoRI. Correct insertion of the two fragments into pBluescript SK(-) was confirmed by dideoxynucleotide sequencing using the Sequenase DNA sequencing kit (United States Biochemical). The resulting plasmid was called p5BCATA. The remaining coding sequence of CAT and 1.13 kilobases of sequence downstream of the nos gene were isolated from pMZRI (a gift from B. Volcani, Scripps Institute, La Jolla, CA) by digestion with EcoRI and HindIl and cloned into pSBCATA to give the construct p5BCATB. SSU5B sequence from position -%5 to position +20 was isolated from pXB985 by digestion with Xba I and BamHI and cloned into p5BCATB to give the construct p5BCATNOS. Deletions of the promoter were made either by digestion of pXB985 at convenient restriction sites (Sau3AI at position -83; Dra I at position -739) or by digestion with BAL-31 exonuclease (resulting in deletions to positions -480, -397, -301, -205, and -74) and cloning the resulting fragment into the BamHI site of p5BCATB. Dideoxynucleotide sequencing was used to determine the endpoints of the digested promoter. Bombardment and Irradiation. Monolayers (4 cm in diameter) of dark-treated etiolated Lemna fronds were bombarded using the Biolistics Particle Delivery System (PDS 1000, Du Pont) as described (24, 25). Each sample was bombarded with 1.25 mg of 1.2-,um tungsten particles coated with 8 gg of supercoiled plasmid DNA. Control samples were bombarded with uncoated particles. A single layer of cheesecloth was placed over the sample to prevent fronds from being dislodged. All manipulations were performed under a dim green safelight. Immediately (within 1 min) after bombardment all samples were irradiated with 2 min of FR to counteract any possible effect of the light produced by the discharge of the apparatus. Samples were then either placed in darkness or immediately illuminated with 2 min of R or with 2 min of R followed by 2 min of FR before returning to darkness. For some experiments, 2 min of R or 2 min of R and 10 min of FR treatments were given every 2 hr. Samples were incubated at 25°C and harvested 16 hr after bombardment unless otherwise stated. Many of the fronds showed visible signs of damage after bombardment. It is not known how many individual cells are responsible for the observed activity.

Proc. Natl. Acad. Sci. USA 88 (1991)

CAT Activity Assays. The entire 4-cm monolayer sample was harvested for each assay. Fronds were ground in 400 Al of ice-cold 0.25 M Tris Cl, pH 7.5/5 mM EDTA/0.5 mM phenylmethylsulfonyl fluoride/1 mM dithiothreitol using a ground-glass homogenizer. The homogenate was centrifuged at 14,000 x g at 40C for 10 min to remove debris. Protein concentrations in the supernatant were determined using the Bradford assay method (Bio-Rad). CAT activity in the supernatant was determined as described (30) except that the incubation at 370C was extended to 90 min. Equal volumes (200 1.l) of clarified extract were used in each assay, and results were expressed as [14C]chloramphenicol acetylated per mg of protein per hr after correction for background activity from control samples that had been bombarded with uncoated tungsten particles. Activity of control samples was unaffected by light treatments.

RESULTS Optimization of the Transient Assay System. Optimal conditions for the transient assay in Lemna were established using etiolated fronds bombarded with pSBCATNOS DNA. CAT activity from fronds placed in constant white light after bombardment was first detectable at 6-8 hr and reached a maximum by 16-24 hr (19). Incubation for up to 72 hr produced no further change in activity, indicating that CAT activity produced during the transient assay was not degraded during the incubation period. Therefore, a 16-hr incubation after bombardment was used for all assays. Previous reports of transient assay systems using the particle gun have used a constitutive reference gene cobombarded with the test construct to compensate for large variations in expression observed between experiments (24, 25). In the Lemna system cobombardment with ubi-Luc, a constitutive maize ubiquitin promoter fused to the luciferase reporter gene (25), led to reduced expression of the p5BCATNOS construct. One possible explanation for this observation is that the maize ubiquitin promoter competed for binding factors with the Lemna SSU5B promoter. UbiLuc was, therefore, unsuitable as a reference gene. Additionally, we observed considerably less variation between bombardments with Lemna than was seen with rice seedlings; we attribute this consistency to the uniform target presented by the monolayer of fronds. Therefore, we were able to use repeated identical assays to quantify gene expression without an internal reference gene. Expression due to the maize ubiquitin promoter was reported to be unaffected by light when introduced into rice seedlings (25). Bombardment of Lemna fronds with a construct consisting of the maize ubiquitin promoter fused to the CAT gene with a nos 3' untranslated region (ubi-CAT) gave high levels of CAT expression. Fig. 1A shows that no significant difference in CAT activity was observed between light- and dark-treated samples. In contrast, CAT expression from p5BCATNOS was very low in fronds incubated in the dark and substantially higher in those incubated in white light. Therefore, this difference represented light-regulated expression rather than a general overall increase in expression in light. The SSUSB Promoter Confers Phytochrome Regulation on a Reporter Gene. In vitro transcription run-on experiments have shown that transcription of the SSU5B gene is reduced to undetectable levels in dark-treated plants stimulated by a single R treatment, reaching a maximum 2 hr after illumination, but is undetectable after 4 hr (P. A. Okubara and E.M.T., unpublished results). To determine whether the expression of the p5BCATNOS construct was regulated by phytochrome, etiolated Lemna fronds were bombarded with p5BCATNOS DNA and exposed to a variety of light treatments. The results from these experiments are shown in Fig.

Botany: Rolfe and Tobin

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Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 1. Expression of ubi-CAT and p5BCATNOS DNAs after light treatments. Etiolated Lemna fronds were bombarded with ubi-CAT DNA (shaded bars) or p5BCATNOS DNA (hatched bars) and irradiated with 2 min of FR. (A) Samples were placed in darkness (bars D) or constant white light (bars CWL). (B) Samples were irradiated once with 2 min of R (bar R) or 2 min of R followed by 2 min of FR (bar RFR). Other samples were irradiated every 2 hr with 2 min of R (bar R*) or 2 min of R followed by 10 min of FR (bar RFR*) or were placed in darkness (bar D). CAT activity (pmol per mg per hr) was measured 16 hr after bombardment and corrected for background activity. Error bars are the SEM for three to six assays. Note that the scale for ubi-CAT differs from that for p5BCATNOS. various

1B. All samples received 2 min of FR after bombardment because the flash of light produced by the particle gun would convert a proportion of the phytochrome to the active Pfr form. Plants that were placed in darkness showed a low level of expression that was stimulated approximately 12-fold by a single 2-min R illumination. If FR immediately followed the R, little stimulation was observed. No significant increase in activity was observed over the R level when plants received either R treatments every 2 hr (R*) or constant white light (CWL). A single light treatment immediately after bombardment was, therefore, used in all subsequent assays. Localization of Regulatory Elements in the SSU5B Promoter. A series of 5' deletions of the SSU5B promoter was analyzed in the transient assay system to determine their responsiveness to phytochrome action after a single light treatment. Fig. 2 shows that deletion of the promoter to position -480 from the start of transcription had no significant effect on the level of expression compared to the 1-kilobase promoter. Deletion to position -397 resulted in a substantial decrease in the level of R-induced expression; further deletion to position -301 restored this expression. These data can best be explained by the presence of an upstream activating sequence between -480 and -397 and a silencer element between -301 and -397. Constructs that contained promoter sequences extending to position -205 showed a lower level of phytochrome-regulated expression, thus revealing the presence of a second upstream activating sequence between -301 and -205. Although the level of R-induced expression was modified by the presence of the upstream activating sequences and the putative silencer, all constructs that included sequence 205 nucleotides upstream of the transcription start showed phytochrome regulation. Therefore, elements upstream of position -205 were not required to obtain phytochrome regulation. Deletions to positions -83 and -74 gave a low level of expression that was not phytochrome regulated. Deletion of the entire promoter (position +20) resulted in expression indistinguishable from background levels. Thus, we conclude

-739

-480

-397

-301

-205

-83

-74

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FIG. 2. 5' deletion analysis of the SSU5B promoter. The 5' deletion constructs were assayed for phytochrome-regulated CAT activity (pmol per mg per hr) by using the transient assay system. The numbers refer to the 5' end of the promoter relative to the start of transcription. After bombardment, samples were irradiated with 2 min of FR and then placed in darkness (open bars) or irradiated with 2 min of R (hatched bars) or with 2 min of R followed by 2 min of FR (shaded bars). After a 16-hr incubation in darkness samples were assayed for CAT activity as in Fig. 1. Error bars are the SEM for six to eight bombardments.

that elements located between positions -205 and -83 were sufficient for conferring phytochrome-regulated expression.

DISCUSSION In this study we have shown that a transient gene expression system using particle bombardment of Lemna fronds can be used to examine phytochrome-regulated gene expression. Furthermore, we used this system to carry out analysis of a deletion series of the SSU5B promoter that can direct phytochrome-regulated expression of a reporter gene in response to a single brief R treatment. The level of CAT activity observed at the end of the 16-hr incubation period after bombardment results from a combination of processes that include transcription of the introduced DNA, processing and stability of the transcribed RNA, translation of the mRNA, and stability of the CAT enzyme. Any of these could be phytochrome regulated. However, the constructs deleted to positions -73 and -84 showed a low level of CAT activity that was not phytochrome regulated. There is no reason to believe that the mRNA transcribed from these constructs differed from mRNA from the full-length construct; therefore, we conclude that the differences in CAT expression seen in response to R resulted from changes in

transcription. Transcription of the native SSU5B gene in vivo increases by 2 hr after a single treatment with R and has declined to an undetectable level by 4 hr after illumination. Here we found that multiple treatments with R (Fig. 1B, bar R*) did not lead to a significant increase in CAT activity over that observed with a single R treatment. Therefore, it is possible that the DNA introduced by particle bombardment may not be transcribed for longer than 4 hr. The deletion series analysis showed that the sequence extending from position -83 to position -205 was necessary to confer phytochrome regulation on a reporter gene. This result correlates well with studies of the DNA binding factor LRF-1 present in Lemna nuclear extracts. LRF-1 interacts with the SSU5B promoter within this region in a lightregulated manner (9). Sequence comparisons have led to the identification of three conserved regions within the SSU5B promoter (boxes X, Y, and Z), as shown schematically in Fig. 3. In vitro binding studies have shown that the LRF-1 binding site overlaps box X (9). The possible role of box Y in

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Proc. Natl. Acad. Sci. USA 88 (1991) 5B leAder

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FIG. 3. Conserved elements in the SSU5B promoter. Conserved elements in the SSUSB promoter identified by sequence homology are shown as boxes X, Y, and Z. The region protected from DNase I digestion by the Lemna nuclear factor LRF-1 is shown as box LRF-1. The large arrow marks the start of transcription in vivo. The relative position of the TATA box is shown. The endpoints of the 5' deletion constructs are marked by vertical arrows and the distance relative to the start of transcription is indicated.

4.

phytochrome-regulated expression remains to be investigated. The construct with promoter sequence extending to position -83 contained box Z but was not phytochrome regulated. Therefore, box Z alone is not sufficient to direct phytochrome-regulated expression. In pea, the rbcS 3A gene contains a binding site known as box II for the DNA binding protein GT-1. It has been reported that a tetrameric box II sequence placed upstream of a truncated cauliflower mosaic virus 35S promoter conferred light-dependent expression of a reporter gene when introduced into tobacco (31). It is of interest that no sequence showing greater than 53% identity with box II is present in the Lemna SSU5B promoter region extending to position -205. Therefore, the box II sequence is not required for phytochrome regulation of gene expression in Lemna. We have found evidence for at least two activating sequences and one silencer upstream of position -205. The presence of these elements was not required to confer phytochrome regulation on transcription, but they did modulate the level of R-induced expression. Whether binding of factors to these elements occurs and is phytochrome regulated remains to be determined. The presence of multiple regulatory elements in the promoters of other light-regulated genes has been noted by several groups (10, 11, 13, 17), although the possible interactions of factors binding to these elements with those required for phytochrome regulation have yet to be demonstrated. No obvious homologies between the Lemna SSUSB promoter and sequences containing upstream activating sequences and silencers from other lightregulated genes were observed. The development of the transient assay system in Lemna and its sensitivity in detecting changes resulting from a single R treatment will allow mutated promoters to be investigated rapidly. Such studies should allow close correlations to be made between DNA elements identified by in vitro binding experiments and phytochrome-regulated transcription in

8.

vivo. We thank Dr. Peter Quail and Dr. Wesley Bruce for their help in developing this transient assay system. Ubiquitin constructs were kind gifts from Dr. Alan Christensen and pMZR1 was from Dr. Ben Volcani. We thank Lu Huang for maintaining Lemna cultures and other members of our laboratory for help during preparation of this manuscript. This research was supported by National Institutes of Health Grant GM-23167 (E.M.T.) and Biomedical Research Support Grant RR07009-23 to University of California, Los Angeles, CA. S.A.R. was supported in part by a European Molecular Biology Organization Fellowship. 1. Quail, P. H. (1983) in Encyclopedia of Plant Physiology (NS)

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29. 30.

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