Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 492-499
doi:10.1111/j.1745-7270.2006.00188.x
OsFY, a Homolog of AtFY, Encodes
a Protein that Can Interact with OsFCA-g
in Rice (Oryza sativa L.)
Qi LU, Zheng-Kai XU, and
Ren-Tao SONG*
Shanghai Key Laboratory of
Bio-energy Crop, School of Life Sciences, Shanghai University, Shanghai 200444,
China
Received: March 13,
2006
Accepted: April 11,
2006
*
Corresponding author: Tel, 86-21-66135167; Fax, 86-21-66135163; Email,
Abstract FCA and FY are flowering time related genes
involved in the autonomous flowering pathway in Arabidopsis. FCA
interacts with FY to regulate the alternative processing of FCA pre-mRNA.
The FCA/FY interaction is also required for the regulation of FLC
expression, a major floral repressor in Arabidopsis. However, it is not
clear if the regulation of this autonomous flowering pathway is also present in
monocot plants, such as rice. Recently, alternative RNA processing of OsFCA was
observed in rice, which strongly suggested the existence of an autonomous
flowering pathway in rice. In this work, we cloned the cDNA of the autonomous
flowering pathway gene OsFY from rice. The predicted OsFY protein
contained a conserved 7 WD-repeat region and at least two Pro-Pro-Leu-Pro
motifs compared to Arabidopsis FY. The protein-protein interaction
between OsFY and OsFCA-g, the key feature of their
gene function, was also demonstrated using the yeast two-hybrid system. The
GenBank database search provided evidence of expression for other autonomous
pathway gene homologs in rice. These results indicate that the autonomous
flowering pathway is present in monocots, and the regulation through FY and
FCA interaction is conserved between monocots and dicots.
Key words OsFY; autonomous flowering pathway; OsFCA; protein
interaction; yeast two-hybrid
The mechanism of flowering has
been mainly studied in Arabidopsis. There were four genetically
separated flowering promotion pathways demonstrated: the photoperiod,
gibberellin, vernalisation and autonomous pathways [1–4].
The autonomous pathway is comprised of at least seven genes: FCA, FPA,
FY, FLD, LD, FVE and FLK. Mutations in any
of these genes could increase the expression of FLC and cause the delay
of flowering [2,4].
In rice, the floral transition is induced by the photoperiod pathway.
Many homologous flowering time genes involved in the Arabidopsis
photoperiod pathway are also found in rice. For example, OsGI, Hd1 (Se1)
and Hd3a from rice are homologous to GI, CO, and FT
in Arabidopsis [5]. It is likely that rice lacks the vernalisation
pathway because it was evolved from subtropical primitive grasses with no
vernalisation requirement. Consistent with this, ortholog genes of the Arabidopsis
vernalisation pathway have not been identified in rice [6].In Arabidopsis, FY is a flowering time gene in the
autonomous pathway. FY belongs to a highly conserved eukaryotic protein group,
represented by Saccharomyces cerevisiae RNA 3‘ end-processing
factor, Pfs2p [7]. FY interacts with FCA to control the Arabidopsis
floral transition [8,9]. FY is a protein with highly conserved 7 WD-repeat
region and several Pro-Pro-Leu-Pro (PPLP) motifs. The first PPLP motif is
invariant among the FYs from different plant species [7]. The PPLP motif was
predicted to interact with the WW domain of FCA. The FY-FCA complex bound FCA
pre-mRNA when the FCA-g was excessive in Arabidopsis, promoted premature cleavage
and polyadenylation at a promoter-proximal site in intron 3 of its own
pre-mRNA, and resulted in the production of FCA-b, which acts as
a nonfunctional truncated transcript [7,10]. In rice, OsFCA-b, the product
of alternative splicing and polyadenylation from OsFCA, was
also observed [11], suggesting that the OsFY gene, as well as the
interaction between OsFY and OsFCA, might also be present in rice. In this study, we report the isolation of OsFY cDNA, which
contains the full-length encoding region of OsFY, and demonstrate that
OsFY can interact with the large fragment of OsFCA-g.
Materials and Methods
Plant materials, plasmids and
strains
Rice seedlings of Oryza sativa L. cv. Nipponbare were
hydroponically grown in a growth cabinet for 3 weeks at 30 ?C. The yeast strain
EGY48 and plasmids pEG202 (bait plasmid), pJG4-5 (target plasmid) and pSH18-34
(reporter plasmid) were kindly provided by Dr. Jing-Liu ZHANG (National
Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai, China). The plasmid pGEMT-rFCA-1 containing the
full length of OsFCA-g cDNA was kindly provided by Dr. Jin-Shui YANG (Institute of
Genetics, Fudan University, Shanghai, China).
Prediction of full-length
encoding region of OsFY
Through the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/)
using an Arabidopsis FY sequence, two predicted OsFY mRNAs known
as AK111493 and Xm_463744 were found and retrieved from GenBank. As the two OsFY
entries were inconsistent at the sequence level, we carried out the OsFY
mRNA prediction again. Through a BLAST search using AK111493, the OsFY genomic
DNA sequence was obtained from BAC clone B1147A04 (chromosome I, Genbank
accession No. AP003735). We predicted OsFY mRNA using HMM-based gene
structure prediction with the monocot setting at http://www.softberry.com/berry.phtml.
The predicted mRNA (totalling 2558 bp in length) was different to AK111493 and
Xm_463744.
Cloning of OsFY cDNA
Total RNA (5 mg) was isolated from leaves and stems of rice seedlings (four-leaf
stage). In accordance with the predicted OsFY mRNA sequence, a gene
special primer OsFY/P2 (5‘-GTGCTGCAGTTACCGCATGGAAAATAGG-3‘) (Fig.
1), located downstream of the predicted OsFY open reading frame
(ORF), was designed to synthesize the first-strand cDNA using SuperScript III
Reverse Transcriptase Kit (Invitrogen, Carlsbad, USA). The 3‘ portion
of OsFY cDNA was amplified by polymerase chain reaction (PCR) with the
primers OsFY/P2 and OsFY/P3 (5‘-CAGGGCGTCGGCTTATTACGGGAT-3‘) (Fig.
1), then cloned into the pSK-T vector to generate pSK-T-3‘–OsFY.
The 5‘ portion of OsFY cDNA was amplified by PCR with the primers
OsFY/P1 (5‘-CGCACGGATCCAAAACCCTAGCTC-3‘) and OsFY/P4 (5‘-CCAGTGACCATCCAGTTCTCATTGT-3‘)
(Fig. 1). The 5‘ end of OsFY cDNA was rich in G and C. It
was only amplified successfully using Pyrobest DNA Polymerase (TaKaRa, Dalian,
China) under the following conditions: 6% dimethylsulfoxide in a 20 ml PCR reaction
system; 3 min at 94 ?C; 20 cycles of 30 s at 94 ?C, 30 s at 64 ?C, 1 min at 72
?C. The product was 1:50 diluted and used as the template for the second round
of PCR under the same conditions. The 5‘ end of OsFY cDNA was
cloned into pSK-T vector to generate pSK-T-5‘–OsFY. The pSK-T-3‘–OsFY
and pSK-T-5‘–OsFY were sequenced. The 3‘ portion of
the OsFY fragment was released by cutting pSK-T-3‘–OsFY
with EcoRI and KpnI, and cloned into the same sites of pSK-T-5‘–OsFY.
The resulting plasmid, containing the full length of the OsFY encoding
region, was named pSK-T-OsFY.
OsFY and OsFCA interaction
analysis
The pSK-T-OsFY was cut with EcoRI and NotI to
obtain the 3‘ fragment of OsFY, which would be cloned into
pEG202 (bait plasmid) to make pEG202-3‘–OsFY. The 5‘
fragment of OsFY was prepared by PCR with primers of OsFY/YP3 (5‘-GAGAATTCGCGGAGATGATGCAGCAGC-3‘)
and OsFY/P5 (5‘-AAGATTGGCCATTCCATAGGGTGA-3‘) (Fig. 1).
The 5‘ fragment was then cut with EcoRI, and cloned into pEG202-3‘–OsFY
to form pEG202-OsFY.The large fragment (305–2258 bp) of OsFCA-g cDNA (AY274928)
was prepared by amplifying pGEMT-rFCA-1 [12] by the primers of OsFCA-g5/P4 (5‘-TATAGAATTCGGCGGCGGCGAGTACG-3‘)
and OsFCA3/P5 (5‘-AGGAGAATTCAACTTTTCCAAGCACGT-3‘). We were not
able to get the very 5‘ end of the OsFCA exactly due to its high
GC content. The large fragment of OsFCA-g cDNA, containing all the predicted essential protein sequence for
FY-FCA interaction, was fused to pJG4-5 (target plasmid) to make pJG4-5‘–OsFCA-g.Following the instructions of the yeast two-hybrid system DupLEX-A
(Origene, Rockville, USA), pEG202-OsFY and pSH18-34 were transformed
into yeast strain EGY48 (MATa trp1 his3 ura3 leu2::6 LexAop-LEU2), and the autoactivation potential of the bait was tested to be
negative. Then pJG4-5-OsFCA-g was transformed into EGY48 containing the
plasmids pEG202-OsFY and pSH18-34 to analyze the interaction between
OsFY and OsFCA. Positive transformants were screened from the YNB(glu)–ura–his–trp plates. Each
positive colony was diluted in sterile distilled water and then plated onto
the YNB(gal)–ura–his–trp–leu plate to test the expression of reporter gene LEU2. The
expression of reporter gene LacZ was tested by re-streaking
positive colonies to the YNB(gal)–ura–his–trp+X-gal plate.
Results
Isolation of FY
ortholog from rice
Oryza sativa L. cv. Nipponbare was used in
this study because its genome was sequenced and available in GenBank [13]. The OsFY
mRNA had not been identified experimentally before. Through BLAST, two
predicted OsFY mRNAs known as AK111493 and Xm_463744 were found.
However, the two predicted OsFY mRNA sequences were not consistent with
each other. We obtained the genomic sequence of OsFY by BLAST using the
rice genome sequences with AK111493. The mRNA of OsFY was predicted by
FGENESH (http://www.softberry.com/berry.phtml).
The predicted mRNA was 2558 bp in length, 776 bp longer than Xm_463744, and had
a different ORF to AK111493.Based on the predicted OsFY mRNA sequence, a gene-specific
primer OsFY/P2, which was downstream of the coding region, was designed to
synthesize the first-strand cDNA. Due to the high GC content (approximately
80%) in the 5‘ end sequence, the OsFY cDNA was first cloned as
two separate 5‘ and 3‘ overlapping fragments. The overlapping
fragments were then fused together to form a single piece containing the
full-length coding region of OsFY.The OsFY cDNA we cloned was 2308 bp in length. The sequence
was identical to our predicted OsFY cDNA, and different from the two
entries in GenBank, AK111493 and Xm_463744. The OsFY cDNA derived from
this study was submitted to GenBank under accession No. DQ132809.
Analysis of OsFY cDNA
The OsFY cDNA shared 57% nucleotide identity with the Arabidopsis
FY gene. We confirmed that our OsFY cDNA contained the full-length
coding region because there was an in-frame stop codon (TAG, nt 1921) right
before the predicted start codon (nt 52–54). The predicted ORF (nt
52–2205)
(Fig. 2) consisted of 18 exons [Fig. 3(A)], which was
consistent with the Arabidopsis FY gene, and did not show any
changes in the number or size of the exons. Hence, the FY gene structure
was evolutionally conserved in dicots and monocots.Protein motifs of OsFY were predicted by PROSITE at http://www.expasy.org. Similar to Arabidopsis
FY, the OsFY also contained one 7 WD-repeat region and at least two PPLP motifs
[Fig. 3(B)]. The 7 WD-repeat and the first PPLP motif from OsFY were
highly conserved in plants, with the WD-repeat region having 80% nucleotide identity
to Arabidopsis and 96% nucleotide identity to another monocot species,
ryegrass. In the monocot plants rice and ryegrass, the third WD domain (for
example, a.a. 235–276 of OsFY) was immediately linked to the fourth WD domain (for
example, a.a. 277–318 of OsFY), but in Arabidopsis, these two WD domains were
separated by 19 a.a. linker. A similar case was found at the region between
the 7 WD-repeat and the first PPLP motif, but in Arabidopsis there was
an extra sequence of approximately 40 a.a. compared with that of rice and
ryegrass. The C-terminal region of OsFY was less well conserved, sharing only
37% identity with Arabidopsis and 71% identity with ryegrass. However,
the first of the PPLP motifs in the C-terminal region was notably highly conserved
in plants, as shown in Fig. 4, which was consistent with previous
research [7].
OsFY can interact with the
large fragment of OsFCA-g
In rice, there were different forms of OsFCA, but only OsFCA-g contained complete
conserved domains (two RNA recognition motifs and one WW domain). The WW domain
of OsFCA-g shares approximately 93% a.a. identity with Arabidopsis
FCA-g protein [11]. The PPLP motif in FY was predicted to interact with
the WW domain of FCA [7]. The conservation of PPLP motifs of FY in plants
suggested the conservation of interaction between FY and FCA in rice. The
yeast two-hybrid system was used to identify the interaction of OsFY and
OsFCA-g in rice. Two-hybrid plasmids, pEG202-OsFY and pJG4-5-OsFCA-g, were
constructed and transformed into yeast strain EGY48 with reporter plasmid
pSH18-34 which contained reporter gene LacZ. If interaction occurred
between OsFY and OsFCA-g, the reporter genes LacZ and LEU2 would be induced
and the positive transformants could either turn blue on the YNB(gal) –ura–his–trp+X-gal plates
or grow on the YNB(gal)–ura –his–trp–leu plates. The result indicated that OsFY interacted with the large
fragment of OsFCA-g (Fig. 5).
Rice has other autonomous pathway
components homologs
In an attempt to search for other autonomous pathway components in
rice, we used Arabidopsis autonomous pathway gene products, such as FPA,
FVE, FLD, LD and FLK, to BLAST the rice genome as well as the rice expressed sequence
tags (ESTs). Although only FCA and FY have been isolated from rice so far, all
other components have their corresponding ESTs and gene homologs in rice (Table
1). They had different sequence homologs to their Arabidopsis
counterparts. For example, FCA, FY and FLK were approximately 40%. This number
was good enough to maintain the conserved function, as demonstrated by OsFY and
OsFCA-g in this study. FPA and LD had a slightly lower homolog,
approximately 30%, whereas FVE and FLD had a higher homolog of approximately
70%. The data suggested that different genes in the autonomous flowering
pathway had evolved at a different rate.
Discussion
In Arabidopsis, a series of mutants such as fca, fpa,
fy, fld, ld, fve and flk, could delay flowering
regardless of photoperiods. This late-flowering phenotype could be overcome by
vernalisation, making them different from the other three flowering pathways.
All of these genes were classified as the autonomous promotion pathway genes [2,3].
Among them, FCA, FPA and FLK were RNA-binding proteins. FY was a 3‘-end
RNA processing factor, and LD was a homeodomain protein that might interact
with RNA or DNA [14–17]. All genes
in this pathway regulated FLC expression through
different mechanisms. Their gene functions suggested that post-transcriptional
regulation was a very important mechanism to promote floral transition in this
pathway. The protein-protein interaction between FCA and FY was a key feature
of the function of this flowering pathway, therefore it was the emphasis of
this study.Because of the inconsistencies of two previously predicted OsFY
mRNA sequences in the GenBank database, we carried out the OsFY gene
prediction again, and isolated the cDNA of OsFY from rice RNAs.
Winichayakul et al. also isolated FY cDNA (AY654583) from
ryegrass (Lolium perenne L.), another monocot plant [18], but the
sequence did not contain the full-length encoding region, and the predicted LpFY
(AAT72461) lacks the 5‘ end. However, the OsFY cDNA isolated in
this study represented a cDNA containing the FY full-length encoding
region of monocots. This enabled us to discover some sequence features between
dicot and monocot FYs. In general, dicot and monocot FYs show very high
sequence homology at the WD-repeat region and the first PPLP motif, but
relatively low homology at the C-terminal region. In particular, the second
PPLP motif of monocot FY was at a very different position compared to dicot FY
(Fig. 4). Dicot FY had two extra sequence linkers, one between two WD domains,
and the other between the WD domain and PPLP motif, compared with monocot FY.In Arabidopsis, FY-FCA interaction is required for downregulating FLC
expression and autoregulating FCA active mRNA. It is not yet known
whether the regulation of FLC pre-mRNA is direct or if an intermediate
RNA is recognized by FY-FCA [7,19]. In this study, we demonstrated that OsFCA-g can interact
with OsFY using the yeast two-hybrid system. Winichayakul et al. also
demonstrated that the PPLP motif of ryegrass FY protein could interact with
the WW domain of AtFCA by pull-down assay, despite the ryegrass FY from
their study missing the N terminal portion [18]. These data indicate that
FY-FCA interaction was conserved between monocots and dicots. We also
constructed pJG4-5-OsFCA-g-ww, which only
contained the OsFCA-g WW domain and some flanking sequences
(totalling approximately 600 bp), and tested its interaction with OsFY in the
yeast two-hybrid system. Only weak interaction was detected, as indicated by
very light blue color staining on YNB(gal)–ura–his–trp+X-gal plates (data not
shown). Therefore, although the WW domain was essential for the FY-FCA
interaction, other sequences in FCA could influence the interaction as well.When we searched the rice sequences in the GenBank database for
other autonomous pathway genes, such as FPA, FVE, FLD, LD,
and FLK, we found all of them not only in the genomic sequences, but
also in EST sequences. These data indicated that all autonomous pathway genes
are expressed in rice. Together with the fact that OsFY and OsFCA could perform
protein-protein interactions, which is required for autonomous flowering
pathway function, the autonomous flowering pathway was suggested to be present
in rice.The Arabidopsis autonomous pathway repressed the expression
of floral repressor FLC, then upregulated SOC1 and FT [20]
and promoted flowering. Downregulation of SOC1 by FLC might constitute
an important downstream activity of FLC [2,21]. Although the FLC
ortholog has not been found in rice, overexpression of the Arabidopsis FLC gene
in rice did cause late flowering and delayed the upregulation of rice OsSOC1
[22]. Lee et al. determined that the ectopic expression of OsFCA,
as driven by the 35S promoter, caused Arabidopsis fca-1 mutants
to show early flowering behavior [11]. The constitutive expression of OsFCA
altered endogenous SOC1 expression patterns, but with no concomitant reduction
in the levels of FLC mRNA [11], so SOC1 levels might be downregulated to bypass
FLC [11,21,23]. These results suggest that, despite rice lacking the FLC
homolog, the autonomous flowering pathway could be functioned by upregulating OsSOC1
expression to promote flowering.
Acknowledgements
We would like to thank Dr. Xi-Ling DU, Dr. Jun LIU and Dr. Ping LI
for their help with the experiment. We would also like to express our
appreciation to Dr. Jing-Liu ZHANG and Dr. Jin-Shui YANG for providing
experimental materials.
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