Planta (2008) 227:1377–1388
DOI 10.1007/s00425-008-0709-1
O R I G I N A L A R T I CL E
A maize CONSTANS-like gene, conz1, exhibits distinct diurnal
expression patterns in varied photoperiods
Theresa A. Miller · Elizabeth H. Muslin ·
Jane E. Dorweiler
Received: 21 December 2007 / Accepted: 5 February 2008 / Published online: 27 February 2008
© Springer-Verlag 2008
Abstract Maize (Zea mays ssp. mays L.) was domesti-
cated from teosinte (Z. mays L. ssp. parviglumis Iltis &
Doebley), a plant requiring short day photoperiods to
Xower. While photoperiod sensitive landraces of maize
exist, post-domestication breeding included eVorts to grow
maize in a broad range of latitudes. Thus, modern maize is
often characterized as day-neutral because time to Xower is
relatively unaVected by photoperiod. We report the Wrst
identiWcation of maize constans of Zea mays1 (conz1), a
gene with extensive sequence homology to photoperiod
genes CONSTANS (CO) in Arabidopsis (Arabidopsis thali-
ana (L.) Heynh.) and Heading date1 (Hd1) in rice (Oryza
sativa L.). conz1 maps to a syntenous chromosomal loca-
tion relative to Hd1. Additionally, conz1 and two maize
homologs of another photoperiod gene exhibit diurnal
expression patterns notably similar to their Arabidopsis and
rice homologs. The expression pattern of conz1 in long
days is distinct from that observed in short days, suggesting
that maize is able to discern variations in photoperiod and
respond with diVerential expression of conz1. We oVer
models to reconcile the diVerential expression of conz1
with respect to the photoperiod insensitivity exhibited by
temperate inbreds.
Electronic supplementary material The online version of this
article (doi:10.1007/s00425-008-0709-1) contains supplementary
material, which is available to authorized users.
Sequence data from this article can be found in the GenBank (http://
www.ncbi.nlm.nih.gov/) data library under the following accession
numbers:conz1 mRNA: EU098139, EU098140; gigz1A: BK006299;
gigz1B: BK006298.
T. A. Miller · E. H. Muslin · J. E. Dorweiler (&)
Department of Biological Sciences, Marquette University,
P. O. Box 1881, Milwaukee, WI 53201-1881, USA
e-mail: jane.dorweiler@marquette.edu
Keywords Circadian · Flowering · qPCR · Teosinte · Zea
Introduction
The transition from vegetative to reproductive growth is an
important developmental switch in angiosperms. Many
genetic and molecular components underlying the Xoral
transition have been characterized in Arabidopsis (Arabid-
opsis thaliana (L.) Heynh.) and rice (Oryza sativa L.).
Extensive work in Arabidopsis has identiWed a complex
network of both activators and repressors of this transition.
The molecular components function in several regulatory
pathways: autonomous, gibberellin, photoperiod and ver-
nalization pathways. Each independent pathway works in
response to diVerent environmental and endogenous sig-
nals. These pathways converge to regulate the expression of
Xoral integrators, namely FLOWERING LOCUS T (FT),
SUPRESSOR OF CONSTANS1 (SOC1) and LEAFY (LFY)
in Arabidopsis (Parcy 2005), ultimately responsible for the
transition to reproductive growth. These networks reveal an
intricate system of checks and balances that ensure promo-
tion of Xowering when environmental and endogenous con-
ditions are optimal for successful generation of progeny. A
growing body of evidence suggests that a majority of these
Xoral regulators and their networks are well conserved in
rice (reviewed in Izawa et al. 2003; Izawa 2007a).
In contrast to rice and Arabidopsis, little is known about
the molecular mechanism underlying this transition in
maize (Zea mays L. ssp. mays) despite its economic impor-
tance. Characterization of the molecular components of the
maize Xoral network has begun relatively recently and in
most cases was aided by homology with previously identi-
Wed genes in Arabidopsis. Genes in the maize network
include delayed Xowering1 (dlf1), Z. mays Floricaula/
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1378
Leafy genes (zX1 and zX2), dwarf8 (d8), and indeterminate1
(id1) (Colasanti et al. 1998; Thornsberry et al. 2001; Bom-
blies et al. 2003; Muszynski et al. 2006). Several EMS alle-
les of dlf1 were isolated and characterized by M. G. NeuVer
over a decade ago. This late Xowering mutant produces four
to six additional leaves relative to wild-type siblings (Neu-
Ver et al. 1997). Recent characterization, including identiW-
cation of Mutator insertion alleles, enabled cloning of dlf1,
which was found to be homologous to Arabidopsis FD
(Muszynski et al. 2006). In Arabidopsis, a protein complex
comprised of FD and the Xoral integrator FT is required to
promote the expression of meristem identity genes that
cause the shoot apical meristem to switch from vegetative
to reproductive growth (Abe et al. 2005).
zX1 and zX2 are orthologous to two meristem identity
genes, FLORICAULA in Antirrhinum majus L. (snap-
dragon) and LEAFY in Arabidopsis (Bomblies et al. 2003).
The carboxy-termini of the zX genes are most similar to
LEAFY, a transcription factor with reported DNA-binding
capability (Weigel et al. 1992; Parcy et al. 1998; Busch
et al. 1999; Wagner et al. 1999). These genes all share con-
served proline-rich, leucine-rich, basic and acidic domains
(Coen et al. 1990; Weigel et al. 1992; Bomblies et al.
2003), although the biochemical functions of these domains
remain unknown.
While dlf1and the zX genes function immediate to and
during the Xoral transition, respectively, d8 is predicted to
work farther upstream in the Xoral pathway. d8 was origi-
nally characterized as a dominant, dwarf mutant that was
insensitive to gibberellic acid treatment (Phinney 1956).
d8 encodes a DELLA protein (Thornsberry et al. 2001)
orthologous to GIBBERELLIN INSENSITIVE (GAI) in
Arabidopsis which modulates gibberellic acid responses
(Peng et al. 1999). Analysis of intragenic sequence poly-
morphisms among d8 alleles in a diverse set of maize
lines identiWed a signiWcant association with Xowering
time variation (Thornsberry et al. 2001; Andersen et al.
2005).
Unlike the maize genes mentioned above, id1 has no
clear Arabidopsis ortholog (Colasanti et al. 2006) and, thus,
the relative position of id1 within the Xoral induction net-
work is unclear. ID1 protein is the founding member of a
family of plant-speciWc zinc-Wngers (Colasanti et al. 1998)
and has DNA binding capabilities which suggest a function
in transcriptional regulation (Kozaki et al. 2004). The id1
mutant exhibits an extensive vegetative phase, and while
the initial report suggested a requirement for short days to
eventually Xower (Singleton 1946), more recent reports
suggest that id1 transcript and protein levels do not Xuctu-
ate in relation to light–dark cycles (Wong and Colasanti
2007) consistent with id1 acting independently from the
photoperiod pathway (Coneva et al. 2007). Rather, it is
thought that id1 may act in the autonomous pathway (Cola-
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Planta (2008) 227:1377–1388
santi and Sundaresan 1996). Based on genetic and expres-
sion data, it has recently been proposed that id1 activates
dlf1 (Muszynski et al. 2006), albeit not necessarily in a
direct manner.
With the exception of id1, the aforementioned examples
demonstrate the usefulness of Arabidopsis as a framework
from which to expand our knowledge of Xowering in
maize. Additionally, rice is rapidly developing as a mono-
cot model for the study of Xowering time, particularly with
respect to the photoperiod pathway. This has proven infor-
mative given that rice and Arabidopsis have opposite
responses to photoperiod. Arabidopsis is a long day plant,
Xowering much more rapidly in long days than in short
days, whereas rice is a short day plant. Despite this distinc-
tion, the speciWc molecular components involved in the
photoperiod pathway are strikingly conserved, although
some appear to diVer in their mode of regulation. Photope-
riod sensitivity in Arabidopsis and in rice is largely depen-
dent upon the homologous Xoral regulators CONSTANS
(CO) (Putterill et al. 1995, 2004; Parcy 2005) and Heading
Date1 (Hd1) (Yano et al. 2000; Hayama et al. 2003; Imaiz-
umi and Kay 2006), respectively. CO has been shown to
function as a Xoral activator in inductive long days, but has
no function in short days (Putterill et al. 1995). Similarly,
HD1 is suggested to function as a Xoral activator under
inductive short days, but is distinct from CO in that under
non-inductive photoperiod conditions it functions as a Xoral
repressor (Hayama et al. 2003).
Transcriptional regulation of CO and Hd1 in response to
photoperiod results in circadian patterns of expression.
Photoperiod serves as an important environmental cue in
setting the circadian clock. GIGANTEA (GI) in Arabidopsis
and OsGI in rice are direct outputs of the circadian clock
and promote the expression of CO and Hd1, respectively
(Fowler et al. 1999; Park et al. 1999; Cremer and Coupland
2003; Hayama and Coupland 2003; Mizoguchi et al. 2005).
As a result, a distinct circadian rhythm characterizes the
expression of both GI/OsGI and CO/Hd1. These genes tend
to exhibit a single prominent peak of expression per 24-h
period, with the expression of GI/OsGI preceding CO/Hd1
by approximately 3–6 h (Park et al. 1999; Suarez-Lopez
et al. 2001; Hayama et al. 2002; Shin et al. 2004). One
exception is a subtle, but reproducible, biphasic pattern of
CO expression when Arabidopsis is entrained in inductive
long days (Suarez-Lopez et al. 2001). This pattern of
expression can be accounted for by CDF1 (CYCLING
DOF FACTOR 1) repressing CO expression during the Wrst
half of the day (Imaizumi et al. 2005), and then FKF1
(FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) works
with GI to relieve the repression of CO by CDF1 later in the
day (Sawa et al. 2007).
The importance of the photoperiod pathway in the Xoral
network of Arabidopsis and rice as evidenced by GI/OsGI
Planta (2008) 227:1377–1388
and CO/Hd1 prompted us to investigate the role of the
photoperiod pathway in maize. Maize was domesticated
from teosinte (Z. mays L. ssp. parviglumis Iltis & Doeb-
ley), a short day plant (Emerson 1924). While breeding
eVorts have continued to select for insensitivity to photo-
period in regards to Xowering, numerous tropical land-
races of maize remain sensitive to photoperiod and Xower
faster in response to short days. Furthermore, although the
majority of maize inbreds are generally characterized as
day-neutral, a large number of temperate inbreds retain
some detectable level of photoperiod sensitivity (Francis
et al. 1969; Russell and Stuber 1983; Mungoma and Pollak
1991; Ellis et al. 1992; Bonhomme et al. 1994; Moutiq
et al. 2002). Indeed, meta-analyses of numerous QTL stud-
ies involving a wide array of inbred lines, and extrapola-
tion of consensus Xowering time QTLs to syntenous
regions of rice, suggested that several orthologs of photo-
period pathway genes may continue to make important
contributions toward Xowering time variation in maize
(Chardon et al. 2004). Aside from studies of a few circa-
dian clock components and speciWc photoreceptors, to our
knowledge there have been no studies of maize genes
directly involved in the photoperiod pathway to date. Fur-
thermore, it is not known how breeding eVorts have ren-
dered maize relatively insensitive to photoperiod. We
report here the identiWcation of maize genes with sequence
homology to two photoperiod genes in Arabidopsis (GI
and CO) and rice (OsGI and Hd1). We further characterize
these genes and show that the mRNA of the maize genes is
expressed in diurnal patterns, similar to the transcriptional
regulation of their homologs. The expression proWle of the
maize CONSTANS homolog, conz1, is diVerent in long-
versus short-days, suggesting that maize diVerentiates
photoperiod at the transcriptional level. Finally, we dem-
onstrate that the conz1 expression pattern diVers between
teosinte and maize under long-days, which is the non-
inductive photoperiod for teosinte, and discuss the impli-
cations of these data.
Materials and methods
Plant materials
Zea mays ssp. mays L. (maize) inbred B73 from the Maize
Genetics COOP Center at S-123 Turner Hall, 1102 South
Goodwin Avenue, Urbana, IL 61801-4730, USA; http://
www.maizegdb.org was used in a majority of the experi-
ments. One experiment (Fig. 8) also used Z. mays L. ssp.
parviglumis Iltis & Doebley (teosinte, Maiz de Huiscatote)
accession #566688 from USDA GRIN at Regional Plant
Introduction Station, Ames, IA 50011-1170, USA; http://
www.ars-grin.gov.
1379
Plant growth conditions
Seed from maize inbred B73, the genome of which is cur-
rently being sequenced, were planted in individual plastic
pots and were grown for 3 weeks in a Percival growth
chamber (PGC-15.5-AR). Plants were grown in either short
days (8 h light, 16 h dark) or long days (16 h light, 8 h
dark). The temperature in the chamber was maintained at
26°C during light, dark and constant light conditions for all
photoperiod experiments. Light in the chamber measured
700–900
mol m¡2 s¡1. A subsequent experiment repli-
cated the results for B73 under comparable long day condi-
tions and simultaneously tested teosinte.
Tissue collections and sample preparation
After 3 weeks of growth, tissue samples were taken from
plants every 3 h for a 48 h period. Each tissue sample con-
sists of three paper-hole punches from the newest leaf of
three diVerent, randomly-selected plants. Two biological
replicates were collected at the same time; thus the replicate
consisted of tissue from three plants distinct from those
used in the Wrst replicate sample. The tissue samples were
collected in pre-chilled tubes, placed immediately on dry
ice, and then stored at ¡80°C.
Total RNA was extracted from each tissue sample using
TRIzol® from Invitrogen (Cat. No. 15596-018) following
manufacturer’s protocol including the optional step for
removal of excess polysaccharides. Each RNA sample was
treated with Ambion’s TURBO DNA-free™ (Cat. No.
1907) following manufacturer’s “Rigorous Treatment” pro-
tocol in an eVort to remove any residual genomic DNA
(gDNA). DNase treated RNA was subjected to reverse
transcriptase reactions using oligo-dT primer and M-MLV
Reverse Transcriptase (Promega Cat. No. M1705) as fol-
lows: 1,000 ng total RNA, 1
L 10 mM oligo-dT and dH2O
(to 16
L) were mixed and incubated at 70°C for 5 min and
Xash cooled on ice. M-MLV Reaction buVer, 2
L 2.5 mM
dNTP and 200 Units M-MLV enzyme were added to the
reaction and incubated at 37°C for 60 min followed by
15 min at 70°C.
Real time PCR
Quantitative Real-Time PCR (qPCR) was performed on a
Bio-Rad MyIQ Single Color thermocycler with Bio-Rad
SYBR Green Supermix (Cat. No.170-8884). Reaction vol-
umes were 25
L and consisted of 12.5
L SYBR Green
Supermix, 2
L 5 mM primer mix (forward and reverse),
and cDNA template (10.5
L of a 40:1 dilution of the RT
reaction). Each reaction was performed in triplicate using
the following conditions: 10 min activation at 95°C, 40
cycles of three-step ampliWcation (10 s at 95°C, 20 s at the
123
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determined optimal annealing temperature (varies by
primer pair) and 20 s at 72°C), and a Wnal extension of
1 min at 72°C followed by the melt curve (80 cycles of 7 s
at increasing temperatures starting at 55°C and increasing
by 0.5°C at each cycle).
Primer design
Primers were designed for PCR using Gene Runner v. 3.05
(Hastings Software, Inc.), which facilitated in silico pre-
screening to eliminate pairs with stable hairpin loops,
dimers, bulge loops and internal loops. Experimentally
optimal primers were identiWed based upon their ability to
meet several standards: (1) Robustness—successful ampli-
Wcation over a range of annealing temperatures, (2) Speci-
Wcity—generation of a single signiWcant peak in the melt
curve and (3) Consistency—highly reproducible Ct values
within the reactions of a triplicate.
The primers used for conz1 are JD170f: 5⬘-ggtcagtgctt-
acacagattc-3⬘, JD170r: 5⬘-gagcttggcatgtctgtc-3⬘; for gigz1A
are JD242f: 5⬘-gatcactgacatattgctagcc-3⬘, JD242r: 5⬘-ccaga
tcctcggctgc-3⬘; for gigz1B are JD241f: 5⬘-ctataggaaatgaa
tctgacc-3⬘, JD241r: 5⬘-ataagttgtgggtgctttc-3⬘; for ubi1 are
JD82f: 5⬘-gtttaagctgccgatgtgcctg-3⬘, JD82r: 5⬘-gacacgactca
tgacacgaacagc-3⬘; and for
-tub are JD83f: 5⬘-agaactgc
gactgcctccaagg-3⬘, JD83r: 5⬘-agatgagcagggtgcccattc-3⬘.
Where possible, primers were designed to span an intron of
the respective gene such that any residual gDNA would
generate a diVerent size product from the cDNA product
and would be visible on the melt curve. In these cases, no
(or negligible) products from gDNA were visible.
Data analysis
The results of Real-Time PCR were analyzed using
DART-PCR (Peirson et al. 2003), a program that facili-
tates the analysis of the kinetics of each reaction per-
formed. The average ampliWcation eYciency of each
primer pair was determined, and primers performing
poorly were replaced. The average eYciency of all of the
primer pairs discussed in this study ranged between 0.95
and 1.0. DART-PCR then uses the ampliWcation eYciency
of each primer pair to calculate a value (R0) representative
of the initial template concentration. The R0 values for
ubiquitin and experimental genes were used to calculate
expression levels relative to ubiquitin1 (ubi1). ubi1 was
used as a normalization control because independent trials
of several housekeeping genes had found it to be the most
reproducible in our hands across various genotypes and
tissues (J. Nobler and J. Dorweiler, unpublished results).
The only exception was the teosinte experiment, where

-tubulin was used because it proved more reproducible
than ubi1.
123
Planta (2008) 227:1377–1388
Results
IdentiWcation of a maize CONSTANS homolog
We searched publicly curated databases (MaizeGDB, http:/
/www.maizegdb.org/, MAGI, http://www.plantgenom-
ics.iastate.edu/maize/, and PlantGDB, http://www.plant-
gdb.org/) and the DuPont/Monsanto/Ceres Maize Sequence
Information Sharing Program database (http://www.maiz-
eseq.org/) for sequences with strong similarity to Arabidop-
sis thaliana CONSTANS (CO) and Oryza sativa Heading
Date1 (Hd1). These searches identiWed several sequences
predicted to encode proteins similar to CO and Hd1. Most
of the sequences were incomplete, lacking some of the con-
served domains, and appeared to be mis-spliced transcripts
(see below), however, one assembled DuPont sequence,
49498.1 (accession #EU098139), appeared to be full-
length. Primers were designed to the 5⬘ and 3⬘ UTRs of
49498.1 for independent ampliWcation, cloning and
sequencing of the entire coding region (accession
#EU098140). Translation of the full sequence revealed
65% amino acid identity with HD1 (Fig. 1) and the pres-
ence of B-box-type zinc Wngers and a CCT domain (Fig. 1),
both characteristic domains of CO, Hd1 and the CO-like
protein families (GriYths et al. 2003). The B-box regions
of the predicted maize protein and HD1 are 89% identical;
the CCT domains are 93% identical. Furthermore, both
B-box type zinc Wngers of the predicted maize protein
retain the Wve requisite and correctly spaced Cysteine resi-
dues (Fig. 1). Despite the divergence of monocots and
dicots, the maize sequence also maintains signiWcant simi-
larity with the functional domains of the Arabidopsis CON-
STANS protein: the B-boxes are 69% identical and the
CCT domains are 83% identical. Given the potential for
this transcript to encode a protein homologous to CO and
Hd1, in contrast to the aforementioned incomplete or poten-
tially mis-spliced transcripts, we will refer to this transcript
herein as the “normal” transcript. In an eVort to determine
where the maize gene resides, additional primers were
designed to the normal transcript and PCR was performed
using genomic DNA from the Oat–Maize addition lines
(Riera-Lizarazu et al. 1996; Ananiev et al. 1997) as tem-
plates. This assay demonstrated that conz1 is located on
chromosome 9 because a positive PCR product was
observed only from the Oat–Maize line containing maize
chromosome 9 (Fig. 2).
The maize homolog and Hd1 map to syntenous
chromosomal locations
Given that CO is part of a large gene family (Robson et al.
2001; GriYths et al. 2003) we wanted to verify that we
were working with the evolutionary ortholog of CO and
Planta (2008) 227:1377–1388 1381

B-box 1
AtCO : MLK--------QESN----DIGSGENN-----RARPCDTCRSNACTVYCHADSAYLCMSCDAQVHSANRVASRHKRVRV : 62
OsHd1 : MNYNFGGNVFDQEVGVGGEGGGGGEGSGCP--WARPCDGCRAAPSVVYCRADAAYLCASCDARVHAANRVASRHERVRV : 77
conz1 : MDYNFDTSVLDEDVA----GRGGREGS-CPPAWARACDGCRAAPSVVYCHADTAYLCASCDSRVHAANRVASRHERVRV : 74
B-box 2
AtCO : CESCERAPAAFLCEADDASLCTACDSEVHSANPLARRHQRVPILPISGNSFSSMTTTHHQSEKTMTDPEKRLVVDQEEG : 141
OsHd1 : CEACERAPAALACRADAAALCVACDVQVHSANPL------------PAITIPATSVLAEAVVATAT------------- : 131
conz1 : CEACECAPAVLACRADAAALCAACDAQVHSANPLAGRHQRVPVLPLPAAAVPAASVLAEATATAAS------------- : 140

AtCO : EEGDKDAKEVASWLFPNSDKNNNNQNN------------------GLLFS--DEYLNLVDYNSSMDYKFT----GEYSQ : 196

OsHd1 : VLGDKD-EEVDSWLLLSKDSDNNNNNNNNNDNDNNDNNNSNSSNNGMYFGEVDEYFDLVGYNSYYDNRIENNQDRQYGM : 209
conz1 : VAGDKD-EEVDSWLLLTKDPDDDDKNHNCSSNNNNNN---ISSNTSTFYADVDEYFDLVGYSSYCDNHINSNT-KQYGM : 214

AtCO : H-----QQNCSVPQTSY----GGDRVVPLKL-----EESRGHQCHNQQNFQFNIKYGSSGTHYNDNGSINHNAYISSME : 261

OsHd1 : HEQQEQQQQQQEMQKEFAEKEGSECVVPSQITMLSEQQHSGYG-VVGADQAASMTAGVSA--YTDSIS-NSISF-SSME : 283
conz1 : ------QEQQLLLHKEFGDKEGSEYVVPSQV----GQQQSGYHRVIGTEQAASMTPGVSA--YTDSIS-NSISYSSSME : 280
CCT
AtCO : TGVVPESTACVTTASHP--RTPKGTVEQQ-PDPASQMITVTQLSPMDREARVLRYREKRKTRKFEKTIRYASRKAYAEI : 337
OsHd1 : AGIVPDST-VIDMPNSRI-LTPAGAINLF-SGPSLQM--SLHFSSMDREARVLRYREKKKARKFEKTIRYETRKAYAEA : 357
conz1 : VGIVPDNMATTDMPSSGILLTPAGAISLFSSGPPLQM--PLHLASMDREARVLRYREKKKSRKFEKTIRYATRKTYAEA : 357

AtCO : RPRVNGRFAKR--EIEAE-EQGFNTMLMYNTG-YGIVPSF : 373

OsHd1 : RPRIKGRFAKR-SDVQIEVDQMFSTAAL-SDGSYGTVPWF : 395
conz1 : RPRIKGRFAKRSSDMDDEVDQMFSAAALSSDGSYGTVPWF : 397

Fig. 1 Alignment of the CONSTANS homologs from Arabidopsis (At- ilies are boxed. Amino acids conserved among all three proteins are
CO), rice (OsHd1), and maize (conz1). The conserved B-Box and CCT shaded in black. Amino acids identical between OsHd1 and conz1 are
domains characteristic of the CONSTANS/CONSTANS-like gene fam- shaded in gray

M
M M M bp
1 2 3 4 5 6 7 8 9 10
Oat
2000
1000
Maize 750
500
Fig. 2 PCR of Oat–Maize Addition lines. An inverted image of an
ethidium-bromide stained gel reveals PCR products obtained from
gDNA of the oat parental lines and the maize parental lines (left). PCR
products were obtained from gDNA of the Oat–Maize Addition lines
(right). The number above each lane corresponds to the maize chromo-
some introduced into that oat line; (M) corresponds to the molecular
weight marker of which relevant sizes are designated (far right) in base
pairs (bp)
Hd1 as opposed to a closely related family member. In this
context, we use the term ortholog as Wrst deWned by Fitch
(1970) to describe genes related through speciation from a
common ancestor, rather than a gene family member cre-
ated via duplication and speciation. In this sense, orthologs
may not necessarily retain the same function. To assess the
evolutionary relationship of the normal transcript to Hd1,
we made use of the synteny between the maize and rice
genomes. The Hd1 locus of rice (LOC_Os06g16370) is on
chromosome 6. Using the Hd1 locus as an anchor, the
Comparative Map Viewer (CMAP) function of Gramene
(http://www.gramene.org) enabled us to identify two con-
tigs of maize BAC clones, both mapping to chromosome 9,
based on presence of a conserved molecular marker,
PCO105988 (ctg362 and ctg366, AGI FPC Map October
2004; http://www.genome.arizona.edu/fpc/maize/). Using
individual BAC clones predicted to contain PCO105988 as
templates, PCR was performed with the gene-speciWc prim-
ers used on the Oat–Maize addition lines (see above). A
positive PCR product of the correct size was ampliWed from
BAC clone ZMMBBc0036D18 (data not shown). Sequenc-
ing of this BAC clone is in progress as part of the Maize
Genome Sequencing Project (accession #AC189064). The
sequence of the clone aligns perfectly with the cDNA
sequence we identiWed. The signiWcant sequence similarity
the maize gene shared with CO and Hd1 and the fact that
the maize gene lies in a syntenous location to Hd1 is consis-
tent with these genes deriving from a common ancestor
through speciation. Thus, we have designated this gene
conz1 for constans of Zea mays1.
Expression of a conz1 paralog has not been detected
In addition to the aforementioned normal, full-length tran-
script, our initial screens identiWed several additional EST

123
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a
b
c
d
e
f
Fig. 3 Several diVerentially spliced transcripts originate from the
conz1 locus. Diagram depicting the intron/exon structure of the conz1
locus, including several diVerentially spliced transcripts, relative to the
genomic sequence. a The conz1 gene is composed of two exons sepa-
rated by an intron of 298 bp. The location of primers used in assays
shown in Figs. 2, 4, 5 and 8 is indicated by arrows. Black boxes delin-
eate the ORF, the translation of which is shown in Fig. 1. The purple
and blue boxes indicate the location of the B-boxes and CCT domain,
respectively. b through e At least 33 EST sequences (Supplementary
Table S1) support the presence of alternative transcripts originating
roughly 3 kb upstream of the Wrst exon of conz1, although completely
bypassing that exon, and using one of several possible acceptor sites
for the last exon. The canonical acceptor site, used in the normal tran-
sequences in publicly curated databases. The majority of
these transcripts are currently annotated as Hd1 homologs.
To clarify the nature of these transcripts and to identify the
relationship of these transcripts, if any, with the normal
transcript, we did extensive bioinformatic work including
BLAST searches and alignments. Relative to the normal
transcript, these sequences were predicted to encode nearly
identical CCT domains but had completely diVerent 5⬘
ends. Given the ancient allotetraploid origin of maize (Gaut
and Doebley 1997), one possibility was that these
sequences may originate from a conz1 paralog. Instead, we
found that all of these sequences correspond to the same
BAC clone that harbors the conz1 locus, ZMMBB
c0036D18 (accession #AC189064). These sequences
appear to represent alternatively spliced transcripts lacking
the Wrst coding exon, which encodes the B-boxes, instead
consisting of extra sequences from farther upstream spliced
directly to the second coding exon, encoding the CCT
domain, with some variation as to the splice acceptor site
used (Fig. 3; Supplementary Table 1). The upstream
sequences contain stop codons in all three reading frames.
The longest contiguous ORF in the 5⬘ most portion of these
alternative transcripts is 156 amino acids, but lacks a start
codon (Fig. 3b). Each of these alternative transcripts has the
potential to encode the entire CCT domain within the sec-
ond coding exon, as well as some additional N-terminal
amino acids upstream of the CCT domain (Fig. 3). Given
that alternative splicing is a common mechanism of post-
transcriptional regulation (reviewed in Reddy 2007), we
evaluated the abundance of these transcripts by real-time
PCR (data not shown) and found the same pattern of
mRNA abundance when compared with the pattern of the
123
Planta (2008) 227:1377–1388
200bp
script and in those represented by d, is indicated by the vertical line.
Some ESTs are limited to one side of this splice junction, and thus are
shown only as a light gray outline, whereas the shaded yellow boxes
depict one or more ESTs that are spliced as shown (See Supplementary
Table S1 for additional details). The positions of stop codons are indi-
cated by vertical red lines within the yellow box when in-frame relative
to the CCT domain, and above or below the box when frame-shifted
§1, respectively. There are numerous additional stop codons 3⬘ of the
normal stop codon (not shown). The position of the 5⬘ most ATG in-
frame relative to the CCT domain is indicated by a green vertical line.
f Contiguous genomic sequence (AC189064) corresponds to all of the
above transcripts
normal transcript (see below). Thus, we are unable to sug-
gest particular signiWcance for the alternative splicing,
although more experiments are needed. Circumstances in
which a majority of transcripts are alternatively or incor-
rectly spliced are not novel; for instance, it has also been
observed for the CO ortholog in Pharbitis nil (Japanese
Morning Glory; Liu et al. 2001), PnCO. Because the
incomplete ESTs we identiWed map to the same genomic
locus as conz1 and because no additional full-length or par-
tial ESTs show higher predicted amino acid similarity to
CO or Hd1 than conz1, there is currently no evidence for
expression of a conz1 paralog.
conz1 expression in short days has diurnal Xuctuations
To further test the hypothesis that conz1 is homologous to
CO and Hd1, we set out to determine if conz1 expression is
similar to that of CO and Hd1. Because our RT-PCR assays
measure mRNA abundance, which could reXect diVerences
in transcription or in mRNA stability, we generally use the
term expression in this manuscript. Maize plants (inbred
B73) were germinated and entrained in short day photoperi-
ods for 3 weeks. To assay conz1 expression, leaf samples
were collected every 3 h over a 96-h period: 48 h of short
day conditions followed by 48 h of constant light. Two
independent samples, consisting of pools of tissue from
three diVerent plants each, were collected for each time
point. Expression was assayed with real-time RT-PCR
using primers demonstrated to be speciWc and reproducible
(see “Materials and methods”). Real-time PCR was used as
a sensitive assay to detect and quantify the rare conz1 tran-
script. The low level of conz1 transcript is not novel, as
Planta (2008) 227:1377–1388
conz1 Expression in Short Days
0.035
0.030 a
Expression (per ubi1)
0.025 b
0.020
0.015
0.010
0.005
0.000
Light Condition
Fig. 4 Expression patterns of conz1 in two independent replicates
(a, b) reXect a diurnal rhythm in plants grown in short days (8 h light,
16 h dark). Expression as determined by Real-Time RT-PCR was nor-
malized to ubiquitin1 in all cases. Each data point represents expres-
sion of conz1 from a pool of three diVerent individuals grown
concurrently. Light conditions are indicated by the colored bar at the
bottom of the graph, with the hatched boxes representing constant light
condition corresponding to subjective dark
both CO and Hd1 transcripts are rare and undetectable by
RNA gel blot (Putterill et al. 1995; Yano et al. 2000). Prim-
ers for conz1 expression analyses span the 298 bp intron of
the normal transcript (as shown in Fig. 3a). All reactions
were performed in triplicate, and normalized relative to
ubiquitin. Consistent with the expression pattern seen for
CO and Hd1 in short days, the expression of conz1 in short
days exhibits diurnal Xuctuations, peaking in the dark
period at about 15 h “post-dawn” (Fig. 4).
Expression pattern of conz1 is distinct in long days
Because Xowering in maize is generally thought to be
insensitive to day-length, we considered several hypothe-
ses. Relative insensitivity to daylength may have been
achieved by a loss of function: either via inability to per-
ceive daylength, or disruption of the regulatory cascade
linking daylength to Xoral induction. Alternatively, the per-
ception of daylength may be intact, but other (i.e., gain of
function) modiWcations may have enabled maize to Xower
despite varied photoperiods. To explore these possibilities,
we examined the conz1 expression pattern in varied day-
lengths. A comparable circadian experiment to that
described above was repeated under long day photoperiods.
The expression of conz1 in long days was strikingly distinct
from that seen in short days. Whereas conz1 expression in
short days peaked once per 24-h period, the expression of
conz1 in long days peaked twice. The Wrst long day peak
occurred at roughly the same time as the peak in short days:
12–15 h after dawn (Fig. 5). Expression then decreased dra-
matically at 18 h after dawn only to increase again at 21 h
after dawn. The distinct conz1 expression patterns seen in
1383
conz1 Expression in Long Days
0.06
a
0.05 b
Expression (per ubi1)
0.04
0.03
0.02
0.01
0.00
Light Condition
Fig. 5 Expression patterns of conz1 in two independent replicates
(a, b) reXect a diurnal rhythm in plants grown in long days (16 h light,
8 h dark). Expression as determined by Real-Time RT-PCR was nor-
malized to ubiquitin1 in all cases. Each data point represents expres-
sion of conz1 from a pool of three diVerent individuals grown
concurrently. Light conditions are indicated by the colored bar at the
bottom of the graph, with the hatched boxes representing constant light
condition corresponding to subjective dark
short- and long-day photoperiods, despite characterization
of maize as day-neutral, led us to explore this photoperiod
response more extensively.
An upstream component of the photoperiod pathway
is conserved in maize
We asked whether the distinct expression pattern seen in
long days could be explained by the expression of genes
upstream in the photoperiod pathway. To begin to address
this question, we returned to the databases in search of a
homolog (or homologs) of GIGANTEA [GI in Arabidopsis
(Fowler et al. 1999); OsGI in rice (Hayama et al. 2003)].
We identiWed two putative homologs (Supplementary
Figs. S1, S2), consistent with the ancient allotetraploid ori-
gin of maize (Gaut and Doebley 1997). For convenience,
these genes are referred to as gigantea of Zea mays1 A and
B (gigz1A and gigz1B), respectively. Supplementary
Fig. S1, published as supporting material online, shows the
amino acid alignment for GI, OsGI, and the two maize
genes, and Supplementary Fig. S2 shows the intron/exon
structure of the two maize loci. Using the same cDNA tem-
plates as used in the conz1 photoperiod experiments, PCR
was performed with primers designed to speciWcally
amplify gigz1A or gigz1B. Both genes exhibited diurnal
Xuctuations in expression in short days (Fig. 6) and in long
days (Fig. 7). In short days, gigz1A and gigz1B expression
began to increase during the light at 6–9 h after dawn and
remained high throughout the light phase and into the dark
(Fig. 6)—thus the initial increases in gigz1A and gigz1B
expression occurred 6 h before the peak in conz1 expres-
123
1384
Expression of GIGANTEA homologs in Short Days
0.35
gigz1A
0.3
Expression (per ubi1)
gigz1B
0.25
0.2
0.15
0.1
0.05
0 0
Light Condition
Fig. 6 Expression patterns of gigz1A and gigz1B reXect a diurnal
rhythm in plants grown in short days (8 h light, 16 h dark). Expression
as determined by Real-Time RT-PCR was normalized to ubiquitin1 in
all cases. Each time point represents expression of gigz1A and gigz1B
from a pool of three diVerent individuals grown concurrently. Light
conditions are indicated by the colored bar at the bottom of the graph,
with the hatched boxes representing constant light condition corre-
sponding to subjective dark
sion, consistent with the idea that GI is upstream of CO in
the photoperiod pathway (Mizoguchi et al. 2005). Simi-
larly, in long days, gigz1A and gigz1B showed a single peak
in expression, at 12–15 h post dawn (Fig. 7), 6–9 h before
the second peak in conz1 expression. Thus, this single peak
in gigz1A and gigz1B expression may account for the sec-
ond peak of conz1 expression, but fails to account for the
Wrst peak. It is also worth noting that the peak of gigz1A
and gigz1B expression occurred later in long days than it
did in short days. The distinct long versus short day pat-
terns of conz1 and the diVerent peak time of the gigz gene
expression support the idea that maize both perceives var-
ied day lengths and transmits that signal to genes predicted
to function within the photoperiod pathway. Given that
maize is insensitive to daylength, it remains to be seen
whether this pathway still functions in maize, or represents
a vestigial remnant of an ancestral regulatory pathway.
conz1 expression in long-day grown teosinte resembles
conz1 expression in short-day grown B73
Even though the expression of gigz1A and gigz1B did not
account for the biphasic conz1 expression pattern in long
days, we reasoned that this diVerence in expression of
conz1 could be related to the insensitivity of B73 to photo-
period with regards to Xowering. We thus asked whether
the expression of conz1 in photoperiod-sensitive teosinte
would be distinct from that observed in photoperiod insen-
sitive B73. To address this question, we obtained seed and
grew teosinte plants (alongside maize B73, for replication)
under non-inductive long-day photoperiods as done previ-
ously. RNA was collected as described for the previous
123
Planta (2008) 227:1377–1388
Expression of GIGANTEA homologs in Long Days
0.5 1.6 0.06
gigz1A
1.4
Expression (per ubi1)
0.05
0.4 gigz1B
1.2
0.04
0.3
1
0.8 0.03
0.2 0.6
0.02
0.4
0.1 0.01
0.2
0 0
Light Condition
Fig. 7 Expression patterns of gigz1A and gigz1B reXect a diurnal
rhythm in plants grown in long days (16 h light, 8 h dark). Expression
as determined by Real-Time RT-PCR was normalized to ubiquitin1 in
all cases. Light conditions are indicated by the colored bar at the bot-
tom of the graph, with the hatched boxes representing constant light
condition corresponding to subjective dark.
experiments and was reverse transcribed. The primers used
to amplify conz1 from B73 were able to amplify conz1
from teosinte (amplicon size, melt curve and ampliWcation
eYciency were all comparable to that seen for B73; data
not shown) and were, thus, used in real-time PCR reactions
as previously described. In this experiment, ampliWcation
of ubiquitin1 (ubi1; as done previously with B73) was less
than ideal (exhibiting higher variations in ampliWcation
eYciency, melt curves and Ct values among data points
than all prior experiments), such that normalization of
conz1 per ubi1 made it diYcult to conWdently discern a pat-
tern of conz1 expression. In contrast,
-tubulin (
-tub)
ampliWed well in teosinte samples (exhibiting tight melt
curves and mean ampliWcation eYciencies of 1.0 § 0.014
SE), therefore the expression of conz1 was normalized to

-tub for this experiment. As shown in Fig. 8, conz1
expression in teosinte grown in long-days has one major
peak per 24-h period. Despite poor ubi1 ampliWcation, the
general trend observed upon normalization of conz1
expression to ubi1 is still consistent with the pattern seen
using
-tub (data not shown). The overall pattern of one
peak per day is similar to conz1 expression for B73 grown
in short days, and to the expression of Hd1 in short-day rice
(Kojima et al. 2002, Hayama et al. 2003, Shin et al. 2004).
Thus, the expression of conz1 in long-day grown B73 has
been altered relative to teosinte.
Discussion
We have cloned and characterized the expression of con-
stans of zea mays1 (conz1), a maize gene with signiWcant
sequence similarity to CONSTANS (CO) from Arabidopsis
and Heading date1 (Hd1) from rice. The conz1 gene is in a
Planta (2008) 227:1377–1388
conz1 Expression in Teosintein Long Days
Avg. Expression (per b-tubulin)
1.6
1.4
1.2
1 gigz1B may act upon conz1 to induce the Wrst peak in conz1
0.8
0.6
0.4
0.2
0 et al. 2007).
0 10 20 30
Hours Post-Dawn
Fig. 8 Average expression of conz1 in teosinte from two independent
replicates reXects a diurnal rhythm in plants grown in long days (16 h
light, 8 h dark). Expression as determined by Real-Time RT-PCR was
normalized to
-tubulin in all cases. Each data point represents the
average expression (§SE) of conz1 from two replicates each consist-
ing of a pool of three diVerent individuals grown concurrently. Num-
bers at the bottom of the graph represent the number of hours since the
initial dawn at the beginning of the experiment. Light conditions are
indicated by the colored bar at the bottom of the graph
syntenous location relative to Hd1 and, similar to both Hd1
and CO, exhibited a diurnal expression pattern. Thus, all of
our data strongly supports conz1 as orthologous to Hd1 and
CO. Additionally, we characterized the expression of
gigantea of Zea mays1A and B (gigz1A and gigz1B), homo-
logs of genes upstream of CO and Hd1 in the photoperiod
pathway.
gigz1A and gigz1B have virtually identical expression
patterns and their expression is consistent with Arabidopsis
GIGANTEA (GI) and rice OsGI. Regardless of photoperiod,
gigz1A and gigz1B expression peaks during the light phase.
In short days, gigz1A and gigz1B expression began to
increase at 6–9 h after dawn and remained elevated into the
dark period (Fig. 6). The expression of conz1 in short days
followed the expression of gigz1A and gigz1B by approxi-
mately 6 h, peaking in the dark at 15 h post-dawn (Fig. 4).
This pattern is consistent with that seen in Arabidopsis and
rice, where GI and OsGI expression precede the expression
of CO and Hd1, respectively (Suarez-Lopez et al. 2001;
Hayama et al. 2002; Kojima et al. 2002; Hayama and
Coupland 2003).
Unlike the situation in short days, where the expression
of gigz1A and gigz1B correlates with the subsequent
expression of conz1, the expression of gigz1A and gigz1B
in long days does not readily predict the expression pattern
of conz1. In long days, gigz1A and gigz1B showed one peak
in expression in the light phase, at 12–15 h post dawn
(Fig. 7). conz1 expression in long days, however, had two
peaks per day (Fig. 5). The Wrst conz1 peak occurred in the
light phase at 15 h post-dawn, coincident with gigz1A and
gigz1B expression. However, the gigz1A and gigz1B peak
1385
occurred 6–9 h before the second peak in conz1 expression.
Thus, the expression of these maize GI homologs does not
account for the presence of an additional peak in conz1
expression. A regulatory factor in addition to gigz1A and
expression in long day photoperiods. One possible regula-
tory factor is a homolog of FKF1. In Arabidopsis, FKF1
has recently been shown to form a complex with GI that is
required for relieving the repression of CO by CDF1 (Sawa
40 The similar expression of gigz1A/B and of conz1 in short
days to the expression of homologous genes in photoperiod
sensitive plants was expected given that maize was domes-
ticated from teosinte, which requires short days to Xower
(Emerson 1924). The dramatic diVerence in conz1 expres-
sion in long-days (compared with the expression in short-
days), however, was striking, as modern maize is relatively
insensitive to photoperiod with regards to Xowering. Thus,
the expression data presented here suggest that modern
maize is able to discern variations in photoperiod despite
being insensitive to photoperiod in terms of Xowering. The
diurnal Xuctuations seen in our data are consistent with the
expression of the homologous photoperiod pathway genes
in daylength sensitive species, like Arabidopsis and rice.
This suggests the possibility that the photoperiod pathway
may continue to inXuence Xowering in maize. Indeed
regions of maize chromosomes syntenous with both Hd1
and OsGI in rice are associated with Xowering time QTL
(Chardon et al. 2004). However, temperate inbreds have
undergone relatively recent selective pressures aimed at
growing maize in a broader range of latitudes and are, thus,
relatively insensitive to photoperiod (Francis et al. 1969;
Russell and Stuber 1983; Mungoma and Pollak 1991; Ellis
et al. 1992; Bonhomme et al. 1994; Birch et al. 1998). With
such recent selection for relative insensitivity to daylength
in the regulation of Xowering, diurnal Xuctuations within
this pathway could represent vestigial patterns of gene
expression that no longer inXuence Xowering. In order to
determine if the long-day expression pattern is ancestral or
unique to the photoperiod insensitive inbreds, we looked at
conz1 expression in the short day progenitor of maize.
conz1 expression in teosinte grown in non-inductive long
days exhibits only a single peak per 24 h period (Fig. 8).
This suggests that changes have occurred within the photo-
period pathway during the domestication of maize from
teosinte, possibly in response to the selective pressure
towards daylength insensitivity. Precedence for this idea
comes from other grasses including Avena and O. sativa.
Selective breeding of oat (A. sativa) led to daylength insen-
sitive lines of oat in which a single gene, designated Di1,
confers the daylength insensitive trait (Burrows 1986).
Comparative mapping has recently shown that the Di1
locus is syntenous with the Hd1 locus in rice (Locatelli
123
1386
et al. 2006). Furthermore, in rice, selective pressures to
grow rice in a broader range of latitudes led to changes in
Xowering time and, speciWcally, the Hd1 and Ehd1 loci (see
below; reviewed in Izawa 2007b).
Photoperiod insensitivity in temperate inbreds, like B73
used here, could be achieved in a variety of ways, including
inactivating, overriding or modifying the photoperiod path-
way. For instance, the photoperiod pathway could still
specify that maize be sensitive to daylength, but another
pathway, such as an autonomous pathway could override
the input from the photoperiod pathway and initiate the
developmental transition regardless of photoperiod. This
model is consistent with maize inbreds, including B73,
Xowering at approximately the same time regardless of the
photoperiod in which they were grown (Birch et al. 1998).
Alternatively, other conz1-independent factors may be
aVecting Xowering. In rice, for example, Ehd1 works in
response to short days (preferentially) and long days to ini-
tiate Xowering independently of Hd1 (Doi et al. 2004).
Thus far there have been no reports of an Ehd1 homolog in
maize. Our results demonstrate that maize still perceives
diVerences in daylength (as shown by the distinct patterns
of conz1 expression and by the diVerential timing of gigz1
peaks relative to dawn in diVerent photoperiods), but we
cannot rule out the possibility of another pathway ulti-
mately overriding the input from the photoperiod pathway,
the possibility of post-transcriptional modiWcations made to
components of the pathway that aVect Xowering, nor the
possibility of mutations that disrupt the potential for activa-
tion of a Xoral integrator by the photoperiod pathway.
The expression patterns of conz1 in short- and long-day
photoperiods suggest that maize, like Arabidopsis and rice,
is able to discern variations in day-length. In other plants,
critical aspects that diVerentiate the inductive versus non-
inductive photoperiods occur at the post-translational level
(Yanovsky and Kay 2003; Hayama and Coupland 2004;
Putterill et al. 2004). In Arabidopsis, the CO protein is
ubiquitinated and rapidly degraded in the dark (Valverde
et al. 2004), so mRNA expression and translation must be
coincident with light for the protein to be stable. The CO
protein then is able to induce expression of FT protein,
which, together with FD, induces expression of Xoral meri-
stem identity genes (Abe et al. 2005). This situation only
occurs in long days, whereas in short days, CO transcrip-
tion peaks in the dark period and Xowering does not occur
(Suarez-Lopez et al. 2001). In rice, however, the model is
diVerent. Hd1 expression peaks during the dark and the pro-
tein appears to behave as a Xoral activator under inductive
short days, positively regulating expression of Heading
Date3a (Hd3a), the FT homolog (Hayama et al. 2003). Yet
lack of Hd1 function leads to early Xowering in long days,
implying that HD1 protein is stable and serves as a repres-
sor of Xowering in the non-inductive photoperiod (Hayama
123
Planta (2008) 227:1377–1388
et al. 2003). Careful analysis of a loss-of-function conz1
mutant would reveal if Xowering time in maize is altered in
either photoperiod. The phenotype of a conz1 mutant would
suggest function of CONZ1 as Xoral activator, repressor,
both, or unrelated to Xowering. Searches of available
mutant lines have, thus far, failed to identify such a mutant.
Assaying CONZ1 protein levels in varying photoperiod
conditions will give an indication as to the possible func-
tion of the CONZ1 protein in Xowering, based on the pro-
tein stability models in Arabidopsis and rice. EVorts to
generate an anti-CONZ1 antibody are in progress, such that
future analyses will reveal if CONZ1 stability is inXuenced
by light.
The data presented here suggest the possibility that
maize is not day-neutral with regards to perception of day-
length, but has modiWed the photoperiod pathway down-
stream of conz1 transcription. Alternatively, it is possible
that maize may have developed relative insensitivity to
photoperiod by activating an alternative Xowering pathway
to override input from the photoperiod pathway. Determin-
ing how the regulation of conz1 has been altered could
facilitate breeding strategies (e.g. using marker assisted
selection) such that photoperiod insensitivity of temperate
inbreds could be maintained while introducing valuable
traits from photoperiod sensitive landraces of maize, or
even suggest strategies for establishing day-neutral lines of
other important photoperiod sensitive crops.
Acknowledgments We thank Ron Phillips (University of Minne-
sota) and his research group for providing the Oat–Maize Addition
lines. Also, we thank the DuPont/Monsanto/Ceres Maize Sequence
Information Sharing Program (available at http://www.maizeseq.org/)
for sequence information used to identify conz1. Our thanks also go to
Judd F. Hultquist, K. Dale Noel, Gail L. Waring (Marquette Univer-
sity) and several anonymous reviewers for their critical reading of this
manuscript and for their helpful suggestions. This project was sup-
ported by the National Research Initiative of the USDA Cooperative
State Research, Education and Extension Service, grant number 2004-
35301-14495.
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