Plant Physiol.-1997-Childs-611-9.pdf

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Plant Physiol. (1997) 113: 61 1-619

The Sorghum Photoperiod Sensitivity Gene, Ma,,Encodes a Phytochrome B'Kevin L. Childs, Frederick R. Mil ler , Marie-Michle Cordonnier-Pratt, Lee H. Pratt,

Page W. Morgan, and John E. Mullet*Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-21 28 (K.L.C.,

J.E.M.); Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843-2474(F.R.M., P.W.M.); and Department of Botany, University of Georgia, Athens, Georgia 30602 (M.-M.C.-P., L.H.P.)

The Ma, gene i s one of s i x genes that r egulate the photop eriodicsensitivity of f lowering in sorghum (Sorghum bicolor [L.] Moench).The masRmu tation of this gene causes a p henoty pe that i s similar toplants that are known to lack phytochrome B, and masR sorghumlacks a 123-kD phytochrome that predominates in l ight-grownplants and that i s present in non-magRplants. A popu lation segre-gating for Ma, and masRwas created and used to identify tworandomly ampl i f ied polymorphic DNA markers l inked to Ma,.These two markers were c loned and mapped in a recombinantinbred p opulat ion as restr ic t ion fragment length polymorphisms.cDNA clones of P H Y A and P H Y C were cloned and sequenced froma cDNA library prepared from g reen sorghum leaves. Using agenome-walk ing technique, a 7941 bp partia1 sequence of PH YBwas determined from genomic DNA from masRsorghum. PH YA,PH YB, and P H Y C all m apped to th e same linkage group. The Ma,-l inked markers mapped with PH YB more than 121 centimorgansfrom P H Y A and P H Y C . A frameshif t mutat ion resul t ing in a prema-ture stop codon was found in the PH YB sequence from magR or -ghum. Therefore, we co nclud e that the Ma, locus in sorghum i s aPH YB gene that encodes a 123-kD phytochrome.

The transition from vegetative to reproductive growth isthe result of the activation of genes responsible for inflo-rescence and floral organ formation. These genes, whichcontrol apex identity and floral organ morphogenesis, arestrictly regulated, since their improper expression resultsin abnormal flowers and inflorescences (Okamuro et al.,1993; Veit et al., 1993). The initial activation of these genesis usually the result of environmental cues that indicate anappropriate time to flower. The mechanisms by whichenvironmental factors activate inflorescence and floral or-gan production are complex and many genes are known tobe involved in the transduction of environmental signalsthat regulate flowering (Bernier et al., 1993; Coupland,1995).

Of all of the environmental factors that are sensed byplants, daylength is probably the most important in induc-ing flowering. The phenomenon whereby daylength regu-lates flowering is referred to as photoperiodism. Photope-'Supported by U.S. Department of Agriculture competitive* Corresponding author; e-mail [email protected]; faxgrants 94-3730070331 to J.E.M. and 93-03030 to M.-M.C.-P.

1-409-862-4718.611

riodic control of flowering allows plants to coordinate theirreproduction with the environment or with other membersof their species. An unders tanding of the effect of day-length on reproductive development has agronomic impor-tance because the ability to alter flowering time allows thecultivation of a species in environments that differ greatlyfrom the one in which it originally evolved.

Our understanding of photoperiodism has historicallyrelied upon a physiological examination of the phenome-non. Recently, genetic analysis of floral induction has pro-vided new insights into this process. In the LD plant Ara-bidopsis thaliana a series of genes has been recognized thatinfluences flowering time, and these genes have been cat-egorized into six phenotypic groups based on earliness orlateness in flowering in response to short days, long days,and vernalization; (Coupland, 1995). The existence of theseseparate phenotypic classes suggests the existence of sev-era1 pathways that regulate photoperiod sensitivity. A sim-ilar process has been used in the study of photoperiodicsensing in the SD grass, sorghum (Sorghum bicolor [L.]Moench). A series of six maturi ty genes has been recog-nized to alter flowering time in sorghum: Ma,, Ma,, Ma,,Ma,, Ma,, and M a , (Quinby, 1967; F.R. Miller, unpublisheddata). The first four maturity genes cause long days toinhibit flowering but allow early flowering under shortdays. Of these four genes, mutations at M a , cause thegreatest reduction in sensitivity to long days. Mutations atMa,, Ma,, and M a , generally have a more modest effect onsensitivity to long days (Quinby, 1967). However, evensorghum with the recessive alleles ma,, ma2, or ma, flowerlater under long days than under short days (Pao andMorgan, 1986). The genes M a , and M a , represent a specialcase because only when they are both present in the dom-inant form will they very strongly inhibit floral initiationregardless of daylength (F.R. Miller, unpublished data).The sorghum photoperiod mutants constitute a phenotypicclass that corresponds to one of the early-flowering pheno-typic classes of A. thaliana. Mutations in any of the sorghummaturity genes or the A. tkaliana genes E L F , ESD, COP1,

Abbreviations: cM, centimorgans; LD, long-day; QTL, quantita-tive trait loci; RACE, rapid amplification of cDNA ends; RAPD,randomly amplified polymorphic DNA; RFLP, restrictionfragment-length polymorphism; RI, recombinant inbred; RT, re-verse transcriptase; SD, short-day.


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and P H YB cause decreased sensitivity to inhibitory photo-periods (Coupland, 1995).

It is interesting that mutations in sorghum M a, and A.tkaliana PHYB both reduce sensitivity to noninductive day-lengths, since we hypothesized that M a , codes for PhyB.This hypothesis was based on work done with the drastic,pleiotropic mutation magR.Sorghum plants with magRhavea greatly reduced photoperiod sensitivity compared withM a , plants and will flower early under any photoperiod(Pao and Morgan, 1986; Childs et al., 1995).The phenotypecaused by the magR mutation is characteristic of mutantsthat are known to lack PhyB: phyB (A. kaliana), ein (Brassicarapa), lh (Cucumis sativus) , and tri (Lycopersicon esculentum)(Devlin et al., 1992; Lpez-Juez et al., 1992; Reed et al., 1993;van Tuinen et al., 1995). Because of the similari ties of magRsorghum to these mutants known to lack PhyB, it was ourhypothesis that the M a , locus encodes a P H YB gene (Childset al., 1991, 1992, 1995).

To provide evidence that M a , is synonymous withP H YB , we have mapped P H Y A , P H Y B , P H Y C , and Ma,-linked molecular markers. We sequenced the three phyto-chrome genes and demonstrate that magR sorghum con-tains a mutation in P H YB . The ability of PhyB to regulateflowering similarly in both a LD dicot and a SD monocot isdiscussed.

MATERIAL S A N D MET HO DSThe cultivars of Sorgkum bicolor (L.) Moench used in

these experiments were 58M (ma,R), 100M (Ma3) ,BTx 623,and IS 3620C. 58M and lOOM were grown in a greenhouseand crossed to produce heterozygous F, plants, whichwere grown and self-pollinated to produce F, seed. F,plants were grown in the field and scored for days-to-flowering. A11 F, plants were self-pollinated. F, familyrows were grown to confirm the phenotype of early-flowering F, plants and to differentiate heterozygous and

homozygous late-flowering F, plants. For mapping withinthis Ma,/ magRsegregating population, reserved seed fromF, families were planted in a growth room, and DNA wasextracted from the combined tissue of severa1 memberswithin each family.

ldent i f icat ion o f Ma,-Linked RAPD MarkersDNA was extracted and RAPD-PCR was performed as

described by Pammi e t al. (1994). Four-hundred-eighty10-mer primers (Operon Technologies, Alameda, CA) wereused to screen for RAPDs. RAPD-PCR products were ra-diolabeled by the incorporation of [32P]dCTP n the reac-tion. Radiolabeled products were separated on sequencinggels that were dried on blotting paper and exposed to film.Polymorphisms were detected by visual examination of theautoradiographs.

Polymorphic DNA bands were extracted from the driedacrylamide gel by incubating at 37C for 12 h with elutionbuffer consisting of 0.5 M ammonium acetate, 10 mM mag-nesium acetate, 1mM EDTA, 0.1% SDS (Ogden and Adams,1987). The DNA was precipitated with ethanol after the

addition of tRNA and then cloned into T-tailed pBluescriptI1 (SK+) (Stratagene) (Holton and Graham, 1991).

Clon ing o f PHYA an d P H Y CDegenerate primers corresponding to the conservedamino acid sequences DIPQA and ACEFL were created to

allow PCR amplification of partial phytochrome genomicsequences from sorghum DNA (Pratt et al., 1995). Theproducts from this reaction were cloned into pBluescript I1(KS-), and individual clones were sequenced. Truncatedclones derived in this manner, which corresponded toP H Y A and P H Y C , were used to screen a AgtlO cDNAlibrary created using RNA isolated from green sorghumleaf tissue (Sambrook et al., 1989). cDNA clones for P H Y Aand P H Y C were subcloned into pBluescript (SK+) andsequenced.

phyB SequencingTo obtain the sequence of P H YB , the DIPQA and ACEFL

primers were used to amplify by PCR a 359-bp P H YBfragment from sorghum that was cloned into pBluescript I1(KS-). From the sequence of this clone, a gene-specificprimer was made that was used in a 3 RACE reactionusing cDNA from light-grown magRsorghum (Frohman etal., 1988). cDNA was synthesized from 1 pg of poly(A)RNA using a dT-AMP primer (5-GCTCGAGTC GAC-CGCTTT TTTTTTTTT-3) n a 24-pL reaction containing 50mM Tris-HC1, pH 8.3, 50 mM KC1, 10 mM MgCl,, 0.5 mMspermidine, 10 mM DTT, 2 mM each dNTP, 15 pmol ofdT-AMP primer, 10 units of RNasin, and 10 units of avianmyeloblastosis virus RT incubated at 42C for 2 h. Thegene-specific primer (5-CTCCTCGGC AATGAAGTT GT-3, nucleotides 801-820 [nucleotide numbering refers to thenucleotides of the final sequences submitted to GenBank])and a 3 specific primer, AMP (5-GCTCGAGTC GACCGC-3), were used in a PCR reaction with the first-strand cDNAand resulted in a 1.3-kb fragment that was cloned intoT-tailed pBluescript (SK+). This clone was sequenced andfound to be homologous to P H YB . This partial clone isreferred to as pBSB18. Because the same size fragment wasobtained by 3 RACE when using cDNA from either M a , ormagR sorghum (data not shown), it was assumed that thetruncated 3 RACE product was due to interna1 annealingof the dT-AMP primer. Later, the region of P H Y B corre-sponding to pBSB18 was amplified from genomic DNA,and the PCR product was directly sequenced to confirm theaccuracy of the 3 RACE clone.

To obtain additional sequence 5 and 3 to the initial 1.7kb of sequence, a genome-walking technique was used(Siebert et al., 1995). Briefly, genomic DNA from magRsorghum was digested in separate reactions with blunt-endendonucleases: DraI, EcoRV, PvuII , ScaI, and SmaI. Theends of the DNA in each digested pool were ligated to anadaptor that had been created by the annealing of twooligonucleotides: adaptor L (5-CTAATACGA CTCAC-TATA GGGCTCGAG CGGCCGCCC GGGCAGGT-3) andadaptor S (5-PO3-ACCTGCCC-NH2-3).Long PCR wasthen performed using a commercial enzyme mix (Elongase,


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Phytochrome B Sorghum bicolor Mutant 613

Life Technologies) and adaptor- and gene-specific pr imers.The procedure involved two rounds of PCR in which theproducts from the first reaction were diluted 1:100 andused in the second reaction. Thermocycling conditions con-sisted of an initial 1-min, 94C denaturation followed by 35(primary PCR) or 20 (secondary PCR) cycles of denatur-ation for 30 s at 94"C, annealing/extension for 6 min at68"C, and a final extension of 15 min at 68C. Nestedprimers were used for each set of pr imary and secondaryreactions (Table I) . The products from the secondary PCRreaction were precipitated with ethanol and resuspendedfor loading onto a 1% low-melting-point agarose gel. Afterelectrophoresis and visualization under UV , the predomi-nant band resulting from each adaptor-ligated pool ofDNA was excised and purified from the agarose usingQiaquick spin columns (Qiagen, Chatsworth, CA). ThePCR products were then sequenced directly.Sequencing

Initially, sequencing of plasmid clones was done usingSequenase version 2.0 (United States Biochemical). For themajority of the sequencing, sequence data were generatedusing either the ABI 373a or the ABI 377 with samplesprepared with the ABI Dye Terminator Cycle SequencingReady Reaction kit (Perkin-Elmer).RT-PCR

RT-PCR was done as described by Ohtsuki et al. (1994).RNA was extracted from both green and etiolated leaves(Childs et al., 1995). RNA and cDNA were prepared asdescribed above except that for cDNA synthesis 1 pg ofpoly(A) RNA was used and 2 p~ random hexamers (Phar-macia) were substituted for the oligo(dT)-AMP primer. Am-plification reactions were similar to the RAPD reactionsexcept that cDNA corresponding to 10 ng of initial poly(A)RNA was used as the target DNA, no [32P]dCTPwas used,and 1.3 PM of each of two gene-specific primers were in-cluded (for P H Y A : 5'-GCTGAGATC ACCAAACCT GG-3',nucleotides 1000-1019 and 5'-GCATCCTTC TGTTGTCATCC-3', nucleotides 1891-1872; for P H Y B : 5'-TGTCATGGGGACTCAACA GG-3', nucleotides 1156-1173 and 5'-CATCTCTCT TGCTACAGA GC-3', nucleotides 1599-1570;for P H Y C : 5'-CTGGTCGTCT GCCATCATAC-3', nucleo-

tides 1395-1414 and 5'-CGTGGATGCA TCTTCCTGCC-3',nucleotides 1929-1948). The reaction products were sepa-rated by electrophoresis on a 0.8% agarose gel and visual-ized with UV light.Mapping

An RI population derived from a cross of cultivars BTx623 and IS 3620C was used for the mapping of markersonto a genomic map of 1462 cM (Y. Peng, K.S. Schertz, andG.E. Hart, unpublished data). This population consisted of137 F, and F, lines and has been used for the creation of agenomic map comprising primarily sorghum and Zea maysRFLP markers. Severa1 plants f rom each family weregrown, and harvested tissues from each family were com-bined for DNA extractions.

For RFLP mapping, the P H Y B partia1 clone pBSB18, a476-bp BglII fragment of the the PHY C cDNA clone, andclones of the Ma,-linked markers were used in Southernhybridizations. Southern blots prepared after the digestionof DNA from each of the RI lines with either HindIII or XbaIwere kindly provided by Dr. Gary Hart. Probes were pre-pared by random primer labeling (Feinberg and Vo-gelstein, 1983), and hybridization and washing were asdescribed by Sambrook et al. (1989).

A single-nucleotide polymorphism that distinguishedBTx 623 from IS 3620C was found i n a region correspond-ing to the very 3' terminus of the P H Y A cDNA clone(nucleotide 3559). This polymorphism resulted in the cre-ation of a DdeI restriction site in the IS 3620C parent thatwas absent in the BTx 623 parent. DNA from each of the RIlines was used in PCR reactions to amplify the regioncontaining the site of this polymorphism (primers: 5'-TGTTGGTGG CTCTGTTG-3', nucleotides 3333-3349; and5'-TGCCAGTAC AACTTTCC-3', nucleotides 3782-3766).The resulting 449-bp products were digested with DdeI,electrophoresed on a 3% agarose gel (Metaphor, FMC,Rockland, ME), and visualized under UV.Mapping Data Analysis

The RI population used for mapping has been usedpreviously to create a detailed genetic map of sorghum (Y.Peng, K.S. Schertz, and G.E. Hart, unpublished data). Ourdata were incorporated into this existing map using Map-

Table 1. List of prime r sets used for flanking PCR to obtain PHYB gene sequencePrimer Name" Sequence Nucleotides

Adaptor primers 5' - GGATCCTAAT ACGACTCACT ATAGGGC- 3' ma.'5' - AATACGACTC ACTATAGGGC TCGAGCGGC- 3' n.a.5' - TCTGTAGCAA GAGAGATGGT TCGGTTGATAG- 3' 1804 to 183425' - GATGGTTCGG TTGATAGAGA CAGCAACAGTAC- 3' 1818 to 1849

83 5' - GACCGCCAAC ACAAGGAGCA AAATGGATGG- 3' 3200 to 32295' - AGCAAAATCC ATGGTAAATC CATTGGCGCC- 3' 321 6 to 3245

B4 5' - AAGCTGCAAT TCCATGTTGA GCTGCAGCCC- 3' 936 to 9075' - GCAGCCCAGA TGCCTGCATG AGAAACTCGC- 3' 91 3 to 884

4257 to 42865' - ACTGTACAGT AACTGGCATG CGTCTGGCTG- 3' 4265 to 4294

B5 5' - ACCATCATAC TGTACAGTAA CTGGCATGCG- 3'a The adaptor primers were common to each set of nested reactions. The p rimer names refer to the section of the gene amplified by that primer

set (see Fig. 2). ' .a., N o t applicable.


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maker Macintosh version 2.0 (Lander et al., 1987). Ourmarkers were assigned to an existing linkage group usingthe "near" command and then placed in the most appro-priate order using the "try" command. The "ripple" com-mand confirmed marker order and indicated that markershad been placed with a LOD>3. Recombination distanceswere determined with the Kosambi function (Kosambi,1944).

RESULTSThe near-isogenic cultivars 100M (Ma,) and 58M

were screened for RAPD markers using 480 random 10 -mers. Because of the near-isogenic nature of these twocultivars, only 21 RAPD markers were found to differenti-ate the two cultivars (data not shown). A population seg-regating for M a , and magRwas made by crossing lOOM and58M. Forty F, families were used to determine that threeRAPD markers cosegregated with Ma,. Two of these Ma,-linked RAPD markers, op-k20a and op-kZOc, were cloned.

SorghumLinkage Group Achromosome 1

16.7-op-k2Oa

12.3-hVbf ___Maize

15.3-8.0-

I chromosome 5QLnGmcsidmah91

Figure 1. Linkage maps showing positions of PHYA, PHYB, PHYC,and Ma,-linked markers and showing the synteny between S. bicolorand Z. mays linkage groups. C ene-specific markers for PHYA, PHYB,and PHYC and Ma,-linked markers op-kZOa and op-kZOc weremapped in a sorghum RI popula tion that has been used previously tocreate a map of RFLP markers (Y. Peng, K.S. Schertz, and G.E. Hart,unpublished data). Data from PHYA, PHYB, PHYC, and the Ma,-linked markers were incorporated into the RFLP map. The Z. mayslinkage groups shown were created for each linkage group by usingdata p rovided to Agricultura1 G enome lnformation S erver's M aizeD Bby Brookhaven National Laboratory. For Z. mays chromosomes 1and 9, data were from Brookhaven Nationa l Laboratory Ma y 93 mapscreated from a C 159 X TX303 RI popu lation (Burr et al., 1988). Fori mays chromosome 5, data were used from a Brookhaven N ationa lLaboratory May 93 map made from a T232 X CM37 RI population(Burr et al., 1988). Markers that are underlined were shown byP hillips et al. (1992) to be linked to QTL for flowering time in Z.mays. The numbers in the diagram indicate c M distances betweenmarkers. For the Z. mays linkage groups presented, o nly the markersshown were used to create the maps shown.

Table II. Comparison o f derived amino a cid sequences from sor-ghum phytochromes A and C an d partia/ phytochrome Bsequences

The percent identity values were obtained b y comparing derivedprotein translations using the Bestfit program from the WisconsinPackage (Genetics Computer G roup, M adison, W I).

Phytochromes PhyA PhyC Partia1 PhyB

Maize P hyARice PhyARice PhyBArabidopsis PhyAArabidopsis PhyBArabidopsis PhyCArabidopsis P hyDArabidomis PhvE

96.588.250.264.449.050.549.145.8

% identity52.3 50.353.1 50.553.0 91.156.4 53.551.9 75.059.4 51.749.1 58.651.8 71.a

M a, was then mapped in an RI population of 137 linesresulting from a cross between BTx 623 and IS 3620C usingop-k20a and op-k2Oc as probes to detect RFLPs. The Ma,-linked RAPD markers mapped within 11.6 cM of eachother on sorghum linkage group A (Fig. 1) (Y. Peng, K.S.Schertz, and G.E. Hart, unpublished data).

We determined the sequences of cDNA clones of P H Y Aand P H Y C . The P H Y A and P H Y C clones are 3805 and 3755bp in length, respectively. The open reading frames ofthese two clones are 3393 and 3405 bp long, respectively. A7941-bp sequence of P H Y B was determined from genomicDNA from sorghum. Based on comparison withP H Y B from Oryza sativa, the sorghum sequence beginswithin 500 bp of the 5' end of the gene and extends to 1076bp past the end of the stop codon. The gene is made up ofat least four exons and three introns (Fig. 2). The partia1P H Y B sequence encodes 1036 amino acids.

The identification of the sequences as P H Y A , P H Y B , andP H Y C was based on a comparison of the amino acid se-quences of the encoded proteins to known phytochromesby using the Bestfit program from the Wisconsin Package

B4-Pvuf--B4-Sma

s12c-

BS-SmaBS-Pvu

F

wBS-Sca______ .+c

pBSB18 BS-Dra____)B2-Sca____)

B3-Eco-igure 2. Diagram o f the PHYB gene and the mutation that is presentin ma3R.Diagram o f the PHYB gene showing exons, introns, a nd theuntranslated region. Boxes, exons; thick lines, introns; thin line, theuntranslated region; *, the position of the point mutation found inmajR sorghum; O, the area within the last intron that i s highlyhomologous to noncoding regions found in severa1 other sorghumgenes. The arrows indicate steps used in walking across this gene.The primers used for each step in the walk are indicated in Table I.The fragment S12 shows the pos ition of the 35 9-bp fragment used tobegin the sequencing of this gene.


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tween sorghum and other plants known to containP H Y B mutations. Like other P H Y B mutants, magR sorghumis relatively insensitive to photoperiod, displays an elon-gated growth pattern, has a reduced chlorophyll content,and lacks a deetiolation response to high-irradiance redlight (Childs e t al., 1991,1992,1995; Goto et al., 1991; Devlinet al., 1992; Lpez-Juez et al., 1992; McCormac et al., 1993;Reed et al., 1993; van Tuinen et al., 1995).In this report, twoMa,-linked markers.and P H Y B are shown to map to thesame location in the sorghum genome (Fig. 1).Furtherproof that Ma, codes PhyB was obtained by sequencingP H Y B from magR sorghum that lacks a 123-kD phyto-chrome predominating in green seedlings (Childs et al.,1992). If P H Y B codes for the 123-kD sorghum phyto-chrome, then the sequence of P H Y B from magR shouldpossess a mutation that would cause the absence of PhyB.In fact, a frameshift mutation in the 3 terminus of P H Y B inmagR sorghum was found (Figs. 2 and 4). The mutationresults in a premature stop codon shortly after the muta-tion. This mutation is not present in P H Y B from Ma, sor-ghum (Fig. 4). The portion of PhyB in magR sorghum that isnot translated is presumed to contain regions that arenecessary for dimerization and biological activity (Cherryet al., 1993; Edgerton and Jones, 1993).

Similar mutations have been demonstrated in P H Y B ofA. tkaliana, and these A. thaliana mutants (37-5 and 49-8)also lack immunologically detectable PhyB (Wagner andQuail, 1995). Although we have been unable to obtain asmall portion of the 5 end of the P H Y B gene, it is demon-strated here that the sequence. that we have obtained isexpressed. Using RT-PCR, a portion of the P H Y B transcriptwas amplified from both Ma, and magR sorghum. Theamplified product not only contains the mutation found inthe genomic sequence, but it lacks a 1.6-kb intron thatwould have been amplified if genomic DNA had been usedin the reaction. Because of the physiological similaritiesbetween magR sorghum and known P H Y B mutants, be-cause MaJinked markers map with P H YB , and because aframeshift mutation occurs in the P H Y B gene ofphytochrome-deficient majR plants, we conclude that Ma,is synonymous with P H Y B . Given that the identity of thissorghum maturity gene is now known, Ma, and ma,* willnow be referred to as P H Y B and pkyB-1, respectively.

In the course of this work, we obtained two full-lengthphytochrome cDNA clones, P H Y A and P H Y C , and a partia1P H Y B genomic sequence from sorghum. The sequence ofP H Y B was resolved by utilizing a new technique calledgenome walking, which involved sequencing the productsof PCR amplification of DNA flanking a known sequence.We are confident that the use of this technique resulted inthe determination of contiguous sequence for four reasons.First, long gene-specific primers were used to anchor theamplification reaction to the site in the genome in whichwe were interested (Table I). Thus, the likelihood of anunrelated region of the genome being amplified using sucha specific primer was minimal. Second, the gene-specificprimers were designed so that the resulting amplificationproducts overlapped with known sequence. This allowedus to determine that each step of a genome walk was

related to the starting point of that walk. Third, PCR wasperformed using a polymerase mix that contained a smallamount of a polymerase with 3 to 5 exonuclease proof-reading activity. Such activity can reduce the incorporationof inappropriate bases by 10-fold compared with poly-merases without proofreading activity (Eckert and Kunkel,1991; Scott et al., 1994). Finally, PCR products were directlysequenced to further reduce the likelihood of polymerase-induced errors being incorporated into the final sequence.For these reasons, we conclude that the sequence that wehave reported as P H Y B is accurate and represents a singlecontiguous region of the sorghum genome.

The isolation of P H Y A , P H Y B , and P H Y C allowed adetermination of their map positions, which indicated thatonly P H YB , and not P H Y A or P H Y C , could be Ma, (Fig. 1).Additionally, the sequencing of these genes allowed theiridentification. Derived amino acid sequence comparisonsindicate that sorghum P H Y A and P H Y B are most similar toother monocot P H Y A s and P H Y B , respectively (Table 11).There are no full-length monocot P H Y C s to which thesorghum P H Y C can be compared. However, of the phyto-chromes from A. tkaliana, sorghum P H Y C is most similar toA. tkaliana P H Y C (Table 11). Additionally, phylogeneticanalysis indicates that the sorghum and A. thaliana P H Y C sare more closely related to each other than to any otherphytochrome (data not shown). To further confirm thatthese sorghum phytochrome genes have been identifiedcorrectly, the expression of the genes was examined i netiolated and green leaves of sorghum using RT-PCR(Fig. 3). Sorghum P H Y A mRNA leve1 was reduced in greenbut not in etiolated leaves. P H Y B and P H Y C mRNA levelswere similar in both etiolated and green leaves. Theseresults are consistent with the expression of P H Y A , P H Y B ,and P H YC in other plants (Dehesh et al., 1991; Clack et al.,1994). Based on amino acid similarities and expressionpatterns, we conclude that we have correctly identifiedthese sorghum genes as P H Y A , P H Y B , and P H Y C , and wenote that this is the first report of a complete P H YC se-quence from a monocot.

It is now possible to state that the aberrant phenotype ofpkyB-1 sorghum is due to the lack of a 123-kD PhyB. Thismeans that in both monocots and dicots, PhyB controlsshoot elongation, chlorophyll content, and red-light-high-irradiance-response-mediated deetiolation (Childs et al.,1991, 1992, 1995; Goto et al., 1991; Devlin et al., 1992;Lpez-Juez et al., 1992; McCormac et al., 1993; Reed et al.,1993; van Tuinen et al., 1995). This degree of conservationof developmental regulation between monocots and dicotsis not surprising given that photoperception is so impor-tant to sessile, photosynthetic organisms that begin theirlives below ground. However, it is surprising to find thatPhyB deficiency in both SD sorghum and LD A. tkalianacauses relative photoperiod insensitivity and early flower-ing (Pao and Morgan, 1986; Halliday et al., 1994; Childs e tal., 1995). This observation can be explained if PhyB playsa common role in photoperiod sensing in both plants.Thomas and Vince-Prue (1995) suggest that a light-dominant (LD) response pathway and a dark-dominant(SD) response pathway coexist in plants to regulate photo-


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Phytochrome B Sorghum bicolor Mutant 619

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