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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 492-499 |
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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,
[email protected]
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 pre�mature 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'-GTGCTGCAGTTACCGCATGGAAAA�TAGG-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'-CGCACGGATCCA�AAACCCTAGCTC-3') and OsFY/P4 (5'-CCAGTG�ACCATCCAGTTCTCATTGT-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'-GAGAATTCGCGGAGATGATGC�AGCAG�C-3') and OsFY/P5 (5'-AAGATTGGC�CAT�TCC�ATAGGG�TG�A-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'-TAT�AGAATTCGGCGGCGGCGAGTACG-3') and OsFCA3/P5 (5'-AGGAGAATTCAACTTTTCCAAGC�ACG�T-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 inter�action� 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 inter�action 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 demon�strated 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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