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The Plant Cell, Vol. 5, 757-768, July 1993 O 1993 American Society of Plant Physiologists

Phytochrome A Null Mutants of Arabidopsis Display a Wild-Type Phenotype in White Light

Garry C. Whitelam,' Emma Johnson,' Jinrong Peng,b Pierre Carol,b Mary L. Anderson,b9' John S. COWI,~~* and Nicholas P. HarberdbV3 a Department of Botany, University of Leicester, University Road, Leicester LEI 7RH, United Kingdom

Cambridge Laboratory, John lnnes Centre, Colney, Norwich NR4 7UJ, United Kingdom

Phytochrome is a family of photoreceptors that regulates plant photomorphogenesis; the best-characterized member of this family is phytochrome A. Here, we report the identification of nove1 mutations at three Arabidopsis loci ( fhyl, fhy2, and fhy3) that confer an elongated hypocotyl in far-red but not in white light. fhy2 mutants are phytochrome A deficient, have reduced or undetectable levels of PHYA transcripts, and contain structural alterations within the P HYA gene. When grown in white light, fhy2 mutants are morphologically indistinguishable from wild-type plants. Thus, phytochrome A appears to be dispensable in white light-grown Arabidopsis plants. fhy2 alleles confer partially dominant phenotypes in far-red light, suggesting that the relative abundance of phytochrome A can affect the extent of the far-red-mediated hypocotyl growth inhibition response. Plants homozygous for the recessive fhyl and fhy3 mutations have normal levels of functional phytochrome A. The FHYl and FHY3 gene products may be responsible for the transduction of the far-red light signal from phytochrome A to downstream processes involved in hypocotyl growth regulation.

INTRODUCTION

Phytochrome is a regulatory photoreceptor that controls plant growth and development in response to signals from the light environment. Phytochrome is composed of a linear tetrapyr- role chromophore attached to an apoprotein moiety and exists in two stable photointerconvertible forms, Pr and Pfr. Photochro- micity between inactive, red light (R)-absorbing Pr and active, far-red light (FR)-absorbing Pfr endows phytochrome with the capacity to sense the relative amounts of R and FR (Smith and Whitelam, 1990). It has recently been established that higher plants possess multiple, discrete phytochrome species, the apoprotein components of which are the products of a small family of divergent genes called PHYA, PHYB, PHYC, PHYD, and PHY€(for review see Quail, 1991). Through comparisons of peptide sequence and deduced amino acid sequence, it is now accepted that phytochrome A, the apoprotein of which is the PHYA gene product, is equivalent to the well- characterized light-labile or type I phytochrome (Quail, 1991).

Phytochrome A accumulates to relatively high levels in etiolated plant tissues, but its levels are rapidly depleted upon exposure of such tissue to R or white light (W). This depletion is due to light-induced degradation of Pfr and to light-mediated

Current address: Nottingham Arabidopsis Stock Centre, Department of Life Sciences, University of Nottingham, University Park, Notting- ham NG7 2RD, United Kingdom. * Current address: John lnnes Institute, John lnnes Centre, Colney, Norwich NR4 7UH, United Kingdom.

To whom correspondence should be addressed.

down regulation of PHYA gene expression (Quail, 1991). Al- though phytochrome A is still present in fully deetiolated plants, type II or light-stable phytochromes, such as phytochromes 6 and C, predominate (Smith and Whitelam, 1990; Quail, 1991).

The existence of multiple phytochrome species together with the diversity of phytochrome responses and response modes have led to the suggestion that different phytochromes may have discrete roles or functions (Smith and Whitelam, 1990). Current understanding of phytochrome function has been greatly advanced through the analysis of the long hypocotyl mutants of Arabidopsis (hyl to hy7) that display an elongated hypocotyl when grown in W (Koornneef et al., 1980; Chory et al., 1989; Chory, 1991). These mutants have lost the capacity for the light-mediated inhibition of hypocotyl growth exhibited by wild-type individuals. hyl, hy2, and hy6 mutants appear to be chromophore depleted, thus causing a corresponding depletion in the levels of active phytochrome (both light-labile and light-stable phytochromes; Choryet al., 1989; Parks et al., 1989; Parks and Quail, 1991). hy3 mutations map to the PHYB gene, and hy3 mutants are specifically deficient in phytochrome B activity (Nagatani et al., 1991; Somers et al., 1991; Reed et al., 1993). Thus, phytochrome 6 is the phytochrome species predominantly responsible for W-mediated hypocotyl growth inhibition, and hyl, hy2, and hy6 mutants probably exhibit an elongated hypocotyl in W because of a reduction in phytochrome 6 activity.

hy3 seedlings display normal inhibition of hypocotyl growth in response to prolonged FR but lack a hypocotyl growth

758 The Plant Cell

inhibition response to R (Koornneef et al., 1980; McCormac et al., 1993). This observation is fully consistent with physiological studies indicating that light-labile phytochrome is responsible for the FR-mediated hypocotyl growth inhibition of etiolated seedlings (the so-called FR high-irradiance response) and that a light-stable phytochrome mediates the effect of R (e.g., Beggs et al., 1980). Thus, phytochrome A null mutants might be expected to exhibit an elongated hypocotyl when grown in FR. Two recent papers have described the isolation of ethylmethane sulfonate (EMS)-induced Arabidopsis mutants (hy8 and frel) that exhibit an elongated hypocotyl in FR and are deficient in functional phytochrome A (Nagatani et al., 1993; Parks and Quail, 1993). Here, we describe the isolation and characterization of further mutants that display an elongated hypocotyl in FR (but not in W). Analysis of these mutants has enabled us to extend the conclusions of previous studies (Nagatani et al., 1993; Parks and Quail, 1993). The mutations identified belong to three distinct complementation groups (fhyl, fhy2, and fhy3). Physiological and molecular experiments showed that the fhy2 mutants are phytochrome A deficient and carry mutations in the PHYA gene. fhyl and fhy3 identify previously undescribed genetic loci that may play a role in the transduction of the signal from phytochrome A to subsequent steps in the FR response pathway.

Because phytochrome is an important regulatory molecule, it might be expected that alterations in the abundance of phytochrome would have significant effects on the processes it controls. To investigate this possibility, we have studied the effect of heterozygosity for mutations conferring phytochrome A deficiency (fhy2-7, fhy2-2) on hypocotyl elongation in FR. The results of these experiments show that these alleles display partial dominance and suggest that reduced phytochrome A abundance in fhy2 heterozygotes results in a significant length- ening of the hypocotyl of plants grown in FR.

RESULTS

lsolation and Complementation Analysis of Mutations Conferring an Elongated Hypocotyl in FR

Mutants displaying an elongated hypocotyl in FR were isolated from EMS and y-irradiation-mutagenized M2 populations of Arabidopsis (for details see Methods). These mutants were then transplanted to soil, grown in W, and allowed to self- pollinate. The resultant (M3) seedlings were then tested for the occurrence of elongated hypocotyls in FRand W. Mutant lines displaying elongated hypocotyls in W were excluded from further analysis. Four independent mutant lines displaying elongated hypocotyls only in FR were studied further. Com- plementation analysis of these four mutant lines was performed by examining the phenotypes of F, double heterozygotes obtained in crosses between them (all possible combinations). These experiments showed that the four mutant lines fel1 into

three complementation groups, fhyl (for far-red elongated hypocotyl), fhy2 (at which two alleles, fhy2-7 and fhy2-2, were identified), and fhy3 (data not shown). fhy7 fhy3 double heterozygotes displayed a hypocotyl of a length that was indistinguishable from that of the wild type when grown in FR. fhy7 and fhy3 are therefore complementing mutations at two separate genetic loci, FHYl and FHY3, respectively. fhy2-7 fhy2-2 double heterozygotes displayed an elongated hypocotyl of a length that was indistinguishable from that of either mutant homozygote parent when grown in FR. The fhy2-7 and fhy2-2 mutant lines therefore contain noncomplementing, allelic mutations at the FHM locus. Analysis of fhyl fhy2 and fhy2 fhy3 double heterozygotes was more complex due to the partial dominance exhibited byfhy2 alleles (see below). These double heterozygotes displayed a slightly more elongated hypocotyl than the wild type in FR. However, in both cases, the degree of hypocotyl elongation exhibited by the double heterozygote in FR was not as great as that exhibited by either parenta1 homozygote. Thus, the fhy2-7 and fhy2-2 alleles were both assigned to a complementation group distinct from those of fhyl and fhy3.

The fhyl and fhy2 mutants were isolated from the r-ray- treated MP population, and the fhy3 mutant was isolated from the EMS-treated M2 population. We chose to designate these loci fhy so as to clearly distinguish them from previously described hy loci (hyl to hyq Koornneef et al., 1980; Chory et al., 1989; Chory, 1991), at which mutations conferring an elongated hypocotyl in W have been obtained. Because of their profoundly differing photosensitivities, it seems unlikely that any of the fhyl to fhy3 mutations are allelic to any of the hy l to hy7 mutations. For reasons that will become apparent, it is very likely that the fhy2 mutations are allelic to mutations at the hy8 and frel loci that confer a deficiency in functional phytochromeA(Nagatani et al., 1993; Parks and Quail, 1993). However, because complementation tests with hy8 and frel alleles have not been performed, it would be premature to reas- sign the fhy2 mutations to the hy8 or frel complementation groups at present.

Phenotypes of fhyl, fhy2, and fhy3 Mutants

Plants homozygous for the fhy2-7 and fhy2-2 mutations display a hypocotyl phenotype strikingly different from that of the wild type when grown in FR. In these conditions, fhy2 mutant homozygotes are almost identical to dark-grown wild-type plants, exhibiting an extremely elongated hypocotyl, closed hypocotyl hooks, and unexpanded cotyledons. In contrast, wild- type plants have short hypocotyls and expanded cotyledons when grown in FR. fhy2 mutant homozygotes do not differ significantly from wild-type plants when grown in W or R. These data are presented in Figures 1 and 2. When grown in standard glasshouse conditions, fhy2 mutants are morphologically indistinguishable from the wild type at all stages of the life cy- cle (data not shown). Plants homozygous for fhy7-7 and fhy3-7

Phytochrome A Mutants of Arabidopsis 759

Figure 1. Phenotypes Exhibited by ftiy Mutants and Their Correspond-ing Wild Types.

Seeds of fhyl, fhy2-1, fhy2-2, and fhy3 and their corresponding wildtypes (wtL Landsberg erecta for fhyl and fhy2; wtc Columbia for fhy3)were sown on agar plates and germinated in the dark for 1 day, asdescribed in Methods. Seedlings were grown for an additional 4 daysin the dark (D) or under continuous R, continuous FR, continuous B,or continuous W.

also display an elongated hypocotyl selectively in FR (and notin R or W; Figures 1 and 2). Cotyledon expansion and hypocotylhook opening are inhibited in FR-grown fhyl-1 and fhy3-1 mu-tants (Figure 1). Compared with their respective wild types,blue light (B)-treated fhyl, fhy2, and fhy3 mutant hypocotylsare all slightly elongated (Figures 1 and 2). That light-labilephytochrome (phytochrome A) mediates at least some of theeffects of B on the inhibition of hypocotyl elongation has beenconcluded from earlier physiological experiments (Beggs etal., 1980).

FHY2 Maps in the Vicinity of PHYA

To obtain the chromosomal locations of the FHY1 and FHY2loci, we mapped them with respect to the site of insertion ofthe T-DNA in transformant line A264. This line carries a T-DNAinserted into the top arm of chromosome 1 (Peng and Harberd,1993) within DMA spanned by yeast artificial chromosomesEG11A5 and EG12E7 (Grill and Somerville, 1991; C. Recknageland G. Coupland, unpublished data). EG11A5 and EG12E7

also both contain PHYA, the structural gene for the phytochromeA apoprotein (J. Peng and N. P. Harberd, unpublished data).Because EG11A5 and EG12E7 contain Arabidopsis DNA in-serts of ~150 kb (Hwang et al., 1991; J. Peng and N. P. Harberd,unpublished data), it follows that the T-DNA insertion site islikely to be within 150 kb of PHYA.

Plants homozygous for fhyl were crossed with plantshomozygous for the T-DNA. The resulting FT plants were

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Figure 2. Photocontrol of Hypocotyl Elongation in Wild-Type and fhyMutant Arabidopsis Seedlings.

Seeds of fhyl, fhy2-1, fhy2-2, and fhy3 and their corresponding wildtypes (as given in Figure 1) were sown on agar plates, germinated inthe dark for 1 day, and grown for an additional 4 days in the dark (D)or under continuous R, continuous FR, continuous B, or continuousW (as shown in Figure 1). The mean hypocotyl lengths of at least 30seedlings from each light treatment are plotted. Error bars representthe standard errors of the means.

760 The Plant Cell

~

Table 1. Segregation of Phenotypic Classes Obtained following the Backcross of fhylh'hyl; KVKS x fhvl/FHY7; KVK"

~~ ~~ ~ ~

Phenotypes Tallb ShortC

Resistantd Sensitivee Resistant Sensitive

41 45 41 40

kanamycin resistance conferred by the T-DNA (A264), is dominant to kanamycin sensitivity (Ks).

fhylh'hyl homozygote, elongated hypocotyl in FR. fhyVFHY7 heterozygote, normal length hypocotyl in FR. Kanamycin resistant.

e Kanamycin sensitive.

backcrossed with plants homozygous for fhyl. The backcross progeny were used to score segregation for kanamycin resistance (conferred by the T-DNA) and an elongated hypocotyl in FR (conferred by fhy7). These data are presented in Table 1. As expected, an approximate segregation of 1 plant displaying an elongated hypocotyl in FR (tall; fhyVfhy7) to 1 plant displaying a wild-type hypocotyl length in FR (short; FHY7Bhy-I) was observed. In both of the tal1 and short classes, approximately one-half of the plants were kanamycin resistant and approximately one-half were kanamycin sensitive. Thus, fhy7 segregated independently of the T-DNA, showing that fhy7 is not linked to the T-DNA insertion site and is not in the vicinity of PHYA.

Plants homozygous for fhy2-7 were crossed with plants homozygous for the T-DNA. The resulting F1 plants were permitted to self-pollinate. Fp populations were used to score for kanamycin resistance and for an elongated hypocotyl in FR. As expected, an approximate segregation of 3 plants displaying a relatively short hypocotyl in FR (fhy2-VFHY2, FHWFHY2) to 1 plant displaying an elongated hypocotyl in FR (tall; fhy2- 7Ifhy2-7) was observed (3136 short, 974 tall). Of the 974 tal1 plants observed, none was resistant to kanamycin. Thus, recombinant plants displaying an elongated hypocotyl in FR (due to homozygosity for fhy2-7) and kanamycin resistance (conferred by the T-DNA) were not detected. These data give a maximum estimate linkage map distance of 0.05 centimor- gans (cM) between fhy2-7 and the T-DNA. Thus, fHY2 is closely linked with the T-DNA insertion site and with PHYA, and fhy2-7 may well be a mutant allele of PHYA. This latter conclusion is strengthened by the physiological and molecular analyses presented below.

Spectroscopic and lmmunochemical Analysis of Phytochrome in fhyí, fhy2, and fhy3 Mutants

In vivo spectrophotometric measurements revealed that etiolated fhyl-7 and fhy3-7 seedlings possess levels of spegrally active phytochrome that do not differ appreciably from those of their respective wild types. On the other hand, as shown in Figure 3, etiolated fhy2-7 and fhy2-2 seedlings contain levels

of spectrally active phytochrome that are below the limit of detection of the spectrophotometer. Because the spectral activity of phytochrome from etiolated tissues is very largely determined by the abundance of light-labile phytochrome (e.g., Brockmann and Schafer, 1982; Somers et al., 1991), it can be concluded that fhy2 seedlings are deficient in spectrally active phytochrome A.

This conclusion is supported by immunoblot analyses of phytochrome A polypeptide levels in wild-type and fhy seedlings. Whereas extracts of etiolated fhyl-7 and fhy3-7 seedlings contain more or less wild-type levels of immunochemically detectable phytochrome A polypeptide, no detectable staining is observed for extracts of either fhy2-7 or fhy2-2 seedlings, as shown in Figure 4. Thus, the fhy2-7 and fhy2-2 mutations appear to reduce phytochrome A abundance to levels below the limit of detection, whereas phytochrome A levels appear normal in fhy7-7 and fhy3-7 seedlings.

lsolation and Characterization of Arabidopsis Genomic DNA Clones Containing the PHYA Gene

To obtain hybridization probes for use in RNA and DNA gel blot analyses of the fhy mutants, Arabidopsis genomic DNA

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L Figure 3. In Vivo Spectral Detection of Phytochrome in WildType and fhy Mutant Seedlings.

Seeds of fhy7, fhy2-7, fhy2-2, and fhy3 and their corresponding wild types (wtL Landsberg erecta for fhy7 and fhy2; wtc Columbia for fhy3) were germinated in the dark for 6 days, and phytochrome contents of the seedlings were determined by in vivo spectrophotometry, as described in Methods. The error bars represent standard deviations of the means of three samples.


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45

clones containing PHYA were isolated from a genomic DMAclone library (see Methods). Two such clones, KPHYA81 andXPHV5H91, were selected for further analysis. Restriction frag-ment maps of XPHV5A81 and XPHYA91 are shown in Figure 5.Hybridization of oligonucleotide probes (phyAS' and phyA3';see Methods) containing sequence close to the 5' and 3' endsof the phytochrome A cDNA (see Methods; Sharrock and Quail,1989) to digests of XPH//H81 and XPHM91 DNA permitted theidentification of approximate 5' and 3' ends of the PHYA gene,as shown in Figure 5. The entire PHYA gene together with 5'and 3' flanking genomic DNA is contained within the overlap-ping XPHVB81 and XPHV/491 clones. DNA from XPHY5H81 andXPHV/491 was used as hybridization probes in the RNA andDNA gel blot experiments described below.

29-

Figure 4. Immunological Detection of Phytochrome A in Wild-Type andfhy Mutant Seedlings.Protein extracts, enriched for phytochrome by ammonium sulfateprecipitation, were prepared from 6-day-old etiolated wild-type (wtL,Landsberg erecta; wtc, Columbia) and fhy mutant seedlings. Eachlane was loaded with 25 ng of protein.

Analysis of PHYA mRNA Transcripts in fhy2 Mutants

The abundance of PHYA mRNA in fhy2-1 and fhy2-2 mutanthomozygotes was analyzed in RNA gel blot experiments. Etio-lated plant material was used in these experiments becausePHYA transcripts are known to accumulate to relatively highlevels in these conditions (Sharrock and Quail, 1989). TotalRNA was isolated from etiolated wild-type and mutant seedlings

PHYA

1 i X. Z

XPHYA81

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5' 3*'

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1XPHYA91

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Figure 5. Restriction Fragment Maps of Genomic DNA Clones Containing PHYA.

The inserts of XPHV5481 and XPHM91 are Arabidopsis genomic DNA fragments containing the PHYA gene. Restriction fragment maps of theseclones are compared as shown. The position of the PHYA gene is based on approximate 5' and 3' transcript end positions determined usingoligonucleotides phyAS' and phyA3'(see Methods). The Sail site at the ends of both clone inserts is from the cloning site of the XFIX II cloningvector. The Hindlll site shown very close to, and 3' of, the phyAS' position may represent a sequence polymorphism between Landsberg erecfaand Columbia DNA because this site is not present in the phytochrome A cDNA sequence (Sharrock and Quail, 1989).

762 The Plant Cell

i

— 4.0kb

— 1.2kb

of the PHYA gene (see Figure 5). This restriction fragment al-teration cannot be due to the occurrence of a mutation creatinga novel EcoRI site within the PHYA gene in fhy2-1 becausethe sum of the sizes of the new fragments observed (9.1 +1.3 = 10.4 kb) would then be expected to be equal to 6.2 kb.Furthermore, \PHYA91 also detects novel fragments in Hindllldigests of genomic DMA from fhy2-1 (data not shown). Thus,the fhy2-1 mutant line contains a rearrangement mutation ofPHYA. As expected, restriction fragment alterations were notobserved when EcoRI digests of DMA from fhy1-1 and fhy3-1were probed with the PHYA gene (data not shown).

Experiments to define more precisely the site of the rear-rangement mutation in the PHYA gene of the fhy2-1 mutantare shown in Figure 8. Probes 1, 2, and 3 are internal DMAfragments purified from an Arabidopsis phytochrome A cDNA(Sharrock and Quail, 1989; for locations of probes 1, 2, and3, see Figure 8A). Probe 1 hybridizes with the 9.1-kb, probe2 with both the 9.1- and 1.3-kb, and probe 3 with the 1.3-kb

Figure 6. Expression of PHYA Transcripts in fhy2 Mutants and the WildType.A radiolabeled Sail fragment (purified from XPHVH81; see Figure 5)containing the Arabidopsis PHYA gene plus ~4 kb of flanking DNAdetected an ~4-kb PHYA message in total RNA isolated from dark-grown FHY2 (wild-type) seedlings. This message was absent in dark-grown fhy2-1 and present at greatly reduced abundance in dark-grownfhy2-2 mutant seedlings. The probe also detected a 1.2-kb RNA spe-cies (identity unknown, possibly homologous to DNA flanking PHYA),which served as a control for equal sample loading.

9.1 kb —

I— 6.2 kb

(see Methods). A radiolabeled hybridization probe containingthe PHYA gene (a subfragment of XPWVA81) identified a 4-kbtranscript that was present in the wild type, present at greatlyreduced levels in fhy2-2, and not detectable in fhy2-1, as shownin Figure 6. The levels of PHYA transcript in fhy1-1 and fhy3-1were not significantly different from that of the wild type (datanot shown).

fhy2-1 Mutants Show a Structural Rearrangement ofthe PHYA Gene

Irradiation mutagenesis of Arabidopsis often generates dele-tion (Wilkinson and Crawford, 1991) or rearrangement (Shirleyet al., 1992) mutations. To determine if the PHYA gene in theirradiation-induced fhy2 mutants had sustained any such al-terations, its structure was investigated in DNA gel blotexperiments. The data are presented in Figures 7 and 8. A hy-bridization probe containing the entire Arabidopsis PHYA gene(XPHV/491) detected novel EcoRI fragments (9.1 and 1.3 kb) inDNA from fhy2-1. These novel fragments replace a 6.2-kb EcoRIfragment seen in DNA from the wild type (FHY2) and fhy2-2(Figure 7). The 6.2-kb fragment seen in FHY2 contains most

— 3.5 kb

1.3 kb —

Figure 7. Identification of Restriction Fragment Alterations in a fhy2Mutant.A radiolabeled genomic X DNA clone (XPHVH91; insert size 14.5 kb)containing the entire Arabidopsis PHYA gene plus flanking DNA (seeFigure 5) detected restriction fragment alterations in EcoRI-digestedgenomic DNA from fhy2-1 with respect to EcoRI-digested DNA fromfhy2-2 and the wild type (FHY2). The differences between fhy2-1 andFHY2 are not due to partial digestion of DNA because a control genomicDNA fragment detected identical restriction fragment patterns in alllanes when hybridized with the same filter (data not shown). The 6.2-and 3.5-kb EcoRI fragments detected by XPHVA91 in FHY2 DNA areequivalent to the internal 6.2- and 3.5-kb EcoRI fragments of XPHVH91(see Figure 5). The remaining unlabeled fragment (in all samples) isdetected by DNA flanking the 3.5-kb EcoRI fragment at the 3' extremityof XPHV/491. The DNA flanking the 6.2-kb EcoRI fragment at the 5' ex-tremity of XPHVH91 may be of insufficient length to give a detectablesignal (see Figure 5).


P/jyA cDNAATG Hind III FcoRV Eco R\ TAG

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•^623 bp +- -^—766 bp —*- -^—————

1 2Probe used in (B)

Site of rearrangement

Likely intron position in the genomic sequence

-1240bp-

3

x~\

1-9.1 kb-l

6.2 kb-l 1—6.2 kb —

.5 kb —I

1.3 kb—I —1.3 kb

Figure 8. Location of the Rearrangement Junction in the PHYA Gene in fhy2-1.

(A) Restriction fragment map of the Arabidopsis phytochrome A cDNA (calculated from sequence data; Sharrock and Quail, 1989), identifyingfragments 1, 2, and 3 used in the DMA blot hybridization experiments shown in (B). The positions of the translation start (ATG) and stop (TAG)codons are as shown. The likely positions of introns in the genomic DMA sequence of PHYA (based on the fact that intron positions are conservedin the PHYA genes of oat, maize, rice, and pea [Hershey et al., 1987; Sato, 1988; Cristensen and Quail, 1989; Kay et al., 1989] and in the PHYBgene of Arabidopsis [Reed et al., 1993]) are as indicated. The highlighted region shows the EcoRV-Kpnl fragment within which the rearrangementjunction in fhy2-1 lies.(B) Radiolabeled subfragments of the Arabidopsis phytochrome A cDNA (probes 1, 2, and 3; see above) also detected restriction fragment differ-ences between EcoRI digests of DMA from fhy2-1 and FHY2. In addition to the 6.2-kb EcoRI fragment in FHY2 (1.3 kb in fhy2-l), probe 3 alsodetects the 3.5-kb EcoRI fragment (see Figure 5) because it overlaps the EcoRI site in the cDNA sequence. Faint hybridization to a low molecularweight fragment (seen in both FHY2 and fhy2-1) may be due to cross-hybridization with additional phytochrome or phytochrome-related sequencesin the genome.

764 The Plant Cell

EcoRl fragments originally seen in DNA from fhy2-7 with the hPHYA91 probe (Figure 8B). Thus, probe 1 lies 5' of the rearrangement, probe 2 spans the rearrangement junction, and probe 3 is 3'of it. The rearrangementjunction in fhy2-7 therefore lies within the EcoRV-Kpnl fragment highlighted in Figure 8A and is close to the center of the transcribed region of PHYA.

The rearrangement in fhy2-7 can occur in one of two ways. First, insertion of DNA into PHYA at the rearrangement junction site identified above may have occurred. To create the nove1 9.1- and 1.3-kb EcoRl fragments described above, the mini- mum size of this possible insertion is 4.2 kb (such that this insertion itself carries a single EcoRl site). Second, an inter- stitial inversion (with one breakpoint junction located in the center of PHYA, the other at an unknown site) may have occurred. 60th of these types of rearrangement would be expected to create a null allele.

fhy2 Mutations Are Partially Dominant

To determine whether the fhy2-7 and fhy2-2 mutations are dominant or recessive, plants homozygous for these mutations were backcrossed to the Landsberg erecta progenitor strain (FHY2IFHY2). Hypocotyl elongation in FR of the resulting heterozygotes (fhy2-VFHY2; fhy2-VFHY2) was compared with that

12 I m

Figure 9. fhy2Alleles Confer a Partially Dominant Phenotype in FR.

Wild-type (FHY2/FHY2), fhy2 heterozygote (fhy2-VFHY2 and fhy2- 2/FHY2), and fhy2 homozygote (fhy2-7Iyhy2-7 and fhy2-Mhy2-2) seeds were sown on agar plates, germinated in darkness for 1 day, and then grown for an additional4 days under continuous, low photon fluence rate FR (1 pmol m+ sec-l). Data are plotted as the mean hypocotyl lengths of 12 to 15 individuals of each genotype, and the bars represent the standard errors of the means.

of mutant (fhy2-7Bhy2-7; fhy2-Hhy2-2) and wild-type (FHY2/ FHY2) homozygotes. The data are presented in Figure 9. Al- though they are nowhere near as elongated as mutant homozygote hypocotyls, the hypocotyls of heterozygotes (fhy2-VFHY2 and fhy2-2FHY2) are significantly longer than those of wild-type homozygotes (FHYUfHY2) when grown in FR. Thus, the fhy2-7 and fhy2-2 mutations confer partially dominant phenotypes in FR. The phytochrome A-deficient hy8 and frel mutants were found to confer recessive phenotypes in FR (Nagatani et al., 1993; Parks and Quail, 1993). This apparent discrepancy with our results may be explained by differences in duration or fluence rate of the FR exposures used. In addition, the effects of heterozygosity for mutations conferring phytochrome A deficiency are subtle and require the observation of a number of heterozygous individuals for a convincing demonstration of their existence.

Similar experiments showed that fhyl-WHY7 and fhy3-7/ FHY3 heterozygotes display hypocotyl lengths in FR that are indistinguishable from that of wild type (data not shown). Thus, the fhyl-7 and fhy3-7 mutations confer recessive phenotypes in FR.

DlSCUSSlON

This paper describes the isolation and characterization of Arabidopsis mutants that display an elongated hypocotyl phenotype in FR but not in W. These mutants fall into three complementation groups, identifying the FHY7, FHM, and FHY3 loci. Previous publications on this subject have described the identification of mutants defining single complementation groups (hy8 and fre7; Nagatani et al., 1993; Parks and Quail, 1993) that are probably equivalent toFHY2. A possible reason for our success in identifying the fhy7 mutant is that the agar medium used in our mutant screening experiments lacked sucrose. Exogenous sucrose affects hypocotyl growth and partially suppresses fhy7 phenotype (G. Whitelam, unpublished data) and so may have masked the presence of mutants equivalent to fhyl in the previous mutant screens (Nagatani et al., 1993; Parks and Quail, 1993). In addition, the hypocotyl elongation displayed by fhyl in FR, although significantly greater than that of the wild type, is considerably less dramatic than that of the fhy2 mutants. It is possible that, because of their less severe phenotypes, such mutants were simply overlooked in previous experiments. The fhy3 phenotype is not suppressed by exogenous sucrose (G. Whitelam, unpublished data). At present, we cannot explain why previous experiments failed to identify mutants equivalent to fhy3.

The results of our experiments showed that the fhy2-7 and fhy2-2 mutant lines contain mutant alleles of PHYA. Phyto- chrome A is undetectable in fhy2-2, and this mutant accumulates PHYA transcripts at levels that are greatly reduced with respect to that of the wild type. The fhy2-7 mutant line lacks detectable phytochrome A, lacks detectable PHYA transcripts, and contains a structurally altered PHYA gene. This alteration


interrupts the PHYA transcriptional unit, suggesting that fhy2-7 is a phytochrome A null mutant. These observations conclu- sively demonstrate that seedlings homozygous for mutations at PHYA exhibit an elongated hypocotyl when grown in FR.

When grown in FR, fhy2 mutants are almost indistinguishable from wild-type plants grown in the dark. This characteristic distinguishes these mutants from previously characterized phytochrome-related hy mutants (hy7, hy2, hy3, and hy6). These mutants display long hypocotyls when grown in W. However, the length of these hypocotyls is notas great as that observed in dark-grown (etiolated) seedlings, possibly because of the action in W of photoreceptors (E, UV-A and UV-B receptors) other than phytochrome. The fact that FR-grown fhy2 mutants mimic etiolated wild-type plants implies that phytochrome A is the principal active photoreceptor in FR.

With the possible exception of hy5, none of the hy mutants appears to be a phytochrome-related signal transduction chain mutant (Chory, 1992). We have identified two nove1 mutations (fhyl-7 and fhy3-7) that confer an elongated hypocotyl in FR (but not in W) and do not belong to the fhy2 (PHYA) complementation group. Unlike the fhy2 mutants, fhy7-7 and fhy3-7 confer recessive phenotypes in FR. The FHY7 and FHY3 loci appear not to be extragenic regulators of PHYA because levels of spectrophotometrically and immunochemically detectable phytochrome A appear to be normal in both fhyl-7 and fhy3-7 mutants. Because fhyf-7 and fhy3-7 display an elongated hypocotyl in FR, but a hypocotyl indistinguishable from the wild type in R or W, it seems likely that these mutations specifically affect components of a phytochrome A signal transduction pathway. Thus, the FHYl and FHY3 gene products do not appear to be involved in the transduction of phytochrome B signals. In addition, it is clear that the fhy7 and fhy3 mutants differ from hy5 mutants, which display a long hypocotyl in all light regimes (Koornneef et al., 1980; Chory, 1992). Hypocotyl elongation and inhibition of cotyledon expansion of the fhy7-7 and fhy3-7 mutants in FR are not as pronounced as in the fhy2 (phytochrome A-deficient) mutants. The fhyl-7 and fhy3-7 mutations may be reduced-function rather than loss-of-function mutations, thus reducing but not com- pletely abolishing FR-mediated hypocotyl growth inhibition. Alternatively, the FHYl and FHY3 gene products may not have sole responsibility for the signal transduction steps that they identify, Further analyses of the functions of FHYl and FHY3 are in progress.

The analysis of plants heterozygous for fhy2 alleles shows that FR-grown hypocotyls of fhyYFHY2 plants are longer than those of FR-grown FHYYFHY2 plants. Thus, the fhy2 alleles are dominant over the wild-type allele (FHY2). Because FR- grown fhyYFHY2 hypocotyls are much closer in length to FR- grown FHYYFHY2 hypocotyls than to FR-grown fhy2/fhy2 hypocotyls, the fhy2 alleles are partially dominant. The molecular analyses presented above indicate that the fhy2 mutations are loss-of-function or reduced-function alleles of PHYA. Dominant loss-of-function alleles exert their effects because the wild-type allele is needed in two copies to confer the wild-type phenotype. This usually reflects a close relationship

between the leve1 of wild-type gene product and phenotype. Thus, the fhy2 alleles probably display partia1 dominance because phytochrome A is less abundant in fhyYFHY2 heterozygotes than in FHYYFHY2 homozygotes, and because the FR-mediated hypocotyl growth inhibition response is sensitive to this difference in abundance.

We have shown that the fhy2 mutants contain mutations at PHYA. We will therefore be referring to fhy2-7 and fhy2-2 as phyA-7 and phyA-2, respectively, in subsequent publications. Perhaps the most dramatic implication of our findings is that, when grown in W, phytochrome A-deficient mutants have a phenotype that is indistinguishable from that of the wild type (see also Nagatani et al., 1993; Parks and Quail, 1993). Be- cause fhy2-7 is likely to be a phytochrome A null mutation, our observations suggest that this normal phenotype cannot be attributed to the presence of low levels of phytochrome A conferred by an incompletely null mutation. Of all of the phytochrome species, phytochrome A is the best characterized in terms of its spectral, physiological, and molecular proper- ties. Our experiments show that phytochrome A is dispensable and plays a relatively minor role in the photomorphogenesis of Arabidopsis plants grown in W.

METHODS

Arabidopsis Lines and Plant Maintenance

The Arabidopsis tbaliana Landsberg erecta laboratory strain was obtained from Maarten Koornneef (Wageningen Agricultura1 University, The Netherlands). The T-DNA transformant line A264 was obtained from George Coupland (Cambridge Laboratory, John lnnes Centre, Norwich, UK). The genetic nomenclature used in this paper follows the conventions established at the Third lnternational Arabidopsis Meet- ing (East Lansing, MI, April 1987; Koornneef and Stam, 1992). Genotypes are italicized, the wild-type genotype is capitalized (e.g., FHY2), the mutant genotype is represented in lowercase letters, and independent alleles are given identifying numbers (e.g., fhy2-7 and

For routine plant rnaintenance, seeds were imbibed on rnoistened filter paper at 4OC for 4 days (to break dormancy) and then planted on “Arabidopsis mix”(2 parts Levington’s M3 potting compost to 1 part gritlsand). Plants were then grown in standard glasshouse conditions at 15 to 20OC. Transgenic plants were grown according to United King- dom Ministry of Agriculture, Fisheries and Food (MAFF) regulations (License Number PHF 1418/8/22).

fhy2-2).

y-lrradiation Mutagenesis

y-lrradiation mutagenesis was performed using the cesium-137 source at the University of Nottingham, United Kingdom. Approximately 10,000 dry Landsberg erecta seeds were exposed to 90-kR y rays. The seeds were then chilled and planted (as described above) in 10 batches of 4 0 0 0 seeds each. The plants were allowed to self-pollinate, and MP seeds were then bulk harvested from each batch.

766 The Plant Cell

lsolation of Mutants Displaying an Elongated Hypocotyl in Far-Red Llght

The y-ray-treated M2 material and the additional ethylmethane sulfonate (EMS)-treated M2 material (Columbia glabrous; Lehle Seed Co., Tucson, AZ) were screened for mutants displaying an elongated hypocotyl in far-red light (FR). Seeds, in batches of 40,000, were evenly sown onto the surface of 0.8% agar in mineral salts (Whitelam et al., 1992) in clear Perspex boxes that were 12 x 22 x 7 cm deep. The seeds were allowed to imbibe for 4 days at 4OC in the dark and induced to germinate by exposure to white fluorescent light (W; photon fluence rate, 400 to 700 nm, 150 pmol m-2 sec-I) at 2OoC (for 30 min). The seeds were allowed to germinate for 1 day in the dark at 22% and then transferred to continuous, broad band FR for 4 days. The FR source consisted of the output of water-cooled 100 W incandes- cent lamps filtered through black Plexiglass (type FRF 700; Westlake Plastics, Lemmi Mills, PA) providing a photon fluence rate, 700 to 800 nm, of 11 pmol m-2 sec-’ and the same spectral photon fluence rate distribution as the FR source described by Rich et al. (1987).

Putative long hypocotyl mutants were transplanted to soil and grown in a controlled environment room at 2OoC under continuous white fluorescent light (photon fluence rate, 400 to 700 nm, 170 pmol m+ sec-l) to promote rapid flowering, and allowed to self-pollinate. M3 seed were germinated as described above, and the resultant seedlings were grown under the following light regimes: continuous FR (as described above); continuous W (cool-white fluorescent tubes, photon fluence rate, 400 to 700 nm, 100 pmol sec-I); continuous red light (R; output of warm-white fluorescent tubes filtered through a 1-cm-deep solution of copper sulfate [1.5O/o wlv] and one layer of No. 406 primary red cinemoid [Rank Strand, Isleworth, UQ, photon fluence rate, 600 to 700 nm, 6 pmol m-2 sec-I); continuous blue light (B; output of cool-white fluorescent tubes filtered through one layer of No. 419 dark blue cinemoid [Rank Strand], photon fluence rate, 400 to 500 nm, 6 pmol m-2 sec-l).

The mutants isolated were designated fhyl-7, fhy2-7, fhy2-2, and fhy3-7; fhy is for far-red elongated hypocotyl. The fhy2-7 and fhy2-2 mutations were isolated from different batches of y-ray-treated M2 seed. They are, therefore, the products of independent mutagenic events at the fHY2 locus.

Llnkage Mapping

In the genetic mapping experiments, F1 and F2 material was screened for individuals displaying an elongated hypocotyl in FR as described above, except that kanamycin (50 mglL) was incorporated into the agar medium. Following 3 days of continuous FR, mutant individuals displaying an elongated hypocotyl were clearly visible (the kanamycin does not affect hypocotyl elongation in FR). The plates were then placed in W for 3 days during which time the kanamycin-resistant plants de- veloped expandedgreen cotyledons, whereas the kanamycin-sensitive plants did not. The numbers of individuals displaying an elongated hypocotyl in FR andlor kanamycin resistance were recorded; genetic linkage distances were calculated as described by Koornneef and Stam (1992).

In Vivo Phytochmme Spectmphotometry

Seeds were germinated as described above, and seedlings were grown for 6 days in absolute darkness. Phytochrome photoreversibility was

measured using a dual wavelength spectrophotometer (DW 2-A; Aminco, Silver Spring, MD) with the measuring beams set at 660 and 730 nm. Samples consisted of 0.8 g of whole seedlings gently packed into a 2.3-mm light path cuvette.

Phytochrome Extraction and lmmunoblot Analysis

Phytochrome-enriched extracts of 6-day-old etiolated seedlings were prepared using the procedure described by Somers et al. (1991). Pro- tein extracts were separated on denaturing 8% polyacrylamide gels using the buffer system of Laemmli (1970) and electroblotted onto nitrocellulose. lmmunostaining of phytochrome A was performed as described by McCormac et al. (1992) using spent culture medium from the monoclonal hybridoma LAS32 as primary antibody.

lsolation of Genomic DNA Clones Containing PHYA

Genomic DNA was isolated from frozen Arabidopsis tissue (homozygous for the gibberellin-insensitive [gail mutation, Landsberg erecta background) and purified by CsCl banding (Hauge and Goodman, 1992). This DNA was partially digested with SauSA, and a subsequent partial fill-in reaction was performed with dGTP and dATF‘, resulting in 5’-GA overhangs. This DNA was ligated into a hFlX II vector (Stratagene), which had been digested with Xhol, followed by partial fill-in with dTTP and dCTP to create 5’-TC overhangs. The resulting phage library was screened using a degenerate 44-mer oligonucleotide (NH2) containing a region of sequence strongly conserved between PHYA, PHYB, and PHYC (encoding amino acids 301 to 315 in the sequences shown in Sharrock and Quail, 1989). The oligonucleotide was end labeled using T4 polynucieotide kinase and y-=P-ATP (Sambrook et al., 1989). Hybridizing phages were plaque purified and sorted into clones containing PHYA, PHYB, or PHYC by restriction fragment map ping. Clones 1IPHYA81 and hPHYA91 were shown to contain the PHYA gene by the following criteria. First, EcoRl digestion of these clones produced a 6.2-kb fragment that hybridized with the NH2 oligonucleotide, as expected for PHYA (Sharrock and Quail, 1989). Second, oligonucleotide probes derived from the 5‘ and 3’ end regions of the PHYA sequence (Sharrock and Quail, 1989) hybridized to XPHYA81 and WHYASl DNA. These oligonucleotides were phyA5’ (SAGGTGG- TGATCGAGGCCAAG-3: the final G is 111 bp upstream of the starting ATG codon in the phytochrome AcDNA sequence; Sharrockand Quail, 1989) and phyA3‘(5’-ACATcTTcT~TGAAcTccTT-3: the first A is 174 bp downstream of the TAG stop codon in the phytochrome A cDNA sequence; Sharrock and Quail, 1989). These oligohucleotides are not homologous to the PHYB or PHYC sequences (Sharrock and Quail, 1989; Reed et al., 1993), thus confirming that hPHYA81 and XPHYA91 contain PHYA and not PHYB or PHYC. DNA from phages XPHYA81 and 1IPHYA91 was used in the hybridization experiments described below.

Analysis of the PHYA Gene Transcrlpts

Seeds were imbibed for 7 days at 4OC in the dark. The seeds were then given a 10-min pulse of W to induce germination and were then grown in the dark for an additional 5 days. The etiolated seedlings were harvested in green light and immediately frozen in liquid nitro- gen. Up to 0.2 g of frozen tissue was ground. to a fine powder and resuspended in 2 mL of extraction buffer (150 mM LiCI, 5 mM EDTA, 5% SDS, 80 mM Tris-HCI. pH 9.0). Following two phenollchloroform


extractions, nucleic acids were precipitated with one-tenth volume of 3 M sodium acetate and one volume of isopropanol. The pellet was resuspended in 3 M LiCl on ice for 10 min. The RNA was pelleted again by centrifugation, washed with 75% ethanol, dried, and resuspended in H20.

Approximately 10 pg of RNA was fractionated on a 1.3% agarose gel (containing 0.66 M formaldehyde) and transferred onto Hybond-N membrane (Amersham lnternational) via capillary action. The RNA was hybridized with an 11.5-kb Sal1 genomic DNA fragment (from hPHYA81) containing the Arabidopsis PHYA gene (labeled with phosphorus-32 by random primer extension; Feinberg and Vogelstein, 1983). Hybridiration conditions were as described previously (Peng and Harberd, 1993).

ldentlflcation of Restrlction Fragment Alterations in PHYA

Arabidopsis genomic DNA was prepared using a miniprep method (modified from Tai and Tankley, 1991) and digested with excess EcoRI. The DNA digests were separated on an 0.8Vo agarose gel and transferred onto a Hybond-N membrane via capillary action. The Arabidopsis phytochrome A cDNA (Sharrock and Quail, 1989) was obtained from Peter Quail (Plant Gene Expression Center, Albany, CA). Subfragments of the phytochrome A cDNA obtained following restriction endonuclease cleavage were fractionated through 0.8% low melting point agarose in TBE (Sambrook et al., 1989), 1 ng mL-l ethidium bromide. The fragments required were purified from the gel. hPHYA91 DNA and phytochrome A cDNA subfragments were labeled via random primer extension (Feinberg and Vogelstein, 1983) and used in hybridization experiments as described previously (Peng and Harberd, 1993).

ACKNOWLEDGMENTS

We thank Maarten Koornneef for providing plant material; Bernard Mulligan and Mark Aarts for y irradiation; George Murphy for construction of oligonucleotides; George Coupland for T-DNA transformant line A264; Peter Quail for the phytochrome A cDNA; Paul Sinacola for as- sistance with plant care and cross-pollinations; Marie Bradley for comments on the manuscript. PC., M.L.A., and J.S.C. were supported by Agricultura1 and Food Research Council (AFRC) PMB Grant PG208/520. This work was also supported by AFRC Grant LR91/519 (linked research grant to G.C.W. and N.P.H.). E.J. was supported by a studentship from the Science and Engineering Research Council, and J.P was supported by a studentship from the British Council (Sino- British Friendship Scholarship Scheme).

Received April 1, 1993; accepted May 27, 1993,

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DOI 10.1105/tpc.5.7.757 1993;5;757-768Plant Cell

G C Whitelam, E Johnson, J Peng, P Carol, M L Anderson, J S Cowl and N P HarberdPhytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light.

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