phytochrome-mediated light regulation of phya- and phyb-cus … · plant physiol. (1995) 107:...

Plant Physiol. (1995) 107: 523-534

Phytochrome-Mediated Light Regulation of PHYA- and PHYB-CUS Transgenes in Arabidopsis thaljana Seedlings’

David E. Somers’ and Peter H. Quail*

Department of Plant Biology, University of California, Berkeley, Berkeley, California 94720; and Plant Gene Expression Center, United States Department of Agriculture, Agricultura1 Research Service,

Albany, California 9471 O

Phytochrome wild-type gene-P-glucuronidase (PHY-GUS) gene fusions were used in transgenic Arabidopsis to compare the activity levels and light regulation of the PHYA and PHYB promoters and to identify the photoreceptors mediating this regulation. In dark- grown seedlings, both promoters are 4-fold more active in shoots than in roots, but the PHYA promoter is nearly 20-fold more active than that of PHYB in both organs. In shoots, white light represses the activities of the PHYA and PHYB promoters 10- and Z-fold, respectively, whereas in roots light has no effect on the PHYA promoter but increases PHYB promoter activity 2-fold. Conse- quently, PHYA promoter activity remains higher than that of PHYB in light in both shoots (5-fold) and roots (1 1-fold). Experiments with narrow-waveband light and photomorphogenic mutants suggest that no single photoreceptor i s necessary for full white-light-directed PHYA repression in shoots, but that multiple, independent photoreceptor pathways are sufficient alone or in combination. In contrast, phytochrome B appears both necessary and sufficient for a light-mediated decrease in PHYB activity in shoots, and phytochrome A mediates a far-red-light-stimulated increase in PHYB promoter activity. Together, the data indicate that the PHYA and PHYB genes are regulated in divergent fashion at the transcriptional level, both developmentally and by the spectral distribution of the prevailing light, and that this regulation may be important to the photosensory function of the two photoreceptors.

Photoregulation of gene expression in higher plants is an evolutionary consequence of light-dependent growth and development. The establishment and maintenance of energy reserves, through photosynthesis, requires the assem- bly of a highly complex light-harvesting and electron- transfer apparatus. It is not surprising, then, that transcript accumulation for many of the nuclear genes encoding chloroplast proteins is light inducible (Thompson and White, 1991). Two gene families in particular, ribulose-1,5- bisphosphate carboxylase genes and Chl a/b-binding pro-

l This work was supported by Department of Energy Office of Basic Energy Sciences grant No. DE-FG03-92ER13742, U.S. De- partment of Agriculture-National Research Initiative Competitive Grants l‘rogram grant No. 92-37301-7678, and U.S. Department of Agriculture/Agricultural Research Service Current Research In- formation Service grant No. 5335-21000-006-00D.

Present address: Department of Biology, Gilmer Hall, Univer- sity of Virginia, Charlottesville, VA 22901.

* Corresponding author; e-mail [email protected]; fax 1-510-559-5678.

tein genes, have been the primary subjects of many studies of light-regulated gene expression (Tobin and Silverthorne, 1985; Dean et al., 1989; Wanner and Gruissem, 1991; Batschauer et al., 1994).

In contrast, fewer genes have been identified whose expression is repressed by light. The Pchlide reductase, Asn synthase, and phyA genes are among those with known functions that show a marked reduction in transcript abundance when dark-grown plants are illuminated (Quail, 1991). Of these, the light-labile photoreceptor phyA is the best studied. In oat (Avena sativa) and rice (Oryza sativa) the reduced PHYA mRNA levels in red or far-red light largely result from a strong and rapid decrease in transcription (Lissemore and Quail, 1988; Kay et al., 19891, which is most likely regulated via the phyA signal transduction pathway (Quail, 1994). In pea (Pisum sativum), only one of the three PHYA transcripts produced is reversibly decreased by red light and increased by far-red light (Sato, 1988). Short periods of red light do not detectably reduce PHYA mRNA levels in tomato and Arabidopsis thaliana, although contin- u o u ~ white light is effective (Sharrock and Quail, 1989; Somers et al., 1991; Quail, 1994). These examples suggest that photoregulation of PHYA activity may differ between the graminacious monocots and at least some dicots.

In contrast, PHYB transcript accumulates in rice, potato (Solanum tuberosum), and Avabidopsis to similar levels, re- gardless of light treatment (Sharrock and Quail, 1989; De- hesh et al., 1991; Somers et al., 1991; Heyer and Gatz, 1992; Clack et al., 1994). This suggests a light-independent con- stitutive transcription of this gene in both monocots and dicots. Consistent with this idea, red-light treatment of dark-grown Arabidopsis seedlings has little effect on PHYB polypeptide levels (Somers et al., 1991). However, a de- tailed quantitation of mRNA and polypeptide gene products for this phytochrome in Arabidopsis is still lacking.

We wished to examine the expression levels and transcriptional photoregulation of PHYA and PHYB genes in Avabidopsis to address the following questions: (a) what are the relative activities of the PHYA and PHYB promoters in young seedlings and are there organ-specific differences in the expression levels of these two genes; (b) are the activities of the PHYA and PHYB promoters light regulated,

Abbreviations: PHY, phytochrome wild-type gene; phy, phytochrome mutant gene; PHY, phytochrome apoprotein; phy, phytochrome holoprotein.

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524 Somers and Quail Plant Physiol. Vol. 107, 1995

and, if so, to what degree; (c) which photoreceptors mediate any observed light effects; and (d) how do these findings relate to the distinct photophysiological roles that are emerging for these two phytochromes?

Our approach to these questions was to use PHY promoter-gusA (GUS) reporter gene fusions transformed into Arabidopsis. GUS enzyme assays have the advantage of being rapid, facile, and amenable to quantitation (Jefferson et al., 1987). Since small amounts of tissue can be sampled, a high degree of spatial resolution is possible, allowing quantitative intra-seedling and intra-organ assessments of promoter activity. A previous report of pea PHYA-GUS expression in petunia similarly exploited this advantage (Komeda et al., 1991). In addition, as reporters of promoter activity, PHY-GUS transgenes provide the means to separate the contribution of transcription from that of mRNA stability as the two main determinants of final phytochrome mRNA abundance.

Here we have examined the expression levels of PHYA- and PHYB-GUS reporter genes in seedlings under narrow- waveband light and in different photoreceptor-deficient genetic backgrounds to determine the effects of light on the activity of both phytochrome promoters in each organ, and to identify the photoreceptor(s) mediating such effects.

MATERIALS AND METHODS

lsolation of PHYA and PHYB Promoter Sequences

A probe made from the 5' region of the Arabidopsis thaliana PHYA cDNA (Sharrock and Quail, 1989) was used to identify a recombinant phage from a A ZapII (Stratagene, La Jolla, CAI Arabidapsis (Columbia) genomic library. A 6-kb plasmid insert was excised and comparison of nucleotide sequences from its 3' region to the 5'.end of the PHYA cDNA confirmed that this clone contained the upstream sequences of the PHYA gene. The complete nucleotide sequence of the Arabidopsis PHYA gene, including the regions used in the constructs of this study, has recently been reported (Dehesh et al., 1994).

An EMBL3 A recombinant phage from an Arabidopsis (Columbia) genomic library containing an 11.9-kb insert was identified using a probe from the coding region of the PHYB cDNA (Sharrock and Quail, 1989). A 2.3-kb SalI fragment containing upstream genomic sequence co-linear and overlapping with the PHYB cDNA sequence was further identified within this clone by hybridization to a 32P- labeled single-stranded oligonucleotide (5'-ATGGTTTC- CGGAGTCGGG-3') designed from the published PHYB cDNA sequence (Sharrock and Quail, 1989) and by complete nucleotide sequencing (dideoxy chain termination) by standard techniques (Sambrook et al., 1989).

Promoter-CUS Plasmid Constructions

A 2.9-kb sequence from the PHYA gene was ligated to the gusA coding region (GUS) contained within the Agrobacterium transformation vector pCIT20. The deriva- tion and construction of the modified binary vector to contain a promoterless GUS-NOS3' cassette (TC18) has been previously described (Caspar and Quail, 1993). The

PHYA sequence used resulted from a BamHI/SalI digest of the original 9-kb A ZapII-derived plasmid that had been linearized first with NdeI and further modified by the ligation of a 115-bp NdeI/SalI PCR fragment derived from the PHYA genomic sequence between +962 and + 1077 bp (Dehesh et al., 1994) to create a nove1 3' SalI restriction site. This modified PHYA fragment was ligated into a BgWSalI site of TC18, which was created from a partia1 SalI digest of the plasmid. The final chimeric PHYA-GUS gene consisted of 2.9-kb of PHYA upstream sequence, including 0.9 kb of intron from the 5' untranslated region, and coded for the first 8 amino acids of the PHYA polypeptide fused in frame with the sequence for 12 amino acids from the polylinker of TC18 and the entire GUS coding region.

A similar strategy was used to create the PHYB-GUS construct in pCIT20. A 2.1-kb ClaI/XhoI fragment was iso- lated from the original 2.3-kb Sal I /Sa l I PHYB genomic DNA subcloned into Bluescript -KS (Stratagene). A 49-bp oligonucleotide with XhoI half sites at each end was syn- thesized to match the sequence beween the XhoI site in the 5' untranslated region (+2254) and the translation start (+2303) of the PHYB gene. TC18 was cut at the ClaI/XhoI site and the final PHYB-GUS gene chimera was completed by three-way ligation. The final gene product contained the first 2 PHYB amino acids plus 22 amino acids coded by the TC18 polylinker appended to the NH, terminus of the GUS polypeptide.

Plant Transformations

The PHYA-GUS and PHYB-GUS reporter genes. were transformed into A. thaliana (ecotype No-O) via a root transformation protocol (Valvekens et al., 1988) modified according to Caspar and Quail(1993). Ten to 12 independent transgenic lines were obtained for each construct and seg- regation analysis was used to determine the number of T-DNA loci. The degree of transgene expression (GUS activity) did not correlate with the number of T-DNA loci, although quantitative analyses were conducted primarily with homozygous single-locus lines.

Plant Crowth Conditions

Surface sterilization and stratification of wild-type and transgenic seed was according to Somers et al. (1991). A11 plants were grown on germination medium (Valvekens et al., 1988) in either continuous darkness or under the appropriate light conditions in which a11 organs of the seedling were exposed to the prevailing light regime. White fluorescent (48 mmol m-' s-'), red (51 mmol m-' s-'), and far-red (30 mmol m-'s-') light sources were as described by Parks and Quail (1993). The blue light source (11 mmol m-' s- ' ) was constructed according to Gallagher et al. (1988).

Fluorometric CUS Assays

Fluorometric assays were performed on soluble protein extracts prepared in GUS extraction buffer (50 mM Na phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mM P-mercaptoethanol) from homogenate of

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pooled intact seedlings or organs. The crude extract was clarified by centrifugation in a microfuge (Eppendorf, Fre- mont, CA) at 14,000 rpm for 10 min. Aliquots were assayed over a 20- to 30-min time course in GUS reaction buffer (GUS extraction buffer, 1 mM 4-methylumbelliferyl p-D- glucuronide, 20% methanol [v/v]) and then diluted 1:lO into 0.2 M Na,CO, to stop the reaction. Fluorescence was determined on a Perkin-Elmer (Norwalk, CT) LS-30 lumi- nescence spectrometer (excitation wavelength, 365 nm; emission wavelength, 455 nm). Protein determinations were according to Bradford (1976).

Genetic Methods

The following mutant alleles were used as pollen accep- tors in crosses with PHY-GUS transgenic lines: hy3-Bo64 (phyB-2); hy4 (2.23N); hy5 (Ci88); ky6-2; and hy8-2 (phyA- 202). F, progeny were selfed and F, seeds were grown and selected on hygromycin for the presence of the transgene and also scored for the appropriate mutant phenotype. Resistant plants were selfed and seeds from individual F, progeny were tested for homozygosity of both the transgene and mutant phenotypes. Since four different ecotypic backgrounds were involved in these crosses (Landsberg erecta: hy3, hy4, hy5; Columbia: hy6; RLD: hy8; No-O: transformants), the results within each line were normalized to the GUS activity of the dark level (100%) to allow relative comparisons between lines.

RESULTS

Promoter-GUS Transgenic Plants

We transformed Arabidopsis (No-O ecotype) with two different chimeric promoter-reporter gene constructs. Both consisted of translational fusions between the respective 5' upstream regions from the genomic sequences of the PHYA and PHYB genes and the coding region of the gusA gene.

Figure 1 shows that a 0.9-kb intron resides within the 2.9 kb of the PHYA upstream region (Dehesh et al., 1994) that was used in the PHYA-GUS gene fusion. This structural arrangement is shared among a11 PHYA genes sequenced to date (Quail, 1994) and may have a regulatory function. Three differently sized transcripts have been mapped (Dehesh et al., 1994), and about 1.7 kb of DNA upstream of the most 5' transcription start site is retained in the reporter gene construct (Fig. 1). Ten independent transgenic plants were obtained via Agrobacterium-mediated transformation, and most expressed the GUS transgene in a qualitatively similar way. Exceptions are noted below.

The PHYB-GUS reporter gene included 2.1 kb of genomic sequence that lies 5' of the PHYB coding region (Fig. 1). The nucleotide sequence of this region and that corresponding to the primary transcript from the Columbia ecotype is nearly identical to that reported for the Landsberg ecotype (Reed et al., 1993). Three differences (Columbia/Lands- berg) were found, at bases 744 (C/G), 1182 (C/G), and 1677 (-/A), and a11 lie upstream of the known transcribed sequence (Sharrock and Quail, 1989). The 2.1-kb PHYB genomic sequence fused to the GUS gene in this study is identical to that which, when fused to the PHYB cDNA and

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Figure 1. PHY-GUS gene fusion constructs. Ahout 2 to 3 kh of genomic sequence cloned from directly upstream of the coding regions of PHYA and PHYB were translationally fused to the CUS coding sequence as shown. Cross-hatching indicates PHY-derived sequence; open areas show linker-derived sequence; line shows intron; shaded areas show nopline synthase (NOS) 3' sequence; and black areas indicate GUS coding sequence. 4ATC, Phytochrome translation start site. Numbers above the bars show the numher of amino acids added to the N terminus of GUS, except where otherwise indicated.

transformed into a PHYB-nu11 mutant (hy3-Bo64; phyB-2) (Somers et al., 1991; Reed et al., 1993), results in restoration of the wild-type phenotype (Wester et al., 1994). This result strongly suggests that a11 the regulatory sequences necessary and sufficient for the proper temporal and spatial expression of the PHYB gene are contained within these 2.1 kb. A similar experiment with the PHYA promoter has not been performed. With some exceptions, the 10 independent transgenic plants obtained via Agrobacterium-mediated transformation expressed the PHYB-GUS transgene in qualitatively similar ways.

PHYA and PHYB Promoters Differ in Activity and Are Differentially Light Regulated in Shoots and Roots

In dark-grown Arabidopsis plants, PHYA mRNA and PHYA polypeptide accumulate to much higher steady-state levels than do the respective gene products of PHYB (Shar- rock and Quail, 1989; Somers et al., 1991). This could result from greater transcriptional activity of the PHYA promoter in the dark, relative to PHYB promoter activity, or to differences in mRNA stability. Figure 2 demonstrates that the level of GUS enzyme activity in 7-d-old dark-grown PHYA- GUS seedlings was approximately 10- to 20-fold greater than the level of GUS enzyme activity in PHYB-GUS transformants grown under the same conditions. These findings are consistent with the conclusion that the greater accumu-

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Figure 2. Specific activity of CUS in whole-seedling extracts of 7-d dark- and white-light-grown seedlings transformed with PHYA-CUS or PHYB-GUS. Whole-seedling extracts from two independently derived PHYA-GUS lines (19-1-1-6, 19/26-1) (A) and two independently derived PHYB-GUS lines (Bl-8, 83-5) (B) were assayed for GUS activity in dark-grown (filled) and light-grown (cross-hatched) plant extracts. Twenty-five to 35 light-grown seedlings and 50 to 70 dark-grown seedlings were assayed. The ratio of GUS activity from dark-grown extracts to that of light-grown extracts (D/L) is shown at each pair of results. Activity is expressed as nmol of 4-methylumbelliferone (MU) produced min-' mg-' soluble protein.

lation of endogenous PHYA gene products in the dark is due entirely to a dark transcription rate higher than that of PHYB. Furthermore, when GUS activity levels in dark- and light-grown seedlings are compared, it is apparent that the two types of transgenes responded very differently to light. Compared to etiolated plants, PHYA-GUS expression was 6- to 8-fold lower in light-grown seedlings (Fig. 2A). The ratio of GUS activity in the dark to GUS activity in the light (D/L) was very similar between the two PHYA-GUS lines (D/L: 6.1/7.9, Fig. 2A). In contrast, both PHYB-GUS transformants showed only a 1.5- to 2-fold lower expression level of the transgene in light compared to dark (D/L: 1.8/1.6, Fig. 28). The relative similarity in this ratio between the two lines of each pair indicates that the effects on transgene activity of variables such as genome position affected expression in light and dark equally in these two sets of plants. Despite the stronger decline in the expression of PHYA-GUS than PHYB-GUS induced by light in these seedlings, the net activity of the PHYA-GUS transgene remains an average of about 6-fold higher than that of PHYB-GUS in the light (Fig. 2).

To confirm and extend these results, we examined a larger number of transgenic lines to a greater degree of spatial resolution. Figure 3 shows the GUS enzyme activity in the shoots and roots of the six independent PHYA-GUS and PHYB-GUS transgenic lines grown in constant light or constant darkness for 7 d. In dark-grown seedlings, although there was considerable variation in absolute expression levels (>lO-fold) among the members of the two sets of transgenic lines, the GUS activity levels from a11 six PHYA-GUS transformants were consistently higher (averaging 19 times greater) than the GUS activity levels in the six PHYB-GUS transformants in both roots and shoots (Fig. 3, A, B, D, and E; Table I). In addition, the relative level of GUS activity averaged 4 times higher in shoots than in roots (shoot/root ratio) for each transgene (Table I).

Organ-specific effects of light were observed on the activity of both PHY promoters. In shoots, five of six independent lines expressed the PHYA-GUS transgene at an average of 9-fold lower in the light than in the dark (range, 5- to 13-fold) (Fig. 3A). In marked contrast, however, GUS enzyme activity was nearly the same in light- and dark- grown roots in four of five lines (mean D/L: 0.9) (Fig. 3D). The single anomaly, 26-1-5, appeared to have 2.5 times higher expression in the dark in both organs relative to other lines with similar levels of enzyme activity in white light (Fig. 3, A and D). Results from the remaining transformants indicate a strong light-directed down-regulation of the PHYA promoter activity in shoots and the absence of such a response in roots. As a result, the average shoot-to- root ratio of GUS activity (S/R) in dark-grown seedlings was 4 compared to 0.4 in light-grown seedlings (Table I). These results also show that the effects of light on the GUS activity levels from whole-seedling extracts (Fig. 2) largely reflected the effects on the shoot compartment alone.

The light-regulated expression of the PHYB-GUS transgene among six independent transformants differed in both degree and direction from the above results. GUS activity in the shoots of light-grown plants was 2 to 3 times lower than in dark-grown seedlings (mean D/L: 2.3) (Fig. 3B). The converse was true in the roots, where GUS activity in light-grown plants appeared to be about twice that of dark-grown seedlings (mean D/L: 0.5) (Fig. 3E). Although these differences between light and dark expression levels in the roots and shoots of the PHYB-GUS transgenics appear small, the consistency of response among the six lines suggests that these results are an accurate reflection of the activity of the PHYB promoter region. These data also show that, as for PHYA-GUS, the shoot compartment dom- inated in determining the net effect of light on PHYB-GUS expression observed in whole seedling extracts (Fig. 2). A 35s-GUS transformant (Deng et al., 1991) tested in the same way showed no significant light-mediated expression differences in either the shoots (D/L: 1.2) or roots (D/L: 0.8) (Fig. 3, C and F), consistent with previous reports (Nagy et al., 1985).

Despite the nearly 10-fold lower level of GUS activity in white-light-grown shoots of PHYA-GUS transformants relative to that in etiolated seedlings, the absolute GUS activity level still averaged 5-fold higher than that found in green PHYB-GUS shoots (Fig. 3, A and B; Table I). Simi- larly, in light-grown roots GUS activity was 11-fold higher in PHYA-GUS transformants than in PHYB-GUS plants (Fig. 3, D and E; Table I).

PHYA and PHYB Promoter Activities Respond to Dark Adaptation of Light-Grown Seedlings

We wished to determine whether the transcriptional repression of PHY-GUS expression in the light could be reversed by shifting light-grown plants into darkness. Al- though such dark-adapted plants are not fully equivalent to light-developed plants left in the light, an increase in GUS activity in the dark-shifted seedlings, relative to plants maintained in the light, would be consistent with the

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Figure 3. Specific GUS activity in shoots and roots of six independently derived PHYA-GUS (A and D) and PHYB-GUS (B and E) transformants and one 35s-GUS transformant (C and F). Plants were grown in continuous dark (filled) or continuous light (cross-hatched) for 7 d. Twenty-five to 35 light-grown seedlings and 50 to 70 dark-grown seedlings from each condition and line were pooled and assayed for GUS activity. Shoots (A-C) and roots (D-F) were assayed separately, and for each compartment the ratio of GUS activity from dark-grown to that of light-grown extracts (D/L) is shown beneath each transgenic line designation (line). Shaded boxes show mean CUS activity for all six lines for the given condition and compartment; the D/L of the means i s shown below. Axes scale is identical in B and C and in E and F; note differences from A and D. In 19-1-4 the sample of root extracts was lost. Lines with error bars (?SE) show results of two to four paired (shoot and root; light and dark) trials; remaining lines were tested once. Units are nmol of product (methylumbelliferone) produced min-’ mg-’ total soluble protein.

,

possibility of direct promoter responsiveness to light as distinct from indirect effects of light on development.

Seedlings grown for 7 d under continuous light were shifted into darkness for 2,4, or 8 additional days and GUS activity in shoots and roots was assayed independently. Over the 8-d period, the leve1 of GUS activity Tose 4-fold in the dark-shifted PHYA-GUS lines, but never attained the more than 15-fold difference between 15-d continuous dark- and continuous light-grown plants (Fig. 4A). These results differed from the expression of a pea PHYA-GLIS

transgene in petunia, in which 7-d-old light-grown seedlings attained, after 7 d in the dark, GUS activity levels near that of etiolated plants of the same age (Komeda et al., 1991).

After 8 d, PHYB-GUS expression levels in dark-shifted plants had rísen to nearly 2.5 times that of the light-grown controls (Fig. 4B). By comparison, expression of 35s-GLIS in light-grown and dark-shifted shoots remained essentially identical throughout the experiment (Fig. 4C). These results suggest that the activity of the PHYA and PHYB

Table 1. Average GUS enzyme activity levels in 7-d-old PHYA-GUS and PHYB-GUS transgenic seedlings (nmol methylumbelliferone min- ’ mg- ’ protein) NB, Ratio of the average GUS enzyme activity in PHYA-GUS lines to the average GUS enzyme activity in PHYB-GUS lines in the shoots and

roots of light- and dark-grown plants; S/R, ratio of CUS activity in shoots to GUS activity in roots for each light condition and type of transgenic plant.

Shoots Roots S I R Crowth Conditions

PH YA PH YB NB PH YA PH Y6 NB PH YA PHYB

Dark grown 388 20 19 94 5 19 4 4 Light grown 43 9 5 114 10 11 0.4 1

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promoters remain light responsive in the shoots of green seedlings, whereas expression of the 355 promoter is independent of light. The apparent rise in dark expression levels over the 15-d trial for a11 transformant types may be related more to the gradual decrease in total protein levels (per seedling) within etiolated tissue (data not shown) than to an actual increase in GUS enzyme levels. Similar obser- vations have been made previously (Caspar and Quail, 1993).

The dark-shifted roots of the PHYA-GUS and 35s-GUS transformants showed slightly higher levels of GUS enzyme activity than those of the light-grown controls (Fig. 4, A and C), whereas GUS activity in light-grown and dark- shifted roots of PHYB-GUS lines were essentially identical over the period of time tested (Fig. 4B). These results support the above contention that significant light regulation of PHYA and the cauliflower mosaic virus 35s promoter is lacking in roots. For PHYB-GUS, a decrease in GUS activity would be expected if promoter activity is lower in the dark, as suggested in Figure 3E. However, the long half-life of the GUS protein (Jefferson et al., 1987) and the narrowing of the difference between dark-grown and light-grown expression levels over the extended trial period may explain why this expected decrease was not observed in the roots of the dark-shifted plants.

Photoregulation of PHY Promoter Activity under Constant Narrow-Waveband Light

Figure 5 compares the responses of three types of GUS reporter constructs in the cotyledons, hypocotyl, and roots of 7-d-old seedlings grown under one of five different continuous light conditions. The partitioning of the shoot into cotyledons and hypocotyl extended our previous results to show that, despite their different morphology, light regulation of both PHY promoters was similar in the two organs (Fig. 5, A and B). The strong decrease in PHYA-GUS expression in white-light-grown shoots (Fig. 3 ) was equally apparent in both the cotyledons and hypocotyl (about 6- to Y-fold), and red light alone was al- most as effective (about 4- to 5-fold) (Fig. 5A). Blue and far-red light were less effective, reducing PHYA-GUS expression in these organs to only 2-fold or less than dark expression levels.

The cotyledons and hypocotyls of PHYB-GUS transformants also responded in parallel to the different light treatments (Fig. 58). The 2-fold reduction of PHYB expression in the shoot directed by white light (Fig. 3) was observed in both organs, and red light was equally as effective as fluorescent white light (Fig. 5B). Surprisingly, however, both blue and far-red light induced PHYB-GUS

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Figure 5 . Relative GUS activity in three seedling compartments of PHY-GUS and 35s-GUS transformants after growth in one of five different light regimes. PHYA-CUS (A), PHYB-CUS(B), and 35s-GUS (C) seedlings were grown for 7 d in continuous darkness, white fluorescent, red, blue, or far-red light. Cotyledons, hypocotyls, and roots were harvested and pooled from 25 to 35 light-grown seedlings or 50 to 90 etiolated seedlings. All results were normalized to dark-grown hypocotyl activity (1 00%) for each trial before averaging. Two to three independent PHY-GUS transgenic lines and one 35s- GUS line were assayed for each data set and results are t h e means of at least three trials. Bars show SE

expression to levels 2 to 3 times higher than that seen in dark-grown plants (Fig. 5B). The equivalent effects of white and red light on the reduction of PHYB-GUS transgene activity, despite the presence of blue light in the fluorescent source, indicates the greater effectiveness of red light over blue light in affecting this response.

In roots of both sets of PHY-GUS transformants, none of the light treatments had effects on GUS levels as marked as those observed in shoots (Fig. 5, A and B). As noted above, levels of PHYA-GUS expression in dark- and light-grown roots were very similar, and the same was true under the narrow-waveband sources (Fig. 5A). For PHYB-GUS ex-

pressers, this data set did not show the 2-fold-higher GUS levels in white-light-grown roots noted earlier (Fig. 3) (although see below), and the other light treatments were even less effective (Fig. 5B). The reason for this discrepancy is unknown.

GUS activity in a 35s-GUS transformant was assayed in parallel to the PHY-GUS trials to control for possible indirect effects of the light conditions on the veracity of the assay. Figure 5C shows that GUS activity levels within each organ type in this line were essentially identical under a11 five growth conditions, indicating that the PHY-GUS results reflect intrinsic promoter activity.

Photoregulation of PHY Promoter Activity in Photoreceptor-Deficient Mutant Backgrounds

We also determined transgene expression in plants deficient or absent in one or more photoreceptors or compo- nents of the light transduction pathway. phyB-1 contains a lesion in the PHYB gene (Reed et al., 1993), causing a deficiency in PHYB mRNA and polypeptide abundance (Somers et al., 1991). HY4 codes for a putative blue-light receptor (Ahmad and Cashmore, 1993). hy5 appears deficient in a putative component of the light-signaling pathway (Koornneef et al., 1980; Ang and Deng, 1994). hy6 is thought to be deficient in a11 phytochromes due to a lesion presumed to be in the chromophore biosynthetic pathway (Chory et al., 1989; Nagatani et al., 1993). The phyA-202 mutation resides in the PHYA gene (Dehesh et al., 1993) and causes a loss of PHYA mRNA and polypeptide (Parks and Quail, 1993). If a white-light-grown PHY-GUS transformant in a mutant background shows GUS activity at or near the level of dark-grown wild-type plants, we can infer a necessary role for that component in light-mediated transgene expression.

Figure 6 shows the results of expression of the PHYA- GUS transgene in the five different mutant backgrounds. In each case the degree of light-induced repression of PHYA activity in the cotyledons and hypocotyl was very similar to the level in the wild type (Fig. 6, A and B). These results imply that neither of the two phytochromes nor the putative HY4 blue-light receptor is alone necessary to mediate PHYA-GUS down-regulation in white light. However, it is still possible that separate pathways lead from these photoreceptors, which alone are sufficient (see "Discussion"). In roots, where there was no previous evidence of the photoregulation of PHYA-GUS activity, the results were the same in a11 backgrounds (Fig. 6 0 .

PHYB-GUS expression in the same mutant backgrounds contrasted with these results (Fig. 6). In both organs of the shoot, PHYB expression in the hy6 background in white light was the same or slightly greater than that in the dark (Fig. 6, A and B). This result implies that one or more phytochromes are necessary for essentially a11 the white- light-mediated reduction in PHYB promoter activity and that other photoreceptors cannot substitute in the absence of phytochrome. Since GUS activity was at wild-type levels in the phyA mutants and in the hy4 and hy5 mutant backgrounds, none of these mutations appear to be in the

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Figure 6. Relative GUS activity of the PHY-GUS transgene in different mutant backgrounds. Transformant 19-1-1-6 (PHYA-GUS) and transformants B1-8 and 83-5 (PHYB-GUS) were crossed into the five mutant backgrounds, and homozygotes for both the transgene and mutant loci were assayed for GUS activity in the cotyledons (A), hypocotyls (B), and roots (C) of 7-d dark-grown (filled) and white-light-grown (cross-hatched) plants. Organs from 25 to 35 light-grown seedlings or 50 to 90 etiolated seedlings were pooled for each trial. Since the genetic backgrounds of the five different F, families were nonisogenic, the data pair for each organ (dark activity, light activity) of each trial was normalized to the dark level (100%) before averaging to facilitate comparison among the different mutant strains. Results are the means of at least three trials. Bars show SE.

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phototransduction pathway that regulates PHYB expression in the shoots.

Furthermore, it is phyB itself that appears to be largely responsible for mediating PHYB down-regulation. GUS enzyme activity levels of PHYB-GUS in the shoots of phyB (hy3) mutants were equivalent, or nearly so, in both light and dark (Fig. 6, A and B). The pkyB allele used here, pkyB-l (hy3-B064), lacks detectable PHYB polypeptide (Somers et al., 1991; see "Discussion") and is probably a nu11 PHYB mutation (Reed et al., 1993). The ky6 lesion, however, is undefined and appears to possess a limited amount of physiologically active phytochrome (Chory et al., 1989). Hence, the observation of higher GUS activity levels in the ky6 background than in the ky3 mutant (Fig. 6, A and B) suggests that there may be a limited role for another phytochrome other than phyB.

Relative to dark-grown plants, GUS activity in roots of PHYB-GUS lines was 2-fold higher in white-light-grown seedlings in the wild-type, pkyA, and hy6 backgrounds. In contrast, in the pkyB and ky4 mutant backgrounds, GUS enzyme activity levels in white-light-grown plants were essentially equal to the levels in dark-grown plants (Fig. 6C), implying that the presence of at least the PHYB and HY4 photoreceptors together are required for full induction of transgene activity. Since the hy6 mutant is presum- ably deficient in a11 phytochromes, the presence of wild- type levels of GUS enzyme activity in this background (Fig. 6C) suggests that enough chromophore is made to main-

PHYA-GUS

phyBhy4 hy5 hy6 p h y A &t

PHYB-GUS

tain sufficiently high levels of phyB in roots to co-mediate PHYB induction. The presence of the HY5 gene product also appears to be required for light-induced PHYB expression in the roots.

DI SC U SS I ON

Transcriptional Activity and Photoresponsiveness of PH YA and PHYB Promoters

The evidence presented here indicates that the PHYA and PHYB genes of Arabidopsis are regulated at the transcriptional level both developmentally and by light. How- ever, there are strong differences in the transcriptional activities of the two promoters and in their responsiveness to the two regulatory parameters (summarized in Table I). In dark-grown seedlings, the PHYA promoter is nearly 20-fold more active than the PHYB promoter in both shoots and roots. Moreover, each promoter is 4-fold more active in shoots than in roots. In light-grown seedling shoots, PHYA promoter activity is 9-fold lower that in the dark, whereas PHYB promoter activity decreases only 2-fold. By contrast, in light-grown seedling roots, PHYA promoter activity is unchanged from that in the dark and PHYB promoter activity increases 2-fold. The net consequences of these light-induced changes are 3-fold. First, despite strong down-regulation, the PHYA promoter remains 5-fold more active than that of PHYB in seedling shoots in the light.

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Second, despite an increase in PHYB activity, the PHYA promoter remains 11-fold more active than that of PHYB in seedling roots in the light. Third, PHYA promoter activity remains twice as active in shoots as in roots of light-grown seedlings, whereas PHYB is about equally active in both organs. In addition, within the shoot each promoter is equivalently active and light responsive in cotyledon and hypocotyl tissue (Fig. 5).

The validity of the above interpretations of relative promoter activity assumes that the fluorometric quantitation of the PHY-GUS transgene activities reported here faith- fully reflect the activities of the corresponding endogenous promoters. A number of considerations suggest that to the first approximation this is so. First, the PHYA-GUS and PHYB-GUS constructs were identical throughout the open reading frame and 3' untranslated regions (see "Materials and Methods"), where determinants of mRNA stability are considered to reside (Gallie, 1993; Sachs, 19931, thus sug- gesting that the differences in GUS activity observed between the two sets of transgenic lines should be largely ascribable to the differential expression of the respective PHY promoters. On the other hand, it is still possible that the different 5' untranslated regions of the two transgenes or the intron in the PHYA gene may also account for part of the observed differences. As well, sequences within the coding region and native 3' untranslated regions of the endogenous phytochrome genes may result in expression and regulation patterns different from those described here using reporter genes.

Second, to avoid as much as possible complications aris- ing from the reported long half-life (about 50 h) of GUS (Jefferson et al., 1987), we used seedlings grown only in continuous dark or continuous light from germination on- ward, or plants grown first in continuous light and then shifted to darkness. These protocols were designed to provide light-imposed preemptive repression of GUS accumulation that otherwise occurs in comparable dark controls. This precludes the necessity of following light-induced reductions in the levels of previously accumulated GUS enzyme upon transfer of dark-grown seedlings to the light.

Third, the absence of significant effects of light on 35S- GUS activity (Figs. 3 and 5) indicates the absence of light- induced posttranscriptional changes in GUS mRNA or protein levels through altered stability or altered specific activity through changes in general protein levels. The data strongly indicate, therefore, that the PHY sequences in the PHY-GUS transgenes are selectively light responsive.

Fourth, and most direct, our results are in close agree- ment with the relative mRNA abundances suggested for PHYA and PHYB in dark-grown and light-grown Arabidop- sis seedlings from RNA blot analysis (Sharrock and Quail, 1989; Clack et al., 1994). In these studies, direct quantitative comparisons of PHYA and PHYB mRNA abundances could only be approximated because of differences in probe specific activity and blot exposure times. Nevertheless, the data suggested that in both continuous dark- and contin- u o u ~ light-grown seedlings PHYA mRNA levels were many-fold higher than those of PHYB (Sharrock and Quail, 1989; Clack et al., 1994). Our present data thus strongly

suggest that the previously reported differences in steady- state mRNA abundances for the two phytochromes are probably determined primarily by different transcription rates of the two genes. Similarly, the magnitude of the light-imposed repression of PHYA-GUS expression can account for the previously reported difference in PHYA mRNA levels from light- and dark-grown Arabidopsis seedlings (Sharrock and Quail, 1989; Clack et al., 1994). Hence, these results suggest that photocontrol of the transcription rate may be the primary determinant of steady-state PHYA mRNA levels in Arabidopsis shoots. Two previous studies using PHYA-GUS or PHYA-CAT reporter genes in transgenic petunia (Komeda et al., 1991) and tobacco (Adam et al., 1994), respectively, also concluded that PHYA expression is controlled primarily through transcription. A similar situation is found in oat, where run-on transcription experiments with nuclei from shoots demonstrate a rapid and strong down-regulation of PHYA promoter activity in the light (Lissemore and Quail, 19881, whereas mRNA stability is light independent (Seeley et al., 1992).

In oat root tissue, PHYA polypeptide levels decrease in light much less severely than in shoots (Wang et al., 1993a). Our present results with PHYA-GUS expression in Arabi- dopsis roots suggest that this may result primarily from the lack of transcriptional photoregulation in oat, leaving only the intrinsic instability of PfrA as the cause of the light/ dark difference in phyA protein (Fig. 3D). This striking difference in transcriptional regulation between root and shoot was unanticipated and suggests that critica1 factors governing the light-directed decrease in PHYA transcription are missing in roots, and/or that positive factors pro- moting high levels of transcription in the dark are present only in the shoots. The 4-fold-lower expression level of PHYA in the roots than in the shoots of dark-grown seedlings is consistent with the latter possibility.

Previous studies based on mRNA abundance have concluded that PHYB expression is not significantly light regulated in Arabidopsis (Sharrock and Quail, 1989; Clack et al., 1994). Our consistent observation of a 2-fold light-directed decrease in PHYB-GUS expression in the shoot (Fig. 38) does not suggest a serious discrepancy between these two sets of results, since the endogenous PHYB mRNA levels were not rigorously quantitated in these previous studies. In oat, the level of one species of light-stable phytochrome polypeptide is 2-fold lower in light-grown shoots than in dark-grown shoots (Wang et al., 1993a, 1993b), which is also consistent with a light-induced decrease in gene transcription.

Although a 2-fold change in photoreceptor abundance may appear small, just such an increase in phyB levels has been shown to significantly affect seedling phenotype. Photobiological experiments have shown that only a 50% increase in PfrB levels strongly inhibits wild-type Arabidop- sis hypocotyl elongation (McCormac et al., 1993). In addition, when rice phyB was expressed in Arabidopsis 2.5-fold over the levels of native phyB, hypocotyl length and petiole morphology were strongly affected relative to wild type under the same conditions (McCormac et al., 1993). A doubling of the endogenous PHYB polypeptide levels was

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achieved in Arabidopsis plants transformed with a PHYB cDNA regulated by the native PHYB promoter (Wester et al., 1994). Cell growth, chloroplast development, and ro- sette development at time of flowering were a11 affected when plants were grown under continuous white light. Hence, in Arabidopsis these processes appear to be ex- tremely sensitive to phyB levels in the shoots, and even a 2-fold light-induced down-regulation may have signifi- cance in a natural context (see below).

Diverse Photoreceptors Mediate the Light Responsiveness of PHY Promoters

The increase in activity of the PHYA-GUS and PHYB- GLTS transgenes when light-grown plants are shifted to the dark suggests that light regulation of PHY gene transcription, and hence, phytochrome polypeptide levels, may be part of the normal diurna1 cycle of the plant. Since vegetative shade and reflected light in the natural environment strongly affect light quality and, consequently, plant development (Smith, 19941, further insight into the adaptiveness of PHY photoregulation can be gained by knowing which photoreceptors mediate the changes in PHY gene activity. The results of our two approaches to this question suggest that this regulation is complex and, in some cases, divergent between the two PHY genes.

The negative photoregulation of PHYA in the shoot appears to occur primarily, but not exclusively, through a red-light-absorbing photoreceptor(s). The equal effectiveness of red and white light in reducing PHYA-GUS transgene activity suggests a light-stable (type 2) phytochrome as the primary receptor, although Chl cannot be entirely excluded. The partia1 effectiveness of blue and far-red light also implicate other photoreceptors, including phyA, in repression of PHYA activity. In oat there is clear evidence that a light-labile phytochrome (probably phyA) is pre- dominantly, if not exclusively, responsible for transcriptional repression of PHYA (Lissemore and Quail, 1988). However, in white light the level of decrease in PHYA-GUS activity in a11 the mutant backgrounds was similar to that of the wild type, implying that no one photoreceptor is necessary but that two or more alone, or additively, are sufficient to mediate full repression of PHYA expression in white light in Arabidopsis. The wild-type level response of the transgene in the hy6 background suggests that some component of fluorescent white light other than red light (via phytochrome) can repress PHYA expression. Since blue light alone was only partially effective, the small component of UV-A present in these lamps may be responsible. It is also likely that a small amount of photoactive phytochrome remains in the ky6 mutants (Chory et al., 1989), and it is possible that this amount may be sufficient to fully mediate the light effects.

Photoregulation of PHYB in the shoots appears less complex than that of PHYA. Notwithstanding the slight difference between the activity levels in the pkyB (hy3) and ky6 backgrounds (see "Results"), results from the photophysiological experiments and from the mutant background assays together implicate phyB as the primary receptor necessary and sufficient for mediating most of the decrease in

PHYB expression in white light. Preliminary results have also shown that PHYB-GUS expression under red light in the phyB background was similar to the GUS activity level in etiolated seedlings (data not shown). In oat the levels of one or more light-stable phytochromes increased in abundance after end-of-day far-red irradiation (Stewart et al., 1992). Since the end-of-day far-red response is phyB mediated (Smith, 1994), this result is also consistent with phyB- mediated regulation of PHYB transcription.

The increase in PHYB-GUS activity in shoots grown under continuous far-red light, relative to the dark level, appears to result from a far-red high-irradiance response, which implies mediation entirely through phyA (Mancinelli, 1994; Smith, 1994). Preliminary results of experiments done in the pkyA background under far-red and blue light showed that induction of PHYB-GUS was lacking and GUS enzyme activity was at dark levels (data not shown). This further implies that phyA is necessary for both the blue and far-red light induction of PHYB activity.

The opposing effects that different wavelengths have on PHYB expression support the contention that phyB levels could be a critical parameter in the light-sensing repertoire of the plant (Wester et al., 1994). Unlike the rapid degradation of PfrA in the light, the relative stability of the phyB polypeptide (Somers et al., 1991) suggests that regulation of phyB levels occurs through a different mechanism. Based on our results, the primary means appears to be through a light-modulated control of gene transcription. Between the extremes in phytochrome photoequilibria of growth in monochromatic red light and monochromatic far-red light, transcriptional activity of the PHYB promoter ranged 4- to 5-fold in shoots (Fig. 5B). If PHYB mRNA abundance and translatability are directly proportional to promoter activity, the resulting range of possible phyB levels would be well within that found to markedly affect plant growth (Wagner et al., 1991; McCormac et al., 1993; Wester et al., 1994). Hence, under natural conditions the inductive capacity of far-red (and blue) light and the re- pressive capacity of red light might act in opposition to set PHYB transcription, and hence PHYB holoprotein, to a level that reflects the red/far-red ratio of the ambient light environment and that then signals appropriate developmental responses. For example, in the far-red-rich light environment under vegetative shade, phyB levels in the shoot may rise through a phyA-mediated increase in PHYB transcription. This could have the effect of increasing the sensitivity of the seedling to the diminished red light levels present under a canopy. Accurate determinations of phyB protein levels in plants grown under different light quali- ties would be necessary to test this hypothesis.

Our results show that in the shoot, PHYA transcription in the light is also highest under far-red light. In fact, the effects of the four different light conditions on the transcriptional activities of the two transgenes in shoots were very similar (lowest in white light, highest in far-red light). This similar correlation between spectral quality and transcriptional activity for the PHYA and PHYB genes is consistent with the finding that in some cases (e.g. Chl a/b- binding gene expression) the two photoreceptors mediate a

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coordinate response to light (Reed e t al., 1994). The persis- tently high leve1 of PHYA promoter activity i n white light, relative t o PHYB, is also consistent with the increasing evidence from physiological experiments with phyA and phyB mutants that the levels of phyA maintained in light- grown plants (Somers e t al., 1991; Wang et al., 1993a) are physiologically active i n processes such a s suppression of hypocotyl elongation and day length perception (Johnson et al., 1994; Reed et al., 1994). Very high levels of endogenous PHYA transcription in the light may be the only way t o maintain physiologically significant levels of PfrA in white light in the face of its inherent instability.

Our finding that PHYA a n d PHYB appear t o be co- expressed i n most cells throughout development (Somers and Quail, 1995), together wi th the present data, support the notion that the photosensory functions of the two photoreceptors may be determined, in part, by organ-specific differences in the photoregulation of PHYA a n d PHYB transcription.

ACKNOWLEDCMENTS

We thank Tim Caspar for providing the promoterless-GUS plasmid (TClS), and Dr. Caspar and Xing-Wang Deng for the Arubi- dopsis 35s-GUS seed. We are also grateful to Patti Taranto and Jose Gutierrez for their efforts in sequencing the PHYB promoter. Julie Montgomery provided welcome advice on the use of the lumines- cence spectrometer and in discussions on the interpretation of the GUS enzyme assays.

Received July 5, 1994; accepted November 15, 1994. Copyright Clearance Center: 0032-0889/95/l07/0523/12.

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