Download Functional Analysis of Three Lily (Lilium longiflorum) APETALA1

Plant Cell Physiol. 49(5): 704–717 (2008)
doi:10.1093/pcp/pcn046, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Functional Analysis of Three Lily (Lilium longiflorum) APETALA1-like
MADS Box Genes in Regulating Floral Transition and Formation
Ming-Kun Chen, I-Chun Lin and Chang-Hsien Yang *
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, ROC
to act as floral meristem identity genes (Angenent et al.
1992, Angenent et al. 1994, Pnueli et al. 1994, Rounsley
et al. 1995, Mandel and Yanofsky 1998, Pelaz et al. 2000).
Interestingly, A and E function genes were grouped in the
same AP1/AGL9 subfamily of MADS box genes by phylogenetic analysis (Theissen 2001, Theissen and Saedler 2001).
Mutation in A function genes such as AP1 of Arabidopsis
and SQUAMOSA (SQUA) of Antirrhinum often causes
partial flower transformation into inflorescence shoots
along with alterations in sepal and petal organ identity
(Huijser et al. 1992, Bowman et al. 1993, Schultz and
Haughn 1993, Gustafson-Brown et al. 1994). Genes such as
CAULIFLOWER (CAL) encode proteins with partially
redundant activities with AP1 in Arabidopsis (Kempin et al.
1995). In ap1/cal double mutants, the floral meristem was
completely transformed into an inflorescence meristem and
resulted in the generation of additional meristems in a
spiral phyllotaxy (Bowman et al. 1993, Kempin et al. 1995).
In addition, a FRUITFULL (FUL, previously described as
AGL8) gene of Arabidopsis has also been thought to have
partially redundant activities with AP1 in Arabidopsis,
since the ap1 cal ful triple mutant lost all floral meristem
characters and flowers were not developed (Ferrandiz
et al. 2000).
Mutation in E function genes such as SEPALLATA3
(SEP3) of Arabidopsis caused a phenotype similar to that
observed with intermediate alleles of AP1 (Pelaz et al. 2001).
Mutation in three E function genes SEP1/SEP2/SEP3
caused the conversion of entire flower organs into sepals
(Pelaz et al. 2000). The flowers were completely converted
into leaf-like structures in the sep1 sep2 sep3 sep4 quadruple
mutants (Ditta et al. 2004, Malcomber and Kellogg 2005).
The A and E functional genes have also been thought
to play a role in regulation of floral transition (Mandel et al.
1992, Bowman et al. 1993, Mandel and Yanofsky 1995b,
Purugganan et al. 1995, Mouradov et al. 1998, Liljegren
et al. 1999, Yu and Goh 2000, Jack 2001, Kater et al. 2006).
This assumption was supported by the ectopic expression of
A or E function genes in transgenic plants. Ectopic
expression of AP1 or its orthologs produced an extremely
early flowering phenotype and the conversion of inflorescence to the terminal flower structure in Arabidopsis
(Mandel and Yanofsky 1995b, Kyozuka et al. 1997).
Three cDNAs showing a high degree of homology to the
SQUA subfamily of MADS box genes were isolated and
characterized from the lily (Lilium longiflorum). Lily MADS
Box Gene 5 (LMADS5) showed high sequence identity to oil
palm (Elaeis guineensis) SQUAMOSA3 (EgSQUA3).
LMADS6 is closely related to LMADS5 whereas LMADS7
is more related to DOMADS2, an orchid (Dendrobium) gene
in the SQUA subfamily. The expression pattern for these three
genes was similar and their RNAs were detected in vegetative
stem and inflorescence meristem. LMADS5 and 6 were highly
expressed in vegetative leaves and carpel, whereas LMADS7
expression was absent. Ectopic expression of LMADS5, 6 or 7
in transgenic Arabidopsis plants showed novel phenotypes by
flowering early and producing terminal flowers. Homeotic
conversions of sepals to carpelloid structures and of petal to
stamen-like structures were also observed in 35S::LMADS5,
6 or 7 flowers. Ectopic expression of LMADS6 or LMADS7
was able to complement the ap1 flower defect in transgenic
Arabidopsis ap1 mutant plants. These results strongly
indicated that the function of these three lily genes was
involved in flower formation as well as in floral induction.
Furthermore, the ability of lily LMADS6 and 7 to complement
the Arabidopsis ap1 mutant provided further evidence to
show that the conserved motifs (paleoAP1 or euAP1) in the
C-terminus of the SQUA/AP1 subfamily of MADS box genes
is not strictly necessary for their function.
Keywords: APETALA1 — Arabidopsis thaliana — Floral
formation — Lilium longiflorum — MADS box genes —
SQUAMOSA.
Abbreviations: CaMV, cauliflower mosaic virus; RACE,
rapid amplification of cDNA ends; RT–PCR, reverse transcription–PCR.
Introduction
The ABCDE model predicted the interaction of five
classes of MADS box genes in regulating flower development in various plant species (Theissen 2001, Theissen and
Saedler 2001). In addition to the involvement of flower
formation, A and E function genes have also been thought
*Corresponding author: E-mail, [email protected]; Fax, þ886-4-2285-3126.
704
Function of lily APETALA1-like genes
Overexpression of the SQUA-like gene OsMADS14 in rice
produced an early flowering phenotype at the callus or
young plantlet stage (Jeon et al. 2000). Ectopic expression
of OsMADS18 not only resulted in an early flowering
phenotype but also precociously initiated the formation of
axillary shoot meristems (Fornara et al. 2004). A similar
early flowering phenotype was generated in transgenic
tobacco plants ectopically expressing the MdMADS2 gene
of apple or OMADS1 of orchid (Sung et al. 1999, Hsu et al.
2003).
Similar phenotypes were also observed in transgenic
plants ectopically expressing SEP3 or its orthologs (Honma
and Goto 2001, Pelaz et al. 2001, Ferrario et al. 2003, Tzeng
et al. 2003). This early flowering phenotype is significantly
enhanced in transgenic plants ectopically expressing both
SEP3 and AP1 (Pelaz et al. 2001). Further analysis
indicated that AP1/CAL or one of its orthologs, such as
OsMADS18, was able to interact with SEP3 or SEP1
proteins (Honma and Goto 2001, Pelaz et al. 2001, Fornara
et al. 2004, de Folter et al. 2005). A recent study indicated
that the K domain and C-terminal region of AP1/CAL
genes were crucial for their ability to interact with other
proteins during meristem fate and floral organ determination (Alvarez-Buylla et al. 2006). These results clearly
revealed that A and E functional genes interact each other
to regulate flower initiation in Arabidopsis (Pelaz et al.
2001, Malcomber and Kellogg 2005).
On comparison with dicots, relatively few A function
MADS box genes in the SQUA subclade were characterized
in monocots (Theissen et al. 2000, Theissen 2001, Kater
et al. 2006). Among monocots, research on genes of the
SQUA subclade was mostly limited to maize, rice and
orchid (Mena et al. 1995, Schmidt and Ambrose 1998,
Moon et al. 1999, Sung et al. 1999, Kyozuka et al. 2000,
Theissen et al. 2000, Yu and Goh 2000, Hsu et al. 2003).
SQUA-like genes tend to have gene duplication in monocots. For example, at least five different SQUA-like genes
have been isolated from maize (Fischer et al. 1995, Mena
et al. 1995, Theissen et al. 2000). In the rice genome, there
are at least four genes that encode SQUA-like proteins
(Moon et al. 1999, Jia et al. 2000, Kyozuka et al. 2000).
These genes may have redundant function since mutation in
rice OsMADS18 alone by RNA interference (RNAi)mediated silencing did not produce any mutant phenotype
(Fornara et al. 2004). However, no further functional
analyses have been carried out for these genes. Therefore,
the isolation and characterization of more SQUA-like genes
from monocots is necessary. Despite its economic importance in the cut flower market, only B, D and E function
MADS box genes have been reported for the monocot lilies
(Theissen et al. 2000, Tzeng and Yang 2001, Tzeng et al.
2002, Winter et al. 2002, Tzeng et al. 2003, Tzeng et al.
2004). In this study, we report the isolation and functional
705
analysis of three putative SQUA-like MADS box genes from
lily (Lilium longiflorum). The exploration of the possible
evolution for the AP1/AGL9 group of MADS box genes in
plant species is also discussed.
Results
Isolation of LMADS5, 6 and 7 cDNAs from lily
Three AP1-like MADS box genes were isolated from
lily by using reverse transcription–PCR (RT–PCR) and the
50 -RACE (rapid amplification of cDNA ends) strategy. The
cDNA sequence of Lily MADS Box Gene 5 (LMADS5)
encodes a 252 amino acid protein that showed a high
sequence identity (57%) and similarity (68%) to oil palm
EgSQUA3 (Adam et al. 2006) (Fig. 1). In the MADS box
domain, 88% (51/58) and 98% (57/58) of amino acids are
identical or similar between LMADS5 and EgSQUA3
(Fig. 1). LMADS5 also showed 88% (51/58) identity and
96% (56/58) similarity to AP1 in the MADS box domain
(Fig. 1). In addition to the MADS box domain, a putative
protein dimerization K box domain, which showed 62%
(64/102) and 47% (48/102) identity to EgSQUSA3 and AP1,
was found in the middle of the protein (Fig. 1). The high
sequence identity between LMADS5 and the AP1 orthologs
from various plant species suggests that LMADS5 is the
putative AP1 ortholog of lily.
The cDNA sequence of Lily MADS Box Gene 6
(LMADS6) encodes a 250 amino acid protein that showed
62 and 61% identity to LMADS5 and oil palm EgSQUA3,
respectively (Adam et al. 2006) (Fig. 1). In the MADS box
domain, 87% (51/58) and 96% (56/58) of amino acids are
identical or similar between LMADS6 and LMADS5
(Fig. 1). Further, 93% (54/58) and 98% (57/58) of amino
acids are identical or similar in the MADS box domain
between LMADS6 and EgSQUA3 (Fig. 1). In the K box
domain of LMADS6, 63% (65/102) and 66% (68/102)
identity was found with LMADS5 and EgSQUA3, respectively (Fig. 1). The high sequence similarity of LMADS6
and LMADS5 indicates that LMADS6 is also a possible
AP1 ortholog of lily.
The cDNA sequence of Lily MADS Box Gene 7
(LMADS7) encodes a 244 amino acid protein that showed a
high sequence identity (61%) and similarity (73%) to
DOMADS2, an orchid (Dendrobium) gene in the SQUA
subfamily (Yu and Goh 2000) (Fig. 1). LMADS7 also
showed 51% identity and 71% similarity to LMADS5.
In the MADS box domain, 91% (53/58) and 97% (56/58) of
amino acids were identical or similar between LMADS7
and DOMADS2 (Fig. 1). Eighty-four percent (49/58) of the
amino acids are identical in the MADS box domain
between LMADS7 and LMADS5 (Fig. 1). LMADS7
showed 56% (56/100) and 48% (48/100) identity to
DOMADS2 and LMADS5 in the K box domain of the
706
Function of lily APETALA1-like genes
10
20
30
40
50
60
70
....|....|....|....|....|....|....|....|....|....|....|....|....|....|
LMADS5
LMADS6
LMADS7
EgSQUA1
EgSQUA3
DOMADS2
OsMADS14
OsMADS18
AP1
MGRGKVQLKRIENKISRQVTFSKRRPGLLKKAHEISVLCDAEVALIVFSTRGKLYEYSADSSMERILERY
....R..........N.........S...................V...AK...F...T.A.........
....R........NVN.........S..................A....SK..I....T.....K.....
....R...R......N.........S....................I...K......AT..C........
....R........E.N.........S..................VV....K.......T.....K.....
....R..........N.........S.......................NK.......T.....K.....
...............N.........S......N.............I...K......AT..C.DK.....
....P...R......N.........N...............D........K.....F.SH....G.....
....R..........N.........A...................V...HK...F...T..C..K.....
LMADS5
LMADS6
LMADS7
EgSQUA1
EgSQUA3
DOMADS2
OsMADS14
OsMADS18
AP1
ELYSQAETVVTDTYPEAQGNWCQEYGRLKAKVETLQKSQRHLMGEQLEDLTLKQLQQLEQQLEVSFKHVR
.R.....RA.KQGDT.S..S..L..S.....IDV...R..Q......DSC...EI........TGL..I.
.D..H..GA.STNTQS-EVS.DY..SK..S.A.V...N..Q......DN.SI.E.N.....I.I.L.NT.
.R.TY..KALISSG..L.....H.F.K......A..............P.N..E.........S.L..I.
RQ..N..KALAQGD.GP..S.LH.F.E..S...A...C.........DS.A..E......R..SALR.I.
.R..Y..RALFSNEANP.AD.RL..NK...R..S.............DS.SI.E..R......S.L.FI.
.R..Y..K.LISAESDT.....H..RK.......I..C.K.....D..S.N..E.........N.L..I.
QR..FD.RA.LEPNT.D.E..GD...I..S.LDA......Q.L....DT..I.E.....H...Y.L..I.
.R..Y..RQLIAPESDVNT..SM..N.....I.L.ERN...YL..D.QAMSP.E..N.....DTAL..I.
80
90
100
110
120
130
140
....|....|....|....|....|....|....|....|....|....|....|....|....|....|
150
160
170
180
190
200
210
....|....|....|....|....|....|....|....|....|....|....|....|....|....|
LMADS5
LMADS6
LMADS7
EgSQUA1
EgSQUA3
DOMADS2
OsMADS14
OsMADS18
AP1
SRKHQLLFDSITELQRTEKLLREQNTIMEKKLMEFQK--MEALTQQDHWDIRGQ-QPMTR-PPPFLMPHL
...N......L.....K.RS.Q.E.KAL..V.Q.HKA---K....WEE-QQQ..PHTS.CL.SFL.PVEH
..RSKVML.T.S.F.SK..S.K...K-FL.EMIDK-----K.MDLSQPVENQS.QSMSSSL..LLPVSDP
T..C..M.E..S...KK..S.Q...KML..E...K..--VK..N..AP.EQQ.PP.TSSSS.TS..IGDS
...N.......A..R.K..S.Q...C.L..R.V.SSA---G.QNEHP.CERQS.PRTSSSS.L...VTDS
...T..ILH..S...KM..I.L...KTL..EIIAKE.--AK..V.HAP.EKQN.S.YSSAL..VISDSVP
...S..MLE..N....K..S.Q.E.KVLQ.E.V.K..--VQ--K..VQ..QTQP-.TSSSS-SS.M.REA
.K.N....E..S...KK..S.KN..NVLQ.-...TE.EKNN.IINTNREEQN.A-T.S.SS.T.VTA.DP
T..N..MYE..N...KK..AIQ...SMLS.QIK.RE.-ILR.QQE.WDQQNQ.HNM.PPLP.QQHQIQ.P
LMADS5
LMADS6
LMADS7
EgSQUA1
EgSQUA3
DOMADS2
OsMADS14
OsMADS18
AP1
-HPVQNAGIYPERGSSSSDADEGGAEQPLMRVGSSSLPPWMLRHVNR-L.TL.I.N.QA.DNGP--EN..AEA..MAQTD.NK......SR..G-LLEG.ISN.QGGAVE--EEAPEPQ---ERISNC.T.....I.DL.G-L.TL.I.T.QCS.NEHGEEAAQPQ---VRIGN-.L......S.L.G-F.TLHVRSNQA..--------------------T...A......SG-T.TS--RTFQA.ANE--EESPQPQ---.RVSN-TL......S.M.GQ
-L.TT.ISN..AAAGERIEDVAA.QP.HVRIG----......S.I.G-I.TT.NSQSQP...GE.E.QPSP.QA-----.N.K.......TSHTYMLSHQPSPFLNM.GLYQED.PMAMRRNDLELTLEPVYNCN.GCFAA-
220
230
240
250
....|....|....|....|....|....|....|....|....|...
Fig. 1 Alignment of the amino acid sequence of LMADS5, 6 and 7, and the related SQUA group of MADS proteins such as EgSQUA1 and
EgSQUA3 (oil palm), DOMADS2 (Dendrobium), OsMADS14 and OsMSADS18 (rice), and AP1 (Arabidopsis). The first underlined region is
the MADS box domain, whereas the second underlined region is the K box domain. The boxed region represents a highly conserved
LPPWML (paleoAP1) motif among the SQUA group of MADS proteins. Amino acid residues identical to LMADS5 in this alignment are
indicated by dots. Dashes were introduced to improve alignment. The amino acid sequences were aligned by the BIOEDIT program (http://
www.mbio.ncsu.edu/BioEdit/bioedit.html) using CLUSTALW MULTIPLE ALIGNMENT with default parameters.
protein (Fig. 1). The sequence similarity between LMADS7
and DOMADS2 indicates that LMADS7 is a lily AP1-like
gene in the SQUA subfamily. The alignments of amino acid
sequence shown in Fig. 1 and sequences of several other
MADS box genes were used to construct a phylogenetic tree
for the SQUA group of genes (Fig. 2).
Identification of the LPPWML motif for LMADS5, 6 and 7
When the sequences of the LMADS5, 6, 7 and other
SQUA-like genes were further compared, a highly conserved
motif (LPPWML) in the C-terminal region of the proteins
was identified (Fig. 3). This LPPWML motif was conserved
in most SQUA-like genes of monocots tested (Fig. 3,
monocots SQUA), whereas it showed sequence diversification by a change of 1–2 amino acids in most SQUA-like
genes of magnoliids and the AGL8/FUL subgroup of
SQUA-like genes of eudicots (Fig. 3, magnoliids SQUA
and eudicots FUL). In contrast, this motif was completely
absent in the AP1/SQUA subgroup of SQUA-like genes
of eudicots (Fig. 3, eudicots AP1). When the C-terminal
region of proteins in the AGL2 subfamily in the same
AP1/AGL9 group of MADS box genes was examined,
a region corresponding to the LPPWML motif was also
Function of lily APETALA1-like genes
707
OsMADS18 (Oryza sativa)
1000
TaAGL10 (Triticum aestivum)
113
LMADS5 (Lilium longiflorum)
169
414
LMADS6 (Lilium longiflorum)
EgSQUA3 (Elaeis guineensis)
LMADS7 (Lilium longiflorum)
666
DNMADS1 (Dendrobium nobile)
1000
DOMADS2 (Dendrobium grex)
177
EgSQUA1 (Elaeis guineensis)
683
TvFL1 (Tradescantia virginiana)
817
Monocots
SQUA
OsMADS15 (Oryza sativa)
995
TaAGL29 (Triticum aestivum)
996
OsMADS14 (Oryza sativa)
515
TaAGL25 (Triticum aestivum)
999
WAP1 (Triticum aestivum)
KcAP1c (Crocus sativus)
998
KcAP1a (Crocus sativus)
999
KcAP1b (Crocus sativus)
MADS669 (Saururus chinensis)
228
406
MgAP1 (Magnolia grandiflora)
1000
MpMADS15 (Magnolia praecocissima)
307
PEamAP1 (Persea americana)
417
Magnoliids
SQUA
HcAP1 (Houttuynia cordata)
447
MADS600 (Asarum caudigerum)
733
EUplFL1 (Euptelea pleiosperma)
FUL (Arabidopsis thaliana)
904
950
PsFUL (Pisum sativum)
MADS5 (Betula pendula)
852
690
Eudicots
FUL
MdMADS2 (Malus x domestica)
784
NsMADS1 (Nicotiana sylvestris)
517
1000
NtMADS11 (Nicotiana tabacum)
VAP1 (Vitis vinifera)
989
SQUA (Antirrhinum majus)
606
PsPIM (Pisum sativum)
636
AP1 (Arabidopsis thaliana)
738
Eudicots
AP1
CsMADS (Citrus sinensis)
1000
AP1-c (Brassica oleracea)
595
SaAP1 (Sinapis alba)
Fig. 2 Phylogenetic analysis of SQUA-like MADS box genes. Based on the amino acid sequence, LMADS5 and LMADS6 were highly
related to oil palm EgSQUA3 whereas LMADS7 was closely related to DOMADS2. LMADS5, 6 and 7 proteins are underlined. The names
of the plant species for each MADS box protein are listed behind the protein names. Amino acid sequences of SQUA-like MADS box genes
were retrieved via the NCBI server (http://www.ncbi.nlm.nih.gov/). The multiple sequence alignment was performed using the DDBJ
ClustalW 1.83 program with default parameters (Thompson et al. 1994, Miyazaki et al. 2004). The distance matrices for the aligned
sequences with all gaps ignored were calculated using the Kimura two-parameter method (Kimura 1980), and the phylogenetic tree was
generated by the neighbor-joining (NJ) method after alignment (Altschul et al. 1990). The tree figure was generated using the TREEVIEW
program, version 1.6.6. Numbers on major branches indicate bootstrap percentages for 1,000 replicate analyses.
708
Function of lily APETALA1-like genes
10
20
30
....|....|....|....|....|....|....|...
OsMADS18
TaAGL10
LMADS5
LMADS6
EgSQUA3
LMADS7
DNMADS1
DOMADS2
EgSQUA1
TvFL1
OsMADS15
TaAGL29
OsMADS14
TaAGL25
WAP1
KcAP1c
KcAP1a
KcAP1b
MADS669
MgAP1
MpMADS15
PEamAP1
HcAP1
MADS600
EUplFL1
FUL
PsFUL
MADS5
MdMADS2
NsMADS1
NtMADS11
VAP1
SQUA
PsPIM
AP1
CsMADS
AP1-c
SaAP1
OsMADS45
TaAGL16
TaAGL30
TaMADS1
OsMADS24
SEP1
SEP2
PsSEP1-2
SEP4
LMADS3
PsMTF1
OsMADS34
LMADS4
OsMADS1
SEP3
TaAGL28
OsMADS5
------RGSGESEAQPSPAQAGNSKLPPWMLRTSHT--------.G.NP.P.......N..N......S.ISSR------SDAD.GG.EQPLMRV.S.S.......HVNR-------GPEN.GAEAQPM..TDSN.......SRVNG-------VTDSFPTLHVRSN..RGTS..A....HVSG-------GAVE.EAPE.QERISNC.T.....I.DLNG-------AIAN.E.SPQAQLRVS.TL......GHMNG--------RAN.E.SPQPQLRVS.TL......SHMNGQ------GNEHGE..AQPQVRI...L......SHLNG--FQGRTNAVEAE.N.QPXMRICS.L......------------AAAAAAV.AQGQV.LRIGG......SHLNA-------EAAAAAA.PGQQ..LRIGG......SHLNA-------A.ERIEDVAAGQP.HVRIG......SHING-------T.ERAED.AVQ.QAPPRTG.....VSHING-------A.ERAGD.AVQ.QAPPRTG..L..VSHING-------PARST.DEDRPLP.VNS...L...I.SVNG-------GS.E.DYEARPLPP.NSNR.....I.SANE-------GS.E.DNEARLLPPVNRNR.....V.SANE-------GTAT.TGEKEEAHPGH.TVMLA.L..S.NN-------Q.NEVE.EGAR.PART..LM....V.HVNE-------Q.NEVE.EGAR.PART..LM......HVNE------VGVAA.E.VGAR.LART..LV......HIN--------GTEIREQEAAR.LAHS..QM.A....HASE-------.NTETEKQGDR.HSRS..GI.A...SHMND-------....SE.EVAQ.HART.TLM......HVNE---------V.GENGGA.SLTEP..L..A....PTT.NE
------CSPQDRGDNEGSQTQS.AL.......PIND-------ARGNGRVDEGT.PHRA.AL.......HLNQ-------GDNQQYGDETPTPHRP.ML..A....HLNE-------A.DNGEVEGS.RQ.QQ.TVM......HLNG-------A.DNGEVEGS.RQ.QQ.TVM......HLNN-------YQGEAHG.RRNELDLTLEPIY.SH.GCFT.-------E.E.AN.DRRNELDLTLDS.YSCH.GCFAA-----GNYREEAP.MGRNELDLTLEP.YTCH.GCF---------QEDDPMAMRRNDLELTLEPVYNCN.GCFAA-------QEDDPMAMRRNDLELTFEPVYNCN.GCFAA-------QEEDPMAMRRNDLDLSLEPVYNCN.GCFAA-------QEEDPM.MRRNDLDLSLEPVYNCN.GCFAA--EPTLQI.YPAEHHEAMNSACM.TYM...-.P-----AAGEPTLHI.YPPEPLNSSCMTT-FM...-.P-----AAGEPTLHI.YPPESM.NSCMTT-FM...-.P------TTEPTLQI.YTQE.INN.CVAA.FM.T.-.P-----AAAEPTLQI.FTPE.MNNSCVTA-FM.T.-.P------YDNPVCSEQITATTQAQ..Q..GYI.G...-------YSHPVCSEQMAVTVQGQS.Q..GYI.G...-------YRYNNVA.DQIASTSQAQ.QV.GFV.G...-------SSHYNHNPANATNSATTS.NV.GFF.G..V-------TLQIGYPPDQITIASG.GPS.S.Y..G.-.A------TLQIGYHQ.DPGSVVTAGPSM.NYMGG.-.P------HFFQAL.LHAVDVNQP..PPPGGYP.E..A------LQIGFHPVGIDQLNNGVSP.N.DDYA.A..-------HHQAYMDHLSNEA.DMVAHHPNEHIPSG.I--------------.YQGQQDGMGAGPSV.NYMLG.LPYDTNSI-AGEPTLHI.YPPESL.NSCMTT-FM.RGC.E-----------SEANQEFLHHAICDPSLHIGYQAYMDHLNQ--
observed (Fig. 3). Various numbers of amino acid changes
were observed in this LPPWML motif of most AGL2-like
genes (Fig. 3, AGL2-like).
Monocots
SQUA
Magnoliids
SQUA
Eudicots
FUL
Eudicots
AP1
AGL2like
Fig. 3 Alignment of the amino acid sequence in the C-terminus of
SQUA-like and AGL2-like MADS proteins in the AP1/AGL9 group
of MADS box genes. The boxed region represents a highly
conserved sequence (LPPWML) for SQUA-like and AGL2-like
proteins. This region experienced sequence change in SQUA-like
proteins in magnoliids and eudicots, and AGL2-like MADS proteins
in both monocots and dicots. Amino acid residues identical to
OsMADS18 in this alignment are indicated by dots. The names of
the LMADS5, 6 and 7 proteins are underlined. The 30 amino acids
of the C-terminus of all sequences were aligned by the BIOEDIT
program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) using
CLUSTALW MULTIPLE ALIGNMENT and optimized manually.
The MADS-box genes used in this study included AP1
(NM_105581), FUL (NM_125484), SEP1 (NM_121585), SEP2
(NM_111098), SEP3 (NM_102272) and SEP4 (NM_126418) from
Arabidopsis thaliana; AP1-c (AJ505846) from Brassica oleracea;
CsMADS (EF185417) from Citrus sinensis; DNMADS1 (EF535598)
Gene expression for LMADS5, 6 and 7
To explore the relationships between sequence homology and the expression pattern for three lily AP1-like genes,
RNA expression was analyzed by Northern analysis
(Fig. 4). The expression pattern and the strength of the
expression for LMADS5 were extremely similar to those
observed for LMADS6. As shown in Fig. 4, LMADS5 and
6 mRNA was detected in vegetative stem, vegetative leaf
and inflorescence meristem. When floral organs from
10 mm floral buds were examined, LMADS5 and 6
mRNAs were strongly detected in carpels and weakly
detected in the three other floral organs. In contrast to
LMADS5 and 6, LMADS7 was only expressed in vegetative
stem and inflorescence meristem but was absent in
vegetative leaf. The expression of LMADS7 in vegetative
stem and inflorescence meristem was relatively weaker than
that of LMADS5 and 6. In addition, LMADS7 expression
was not detected in any of the four organs of the flower.
Ectopic expression of LMADS5, 6 or 7 causes early flowering
and homeotic conversion of flowers in transgenic Arabidopsis
plants
To investigate the function of LMADS5, 6 and 7, the
cDNAs of these three genes driven by the cauliflower
mosaic virus (CaMV) 35S promoter were transformed
into Arabidopsis plants for functional analysis.
from Dendrobium nobile; DOMADS2 (AF198175) from
Dendrobium grex; EgSQUA1 (AF411840) and EgSQUA3
(AF411842) from Elaeis guineensis; EUplFL1 (DQ656558) from
Euptelea pleiosperma; HcAP1 (AB089153) from Houttuynia
cordata; KcAP1a (AY337928), KcAP1b (AY337929) and KcAP1c
(AY337930) from Crocus sativus; LMADS3, LMADS4, LMADS5,
LMADS6 and LMADS7 from Lilium longiflorum; MADS5 (X99655)
from Betula pendula; MADS600 (AJ419956) from Asarum caudigerum; MADS669 (AJ419690) from Saururus chinensis; MdMADS2
(DQ205652) from Malus x domestica; MgAP1 (AY821777) from
Magnolia grandiflora; MpMADS15 (AB050657) from Magnolia
praecocissima; NsMADS1 (AF068725) from Nicotiana sylvestris;
NtMADS11 (AF385746) from Nicotiana tabacum; OsMADS1
(L34271), OsMADS5 (U78890), OsMADS14 (AF058697),
OsMADS15 (AF058698), OsMADS18 (AF091458), OsMADS24
(OSU78892), OsMADS34 (AB003324) and OsMADS45
(OSU78891) from Oryza sativa; PEamAP1 (DQ398019) from
Persea americana; PsFUL (AY884287), PsMTF1 (AJ223318),
PsPIM (AJ291298) and PsSEP1-2 (AY884290) from Pisum sativum;
SaAP1 (X81480) from Sinapis alba; SQUA (X63701) from
Antirrhinum
majus;
TaAGL10
(DQ512331),
TaAGL16
(DQ512334), TaAGL25 (DQ512342), TaAGL28 (DQ512345),
TaAGL29 (DQ512346), TaAGL30 (DQ512348), TaMADS1
(AF543316) and WAP1 (AB007504) from Triticum aestivum;
TvFL1 (AY306190) from Tradescantia virginiana; and VAP1
(AY538746) from Vitis vinifera.
Function of lily APETALA1-like genes
vs
vl
im
se
pe
st
ca
LMADS5
LMADS6
LMADS7
rRNA
Fig. 4 Detection of expression of LMADS5, 6 and 7. Total RNA
was isolated from the vegetative stem before flowering (vs),
vegetative leaves (vl), inflorescence meristem (im), sepal (se),
petal (pe), stamens (st) and carpel (ca) of 10 mm long floral buds. In
these 10 mm long floral buds, the interior organs were still enclosed
by the sepals and petals. The size of the anther increased
significantly (about 5 mm long) and most of the stamen was still
made up of this developing anther. The stigma, style and ovary of
the carpels have clearly differentiated at this stage (Tzeng and Yang
2001). For Northern hybridization, LMADS5-, 6- and 7-specific
DNA probes (without a MADS box domain) were used. The result
indicated that LMADS5 and LMADS6 mRNAs were detected in the
vegetative organs, inflorescence meristem and carpels, whereas
LMADS7 mRNA was detected in the vegetative stem and
inflorescence meristem, and was absent in all flower organs.
rRNA stained in an ethidium bromide gel before blotting and
hybridization was used to show the amount of RNA used for
Northern analysis.
Eight independent 35S::LMADS5 transgenic Arabidopsis
T1 plants were obtained. One plant was phenotypically
indistinguishable from wild-type plants, whereas the other
seven plants showed identical phenotypes by flowering
earlier than wild-type plants and producing only 5–6 small
curled rosette leaves (Fig. 5A). At the same stage, wild-type
plants produced only round rosette leaves and remained in
vegetative development (Fig. 5A). In contrast to wild-type
plants, terminal flowers composed of 2–3 carpels were
produced in these 35S::LMADS5 plants at the end of either
the main inflorescence or the branches (Fig. 5B, C). When
the flower organs were examined, homeotic conversion of
sepals into carpel-like structures (Fig. 5B, D, E) and petals
into stamen-like structures (Fig. 5B, C, E) was observed.
Stigmatic papillae (Fig. 5F, G) and ovules (Fig. 5G) were
clearly observed at these carpel-like structures. The second
whorl organ petals with staminoid sectors (Fig. 5G, H) were
produced in these 35S::LMADS5 flowers.
Nine and 19 independent 35S::LMADS6 and
35S::LMADS7 transgenic Arabidopsis T1 plants were
obtained. All plants showed identical phenotypes to those
observed in 35S::LMADS5 plants by flowering earlier and
producing fewer rosette leaves than wild-type plants
(Fig. 5I, M). In addition, similar terminal flowers and
homeotic conversion of sepals into carpel-like structures
with stigmatic papillae (Fig. 5J, N, P, O) and ovules
(Fig. 5N, O), and of petals into stamen-like structures with
staminoid sectors (Fig. 5K, L, P, Q, R, S) were also
observed in these 35S::LMADS6 and 35S::LMADS7 plants.
709
To explore whether the phenotype correlated with
LMADS5, 6 and 7 expression in 35S::LMADS5, 6 and 7
transgenic plants, RT–PCR analysis was performed. As
shown in Fig. 6A, high LMADS5 expression was observed in
the transgenic plants with the severe phenotype. In contrast,
LMADS5 expression was undetectable in 35S::LMADS5
transgenic plants indistinguishable from wild-type plants.
A similar result with higher expression of LMADS6 and
7 was also observed in the severe phenotype 35S::LMADS6
(Fig. 6B) and 35S::LMADS7 (Fig. 6C) transgenic plants
than in the transgenic plants with the less severe phenotype.
This result indicated that the phenotypes generated in
35S::LMADS5, 6 and 7 transgenic Arabidopsis was due to
the ectopic expression of the lily LMADS5, 6 and 7 genes.
Ectopic expression of LMADS6 and LMADS7 rescued the
ap1 phenotype in ap1 mutants
To investigate further the function of LMADS6 and
LMADS7, the cDNAs of these two genes driven by the
CaMV 35S promoter were transformed into ap1-10 (strong
allele), ap1-11 (intermediate allele) and ap1-12 (weak allele)
plants for complementation analysis. All lines of transgenic
plants flowered earlier than ap1 mutants by producing only
5–6 small curled rosette leaves (Fig. 7A–C). The ap1 mutation often causes partial transformation of flowers into
inflorescence shoots (Bowman et al. 1993, Gustafson-Brown
et al. 1994). When flowers were analyzed, the sepals were
converted to bracts or were entirely absent in the strong
ap1-10 allele (Fig. 7D, E). The sepals were usually bract- or
petaloid-like in the intermediate ap1-11 (Fig. 7F, G) or weak
ap1-12 (Fig. 7H, I) alleles. In the strong ap1-10 allele, several
secondary flowers were occasionally formed in the axils
of the transformed sepals, as shown in Fig. 7D and E.
The tertiary flowers usually developed from secondary
flowers and resulted in these branch-like structures. In
35S::LMADS6 or 35S::LMADS7 transgenic ap1-10, -11 and
-12 plants, the defect in sepal formation was clearly restored
(Fig. 7J–O) and the secondary flowers or branch-like
structures were not observed in transgenic ap1-10 plants
(Fig. 7J, K).
The formation of second whorl organs (petals) was
also clearly altered in ap1 mutants. Approximately 74, 67
and 75%, respectively, of flowers in ap1-10 (Fig. 7D, E;
Table 1), ap1-11 (Fig. 7F, G; Table 1) and ap1-12 (Fig. 7H,
I; Table 1) plants did not produce any petals. When
35S::LMADS6 was introduced into these ap1 mutants,
an approximately 30 and 7% increase of petal production
was observed in ap1-10 and ap1-11 flowers (Table 1).
One hundred percent of the flowers produced petals in
35S::LMADS6 ap1-12 plants (Table 1). Flowers with four
petals increased from 1.3 to 82% in these 35S::LMADS6
ap1-12 plants (Table 1). A similar effect for 35S::LMADS7
was also observed in three lines of transgenic ap1 mutants.
710
Function of lily APETALA1-like genes
A
B
C
ps
ps
sc
F
E
D
G
H
ps
stg
ps
stg
sc
ps
o
sc
o
sc
I
L
K
J
fb
ps
stg
ps
sc
ps
M
P
N
fb
ps
sc
stg
o
Q
O
ps
R
ps
S
ps
stg
o
Fig. 5 Phenotypic analysis of transgenic Arabidopsis plants ectopically expressing LMADS5, 6 or 7. (A) A 25-day-old 35S::LMADS5 plant
(left) grown on soil flowered earlier than the wild-type plant (right) after producing only six curled rosette leaves. At this stage, the wild-type
plant did not flower and produced only round rosette leaves. (B) The terminal flower consisting of two flowers was produced in a
35S::LMADS5 plant. Sepal–carpel-like (sc) and petal–stamen-like (ps) structures were observed in this terminal flower. (C) A petal–stamenlike (ps) structure produced in a terminal flower of a 35S::LMADS5 plant. (D) A 35S::LMADS5 flower produced a carpel-like sepal (sc).
The second whorl petals were absent or were converted into stamen-like structures. (E) Sepal–carpel-like (sc) and petal–stamen-like (ps)
structures were observed in a 35S::LMADS5 flower. (F) Close-up of the sepal–carpel-like structure in (B). Stigmatic papillae (stg) were
observed in this sepal–carpel-like structure. (G) Close-up of the sepal–carpel-like (sc) and petal–stamen-like (ps) structures in (E). Ovules (o)
and stigmatic papillae (stg) were observed in this sepal–carpel-like structure. (H) Close-up of the petal–stamen-like (ps) structure in (C).
(I) An 18-day-old 35S::LMADS6 plant (right) grown on medium flowered earlier than the wild-type plant (left) after producing only two
rosette leaves. fb, floral buds. (J) A sepal–carpel-like structure (sc) produced in a 35S::LMADS6 flower. Stigmatic papillae (stg) were
observed in this sepal–carpel-like structure. (K) Petal–stamen-like (ps) structures produced in a 35S::LMADS6 flower. (L) Close-up of the
petal–stamen-like structure in (K). (M) An 18-day-old 35S::LMADS7 plant (right) grown on medium flowered earlier than the wild-type
plant (left) after producing only three rosette leaves. fb, floral buds. (N) The terminal flower consisting of two flowers was produced in
the 35S::LMADS7 plant. A sepal–carpel-like structure with stigmatic papillae (stg) and ovules (o) was observed in the terminal flower.
(O) Close-up of the sepal–carpel-like structure in (N). Stigmatic papillae (stg) and ovules (o) were observed in this sepal–carpel-like
structure. (P) A sepal–carpel-like structure (sc) and a petal–stamen-like (ps) structure produced in the 35S::LMADS7 flower. (Q) Close-up of
the petal–stamen-like (ps) structure in (P). (R) A petal–stamen-like (ps) structure produced in the 35S::LMADS7 flower. (S) Close-up of the
petal–stamen-like (ps) structure in (R).
Function of lily APETALA1-like genes
WT
A
L5-2
L5-7
L5-1
LMADS5
rRNA
WT
B
L6-1-1
L6-6-1
L6-1-2
L6-6-2
LMADS6
ACT
WT
C
L7-7-1 L7-13-1 L7-7-2 L7-13-2
LMADS7
ACT
Fig. 6 The detection of LMADS5, 6 and 7 expression in transgenic
Arabidopsis plants. (A) Total RNAs isolated from three 14-day-old
35S::LMADS5 transgenic Arabidopsis plants and from one untransformed wild-type plant were used as template. L5-2 and L5-7
showed early flowering whereas L5-1 was wild type like
35S::LMADS5 transgenic plants. The expression of LMADS5 was
clearly high in L5-2 and L5-7. LMADS5 expression was undetectable in L5-1 and untransformed wild-type plants. rRNA stained in
an ethidium bromide gel was used to show the amount of RNA
used for each RT–PCR. (B) Total RNAs isolated from two severe
phenotype (L6-1-1 and L6-6-1) and two less severe phenotype
(L6-1-2 and L6-6-2) 42-day-old 35S::LMADS6 transgenic
Arabidopsis plants, and from one untransformed wild-type plant
were used as template. The result indicated that LMADS6 was
clearly more highly expressed in L6-1-1 and L6-6-1 than in L6-1-2
and L6-6-2 transgenic plants. A fragment of the ACTIN (ACT ) gene
was amplified as an internal control. (C) Total RNAs isolated from
two severe phenotype (L7-7-1 and L7-13-1) and two less severe
phenotype (L7-7-2 and L7-13-2) 42-day-old 35S::LMADS7 transgenic Arabidopsis plants, and from one untransformed wild-type
plant were used as template. The result indicated that LMADS7 was
clearly more highly expressed in L7-7-1 and L7-13-1 than in L7-7-2
and L7-13-2 transgenic plants. A fragment of the ACTIN (ACT )
gene was amplified as an internal control.
An approximately 23% increase in petal formation was
observed for ap1-10 flowers. One hundred percent of the
flowers produced petals in 35S::LMADS7 ap1-11 and
35S::LMADS7 ap1-12 plants, and almost all the flowers
(94 and 97%, respectively) produced four petals, as
observed in wild-type flowers (Table 1). There were only
1.6 and 1.3% of the flowers with four petals in the ap1-11
and ap1-12 mutants, respectively (Table 1). The result
clearly indicates that LMADS6 and LMADS7 were able to
rescue the ap1 mutant phenotype in transgenic plants.
Discussion
To investigate the role of SQUA subfamily of
MADS box genes in regulating lily (L. longiflorum) floral
711
transition and flower development, three SQUA-like genes
were identified and characterized in this study. The presence
of three SQUA-like genes in lily is not surprising since large
numbers of similar SQUA-like genes were also observed in
other monocots such as maize and rice (Fischer et al. 1995,
Mena et al. 1995, Moon et al. 1999, Jia et al. 2000, Kyozuka
et al. 2000, Theissen et al. 2000).
The expression patterns for these three lily SQUA-like
genes were interesting. First, the pattern was almost
identical for LMADS5 and LMADS6 (Fig. 4). This result
strongly demonstrated that they were increased through
gene duplication events and possibly had a similar function
in lily. Only about 62% identity was observed among these
two MADS proteins (Fig. 1), thereby indicating significant
sequence diversification after duplications. Although a large
sequence diversity was observed, the transcriptional regulation of these two genes is highly conserved during evolution.
This result is quite different from that in maize or rice in
which large numbers of SQUA-like genes have functional
diversification by having a broad range of expression
patterns (Theissen et al. 2000, Kater et al. 2006).
LMADS7 showed 51 and 58% identity to LMADS5 and
LMADS6, respectively. However, LMADS7 showed a
different expression pattern from that of LMADS5 and
LMADS6; for example, its expression was absent in
vegetative leaf and carpel where LMADS5 and 6 were
highly expressed (Fig. 4). This indicated the possibility of a
small functional diversification for LMADS5, 6 and 7.
LMADS7 might be separated from LMADS5 and 6
through an early duplication event, whereas the second
duplication event further separated LMADS5 and
LMADS6. This assumption was supported by the phylogenetic analysis as shown in Fig. 2.
The second interesting characteristic of LMADS5,
6 and 7 is their spatial and temporal expression pattern.
Most genes in the SQUA subfamily were expressed in early
floral meristem and flower primordia, as seen for the typical
A function genes AP1 and SQUA (Huijser et al. 1992,
Mandel et al. 1992). However, a diversity of gene expression
in flowers was observed for genes in this subfamily. For
example, mRNAs for AP1 and SQUA were only detected in
sepal and petal of mature flowers (Huijser et al. 1992,
Mandel et al. 1992, Kempin et al. 1995). RAP1A and RAP1B
of rice were only expressed in lemma/palea (corresponding
to dicot sepals) and lodicules (corresponding to dicot petals)
of flowers (Kyozuka et al. 2000). A similar pattern was
observed for maize ZAP1 which is only expressed in the
floret (lodicules, lemma and palea) of developing ear and
tassel (Mena et al. 1995). In contrast, OsMADS14, another
rice AP1-like gene, was expressed in lemma and palea,
weakly detected in stamen and carpel, but undetectable
in lodicules (Moon et al. 1999). DOMADS2 was only
expressed in column and ovary (Yu and Goh 2000),
712
Function of lily APETALA1-like genes
C
B
A
D
E
J
K
F
sf
L
G
H
N
M
I
O
p
p
s
s
p
s
Fig. 7 Phenotypic analysis of Arabidopsis ap1 mutants ectopically expressing LMADS6 or LMADS7. (A) A 34-day-old 35S::LMADS6/
ap1-10 plant (right) grown on soil flowered significantly earlier than an ap1-10 plant (left) after producing only six curled rosette
leaves, and flowered around 20 d after germination. At this stage, the ap1-10 plant just starts to bolt after producing nine rosette leaves.
(B) A 34-day-old 35S::LMADS7/ap1-11 plant (right) grown on soil flowered earlier than an ap1-11 plant (left) after producing only six
curled rosette leaves, and flowered around 20 d after germination. At this stage, the ap1-11 plant just starts to bolt after producing 10 rosette
leaves. (C) A 34-day-old 35S::LMADS7/ap1-12 plant (right) grown on soil flowered earlier than an ap1-12 plant (left) after producing only
five curled rosette leaves, and flowered around 20 d after germination. At this stage, the ap1-12 plant did not flower and produced only
round rosette leaves. (D) The inflorescence of an ap1-10 mutant. (E) An ap1-10 mutant flower. The sepals were bract like, and several
secondary flowers (sf) were formed in the axils of the transformed sepals. The petals were absent. (F) The inflorescence of an ap1-11
mutant. (G) An ap1-11 mutant flower. The petals were absent in this flower. (H) The inflorescence of an ap1-12 mutant. (I) An ap1-12
mutant flower. The petals were absent in this flower. (J) The inflorescence of a 35S::LMADS6/ap1-10 transgenic plant. The sepals and petals
were normally formed and the secondary flowers were not produced. (K) Close-up of a 35S::LMADS6/ap1-10 flower with normally
developed sepals (s) and petals (p). (L) The inflorescence of a 35S::LMADS7/ap1-11 transgenic plant. The sepals and petals were normally
formed. (M) Close-up of a 35S::LMADS7/ap1-11 flower with normally developed sepals (s) and petals (p). (N) The inflorescence of a
35S::LMADS7/ap1-12 transgenic plant. The sepals and petals were normally formed. (O) Close-up of a 35S::LMADS7/ap1-12 flower with
normally developed sepals (s) and petals (p). The petals were slightly shorter than those in wild-type flowers.
NtMADS11 (tobacco) was expressed in sepals, carpels and
petals, whereas TOBM1 and TOBM2 of tobacco (Yan et al.
2000) and MdMADS2 of apple (Sung et al. 1999) were also
expressed in all four whorls of mature flowers. In contrast to
other A function AP1/SQUA genes described elsewhere, in
flowers, LMADS5 and 6 mRNAs were strongly expressed in
carpels and almost undetectable in the other three flower
organs, whereas LMADS7 expression was completely absent
in all four flower organs (Fig. 4).
In addition to the unique expression pattern in flowers,
LMADS5 and 6 were also specifically expressed in inflorescence meristem and vegetative tissue such as stem and
leaves (Fig. 4). Although its expression was absent in leaves,
LMADS7 still showed a similar expression pattern to
LMADS5 and 6 by specificy expression in inflorescence
meristem and vegetative stem (Fig. 4). Genes such as
MdMADS2 and DOMADS2 were also expressed in inflorescence meristem. However, they were not expressed in
stem or leaves (Sung et al. 1999, Yu and Goh 2000).
NtMADS11 has been reported to be expressed in stem and
mature leaves after flowering but not in mature leaves before
flowering (Jang et al. 2002). OsMADS18 of rice is expressed
in roots, leaves, inflorescences and developing kernels
(Masiero et al. 2002, Fornara et al. 2004). Therefore, the
expression pattern distinguished LMADS5, 6 and 7 from
most other genes in the SQUA subfamily.
Function of lily APETALA1-like genes
713
Table 1 The percentage of flowers containing different number of petals in ap1 mutants and 35S::LMADS6 and 7
transgenic ap1 plants
No. of petals
Genotype
ap1-10
4
1–3
0
35S::L6
ap1-10
a
35S::L7
ap1-10
b
ap1-11
35S::L6
ap1-11
35S::L7
ap1-11
ap1-12
35S::L6
ap1-12
35S::L7
ap1-12
2 3.2 5.7 4.8
6.1 6.2
1.6 3.5
3.4 6.2 94.4 7.9 1.3 2.6 81.5 22.3 97.2 4.4
24.1 6.5 53.9 17.6 43.3 13.2 31.6 17.1 36.2 6.8 5.64 7.9 24.2 5.9 18.5 22.3 2.8 4.4
73.9 6.9 40.5 19.1 50.6 10.2 66.8 19.7 60.4 9.3
0
74.5 7.0
0
0
a
L6 ¼ LMADS6.
L7 ¼ LMADS7.
b
The detection of LMADS5, 6 and 7 RNA at high levels
in inflorescence meristem, stem or leaves before flowering
indicated that these three genes should have a function in
flower induction and initiation. In Arabidopsis, three genes
(FUL/AGL8, AP1 and CAL) in the SQUA subfamily were
identified. FUL/AGL8 was highly expressed only in the
inflorescence apical meristem before flowering (Mandel and
Yanofsky 1995a), whereas AP1 and CAL were expressed
later in floral meristem and flower primordia (Mandel et al.
1992, Kempin et al. 1995). It seems that the timing of the
expression of LMADS5, 6 and 7 in lily was similar to that of
FUL/AGL8, AP1 and CAL in Arabidopsis. Interestingly,
mRNA of DOMADS2, the characterized SQUA-like gene of
monocot orchid, was detected much earlier than FUL/AGL8
of Arabidopsis in both inflorescence and floral meristem
(Yu and Goh 2000). Do these results reveal anything about
the functional evolution of SQUA-like genes?
Interestingly, when the sequences of the SQUA-like
genes were compared, a conserved LPPWML (paleoAP1)
motif in the C-terminal region of the proteins was identified
(Fig. 3; Vandenbussche 2003, Litt and Irish 2003). This
LPPWML (paleoAP1) motif was completely conserved in
SQUA-like genes of monocots and showed sequence
diversification in SQUA-like genes of dicots (Fig. 3). For
example, in dicots, one or two amino acid changes
were observed in this LPPWML (paleoAP1) motif in the
FUL/AGL8 subgroup of SQUA-like genes, and this motif
was completely absent and changed to a euAP1 motif in the
euAP1 subgroup of SQUA-like genes (Fig. 3). It had been
thought that the conversion of a paleoAP1 to a euAP1 motif
in the AP1 subgroup was due to a translational frameshift
mutation (Vandenbussche 2003, Litt and Irish 2003).
Based on the conservation of the LPPWML
(paleoAP1) motif, it has been postulated that the
AP1/AGL9 group of MADS box genes such as SQUAlike and AGL2-like genes might have evolved through gene
duplication from an ancestral gene that contained a
(paleoAP1) motif and regulated both the transition from
vegetative to reproductive development and floral formation (Fig. 8). In monocots such as lily and rice, the
LPPWML (paleoAP1) motif and the function of the
ancestral gene were highly conserved among those duplicated SQUA-like genes (Fig. 8). Therefore, the ability to
regulate both floral initiation and flower formation was
retained in genes such as LMADS5, 6 and 7. In contrast, the
LPPWML (paleoAP1) motif and the function of those
duplicated genes in dicots, such as Arabidopsis, experienced
more sequence changes and diversification after duplications than those in monocots. In dicots, the SQUA gene was
further duplicated into FUL/AGL8-like and euAP1-like
genes in which one or two amino acid changes in the
LPPWML (paleoAP1) motif were observed in FUL/AGL8like genes whereas the LPPWML (paleoAP1) motif was
converted into the euAP1 motif in euAP1-like genes
(Fig. 8). This diversification strongly correlated with the
separation of the ancestral gene’s functions (floral initiation
and flower formation) into two groups of genes (FUL/
AGL8 and euAP1 genes) in dicots.
Not surprisingly, functional analysis produced useful
results to interpret the possible role of LMADS5, 6 and 7 in
both flower initiation and flower formation. The early
flowering phenotype and the production of terminal flowers
in 35S::LMADS5, 6 and 7 transgenic Arabidopsis plants
were similar to those observed in transgenic plants ectopically expressing A function MADS box genes (Mandel and
Yanofsky 1995b, Kyozuka et al. 1997). Therefore, our result
suggested that part of the function of LMADS5, 6 and 7
should be an involvement in floral initiation. However,
flowers produced in 35S::LMADS5, 6 or 7 transgenic plants
have flower organ conversions such as carpelloid sepals and
staminoid petals which were not observed in 35S::AP1
transgenic plants. Interestingly, similar alterations of carpel–
sepal and stamen–petal were also observed in transgenic
Arabidopsis ectopically expressing EAP1 of Eucalyptus
(Kyozuka et al. 1997). In addition, it has been reported
that ectopically expressed OsMADS18 of rice in Arabidopsis
also resulted in an ap1 mutant phenotype (Fornara et al.
2004). One explanation for this result is the possible
generation of a dominant-negative mutation for A function
genes in Arabidopsis.
714
Function of lily APETALA1-like genes
LPPWML motif
variable
LPPWML motif lost
caused by frame shift
LPPWML motif
variable
LPPWML motif
variable
LPPWML motif
retained
SEP
Eudicots
AP1
Eudicots
FUL
Magnoliids
SQUA
Monocots
SQUA
Dicots
Ancestral E
(AGL2) gene
Monocots
Ancestral A
(SQUA) gene
LPPWML
motif
In C terminus
Ancestral AP1/AGL9 gene
Fig. 8 The possible evolution of the AP1/AGL9 group of MADS box genes. Ancestral forms of SQUA-like and AGL2-like genes were
generated by gene duplication from an ancestral AP1/AGL9 gene containing a conserved sequence with a core LPPWML (paleoAP1) motif.
Many genes were further generated through gene duplication from these two SQUA-like (A function) and AGL2-like (E function) genes.
In monocots, the sequence of this LPPWML (paleoAP1) motif was highly conserved in duplicated SQUA-like genes and experienced
a sequence change in duplicated AGL2-like genes. In dicots, the sequence of this LPPWML (paleoAP1) motif was changed in both
SQUA-like and AGL2-like genes. Based on the conservation of the LPPWML (paleoAP1) motif, the ancestral forms of SQUA-like and
AGL2-like genes in dicots were probably an FUL/AGL8-like and SEP-like gene, respectively since 3–4 amino acids were conserved in their
LPPWML (paleoAP1) motif. Further gene duplication from a FUL/AGL8-like gene produced SQUA/AP1 genes in which the LPPWML
(paleoAP1) motif was completely converted into an euAP1 motif due to the translational frameshift. The dashed line indicated the time of
the separation of monocots and dicots from angiosperms. On the basis of the conservation of the LPPWML (paleoAP1) motif, the
duplication event generating FUL/AGL8-like and euAP1-like genes was likely to have occurred in dicots after the separation of monocots
and dicots.
The function of LMADS5, 6 and 7 in flower formation
was further revealed by functional complementation analysis. When LMADS6 and 7 were ectopically expressed in
Arabidopsis ap1 mutants, the restoration of the mutant
phenotype by producing normal sepal and petal organs in
ap1 mutants was observed in transgenic plants (Fig. 7).
These results strongly support that LMADS5, 6 and 7 are
lily A function MADS box genes that are able to regulate
sepal and petal formation. This complementation experiment also raised an interesting question regarding the
function of the C-terminal region of SQUA-like proteins.
Since the euAP1 motif was completely different from the
LPPWML (paleoAP1) motif and was only present in AP1
genes in core eudicots, this indicate its functional importance for regulating floral development specifically seen in
the euAP1 clade (Vandenbussche 2003, Litt and Irish 2003).
In euAP1 sequences, an acidic transcription activation
domain and a farnesylation signal (CFAT/A) were identified in the C-terminal euAP1 motif. It is reasonable to
speculate that the euAP1 motif may correlate with the
function of fixation of the floral structure especially of the
sepal and petal for the euAP1 genes (Litt and Irish 2003).
This assumption was supported by the ability of PEAM4,
the euAP1 gene from Pisum sativum, to rescue the mutant
phenotype in the Arabidopsis strong ap1-1 mutant background (Berbel et al. 2001). However, ectopic expression of
two FUL/AGL8-like genes, NtMADS11 from Nicotiana
tabacum (Jang et al. 2002) and LtMADS2 from Lolium
temulentum (Gocal et al. 2001), lacking the euAP1 motif
entirely, was also able partially to rescue the strong ap1
mutants. This indicated that the euAP1 motif is expendable
for the euAP1 genes in the regulation of sepal and petal
formation. Since lily LMADS6 and 7 contained a more
conserved LPPWML (paleoAP1) motif than that in
FUL/AGL8-like genes, the ability of lily LMADS6 and 7
partially to complement the strong ap1-10 mutant and
almost completely complement the weak/intermediate
ap1-12 and -11 mutants provided further evidence to show
Function of lily APETALA1-like genes
that the conserved motifs in the C-terminus of the SQUA/
AP1 subfamily of MADS box genes is not strictly required
for their function.
In summary, three putative A function SQUA-like
MADS box genes LMADS5, LMADS6 and LMADS7 were
characterized from lily (L. longiflorum). The high similarity
of the expression pattern of these three genes indicated that
their function is highly conserved during evolution. The
detection of their mRNA in vegetative leaf, inflorescence
meristem and floral organs indicated their function in
both floral transition and floral formation. This assumption
was supported by the alteration of floral induction as
well as floral formation in 35S::LMADS5, 6 or 7 transgenic
Arabidopsis plants. The characteristics of these three genes
provide useful information for the understanding of the
relationships among the SQUA-like MADS box genes in
regulating flower development. Furthermore, the ability of
lily LMADS6 and 7 to rescue Arabidopsis ap1 mutants
observed in this study provides new ideas for the function of
the conserved motif identified in the C-terminal end of the
SQUA/AP1 family of MADS proteins in plants.
Materials and Methods
Plant materials and growth conditions
Plants of lily (L. longiflorum Thunb. cv. Snow Queen) used in
this study were grown in the field in Tein Wei County, Chang Haw,
Taiwan. The Arabidopsis ap1 mutant lines (ap1-10, ap1-11 and
ap1-12) were obtained from the Arabidopsis Biological Resource
Center, Ohio State University, Columbus, OH, USA. Seeds for
Arabidopsis were sterilized and placed on agar plates containing
1/2 Murashige and Skoog medium (Murashige and Skoog 1962)
at 48C for 2 d. The seedlings were then grown in growth chambers
under long-day conditions (16 h light/8 h dark) at 228C for 10 d
before being transplanted to soil. The light intensity of the growth
chambers was 150 mE m–2 s–1.
Cloning of cDNA for LMADS5, LMADS6 and LMADS7 from lily
Total RNAs isolated from a mixture of 2, 10 and 30 mm long
floral buds of lily were used for cDNA synthesis as described by
Tzeng and Yang (2001). Synthesized cDNA fragments of 1–1.5 kb,
the MADS box degenerate primer M7 (50 -GCTCTCTGTNCTIT
GYGAYGC-30 ) and the K box degenerate primer K1 [50 -GGAA
TTCTCAGC (A/G/T)AT(C/T)TTNGC(C/T)CT-30 ] were used in
PCR experiments (Tzeng and Yang 2001). PCR products containing the partial sequence for LMADS5 and LMADS7 that showed
high sequence identity to AP1-like MADS box genes were
identified.
Internal gene-specific primers were designed for LMADS5
and LMADS7 for 30 -RACE by using the SMARTTM RACE cDNA
Amplification kit (BD Biosciences Clontech, Palo Alto, CA, USA).
The gene-specific primer for 30 -RACE of LMADS5 was 50 -GACT
CCAGCATGGAAAGGATTCTCG-30 . The gene-specific primers
for 30 RACE of LMADS7 was 50 -GTCGGGCTCATTGTCTT
CTC-30 . The 30 -RACE of LMADS5 identified partial sequences for
two genes, one for LMADS5 as expected and the other containing a
sequence distinct from LMADS5 which was named LMADS6.
The gene-specific primer for 50 -RACE of LMADS5 was 50 -CTCC
715
CCCATGAGTTGCCGTTG-30 . The cDNAs for LMADS5 were
obtained by PCR amplification using the forward primer L1-3:
50 -CTCTGGATCCCCTCTCATCATCACA-30 and reverse primer
L1-2: 50 -GAAACACTGGGATCCACTGCCCATG-30 . The genespecific primer for 50 -RACE of LMADS6 was 50 -ACGGGCAAGA
GGAAGGAGGGCAAGCAGG-30 . The cDNAs for LMADS6
were obtained by PCR amplification using the forward primer
L2-3: 50 -GCGGATCCCTCATCCATCTCAATA-30 and reverse
primer L2-6: 50 -GCGGATCCAGTCTCAGAAGAAGATCCT
GA-30 . The gene-specific primer for 50 -RACE of LMADS7 was
50 -AGGCAAAAGGGGTGGCAACGAGGAGC-30 . The cDNAs
for LMADS7 were obtained by PCR amplification using the
forward primer L3-7: 50 -GGGATCCTCTAAATTAGGGTTTC
CTGATCA-30 and reverse primer L3-6: 50 -GTGGATCCGGGT
TCTGCCAACA-30 . The specific forward and reverse primers for
LMADS5, 6 and 7 contained the generated BamHI recognition site
(50 -GGATCC-30 , underlined) to facilitate the cloning of the cDNA.
Northern blot analysis
A 10 mg aliquot of total RNA isolated from various organs
or tissues of lily was electrophoresed in formaldehyde–agarose
gels and transferred to Hybond Nþ membranes (Amersham
Biosciences, Buckinghamshire, UK). The membranes were prehybridized for 30 min and hybridized with a 32P-labeled DNA
probe overnight at 658C in the same solution [0.25 M Na2HPO4,
pH 7.2, and 7% (w/v) SDS] and then washed twice each in solution
1 [20 mM Na2HPO4, pH 7.2, and 5% (w/v) SDS] and solution
2 [20 mM Na2HPO4, pH 7.2, and 1% (w/v) SDS] for 30 min per
wash. The blots were then air dried, covered with plastic wrap, and
autoradiographed. The DNA probes specific for LMADS5,
LMADS6 or LMADS7 were partial cDNA fragments (without a
MADS box) amplified from a cDNA clone through PCR.
Plant transformation and analysis of transgenic plants
A BamHI fragment containing the full-length cDNA for
LMADS5, 6 or 7 was cloned into the binary vector PBI121 (BD
Biosciences Clontech) under the control of the CaMV 35S
promoter. The orientation of the sense constructs was determined
by PCR and they were used for further plant transformation.
Arabidopsis plants were transformed using a vacuum infiltration
method as described elsewhere (Bechtold et al. 1993). Transformants which survived in the medium containing kanamycin
(50 mg ml–1) were further verified by RT–PCR analyses.
RT–PCR
Total RNA (1 mg) from wild-type and transgenic Arabidopsis
was used for cDNA synthesis by reverse transcription of a 20 ml
reaction mixture using the BcaBESTTM RNA PCR system
(TAKARA SHUZO CO. LTD., Shiga, Japan). A 5 ml aliquot of
cDNA from the reverse transcription reaction was used for PCR
(denaturation at 958C for 60 s, annealing at 558C for 90 s, and
extension at 728C for 120 s). Primers used for RT–PCR were U1-1
(50 -GGGAATTCTGCTTAGTTTCACTAT-30 ) and U1-2 (50 -TTA
CCATCACAGGCTTTTTGATCT-30 ) for LMADS5; L2-3 (50 -GC
GGATCCCTCATCCATCTCAATA-30 ) and L2-6 (50 -GCGGATC
CAGTCTCAGAAGAAGATCCTGA-30 ) for LMADS6; L3-7
and
(50 -GGGATCCTCTAAATTAGGGTTTCCTGATCA-30 )
L3-6 (50 -GTGGATCCGGGTTCTGCCAACA-30 ) for LMADS7;
and ACT-1 (50 -ATGAAGATTAAGGTCGTGGCA-30 ) and
ACT-2 (50 -TCCGAGTTTGAAGAGGCTAC-30 ) for the internal
control ACTIN. PCR products were analyzed by electrophoresis
in 1% agarose gels.
716
Function of lily APETALA1-like genes
Funding
The Council of Agriculture and National Science
Council, Taiwan, ROC (grant Nos. 94AS-5.1.4-FD-Z1
and NSC94-2311-B-005-001 to C.-H.Y); the Ministry of
Education (grant 5Y/50B).
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(Received December 17, 2007; Accepted March 17, 2008)