Plant Physiol. (1999) 120: 747-756

Regions of the Pea Lhcb1*4 Promoter Necessary for Blue-Light Regulation in Transgenic Arabidopsis¹

Kevin M. Folta and Lon S. Kaufman^*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607

	ABSTRACT

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Pea (Pisum sativum) and Arabidopsis contain similar, if not identical, blue-light (BL)-responsive systems that alter expression of specific members of the Lhcb (light-harvesting chlorophyll-binding) gene family. In both plants a single, short pulse of low-fluence BL (threshold = 10¹ µmol m²) causes an increase in the rate of transcription from specific members of the Lhcb gene family in etiolated seedlings. Constructs of the BL-regulated pea Lhcb1*4 promoter (PsLhcb1*4) were created, which altered sequences previously implicated in light responses, deleted the 5-promoter sequence, or removed the 5-untranslated region. These constructs were tested for BL induction in transgenic Arabidopsis. The PsLhcb1*4 promoter deletions to 150 bp maintained normal fluence response, time course, and reciprocity characteristics. The 5- untranslated region contained enhancer elements, but was not necessary for BL induction. The 95 to +2 promoter was capable of responding to BL, whereas sequences from 50 were not. Promoters that lack conserved light-regulatory elements or sequences directly implicated in phytochrome and circadian responses retained BL activity, suggesting that the low-fluence BL response utilizes regions of the promoter independent of those that modulate the phytochrome and circadian responses.

	INTRODUCTION

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Plants possess multiple photomorphogenic systems designed to sense and respond to light signals. These systems identify specific wavelengths and quantities of light, and tailor programs of gene expression in concert with the developmental state of the plant and prevailing environmental conditions. These photomorphogenic systems include the red/far-red responsive phytochrome systems (Furuya and Schafer, 1996) and several independent BL/UV systems (Young et al., 1992; Kaufman, 1993; Jenkins, 1997).

Genetic and physiological analyses have identified a series of Arabidopsis mutants with defects in perception/transduction of BL signals. The BL mutants include plant lines with defects in the BL photoreceptors CRY1 (Ahmad and Cashmore, 1996), CRY2, (Ahmad et al., 1998), and NPH1 (Liscum and Briggs, 1995; Huala et al., 1997), which are responsible for BL-mediated suppression of hypocotyl elongation and phototrophic curvature, respectively. icx1 mutants exhibit abnormally high transcript levels of flavonoid-biosynthesis genes and increased production of anthocyanins, suggesting that ICX1 may be a negative regulator of the BL/UV systems (Jackson et al., 1995). The persistence of known BL responses, including the induction of Lhcb (light-harvesting chlorophyll-binding) gene transcription by the BLF system (threshold = 10¹ µmol m²) in pea (Pisum sativum) and Arabidopsis, PsLhcb and AtLhcb, respectively, indicates the presence of additional BL photomorphogenic systems (Gao and Kaufman, 1994).

In pea and Arabidopsis the steady-state level of Lhcb transcript is dependent upon the total fluence of BL received. Excitation of the BLF system increases the rate of transcription of specific members of the PsLhcb1 and AtLhcb1 gene families. This increase in the rate of transcription occurs in response to a single pulse of BL with a threshold of 10¹ µmol m². The BL-enhanced rate of transcription continues to increase with increasing fluences of BL, reaching a maximum transcription rate at 10⁴ µmol m². The response is immediate, does not require protein synthesis, and probably acts through a heterotrimeric G protein (Warpeha and Kaufman, 1990; Marrs and Kaufman, 1991).

Only specific members of the Lhcb1 gene family are induced by BL. Pea contains at least eight members of this gene family (Alexander et al., 1991). The PsLhcb1*1 and Lhcb1*4 genes are regulated by BL, whereas the Lhcb1*2 and Lhcb1*3 gene are not (White et al., 1995; Tilghman et al., 1997). The Lhcb genes of Arabidopsis are also expressed differentially in response to various light signals. Gao and Kaufman (1994) reported that the AtLhcb1*3 gene is induced by BLF in etiolated seedlings, whereas the AtLhcb1*1 and AtLhcb1*2 genes are not. Sun and Tobin (1990) determined that all three of the AtLhcb1 genes are phytochrome responsive in etiolated Arabidopsis seedlings.

Tilghman et al. (1997) introduced 1.3 kb of the BL-regulated PsLhcb1*4 promoter placed upstream of a GUS reporter gene into Arabidopsis and illustrated that the transgene functioned correctly in response to BL with respect to time course, fluence response, and reciprocity. These results indicated that Arabidopsis is a suitable host with which to study transgenic promoter constructs of the PsLhcb1*4 gene, and demonstrated that the AtLhcb1*3 promoter from -200 bp is inducible by the BLF system.

The 100 to +1 region of the BL-regulated PsLhcb1*4 and the AtLhcb1*3 promoters are more similar to each other than to the comparable region of non-BL-regulated Lhcb promoters (for review, see Arguello-Astorga and Herrera-Estrella, 1998). The 100 to +1 region contains sequence elements implicated in light regulation, including elements that confer responses to phytochrome (Kehoe et al., 1994; Keningsbuch and Tobin, 1995) and circadian induction (Anderson and Kay, 1995). Regions of Lhcb promoters regulated by BL have yet to be identified. The goal of the present study was to utilize transgenic Arabidopsis to determine the region(s) of the pea Lhcb1*4 promoter and/or 5-UTR necessary for induction of the BLF response.

Our data indicate that truncated versions of the PsLhcb1*4 gene maintain the normal photobiological characteristics of the full-length promoter. The basal response of the BLF system utilizes sequences in the PsLhcb1*4 gene between 95 and +2, and sequences present upstream of 95 or in the 5-UTR enhance transcription directed by this promoter. Previously defined elements, identified for their roles in phytochrome and circadian regulation, are not necessary for the BL response.

	MATERIALS AND METHODS

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Plant Material

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All planting for photophysiological assays was done under a green safelight (Warpeha and Kaufman, 1989). Seeds were surface-sterilized in 60% (v/v) commercial bleach for 10 min, rinsed three times in at least 10 volumes of water, and then resuspended in 0.5× Murashige-Skoog medium and 0.8% (w/v) low-melting-point agarose. The Murashige and Skoog/agarose/seed suspension was plated onto 50 mL of solidified 0.5× Murashige and Skoog medium containing 0.8% (w/v) agarose in Phytatrays (Sigma), and were then stratified at 4°C for 48 h. Following stratification, the seeds were irradiated with 16 µmol m² white light for 10 min to synchronize germination, and were then grown in absolute darkness at 22°C for 6 d.

PsLhcb1*4 Deletion and Mutagenized Constructs

Independent promoter constructs representing 5 promoter deletions of the pea Lhcb1*4 promoter were generated using PCR with Pfu polymerase (Stratagene). Primers were designed to produce promoter fragments extending from 281, 250, 200, 150, 100, 50, or +1 on the upstream end to +64 on the downstream end, and from 281 and 100 on the upstream end to +2 on the downstream end. The primers were designed with PstI and BamHI sites (upstream and downstream, respectively) to allow directional insertion of promoter fragments upstream of the GUS reporter gene in pBSK101.3 vector.

The REP7550 construct contains the PsLhcb1*4 promoter from 281 to +2, wherein the sequence between 75 and 50 has been replaced by a nonsense sequence. The region between 75 and 50 contains several highly conserved sequence elements that have been identified in the promoters of light-regulated genes: a sequence referred to as "G-box like" (Arguello-Astorga and Herrera-Estrella, 1996) and a double-GATA sequence known as the "I box" (Borello et al., 1993). These elements have been previously implicated in phytochrome (Kehoe et al., 1994) and circadian (Anderson and Kay, 1995) regulation of Lhcb1 genes. Both of these conserved sequences exist in the BL-regulated pea and Arabidopsis Lhcb1 promoters between 75 and 50. This region was replaced with a nonsense sequence using PCR-based overlap extension mutagenesis (Ho et al., 1989) utilizing primers containing nonsense sequence between 75 and 50. The mutagenized promoters were placed upstream of GUS in the pBSK101.3 vector into the PstI and BamHI sites.

Promoter/GUS fusions were moved into a modified form of the plant-transformation vector pPZP100 (Hajdukiewicz et al., 1994) called pBL. The pBL vector contains a 35S-Bar cassette conferring basta resistance to transformed plants (Bechtold et al., 1993), which is oriented toward the right border. A reference construct containing a copy of the Arabidopsis 1.3-kb Lhcb1*3 promoter fused to the luciferase reporter is present 3 of the left border, and was designed for use in future transcription assays. Experimental constructs containing pea Lhcb1*4 promoter/5-UTR deletions fused to GUS were introduced into unique PstI and XhoI sites in an opposite orientation to and upstream of the 35S::Bar cassette.

Transformation

Treatment and Analysis

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Fluence response, time course, and reciprocity experiments were performed on transgenic lines containing the 281 to +64 (with 5-UTR) promoter, the 281 to +2 promoter (without 5-UTR), and the 150 to +64 promoter (with 5-UTR). Fluence-response experiments were performed by treating 6-d-old dark-grown plants with a single pulse of BL at a fluence ranging from 10¹ to 10⁴ µmol m², or with a mock pulse. In these experiments tissue was harvested 2 h after termination of the pulse.

Time-course experiments consisted of treating 6-d-old dark-grown plants with a single pulse of BL with a total fluence of 10² µmol m². The plants were then harvested at 5, 15, 30, 60, and 120 min after the pulse.

Reciprocity experiments involved treating 6-d-old dark-grown seedlings with a single pulse of BL at 10² µmol m² delivered in 5, 20, 100, or 1110 s. Plants were harvested 2 h after the completion of the BL pulse.

The fha1 and nph1 mutants were irradiated with either 10² µmol m² or a mock pulse. Plant tissue was harvested 2 h after the completion of the irradiation.

RNA was extracted and used for northern blotting, as previously described (Tilghman et al., 1997), or for slot blotting for quantitative analysis. Transcript accumulation was quantified on a phosphor imager (Molecular Dynamics, Sunnyvale, CA).

	RESULTS

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REP7550 Response to BL and Phytochrome Induction

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View larger version (47K): [in this window] [in a new window]   Figure 1. The region from 75 to 50 of the PsLhcb1*4 gene is not necessary for phytochrome or BLF regulation. Six-day-old dark-grown transgenic Arabidopsis seedlings containing the mutagenized pea Lhcb1*4 promoter construct REP7550, in which the sequence between 75 and 50 was replaced by a nonsense sequence fused to GUS, were irradiated with a single pulse of either BL or red light (RL) with a total fluence of 102 µmol m2 or a mock pulse (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed simultaneously for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively.

The substitution of the 75 to 50 region failed to eliminate the response to BL, indicating that the sequences necessary for the PsLhcb1*4 gene to respond to excitation of the BLF system are not within this region. A series of constructs with truncated upstream regions was created to help define upstream sequences necessary for the BLF response. PsLhcb1*4 promoter constructs were made extending from 281, 250, 200, 150, and 50 on the upstream end to +64 (where +1 represents the site of transcription initiation and +1 to +64 represents the 5-UTR) on the downstream end.

Although we tried on several occasions, we were unable to obtain transgenic lines containing a promoter construct extending from 100 on the upstream end to +64 on the downstream end. In each case we were able to obtain basta-resistant, transformed seedlings; however, the region between 100 and +64 had undergone a rearrangement. This rearrangement was not present in the Agrobacterium tumefaciens strain used to effect the transformation. As a consequence, we eliminated the 5-UTR (+2 to +64) from the construct. The integrated construct, extending from 100 to +2, did not exhibit these rearrangements.

Constructs containing the PsLhcb1*4 upstream sequence extending to 281, 250, 200, and 150 were all capable of responding to low-fluence BL (Fig. 2). The region from 50 to +64 was not capable of initiating a BLF response. Constructs representing the upstream region from 281 or 100 to +2 (i.e. without the 5-UTR) were also capable of responding to a BL pulse of 10⁴ µmol m² (Fig. 3).

View larger version (51K): [in this window] [in a new window]   Figure 2. Northern analysis of GUS mRNA accumulation from truncated Lhcb1*4 promoters. Six-day-old dark-grown transgenic Arabidopsis seedlings containing truncated pea Lhcb1*4 promoter constructs fused to GUS were irradiated with a single pulse of BL with a total fluence of 104 µmol m2 or a mock pulse (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed simultaneously for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The pea Lhcb1*4 promoters (as indicated on the figure) extend from +65 on the downstream end (where +1 represents the site of transcriptional initiation and +65 represents the A from the ATG) to 281, 200, 150, 50, or 1 on the upstream end.

View larger version (63K): [in this window] [in a new window]   Figure 3. Steady-state levels of GUS and Lhcb RNA in response to 104 µmol m2 BL from constructs lacking the Lhcb 5-UTR. Six-day-old dark-grown transgenic Arabidopsis seedlings containing truncated pea Lhcb1*4 promoter constructs fused to GUS were irradiated with a single pulse of BL with a total fluence of 104 µmol m2 or a mock pulse (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed simultaneously for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The pea Lhcb1*4 promoters (as indicated on the figure) extend from +2 on the downstream end (where +1 represents the site of transcriptional) to either 281 or 100 on the upstream end.

The relative promoter strength of these constructs was measured by comparing the induction of the transgene to the induction of the endogenous BL-responsive AtLhcb gene in the same plant line following BL treatment (Fig. 4). The constructs extending from 281, 250, and 200 exhibited a response to 10⁴ µmol m² BL that was identical to that of the endogenous Lhcb1 promoter. The construct extending to 150 directs BL expression at 43% of the level of the constructs containing sequence up to and beyond 200. Based on these data, it is likely that enhancer sequences lie in this 150 to 200 region.

View larger version (24K): [in this window] [in a new window]   Figure 4. Relative induction of GUS RNA from Lhcb1*4 truncated promoters. Six-day-old dark-grown Arabidopsis seedlings from each of 10 independent transgenic lines for each truncated pea Lhcb1*4 promoter construct indicated on the figure, were grown separately and irradiated with a single pulse of BL with a total fluence of 104 µmol m2 or were given no light (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed simultaneously for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The resulting BL-induced change in GUS RNA in each line was compared with the induction of the endogenous Lhcb gene in that line. The ratio of the two inductions is presented on the figure. Error bars represent SE of the mean.

As noted above, sequences within the pea Lhcb1*4 5-UTR (+1 to +64) are not necessary for the BLF response (Fig. 3). However, removal of this sequence did result in a decrease in the amplitude of the response (Fig. 4), indicating the presence of one or more enhancer elements in the PsLhcb1*4 5-UTR.

Photophysiological Responses

Figure 5A shows the steady-state levels of GUS RNA detected 2 h after a single pulse of BL at various fluences. Data from three independent experiments are presented in Figure 5B. These data illustrate that the promoter deletions to 281 and 150 respond with the same fluence-response characteristics as the endogenous Arabidopsis Lhcb gene, and that the presence or absence of the 5-UTR does not alter the threshold of the response.

View larger version (40K): [in this window] [in a new window]   Figure 5. Fluence response characteristics of transgenic constructs containing the 281 to +2, 281 to +64, and 150 to +64 versions of the pea Lhcb1*4 promoter. A, Six-day-old dark-grown transgenic Arabidopsis containing truncated pea Lhcb1*4 promoter constructs fused to GUS were irradiated with a single pulse of BL with a total fluence of 104 µmol m2 or were given no light (D). RNA was prepared 2 h after the light treatment and analyzed by slot blotting. The resulting blots were probed for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The pea Lhcb1*4 promoters (as indicated on the figure) extend from either 281 or 150 on the upstream end to either +65 (+5-UTR, where +1 represents the site of transcriptional initiation, +1 to+64 represents the pea Lhcb1*4 5-UTR and +65 is the A of the ATG start codon) or +2 (5-UTR) on the downstream end. B, Compilation of three independent experiments. All data points were plotted relative to dark levels (set to 1%) and 104 µmol m2 BL levels (set to 100%) and represent the constructs 281 with a 5-UTR (white bars), 281 without a 5-UTR (light gray bars), 150 with a 5-UTR (dark gray bars), and the endogenous Lhcb gene (black bars). All signals were normalized to rRNA. Error bars indicate SE of the mean.

The time course of GUS RNA accumulation from the PsLhcb1*4 promoter deletion constructs was measured following a single 10² µmol m² pulse of BL. Transcript was measured at 5, 15, 30, 60, and 120 min following the pulse. Figure 6A shows the response for individual lines tested and Figure 6B shows the compilation of data from three independent experiments. RNA from the 281 promoter constructs began to accumulate between 30 min and 1 h after the pulse, coinciding with the response of the endogenous Lhcb1 genes. This was true regardless of the presence or absence of the 5-UTR. The 150 deletion became detectable between 1 and 2 h. This apparent late onset was more likely due to the inability to detect the minimal increases from this low-level promoter at 30 min rather than to the lack of a BLF response. Again, the relative time course of the response was similar to that observed with the full-length and endogenous promoters, suggesting that these deletions were responding correctly to stimulation of the BLF system.

View larger version (30K): [in this window] [in a new window]   Figure 6. Time course of accumulation of transcript derived from transgenic constructs containing the 281 to +2, 281 to +64, and 150 to +64 versions of the pea Lhcb1*4 promoter in response to 102 µmol m2 BL. A, Six-day-old dark-grown transgenic Arabidopsis containing truncated pea Lhcb1*4 promoter constructs fused to GUS, were irradiated with a single pulse of BL with a total fluence of 102 µmol m2. RNA was prepared 0, 5, 15, 30, 60, and 120 min after the light treatment and analyzed by slot blotting. The resulting blots were probed for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The pea Lhcb1*4 promoters (as indicated on the figure) extend from either 281 or 150 on the upstream end to either +65 (+5-UTR, where +1 represents the site of transcriptional initiation, +1 to +64 represents the pea Lhcb1*4 5-UTR, and +65 is the A of the ATG start codon) or +2 (5-UTR) on the downstream end. B, Composite of three independent experiments. Pea Lhcb1*4 promoter constructs extend from 281 with the 5-UTR (), 281 without the 5-UTR (), and 150 with the 5-UTR () were tested and compared with the induction of the endogenous Lhcb genes (). All data points were plotted relative to dark levels (set to 1) and all signals were normalized to rRNA. Error bars indicate SE of the mean.

To determine whether the induction from the three representative promoters obeys reciprocity, steady-state levels of GUS RNA were measured following treatment with a single pulse of BL with a total fluence of 10² µmol m² delivered over various time intervals ranging from 5 to 1110 s. All three promoters respond by producing RNA levels that were very similar, regardless the length of the irradiation (Fig. 7).

View larger version (30K): [in this window] [in a new window]   Figure 7. Reciprocity characteristics for accumulation of transcript derived from transgenic constructs containing the 281 to +2, 281 to +64, and 150 to +64 versions of the pea Lhcb1*4 promoter in response to 102 µmol m2 BL. Six-day-old dark-grown transgenic Arabidopsis containing truncated pea Lhcb1*4 promoter constructs fused to GUS were irradiated with a single pulse of BL with a total fluence of 102 µmol m2 delivered in 5 s (white bars), 20 s (light gray bars), 100 s (dark gray bars), or 1110 s (black bars). RNA was prepared 2 h after the light treatment and analyzed by slot blotting. The resulting blots were probed for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively. The pea Lhcb1*4 promoters (as indicated on the figure) extend from either 281 or 150 on the upstream end to either +65 (+5-UTR, where +1 represents the site of transcriptional initiation, +1 to+64 represents the pea Lhcb1*4 5-UTR, and +65 is the A of the ATG start codon) or +2 (5-UTR) on the downstream end. The results are the average of three independent experiments. All signals were normalized to rRNA. The error bars represent the SE of the mean.

Deletion to 95

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View larger version (82K): [in this window] [in a new window]   Figure 8. BL-induced accumulation of RNA derived from transgenic constructs containing the truncated Lhcb1*4 promoter 95 to +2. Six-day-old dark-grown transgenic Arabidopsis seedlings containing a truncated pea Lhcb1*4 promoter extending from 95 on the upstream end to +2 on the downstream end (where +1 represents the site of transcriptional initiation) fused to GUS were irradiated with a single pulse of BL with a total fluence of 102 µmol m2 or 104 µmol m2 or were given no light (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed simultaneously for GUS and Lhcb RNA, representing expression of the transgene and the endogenous Arabidopsis Lhcb, respectively.

Nph1 and Fha1 Mutants

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View larger version (67K): [in this window] [in a new window]   Figure 9. Induction of endogenous Lhcb RNA in response to BL in recently characterized BL photoreceptor mutants. Six-day-old etiolated seedlings derived from the Arabidopsis mutants nph1 and fha1 were irradiated with 102 µmol m2 or 104 µmol m2 BL or were given no light (D). RNA was prepared 2 h after the light treatment and analyzed by northern blotting. The resulting blots were probed for Lhcb RNA.

	DISCUSSION

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In pea and Arabidopsis the transcription rate of Lhcb1 genes increases in response to a single pulse of BLF (Marrs and Kaufman, 1989; Anderson et al., in review). We previously determined that the BL induction directed by the full-length 1.3-kb pea Lhcb1*4 promoter is normal in transgenic Arabidopsis and occurs independent of phytochrome excitation (Tilghman et al., 1997). Data presented herein indicate that all of the sequences required for a normal BLF response, as characterized by the typical fluence response, time course, and reciprocity, are present within 150 bp of the site of transcription initiation (Figs. 1, 4, and 5). The 5-UTR is not necessary for the response.

The data presented in Figure 8 indicate that the region between 95 and +2 is also capable of responding to BLF, as transcript accumulated following a single BLF pulse, albeit at 36% of the level of the 281 promoter. These data demonstrate that the CCA1-binding sequence is not necessary for the BLF response. Previous studies have illustrated that the CCA1-binding sequence is required for the response to phytochrome (Wang et al., 1997) and the circadian clock (Wang and Tobin, 1998). The data presented in this study demonstrate that this sequence, which is pivotal in other light responses, is not necessary for the BLF response.

The 95 to +2 region contains several highly conserved elements that have been implicated previously in light responses (Donald and Cashmore, 1990). One of these "light-regulatory elements" is represented by a double-GATA sequence (I box), which is present in many light-inducible promoters (Arguello-Astorga and Herrera-Estrella, 1996) including both BL-regulated pea and Arabidopsis Lhcb promoters. Donald and Cashmore (1990) illustrated that this motif is important for expression from the Arabidopsis rbcS-1A promoter. In the context of the full rbcS 1.7-kb promoter, both I boxes were mutagenized, resulting in a >90% reduction of promoter activity (Donald and Cashmore, 1990). Kehoe et al. (1994) demonstrated that this sequence is required for phytochrome regulation of the cab2 promoter from Lemna gibba. The GATA motif has been described as a target of protein-binding activities in response to light regulation through the circadian clock and phytochrome (Anderson et al., 1994). This region also contains a conserved G-box-like element that has been implicated in light responses (Arguello-Astorga and Herrera-Estrella, 1996). The REP7550 construct, which replaces sequence in the pea Lhcb1*4 promoter between 50 and 75, maintains BL activity, indicating that this region is not necessary for BL activity in the etiolated seedling.

Taken together, deletion analysis and light-regulatory element mutagenesis indicate that sequences necessary for BL induction lie in a 20-bp region between 75 and 95 of the PsLhcb1*4 gene. Comparison of the BL-regulated PsLhcb1 and AtLhcb1 genes reveals identity in sequences at and immediately upstream of the CCAAT sequence. The sequence element CCAAT is highly conserved among light-regulated promoters, and is present in the 100 to 80 region in the BL-responsive Lhcb1 promoters in Arabidopsis, pea, and a number of other species. This element is present in the same position relative to the TATA sequence and is preceded by the sequence ACT in both BL-regulated promoters. The ACT/CCAAT sequence is not present in Lhcb1 genes that do not respond to a single pulse of BLF (Fig. 10). It is possible that the BL response is orchestrated through binding of factors and/or activation of factors into this specific ACT/CCAAT region.

View larger version (20K): [in this window] [in a new window]   Figure 10. Comparison of the sequence in the 100 to 50 region of the BL-regulated and non-BL-regulated Lhcb promoters from pea and Arabidopsis. The CCA-1 (Wang and Tobin, 1998), I-box (Donald and Cashmore, 1990), and CCAAT-box domains are indicated on the figure with boxes. Sequence identity to the Arabidopsis Lhcb1*3 promoter are indicated by bold print.

The CCAAT sequence is found in many eukaryotic promoters and is recognized by a set of proteins that can activate transcription. It was recently demonstrated that Arabidopsis contains several homologs to the yeast hap gene family of CCAAT-binding proteins (Edwards et al., 1998). Whereas yeast and vertebrates contain only a single variant of each HAP protein, Arabidopsis contains several different variants of each class. Additionally, while the CCAAT-binding proteins are ubiquitously and constitutively expressed in yeast and vertebrates, the members of the Arabidopsis HAP3 family are expressed in a regulated fashion. Circadian-regulated binding to the CCAAT sequence in the Lhcb1*1 promoter has been described previously (Carre and Kay, 1995), and although this gene contains the CCAAT sequence, it is not BL regulated.

In addition to the enhancer sequences upstream of the site of initiation, enhancers have been identified in the 5-UTR of many genes. The enhancer motif described as a CT box is present in the 5-UTR of several plant nuclear genes, including the PsaF and PetH genes in spinach (Bolle et al., 1994) and the HMG2 gene in tomato (Daraselia et al., 1996). The Lhcb1*4 promoters from 281, 100, and 95, which lack the 5-UTR, still respond to a pulse of BL. However, accumulation of transcript was only 67% of that where the 5-UTR is present. The 5-UTR of the PsLhcb1*4 gene does not contain the CT-box sequence.

BLF-mediated Lhcb transcript accumulation is normal in the BL perception mutants nph1 and fha1 (cry2). Both mutants accumulate Lhcb transcript in response to a pulse of BLF, indicating that neither the product of NPH1 nor the product of FHA1 is necessary for the BLF response. It has been shown previously that the BLF response of Lhcb genes is normal in hy4 mutants (Gao and Kaufman, 1994). These results suggest either the presence of an additional BL receptor or receptors that modulate Lhcb expression, or that Lhcb expression may be activated from multiple, redundant photoperception systems. Recent studies in the cry1/cry2 double mutant indicate that the double mutant may have more ranging effects on light-induced phenomena (i.e. suppression of hypocotyl elongation) than either single mutation alone (Ahmad et al., 1998). This synergism between two CRY photoreceptors, and possibly interaction between the CRY and NPH photoreceptors, may have an influence on Lhcb transcript accumulation, and these possibilities will be tested.

This study illustrates that the PsLhcb1*4 promoter relies on the sequence within 95 of the site of transcriptional initiation to initiate the BLF response, which is not dependent upon the double-GATA (I box) motifs common to many light-regulated promoters. Additionally, it is now clear that the BLF response is triggered by activation of transcription within this 95 region and utilizes enhancer sequences upstream of 95 and sequences in the 5-UTR for high-level expression. The most important aspect of this study illustrates that many of the previously characterized motifs recognized as playing a functional role in phytochrome and/or circadian responses are not required for the BLF response. Ongoing studies will identify the minimal sequences sufficient for inducing the basal BLF response from those now identified in the limited 75 to 95 region of the PsLhcb1*4 promoter.

	FOOTNOTES

   Received December 14, 1998; accepted April 5, 1999.

	ABBREVIATIONS

Abbreviations: BL, blue light. BLF, blue low-fluence light. UTR, untranslated region.

	Acknowledgments

The authors gratefully thank Mary Beth Anderson, John Marsh III, Yevgenia Lapik, and Zhaoming Wang for their useful discussion and critical reading of this manuscript. We also acknowledge Dr. Joseph Kieber and Dr. Keith Woeste for their assistance in the cultivation and transformation of Arabidopsis.

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