JXB Advance Access originally published online on October 16, 2003
Journal of Experimental Botany, Vol. 54, No. 393, pp. 2655-2660,
December 1, 2003
© 2003 Oxford University Press
Mutation in the ap2-6 allele causes recognition of a cryptic splice site
Received 8 May 2003; Accepted 6 August 2003
Department of Biology, University of Western Ontario, 1151 Richmond Street North, London, Ontario N6A 5B7, Canada
* To whom correspondence should be addressed. Fax: +1 519 661 3935. E-mail: skohalmi{at}uwo.ca
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Abstract |
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Mutations in the homeotic gene APETALA2 of Arabidopsis thaliana cause severe developmental alterations, most prominently homeotic floral organ replacements from petals to carpels and petals to stamens in the outer two floral whorls. To date, ten different alleles have been identified conferring phenotypes of various degrees. Of these ten alleles, only three have been characterized at the sequence level. The identification of the sequence alteration in the ap2-6 allele is reported here. In ap2-6 a single G·C to A·T transition occurred at the 3' end of intron 6 (position 1342) which leads to a dinucleotide loss at the mRNA level. This change is consistent with the G·C to A·T transition destroying a conserved dinucleotide motif (AG) required for proper splice recognition and with the resulting recognition of the next available downstream AG dinucleotide which in AP2 is immediately adjacent to the authentic 3' splice site. The dinucleotide loss will cause a frameshift, the translation of three incorrect amino acids and a premature stop codon resulting in a truncation of the AP2 sequence within the AP2-R2 domain. Such a truncation is predicted to impact severely on the function of AP2 and is consistent with the observed phenotype.
Key words: ap2 mutant allele, ap2-6, APETALA2, Arabidopsis, developmental mutant, homeotic mutant, splicing.
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Introduction |
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The Arabidopsis thaliana protein APETALA2 (AP2) is a major developmental regulator of floral organ initiation and identity (Riechmann and Meyerowitz, 1998). AP2 belongs to a large family of proteins that is unique to plants. Members of this family are characterized by having one or two copies of a conserved protein motif, known as the AP2 domain (Jofuku et al., 1994; Okamuro et al., 1997; Weigel, 1995). Proteins carrying an AP2 domain are able to bind DNA (Büttner and Singh, 1997; Hao et al., 1998; Kagaya et al., 1999; Nole-Wilson and Krizek, 2000; Stockinger et al., 1997) and to interact with other proteins (Xu et al., 1998). Within the AP2 domain, the YRG element, a conserved basic and hydrophilic region, is a putative DNA binding site and the RAYD element is predicted to form amphipathic
-helices that have been shown to mediate protein–protein interactions (Beamer and Pabo, 1992; Rost and Sander, 1994; Weigel, 1995). AP2 itself has two AP2 domains (AP2-R1 and AP2-R2), a putative nuclear localization signal within a highly basic region and an acidic and serine-rich region which is analogous to transcription activation sequences (Chelsky et al., 1989; Mitchell and Tjian, 1989; Fig. 1). All these features are consistent with AP2 and similar proteins being transcription factors (Jofuku et al., 1994).
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Genetic studies have shown that AP2 has at least three major roles during development. AP2 is required for the specification of floral meristem identity early in flower development (Bowman et al., 1993; Huala and Sussex, 1992; Irish and Sussex, 1990; Okamuro et al., 1997; Schultz and Haughn, 1993; Shannon and Meeks-Wagner, 1993). AP2 is also required for defining floral organ identity and it is essential for the proper specification of the outer two whorl floral organs, sepals and petals, and as a negative regulator of the floral homeotic MADS box gene AGAMOUS (Bowman et al., 1989; Drews et al., 1991; Komaki et al., 1988; Kunst et al., 1989). When mutated, ap2 flowers display distinct homeotic alterations where sepals are replaced by carpels and petals by stamens. Lastly, AP2 expression is required for normal ovule and seed development (Jofuku et al., 1994; Léon-Kloosterziel et al., 1994; Modrusan et al., 1994; Western et al., 2001).
The ap2 mutant phenotype has been described as early as 1980 (Koornneef et al., 1980). Since then many more alleles have been isolated, ap2-1 to ap2-10 (Bowman et al., 1991, 1989; Jofuku et al., 1994; Komaki et al., 1988; Koornneef et al., 1980; Kunst et al., 1989; Mirza and Saeed, 1998). Based on morphological differences the phenotypes of these alleles can be loosely divided into weak, intermediate and strong. The diversity in phenotypes also indicates that different aspects of the AP2 molecular function might be affected. Amazingly, only three of the mutant alleles have been characterized at the sequence level (Fig. 1: ap2-1: Gly-251 to Ser; ap2-5: Gly-159 and Gln-420 to Glu; ap2-10 T-DNA insertion at position 268; Jofuku et al., 1994). To gain a better insight into AP2 function, the ap2-6 mutant allele was analysed and it is reported here that the ap2-6 mutation is consistent with a mis-splicing event and the formation of a truncated protein.
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Materials and methods |
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Seed source and growth conditions
Arabidopsis wild-type Columbia (Col-0) and ap2-6 seeds (seed stock no. CS3176 and CS6240) were obtained from the Arabidopsis Biological Resource Center. Seeds were sterilized by microwave treatment, suspended in 10% PPM (Preservation for Plant Tissue Culture Media, Plant Cell Technology), germinated on 0.5% BM plant growth medium, transferred into soil and grown under standardized growth conditions (Martínez-Zapater and Salinas, 1998).
DNA and RNA isolations
Arabidopsis genomic DNA and total RNA was isolated using the FastPrep system (Bio/Can Scientific) according to the manufacturer’s instructions. Plant tissue (approximately 100–250 mg) was placed in a FastPrep lysis vial containing a single large lysing matrix bead and lysed for 40 s at speed 4.0 or 5.0 (DNA or RNA). For RNA isolations the FastPrep system was used in combination with 1 ml of RNAwiz (Ambion), the supernatant was transferred to a fresh tube and the extraction was completed according to the RNAwiz instructions.
Cloning strategies
AP2 wild-type or mutant sequences were amplified with gene specific primers (AP2 5' PCR: 5'-AAGGGTCGACAAATGTGG GATCTAAACG-3'; AP2 3' PCR: 5'-GGTGGCGGCCGCTCAA GAAGGTCTCATG-3') and Expand High Fidelity Polymerase (Roche Diagnostics) using either Arabidopsis genomic DNA or RNA as a template. In the case of RNA, PowerScript-Reverse Transcriptase (Clonetech) was used for first strand synthesis. PCR products were ligated into a pGEM-T vector (ProMega) (Sambrook and Russell, 2001) and the DNA sequence of the inserts was determined (Robarts Research Institute, London, Ont., ABI377, Perkin Elmer) for both strands using vector specific primers (T7 forward and reverse primers) and primers recognizing internal AP2 sequences.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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To determine the sequence alteration in ap2-6, genomic DNA was isolated from homozygous mutant plants and used as a template for PCR amplification. Two independently PCR-derived ap2-6 copies using DNA from two different mutant plants were sequenced in both directions. Sequence analysis showed that ap2-6 contained a single G·C to A·T transition (Figs 1, 2a) at position 1342. This type of base substitution is consistent with the ap2-6 mutation being generated by EMS (Haughn and Somerville, 1986; Kunst et al., 1989), a mutagen that almost exclusively induces G·C to A·T transitions (Kohalmi and Kunz, 1988). This G·C to A·T transition in ap2-6 occurred close to the exon 6/7 splice site (Fig. 2a) and it was expected that the mutation would affect proper recognition and splicing of intron 6. To determine if the ap2-6 mutation causes improper splicing, RNA was isolated from homozygous ap2-6 plants and amplified by RT-PCR. Size separation of PCR fragments obtained from wild type and ap2-6 showed that all fragments were approximately 1.3 kb in size, the predicted length of the wild-type AP2 open reading frame (Fig. 2b). This indicates that exon 6 (96 bp in length) was still spliced in ap2-6. cDNA sequence analysis revealed that the ap2-6 sequence lacked an AG dinucleotide compared to the wild type (Fig. 2c). The G·C to A·T transition in ap2-6 changes the 3' splice recognition site dinucleotide of intron 6 from AG to AA (Fig. 2a). This forces the splice apparatus to recognize and cleave adjacent to the next available AG dinucleotide sequence downstream of the authentic 3' splice site. In AP2 the next AG dinucleotide is located immediately downstream of the wild-type recognition site (AGAG; Fig. 2d). This leads to an incorrect splicing of intron 6, which causes a 2 bp deletion in the processed mRNA. The resulting frameshift causes the translation of three incorrect amino acids before a premature stop codon (Fig. 2c). Such cryptic splice sites have been reported for a number of other plant genes. For example, agamous-4 (ag-4) has a G·C to A·T transition changing the conserved 3' splice recognition sequence and as a result exon 6 is skipped (Sieburth et al., 1995). In the CHS (chalcone synthase) gene a similar sequence alteration leads to the recognition and splicing of the next downstream AG dinucleotide (Burbulis and Winkel-Shirley, 1999). The majority of known Arabidopsis splice mutants involve a change in the G of the essential AG dinucleotide at the 3' splice site (Brown, 1996).
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The predicted truncation in ap2-6 occurs in and alters the amino acids of the RAYD element of AP2-R2 (Figs 1, 2c). Lacking a major portion of the RAYD element and the C-terminus of the protein probably interferes severely with the protein and DNA binding capabilities of AP2 and, potentially, other additional functions of the C-terminal portion of AP2. Having such a large truncation of the protein correlates very well with the phenotype displayed by ap2-6 mutant plants, which is one of the most severe of all of the known ap2 mutants (Kunst et al., 1989). In addition, sequence alterations in most of the known ap2 mutant alleles, occur in the AP2-R2 domain (ap2-1, ap2-6 and ap2-10) with the exception of ap2-5, which has two point mutations, one in the AP2-R1 domain and one near the C-terminus of the protein (Fig. 1). To date, no other portion of the AP2 coding region has been found to be altered. One could speculate that an alteration occurring in one of the other protein domains (NLS, SRA) is lethal and hence such mutants would not be recovered. Alternatively, single base pair changes (so far, mostly EMS induced mutant libraries have been screened for ap2 mutants) outside highly conserved domains may be insufficient to produce a mutant phenotype and, consequently, avoid detection.
To determine if mutations in AP2 follow a unique pattern they were compared with mutations identified in other proteins of the AP2 subfamily (Fig. 1). These proteins share the highest sequence similarity in the two AP2 domains and the joining linker region while sequences in the C- and N-terminal regions are much more variable. As for AP2, most mutations have been identified in either the AP2-R1 or AP2-R2 domain and a single mutation in this region is typically sufficient to alter morphology in mutant plants (ANT, GL15, IDS1; Chuck et al., 1998; Elliott et al., 1996; Klucher et al., 1996; Krizek, 1999; Moose and Sisco, 1996). This indicates that the presence of both AP2 domains is essential for proper protein function and it is consistent with the idea that both domains and the conserved linker region are required for DNA binding and protein interactions. The importance of both AP2 domains for DNA binding has been further demonstrated by the identification of many amino acid substitutions in ANT that reduce or prevent DNA binding as shown in a yeast in vivo system and by gel retardation assays (Fig. 1; Krizek, 2003). The distinct nature of both AP2 domains also becomes apparent through experiments that demonstrate that the DNA binding specificity of joined ANT AP2 domains differs from those of its individual AP2 domains (Krizek, 2003; Nole-Wilson and Krizek, 2000). It is interesting to note that several mutations, including some which result in severe protein truncations, have been identified in dual AP2 domain proteins C-terminal to the AP2-R1 repeat (ANT, LIPLESS2 [LIP2] and PHAP2A; Fig. 1) (Keck et al., 2003; Klucher et al., 1996; Krizek, 1999; Maes et al., 2001). The C-terminal protein sequences are often less conserved, but they typically contain domains which have proposed functions analogous to the SRA domain in AP2. This indicates that, at least in some dual AP2 proteins, alterations in the C-terminal region are not lethal. In contrast to findings for AP2, plants carrying a null allele of the proposed AP2 orthologues of Petunia and Antirrhinum (PHAP2A, LIP1 and LIP2) are completely wild type in appearance (Keck et al., 2003; Maes et al., 2001). In Antirrhinum, only a lip1lip2 double mutant shows altered organ structures in the two outer whorls, consistent with a lack of Class A function. However, in contrast to ap2 mutants in Arabidopsis, no homeotic floral organ transformations or effects on the expression pattern of the AGAMOUS orthologue PLENA can be detected (Keck et al., 2003). In addition, the lack of a phenotype in lip and phap2A mutants indicate a certain degree of functional redundancy among dual AP2 domain proteins in Antirrhinum and Petunia. Functional conservation has been demonstrated for at least PHAP2A, which is able to complement the Arabidopsis ap2-1 mutation in respect of both requirement for floral organ identity in the outer two whorls and repression of AG expression (Maes et al., 2001). To determine if mutations in the C- or N-terminal regions of AP2 affect AP2 function similarly to mutations detected in ANT, GL15 or IDS1, or if the function of these regions is, at least in part, redundant, as shown for the AP2 orthologues of Antirrhinum and Petunia (at least 10 dual AP2 domain genes can be identified in the Arabidopsis genome; (The Arabidopsis Genome Initiative, 2000), AP2 deletion proteins lacking either of these domains but containing the remainder of the protein have to be tested in planta.
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Acknowledgements |
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We are grateful to The Arabidopsis Biological Resource Center (ABRC) for the supply of seeds. This research was supported by an NSERC grant. We would like to thank L Kohalmi, K Kovacs and C Payne for critical reading of the manuscript.
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