RESEARCH PAPER |
Short, direct repeats (SDRs)-mediated post-transcriptional processing of a transcription factor gene OsVP1 in rice (Oryza sativa)
1Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, Sichuan University, Chengdu 610064, China
2Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai 200433, China
3Institute of Crop Research, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
4State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China
To whom correspondence should be addressed. E-mail: liuyongsheng1122{at}yahoo.com.cn
Received 2 June 2007; Revised 2 September 2007 Accepted 3 September 2007
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Abstract |
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The various degrees of preharvest sprouting occurring in hybrid rice is a limiting factor in the propagation and production of hybrid rice seeds. The phenotype of sprouted rice is very similar to that of the maize (Zea mays) seed-specific mutation viviparous 1. VP1 has been shown to be a transcription factor essential for seed maturation and dormancy induction. In this study, numerous truncated transcripts of OsVP1 resulting from an unusual post-transcriptional processing, were detected in four rice (Oryza sativa) cultivars. The observed events took place at a region spanning exons 1 to 5, and led to a variety of deletions that resulted in the loss of functional domain and frame-shifts with premature termination by introducing a stop codon. Various proportions of the transcripts expressed in both immature and mature embryos were observed to be incorrectly processed and associated with the genetic variation of preharvest sprouting rates among various rice varieties. In sprouting-susceptible rice cultivars, G46B and HeiB, many more incorrectly processed OsVP1 transcripts were expressed in immature than in mature embryos, indicating that the unusual post-transcriptional processing of the OsVP1 gene was developmentally regulated. In addition, comprehensive sequence analyses demonstrated the presence of paired short direct repeats (SDRs) at the junctions of the unusual excision sites in exons of OsVP1 gene. Site selection for the deletion of exon materials was altered along with the genotypes and developmental stages.
Key words: Alternative splicing, Oryza sativa, preharvest sprouting, short direct repeats, vivipary 1
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The plant hormone abscisic acid (ABA) plays a central role in seed maturation and germination, as well as in adaptation to abiotic environmental stresses (Leung and Giraudat, 1998). Identification of abscisic acid response mutants has provided insight into the molecular components of ABA signalling in the regulation of seed development. There are two classes of ABA response mutants in seeds, insensitive and hypersensitive, and a variety of the corresponding genes have been isolated and shown to encode a number of proteins (Finkelstein et al., 2002). Among these mutants, the maize viviparous 1 (vp1) mutant has the most profound effect on late embryogenesis. Inactivation of this locus leads to disruption of embryo maturation, resulting in the germination of embryos while still attached to the cob (vivipary). Analysis of vp1 mutants has shown that this locus carries out two important functions, both in the promotion of embryo maturation and in the repression of germination (McCarty et al., 1991; Hoecker et al., 1995; Holdsworth et al., 1999).
Since no abnormality in vp1 mutants during the early stage of embryogenesis was observed, VP1 is known to be exclusively involved in regulating ABA-responsive gene expression during the mid- to late phase of embryogenesis (McCarty, 1995). However, maize VP1 mRNA is present at a significant level at 10 days after pollination (DAP) (McCarty et al., 1991), while rice VP1 mRNA is expressed in the embryo as early as 2–3 DAP, corresponding to the mid- to late globular stage without organ differentiation (Hattori et al., 1994; Miyoshi et al., 2002). The function of expressed VP1 mRNA at the early stage of embryogenesis remains unknown.
Embryo maturation and dormancy profoundly affects the seed quality of hybrid rice and is influenced by interactions between the genotype and the environment of the developing grain (Hu et al., 2002). Desiccation (water loss) is an important signal involved in dormancy induction in rice seeds, and ABA responsiveness is considered important for the maintenance of embryo dormancy (Tang et al., 2003; Yong et al., 2003; Zhang et al. 2005). Preharvest sprouting (PHS) occurs with high incidence in the hybrid rice seeds derived from some particular cytoplasmic male sterile lines (i. g., G46A) when grains mature under successive rainy/moist conditions (Yong et al., 2003). This directly deteriorates F1 seed quality in subsequent storage and germination. This physiological disorder manifests a considerable genotypic variation among various rice varieties and the genetic variance was explained by significant additive and dominant effects in an incomplete diallele cross design (Hu et al., 2002). A delicate study demonstrated that missplicing of wheat VP1 genes contributes to susceptibility to PHS in modern hexaploid wheat varieties (McKibbin et al., 2002). However, little is known about the molecular processes accounting for the large genetic variation of preharvest sprouting in rice. The expression and processing of the rice (O. sativa) VP1 (OsVP1) gene is reported here. Four varieties showing different preharvest sprouting rates were used in this experiment. Embryos at different developmental stages were included to analyse the structure and composition of the transcripts. It appears that a substantial proportion of OsVP1 transcripts from the sprouting-susceptible cvs G46B and HeiB were incorrectly processed in exonic regions, leading to the loss of coding sequences.
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Materials and methods |
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Plant materials
Four rice lines (including PHS-resistant cvs Fuhui838 and Lemont, and PHS-susceptible cvs G46B and HeiB, respectively) were used for the examination of expression of OsVP1. For RNA extraction and analysis, embryos from immature (15 d post-anthesis) and mature imbibed dormant seeds (6 h imbibed in water) were collected.
Grain sprouting experiment
To evaluate the grain sprouting rate in different rice cultivars, 50 fully mature, dried grains, as well as three ripening panicles from each line harvested at 4 and 5 weeks post-anthesis, respectively, were incubated in Petri dishes containing two pieces of filter paper moistened with distilled water at 25 °C for 7 d, and the numbers of non-sprouted and sprouted grains visible with the naked eye were scored every 24 h.
RNA isolation and reverse transcription (RT)-PCR
Total RNAs were extracted by using Trizol reagent following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA). Reverse transcription was primed by oligo(dT) by using the First Strand cDNA Synthesis kit (Toyobo, Osaka, Japan). 3' region sequences derived from OsVP1 cDNA (GenBank accession no. AK105441
[GenBank]
) were amplified by PCR with primers designed for the OsVP1 (VP1US, nt 963–982, 5'-CAGCAAGAGCAGCGTGGTCG-3'; VP1DS, nt 1946–1968, 5'-GCCATGCTTATGCTTACCTACCG-3'; VP2US, nt 1088–1110, 5'-TCTCCTCCACGAGCTCCTACACC-3' and VP2DS, nt 1918–1937, 5'-GAGTTGCCTTGCTCCTGCGC-3'). PCR was performed by using Taq DNA Polymerase (Takara, Dalian, China) in MJ MiniTM PCR (Bio-Rad, Hercules, California, USA), following the instruction given by the manufacturer.
Sequencing and data analysis
RT-PCR products were cloned into the pMD18-T Vector (Takara, Dalian, China). Cycle sequencing was performed with the ABI Prism BigDye Terminators v2.0 cycle sequencing reaction kit (Applied Biosystems, Foster City, California, USA). Sequences were determined with an ABI Prism 377 genetic analyser (Applied Biosystems), and edited with the computer program BioEdit v4.7.8 (Hall, 1999; Tom Hall, North Carolina State University). The sequence analysis was performed using the software DNAMEN v5.0 (Lynnon Biosoft Inc., Vandreuil, Quebec, Canada).
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Results |
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Evaluation of grain sprouting rate
To analyse the potential of PHS rates in different varieties, the germination velocity was investigated in a moist condition. As a result, freshly harvested seeds of G46B germinated slightly faster than did HeiB, while the seeds of Lemont and Fuhui838 showed a strong dormancy and did not germinate at all after 7 d incubation in the moist condition (Fig. 1A, B). Similarly, fully mature seeds of G46B and HeiB also germinated much faster than did Lemont and Fuhui838 (Fig. 1C).
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Incorrectly processed transcripts of the OsVP1 gene
To examine the expression pattern of OsVP1, total RNAs were isolated from immature embryos and the mRNAs with 3' poly(A) tail were selected over the oligo(dT)-primed reverse transcription for further analysis by RT-PCR. Primers derived from the coding region of OsVP1 corresponding to a region showing abnormal splicing in wheat VP1 (McKibbin et al., 2002) were used. The results showed that a transcription product with an expected size for correct splicing was detected in all the tested varieties, and surprisingly, distinct transcripts with a smaller size were observed exclusively in the PHS-susceptible cvs G46B and HeiB (Fig. 2). To determine whether the expressed products were derived from the OsVP1 gene, the RT-PCR amplified fragments were cloned and sequenced. The resultant sequencing analysis revealed that all the expressed products were derived from the OsVP1 gene. Sequence data indicates that the precisely spliced OsVP1 transcripts were expressed in all the tested varieties (Table 1). However, a substantial proportion of transcripts expressed in the PHS-susceptible cvs G46B and HeiB were observed to be processed incorrectly (Fig. 2; Table 1). For the PHS-resistant cvs Fuhui838 and Lemont, although aberrant RT-PCR products were undetectable by gel separation, several truncated OsVP1 cDNA clones were identified by sequencing analysis (Fig. 2; Table 1).
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To examine the structure of these aberrant transcripts, a total of 73 OsVP1 cDNA clones were studied in detail. Sequence comparison of the cDNAs revealed a large variation in their structural compositions (Table 1; Fig. 3). A total of 26 different types of transcripts with a unique structural composition were identified and all contained a deletion in the coding region. The deleted material ranged from 177–570 nucleotides in size. The deletion sites in these transcripts started at various positions in exon 1 and terminated at a region spanning exons 1 to 5, leading to variety of truncations of exonic sequence. In total 15 different transcripts encoded derivatives with frame-shifts in the open reading frame (ORF), introducing an early stop codon at different positions, whereas 11 different transcripts showed the potential to encode various truncated OsVP1 proteins. Most of the missing sequences from the truncated transcripts indicated above involved two different exons, and in a few cases the truncation took place within a single exon.
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An experiment was conducted to determine the expression pattern of OsVP1 in mature imbibed dormant seeds. The total RNAs were isolated from mature imbibed dormant embryos for the four varieties examined and transcripts with a 3' poly(A) tail were selected for further analysis by RT-PCR. Primers designed to amplify the corresponding region of OsVP1 were used. The prevalent transcripts detected in all the tested varieties were spliced correctly (Figs 2, 3; Table 1), and transcripts with a variety of deletions at the exonic region were also observed in different varieties (Fig. 3; Table 1).
Selection of the unusual excision sites in exons mediated by sequence elements of short direct repeats (SDRs) in the OsVP1 gene
The above analysis indicated that a large number of OsVP1 transcripts derived from both the immature and mature embryos in several varieties were incorrectly spliced. A total of 51 unusual OsVP1 cDNA clones were sequenced to characterize the structure at the deletion boundaries. Comparisons revealed a unique sequence element, namely, the short-direct repeats (SDRs) duplicated at the 5' and 3' excision sites of individual deletion events (Table 1; Fig. 3). As shown in Fig. 3A, incorrect processing of the transcripts in lane 5 of Fig. 2 resulted in the deletion of 387 nucleotides spanning a partial region of the exon 1. The sequence of GCCGGA at the 3' end was identical to that of the immediate upstream 5' end of the deleted sequence. Various SDRs in G46B (GAGC, CCGGA, CCTCG, CGCCG, GCCGGA, CCATCC, CCGG, GGAGCC) were duplicated at the individual deletion junctions located within exons 1 to 3 of the OsVP1 gene. As summarized in Table 1, 2, 7, 10, and 10 SDRs loci were identified to be involved in the abnormally processing of the OsVP1 gene in Fuhui838, Lemont, G46B, and HeiB, respectively. Taken together, in total 21 and 8 SDR loci were identified in the OsVP1 transcripts derived from immature and mature embryos, respectively. SDRs were very short and mostly ranged in size from 3–7 nucleotides. The length of various of the SDRs can be extended to larger nearly identical sequences (Table 1). For example, two copies of SDR of CAG that are separately located at 5' and 3' deletion sites, respectively, can be extended to larger nearly identical sequences of CAGgAGC/CAGAGC (in which the lowercase g indicates an additional nucleotide occurring in the 5' copy of the SDR). Each type of the SDRs was somewhat unique depending on the position of the deletion sites, and contained GC-rich nucleotides. In many cases, only two copies of individual SDRs were present in a proximal genomic region, and each copy located separately at the 5' or 3' excision boundaries. During the post-transcriptional processing, one copy of the SDRs was excised and the other copy retained in the resultant transcripts. In the case of multiple copies of the SDRs that occurred in the premature mRNA, only two of the copies were recruited by individual deletion events. For instance, three copies of AGCC were present at positions 1286, 1543 of exon 1 and position 67 of exon 2, respectively, and providing three combinations of deletion-site choices, in which the copy AGCC at 1286 bases of exon 1 was selected twice for the 5' excision site, while one of the other two copies was alternatively used for the downstream selection of the 3' excision site (Table 1). A similar situation occurred for the SDRs AGC and GAGC. In addition, 10 out of 26 types of deletion events were observed in a region without intron sequence involved in the premature mRNA, suggesting that sequence elements from the intron might not be required for the unusual processing. Based on the comparison of sequence composition in the SDRs, a total of 26 types of SDRs were identified in OsVP1 in this study, and their positions in the genomic sequences were shown in Fig. 4. All the 5' SDRs were distributed in exon 1 while the 3' SDRs were dispersed in exons 1 to 5.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study suggested that a phenotypic variation of preharvest sprouting among different rice varieties is associated with unusual post-transcriptional processing patterns in the OsVP1 gene. A variety of incorrectly processed transcripts derived from both immature and mature embryos were detected in all varieties tested. The observed events took place at the exonic region spaning exons 1 to 5, resulting in the deletion of an important functional domain and frame-shifts with premature termination by the introduction of stop codons. Extensive incorrectly spliced transcripts have also been observed in the wheat VP1 gene (McKibbin et al., 2002). Analysis of wheat VP1 transcript structure showed many combinations of intron insertion/exon deletions, resulting in termination of the ORF before the B3 domain. The significant reduction of precise functional wheat VP1 gene product has been proposed to contribute to the relatively weak embryo dormancy and preharvest sprouting in hexaploid bread wheat (Triticum aestivum) (McKibbin et al., 2002; Wilkinson et al. 2005). In addition, many previous studies demonstrated that VP1 (or ABI3, the equivalent in Arabidopsis) encodes a seed-specific transcription factor essential for seed maturation and dormancy induction, and loss of function in vp1/abi3 leads to non-dormancy or vivipary (McCarty et al., 1989; Nambara et al., 1992, 1994). A distinct function of VP1/ABI3 is to trans-activate transcription of maturation-associated genes (McCarty et al., 1991; Hattori et al., 1992). Consistent with these observations, the present study revealed that a considerable reduction of precisely processed OsVP1 transcripts may contribute to precocious germination in the sprouting-susceptible varieties. It is noteworthy that some of the OsVP1 transcripts tested in the current experiment may be localized in the nucleus and untranslated, which might limit the strength of the correlation analysis between the RNA populations and phenotypic variation of preharvest sprouting. Therefore, further experiments, including western analysis of rice grains and/or assay of polysomal RNA are needed to validate the findings.
Interestingly, the unusual processing events observed in the rice orthologue of VP1 may be regulated by sequence elements of short, direct repeats (SDRs). Careful inspection of sequence data revealed that the extremely short SDRs could be extended to relatively larger nearly identical sequences (Table 1). For each unusual excision event, only one of the paired SDRs is always deleted precisely. This type of event also occurrs in a maize BADH1 cDNA (accession no. DW475114 [GenBank] ) with two copies of the sequence CGCC located at exon 1 and 2, respectively. In another published rice cDNA (accession no. NM_001052820), a 57-nucleotide deletion was found in exon 1 in the ZAP-like (zinc finger antiviral protein-like) gene and two copies of CCCCTTCC are present at the deletion junctions. Furthermore, extensive SDRs-mediated post-transcriptional processing within the coding sequences has been documented in several stress-responsive genes (Niu et al., 2007; Luo et al., 2007). Therefore, it is proposed that the occurrence of the unusual post-transcriptional processing in exons 1 and 5 of OsVP1 is associated with duplicated SDRs that are exclusively distributed in the vicinity region of the prospective deletions. Sequence data did not show any mutations at OsVP1 locus among the four varieties used, but SDRs-mediated unusual splicing events vary in several varieties and different developmental stages, suggesting that functional differentiation of other specific trans-factors necessary for the unusual SDRs-mediated post-transcriptional processing exist among different rice varieties. In addition, in the PHS-susceptible varieties, much higher abundance of incorrectly spliced OsVP1 transcripts from immature, instead of mature embryos, were detected, indicating the processing events in OsVP1 gene may be subjected to spatial and temporal regulation by the putative trans-activating factors required for the recognition of SDRs.
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Acknowledgements |
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This work was supported by the National Science Foundation of China (Grant No. 30770466), the 973 Program (Grant No. 2006CB100205) from the Chinese Ministry of Science and Technology, biotechnology application projects (Grant Nos. 2006Z-05-0039 and 07KJT-11), from the local government of Sichuan Province, and 985 youth talent program (grant No. 0082204127106) from Sichuan University.
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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ABA, abscisic acid; DAP, days after pollination; ORF, open reading frame; OsVP1, Oryza sativa vivipary 1; PHS, preharvest sprouting; RT-PCR, reverse transcription-polymerase chain reaction; SDRs, short direct repeats.
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