The Plant Cell, Vol. 5, 147-157, February 1993 O 1993 American Society of Plant Physiologists

RESEARCH ARTICLE

Mutations in the Gene for the Red/Far-Red Light Receptor Phytochrome B Alter Cell Elongation and Physiological Responses throughout Arabidopsis Development

Jason W. Reed,' Punita Nagpal,' Daniel S. Poo1e,a9b Masaki Furuya,c9' and Joanne Chorya9* a Plant Biology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92186-5800

Department of Biology, University of California at San Diego, La Jolla, California 92093-0116 Laboratory of Plant Biological Regulation, Frontier Research Programs, RlKEN Institute, Wako-shi, Saitama 351-01, Japan

Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These pho- toreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant pho- toreceptor gene. A number of tissues are abnormally elongated in the hyd(phyt3) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.

INTRODUCTION

Plant development is distinguished by its plasticity, which arises from a close coupling of developmental responses to environ- mental stimuli. As the source of energy for photosynthesis, light is particularly important to plants, and plants modulate their physiology and their development according to prevailing light conditions. Examples of light-mediated responses include seed germination, stem elongation, phototropism of leaves and stems, development of leaves and chloroplasts, stomatal open- ing, and flowering. lncident red, blue, far-red, and UV light a11 elicit subsets of these responses (Cosgrove, 1986; Chory, 1991; Thompson and White, 1991).

The mechanisms of light signal transduction in plants are poorly understood. Responses to red and far-red light are controlled by a family of photoreceptor proteins called phyto- chromes (reviewed by Colbert, 1988; Furuya, 1989; Quail, 1991). Phytochromes are 120-kD soluble proteins that have a covalently linked linear tetrapyrrole chromophore, and exist in two photointerconvertible forms, Pr and Pfr. Pr, the red light-absorbing form, is converted to Pfr, the far-red light-ab- sorbing form, upon absorption of red light. Pfr is thought to be the active form of phytochrome, and can be converted back to Pr by far-red light. Consequently, red light-induced re- sponses mediated by phytochromes are typically reversible

Current address: Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-03, Japan. * To whom correspondence should be addressed.

by far-red light. Most plants examined contain multiple phytochrome isoforms. In Arabidopsis, five phytochrome genes have been identified (Sharrock and Quail, 1989). Three of these, PHYA, PHYB, and PHYC, have been characterized in some detail. The sequences of PhyA, PhyB, and PhyC diverge sig- nificantly, having -60% identity to each other at the amino acid leve1 (Sharrock and Quail, 1989). The most abundant phytochrome in etiolated (dark-grown) seedlings is PhyA (Furuya, 1989; Somers et al., 1991). Whereas PhyA protein levels decrease in the light, PhyB and PhyC are present at equal levels in light- and dark-grown plants (Somers et al., 1991).

Based on their rediar-red reversibility, certain light responses have been hypothesized to be mediated by a phytochrome. These responses include red light-induced seed germination, inhibition of the rate of hypocotyl and stem elongation, induc- tion of chloroplast and leaf development, and induction of expression of genes required for photosynthesis (Cosgrove, 1986; Mullet, 1988; Chory, 1991; Thompson and White, 1991). However, it is not known which of these responses is controlled by which phytochrome species, or to what extent the different phytochrome molecules have overlapping functions. It is also not known when, or in which tissues, the different phyto- chromes are active.

A number of researchers have taken a genetic approach to dissecting light responses. Mutants affected in red light inhi- bition of hypocotyl elongation have been isolated in severa1 plants (reviewed in Chory, 1991; Kendrick and Nagatani, 1991;

148 The Plant Cell

Reed et al., 1992). In Arabidopsis, these mutants are calledhy (for long hypocotyl) (Koornneef et al., 1980; Chory et al.,1989). Mutations in hy1, hy2, hy3, hy5, and hy6 affect inhibitionof hypocotyl elongation by red light, hyl, hy2, and hy6 mutantsalso appear pale, as if affected in the greening response tored light. The hy1, hy2, and hy6 mutants have PhyA apoproteinthat is largely spectrally inactive, suggesting that they are defi-cient in phytochrome chromophore availability (Chory et al.,1989; Parks et al., 1989; Parks and Quail, 1991). These find-ings establish a correlation between deficiencies in redlight-induced responses and a deficiency in active phyto-chrome photoreceptors.

hy3 mutations affect a number of responses that are medi-ated by red and far-red light. hy3 mutants are deficient inelongation responses to supplementary far-red light (Nagataniet al., 1991a; Whitelam and Smith, 1991). Instead, they appearelongated without supplementary far-red light, as if showinga constitutive far-red elongation response. hy3 mutant plantshave also been found to flower earlier than wild-type plants(Goto et al., 1991; Whitelam and Smith, 1991; Chory, 1992),and to have a decreased germination response to red light(Spruit et al., 1980; Cone and Kendrick, 1985). In theserespects, they are similar to the hy1, hy2, and hy6 mutants;however, hy3 mutants differ from the hy1, hy2, and hy6 mu-tants in that they are less pale and have spectrally active PhyA(Chory et al., 1989; Parks et al., 1989).

That hy3 mutations affect various red/far-red light responsessuggests that they alter a phytochrome signaling pathway. Infact, immunological studies have shown that/7/3 mutants havea decreased level of PhyB relative to the wild type (Nagataniet al., 1991a; Somers et al., 1991). Like the hy3 mutants, a cu-cumber mutant called Ih (for tong ftypocotyl) is defective inend-of-day far-red elongation, and this mutant also lacks a pro-tein recognized by an anti-PhyB antibody (L6pez-Juez et al.,1990,1992). It therefore seems likely that PhyB mediates theresponses that are deficient in these mutants.

However, it has not been determined whether the hy3 muta-tions fall in the PHYB gene, whether they affect PhyB synthesisor stability, or whether they affect a downstream componentof PhyB-mediated signal transduction. In this work, we dem-onstrate that the HY3 locus encodes phytochrome B. Inaddition, we describe morphological and physiological pheno-types of hy3 mutants. These phenotypes occur in a numberof different tissues and throughout the life cycle of the plant,indicating that PhyB plays multiple roles in Arabidopsisdevelopment.

RESULTS

hy3 Mutations Fall in the PHYB Gene

Arabidopsis hy3 mutants were isolated by screening for mu-tants having a long hypocotyl when grown in white light(Koornneef etal., 1980). Mutants Bo64,4-117, 8-36, 548,1053,

Ler Bo64 4-117 8-36 548 1053 M4084 464-19

Figure 1. Ten-Day-Old Seedlings of Wild Type and Various hy3 Mutants.

Shown are a wild-type Landsberg erecta (Ler) seedling at the left andseven hy3 mutants. Scale bar = 2 cm.

and M4084 are shown in Figure 1, and were isolated previ-ously after ethylmethane sulfonate (EMS) mutagenesis ofecotype Landsberg erecta seeds (Koornneef etal., 1980). Mu-tant EMS142 was obtained by EMS mutagenesis of ecotypeColumbia seeds. In addition, we obtained one T-DNA inser-tion mutant, 464-19, in ecotype WS from K. Feldman (Universityof Arizona, Tucson) (Figure 1). The resistance to kanamycinconferred by this insertion segregated as a single locus in prog-eny of crosses with HY3+ plants, and all long hypocotyl F2

progeny were resistant to kanamycin (M. Koornneef, personalcommunication; data not shown). Genetic complementationanalyses established that these mutations were in the HY3 lo-cus (data not shown).

Previous work has shown that a hy3 mutation and the PHYBgene both map to chromosome 2 (Koornneef et al., 1980; R.Sharrock, personal communication). We tested whether a hy3mutation mapped to the PHYB locus by measuring the link-age between a hy3 mutation and a restriction fragment lengthpolymorphism detected by the cloned PHYB gene (see below).We crossed hy3 strain Bo64 (in the Landsberg erecfa ecotype)with wild-type plants of the Columbia ecotype, and analyzedthe F2 progeny of this cross having long hypocotyls for pres-ence of the Landsberg or Columbia PHYB polymorphism. Of103 such hy3lhy3 progeny examined, all showed only theLandsberg-specific restriction fragment. Thus, none of the 206chromatids tested showed a recombination event between thehy3 mutation and the PHYB polymorphism. This result estab-lishes that this hy3 mutation is very closely linked to the PHYBgene.

The published cDNA sequence of Arabidopsis PHYB is fromthe Columbia ecotype (Sharrock and Quail, 1989), whereasmost of our hy3 mutants are in the Landsberg ecotype. To ana-lyze our mutants molecularly, we cloned the wild-typeLandsberg PHYB gene and sequenced it, as shown in Figure2 and described in Methods. The sequence of the Landsberggene is virtually identical to the Columbia cDNA sequence,differing only at one position in the coding sequence, at nucleo-tide 3177 (Figure 2), and at one position in the 5' untranslatedsequence, at nucleotide 2257 (Figure 2). The latter polymor-phism creates a Xhol restriction site in the Landsberg gene,which we used for the linkage analysis above. Neither of thesepolymorphisms affected the amino acid sequence of PhyB.We found three introns in the coding sequence (Figure 2) at

HY3 Encodes Phytochrome B 149

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Figure 2. Sequence of the phyB Gene from Ecotype Landsberg efecta.

lntrons are in lowercase letters, G-box consensus sequences are underlined, and polymorphisms compared to the published cDNA sequence from ecotype Columbia are marked with asterisks. One kilobase of sequence at the 5' end has been omitted to conserve space. This sequence has been submitted to GenBank as accession number L09262.

150 The Plant Cell

hy3-4- I 17 hts283 j tyr

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hy3&%4

hv3- 464.19 gln448 + UAG I I . , -

hy3-8-36 lrp552 -3 UGA

cy6357 = chmmphore anachment sae 1 W -

Figure 3. Mutations in the phyB Gene.

We sequenced 6509 bp of the wild-type gene. The published cDNA sequence begins at nucleotide 2207 (Figure 2), and coding sequences are drawn as blocks. The chromophore attachment site is at cysteine- 357. Mutation hy3-4-777is a C-toT mutation at nucleotide position 3145 that changes a histidine codon to a tyrosine codon at amino acid resi- due 283. Mutation hy3-EMS742 is a G-toA mutation at nucleotide 3489 that changes a tryptophan codon to a stop codon at amino acid posi- tion 397. Mutation hy3-6064 is a C-toT mutation at nucleotide position 3640 that changes a glutamine codon to a stop codon at amino acid residue 448. Mutation hy3-8-36 is a G-toA mutation at nucleotide 3954 that changes a tryptophan codon to a stop codon at amino acid position 552. Mutation hy3-464-79 is a T-DNA insertion in a 0.15-kb EcoRI-Dral fragment encompassing nucleotides 5732 to 5877.

amino acid positions corresponding exactly to the introns in genomic PHYA genes previously sequenced from oat, maize, rice, and pea (Hershey et al., 1987; Sato, 1988; Christensen and Quail, 1989; Kay et al., 1989), and to the introns in a PHYB gene from potato (Heyer and Gatz, 1992). It is interesting that PHYA and PHYB genes each have the same conserved exonlintron structure.

We also sequenced -2 kb of DNA upstream of the previ- ously reported PHYB sequence. We found that the published cDNA sequence starts immediately after a natural EcoRl site in our genomic sequence. Because the library from which the cDNA clone was obtained was constructed by adding EcoRl linkers to the cDNA molecules and then digesting these with EcoRl before ligating to a vector (Crawford et al., 1988; Sharrock and Quail, 1989), it seems likely that the actual transcriptional start site lies upstream of the previously reported cDNA se- quence. In the presumptive promoter sequence, we found two G-box sequences (CACGTG) (Williams et al., 1992) at nucleo- tide positions 1830 to 1835 and 1865 to 1870 (Figure 2). Two other sequences, at nucleotide positions 1699 to 1704 and 1808 to 1813, had a single change from this palindromic consensus motif (Figure 2). We sequenced just 158 bp of the 3' noncod- ing region, because our clone extended no further (Methods). The polyadenylation site of the published cDNA sequence is 239 nucleotides downstream of the stop codon (Sharrock and Quail, 1989).

By DNA sequencing, we found point mutations in the PHYB gene from four EMS-generated hy3 mutants, diagrammed in Figure 3. Three alleles, hy3-6064, hy3-8-36, and hy3-EMS742,

have stop codons in the coding sequence (Figure 3). A fourth allele, hy3-4-777, has a missense mutation that causes a histidine-to-tyrosine change at amino acid residue 283 (Fig- ure 3). Gel blot hybridization analysis of genomic DNA from the T-DNA-generated allele hy3-464-79 indicated that it con- tains a T-DNA insertion in the PHYB gene (Figure 3). These results establish unequivocally that the HY3 locus encodes ,phytochrome 6. Henceforth, we refer to the mutations asphyf3.

Phenotypes of phyB Mutants

We found that somephyB mutants had longer hypocotyls than others, as shown in Figure 1 and Table 1. Mutants B064,8-36, M4084, EMS142, and 464-19 had the longest hypocotyls, whereas mutants 4-117,548, and 1053 had hypocotyl lengths intermediate between those of the wild type and the mutants having the longest hypocotyls (Table 1).

This long hypocotyl phenotype was caused by increased cell elongation. Hypocotyl epidermal peels from 13-day-old seedlings of the wild type and two phyB mutants are shown in Figure 4. The epidermal cells from the hypocotyl of null mu- tant 8-36 (Figure 4C) are -2.5 times as long as the same cells from the wild-type plant (Figure 4A; data not shown). Epider- mal cells from the hypocotyl of partia1 loss-of-function mutant 4-117 are intermediate in length (Figure 4B), -1.75 times the mean length of cells of the wild type (data not shown). Cell division in the hypocotyl was unaffected by the mutations, as the wild-type and mutant plants all had approximately the same number of cells along the length of the hypocotyl (data not shown).

Many other tissues in phyB mutant plants also appeared elongated. As shown in Figure 5, phyB mutant plants ha3 more elongated petioles and smaller leaf areas than did wild-type plants (Nagatani et al., 1991a). In addition, we found thatphyB plants were taller than wild-type plants, as shown in Figure

Table 1. Hypocotyl Lengths and Chlorophyll Contents of phyB Mutant Seedlingsa

Strain Length (mm) Seedling Hypocotyl pg Chlorophylll

Wild type (Ler) 2.7 f 0.5 3.9 hY 7 8.4 f 1.2 1 . I

PhyB-8-36 8.3 f 0.5 2.2 phyB-Bo64 8.1 f 0.6 2.0

PhyB-548 6.2 & 0.7 2.0 PhyB-1053 5.5 k 0.6 2.0

1.4 8.1 f 1.3 PhyB-464-19

phyB-M4084 8.0 * 1.1 2.7

PhyB-4-117 5.1 f 0.5 2.6 ~ ~~

a Hypocotyls and chlorophyll contents were measured 12 days after sowing. Hypocotyl lengths are based on measurements of 20 plants, and chlorophyll levels are based on measurements of four plants.

HY3 Encodes Phytochrome B 151

B

et al., 1989; Parks et al., 1989; Parks and Quail, 1991) (Table2). We found that they had even greater apical dominance thanphyB mutant plants, each hy1 plant having just a single stem.However, this stem was shorter than wild-type (and phyB)stems, and the number of siliques was lower. These pheno-types could have arisen because the hy1 mutant has a verylow chlorophyll content (see below), which may limit the growthof this mutant.

Interestingly, we also found morphological differences be-tween roots of wild-type and phyB plants. As illustrated in Figure7, root hairs of 1-week-old seedlings of a phyB mutant werelonger than root hairs of the wild-type strain. Dark-grown seed-lings had very few root hairs, but the root hairs present wereapproximately the same length in the mutant and the wild type(data not shown). This result suggests that perception of lightby PhyB normally inhibits root hair elongation. In contrast, theroots of phyB mutant seedlings were shorter than the rootsof wild-type seedlings, as shown in Table 3. The mean rootlength of phyB seedlings was approximately three-fourths thatof the mean root length of wild-type seedlings (Table 3). Thiswas true even in the dark (Table 3). This observation suggeststhat the shorter roots of phyB mutants did not depend on ab-sorption of light, and raises the possibility that PhyB may havesome activity in the absence of red light activation. Alterna-tively, the light treatment given prior to dark incubation to inducegermination of seeds (see Methods) may have been sufficientto affect subsequent root growth.

200

Figure 4. Hypocotyl Epidermal Peels of Wild-Type and phyB MutantPlants.

(A) Wild-type Landsberg erecte.(B) phyB mutant 4-117.(C) phyB mutant 8-36.Scale bar = 200 urn.

6. Measurements of 6-week-old plants, summarized in Table2, revealed thatpftyB plants had longer main stems than wild-type plants, fewer secondary stems, fewer stem termini (asa result of the decreased number of stems), and fewer siliques(fruits) on the main stem. These phenotypes indicate that thephyB mutants have greater apical dominance than the wildtype.

For comparison, we measured the same parameters for adulthy1 plants, probably deficient in multiple phytochromes (Chory

Figure 5. Three-Week-Old Wild-Type and phyB Plants.

(A) Wild-type Landsberg erecfa (Ler).(B) phyB mutant Bo64.(C) phyB mutant 4-117.(D) phyB mutant 8-36.The mutants have elongated petioles and flower early.

152 The Plant Cell

Figure 6. Five-Week-Old Adult Plants of Wild Type and Three phyBMutants.

The wild-type Landsberg erecta (Ler) plant is at left, and phyB mu-tants Bo64, 4-117, and 8-36 are at right.

In addition to these morphological phenotypes, phyB mu-tants exhibited physiological differences from wild-type plants.In agreement with others (Goto et al., 1991; Whitelam andSmith, 1991), we found that they flowered earlier than the wildtype, during both long and short days (data not shown). Consis-tent with their accelerated flowering, phyB mutant plants hadfewer rosette leaves than wild-type plants (Table 2; Whitelamand Smith, 1991;Chory, 1992). We also found that phyB seed-lings had less chlorophyll per seedling than wild-type seedlings(Table 1; Lifschitz et al., 1990). Furthermore, phyB leaf meso-phyll cells have fewer chloroplasts per cell than wild-typemesophyll cells (J. Chory, unpublished result), suggesting thatthe reduced chlorophyll level may arise partly from a decreasednumber of chloroplasts.

We observed a rough correlation between the chlorophylllevels of the mutants and their hypocotyl lengths. Thus, thepresumed null mutants described above, Bo64,8-36, EMS142,and 464-19, which had the most extreme hypocotyl phenotypes,all had low chlorophyll levels, whereas mutant 4-117, which wehypothesized to carry a partial loss-of-function mutation, hadan intermediate chlorophyll level (Table 1 and data not shown).In no case, however, was the chlorophyll content of a phyBmutant as low as that of the hyl mutant (Table 1).

Expression of the PHYB Gene

We analyzed PHYB mRNA in the mutants by RNA gel blot-ting. As shown in Figure 8A, PHYB mRNA could be detectedin all of the mutants. In the EMS-generated mutants, the mRNAwas the same size as the wild-type mRNA, ~4 kb. In the T-DNAinsertion mutant 464-19, however, the mRNA was substantiallylarger, ~7 kb. Presumably, the greater length of the PHYB tran-script in this strain is caused by T-DNA sequences fused tothe PHYB sequences. In addition, we observed that the levelof PHYB mRNA in the mutants was slightly lower than the wild-type level, except in mutant 4-117, where we observed no obvi-ous difference from the wild-type level (Figure 8A). All of themutants had levels of PHYA (phytochrome A) mRNA approxi-mately equal to the wild-type level (Figure 8A), showing thatthe phyB mutations do not affect expression of the PHYA gene.

We examined PhyB protein levels in these mutants usinga monoclonal antibody raised against the C terminus of thepresumptive PhyB protein from tobacco. It was found previ-ously that this antibody recognizes Arabidopsis PhyB proteinselectively (Nagatani et al., 1991 a). Protein gel blots using thisantibody failed to reveal PhyB protein in any of the phyB mu-tants except for mutant 4-117, which had a level approximatelyequal to the wild-type level (Figure 8B). Because the antibodyused was raised against the C-terminal portion of the protein,it would not have detected a truncated fragment of PhyB, asmight exist in mutants Bo64 and 8-36 (nonsense mutations)or 464-19 (T-DNA insertion allele).

DISCUSSION

We have shown here that Arabidopsis hy3 mutations fall inthe PHYB gene, encoding one of the five red/far-red light-

Table 2. Characteristics of 6-Week-Old Wild-Type, hy1, and phyB Plants8

Strain

Ler/I/7-21.84Nphy8-Bo64a Numbers are

Length ofMain Stem

22.7 ± 2.219.9 ± 1.627.8 ± 2.3

means ( ± standard

Number of Siliques(main stem)

23.3 ± 1.914.3 ± 2.016.7 ± 2.2

deviation) of measurements of 10

Number of Stems

2.7 ± 0.61.0 ± 01.3 ± 0.6

(for hy1) or 15 (for

Number ofTermini

10.7 ± 1.64.8 ± 1.57.3 ± 1.3

Ler and phyB) plants.

Number ofRosette Leaves7.9 ± 1.04.0 ± 1.25.3 ± 1.6

HY3 Encodes Phytochrome B 153

Figure 7. Root Hairs of Wild-Type and phyB Mutant Seedlings.

A root from a wild-type plant is at left, and a root from a phyB-8-36plant is at right. Seedlings were grown for 1 week in white light.

sensing phytochrome proteins in this plant. The various pheno-types of hy3(phyB) plants are thus the downstreammanifestations of alterations in a light signal transduction path-way (or pathways) having PhyB as the initial receptor. Theseare the only mutations shown to lie in a phytochrome gene,and they allow us to assess directly the role of this phytochromein Arabidopsis development.

The very long hypocotyls and the molecular phenotypes ofmutants 8-36, EMS142, Bo64, and 464-19 suggest that thesestrains have null or near null mutations. Mutant 4-117, on theother hand, appears to carry a partial loss-of-function muta-tion because it has an intermediate hypocotyl length. Sincemutant 4-117 contains the normal amount of PhyB protein, mu-tation phyB-4-117 probably changes the functional propertiesof the PhyB molecule. The molecular defect in this mutant,a histidine-to-tyrosine missense mutation, is consistent withthis hypothesis. We do not know the biochemical function af-fected by this mutation, but it is intriguing that it occurs in acharged region that is highly conserved among differentphytochromes from both monocots and dicots (Hershey et al.,1987; Sato, 1988; Christensen and Quail, 1989; Kay et al., 1989;Sharrock and Quail, 1989; Dehesh et al., 1991; Heyer and Gatz,1992; J. C. Lagarias, personal communication), as well as lowerplants (Hanelt et al., 1992; Maucher et al., 1992; Winands etal., 1992). The His-283 residue is conserved in all sequencedphytochromes, including PhyA and PhyC (Sharrock andQuail, 1989).

The multiplicity of phenotypes of phyB mutants underscoresthe importance of light quality in controlling plant development.Shade-avoiding plants inhibit elongation of hypocotyl and othertissues and promote leaf and chloroplast development in re-sponse to red light. In high fluences of far-red light, they growtall, show increased apical dominance, flower early, and makeless chlorophyll (Brown and Klein, 1971; Smith, 1982; Corre,1983; Martinez-Zapater and Somerville, 1990; Chory, 1991;

Thompson and White, 1991; Whitelam and Smith, 1991). Far-redgrowth patterns are constitutive in IhephyB mutants becausethe relevant photoreceptor (PhyB) is missing. Since plant shadeis richer in far-red light than direct sunlight, the various pheno-types of the phyB mutants may represent components of theplant's response to excessive shade (Smith, 1982). Other en-vironmental states affecting red/far-red light ratios, such as theonset of twilight or the latitude at which an individual plantgrows, might have similar effects on plant development (Smith,1982).

Our observation that root hairs are longer in thepftyB mu-tants than in the wild type is particularly intriguing, becauseroots are normally hidden from light. This phenotype may in-dicate that red light normally inhibits root hair growth. Otherresearchers have noted phytochrome effects in roots: for ex-ample, in regulating expression of the asparagine synthetasegene (Tsai and Corruzzi, 1991), and in promotion of gravitropism(Lake and Slack, 1961; Feldman, 1984; Kelly and Leopold,1992). Phytochrome has been found to be expressed in rootcaps of maize (Johnson et al., 1992). In preliminary experi-ments, we failed to observe any effect of light on rootgravitropism in Arabidopsis, and we did not notice any defectin gravitropism in thephyS mutants (J. W. Reed, unpublishedresults).

Comparison of hy3(phyB) mutant phenotypes to hy1, hy2,and hy6 mutant phenotypes shows that the relative contribu-tion of PhyB to phytochrome-mediated responses depends onthe particular response. The hy1, hy2, and hy6 mutants aredeficient in phytochrome chromophore attachment or avail-ability (Chory etal., 1989; Parks etal., 1989; Parks and Quail,1991). Presumably, they are deficient in multiple phytochromespecies. These mutants have hypocotyls only slightly longerthan phyB mutant hypocotyls (Table 1; Koornneef et al., 1980;Chory, 1992; J. W. Reed, unpublished results), flower slightlyearlier than phyB mutants (Table 2; Chory, 1992), have sub-stantially less chlorophyll (Table 1; Koornneef et al., 1980;Lifschitz et al., 1990; Chory, 1992), and have defects in redlight induction of gene expression not exhibited by phyB mu-tants (Chory et al., 1989; Sun and Tobin, 1990). Thus, PhyBappears to play a major role in inhibition of hypocotyl elonga-tion by red light, significant roles in controlling flowering andinducing chlorophyll accumulation, and no part in red light in-duction of gene expression in dark-grown plants. Therefore,phytochromes seem to have distinct but partially overlapping

Table 3. Lengths of Wild-Type and phyB Seedling Roots3

StrainRoot Length(light)

Root Length(dark)

LerphyB-8-36

25.7 ± 7.1 mm17.9 ± 6.2 mm

22.9 ± 6.9 mm16.2 ± 6.8 mm

a Root lengths were measured 8 days after sowing on plates incu-bated vertically. Ler, wild type Landsberg erecfa.

154 The Plant Cell

00

phyB

phyA

rDNA

B

PhyB

- S <s0) O °?

_J CQ CO8 £o V

I 1•'ii.*1'

Figure 8. RNA and Protein Gel Blot Analysis of Wild-Type and phyBMutant Plants.

(A) Blots of RNA from wild-type (Ler) and phyB mutant plants probedwith a PHVB-specific fragment, a PHVW-specific fragment, and aribosomal DMA (rDNA) fragment. The data are from two RNA gel blots,one probed with the PHYB-specific fragment and the rDNA fragment,and the second probed with the PHYiQ-specific fragment. When theblot used for PHYA was reprobed with an rDNA fragment, the appar-ent variation in PHYA transcript levels among strains was found to bedue to unequal amounts of RNA in different lanes (data not shown).The same RNA preparations were used for both blots.(B) Gel blot of proteins from wild-type (Ler) and phyB mutants probedwith the anti-PhyB monoclonal antibody.

functions. Differences in the biochemistry of signal transduc-tion mediated by the various phytochromes might account forthe apparently distinct developmental roles of the differentphytochrome isoforms, or they might be expressed in differ-ent tissues of the plant or at different times during development.

The finding that overexpression of PHYB in transgenicArabidopsis plants caused a short hypocotyl phenotype is con-sistent with the conclusion that PhyB is the principalphytochrome involved in controlling hypocotyl elongation(Wagner et al., 1991). However, overexpression of PHYA genesin tobacco, tomato, or Arabidopsis can also inhibit stem or

hypocotyl elongation (Boylan and Quail, 1989,1991; Keller etal., 1989; Nagatani et al., 1991b; Cherry et al., 1992). Theseresults suggest that PhyA can mediate inhibition of cell elon-gation, at least when overproduced substantially. More carefulcharacterization of the light responses of such transgenic plantshas suggested that the responses seen reflect the unusuallyhigh levels of PhyA present, and that these responses aretherefore aberrant (McCormac et al., 1992). In the PHYA over-expression studies, PhyA appears to be able to play a rolenormally performed by PhyB. Mutants defective specificallyin PhyA have not been described, but if PhyB really is the mostimportant phytochrome in mediating inhibition of hypocotylelongation, we would expect that phyA mutants would not haveelongated hypocotyls in white light screens. These experimentsunderscore the importance of considering the phenotypes ofmutants lacking a particular phytochrome, as well as pheno-types caused by overexpression of a gene for that phytochrome,to reach conclusions regarding biological function.

The biochemical targets of PhyB signal transduction arenot known. Different tissues exhibit different types of de-ficiencies in the phyB mutants. Thus, leaf mesophyll cellsproduce fewer chloroplasts and less chlorophyll in the mutant;apical meristematic cells undergo the switch from vegetativeto inflorescence meristem growth prematurely; and hypocotyl,stem, petiole, and root hair cells elongate more. Perhaps lev-els of (or sensitivities to) some hormone or metabolite thatcontrols these processes are affected in the mutants. It hasbeen shown that an increase in cytosolic calcium levels pre-cedes, and can induce, swelling of etiolated wheat protoplasts(Shacklock et al., 1992), implicating calcium in phytochromesignal transduction. Auxins, gibberellins, and brassinosteroidsare known to cause cell elongation (Cleland, 1987; Metraux,1987; Reid, 1987; Mandava, 1988), and phyB mutations maywell affect synthesis or action of one of these plant hormones.Other researchers have found that a protein correspondingto a phytochrome is missing in a sorghum variant that over-produces gibberellins (Beall et al., 1991; Childs et al., 1991,1992). This variant, which carries the ma3R allele at the MASmaturity locus, flowers early and has elongated leaf sheathsand leaf blades (Pao and Morgan, 1986). These phenotypesare reminiscent of those of the phyB mutants described here.Similarly, an oilseed rape mutant that overproduces gibberel-lins is missing a phytochrome (Rood et al., 1990; Devlin et al.,1992). Farther down the pathway, changes in cell wall proper-ties probably account for the increased cell elongation inhypocotyls, root hairs, and other tissues (Taiz, 1984; Casal etal., 1990; Kigel and Cosgrove, 1991).

The identification of mutations in the PHYB gene will facili-tate more detailed investigation of light signal transduction andlight-regulated development in Arabidopsis. By characterizingadditional point mutations, it will be possible to learn moreabout the biochemical activity of this unique light-absorbingprotein. The mutants will also allow further biochemical andphysiological studies aimed at unraveling the downstream sig-nal transduction pathways initiated by absorption of light byphytochrome.

HY3 Encodes Phytochrome 6 155

METHODS were visualized using an alkaline phosphatase-conjugated goat anti-mouse antibody kit from Bio-Rad.

Plant Growth and Phenotyplc Analysis

Seeds were surface sterilized and plated on Murashige-Skoog/sucrose/ phytagar plates (1 x Murashige-Skoog salts, 2% sucrose, 0.8% phyt- agar [GIBCO]), stored overnight at 4OC, and grown in light chambers (16-hr day/8-hr night) kept at 22OC. Seedlings were transferred to soil 10 days to 2 weeks after sowing and grown in a greenhouse. For root observations, the seeds were sown in a line across MS/sucrose/phytagar plates, and the plates were placed vertically instead of horiztlntally. Dark-grown seedlings were first induced to germinate by treatment with white light for 16 to 24 hr. Chlorophyll measurements were as de- scribed by Chory (1992).

Cloning of phyB and Sequencing of Mutant Alleles

A clone containing phytochrome B (PHY6) gene sequences was de- tected in a library of Arabidopsis thaliana ecotype Landsberg erecta genomic DNA sequences (Voytas et al., 1990) cloned in X Fix (Stratagene), using a probe obtained by polymerase chain reaction (PCR) amplification from genomic DNA. The primers used to amplify the PCR probe were 5‘-GACTCATATGATGGCGGGGGAACAG-3’ (con- tains an introduced Ndel restriction site followed by nucleotides 4216 to 4230; Figure 2) and 5’-GCTCAAAGGATTCTTTATCACCIGACAAAT-3’ (nucleotides 5218 to 5189; Figure 2), and were designed based on the published cDNAsequence of PHYB from ecotype Columbia(Sharrock and Quail, 1989). The PHYB clone obtained was found to lack the 3‘ end of the gene, so we amplified a fragment of Landsberg DNA by PCR using primers complementary to the 3’ end of the published Columbia cDNA sequences. The primers used were 5’TCTGTTTCT- TGCAAATCCCGAGC-9’ (from nucleotide 5088 to nucleotide 5110 in Figure 2) and 5’-GCTCTAGAGCTGAACGCAAATAATCTCCCí3‘ (contain- ing an Xbal site near the 5’end, and nucleotides 6509 to 6487 of the PHYB gene sequence; Figure 2). The clones were sequenced by stan- dard methods. A portion of the gene, including the promoter, was sequenced by Lark Sequencing Technologies, Inc. (Houston, TX). Mu- tant phyB alleles were sequenced by amplifying segments of the gene by PCR, reamplifying these fragments by asymmetric PCR, and se- quencing these products directly as described by Beitel et al. (1990). The mutation in strain 8-36 was localized before sequencing by dena- turing gradient gel electrophoresis (Myers et al., 1987, 1989).

RNA and Protein Analyses

RNA was purified as described previously by Chory et al. (1989). For RNA gel blot hybridizations, a purified 1.1-kb Xbal fragment contain- ing nucleotides 5380 to 6509 (Figure 2; the latter Xbal site came from the PCR primer used to clone the 3’end of the gene, see above) was used as a PHYB probe; a PCR product, made by amplifying Landsberg genomic DNA with two primers designed from the published PHYA cDNA seauence (Sharrock and Quail. 1989). was used as a PHYA Drobe.

ACKNOWLEDGMENTS

We thank Daniel Voytas for the Landsberg erecta chromosomal library; Maarten Koornneef, Janny Peters, Richard Kendrick, and Ken Feldman for identifying and making available the T-DNA insertion allele of hy3; Alan Pepper for designing the PCR primers used to amplify a probe for the phy6 gene; Akira Nagatani for supplying anti-PhyB antibody; Nathan Aguilar for help with plant DNA preps; and Marc Lieberman for plant photography. This research was supported by a grant from the Samuel Roberts Noble Foundation (Ardmore, OK) to the Salk In- stitute (San Diego, CA), and by grants from the U.S. Department of Energy (DOE), the U.S. Department of Agriculture, and the National Science Foundation to J. C. Part of the sequencing work was supported by funds from the Frontier Research Prcgrams, RlKEN Institute. J. W. R. is a DOE-Energy Biosciences Research Fellow of the Life Sciences Research Foundation (Baltimore, MD), and !? N. was partially sup- ported by a grant from Schering-Plough (Kenilworth, NJ). The cover photograph was taken by Marc Lieberman, and the cover artwork was by Liz Grabowski and Cynthia Doane.

Received November 19, 1992; accepted December 21, 1992.

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DOI 10.1105/tpc.5.2.147 1993;5;147-157Plant Cell

J W Reed, P Nagpal, D S Poole, M Furuya and J Choryphysiological responses throughout Arabidopsis development.

Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and

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