Structure and Expression of Maize Phytochrome Family Homeologs

12 Pages·2004·0.24 MB·English

Copyright2004bytheGeneticsSocietyofAmerica
DOI:10.1534/genetics.103.026096
Structure and Expression of Maize Phytochrome Family Homeologs
Moira J. Sheehan,*,† Phyllis R. Farmer* and Thomas P. Brutnell*,1
*BoyceThompsonInstituteand†PlantBiologyDepartment,CornellUniversity,Ithaca,NewYork14853
ManuscriptreceivedDecember23,2003
AcceptedforpublicationApril8,2004
ABSTRACT
To begin the study of phytochrome signaling in maize, we have cloned and characterized the phyto-
chrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and
BAClibraryscreens,weshowthatthePhyA,PhyB,andPhyCgenesareeachduplicatedonceinthegenome
ofmaize.Eachgenepairwaspositionedtohomeologousregionsofthegenomeusingrecombinantinbred
mappingpopulations.Theseresultsstronglysuggestthattheduplicationofthephytochromegenefamily
inmaizearoseasaconsequenceofanancienttetraploidizationinthemaizeancestrallineage.Furthermore,
sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome
genesinmaize.ThroughNortherngelblotanalysisandasemiquantitativereversetranscriptasepolymerase
chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling
tissues.However,expressionfromPhyA1,PhyB1,andPhyC1predominateinallseedlingtissuesexamined.
Dark-grownseedlings expresshigher levelsof PhyAand PhyBthan dolight-grown plantsbut PhyCgenes
are expressed at similar levels under light and dark growth conditions. These results are discussed in
relationtophytochromegeneregulationinmodeleudicotsandmonocotsandinlightofcurrentgenome
sequencingeffortsinmaize.
AS sessileorganismsinanever-changingenvironment, lateplantdevelopment(Casaletal.2003).InArabidopsis,
plants have evolved sophisticated signaling net- detailed mutant and molecular genetic analyses have
works to perceive and respond rapidly to changes in definedrolesforallfivephytochromeapoproteinspres-
the external environment. Higher plants have at least entinthegenome.phyAistheprimaryphotoreceptor
threephotoreceptorsystemsthatenablethemtodetect controlling high-irradiance FR light responses such as
red (R)/far-red (FR), blue (B)/UV-A, and UV-B light hypocotyl elongation, cotyledon expansion, and seed
(Gyulaetal.2003).TheprimaryR/FRphotoreceptors germination (Nagatani et al. 1993; Parks and Quail
ofplants,thephytochromes,arethebestcharacterized 1993; Whitelam et al. 1993). phyB is the primary red
of all light receptor families (Quail 2002). The phyto- lightphotoreceptormediatingmanyoftheshade-avoid-
chrome holoprotein consists of a light-sensing linear ance syndrome responses that result in petiole elonga-
tetrapyrrole chromophore, phytochromobilin, cova- tion,internodeelongation,reducedleafarea,andearly
lentlyattachedtothephytochromeapoprotein(Terry flowering (Reed et al. 1993). phyC mediates seedling
et al. 1993). In angiosperms the phytochrome apopro- responses to red light and represses flowering under
teins are encoded by a small nuclear gene family of at short-day conditions (Franklin et al. 2003; Monte et
least three members (Mathews et al. 1995; Mathews al.2003).AlthoughphyDandphyEarestructurallysimi-
andSharrock1996,1997).Phytochrome-regulatedre- lartophyB,theyarenotfunctionallyredundant(Shar-
sponses include seed germination, seedling deetiola- rock et al. 2003a). Responses mediated by phyD and
tion,leafexpansion,shadeavoidance,andtimetoflow- phyEincludepetioleandinternodeelongationandthe
ering.Astheseresponsescontributetoecologicallyand controloffloweringtime(Aukermanetal.1997;Devlin
agronomically important traits, phytochrome has been et al. 1998, 1999).
thesubjectofintensestudyforseveraldecades(forreviews Despiteour detailedunderstanding ofphytochrome
see Smith 1995; Ballare and Casal 2000).
signaling in eudicots, little is known of monocot light
Overthepastfewyearsdetailedgeneticandbiochemi-
signal transduction. The monocots and, in particular,
calstudiesofeudicotshavegreatlyincreasedourunder-
thegrassesarebyfarthemostagronomicallyimportant
standingofhowphytochromesignalingpathwaysregu-
group of plants. Rice, maize, wheat, and sorghum are
staplefoodcropsthroughouttheworldandhavebeen
underintensehumanselectionforthelast5000–10,000
Sequence data from this article have been deposited with the years.Furthermore,traitssuchasyield,floweringtime,
EMBL/GenBank Data Libraries under accession nos. AY234825–
andstatureareallinfluencedbyred/far-redlight(Smith
AY234830.
1995). Early flowering varieties of barley (Hanumappa
1Correspondingauthor:BoyceThompsonInstitute,CornellUniversity,
TowerRd.,Ithaca,NY14853. [email protected] etal. 1999),sorghum (Childset al.1997), rice(Izawa
Genetics167:1395–1405(July2004)
1396 M.J.Sheehan,P.R.FarmerandT.P.Brutnell
et al. 2000), and maize (Sawers et al. 2002) have all with a 10% (v/v) bleach solution for 15 min, rinsed thor-
oughly,andimbibedovernightinsterilewater.Imbibedseeds
recently been shown to contain lesions in the phyto-
were planted in 12 (cid:1) 6-cell Rootrainer trays (Hummert, St.
chromesignaltransductionpathway.Inmaize,lightre-
Louis)filledwithvermiculiteandgrowneitherincontinuous
sponse is a highly variable trait and a reduced light darkness (D) or under continuous white light (W) at 100
responseisassociatedwithearlyfloweringNorthAmeri- (cid:2)molm(cid:3)2sec(cid:3)1at28(cid:4)for10daysinaConviron(Winnipeg,
can and European inbred lines (Markelz et al. 2003). Manitoba, Canada) incubator. Fluence rates were measured
with an IL1400A radiometer (International Light, Newbury-
Together, these studies indicate that phytochrome re-
port, MA) equipped with a SEL033 silicon probe (detection
sponse pathways contribute to the agronomic perfor-
range:200–1100nm).Thewhitelightsourcewasacombina-
mance of these important crop species. tionofincandescentandcool-whitefluorescentlamps.Tissue
The phytochrome gene family structure in Arabi- was harvested under green safe lights for D-grown tissues or
dopsiscontainsfivemembers(PHYA–E);however,char- ambientlightforW-growntissues.Thesecondandthirdleaf
acterization of the gene family in other eudicots has blades (blade), second and third leaf sheaths (sheath), and
mesocotylsfrom20seedlingswereharvestedandtissuepooled
revealed variation in gene family composition (Howe
priortoRNAextractions.
et al. 1998; Alba et al. 2000; Li and Chinnappa 2003).
Maize(Z.mays)phytochromechromophoreattachmentre-
Unlike eudicots, the phytochrome apoprotein gene gionand3(cid:1)-untranslatedregionprobes:The456-bpPhyAchro-
family in all monocots examined contains only three mophoreattachmentregion(CAR)probewasgeneratedfrom
lineages:PhyA,PhyB/D,andPhyC(MathewsandShar- B73genomicDNAusingprimersTBp23(5(cid:5)-GCTTGCTGCC
AAGGCAATCTCCAA-3(cid:5)) and TBp24 (5(cid:5)-GCTCGGGTTCAT
rock 1996, 1997). In maize, a recent polyploidization
CATCCTCTTCA-3(cid:5))designedtothepublishedPhyAsequence
eventhasresultedinlargesegmentalduplications(Gaut
(GenBankaccessionno.AY260865;ChristensenandQuail
andDoebley1997).Recentmappingexperimentssug- 1989).The1055-bpPhyBCARprobewasgeneratedfromB73
gest that two PhyB sequences (PhyB1 and PhyB2) and genomicDNAwithprimers19356F(5(cid:5)-AACCCGATATGGAT
twoPhyAgenesequences(PhyA1andPhyA2)arosefrom CCACTCCAGGGT-3(cid:5)) and 12417R (5(cid:5)-GGAGTGACACCCA
ATGGATAGTATTTC-3(cid:5)) designed to a partial maize PhyB
this genome duplication and are located in homeolo-
sequence(GenBankaccessionno.AF137332).A293-bpPhyC
gous regions of the maize genome (Christensen and
CAR probe was generated from B73 genomic DNA using
Quail1989;Childsetal.1997;Basuetal.2000).Prelim- primers TBp29 (5(cid:5)-TTTCTGTTTATGAAGAACAAAGTG-3(cid:5))
inary studies also suggest that as many as three copies andTBp30(5(cid:5)-TTGTACTGTGCATGGCAACCATGGGAAG-3(cid:5))
ofPhyC-likegenesmaybepresentinthemaizegenome designedtoapartialmaizePhyCsequence(GenBankaccession
(Basuetal.2000).However,onlythePhyA2geneofmaize no.U61220).PCRwasconductedwith2.5unitsrTaq(Panvera,
Madison,WI),0.04%DMSO,500mmBetaine,1(cid:1)PCRbuffer.
hasbeencharacterizedinanydetail(Christensenand
PCRreactionswereperformedonB73genomicDNAunder
Quail 1989). the followingcycling conditions: 30sec at 94(cid:4),30 secat 62(cid:4),
Toexamineevolutionaryandfunctionalrelationships 4 min at 72(cid:4), 35 cycles; 10-min final extension at 72(cid:4) and 4(cid:4)
between phytochrome gene family members in maize, soak.PCRproductswereclonedintopCR4-TOPOvectorand
adetailedanalysisofphytochromegenefamilystructure transformedintoTOP10Escherichiacolicells(Invitrogen,Carls-
bad,CA)accordingtothemanufacturer’sprotocoltogener-
andexpressionhasbeenperformed.Wereportonthe
ate the clones pPhyA-CAR (PhyA), pPhyB-CAR (PhyB), and
cloningandsequenceanalysisoffull-lengthPhyA,PhyB,
pPhyC-CAR(PhyC).Theidentityofeachclonewasconfirmed
and PhyC genes in maize. Screens of bacterial artificial bysequenceanalysisusingT3orT7sequencingprimers.
chromosome (BAC) clones generated from the stan- Recombinantinbredmapping:DNAfrom94individualsde-
dard maize inbred B73 indicate that the entire phyto- rived from the Intermated B73 (cid:1) Mo17 (IBM) 94 (Lee et al.
2002)populationweredigestedwithEcoRI(PhyAandPhyB)or
chromegenefamilyisduplicatedinmaize,resultingin
HindIII (PhyC) and hybridized to gene-specific probes. Initial
closely related PhyA, PhyB, and PhyC gene pairs. These
hybridizationswereperformedwithCARprobes,whichresulted
sequenceshavebeenmappedtohomeologousregions intwosegregatingpolymorphisms.Thesepolymorphismswere
of themaize genome,strongly suggestingthat duplica- scored and submitted to CIMDE (http://www.maizemap.org/
tioneventsareaconsequenceofanancestraltetraploidiza- CIMDE/cimde.html) to determine the map positions of Phy
tionevent(GautandDoebley1997).Toexaminethe genes. DNA probes derived from the 3(cid:5)-untranslated regions
(3(cid:5)-UTRs) of PhyA, PhyB, and PhyC were then used to distin-
expression of phytochrome gene family members, we
guishhomeologs,onthebasisofdifferentialhybridizationand
developedasemiquantitativereversetranscriptasepoly- restriction site polymorphisms. The 3(cid:5)-UTRs were amplified
merase chain reaction (RT-PCR) assay to differentiate fromB73genomicDNAforeachgeneandtheproductswere
betweeneachPhyhomeolog.InconjunctionwithNorth- clonedintopCR4-TOPOaccordingtothemanufacturer’sspeci-
erngelblotanalysisweshowthatallphytochromegenes fication.The407-bpPhyA13(cid:5)-UTRproductwasgenerated with
ZmPA1_3(cid:5)-UTR-F (5(cid:5)-CCAGCTCCCTCAGAGTTCATTCGGA
are expressed in several maize tissues. The structure
AAG-3(cid:5))andZmPA1_3(cid:5)-UTR-R(5(cid:5)-CCCATATTTAAACTCCA
and function of the maize phytochrome gene family is CACTACTAAC-3(cid:5))andthenclonedintopCR4-TOPOtogen-
discussedinrelationtotheevolutionaryhistoryofmaize erate the clone pPhyA1-3(cid:5)-UTR. The 356-bp PhyA2 3(cid:5)-UTR
and current genome sequencing efforts. productwasgeneratedwithZmPA2_3(cid:5)-UTR-F(5(cid:5)-CTCAGAG
TTCATTCGGAAAGCCACGGTG-3(cid:5)) and ZmPA2_3(cid:5)UTR-R
(5(cid:5)-GATGTGAAACTATGAACAAGGAGGCTG-3(cid:5)) and then
MATERIALS AND METHODS cloned into pCR4-TOPO to generate the clone pPhyA2-3(cid:5)-
UTR.The390-bpPhyB13(cid:5)-UTRproductwasgeneratedwith
Plantmaterialsandgrowthconditions:Seedsfromthemaize ZmPB1_3(cid:5)-UTR-F(5(cid:5)-AGAGGGTGCCTAATTTACGAGAAG-3(cid:5))
(ZeamaysL.ssp.mays)inbredlineB73weresurfacesterilized and ZmPB1_3(cid:5)-UTR-R (5(cid:5)-CAACCGTGTAAGCTGAAACAA
PhytochromeFamilyHomeologsofMaize 1397
CTC-3(cid:5))andthenclonedintopCR4-TOPOtogeneratetheclone protocols; however, several regions were refractory to se-
pPhyB1-3(cid:5)-UTR.The313-bpPhyB2 3(cid:5)-UTRproductwasgener- quence analysis and modifications were made to obtain se-
ated with ZmPB2_3(cid:5)-UTR-F (5(cid:5)-GGTCGATTAGGTAACTTA quencefromCG-andAT-richregionsofthegenes.Namely,
GGTGGTG3(cid:5)) and ZmPB2_3(cid:5)-UTR-R (5(cid:5)-GCCGCCTGTGTAC 0.04%(v/v)DMSOand500mmBetaineweresubstitutedfor
ATAACTATCTG-3(cid:5))andthenclonedintopCR4-TOPOtogen- water in the linear amplification stage and eliminated the
eratetheclonepPhyB2-3(cid:5)-UTR.The410-bpPhyC13(cid:5)-UTRprod- probleminmanycases.Overnight digestionofthetemplate
uctwasgeneratedwithZmPC1_3(cid:5)-UTR-F(5(cid:5)-GAAGTCTCGGT DNA with frequent four-base cutting restriction enzymes
TGTAGCTAGCCAAG-3(cid:5)) and ZmPC1_3(cid:5)-UTR-R (5(cid:5)-GAGAGA (HinfI,MspI,andSau3AI;NewEnglandBiolabs)priortolinear
TCAAGAAAACAGACCTACCG-3(cid:5))andthenclonedintopCR4- amplification was also utilized to obtain promoter sequence
TOPOtogeneratetheclonepPhyC1-3(cid:5)-UTR.The353-bpPhyC2 inhalfoftheBACclones.Theaccessionnumbersofthemaize
3(cid:5)-UTRproductwasgeneratedwithZmPC2_3(cid:5)-UTR-F(5(cid:5)-CCG phytochrome genes are as follows: PhyA1, AY234825; PhyA2,
TGTCAAATTCTCGGGTGTAG-3(cid:5)) and ZmPC2_3(cid:5)-UTR-R AY260865(ChristensenandQuail1989);PhyB1,AY234827;
(5(cid:5)-CAGCTAATCGATCGCGAAGAAAC-3(cid:5)) and then cloned PhyB2,AY234828;PhyC1,AY234829;andPhyC2,AY234830.
intopCR4-TOPOtogeneratetheclonepPhyC2-3(cid:5)-UTR. Predictedpeptidealignments:Proteinsequencealignments
BAClibraryscreens:AmaizeHindIIIBAClibrary,ZmMBBb, were generated from full-length predicted sequences from
wasscreenedtoidentifyallpotentialPhygenesofmaize(Tom- Sorghumbicolor,Oryzasativa,andbothZ.mayshomeologsusing
kinsetal.2000,2002).RadiolabeledCARprobesweregener- MegAligninLaserGene 1.66(DNASTAR).MegAlignClustal
atedbyKlenowpolymerase(Promega,Madison,WI)incorpo- Wparameterswereasfollows:GapPenalty(cid:6)10,GapExten-
rationof[(cid:7)-32P]dCTP(NewEnglandNuclear,Boston)using sion penalty (cid:6) 0.2, protein weight matrix was the Gonnet
random hexamers (Sambrook et al. 1989). Hybridizations Series. Accession numbers of sequences used are as follows:
wereperformedasdescribed(ChurchandGilbert1984)at SbPHYA,AAB41397;SbPHYB,AAB41398;SbPHYC,U56731;
65(cid:4)inabottleincubator(ThermoHybaid,NeedhamHeights, OsPHYA,X14172;OsPHYB,X57563;andOsPHYC,AF141942.
MA)usingextra-largebottles(300(cid:1)70mm,ContinentalLab Domain architecture was defined using the Conserved Do-
Products,SanDiego).ProbeswerepurifiedusingMicroBio- main Architecture Retrieval Tool (CDART; http://www.ncbi.
SpinP-30Trischromatographycolumns(Bio-Rad,Hercules, nlm.nih.gov/Structure/lexington/lexington.cgi?cmd(cid:6)rps)
CA)andincorporationefficiencywasestimatedusingaBeck- withthedefaultparameters.
manLS 5801liquidscintillationcounter (BeckmanCoulter, RNAgelblotanalysis:TotalRNAwasextractedfrom(cid:1)1g
Fullerton,CA).FilterswereexposedtoPhosphorImagescreens oftissueaspreviouslydescribed(VanTunenetal.1988)and
(AmershamBiosciences,Sunnyvale,CA)overnightandscanned quantifiedusingaBeckmanDU530spectrophotometer.Ap-
at50-(cid:2)mresolutionusingtheStorm840scanner(Molecular proximately 20 (cid:2)g total RNA per lane was fractionated on
Dynamics,Sunnyvale,CA).Clonescoringwasperformedman- 6.8%(v/v)formaldehyde,1.5%(w/v)agarosegel.Hybridiza-
uallyaidedbyImageQuant(IQMacv1.2)software(Molecular tions and image analyses were performed as described for
Dynamics).BACcloneswereorderedfromClemsonUniversity DNAblotanalysis(seeabove).
GenomicsInstitute(Clemson,SC;http://www.genome.clemson. SemiquantitativeRT-PCRandDNAgelblotanalysis:Asemi-
edu/orders/),andsinglecolonieswereisolatedforovernight quantitative PCR assay was developed to monitor low abun-
culture.MiniprepsofputativepositiveBACclonesweregrown dancePhygenetranscriptsandtoidentifytranscriptsencoded
in4.0mlLuria-Bertanimedium(LB)with12.5(cid:2)gml(cid:3)1chlor- byhomeologousgenes.FiftymicrogramstotalRNAfromlight-
amphenicol at 37(cid:4) with 250-rpm continuous agitation over- anddark-grownsheath,blade,andmesocotyltissueswasfirst
night. Cells were pelleted and the BACs were isolated using treated with DNaseI (Invitrogen) at 25(cid:4) for 1 hr. RNA was
a standard alkaline lysis protocol (Sambrook et al. 1989). purified using phenol:chloroform:isoamyl alcohol (25:24:1,
DNAisolatedfromputativePhyA,PhyB,andPhyCcloneswas v/v) and precipitated with 95% (v/v) ethanol and 1/10 vol-
digestedwithrestrictionenzymes(NewEnglandBiolabs,Bos- ume3msodiumacetate.TheRNApelletwaswashedovernight
ton)EcoRI(PhyA)orHindIII(PhyBandPhyC)overnightat37(cid:4) at 4(cid:4) in 70% (v/v) ethanol to remove excess salts and RNA
andfractionatedon0.8%(w/v)agarosegels.DNAwastrans- sampleswereresuspendedinpyrocarbonicaciddiethylester
ferred to Hybond-N(cid:8) nylon membranes (Amersham Biosci- (DEPC)-treatedwaterataconcentrationof1.0(cid:2)g(cid:2)l(cid:3)1.Five
ences).DNAgelblotanalysiswasperformedasdescribedabove. micrograms of total RNA was added to first strand cDNA
BAC sequencing: Sequence analysis was performed directly synthesis reactions performed with SuperScript II kit (In-
onBACclonestominimizecloningandTaq-generatederrors. vitrogen)accordingtothemanufacturer’sprotocolalongwith
A single representative BAC clone served as a template for se- “no-RT” controls. After a 2-hr incubation, all samples were
quencingeachofthesixPhygenefamilymembers.Phytochrome treatedwithRNaseH(Invitrogen)accordingtothemanufac-
gene sequence was generated from the following BAC clones: turer’s recommended protocol. The cDNA made from each
PhyA1, ZMBBb0024P21; PhyA2, ZMBBb0020P08; PhyB1, ZMB- sample was diluted 10-fold in DEPC-treated water and 10 (cid:2)l
Bb0022H07; PhyB2, ZMBBb0001I24; PhyC1, ZMBBb0092I04; wasused inPCR amplificationsof phytochromegenes (only
andPhyC2,ZMBBb0026K01.Midiprepculturesweregenerated 5 (cid:2)l was used for Ubi control). To obtain a semiquantitative
forthedesiredBACclonesbydirectinoculationfromplates estimateofrelativetranscriptlevels,PCRreactionswereper-
to 250 ml LB with 12.5 (cid:2)g ml(cid:3)1 chloramphenicol. Cultures formedforalimitednumberofcycles.HotStarTaq(QIAGEN)
were grown overnight at 37(cid:4) at 250 rpm. An alkaline lysis was used according to the suggested PCR protocol: 15-min
midiprepkit(QIAGEN,Valencia,CA)wasusedfollowingthe initial denaturation at 95(cid:4); denaturation at 94(cid:4) for 30 sec,
protocolforverylowcopyplasmidsandfollowingthemodified annealingat55(cid:4)for30sec,1-minextensionat72(cid:4)foreither
protocolofKirschnerandStratakis(1999).BigDyetermi- 20or25cycles;10-minfinalextensionat72(cid:4);and4(cid:4)soak.A
natormix(Perkin-Elmer,Wellesley,MA)andsubsequentpuri- region of the maize Ubi gene was also amplified to control
ficationwereperformedaccordingtothemanufacturer’spro- for equal addition of RNA templates. Amplification of Ubi
tocol for BAC templates. Samples were fractionated on an was as described above except that 15 cycles of PCR were
ABI-3100 (Perkin-Elmer), and sequence analysis was per- performed.Theprimersareasfollows:PhyA-F(5(cid:5)-GAGAGAT
formedusingSequencher4.1.2(GeneCodes,AnnArbor,MI) CCATGAAGCAAAAGGTTTAC-3(cid:5)), PhyA-R (5(cid:5)-GAAGGTTG
andLaserGene1.66software(DNASTAR,Madison,WI).The ACATGCCCAGCTTCCCTGAG-3(cid:5)),PhyB-F(5(cid:5)-GTTTTGGCT
majorityofPhygenesequencewasobtainedbyprimerwalking GACTTCGCTAAGCATG-3(cid:5)), PhyB-R (5(cid:5)-GGACGATGAGGA
initiatedatthecentralCARregionsofthegenes.Mostofthe AGAAACTCCGCTCTG-3(cid:5)),PhyC-F(5(cid:5)-GTCATGCAAGGCAT
Phy gene sequence was obtained with the standard BigDye TCCT TTGGGCAAG-3(cid:5)), PhyC-R (5(cid:5)-GCTGCTGAGCTGGG
1398 M.J.Sheehan,P.R.FarmerandT.P.Brutnell
Figure 1.—DNA gel blot analysis of phyto-
chrome gene family structure. Gene-specific DNA
probesforPhyA(A),PhyB(B),andPhyC(C)were
hybridizedtoDNAdigestedwithEcoRI(E),HindIII
(H), or SacI (S) from two inbred lines, B73 and
W22.
CGACCGGGAACTC-3(cid:5)), UBI-F (5(cid:5)-CTACAACATTCAGAAG mic clones containing all phytochrome genes in maize
GAGAGCAC-3(cid:5)),andUBI-R(5(cid:5)-TCTGCAAGGGTACGGCCA
(seematerialsandmethods).Thelibrary,ZmMBBb,is
TCC-3(cid:5)).
estimated to represent 13.5 genome equivalents on 10
A restriction enzyme digest of PCR products was used to
distinguishhomeologs. Theenzyme MaeIIIwas usedto frac- filters(Tomkins et al. 2000, 2002). Thus, a single gene
tionate PhyA1 from PhyA2 products, AviI was used to distin- is expected to be present 1.3 times on each filter. For
guish PhyB1 and PhyB2, and AvaII was used to distinguish duplicated genes, the expected number of clones per
PhyC1 from PhyC2. Ten microliters of the PCR products was
filter is 2.6.
digestedovernightaccordingtomanufacturer’srecommenda-
To estimate phytochrome gene copy number, tallies
tions(NewEnglandBiolabs).Productswerefractionatedon
2% (w/v) agarose gel and transferred by capillary action in oftotalPhy-containingclonesweremade.SixBACfilters
20(cid:1) SSC to nylon membranes (Amersham). Hybridization weresequentiallyhybridizedwithPhyA-,PhyB-,andPhyC-
and image analysis were as described above for DNA blot CAR probes with the expectation that (cid:1)16 clones (2.6
analysis. Bands werevisualized with [(cid:7)-32P]dCTP-labeled RT- clones(cid:1)6filters)wouldberecoveredforeachhomeo-
PCR products derived from PhyA,PhyB, PhyC, or Ubi-specific
logousgenepair.DNAgelblotanalysisofputativephy-
amplificationproducts.
containingBACclones(seematerialsandmethods)
confirmed the identities of 10 PhyA, 7 PhyB, and 10
RESULTS PhyCclones.Restrictionfragmentlengthpolymorphism
(RFLP)andDNAblotanalysesofBACclonesindicated
CharacterizationofPhygenefamilystructure:Although
that there were two classes of PhyA-, PhyB-, and PhyC-
partialexpressedsequencetag(EST)andpartialgenomic
hybridizingclones.Theseresultsareconsistentwiththe
sequenceforphytochromegenesofmaizehavebeende-
DNAgelblotanalysisandindicatethatduplicatedcop-
scribed, only one family member, PhyA2, has been fully
iesofPhyA(PhyA1andPhyA2),PhyB(PhyB1andPhyB2),
sequenced(ChristensenandQuail1989).Thus,itwas
and PhyC (PhyC1 and PhyC2) are present in the B73
firstnecessarytoidentifyandsequenceallphytochrome
genome.
genefamilymembers.Thecopynumberofeachphyto-
SequenceanalysisofBACclonescontainingPhyloci:
chrome gene was first assessed through DNA gel blot
Full-lengthgenomicsequencesforPhyA1,PhyB1,PhyB2,
analysis. DNA from the inbred lines B73 and W22 was
digestedwiththreerestrictionenzymesandhybridized PhyC1, and PhyC2 were obtained by direct sequencing
to32P-labeledDNAderivedfromtheconservedCARof ofBACclonesasdetailedinmaterialsandmethods.
PhyA, PhyB, or PhyC. The CAR regions (see materials Annotated Phygene sequenceswere depositedin Gen-
and methods) represent the most conserved regions Bank. The full-length PhyA2 sequence was previously
of phytochrome genes (Sharrock et al. 1986) and are reported(ChristensenandQuail1989).Putativepro-
thus ideally suited for use as an ortholog-specific gene moter regions and intron/exon junctions were identi-
probe. The use of several restriction enzymes revealed fied through comparisons of previously published Phy
polymorphic sites between homeologs in two standard gene family members of sorghum (Childs et al. 1997)
inbreds, confirming that all loci are in fact present in and existing maize EST sequences. Comparison of the
more than one copy in the maize genome (Figure 1). predicted genic structure of Phy homeologs reveals a
On the basis of the number of restriction fragments striking conservation of gene organization. Figure 2
detected there appear to be two copies of PhyA, two shows a schematic representation of the phytochrome
copies of PhyB, and two copies of PhyC in at least two gene family in maize. Despite 94% (PHYB and PHYC)
standard maize inbred lines. to 96% (PHYA) shared amino acid identity between
Isolation of genomic BAC clones containing phyto- homeologs, several indels and nucleotide substitutions
chromeloci:ABAClibrarywasscreenedtoidentifygeno- clearlydifferentiateeachPhygenepair.PhyAhomeologs
PhytochromeFamilyHomeologsofMaize 1399
Figure 2.—Schematic of Phy gene structure. Alignments of genomic sequences for each phytochrome gene are shown with
gapsremoved.Exonsarerepresentedbyboxes;solidboxesdenotethe5(cid:5)-and3(cid:5)-UTRswhereknown.Stippledboxrepresents
a1.8-kbretroelementinsertionwithsimilaritytoTY3-gypsy-likeelements.Blacklinesrepresentnoncodingregions.
share94%nucleotideidentityacrossthecodingregion communication).Thatis,phytochromegenesonchro-
ofthegenesandextensivesequencesimilaritythrough- mosome 1 (PhyA1, PhyB1, and PhyC1) were most likely
out putative 5(cid:5)- and 3(cid:5)-UTRs. However, the intron in derived from one ancestral genome and the phyto-
the 5(cid:5)-UTR of PhyA1 is approximately double the size chromegeneslocatedonchromosomes9(PhyB2)and
oftheintroninPhyA2.PhyBhomeologsalsoshare94% 5 (PhyA2 and PhyC2) were likely derived from another
sequence identity in coding regions but show greater ancestralgenomefollowinganancientallopolyploidiza-
heterogeneity in intron size. For instance, PhyB1 con- tionevent(GautandDoebley1997;Wilsonetal.1999;
tainsa1.8-kbretroelementinsertion(nucleotides5876– Gaut 2001).
7713)inintronIIwithsimilaritytoaTy3-gypsy-likeretro- Predictedproteinstructureofphytochromegenefamily
transposon. This sequence is not present in intron II inmaize:AlignmentsofPhygenepairswithphytochrome
ofPhyB2orinPhyBfromsorghum(Childsetal.1997), genesfromriceandsorghumsuggestthatallmaizephyto-
suggestingarelativelyrecentoriginorthatPhyB2isthe chromegenesencodefunctionalproteins(seeFigureS1at
homologofsorghumPhyB.IntronIIIofPhyB1hasalso http://www.genetics.org/supplemental/).BLASTsearches
expandedrelativetointronIIIofPhyB2andPhyBinsor- ofCDART(http://www.ncbi.nlm.nih.gov/Structure/
ghum(Childsetal.1997),althoughthereisnoobvious lexington/lexington.cgi?cmd(cid:6)rps)revealedthepresence
insertion element to account for this increase in size. of several highly conserved domains present in all six
PhyC homeologs have similar intron/exon structure maize phytochrome genes. The chromophore attach-
and show little similarity in 5(cid:5)-noncoding regions but ment site is embedded in a GAF domain found in a
not in the 3(cid:5)-UTRs.
Maize phytochrome homeologs map to syntenic re-
gionsofthegenome:Gene-specificprobesweregener-
ated from the coding region or 3(cid:5)-UTR sequences for
eachofthesixPhygenefamilymembersandpositioned
on theIBM recombinant inbred population(Lee et al.
2002;Figure3).AlthoughtheprobesforphyAandphyB
could not distinguish between homeologs due to the
high sequence similarity in the 3(cid:5)-UTR, restriction site
differences enabled the assignment of RFLPs to one or
the other homeolog. The PhyB1 locus placed on chro-
mosome 1S (1.03) and PhyB2 placed on chromosome
9L(9.05/9.06). PhyC1was positionedto 1L(1.12) and
PhyC2waspositionedto5S(5.02/5.03).Wehavefound
no evidence of a third PhyC copy in the B73 genome
as suggestedby Basuet al.(2000) intheir comparative
mapping study.During the courseof this work,we dis-
coveredthatthepublishedPhyAsequenceislocatedon
chromosome 5 and not chromosome 1 as originally
describedinChristensenandQuail(1989).Tomore
accurately reflect the likely ancestry of this sequence,
Figure 3.—Comparative maps of maize and related grass
thepublishedPhyA1sequence(GenBankaccessionno. species, sorghum, and rice showing synteny relationships
AY260865)wasrenamedPhyA2(P.H.Quail,personal basedonsharedRFLPmarkerplacement.
1400 M.J.Sheehan,P.R.FarmerandT.P.Brutnell
vastarrayofsignaltransductionproteins(Aravindand
Ponting 1997; Ho et al. 2000). Flanking the GAF do-
main is a conserved Phytochrome domain found in all
phytochrome genes and two Per-Arnt-Sim domains
foundinmanysensorytransductionproteins(Ponting
and Aravind 1997; Pellequer et al. 1998; Whitmore
et al. 1998). The conserved histidine kinase-related do-
main that functions as a serine/threonine kinase (Yeh
and Lagarias 1998) and a phytochrome-like ATPase
(HATPase)domainarealsopresentineachofthemaize
phytochromegenefamilymembers.Furthermore,criti-
calaminoacidsrequiredforphyA(Deheshetal.1993;
Reed et al. 1994; Quail et al. 1995; Xu et al. 1995) and
phyBactivityinArabidopsis(WagnerandQuail1995;
KrallandReed2000)arealsoconservedinmaizePHYA1/
PHYA2 and PHYB1/PHYB2 proteins (asterisks in Figure
S1, A and B, at http://www.genetics.org/supplemental/).
Taken together, these data strongly suggest that all six
maize Phy genes have the potential for encoding fully
functional PHY proteins. Figure 4.—RNA gel blot analysis of maize phytochrome
DespitethestrikingsimilarityamongallPhygenehome- transcripts in seedling leaf tissues. Plants were grown for 10
days under continuous white light (W) or under complete
ologs, a number of features differentiate the PhyB gene darkness(D) priorto RNAextraction. Approximately20 (cid:2)g
familyhomeologs(seeFigureS1Bathttp://www.genetics.
oftotalRNAwasloadedforeachsample.Blotswerehybridized
org/supplemental/). Several H repeats and a series of togene-specific3(cid:5)-endprobesforPhyA,PhyB,PhyC,orUbiqui-
G repeats near the amino terminal region of PHYB tin(Ubi).27Sribosomalbandsusedasaloadingcontrolwere
define a hypervariable region among monocot PhyB visualizedwithethidiumbromidestaining.
sequences.Thislikelyreflectsthevariableexpansionof
several microsatellite sequences near the 5(cid:5) end of the ingly, PhyC transcript levels were maintained at similar
PhyB genes. For instance, a variable number of CAC re- levels under light and dark growth conditions, indicat-
peatsaccountforthepresenceofthreetosevenHresi- ing that the mechanisms regulating PhyC transcript ac-
dues near the N terminus of ZmPHYB1, ZmPHYB2, cumulation maydiffer fromthose regulatingPhyA and
SbPHYB,andOsPHYB.Similarly,slippedmispairingor PhyB. In addition to light-induced changes in expres-
polymerase stuttering accompanying DNA replication sion, tissue-specific differences in expression were also
throughlongstretchesofGCresidues(Farabaughand observed. Most notably, transcripts for PhyB and PhyC
Miller 1978) may account for the variable number of accumulated to similar levels in light- and dark-grown
GresiduesneartheNterminusofmonocotPHYB.The mesocotyl tissues and PhyA transcripts were much less
predicted PHYB1 protein is five amino acids shorter abundant in dark-grown mesocotyl tissues relative to
thanPHYB2duetoadditionalGrepeatsattheN-termi- dark-grown blade and sheath tissues.
nalregionoftheproteinanda(cid:8)1-bpframeshift9bases Semiquantitative RT-PCR of phytochrome gene ex-
before the predicted stop codon. pression: To examine the transcriptional profiles of
RNAgelblotanalysisofthephytochromegenefamily: eachPhygene,wedevelopedaRT-PCRassaythatdiffer-
RNA gel blot analysis was used to examine the light entiates between each homeolog (see materials and
regulationofPhygenetranscriptaccumulation(Figure methods). In brief, primers were designed to regions
4).Duetothestrongsequencesimilarityamonghomeo- of perfect identity that are shared between homeologs
logous genes, we were unable to develop gene-specific and that span the last intron to control for genomic
probesthatcouldunambiguouslydiscriminateeachho- DNA contamination. Product lengths ((cid:1)300–450 bp)
meolog.Thus,thetranscriptpoolsexaminedbyNorth- foreachhomeologwerealsoidenticaltopreventselec-
ernanalysisreflecttheaccumulationprofilesofhomeo- tiveamplificationofoneortheotherhomeolog.Alim-
logousgenepairsanddonotaddressthecontributions ited number of PCR cycles were then performed to
bysinglegenefamilymembers.Nevertheless,hybridiza- enablesemiquantitativeanalysisofgeneexpressionpro-
tions with gene-specific probes (see materials and files. cDNA products were digested with restriction en-
methods)detectedtranscriptsof(cid:1)4kbforPhyA,PhyB, zymes that could discriminate between cDNA derived
and PhyC, indicating that mature transcripts for PhyA, from homeologs due to the presence of single nucleo-
PhyB, and PhyC accumulate in seedling tissues. As ob- tide polymorphisms (see schematic in Figure 5). Prod-
servedpreviously(ChristensenandQuail1989),tran- ucts were fractionated on agarose gels and transferred
scriptsencodedbyPhyAweremoreabundantinD-grown to nylon membranes and hybridized to gene-specific
tissues than in W-grown tissues. PhyB transcripts also probes.Therelativeabundancewasestimatedfromden-
accumulated to higher levels in D than in W. Interest- sitometric scans of radiolabeled filters.
PhytochromeFamilyHomeologsofMaize 1401
Figure 5.—Semiquanti-
tative RT-PCR assay of phy-
tochrome gene expression
in seedling leaf tissue and
schematicofassay.RT-PCR
productsforeachgenepair
weredigestedovernightwith
MaeII(PhyA),AviI(PhyB),or
AvaII (PhyC); separated on
agarosegels;andtransferred
to nylon membranes. Frag-
ments resolved on 2% aga-
rosegelareshowninthesche-
matic. Restriction enzyme
cut sites are shown with ex-
pectedsizesoffragments.In-
tronnumberisshownabove
grayarrowheadsanddenotes
locationofsplicedintronsin
each transcript. Gene frag-
mentsweredetectedwithra-
diolabeledRT-PCRproducts
specific to PhyA (A), PhyB
(B),PhyC(C),oraUbiquitin-
specificfragment(D).
AsshowninFigure5,allsixmaizephytochromegenes similartothoseobservedforthePhyAgenes.Expression
are expressed in several seedling tissues including leaf fromonlyoneofthePhyBhomeologspredominatedin
blade, leaf sheath, and mesocotyl, but the light- and allseedlingtissuesexamined,andtranscriptsforPhyB1
tissue-specificpatternsofexpressiondiffersignificantly and PhyB2 were more abundant in D-grown relative to
among homeologous genes. Densitometric analysis of W-grownbladeandsheathtissues.However,bothPhyB1
transcriptprofilesindicatedthatPhyA1transcriptsaccu- and PhyB2 were expressed at similar or slightly higher
mulated to higher levels than PhyA2 transcripts in all levels in W-grown mesocotyl tissue relative to D-grown
tissuesexamined(datanotshown).However,thetissue- mesocotyl tissue.
specific patterns of PhyA1 and PhyA2 expression were AsobservedforPhyAandPhyBhomeologs,transcripts
verysimilartooneanother,andtranscriptsencodedby fromthe phyChomeologlocatedon chromosome1pre-
bothgenesaccumulatedtohigherlevelsinalldark-grown dominated in all seedling tissues examined. PhyC tran-
tissuesexaminedrelativetolight-growntissues.Thesimi- scripts accumulated to slightly higher levels in D-relative
larity between PhyA1 and PhyA2 accumulation profiles toW-grownbladetissuebutaccumulatedtosimilarlevels
maybeattributabletosmallregionsofconservedsequence inD-andW-grownsheathtissues.Together,thesestudies
in promoter and 5(cid:5)-UTR (see Figure S2A at http://www. indicate that phytochrome gene family members are
genetics.org/supplemental/) or the 3(cid:5)-UTR (see Figure underbothtissue-andlight-dependentregulationdur-
S2B at http://www.genetics.org/supplemental/). For ex- ing maize seedling development.
ample, putative phytochrome responsive PE3 and RE1
elements (Bruce et al. 1991) were identified in both
DISCUSSION
PhyA1andPhyA2promoters,indicatingasimilarmode
ofPhyAtranscriptionalcontrol(seeFigureS2Cathttp:// Phytochromes are the primary red/far-red photore-
www.genetics.org/supplemental/). ceptors in higher plants and play an essential role in
Gene expression profiles for the PhyB genes were theregulationofplantdevelopmentinresponsetoenvi-
1402 M.J.Sheehan,P.R.FarmerandT.P.Brutnell
ronmental change (Neff etal. 2000). In higher plants, testedfunctionaldivergenceofeachprotein.Neverthe-
phytochromes are encoded by a small nuclear gene less, the overlapping expression domains of maize Phy
family that varies in size and complexity throughout homeologs are consistent with the possibility of direct
theangiospermlineage.Inmanydiploideudicots(e.g., interaction of Phy homeolog proteins.
Arabidopsis, poplar, tomato, and Stellaria longipes) the The high sequence conservation between homeolo-
phytochromegenefamilyisrepresentedbythreetofive gous phy gene pairs also highlights some of the chal-
genefamilymembers(MathewsandSharrock1997; lenges that face maize genome sequencing projects.
Howe et al. 1998; Alba et al. 2000; Li and Chinnappa SearchesofTheInstituteforGenomeResearch(TIGR)
2003).However,inthemonocotsriceandsorghumthe MaizeGeneIndex(ZmGI)revealedtentativeconsensus
family is represented by three lineages, PhyA, PhyB/D, (TC) sequences for PhyA, PhyB, and PhyC genes of
andPhyC(MathewsandSharrock1996,1997;Childs maize. This database curates assemblies of maize ESTs
et al. 1997). Here we show that in maize the phyto- (TIGRcontigsorTCs)andidentifiedtwopotentialPhyA
chromegenefamilyiscomposedofsixmembers,PhyA1, genes.Forexample,TC185264incorporatesseveralEST
PhyA2,PhyB1,PhyB2,PhyC1,andPhyC2.Eachgenefam- sequences derived from multiple cDNA libraries and
ily member is predicted to encode a functional phyto- includes the previously published PhyA2 sequence
chromeapoproteinandisexpressedinseveralseedling (ChristensenandQuail1989).Comparisonsoftheindi-
tissues. The position of Phy paralogs (e.g., PhyA1 and vidualESTclonesequencesthatconstituteTC185264with
PhyA2) in homeologous regions of the maize genome the PhyA1 and PhyA2 sequences reported here reveal
stronglysuggeststhatthesegeneduplicationsweregen- that both PhyA1 and PhyA2 sequences are represented
eratedasaconsequenceofanancienttetraploidevent in the assembly. A tentative consensus sequence
(Gaut and Doebley 1997; Wilson et al. 1999; Gaut (TC171984) was also derived from ESTs that were all
2001). Thus, the PhyA1, PhyB1, and PhyC1 genes on mostsimilartoPhyA1sequence.Thus,TC185264ismost
chromosome 1 are most accurately defined as the ho- similar to PhyA2 whereas TC171984 is most similar to
meologsofPhyA2,PhyB2,andPhyC2,respectively,pres- PhyA1sequence,butneitherassemblyisidenticaltothe
ent on chromosomes 5S (PhyA2 and PhyC2) and 9L predictedPhyA1genereportedhereortothepreviously
(PhyB2). The striking conservation of sequence and published PhyA2 sequence (Christensen and Quail
overlapping expression patterns of phytochrome ho- 1989).
meologs suggest that functional redundancy of phyto- Attempts to distinguish highly similar gene homeo-
chrome gene action has been maintained during the logssuchasthephytochromegenesarefurthercompli-
evolution and domestication of modern maize. cated by the presence of repetitive elements within
Although the function of any Phy gene has yet to be genes.AsshowninFigure2,anearly2-kbretroelement
determined in maize, it is tempting to speculate on insertion is present in the second intron of PhyB1. Al-
some of the consequences of maintaining such similar though gene islands are anticipated to be scattered
homeologs.Perhapsoneofthemostintriguingpossibili- throughoutaseaofretrotransposonsinmaize(SanMi-
tiesisthatthehomeologscouldformactiveheterodim- guel et al. 1996), the presence of large retroposon se-
ers that mediate responses different from those regu- quenceswithingenescouldproveproblematicingeno-
latedbyhomodimers.Earlystudiesofoatphytochrome micassemblies(Rabinowiczetal.2003).Toenrichfor
proteins indicated that the biologically active forms of gene-richregionsofthegenome,currentmaizegenome
phytochrome function as homodimers (Jones and sequencingprojectsareutilizingmethylfiltrationtech-
Quail 1986). Evidence strongly suggests that these di- nologies and high C t sequences to reduce representa-
o
merizedformsinteractwithnuclearlocalizedtranscrip- tionofrepetitiveLTRretroelements(Rabinowiczetal.
tion factors to mediate phytochrome responses (Saka- 2003).Sincewedidnotexaminethemethylationstatus
moto and Nagatani 1996; Kircher et al. 1999, 2002; of this element,it is difficult to predictwhether or not
Yamaguchi et al. 1999; Huq and Quail 2002; Huq et PhyB1intronsequenceswouldberepresentedinmethyl-
al. 2003). In the case of Arabidopsis phyB, dimerized ation-filteredgenomiclibraries(Rabinowiczetal.1999).
and nuclear localized N-terminal sequences (amino Regardless,therepetitivenatureoftheelementandits
acids1–651)aresufficienttomediateanumberofred largesizewouldlikelyleadtoitsexclusionfromgenomic
light responses in a phyB mutant background (Matsu- assemblies.Recentmethodsdevelopedtolinkgene-rich
shitaetal.2003).Althoughmanyregionsofthemature regions should prove particularly useful in assembling
protein assist or strengthen dimerization, the amino genes such as PhyB1 in maize (Yuan et al. 2002).
acids that are critical for dimerization of Arabidopsis Inadditiontoconfoundingbioinformatics-basedap-
phyAliebetween1116and1161(Quail1997).Dueto proachestogenomeannotation,theduplicationsofthe
thehighdegreeofsequenceidentitybetweenhomeolo- maize Phy gene family also highlight challenges faced in
gous genepairs, especiallywithin thisdimerization do- definingsyntenicrelationshipsamonggrassgenomes.The
main, it is likely that the maize PHY homeologs will relatively recent polyploidization in the maize lineage
also form heterodimers. Clearly, the consequences of has likely contributed to the duplication of (cid:9)80% of
heterodimerization are dependent on the, as yet, un- themaizegenomeascolinearregionsofhomeologous
PhytochromeFamilyHomeologsofMaize 1403
maizechromosomes(Gaut2001).Thesehighlysimilar AlthoughlittleisknownofPhyBorPhyCgeneregula-
sequences have the potential to greatly complicate the tioninmonocots,detailedcharacterizationsoforgan-and
analysisoforthologousrelationshipsinthegrasses.For tissue-specificregulationofphytochromegeneexpression
instance,inarecentstudy,aPhyA-containingBACclone havebeenperformedinseveralmodeleudicots.InArabi-
of maize was characterized as a syntenic region with a dopsis,PHYA,PHYB,PHYC,PHYD,andPHYEtranscripts
PhyA gene sequence from sorghum (Morishige et al. accumulateinseveralplantorgans(Clacketal.1994),
2002). The authors characterized a single maize BAC and promoter-Gus fusions indicate that the promoters
clone and assigned this gene and all predicted gene determine tissue specificity, light induciblity, and mag-
sequences residing on this BAC to chromosome 1L, nitudeofgeneexpression(SomersandQuail1995a,b;
correspondingtothepublishedmappositionofPhyA2 Gooseyetal.1997).However,translationaland/orpost-
(ChristensenandQuail1989).However,aswereport translationalmechanismslargelydeterminetheaccumula-
here,theoriginalassignmentofthishomeologtochro- tionofphytochromeproteinsinArabidopsis(Clacketal.
mosome1wasincorrectandisinsteadlocatedonchro- 1994; Sharrock and Clack 2002; Sharrock et al.
mosome5S.Furthermore,becauseonlyoneofthetwo 2003b).Inpotato,auniformpatternofPhyBexpression
PhyA orthologs was characterized, the previously re- was observed in several mature plant organs (Heyer
ported synteny comparisons made between the sor- and Gatz 1992). In tomato, however, temporal tissue-
ghumand maizePhyAregionsare incomplete(Moris- andorgan-specificdifferenceswereobservedforallphy-
higeetal.2002).Itistemptingtospeculatethatbecause tochrome genes (Hauser et al. 1997, 1998). Also, in
allPhygeneslocatedonchromosome1aremorehighly general, PhyA transcripts are most abundant in several
expressed in seedling tissuesthan are their homeologs seedling and mature plant tissues, with the exception
located on chromosomes 5 and 9, genes tightly linked ofripeningfruit,wherePhyB2andPhyFtranscriptspre-
to thesePhy genesmay showsimilar homeolog-specific dominate(Hauseretal.1997).Intobacco,PHYB1-Gus
biases in gene expression. promoterfusionsalso suggesttissue-specificregulation
An analysis of Phy transcript profiles was conducted ofPHYB1transcriptionindevelopingseedlingsandma-
toexaminepotentiallight-andtissue-specificregulation ture plants. In particular, PHYB1 expression appeared
ofPhygeneactivityinmaize.TranscriptsforbothPhyA1 toberestricted tophloemcellsofthe stem,petiole,and
and PhyA2 accumulated to much higher levels in dark midribtissues(Adametal.1996).Theorgan-andtissue-
relative to white light in blade, sheath, and mesocotyl specific expression profiles of PHYB genes in tomato
tissues. This light-dependent decrease in phyA tran- (Hauseretal.1997)andtobacco(Adametal.1996)and
scriptshasbeenobservedinseveralgrassspecies(Col- maize (this study) contrast with the relatively uniform
bertetal.1983;Hersheyetal.1984;Christensenand patternofPHYBexpressioninArabidopsis(Somersand
Quail 1989; Kay et al. 1989; Dehesh et al. 1991) and Quail1995b)andpotato(HeyerandGatz1992)and
likelyreflectsacommonregulatorymechanismthatacts suggestthattheremaybelittleconservationinthetran-
to maintain high levels of phyA transcript in the dark scriptionalregulationofPHYBandPHYCaccumulation
(Bruceetal.1989).Furthermore,severalelementshave between monocots and dicots.
been defined in monocot PhyA promoters that likely In rice (Dehesh et al. 1991; Basu et al. 2000) and
mediate a high transcription rate of PhyA in the dark sorghum(Childsetal.1997)PhyBandPhyCtranscripts
(Bruce andQuail 1990;Bruce etal. 1991).The pres- are maintained at relatively constant levels under dark
enceofPE3andRE1elementsinthepromoterregion and white light conditions, but nothing is known of
ofmaizePhyAhomeologssuggestsasimilarmechanism tissue- or organ-specific expression profiles. In maize,
of transcriptional control. wehaveshownthattissue-specificaccumulationofPhyB
Despite the common features shared by monocot is light dependent. That is, PhyB transcripts are most
phyA promoters, the kinetics of light-mediated reduc- abundant in blade and sheath tissues of dark-grown
tionsofPhyAtranscriptlevelsdiffersamonggrassspecies plantsbutaccumulatetosimilarlevelsinmesocotyltis-
(Colbert et al. 1985; Lissemore and Quail 1988; suesoflight-anddark-grownplants(seeFigure4).Thus,
Christensen andQuail 1989; Kayet al. 1989).In oat light appears to play an important role in regulating
(LissemoreandQuail1988)andrice(Kayetal.1989) the organ-specific patterns of PhyB gene expression in
there is a rapid decrease in PhyA transcript levels that maize. PhyC transcripts were the least abundant of all
can largely be attributed to a decreased rate of PhyA Phy genes in dark-grown tissues and were maintained
transcription.InmaizethereductioninPhyAtranscript at low levels in light-grown tissues as well.
accumulation is less rapid (Christensen and Quail Theidentificationandsequenceanalysisofallphyto-
1989)andindicatesthatpost-transcriptionalregulation chrome gene family members reported here will now
may also contribute to PhyA transcript accumulation. permittheutilizationofassociationmethodstoexamine
Herewehaveshownthatthislight-dependentreduction the role of phytochrome gene function in this impor-
in PhyA transcript accumulation is intrinsic to PhyA1 tant crop plant. In addition, the elucidation of phyto-
and PhyA2 regulation and operates in several seedling chrome gene family organization in maize will guide
tissues. reversegeneticscreenstodisruptallphytochromegene
1404 M.J.Sheehan,P.R.FarmerandT.P.Brutnell
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