Blue Light–Directed Destabilization of the Pea Lhcb1*4 Transcript Depends on Sequences within the 5' Untranslated Region

Correspondence to: Lon S. Kaufman, lkaufman{at}uic.edu (E-mail), 312-413-2691 (fax)

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Pea seedlings grown in continuous red light accumulate significant levels of Lhcb1 RNA. When treated with a single pulse of blue light with a total fluence >10⁴ µmol m^-2, the rate of Lhcb1 transcription is increased, whereas the level of Lhcb1 RNA is unchanged from that in control seedlings. This RNA destabilization response occurs in developing leaves but not in the apical bud. The data presented here indicate that the same response occurs in the cotyledons of etiolated Arabidopsis seedlings. The blue light–induced destabilization response persists in long hypocotyl hy4 and phytochrome phyA, phyB, and hy1 mutants as well as in far-red light–grown seedlings, indicating that neither CRY1 (encoded by the hy4 locus) nor phytochrome is the sole photoreceptor. Studies with transgenic plants indicate that the destabilization element in the pea Lhcb1*4 transcript resides completely in the 5' untranslated region.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

For normal growth and development, all multicellular organisms rely on the correct temporal and spatial control of gene expression in response to changes in the intracellular, extracellular, and extraorganismal environments. Plants, at once sessile and developmentally indeterminate, are unique in that each change in gene expression must account for long-range developmental planning and short-term environmental responses. As such, plants use a myriad of mechanisms to achieve proper gene expression, including control of RNA stability (Gallie 1993 ; Sachs 1993 ; Abler and Green 1996 ; Barkan and Stern 1998 ; Petracek et al. 1998a ).

RNA sequences responsible for destabilization can reside anywhere within the transcript, although most of the recognized destabilization sequences reside within the 3' untranslated region (UTR). The SAUR (for small auxin up RNA) genes in soybean, mung bean, pea, and Arabidopsis provide good examples of plant genes whose primary means of regulation is defined by a post-transcriptional mechanism (McClure and Guilfoyle 1989 ; Franco et al. 1990 ; Gil et al. 1994 ). In these cases, the regulation of transcript stability is mediated by a 40-base region in the 3' UTR referred to as the downstream (DST) element. Specific sequences/bases within this region appear to be responsible for directing transcript destruction (Newman et al. 1993 ; Abler and Green 1996 ).

Stability elements have been reported to occur within the 5' UTR of several chloroplast-encoded transcripts, and in the case of the Chlamydomonas psbD transcript, significant progress has been made toward identifying the mechanism responsible (Nickelsen et al. 1999 ). Stability elements have also been reported in the 5' UTR of several nuclear-encoded genes, although no clear mechanism for altering stability has been defined. These include the 5' UTR of the wheat Em1 gene, which was identified as a destabilization element during a study of the promoter of that gene (Williamson and Quatrano 1988 ; Marcotte et al. 1989 ).

Light-mediated changes in transcript stability are known to occur for the ferredoxin-1 (Fed-1) mRNA in pea and Arabidopsis (Elliot et al. 1989 ; Dickey et al. 1992 , Dickey et al. 1994 ), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) transcripts in petunia (Thompson and Meagher 1990 ), and the light-harvesting chlorophyll binding (Lhcb) transcripts in pea (Warpeha and Kaufman 1990b ; Marrs and Kaufman 1991 ; Kaufman 1993 ). The best characterized of these, Fed-1 RNA, is stabilized upon the excitation of phytochrome (Dickey et al. 1992 ). Phytochrome-mediated stabilization also requires active translation (Dickey et al. 1994 ), sequences within the first 47 codons of the mRNA (Dickey et al. 1994 ), a CAUU sequence in the 5' UTR (Dickey et al. 1998 ), and active photosynthetic electron transport (Petracek et al. 1998b ).

Members of our laboratory are currently investigating two blue (B) light–mediated responses, the first of which demonstrates transcriptional regulation. Previously, we have shown that peas grown in continuous dim red (R) light or total darkness and subsequently exposed to a single short pulse of B light exhibit an increase in the rate of transcription of specific members of the Lhcb gene family, including the pea Lhcb1*4 gene (Marrs and Kaufman 1989 ; Warpeha et al. 1989 ; White et al. 1995 ). The increased rate of transcription initiates upon irradiation and does not require protein synthesis. This low-fluence response has a threshold to B light at 10^-1 µmol m^-2 (1 sec of full moonlight). At low fluences of B light, increases in fluence result in an increased rate of transcription and the concomitant accumulation of Lhcb RNA.

More recently, we demonstrated that a similar low-fluence B light system operates in Arabidopsis, wherein the Arabidopsis Lhcb1*3 gene is B light regulated (Gao and Kaufman 1994 ). Furthermore, transgenic constructs containing the pea Lhcb1*4 promoter in an Arabidopsis background are subject to regulation by the same B light signals (Tilghman et al. 1997 ). The response in Arabidopsis is not generated by activation of the cryptochrome (CRY1 or CRY2) or nonphototropic hypocotyl (NPH1) receptors (Gao and Kaufman 1994 ; Folta and Kaufman 1999 ).

There is also evidence for a second B light–sensing system in pea, a high-fluence B light system responsible for destabilization of the Lhcb transcript. Six-day-old pea seedlings grown in continuous dim R light and irradiated with a single short pulse of B light with a fluence >10³ µmol m^-2 (1 sec of full sunlight) fail to accumulate Lhcb1*4 transcripts, even though the low-fluence B light system continues to operate and the rate of transcription remains elevated (Marrs and Kaufman 1989 , Marrs and Kaufman 1991 ; Warpeha et al. 1989 ).

Subsequent work has identified that the Lhcb transcript destabilization occurs only in the developing leaves of the dim R light–grown peas and not in the apical bud tissue of either dim R light– or dark-grown seedlings (Warpeha and Kaufman 1990b ). The necessity for the dim R light was shown not to be a function of coexcitation with phytochrome but rather to allow the leaves to reach a specific developmental state in which they were competent to respond to the pulse of high-fluence B light.

In this study, we demonstrate that the high-fluence B light response exists in Arabidopsis and does not function by excitation of the CRY1 receptor. Furthermore, we show that transcripts derived from the pea Lhcb1*4 transgene are also subject to regulation by the high-fluence B light system and that the destabilization element resides in the 5' UTR. However, unlike in pea, the response in Arabidopsis is apparent in dark-grown seedlings. Results from transmission electron microscopy analyses are presented to indicate that the developmental state of the cotyledons of dark-grown Arabidopsis more closely resembles that of the developing leaves of dim R light–grown peas than it does the apical buds of either dim R light– or dark-grown peas. A block in translation can mimic the light-induced destabilization, although activation of the high-fluence B light system itself does not result in a global block in translation.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Dark-Grown Arabidopsis Exhibits a High-Fluence B Light Response
We previously reported that treatment of dark- or R light–grown peas with a single pulse of B light with a total fluence >10^-1 µmol m^-2 has multiple effects, including induction of the pea Lhcb1*4 gene (Marrs and Kaufman 1989 ; Warpeha et al. 1989 ; White et al. 1995 ). This response is termed the low-fluence B light response, and the signaling mechanisms are referred to as the low-fluence B light system. The same response occurs in dark-grown Arabidopsis, wherein both the Arabidopsis Lhcb1*3 gene and a transgene containing the promoter region of the pea Lhcb1*4 gene respond to excitation of the low-fluence B light system (Gao and Kaufman 1994 ; Tilghman et al. 1997 ).

Separately, a second response was identified that occurs in R light– but not in dark-grown pea (Warpeha and Kaufman 1990b ). The response, which follows treatment with fluences of B light >10³ µmol m^-2, is referred to as the high-fluence B light response and is presumed to be generated by a high-fluence B light system. In this case, it was demonstrated that excitation of the high-fluence B light system induces destabilization of the Lhcb transcript. Thus, even though the rate of transcription in response to high-fluence B light is elevated due to excitation of the low-fluence B light system, Lhcb transcript does not accumulate above control levels.

Owing to the precocious nature of cotyledon development in dark-grown Arabidopsis, it is possible that a high-fluence B light response may be apparent in etiolated seedlings. To test for this possibility, we treated 6-day-old dark-grown Arabidopsis seedlings with a single short pulse of B light with a total fluence ranging from 10¹ to 10⁵ µmol m^-2, and measured the steady state levels of transcript derived from the endogenous Arabidopsis Lhcb genes and a transgenic construct containing the pea Lhcb1*4 promoter and 5' UTR.

The results shown in Figure 1A and Figure 1B demonstrate that dark-grown Arabidopsis seedlings, like R light–grown pea seedlings, exhibit a lower steady state level of endogenous Lhcb transcript in response to treatment with high-fluence B light (10⁵ µmol m^-2) compared with low-fluence B light (10⁴ µmol m^-2). The high-fluence B light response was also observed for transcript derived from the transgenic pea Lhcb1*4::ß-glucuronidase (GUS) construct, whereas steady state levels of 35S::GUS transcript were unchanged by B light treatment. Thus, etiolated Arabidopsis seedlings exhibit a high-fluence B light response. It is unclear from these data whether the mechanism is the same as that observed in the R light–grown peas and why dark-grown peas are not competent to respond to treatment with high-fluence B light.

View larger version (27K): [in this window] [in a new window]   Figure 1. Etiolated Transgenic Arabidopsis Seedlings Demonstrate the High-Fluence B Light Response. RNA gel blot analysis of 6-day-old dark-grown transgenic Arabidopsis seedlings containing either the pea (Ps) Lhcb 1*4::GUS or the 35S::GUS transgene. Seedlings were irradiated with a single pulse of B light with a total fluence ranging from 101 to 105 µmol m-2 as indicated and harvested 2 hr after the light treatment. Control seedlings received a mock irradiation (D). Total RNA (10 µg) was probed simultaneously for GUS and the endogenous Arabidopsis (At) Lhcb transcripts. (A) One experimental replicate showing transcript derived from the pea Lhcb1*4::GUS transgene (top) and the 35S::GUS transgene (bottom) as well as from the endogenous Arabidopsis Lhcb genes at each fluence. (B) Numerical results compiled from three independent experimental replicates of relative transcript levels at each fluence. The RNA level in each control sample (D) was set to 1. Error bars represent the standard error of the mean. Solid squares denote pea Lhcb1*4::GUS; open circles, Arabidopsis Lhcb; solid triangles, 35S::GUS.

The high-fluence B light response characteristics of several Arabidopsis mutants as well as transgenic Arabidopsis grown in continuous far-red (FR) light were measured, and the data are shown in Figure 2A and Figure 2B. The lower steady state level of transcript accumulation in response to high-fluence B light compared with low-fluence B light persisted in phyA, phyB, phyA phyB, and hy1 mutants as well as in the FR light–grown transgenic Arabidopsis seedlings containing the pea Lhcb1*4::GUS construct. The high-fluence B light response was also present in hy4, as shown in Figure 3A and Figure 3B, in which the fluence response curve is identical to that of wild-type seedlings, although the amplitude of the response was depressed threefold.

View larger version (43K): [in this window] [in a new window]   Figure 2. The High-Fluence B Light Response Persists in Mutant and FR Light–Grown Arabidopsis Seedlings. (A) RNA gel blot analysis of 6-day-old dark-grown Landsberg erecta (Ler) and phyA, phyB, phyA phyB, and hy1 Arabidopsis mutants. Seedlings were irradiated with a single pulse of low-fluence (LF; 104 µmol m-2) or high-fluence (HF; 105 µmol m-2) B light as indicated and harvested 2 hr after the light treatment. Total RNA (10 µg) was probed for the endogenous Lhcb transcript. (B) RNA gel blot analysis of 6-day-old FR light–grown transgenic Arabidopsis seedlings containing the pea (Ps) Lhcb1*4::GUS transgene. Seedlings were irradiated with a single pulse of low-fluence (LF; 104 µmol m-2) or high-fluence (HF; 105 µmol m-2) B light and harvested 2 hr after the light treatment. Total RNA (10 µg) was probed for both GUS and the endogenous Arabidopsis (At) Lhcb transcript.

View larger version (33K): [in this window] [in a new window]   Figure 3. hy4 Arabidopsis Seedlings Exhibit the High-Fluence B Light Response. RNA gel blot analysis of 6-day-old dark-grown hy4 Arabidopsis seedlings. Seedlings were irradiated with a single pulse of B light with a total fluence ranging from 101 to 105 µmol m-2 as indicated and harvested 2 hr after the light treatment. Control seedlings received a mock irradiation (D). Total RNA (10 µg) was probed for the endogenous Arabidopsis Lhcb transcript. (A) One experimental replicate showing the accumulation of Lhcb transcript in the hy4 mutant background at each fluence. (B) Numerical results averaged from three independent experimental replicates of relative transcript levels at each fluence in the hy4 mutant background (solid symbol). Open symbol, wild type (see Figure 1). The RNA level in each control sample (D) was set to 1. Error bars represent the standard error of the mean.

Three models can account for the high-fluence B light response described above: sequences in the 5' region of the gene, including the 5' upstream region and the 5' UTR, may cause repression of low-fluence B light system–mediated transcription, the 5' UTR may contain a destabilization sequence, or a combination of repression and destabilization may regulate transcript level.

The 5' Upstream Region and the 5' UTR of the Pea Lhcb1*4 Gene Do Not Contain a Repressor Element
To determine whether the 5' upstream region and/or the 5' UTR of the pea Lhcb1*4 gene contain a transcriptional repressor activated by excitation of the high-fluence B light system, we compared the rate of transcription of various promoter/5' UTR constructs in response to a single pulse of low-fluence B light, a single pulse of high-fluence B light, or a mock irradiation. The transgenic constructs consisted of either the pea Lhcb1*4 promoter (position -281 to +2, a region that directs a full and normal low-fluence B light system response in Arabidopsis [ Folta and Kaufman 1999 ]) with the pea Lhcb1*4 5' UTR fused to the GUS coding sequence or the full-length Arabidopsis Lhcb1*3 promoter (-1.3 kb to +1) with the Arabidopsis Lhcb1*3 5' UTR fused to the luciferase (LUC) coding sequence. Both fusions were introduced into the Arabidopsis genome on the same T-DNA fragment (see Methods) and as such are most likely present in the same location and at the same copy number.

The results shown in Figure 4 demonstrate that the rate of transcription for both transgenic constructs, as well as that of the endogenous Arabidopsis Lhcb1 genes, remains unchanged following low-fluence or high-fluence B light treatments. This is emphasized by the compilation of the results from three independent experiments. The ratio of low-fluence- to high-fluence-induced transcription for the pea Lhcb1*4 construct is 0.98 ± 0.11. The ratio for the endogenous Arabidopsis Lhcb1 gene family is 0.89 ± 0.20. Therefore, the high-fluence pulse of light does not activate a transcriptional repressor in either the 5' upstream region or the 5' UTR of either the pea Lhcb1*4 or the Arabidopsis Lhcb1*3 gene. Similar activity of the Arabidopsis Lhcb1*3 construct, located adjacent to and having the same copy number as the pea Lhcb1*4 constructs and the endogenous genes, indicates that the observed B light responses are not a function of copy number or site of integration. The immediate suggestion is that an mRNA destabilization element resides in the pea Lhcb1*4 5' UTR and that it is activated by high-fluence B light.

View larger version (100K): [in this window] [in a new window]   Figure 4. The 5' Upstream Region and 5' UTR of the Pea Lhcb1*4 Gene Do Not Contain a Transcriptional Repressor. Run-on transcription analysis of 6-day-old dark-grown transgenic Arabidopsis seedlings containing the pea (Ps) Lhcb1*4 (-281 to +1)::pea Lhcb1*4 5' UTR::GUS and the Arabidopsis (At) Lhcb1*3 (-1.3 kb to +1)::Arabidopsis Lhcb1*3 5' UTR::LUC transgenes present in the same copy number and location as the pea Lhcb1*4 construct. Seedlings were irradiated with a single pulse of low-fluence (LF; 104 µmol m-2) or high-fluence (HF; 105 µmol m-2) B light and harvested 2 hr after the light treatment. Control seedlings received a mock irradiation (D). Nuclei were tested for accumulation of RNA derived from the endogenous Arabidopsis Lhcb1 gene family, the GUS transgene, the LUC transgene, and the rDNA (loading control).

A Destabilization Signal Resides within the 5' UTR of the Pea Lhcb1*4 Transcript
To determine whether the 5' UTR of the pea Lhcb1*4 transcript contains a destabilization sequence, we compared the steady state levels of transcript derived from the pea Lhcb1*4::GUS transgene described above with transcript derived from a similar construct lacking the 5' UTR.

The results of treatment with low-fluence B light, high-fluence B light, or a mock irradiation are shown in Figure 5. As expected, the transcript containing the 5' UTR as well as the endogenous Arabidopsis Lhcb transcript showed lower accumulation in response to high-fluence B light than in response to low-fluence B light. In contrast, the transcript lacking the 5' UTR showed greater levels of accumulation in response to high-fluence B light than in response to low-fluence B light. We are in the process of creating a linker scanning series to identify sequences within the 5' UTR that are necessary for the destabilization event.

View larger version (73K): [in this window] [in a new window]   Figure 5. The 5' UTR of the Pea Lhcb1*4 Gene Contains a Destabilization Signal. RNA gel blot analysis of 6-day-old dark-grown transgenic Arabidopsis seedlings containing either the pea (Ps) Lhcb1*4 (-UTR)::GUS transgene or the pea Lhcb1*4 (+UTR)::GUS transgene. Seedlings were irradiated with a single pulse of low-fluence (LF; 104 µmol m-2) or high-fluence (HF; 105 µmol m-2) B light and harvested 2 hr after the light treatment. Control seedlings received a mock irradiation (D). Total RNA (10 µg) was probed simultaneously for GUS and the endogenous Arabidopsis (At) Lhcb transcript.

Kinetics of Transcript Accumulation in Response to Low-Fluence and High-Fluence B Light Treatment
To determine the time frame within which the destabilization event initiates, we measured the rate of accumulation of transcript derived from the endogenous Arabidopsis Lhcb gene family and the pea Lhcb1*4::GUS transgene in response to a single pulse of low-fluence B light (10⁴ µmol m^-2) or high-fluence B light (10⁵ µmol m^-2). The data presented in Figure 6B to 6D indicate that accumulation of both the endogenous Lhcb and transgenically derived transcripts initiated between 0.5 and 1 hr after treatment with low-fluence B light. The accumulation rate was linear for the remainder of the 2-hr assay period. These results are consistent with previously published observations of low-fluence B light response kinetics (Gao and Kaufman 1994 ; Tilghman et al. 1997 ).

View larger version (43K): [in this window] [in a new window]   Figure 6. Kinetics of Transcript Accumulation in Response to High-Fluence and Low-Fluence B Light. RNA gel blot analysis of 6-day-old dark-grown transgenic Arabidopsis seedlings harboring the pea (Ps) Lhcb1*4::GUS transgene. Seedlings were irradiated with a single pulse of either low-fluence (LF; 104 µmol m-2) or high-fluence (HF; 105 µmol m-2) B light and harvested 0, 0.5, 1, and 2 hr after the light treatment (as indicated above each lane). Control seedlings received a mock irradiation and were harvested 2 hr later (D). A low-fluence sample, harvested at 2 hr, was included as a control in the high-fluence time course and vice versa. Total RNA (10 µg) was probed simultaneously for GUS and the endogenous Arabidopsis (At) Lhcb transcript. (A) One experimental replicate showing the kinetics of transcript accumulation in response to high-fluence B light. (B) One experimental replicate showing the kinetics of transcript accumulation in response to low-fluence B light. (C) Numerical results averaged from three independent experimental replicates demonstrating the kinetics of endogenous Arabidopsis Lhcb transcript accumulation in response to low-fluence (open circles) and high-fluence (solid circles) B light. Error bars represent the standard error of the mean. (D) Numerical results averaged from three independent experimental replicates demonstrating the kinetics of accumulation of transcript derived from the pea Lhcb1*4::GUS transgene in response to low-fluence (open squares) and high-fluence (solid squares) B light. Error bars represent the standard error of the mean.

In contrast to low-fluence treatment, accumulation in response to high-fluence B light initiated immediately upon irradiation. The rate of accumulation, proceeding at a constant level over the 2-hr period, was approximately twofold slower than the response to low-fluence treatment (2.1- and 2.2-fold for the transgene and endogenous genes, respectively).

Similarity between the Cotyledons of Dark-Grown Arabidopsis and the Developing Leaves of R Light–Grown Peas
We have previously reported the presence of a high-fluence B light response in 6-day-old R light–grown pea (Warpeha and Kaufman 1990b ). This response is limited to the developing leaves of the third node and is not present in the apical bud of either R light– or dark-grown pea. Unlike pea, the high-fluence B light response in Arabidopsis is apparent in dark-grown seedlings. Examination of gross morphology (e.g., cell types and venal development) indicates that the cotyledons of dark-grown Arabidopsis resemble the developing leaves of the third node of R light–grown peas and are dissimilar to the embryonic tissue found in the apical buds of both dark- and R light–grown peas.

Transmission electron microscopy was used to determine similarities between tissues competent to exhibit the high-fluence B light response. The results shown in Figure 7C and Figure 7D indicate that the cotyledons of dark-grown Arabidopsis seedlings and the developing leaves of the third node of R light–grown peas both contain etioplasts, as determined by the presence of a prolamellar body (Hoober 1984 ). In contrast, electron microscopy of the apical buds of both dark- and R light–grown pea reveal the presence of proplastids (Figure 7A and Figure 7B).

View larger version (107K): [in this window] [in a new window]   Figure 7. Transmission Electron Micrographs of 6-Day-Old Dark-Grown Arabidopsis Seedlings and Dark- and Dim R Light–Grown Pea Tissue. Transmission electron microscopy of plastid structure in the following tissues. (A) Apical bud of 6-day-old dark-grown pea. (B) Apical bud of 6-day-old dim R light–grown pea. (C) Developing leaf of the third node of 6-day-old dim R light–grown pea. (D) Cotyledons of 6-day-old dark-grown Arabidopsis seedlings. Bars = 500 nm.

The Role of Translation in the 5' UTR–Mediated Destabilization
Blocking translation can induce many 5' UTR–mediated destabilization events. To test whether this is also the case for the high-fluence B light response, we treated 6-day-old dark-grown transgenic Arabidopsis seedlings with cycloheximide (CHX) followed by a low-fluence B light irradiation. The subsequent accumulation of the endogenous Arabidopsis Lhcb and the pea Lhcb1*4::GUS transcripts was measured. To confirm the action of CHX under these conditions, we also monitored the accumulation of the ACC2 transcript, which encodes 1-aminocyclopropane-1-carboxylate (ACC) synthase and which is known to accumulate in CHX-treated Arabidopsis (Liang et al. 1992 ).

The results shown in Figure 8 demonstrate that CHX treatment increased the levels of the ACC2 transcript (Figure 8B, two leftmost lanes) but caused the destabilization of transcripts derived from both the Arabidopsis Lhcb gene family and the pea Lhcb1*4::GUS transgene (Figure 8A, two leftmost lanes). Destabilization of transcript derived from the Arabidopsis Lhcb gene family also occurred in the hy4 background (data not shown).

View larger version (45K): [in this window] [in a new window]   Figure 8. Induction of the High-Fluence B Light System Does Not Result in a Global Block in Translation. (A) RNA gel blot analysis of 6-day-old dark-grown transgenic Arabidopsis seedlings harboring the pea (Ps) Lhcb1*4::GUS transgene. In the two leftmost lanes, seedlings were treated with CHX (+) or a mock solution (-) and, 1 hr later, irradiated with a pulse of low-fluence B light (102 µmol m-2). The remaining lanes represent a fluence response experiment performed as described in Figure 1. Seedlings received either a single pulse of B light with a total fluence ranging from 101 to 105 µmol m-2 or a mock irradiation (D). Tissue was harvested 2 hr after treatment, and total RNA (10 µg) was probed simultaneously for GUS and the endogenous Arabidopsis (At) Lhcb transcript. (B) The same filter described above was stripped and reprobed simultaneously for Arabidopsis ACC2 and the endogenous Arabidopsis Lhcb transcript.

Induction of the destabilization process by CHX introduces the possibility that the single short pulse of high-fluence B light acts to stop translation either globally or specifically of the transcript containing the pea Lhcb1*4 5' UTR destabilization sequence. To determine whether high-fluence B light causes a global block in translation, we measured the B light fluence response characteristics for the accumulation of ACC2 transcript. The data shown in Figure 8B demonstrate that ACC2 RNA does not accumulate in response to any of the fluences tested; therefore, excitation of the high-fluence B light system does not result in a global block in translation.

	DISCUSSION

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We have previously demonstrated that the rate of Lhcb transcription and consequently the steady state level of Lhcb RNA in etiolated Arabidopsis becomes elevated in response to a single short pulse of low-fluence B light (threshold at 10^-1 µmol m^-2) (Gao and Kaufman 1994 ; Tilghman et al. 1997 ). The signal transduction mechanism regulating this change in Lhcb gene expression is effectively identical to the B low-fluence system originally described for pea (Marrs and Kaufman 1989 , Marrs and Kaufman 1991 ; Warpeha et al. 1989 ; Warpeha and Kaufman 1990b ; Kaufman 1993 ). Separately, pea exhibits a high-fluence B light response, wherein a single short pulse of high-fluence B light (>10³ µmol m^-2) results in an elevated rate of Lhcb transcription (presumably by acting through the low-fluence B light system), with no change in the steady state level of Lhcb RNA (Warpeha and Kaufman 1990b ; Kaufman 1993 ). The implication is that excitation of the high-fluence B light system results in destabilization of the Lhcb RNA. The data presented here demonstrate that Lhcb RNA levels in etiolated Arabidopsis are also regulated by the high-fluence B light system and that the destabilization element resides within the 5' UTR of the pea Lhcb1*4 RNA.

The response persists in phyA, phyB, phyA phyB, and hy1 mutants of Arabidopsis as well as in seedlings grown in continuous FR light. The presence of the response in the phyA phyB double mutant indicates that neither PHYA, which has known sensitivity toward B light, nor PHYB acts as the sole receptor for this response. Growth in continuous FR light results in a photodynamic equilibrium for all phytochromes and should saturate most phytochrome responses. This is especially true for PHYA or type I phytochrome responses, due to the hypersensitivity of PHYA to FR light. Antithetically, the hy1 mutant, which lacks the phytochrome chromophore, should be lacking in most phytochrome responses. The persistence of the high-fluence B light response in both the hy1 mutant and FR light–grown seedlings argues strongly that the response is neither due to phytochrome excitation nor requires phytochrome participation.

It is known that levels of ACC2 transcript are elevated in response to stress (Liang et al. 1992 ). ACC2 transcript levels were not elevated by excitation of the high-fluence B light system, indicating that the high-fluence B light response is not stress induced. Activation of the high-fluence B light system in pea results in several events in addition to the destabilization of the pea Lhcb1*4 transcript (Warpeha et al. 1989 ; Warpeha and Kaufman 1990a , Warpeha and Kaufman 1990b ). These responses were originally identified in R light–grown peas and were shown to be absent from etiolated seedlings. Based on the observations that simple coirradiation (R light plus B light) of etiolated seedlings does not elicit a high-fluence B light response and that high-fluence B light system–induced destabilization of Lhcb RNA occurs in the developing leaves and not in the embryonic apical bud, it was concluded that growth in continuous R light is necessary to allow the pea seedlings to reach a specific developmental state in which they are competent to respond to the high-fluence B light treatment (Warpeha and Kaufman 1990b ).

The lack of a high-fluence B light response in etiolated peas contrasts strongly with the data presented here, wherein etiolated Arabidopsis clearly demonstrates competence to respond to excitation of the high-fluence B light system. The coincidence in competence between the cotyledons of etiolated Arabidopsis and the developing leaves of the third node of R light–grown peas suggests a congruence in the developmental state of these two organs. It is further implied that the apical bud of a 6-day-old etiolated or R light–grown pea seedling is in a more embryonic developmental state than are the cotyledons of a 6-day-old etiolated Arabidopsis seedling. Both of these conjectures are borne out by observation of the gross anatomy of the tissues involved and, more acutely, by the plastid status as demonstrated by the electron microscopy data presented here. It is therefore naive to assume that the developmental status of two different species of plants maintained in an etiolated state will be equivalent.

It is interesting to speculate on the general presence of a two-step developmental program exemplified by competence to respond to excitation of the low-fluence and high-fluence B light systems. In pea, the two systems appear to be activated sequentially, with the low-fluence B light system present in the etiolated state and the high-fluence B light system requiring further development. In Arabidopsis, both systems are present in cotyledons of 6-day-old etiolated seedlings. Brusslan and Tobin 1992 demonstrated that a change in phytochrome responsiveness occurred when comparing the cotyledons of etiolated Arabidopsis seedlings <4 days old with those of etiolated seedlings >4 days old. It would be of interest to determine if the high-fluence B light response is present in the <4-day-old seedlings.

Growth of Arabidopsis in continuous FR light is known to result in the destruction of the etioplast (Barnes et al. 1996 ). The data presented here indicate that the high-fluence B light system remains active under FR light growth conditions. Thus, even though the electron microscopy data indicate that the developmental status of the plastid correlates with the presence of high-fluence B light system activity, the relationship is only temporal and not causal.

The presence of a fully active high-fluence B light system in the cotyledons of etiolated Arabidopsis suggests that the entire signaling and response mechanisms are present before irradiation. However, the standard test for the necessity of additional gene expression, persistence of the response in the presence of CHX, is not possible in this case because the CHX treatment itself mimics high-fluence B light system activity.

The data presented here identify the 5' UTR of the pea Lhcb1*4 transcript as containing a sequence involved in the regulation of transcript stability. There are significantly fewer cases in which destabilization elements have been determined to reside within the 5' UTR of either animal or plant transcripts than there are in which such sequences have been determined to reside in the 3' UTR. Furthermore, there are no specific mechanisms in which destabilization mediated by the elements within the 5' UTR of nuclear-coded transcripts have been described. A general mechanism has been proposed for animal, and subsequently plant, transcripts, wherein the element either directly or indirectly results in a stall in translation, exposure of the RNA, and immediate digestion by an RNase activity (Green 1993 ; Sachs 1993 ; Decker and Parker 1994 ; Abler and Green 1996 ). The observation that CHX treatment results in destabilization of endogenous Arabidopsis Lhcb transcripts and the transgenically derived pea Lhcb1*4::GUS RNA in a manner comparable with that observed in response to excitation of the high-fluence B light system is consistent with the translation stall/exposure mechanism.

The 5' UTR of the pea Lhcb1*4 transcript does not contain any of the recognized destabilization elements associated with the 5' UTR of animal transcripts (Green 1993 ; Sachs 1993 ; Decker and Parker 1994 ; Abler and Green 1996 ). A comparison of the 5' UTR of the pea Lhcb1*4 with those of the Arabidopsis Lhcb1*3, Blec1, and pea Fed-1 transcripts, all known to undergo light-mediated changes in stability, reveals two sequences in common, the ARL element (Caspar and Quail 1993 ) as well as a CAUU sequence (Dickey et al. 1998 ). Their role in the destabilization event, if any, is not known.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Material
Transgenic lines of Arabidopsis thaliana ecotype Columbia containing the cauliflower mosaic virus 35S promoter::ß-glucuronidase (GUS) and Pisum sativum Lhcb1*4 promoter (-1246 to the ATG)::GUS (hereafter and in the text referred to as pea Lhcb1*4::GUS) constructs were previously described (Tilghman et al. 1997 ). Transgenic lines containing the pea Lhcb1*4 promoter (position -281 to +2), either with or without the 64-bp pea Lhcb1*4 5' untranslated region (UTR), were cloned into the BamHI site upstream of the GUS reporter gene, essentially as described by Tilghman et al. 1997 , except that a modified form of pPZP100 (Hajdukiewicz et al. 1994 ) was substituted for pBI101 and transformation was achieved using vacuum infiltration rather than root culture. Each construct also carries a transgene consisting of 1.3 kb of 5' upstream region and the 5' UTR of the Arabidopsis Lhcb1*3 gene fused to luciferase (LUC). A detailed description of the modified pPZP100 and the specific manner in which the transgenes were constructed will be presented elsewhere (Folta and Kaufman 1999 ).

The following Arabidopsis mutants were used: hy1-21.84N and hy4 (Koornneef et al. 1980 ), phyA-201, phyB-5, and phyA-201 phyB-5 (Reed et al. 1994 ), as well as the corresponding wild-type Landsberg erecta. All seed stocks were collected from plants maintained under fluorescent lights at 22°C and stored at 4°C under desiccation until use.

Seed stock of pea cultivar Alaska was obtained from Olds Seed Co. (Madison, WI).

Growth Conditions and Light Sources
For each experimental replicate, 100 µL of Arabidopsis seed was surface-sterilized in 60% commercial bleach/0.01% Triton X-100 for 5 min, rinsed three times with sterile water, and suspended in 4 mL of 1 x Murashige and Skoog top agar (Murashige and Skoog basal salt mixture, 0.5 mg mL^-1 Mes, and 0.8% low-melting-point agarose, pH 5.8). The suspension was quickly poured onto Phytatrays (Sigma) containing 1 x Murashige and Skoog agar (the same medium as above, except that commercial agarose replaced the low-melting-point agarose). Sterilization and planting were performed under a green safelight. After planting, seeds were irradiated with 16 µmol m^-2 sec^-1 of fluorescent white light for 10 min and transferred to absolute darkness. Trays containing hy1, phyA, phyB, phyA phyB, and wild-type Landsberg erecta were stored in the dark at 4°C for 4 days before the short dim white light treatment and subsequent transfer to darkness. Seedlings grown in continuous far-red (FR) light were placed in darkness for 1 day before transfer to growth in FR light at a fluence of ~1.0 W m^-2 (Barnes et al. 1996 ). Dim red (R) light growth was as described by Warpeha et al. 1989 .

Experimental light treatments were administered after 6 days of growth in absolute dark (or FR light) at 21°C. Plants were returned to the dark (or FR light) immediately after treatments until harvest. Control seedlings received the same treatment as irradiated seedlings except that the light source remained turned off (mock irradiation). The blue (B) light source used was identical to that described by Warpeha et al. 1989 .

Fluence Response Experiments
Six-day-old dark-grown Arabidopsis seedlings were irradiated with a total fluence of 10¹, 10², 10³, 10⁴, or 10⁵ µmol m^-2 of B light. (Times of irradiation were 20, 20, 50, 167, or 1110 sec, respectively.) Treated and control seedlings were harvested 2 hr after irradiation under safelight conditions and quick frozen in liquid N₂ for RNA extraction.

Time-Course Experiments
Six-day-old dark-grown transgenic Arabidopsis seedlings were harvested 0, 0.5, 1, and 2 hr after irradiation with 10⁴ µmol m^-2 of B light (low-fluence treatment) or 10⁵ µmol m^-2 of B light (high-fluence treatment).Unirradiated seedlings were harvested at 2 hr as controls. A 10⁴ low-fluence sample, harvested at 2 hr, was included as a control in the 10⁵ high-fluence time course and vice versa. Seedlings were quick frozen for use in RNA extraction.

Cycloheximide Treatments
Six-day-old dark-grown Arabidopsis seedlings were sprayed with 0.5 mL of 20 µg mL^-1 cycloheximide (CHX; Sigma) in 0.1% Tween or a mock solution (0.1% Tween only). One hour after spraying, the seedlings were irradiated with 10² µmol m^-2 of B light and then harvested 1 hr later.

RNA Isolation and Gel Blot Analysis
Frozen tissue was ground to a fine powder in liquid N₂, and total RNA was extracted, electrophoresed, and transferred to GeneScreen (DuPont–New England Nuclear, Beverly, MA), as described previously (Gao and Kaufman 1994 ). Hybridization conditions were also as described by Gao and Kaufman 1994 .

Probes used for RNA gel blot analysis were Arabidopsis Lhcb (2-kb EcoRI-SstI fragment from the Lhcb1*3 genomic clone; Leutwiler et al. 1986 ), GUS (internal 1.8-kb NcoI-BamHI fragment), and 1-aminocyclopropane-1-carboxylate (ACC2; internal 900-bp BamHI fragment from the ACC2 cDNA clone; Liang et al. 1992 ). Transcript lengths of ~0.9 kb for Arabidopsis Lhcb, 1.8 kb for pea Lhcb promoter::GUS fusions, and 1.8 kb for ACC2 were detected.

The relative abundance of specific transcripts was determined by scanning the autoradiographs with an IS1000 digital imaging system (Alpha Inotech Corp., San Leandro, CA) and relating the values back to a standard curve. Loading errors were corrected using densitometry values of rRNA as determined from the ethidium bromide–stained gels.

Isolation of Nuclei and Run-on Transcription Assay Conditions
Six-day-old dark-grown Arabidopsis seedlings were harvested 2 hr after irradiation with 10⁴ µmol m^-2 of B light (low-fluence treatment) or 10⁵ µmol m^-2 of B light (high-fluence treatment) or a mock pulse of B light. Nuclei were harvested from the cotyledon/apical dome portions of the seedlings, essentially as described by Marrs and Kaufman 1989 , omitting the percol gradient step. Run-on transcription assays were performed as described by Marrs and Kaufman 1989 . A more detailed description of the method used to isolate transcriptionally active nuclei from etiolated Arabidopsis has been submitted elsewhere (K. Folta and L.S. Kaufman, manuscript submitted for publication).

Electron Microscopy
Apical tissues from 6-day-old dark- or dim R light–grown pea or Arabidopsis seedlings were fixed for 2 hr in the dark with 2% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 6.9, and then washed in the same buffer. Tissues were postfixed in 2% osmium tetroxide in 0.05 M sodium cacodylate buffer for 2 hr and then washed again in buffer without the osmium. Tissue was dehydrated by passage through a graded ethanol series consisting of 2-hr incubations in 10 25, 40, 50, 65, 85, 95, 100, 100, and 100% ethanol in cacodylate buffer. Tissue was infiltrated with Spurr's epoxy resin by sequential passage in 33, 50, 66, and 100% Spurr's resin in ethanol for 2 hr each, followed by two 12-hr incubations in 100% Spurr's resin.

Tissue was subsequently embedded in Spurr's epoxy resin and cured at 70°C overnight. Cured blocks were trimmed and sectioned on a microtome (Ultracut E; Reichert-Jung, Leica Microsystems, Deerfield, IL) with a diamond knife, and 90-nm sections were collected on 200-mesh copper grids. The grids were stained with uranyl acetate followed by lead citrate, and cells were photographed at 60 kV with a transmission electron microscope (model 1200EX; JEOL, Inc., Peabody, MA).

	ACKNOWLEDGMENTS

We thank Dr. Joanne Chory (Salk Institute, San Diego, CA) for the Arabidopsis mutants phyA-201, phyB-5, and phyA-201 phyB-5. We also thank John F. Marsh for critical reading of the manuscript. This work was supported by a grant from the United States Department of Agriculture (No. 9701418).

Received December 3, 1998; accepted June 16, 1999.

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