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Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae)
Plant Science 241 (2015) 277–285
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Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae) Bo Zhang a,1 , Zhi-xiong Liu b,1 , Jiang Ma a , Yi Song c , Fa-ju Chen a,∗ a b c
Biotechnology Research Center, China Three Gorges University, Yichang City, Hubei 443002, PR China College of Horticulture and Gardening, Yangtze University, Jingzhou City, Hubei 434025, PR China College of Landscape Architecture, Beijing Forestry University, Beijing 100083, PR China
a r t i c l e
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Article history: Received 5 July 2015 Received in revised form 19 October 2015 Accepted 29 October 2015 Available online 2 November 2015 Keywords: AGAMOUS Magnolia stellata Alternative splicing Double flower Floral development
a b s t r a c t Magnolia stellata is a woody ornamental shrub with more petaloid tepals than related plants from family Magnoliaceae. Recent studies revealed that expression changes in an AGAMOUS (AG) orthologous gene could resulted in double flowers with increased numbers of petals. We isolated three transcripts encoding different isoforms of a single AG orthologous gene, MastAG, mastag 2 and mastag 3, from M. stellata. Sequence alignments and Southern blot analyses suggested that MastAG was a single-copy gene in M. stellata genomes, and that mastag 2 and mastag 3 were abnormally spliced isoforms of MastAG. An 144 bp exon skipping in MastAG results in the truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas an 165 bp exon skipping of MastAG produces a truncated mastag 3 protein lacking 6 aa of the K3 subdomain and the completely C terminal region. Expression analyses showed that three alternative splicing (AS) isoforms expressed only in developing stamens and carpels. Functional analyses revealed that MastAG could mimic the endogenous AG to specify carpel identity, but failed to regulate stamen development in an Arabidopsis ag-1 mutant. Moreover, the key domain or subdomain deletions represented by mastag 2 and mastag 3 resulted in loss of C-function. However, ectopic expression of mastag 2 in Arabidopsis produced flowers with sepals converted into carpeloid organs, but without petals and stamens, whereas ectopic expression of mastag 3 in Arabidopsis could mimic the flower phenotype of the ag mutant and produced double flowers with homeotic transformation of stamens into petals and carpels into another ag flower. Our results also suggest that mastag 3 holds some potential for biotechnical engineering to create multi-petal phenotypes in commercial ornamental cultivars. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alternative splicing (AS) refers to the assembly of exons in different ways during pre-mRNA splicing to produce multiple distinct transcript isoforms from a single gene. Alternative splicing is thought to be a major source of proteome complexity and functional diversity [1]. In plants AS is involved in many important biological processes including stress responses [2], development [3] and flowering [4,5]. In Arabidopsis, AS plays an important role in stage transition during floral development [6]. Members of the MADS-box transcription factor gene family in flowering plants are
∗ Corresponding author. E-mail addresses: [email protected] (B. Zhang), [email protected] (Z.-x. Liu), [email protected] (J. Ma), [email protected] (Y. Song), [email protected] (F.-j. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plantsci.2015.10.017 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
well-known for regulation of floral organ development, but found to represent families with low occurrences of AS [7]. AGAMOUS (AG) encodes a key C function MADS-box regulator of stamen and carpel identity as well as floral meristem determinacy [8]. According to the C function, AG is expression in the center of the floral meristem starting at stage 3 giving rise to the stamen and carpel primordia in Arabidopsis flowers, and exclusively in the third and fourth whorls until late in floral development [9,10]. AG is the only gene in Arabidopsis harboring a full C function activity; the ag mutant has completely loss of male and female organ identities and produces double flowers with homeotically transformed stamens to petals and a new ag flower arising in place of the gynoecium [11]. Previous studies suggested that changes in AG orthologous genes expression were involved in all cases of domesticated plants with double flowers [12]. However, AS of AG orthologous genes was previously observed in three ornamental plants with double flowers, including rose (Rosa rugosa) [13], the ranunculid Thalictrum thalictroides
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2.2. Isolation and identification of alternatively spliced isoforms of the MastAG from M. stellata
Fig. 1. Flower of M. stellata.
cultivar ‘Double White’ [14], and Japanese cherry blossom (Prunus lannesiana) cultivar ‘Albo-rosea’ [15]. Double flowers with the numerous extra petals were selected as ornamentals in many plant families during the early domestication and in subsequent breeding programs. Although the genetics of crop domestication processes controlling these events are becoming increasingly understood, the underlying molecular mechanisms of the showy floral phenotype that evolve during natural selection remains unclear. Magnolia stellata that produces double flowers belongs to family Magnoliaceae. As an early-diverging clade of ancestral angiosperms [16], it may provide hints in understanding the origin and early domestication of double flower. The perianth whorl of M. stellata consists of 12–45 petaloid tepals (Fig. 1). In the present work three AG-like mRNAs, two of which result from alternative splicing, were isolated from M. stellata. Detailed sequencing and expression analyses of MastAG and both the alternatively spliced isoforms, mastag 2 and mastag 3, were studied. Ectopic expression of all three AS isoforms under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter was examined for complementation of the Arabidopsis ag-1 mutants. The results provided strong evidence of high conservation of AG orthologs in core eudicot model plants and the woody shrub M. stellata, as well as novel information about the origin and subsequent evolution of double flower phonotypes in basal angiosperms.
Total RNA was extracted from floral buds using an EASYspin Plant RNA Extraction Kit (Aidlab China) following the manufacturer’s protocol. First-strand cDNA was synthesized from 1 g of the DNase I-treated RNA, using an oligo (dT) 15 adaptor primer and M-MLV Reverse Transcriptase (TaKaRa, Japan). MastAG, mastag 2 and mastag 3 cDNA sequences were isolated by PCR amplification using the forward primer MastAGF (5 -GGC GTC TAG AAG GAG AAG AAG CGA-3 ) and the reverse primer MastAGR (5 -A CCC GGG TCT TAT GAC CAT CTG CAT-3 ), respectively. Primers design was based on the sequence of AG orthologous genes from closely species, such as MawuAG (Genbank accession numbers: JN133287) from Magnolia wufengensis, and MaodAG (Genbank accession numbers: JQ326240) from M. odoratissima. PCR was performed with a 5 min 94 ◦ C denaturation step, followed by 30 cycles of 45 s at 94 ◦ C, 45 s annealing at 57 ◦ C, a 1 min extension at 72 ◦ C, and a final extension period of 10 min. Genomic DNA was extracted from juvenile leaves of M. stellata using a modified CTAB method according to the protocol suggested by Doyle and Doyle [17]. Partial genomic DNA sequences of MastAG containing missing sequences of mastag 2 and mastag 3 were isolated by PCR using primers successively: FLAGF1 (5 -TTGGTATATATTGGGCAGATTCAG-3 ) (1–24 nt. of the DNA) and FLAGR1 (5 -CTTCTTAGATCTGATTCTGCTAATGC-3 ) (941–966 nt.); then FLAGF2 (5 -GCGCTTTTACAGAATGCAAACG3 ) (772–793 nt.) and FLAGR2 (5 -CTCTTTCGTTCTCAGTTATCTG-3 ) (2594–2615 nt.); and FLAGF3 (5 -TGATAATATGTACCTCCGTG-3 ) (2063–2082 nt.) and FLAGR3 (5 -ACCCGGGTCTTATGACCATCTGCA3 ) (3894–3917 nt.). Genomic DNA from juvenile leaves of M. stellata was treated with RNase, purified, and then digested with Xba I, Hind III and Afl II (TaKaRa, Japan), respectively. 20 g of digested genomic DNA was loaded in each lane, separated in a 1% agarose gels, subsequently transferred to a HyBond-N+ nylon membrane (Amersham Biosciences, UK), and then fixed by UV-crosslinking (254 nm UV light for 2 min). A 205 bp sequence containing consensus exons and introns of MastAG, mastag 2 and mastag 3 present in the genomic DNA sequence of MastAG (2,462–2,666), was prepared for Southern blot probe by PCR with the forward primer MastAGSF (5 -GCA AAA TGA TAA TAT GTA CCT CCG TG-3 ) and the reverse primer MastAGSR (5 -ATG CCT GAA ATC TAC TGG TGG TG-3 ). This probe was labeled with digoxigenin by using DIG High Prime DNA Labeling and a Detection Starter Kit II (Roche, Mannheim, Germany) according to the manufacturer’s procedure. Prehybridization, hybridization, stringency washes and immunological detection were performed by DIG High Prime DNA Labeling and a Detection Starter Kit II (Roche, Mannheim, Germany) according to the manufacturer’s procedure. 2.3. Sequence alignments and phylogenetic analysis
2. Materials and methods 2.1. Plant material Flower buds at sequential developmental stages were collected from M. stellata growing under natural conditions in Beijing. Juvenile leaves, tepals, stamens and gynoecia were sampled during stamen and gynoecium differentiation well before anthesis, immediately frozen in liquid nitrogen and stored at −80 ◦ C until used. The Arabidopsis ag-1 mutant line (CS25) in ecotype Landsberg background was obtained from the Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH, USA.
Deduced amino acid sequences of MastAG were used in a BLAST search of the Genbank database. During the BLAST searches, multiple C class proteins from various angiosperm lineages were selected for alignment. Phylogenetic trees were constructed with MEGA5.0 software using the Neighbor-Joining Method [18,19]. Genbank accession numbers of the sequence data used were as follows: MastAG (AFH74392); Arabidopsis thaliana, AG (P17839), SHATTERPROOF1 (SHP1, P29381), SHP2 (P29385), SEEDSTICK (STK, Q38836), APETALA1 (AP1, P35631), FRUITFULL (FUL, Q38876), AGL79 (AEE77628), AP3 (P35632), PISTILLATA (PI, P48007), SEPALLATA1 (SEP1, P29382), SEP2 (P29384), SEP3 (O22456), SEP4 (NP 178466); Antirrhinum majus, FARINELLI (FAR, CAB42988), PLENA (PLE, AAB25101); M. wufengensis, MawuAG
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(AEO52692); Magnolia figo, Mafi (AFH74385); Magnolia grandiflora, MagrAG (AFH74382); Manglietia fordiana, Mafo (AFH74387); Prunus serrulata var. lannesiana, PrseSHP (ADG45819), PrseAG (ADK95058), PrseSTK (ADD91578); Camellia japonica, CajaAG (AIP87050); Petunia × hybrid, FBP6 (CAA48635), FBP3 (CAA50549), FBP7 (CAA57311), FBP11 (CAA57445); Solanum lycopersicum, TAGL1 (NP 001234187), TAG1 (Q40168), TAGL11 (AAM33102); Medicago truncatula, MtAGa (AGV55405), MtAGb (AGV55406); Nicotiana benthamiana, NbAG (AFK13159), NbSHP (AFK13160); Meliosma dilleniifolia, MediAG (AAS45686); Euptelea pleiosperma, EuplAG (ADC79699); Saruma henryi, SaheAG (AAS45689); Lilium longiflorum, LMADS10 (AIJ29174); Oncidium Gower Ramsey, OMADS4 (AIJ29176); and Oryza sativa, OsMADS3 (Q40704), OsMADS58 (Q2V0P1). Full-length amino acid sequences containing the MADS, I, K and C domains were aligned with BioEdit 7.0.9 software using the ClustalW program with default settings [20]. Several AG orthologous and mutant proteins from different angiosperm lineages were selected for alignment. Sequence data used were as follows: P. serrulata var. lannesiana, PrseAG (ADK95058), prseag1 (ADK95059) [15]; Thalictrum thalictroides, ThtAG1 (AFI23876), ThtAG18 [14]; and A. thaliana, AG (P17839), ag-4 [21]. 2.4. Expression analysis of alternatively spliced isoforms of the MastAG For semi-quantitative RT-PCR analysis, we used 1 g of total RNA extracted from juvenile leaves, tepals, stamens and carpels of M. stellata in order to synthesize the first-strand cDNA with an oligo (dT) 15 primer as described above. A 2 l cDNA sample from the RT reaction was used for 25 cycles of PCR as follows: 20 s at 94 ◦ C, 20 s at 57 ◦ C and 20 s at 72 ◦ C, preceded by 5 min at 94 ◦ C and followed by 10 min at 72 ◦ C. We analyzed 10 l of total PCR product (25 l) in each reaction by electrophoresis in a 1% agarose gel and photography under UV light. RT-PCR was carried out using the genespecific primers qMastAGF (5 -CGAATACGCCAATAACAGGCATTTG3 ) and qMastAGR (5 -ACATCGTATTCAGGAGCAGGCAAC-3 ) for MastAG; qmastag 2F (5 -GATGCGTATCGGAAGCCAATTCTC-3 ) and qmastag 2R (5 -CCACCACATTACCTTAGCACGGAG-3 ) for mastag 2; and qmastag 2F and qMastAGR for mastag 3. Amplification of M. stellata actin with specific primers qMastactinF (5 CAAGAGCTTGAGACAGCAAAGAGTAG-3 ) and qMastactinR (5 AGATAGACCCTCCAATCCAGACACT-3 ) was used as a positive control. For quantitative analysis, total RNA was isolated from flower buds at various developmental stages and treated as described above. Quantitative real-time RT-PCR with three biological replicates was carried out using an ABI7500 Real-time PCR System. The reaction mixture was cycled as follows: 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s, 60 ◦ C for 35 s, followed by 30 s at 95 ◦ C and 1 min at 60 ◦ C. For the melt curve, we changed the temperature by increments of 0.2 ◦ C/s to 95 ◦ C. Experiments were repeated three times for each sample. Gene-specific primers and the normalization control primers were the same as the primers used for semi-quantitative RT-PCR. 2.5. Vector construction and Arabidopsis transformation Full-length cDNA of MastAG, mastag 2, and mastag 3 were cloned into binary vector pBI121 (BD Biosciences, Clontech) using Xba I and Sma I restriction enzymes under control of the cauliflower mosaic virus 35S promoter in the sense orientation. The 35S::MastAG, 35S::mastag 2, and 35S::mastag 3 constructs were transformed into heterozygous AG/ag-1 Arabidopsis plants (ecotype Landsberg) using the floral-dip method according to the protocol suggested by Clough and Bent [22] via Agrobac-
Fig. 2. Southern blot analyses of MastAG in M. stellata. The genomic DNA from M. stellata was digested with Afl II, Hind III and Xba I, respectively. The MastAG-specific probe was a 205 bp sequence containing a consensus exon and intron of MastAG, mastag 2 and mastag 3 present in the genomic DNA sequence of MastAG.
terium tumefaciens strain GV3101-90, respectively. Plasmid pBI121 (negative control) was also transformed into heterozygous AG/ag1 A. thaliana by the same method described above. Seeds of transgenic Arabidopsis plants were selected on solid 0.5 × MS medium [23] containing 50 g/ml kanamycin at 4 ◦ C for 2 days and then transferred to a greenhouse under long-day conditions (16 h light/8 h darkness) at 22 ◦ C for 10 days. The seedlings were subsequently transplanted into soil. Transgenic A. thaliana lines were confirmed by PCR and quantitative real-time RTPCR with transgene-specific primers. Wild-type, heterozygous AG/ag-1 and homozygous ag-1 transformants were isolated using a dCAPS marker designed with the dCAPS finder program with dCAPS primer (5 -GATATATTAATATATGTTGATAAATCACTTA3 ) and reverse primer (5 -AATCAACTTCCTGCTTAATCGGT-3 ) [24]. PCR was performed with 30 cycles of 30 s at 94 ◦ C, 30 s annealing at 53 ◦ C, a 30 s extension at 72 ◦ C, preceded by a 5 min 94 ◦ C denaturation step and a final extension period of 10 min. The 20 l of the total PCR product (25 l) in each reaction was digested with Afl II (TaKaRa, Japan) for 2.5 h and then separated on a 4% agarose gels, stained, and visualized under UV light. The ag-1 fragment was cleaved by Afl II to reduce the 355 bp fragment to 327 bp. Wild-type and homozygous mutant plants containing the transgenes were analyzed after genotyping. For quantitative real-time RT-PCR analysis, triplicate quantitative assays were performed as described above. Amplification of the A. thaliana -actin with the primers qactinF (5 -GATTTGGCATCACACTTTCTACAATG-3 ) and qactinR (5 GTTCCACCACTGAGCACAATG-3 ) was used as an internal control to normalize all data. The cycle parameters were set at 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s, 60 ◦ C for 35 s, followed by 30 s at 95 ◦ C and 1 min at 60 ◦ C. For the melt curve, we changed the temperature by increments of 0.2 ◦ C/s to 95 ◦ C. 3. Results 3.1. Cloning and sequence alignments of MastAG, mastag 2, and mastag 3 from M. stellata Full-length cDNAs of MastAG, mastag 2, and mastag 3 from M. stellata were obtained by homology-based cloning following the
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Fig. 3. Phylogenetic analysis of C-class MADS-box proteins. The tree was constructed with MEGA5.0 software using the neighbor-joining method. Numbers along the branches are bootstrap values from 1000 replications.
above procedures. The MastAG transcript (Genbank accession number: JQ326243) contained a 672 bp ORF encoding 223 amino acids (aa), whereas the mastag 2 transcript (Genbank accession number: KR017756) contained a 528 bp ORF encoding 175 aa, and the mastag 3 transcript (Genbank accession number: KR017757) con-
tained a 552 bp ORF encoding 183 aa. The genomic sequence of the MastAG (Genbank accession number: KR017758) showed that all three transcripts were derived from a consensus pre-mRNA. A 144 bp exon skipping in the genomic sequence of MastAG produced the mastag 2, and the complete omission of another exon (165 bp)
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Fig. 4. Sequence alignments of MastAG, mastag 2, and mastag 3 with other AG-like MADS domain proteins. The first underlined region represents the MADS domain and the second the K domain. The AG motifs I and II in the C-terminal region are boxed. Amino acid residues identical to MastAG are indicated as dots. Dashes were introduced into the sequence to improve the alignment. The k domain contains three heptad (abcdefg)n repeats represented by k1, k2 and k3 subdomains that usually contain hydrophobic amino acids at positions a and d [25].
produced the mastag 3. That is, mastag 2 and mastag 3 were alternatively spliced isoforms of MastAG. Southern blot analysis with gene-specific probes of MastAG (a 205 bp sequence containing a consensus exon and introns of the three alternative splicing isoforms) revealed that MastAG was a single-copy gene in the M. stellata genomes (Fig. 2). Phylogenetic analyses grouped the MastAG (M. stellata AGAMOUS) into the basal eudicots AG lineage (Fig. 3). Moreover, sequence alignments revealed that the MastAG protein contains a 57 aa MADS domain, a 32 aa I domain (58–89), an 89 aa K domain (90–178) and a 45 aa C domain (179–223) (Fig. 4) [25,26]. Three putative amphipathic ␣-helices with expected hydrophobic amino acids at the a and d positions in heptad (abcdefg) n repeats were identified in the K-domain, namely subdomains K1 (90–111), K2 (124–138) and K3 (146–178) [25]; and the C terminal region harbors highly conserved AG motifs I and II [26]. However, the alternative splicing resulted in a truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas the
truncated mastag 3 contained deletions of 6 aa of the K3 subdomain and completely loss of the C terminal region (Fig. 4).
3.2. Expression analyses of MastAG, mastag 2, and mastag 3 In M. stellata, MastAG, mastag 2, and mastag 3 were exclusively transcribed in developing stamens and carpels, and not in juvenile leaves and tepals (Fig. 5). Moreover, all three transcripts were produced during all stages of stamen and carpel differentiation (Fig. 6). MastAG expression was clearly higher than expression of the other two AS transcripts at corresponding stages during reproductive organ development, and mastag 3 expression was higher than that of mastag 2. Furthermore, the expression levels of all three transcripts increased rapidly as the stamens and carpels increased in size. Their expression levels were highest until the anthers and carpels reached maturity.
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Fig. 5. Expression of MastAG, mastag 2, and mastag 3 in the juvenile leaves (le), tepals (te), stamens (st) and carpels (ca) by semi-quantitative RT-PCR with ACTIN as the control.
3.3. Ectopic expression of MastAG, mastag 2, and mastag 3 in ag-1 Arabidopsis In order to gain further insight into the functional divergence of three alternative splicing isoforms, we attempted to rescue the loss–of–function Arabidopsis ag mutant (ag-1) by MastAG, mastag 2, and mastag 3, respectively. Binary vectors carrying 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 were separately introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were confirmed by quantitative real time RT-PCR (Fig. 7) and mutant alleles were also identified by dCAPS genotyping (Fig. 8). Phenotypes of transgenic lines were assayed in a wild-type and homozygous ag-1 mutant backgrounds to determine whether MastAG, mastag 2, and mastag 3 compensated for the absence of the endogenous AG gene in Arabidopsis, respectively. Among a total of 7 35S::MastAG homozygous ag-1 transformants, three (42.86%) lines displayed strong complementation phenotypes producing flowers consisting of 4 sepals in the first whorl, 8 petals in the second and one fat gynoecium in the third (Fig. 9A-c); and two (28.57%) lines displayed medium rescue flower phenotypes with a terminator of unlimited number of perianth by a central fat gynoecium (Fig. 9A-d); and 2 (28.57%) lines displayed no change in flower structure. However, ectopic expression of MastAG rescued carpel development without complementation of stamen development in the Arabidopsis ag-1 mutant background. In other words, MastAG from M. stellata partially or fully compensated for
the endogenous AG gene of A. thaliana that specified carpel identity, but failed to rescue stamen development. Moreover, Among a total of 27 35S::MastAG transformants in wild-type background, nine (33.33%) lines displayed obvious changes in flower structure with the first whorl sepals converted into carpeloid organs (Fig. 9B-c), ten (37.04%) lines displayed weaker changes in flower structure with smaller petals and shorter anther filaments (Fig. 9Bd), and eight (29.63%) lines displayed no change in flower structure. However, all 12 35S::mastag 2 transformants in homozygous ag1 mutant background displayed no change of flower structure (Fig. 9A-e), whereas among a total of 23 35S::mastag 2 transformants in wild-type background, 15 (65.22%) lines produced flowers with carpeloid sepals bearing ovules, but without petals and stamens (Fig. 9B-e), 8 (34.78%) lines displayed no change in flower structure. All 24 35S::mastag 3 transformants in homozygous ag1 mutant background displayed no change in flower structure (Fig. 9A-f), whereas among a total of 11 35S::mastag 3 transformants in wild-type background, 5 (45.45%) lines displayed obvious changes in flower structure that transformed stamens into petals and carpels into additional ag flowers (Fig. 9B-f), and six (54.55%) lines displayed no change in flower structure. 4. Discussion Double-flower varieties have been grown as ornamental and garden plants for their showy traits since ancient times. However, the molecular mechanisms regulating development of double flowers are not well understood although they are of significant commercial interest. In Arabidopsis, the C-function gene AGAMOUS plays a key role in specifying stamen and carpel identities; AG loss-of-function results in homeotic transformation of stamens into petals and the gynoecium into another ag flower, which leads to double flowers [27]. Increasing numbers of studies suggested that changes in AG orthologous genes expression lead to double flowers in non-model plants [12]. For example, restriction of the RhAG expression domain in the center of the flower increased the number of petals in the double-flower roses [28]. Similar results in cultivated Camellia japonica indicated that contracted and expanded expression of AG orthologous gene CjAG were associated with formation of different types of double flowers [29]. CpAG1 and CpAG2 are AG orthologous genes from Cyclamen persicum. Lack of expression of CpAG1 converted stamens into petals and resulted in a flower with an increase of 5 petals, whereas simultaneous lack of expres-
Fig. 6. Relative expression of MastAG, mastag 2, and mastag 3 in stamens and carpels of M. stellata at various developmental stages. (A) Quantitative real time RT-PCR analyses of expression of three AS transcripts in stamens of M. stellata at different developmental stages. MastAG expression in stamens of 7.1 mm flower buds was used for normalization; (B) quantitative real time RT-PCR analyses of expression of three AS transcripts in carpels of M. stellata at different developmental stages. mastag 3 expression in carpels of 7.1 mm flower buds was used for normalization.
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Fig. 7. The 35S::MastAG, 35S::mastag 2, and 35S::mastag 3 transgenic Arabidopsis in a wild-type and a homozygous ag-1 mutant background confirmed by quantitative real-time RT-PCR (qRT-PCR). (A) Endogenous ag expressed in the homozygous ag-1 mutant Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in a homozygous ag-1 mutant background. ag expression in the homozygous ag-1 mutant Arabidopsis (ho) was used for normalization; (B) endogenous AG expressed in wild-type Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in wild-type background. AG expression in wild-type Arabidopsis (WT) was used for normalization; (C) MastAG, mastag 2 and mastag 3 expressed in homozygous ag-1 mutant Arabidopsis and corresponding transgenic Arabidopsis in a homozygous ag-1 mutant background, respectively. mastag 2 expression in line ho-2, mastag 3 expression in line ho-4, and MastAG expression in line ho-5 of transgenic Arabidopsis were used for normalization; (D) MastAG, mastag 2 and mastag 3 expressed in wild-type Arabidopsis and corresponding transgenic Arabidopsis in wild-type background, respectively. mastag 2 expression in line W-1, mastag 3 expression in line W-6, and MastAG expression in line W-7 of transgenic Arabidopsis are used for normalization, respectively.
Fig. 8. Genotyping of wild-type, heterozygote and homozygous ag-1 mutant A. thaliana (Landsberg erecta) plants by dCAPS. The amplicon of the homozygous ag1mutant Arabidopsis plants was cleaved by Afl II to produce a 327 bp fragment; the amplified fragment from an heterozygote ag-1 Arabidopsis plant was cleaved by Afl II to produce a 355 bp and a 327 bp fragments; the wild-type amplicon was cleaved by Afl II to produce a fragment of 355 bp fragment.
sion of CpAG1 and CpAG2 induced rose-like flowers with multiple petals [30]. On the other hand, alternative splicing of AG orthologous gene resulted in mutant proteins with key domain deletions that enable production of double flowers. For example, exon skipping in the AG orthologous gene PrseAG from P. lannesiana resulted in a mutant prseag-1 protein with the C-terminal AG motifs I and II deletions and production of double flowers, in which stamens were converted into petals or petaloid anthers, and carpels changed into leaf-like organs [15]. Alternative splicing of AG orthologous
gene ThtAG1 from T. thalictroides resulted in mutant proteins with K-domain deletions, and production of double flower in ‘Double White’ cultivar [14]. In M. stellata, three alternative splicing transcripts of AG orthologue were simultaneously expressed in stamens and carpels. Ectopic expression of MastAG rescued carpel development without complementation of stamen development in the Arabidopsis ag-1 mutant (Fig. 9A-c), and converted the first whorl sepals of wildtype Arabidopsis into carpeloid organs bearing ovules (Fig. 9B-c). These results suggested that the functional conservation between AG and MastAG in specifying carpel identity and repression of Aclass gene expression. However, a 144 bp exon skipping in MastAG resulted in a truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain. In addition, a 165 bp exon skipping in MastAG produced a truncated mastag 3 protein with a 6 aa deficiency of the K3 subdomain and completely C terminal deletion. Previous studies revealed that the I domain in MADS-box proteins was responsible for dimerization and protein functional specificity, and the K-domain was essential for protein–protein interactions, and the C-terminal domain was involved in both transcriptional activation and the formation of higher order MADS protein complexes [25,31,32]. These domain omissions or amino acid truncations in key subdomain resulted in the C-function loss or changes in AG orthologous proteins [14,15,21]. In Arabidopsis, the ag-4 mutation with partial loss of the C terminus of the K domain resulted in flowers with normal stamens, but with carpels converted into four sepals [21]. In our study, ectopic expression of mastag 2 and mastag 3 failed to rescued stamen and carpel development in the Arabidopsis ag-1 mutant, respectively (Fig. 9A-e,f). However, 35S::mastag 2 transgenic Arabidopsis in wild-type background produced an interesting flower phenotype with carpeloid sepals bearing ovules, but without petals and stamens (Fig. 9B-e). In addition, 35S::mastag 3 transgenic Arabidopsis in wild-type background produced an interesting flower phenotype with homeotic transformation of stamens into petals and carpels into another
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Fig. 9. Comparison of phenotypes of the wild-type, homozygous ag-1 mutant, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in a homozygous ag1 mutant and wild-type background, respectively. A: Phenotypic comparison of the homozygous ag-1 mutant, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in the homozygous ag-1 mutant background. Flowers of: (A-a) homozygous ag-1 mutant A. thaliana; (A-b) transgenic ag-1 mutant Arabidopsis transformed with the pBI121 vector only (negative control); (A-c) 35S::MastAG transgenic lines with strong complementation phenotypes of ag Arabidopsis, showing 4 sepals in the first whorl, 8 petals in the second and one fat gynoecium in the third; (A-d) 35S::MastAG transgenic lines with medium rescue phenotype, showing a terminator of unlimited number of perianth by a central fat gynoecium; (A-e) 35S::mastag 2 transgenic ag Arabidopsis; (A-f) 35S::mastag 3 transgenic ag Arabidopsis. B: Phenotypic comparison of the wild-type Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in wild-type background. Flower of: (B-a) wild-type Arabidopsis; (B-b) transgenic Arabidopsis transformed with the pBI121 vector only (negative control); (B-c) 35S::MastAG transgenic Arabidopsis showing strong phenotype with the first whorl sepals converted into carpeloid organs; (B-d) 35S::MastAG transgenic Arabidopsis showing weaker phenotype with smaller petals and shorter filament; (B-e) 35S::mastag 2 transgenic Arabidopsis showing strong phenotype with carpeloid sepals, but without petal and stamen; (B-f) 35S::mastag 3 transgenic Arabidopsis showing strong phenotype with transformation of stamens into petals and carpels into another ag flower. sep, sepal; pet, petal; sta, stamen; sti, stigma; ov, ovule. Bars: 1000 m.
ag-like mutant flower (Fig. 9B-f). A similar ag-like flower phenotype was also observed in transformants with a 35S::AG construct encoding a truncated AG protein lacking the C-terminal region in Arabidopsis, indicating that this truncated AG protein inhibited normal AG function; however, the transformants with a 35S::AG construct encoding a truncated AG protein lacking both the K domain and C-terminal region produced determinate flowers with more stamens and carpels, indicating that the truncated AG protein slightly reduced normal AG function [33]. These results provided strong evidence to suggest that both the K domain and the Cterminal region have important and distinct in vivo functions. In M. stellata, mastag 2 and mastag 3 proteins may work together to partially inhibit normal MastAG function, resulting in an increased numbers of tepals and normal reproductive organs. However, the AG orthologous gene from M. wufengensis, a closely related species with fewer tepals, has been identified but no abnormal alternatively spliced transcripts were isolated [34]. Previous studies suggested that AS events resulted in truncated protein isoforms lacking certain functional domains. However, most truncated transcriptional factors lacking certain functional modules may still perform part of their functions and hence may act as dominant-negative regulators [35]. In our study, the flowers phenotypes of MastAG overexpres-
sion in ag-1 and wild-type Arabidopsis provided strong evidence for the C-functional conservation of MastAG. However, the flowers phenotypes of mastag 2 and mastag 3 overexpression in ag-1 and wild-type backgrounds indicated that both the AS isoforms lost their ability to develop stamens and carpels, but may dominant negatively regulate Arabidopsis AG. All these results suggested that the key domain or subdomain deletion resulted in C-function loss of mastag 2 and mastag 3, whereas the expression of both the abnormal splicing transcripts could dominant-negatively regulate MastAG and affect flower phenotype. Acknowledgements We thank Robert McIntosh (Plant Breeding Institute, The University of Sydney, Australia) for critical reading of the manuscript. This research was supported by grants from the Natural Science Foundation of China (Grant No. 31170625 and No. 31370256). References [1] F. Birzele, G. Csaba, R. Zimmer, Alternative splicing and protein structure evolution, Nucleic Acids Res. 36 (2008) 550–558.
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Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae) Bo Zhang a,1 , Zhi-xiong Liu b,1 , Jiang Ma a , Yi Song c , Fa-ju Chen a,∗ a b c
Biotechnology Research Center, China Three Gorges University, Yichang City, Hubei 443002, PR China College of Horticulture and Gardening, Yangtze University, Jingzhou City, Hubei 434025, PR China College of Landscape Architecture, Beijing Forestry University, Beijing 100083, PR China
a r t i c l e
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Article history: Received 5 July 2015 Received in revised form 19 October 2015 Accepted 29 October 2015 Available online 2 November 2015 Keywords: AGAMOUS Magnolia stellata Alternative splicing Double flower Floral development
a b s t r a c t Magnolia stellata is a woody ornamental shrub with more petaloid tepals than related plants from family Magnoliaceae. Recent studies revealed that expression changes in an AGAMOUS (AG) orthologous gene could resulted in double flowers with increased numbers of petals. We isolated three transcripts encoding different isoforms of a single AG orthologous gene, MastAG, mastag 2 and mastag 3, from M. stellata. Sequence alignments and Southern blot analyses suggested that MastAG was a single-copy gene in M. stellata genomes, and that mastag 2 and mastag 3 were abnormally spliced isoforms of MastAG. An 144 bp exon skipping in MastAG results in the truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas an 165 bp exon skipping of MastAG produces a truncated mastag 3 protein lacking 6 aa of the K3 subdomain and the completely C terminal region. Expression analyses showed that three alternative splicing (AS) isoforms expressed only in developing stamens and carpels. Functional analyses revealed that MastAG could mimic the endogenous AG to specify carpel identity, but failed to regulate stamen development in an Arabidopsis ag-1 mutant. Moreover, the key domain or subdomain deletions represented by mastag 2 and mastag 3 resulted in loss of C-function. However, ectopic expression of mastag 2 in Arabidopsis produced flowers with sepals converted into carpeloid organs, but without petals and stamens, whereas ectopic expression of mastag 3 in Arabidopsis could mimic the flower phenotype of the ag mutant and produced double flowers with homeotic transformation of stamens into petals and carpels into another ag flower. Our results also suggest that mastag 3 holds some potential for biotechnical engineering to create multi-petal phenotypes in commercial ornamental cultivars. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alternative splicing (AS) refers to the assembly of exons in different ways during pre-mRNA splicing to produce multiple distinct transcript isoforms from a single gene. Alternative splicing is thought to be a major source of proteome complexity and functional diversity [1]. In plants AS is involved in many important biological processes including stress responses [2], development [3] and flowering [4,5]. In Arabidopsis, AS plays an important role in stage transition during floral development [6]. Members of the MADS-box transcription factor gene family in flowering plants are
∗ Corresponding author. E-mail addresses: [email protected] (B. Zhang), [email protected] (Z.-x. Liu), [email protected] (J. Ma), [email protected] (Y. Song), [email protected] (F.-j. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plantsci.2015.10.017 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
well-known for regulation of floral organ development, but found to represent families with low occurrences of AS [7]. AGAMOUS (AG) encodes a key C function MADS-box regulator of stamen and carpel identity as well as floral meristem determinacy [8]. According to the C function, AG is expression in the center of the floral meristem starting at stage 3 giving rise to the stamen and carpel primordia in Arabidopsis flowers, and exclusively in the third and fourth whorls until late in floral development [9,10]. AG is the only gene in Arabidopsis harboring a full C function activity; the ag mutant has completely loss of male and female organ identities and produces double flowers with homeotically transformed stamens to petals and a new ag flower arising in place of the gynoecium [11]. Previous studies suggested that changes in AG orthologous genes expression were involved in all cases of domesticated plants with double flowers [12]. However, AS of AG orthologous genes was previously observed in three ornamental plants with double flowers, including rose (Rosa rugosa) [13], the ranunculid Thalictrum thalictroides
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2.2. Isolation and identification of alternatively spliced isoforms of the MastAG from M. stellata
Fig. 1. Flower of M. stellata.
cultivar ‘Double White’ [14], and Japanese cherry blossom (Prunus lannesiana) cultivar ‘Albo-rosea’ [15]. Double flowers with the numerous extra petals were selected as ornamentals in many plant families during the early domestication and in subsequent breeding programs. Although the genetics of crop domestication processes controlling these events are becoming increasingly understood, the underlying molecular mechanisms of the showy floral phenotype that evolve during natural selection remains unclear. Magnolia stellata that produces double flowers belongs to family Magnoliaceae. As an early-diverging clade of ancestral angiosperms [16], it may provide hints in understanding the origin and early domestication of double flower. The perianth whorl of M. stellata consists of 12–45 petaloid tepals (Fig. 1). In the present work three AG-like mRNAs, two of which result from alternative splicing, were isolated from M. stellata. Detailed sequencing and expression analyses of MastAG and both the alternatively spliced isoforms, mastag 2 and mastag 3, were studied. Ectopic expression of all three AS isoforms under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter was examined for complementation of the Arabidopsis ag-1 mutants. The results provided strong evidence of high conservation of AG orthologs in core eudicot model plants and the woody shrub M. stellata, as well as novel information about the origin and subsequent evolution of double flower phonotypes in basal angiosperms.
Total RNA was extracted from floral buds using an EASYspin Plant RNA Extraction Kit (Aidlab China) following the manufacturer’s protocol. First-strand cDNA was synthesized from 1 g of the DNase I-treated RNA, using an oligo (dT) 15 adaptor primer and M-MLV Reverse Transcriptase (TaKaRa, Japan). MastAG, mastag 2 and mastag 3 cDNA sequences were isolated by PCR amplification using the forward primer MastAGF (5 -GGC GTC TAG AAG GAG AAG AAG CGA-3 ) and the reverse primer MastAGR (5 -A CCC GGG TCT TAT GAC CAT CTG CAT-3 ), respectively. Primers design was based on the sequence of AG orthologous genes from closely species, such as MawuAG (Genbank accession numbers: JN133287) from Magnolia wufengensis, and MaodAG (Genbank accession numbers: JQ326240) from M. odoratissima. PCR was performed with a 5 min 94 ◦ C denaturation step, followed by 30 cycles of 45 s at 94 ◦ C, 45 s annealing at 57 ◦ C, a 1 min extension at 72 ◦ C, and a final extension period of 10 min. Genomic DNA was extracted from juvenile leaves of M. stellata using a modified CTAB method according to the protocol suggested by Doyle and Doyle [17]. Partial genomic DNA sequences of MastAG containing missing sequences of mastag 2 and mastag 3 were isolated by PCR using primers successively: FLAGF1 (5 -TTGGTATATATTGGGCAGATTCAG-3 ) (1–24 nt. of the DNA) and FLAGR1 (5 -CTTCTTAGATCTGATTCTGCTAATGC-3 ) (941–966 nt.); then FLAGF2 (5 -GCGCTTTTACAGAATGCAAACG3 ) (772–793 nt.) and FLAGR2 (5 -CTCTTTCGTTCTCAGTTATCTG-3 ) (2594–2615 nt.); and FLAGF3 (5 -TGATAATATGTACCTCCGTG-3 ) (2063–2082 nt.) and FLAGR3 (5 -ACCCGGGTCTTATGACCATCTGCA3 ) (3894–3917 nt.). Genomic DNA from juvenile leaves of M. stellata was treated with RNase, purified, and then digested with Xba I, Hind III and Afl II (TaKaRa, Japan), respectively. 20 g of digested genomic DNA was loaded in each lane, separated in a 1% agarose gels, subsequently transferred to a HyBond-N+ nylon membrane (Amersham Biosciences, UK), and then fixed by UV-crosslinking (254 nm UV light for 2 min). A 205 bp sequence containing consensus exons and introns of MastAG, mastag 2 and mastag 3 present in the genomic DNA sequence of MastAG (2,462–2,666), was prepared for Southern blot probe by PCR with the forward primer MastAGSF (5 -GCA AAA TGA TAA TAT GTA CCT CCG TG-3 ) and the reverse primer MastAGSR (5 -ATG CCT GAA ATC TAC TGG TGG TG-3 ). This probe was labeled with digoxigenin by using DIG High Prime DNA Labeling and a Detection Starter Kit II (Roche, Mannheim, Germany) according to the manufacturer’s procedure. Prehybridization, hybridization, stringency washes and immunological detection were performed by DIG High Prime DNA Labeling and a Detection Starter Kit II (Roche, Mannheim, Germany) according to the manufacturer’s procedure. 2.3. Sequence alignments and phylogenetic analysis
2. Materials and methods 2.1. Plant material Flower buds at sequential developmental stages were collected from M. stellata growing under natural conditions in Beijing. Juvenile leaves, tepals, stamens and gynoecia were sampled during stamen and gynoecium differentiation well before anthesis, immediately frozen in liquid nitrogen and stored at −80 ◦ C until used. The Arabidopsis ag-1 mutant line (CS25) in ecotype Landsberg background was obtained from the Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH, USA.
Deduced amino acid sequences of MastAG were used in a BLAST search of the Genbank database. During the BLAST searches, multiple C class proteins from various angiosperm lineages were selected for alignment. Phylogenetic trees were constructed with MEGA5.0 software using the Neighbor-Joining Method [18,19]. Genbank accession numbers of the sequence data used were as follows: MastAG (AFH74392); Arabidopsis thaliana, AG (P17839), SHATTERPROOF1 (SHP1, P29381), SHP2 (P29385), SEEDSTICK (STK, Q38836), APETALA1 (AP1, P35631), FRUITFULL (FUL, Q38876), AGL79 (AEE77628), AP3 (P35632), PISTILLATA (PI, P48007), SEPALLATA1 (SEP1, P29382), SEP2 (P29384), SEP3 (O22456), SEP4 (NP 178466); Antirrhinum majus, FARINELLI (FAR, CAB42988), PLENA (PLE, AAB25101); M. wufengensis, MawuAG
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(AEO52692); Magnolia figo, Mafi (AFH74385); Magnolia grandiflora, MagrAG (AFH74382); Manglietia fordiana, Mafo (AFH74387); Prunus serrulata var. lannesiana, PrseSHP (ADG45819), PrseAG (ADK95058), PrseSTK (ADD91578); Camellia japonica, CajaAG (AIP87050); Petunia × hybrid, FBP6 (CAA48635), FBP3 (CAA50549), FBP7 (CAA57311), FBP11 (CAA57445); Solanum lycopersicum, TAGL1 (NP 001234187), TAG1 (Q40168), TAGL11 (AAM33102); Medicago truncatula, MtAGa (AGV55405), MtAGb (AGV55406); Nicotiana benthamiana, NbAG (AFK13159), NbSHP (AFK13160); Meliosma dilleniifolia, MediAG (AAS45686); Euptelea pleiosperma, EuplAG (ADC79699); Saruma henryi, SaheAG (AAS45689); Lilium longiflorum, LMADS10 (AIJ29174); Oncidium Gower Ramsey, OMADS4 (AIJ29176); and Oryza sativa, OsMADS3 (Q40704), OsMADS58 (Q2V0P1). Full-length amino acid sequences containing the MADS, I, K and C domains were aligned with BioEdit 7.0.9 software using the ClustalW program with default settings [20]. Several AG orthologous and mutant proteins from different angiosperm lineages were selected for alignment. Sequence data used were as follows: P. serrulata var. lannesiana, PrseAG (ADK95058), prseag1 (ADK95059) [15]; Thalictrum thalictroides, ThtAG1 (AFI23876), ThtAG18 [14]; and A. thaliana, AG (P17839), ag-4 [21]. 2.4. Expression analysis of alternatively spliced isoforms of the MastAG For semi-quantitative RT-PCR analysis, we used 1 g of total RNA extracted from juvenile leaves, tepals, stamens and carpels of M. stellata in order to synthesize the first-strand cDNA with an oligo (dT) 15 primer as described above. A 2 l cDNA sample from the RT reaction was used for 25 cycles of PCR as follows: 20 s at 94 ◦ C, 20 s at 57 ◦ C and 20 s at 72 ◦ C, preceded by 5 min at 94 ◦ C and followed by 10 min at 72 ◦ C. We analyzed 10 l of total PCR product (25 l) in each reaction by electrophoresis in a 1% agarose gel and photography under UV light. RT-PCR was carried out using the genespecific primers qMastAGF (5 -CGAATACGCCAATAACAGGCATTTG3 ) and qMastAGR (5 -ACATCGTATTCAGGAGCAGGCAAC-3 ) for MastAG; qmastag 2F (5 -GATGCGTATCGGAAGCCAATTCTC-3 ) and qmastag 2R (5 -CCACCACATTACCTTAGCACGGAG-3 ) for mastag 2; and qmastag 2F and qMastAGR for mastag 3. Amplification of M. stellata actin with specific primers qMastactinF (5 CAAGAGCTTGAGACAGCAAAGAGTAG-3 ) and qMastactinR (5 AGATAGACCCTCCAATCCAGACACT-3 ) was used as a positive control. For quantitative analysis, total RNA was isolated from flower buds at various developmental stages and treated as described above. Quantitative real-time RT-PCR with three biological replicates was carried out using an ABI7500 Real-time PCR System. The reaction mixture was cycled as follows: 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s, 60 ◦ C for 35 s, followed by 30 s at 95 ◦ C and 1 min at 60 ◦ C. For the melt curve, we changed the temperature by increments of 0.2 ◦ C/s to 95 ◦ C. Experiments were repeated three times for each sample. Gene-specific primers and the normalization control primers were the same as the primers used for semi-quantitative RT-PCR. 2.5. Vector construction and Arabidopsis transformation Full-length cDNA of MastAG, mastag 2, and mastag 3 were cloned into binary vector pBI121 (BD Biosciences, Clontech) using Xba I and Sma I restriction enzymes under control of the cauliflower mosaic virus 35S promoter in the sense orientation. The 35S::MastAG, 35S::mastag 2, and 35S::mastag 3 constructs were transformed into heterozygous AG/ag-1 Arabidopsis plants (ecotype Landsberg) using the floral-dip method according to the protocol suggested by Clough and Bent [22] via Agrobac-
Fig. 2. Southern blot analyses of MastAG in M. stellata. The genomic DNA from M. stellata was digested with Afl II, Hind III and Xba I, respectively. The MastAG-specific probe was a 205 bp sequence containing a consensus exon and intron of MastAG, mastag 2 and mastag 3 present in the genomic DNA sequence of MastAG.
terium tumefaciens strain GV3101-90, respectively. Plasmid pBI121 (negative control) was also transformed into heterozygous AG/ag1 A. thaliana by the same method described above. Seeds of transgenic Arabidopsis plants were selected on solid 0.5 × MS medium [23] containing 50 g/ml kanamycin at 4 ◦ C for 2 days and then transferred to a greenhouse under long-day conditions (16 h light/8 h darkness) at 22 ◦ C for 10 days. The seedlings were subsequently transplanted into soil. Transgenic A. thaliana lines were confirmed by PCR and quantitative real-time RTPCR with transgene-specific primers. Wild-type, heterozygous AG/ag-1 and homozygous ag-1 transformants were isolated using a dCAPS marker designed with the dCAPS finder program with dCAPS primer (5 -GATATATTAATATATGTTGATAAATCACTTA3 ) and reverse primer (5 -AATCAACTTCCTGCTTAATCGGT-3 ) [24]. PCR was performed with 30 cycles of 30 s at 94 ◦ C, 30 s annealing at 53 ◦ C, a 30 s extension at 72 ◦ C, preceded by a 5 min 94 ◦ C denaturation step and a final extension period of 10 min. The 20 l of the total PCR product (25 l) in each reaction was digested with Afl II (TaKaRa, Japan) for 2.5 h and then separated on a 4% agarose gels, stained, and visualized under UV light. The ag-1 fragment was cleaved by Afl II to reduce the 355 bp fragment to 327 bp. Wild-type and homozygous mutant plants containing the transgenes were analyzed after genotyping. For quantitative real-time RT-PCR analysis, triplicate quantitative assays were performed as described above. Amplification of the A. thaliana -actin with the primers qactinF (5 -GATTTGGCATCACACTTTCTACAATG-3 ) and qactinR (5 GTTCCACCACTGAGCACAATG-3 ) was used as an internal control to normalize all data. The cycle parameters were set at 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s, 60 ◦ C for 35 s, followed by 30 s at 95 ◦ C and 1 min at 60 ◦ C. For the melt curve, we changed the temperature by increments of 0.2 ◦ C/s to 95 ◦ C. 3. Results 3.1. Cloning and sequence alignments of MastAG, mastag 2, and mastag 3 from M. stellata Full-length cDNAs of MastAG, mastag 2, and mastag 3 from M. stellata were obtained by homology-based cloning following the
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Fig. 3. Phylogenetic analysis of C-class MADS-box proteins. The tree was constructed with MEGA5.0 software using the neighbor-joining method. Numbers along the branches are bootstrap values from 1000 replications.
above procedures. The MastAG transcript (Genbank accession number: JQ326243) contained a 672 bp ORF encoding 223 amino acids (aa), whereas the mastag 2 transcript (Genbank accession number: KR017756) contained a 528 bp ORF encoding 175 aa, and the mastag 3 transcript (Genbank accession number: KR017757) con-
tained a 552 bp ORF encoding 183 aa. The genomic sequence of the MastAG (Genbank accession number: KR017758) showed that all three transcripts were derived from a consensus pre-mRNA. A 144 bp exon skipping in the genomic sequence of MastAG produced the mastag 2, and the complete omission of another exon (165 bp)
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Fig. 4. Sequence alignments of MastAG, mastag 2, and mastag 3 with other AG-like MADS domain proteins. The first underlined region represents the MADS domain and the second the K domain. The AG motifs I and II in the C-terminal region are boxed. Amino acid residues identical to MastAG are indicated as dots. Dashes were introduced into the sequence to improve the alignment. The k domain contains three heptad (abcdefg)n repeats represented by k1, k2 and k3 subdomains that usually contain hydrophobic amino acids at positions a and d [25].
produced the mastag 3. That is, mastag 2 and mastag 3 were alternatively spliced isoforms of MastAG. Southern blot analysis with gene-specific probes of MastAG (a 205 bp sequence containing a consensus exon and introns of the three alternative splicing isoforms) revealed that MastAG was a single-copy gene in the M. stellata genomes (Fig. 2). Phylogenetic analyses grouped the MastAG (M. stellata AGAMOUS) into the basal eudicots AG lineage (Fig. 3). Moreover, sequence alignments revealed that the MastAG protein contains a 57 aa MADS domain, a 32 aa I domain (58–89), an 89 aa K domain (90–178) and a 45 aa C domain (179–223) (Fig. 4) [25,26]. Three putative amphipathic ␣-helices with expected hydrophobic amino acids at the a and d positions in heptad (abcdefg) n repeats were identified in the K-domain, namely subdomains K1 (90–111), K2 (124–138) and K3 (146–178) [25]; and the C terminal region harbors highly conserved AG motifs I and II [26]. However, the alternative splicing resulted in a truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas the
truncated mastag 3 contained deletions of 6 aa of the K3 subdomain and completely loss of the C terminal region (Fig. 4).
3.2. Expression analyses of MastAG, mastag 2, and mastag 3 In M. stellata, MastAG, mastag 2, and mastag 3 were exclusively transcribed in developing stamens and carpels, and not in juvenile leaves and tepals (Fig. 5). Moreover, all three transcripts were produced during all stages of stamen and carpel differentiation (Fig. 6). MastAG expression was clearly higher than expression of the other two AS transcripts at corresponding stages during reproductive organ development, and mastag 3 expression was higher than that of mastag 2. Furthermore, the expression levels of all three transcripts increased rapidly as the stamens and carpels increased in size. Their expression levels were highest until the anthers and carpels reached maturity.
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Fig. 5. Expression of MastAG, mastag 2, and mastag 3 in the juvenile leaves (le), tepals (te), stamens (st) and carpels (ca) by semi-quantitative RT-PCR with ACTIN as the control.
3.3. Ectopic expression of MastAG, mastag 2, and mastag 3 in ag-1 Arabidopsis In order to gain further insight into the functional divergence of three alternative splicing isoforms, we attempted to rescue the loss–of–function Arabidopsis ag mutant (ag-1) by MastAG, mastag 2, and mastag 3, respectively. Binary vectors carrying 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 were separately introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were confirmed by quantitative real time RT-PCR (Fig. 7) and mutant alleles were also identified by dCAPS genotyping (Fig. 8). Phenotypes of transgenic lines were assayed in a wild-type and homozygous ag-1 mutant backgrounds to determine whether MastAG, mastag 2, and mastag 3 compensated for the absence of the endogenous AG gene in Arabidopsis, respectively. Among a total of 7 35S::MastAG homozygous ag-1 transformants, three (42.86%) lines displayed strong complementation phenotypes producing flowers consisting of 4 sepals in the first whorl, 8 petals in the second and one fat gynoecium in the third (Fig. 9A-c); and two (28.57%) lines displayed medium rescue flower phenotypes with a terminator of unlimited number of perianth by a central fat gynoecium (Fig. 9A-d); and 2 (28.57%) lines displayed no change in flower structure. However, ectopic expression of MastAG rescued carpel development without complementation of stamen development in the Arabidopsis ag-1 mutant background. In other words, MastAG from M. stellata partially or fully compensated for
the endogenous AG gene of A. thaliana that specified carpel identity, but failed to rescue stamen development. Moreover, Among a total of 27 35S::MastAG transformants in wild-type background, nine (33.33%) lines displayed obvious changes in flower structure with the first whorl sepals converted into carpeloid organs (Fig. 9B-c), ten (37.04%) lines displayed weaker changes in flower structure with smaller petals and shorter anther filaments (Fig. 9Bd), and eight (29.63%) lines displayed no change in flower structure. However, all 12 35S::mastag 2 transformants in homozygous ag1 mutant background displayed no change of flower structure (Fig. 9A-e), whereas among a total of 23 35S::mastag 2 transformants in wild-type background, 15 (65.22%) lines produced flowers with carpeloid sepals bearing ovules, but without petals and stamens (Fig. 9B-e), 8 (34.78%) lines displayed no change in flower structure. All 24 35S::mastag 3 transformants in homozygous ag1 mutant background displayed no change in flower structure (Fig. 9A-f), whereas among a total of 11 35S::mastag 3 transformants in wild-type background, 5 (45.45%) lines displayed obvious changes in flower structure that transformed stamens into petals and carpels into additional ag flowers (Fig. 9B-f), and six (54.55%) lines displayed no change in flower structure. 4. Discussion Double-flower varieties have been grown as ornamental and garden plants for their showy traits since ancient times. However, the molecular mechanisms regulating development of double flowers are not well understood although they are of significant commercial interest. In Arabidopsis, the C-function gene AGAMOUS plays a key role in specifying stamen and carpel identities; AG loss-of-function results in homeotic transformation of stamens into petals and the gynoecium into another ag flower, which leads to double flowers [27]. Increasing numbers of studies suggested that changes in AG orthologous genes expression lead to double flowers in non-model plants [12]. For example, restriction of the RhAG expression domain in the center of the flower increased the number of petals in the double-flower roses [28]. Similar results in cultivated Camellia japonica indicated that contracted and expanded expression of AG orthologous gene CjAG were associated with formation of different types of double flowers [29]. CpAG1 and CpAG2 are AG orthologous genes from Cyclamen persicum. Lack of expression of CpAG1 converted stamens into petals and resulted in a flower with an increase of 5 petals, whereas simultaneous lack of expres-
Fig. 6. Relative expression of MastAG, mastag 2, and mastag 3 in stamens and carpels of M. stellata at various developmental stages. (A) Quantitative real time RT-PCR analyses of expression of three AS transcripts in stamens of M. stellata at different developmental stages. MastAG expression in stamens of 7.1 mm flower buds was used for normalization; (B) quantitative real time RT-PCR analyses of expression of three AS transcripts in carpels of M. stellata at different developmental stages. mastag 3 expression in carpels of 7.1 mm flower buds was used for normalization.
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Fig. 7. The 35S::MastAG, 35S::mastag 2, and 35S::mastag 3 transgenic Arabidopsis in a wild-type and a homozygous ag-1 mutant background confirmed by quantitative real-time RT-PCR (qRT-PCR). (A) Endogenous ag expressed in the homozygous ag-1 mutant Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in a homozygous ag-1 mutant background. ag expression in the homozygous ag-1 mutant Arabidopsis (ho) was used for normalization; (B) endogenous AG expressed in wild-type Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in wild-type background. AG expression in wild-type Arabidopsis (WT) was used for normalization; (C) MastAG, mastag 2 and mastag 3 expressed in homozygous ag-1 mutant Arabidopsis and corresponding transgenic Arabidopsis in a homozygous ag-1 mutant background, respectively. mastag 2 expression in line ho-2, mastag 3 expression in line ho-4, and MastAG expression in line ho-5 of transgenic Arabidopsis were used for normalization; (D) MastAG, mastag 2 and mastag 3 expressed in wild-type Arabidopsis and corresponding transgenic Arabidopsis in wild-type background, respectively. mastag 2 expression in line W-1, mastag 3 expression in line W-6, and MastAG expression in line W-7 of transgenic Arabidopsis are used for normalization, respectively.
Fig. 8. Genotyping of wild-type, heterozygote and homozygous ag-1 mutant A. thaliana (Landsberg erecta) plants by dCAPS. The amplicon of the homozygous ag1mutant Arabidopsis plants was cleaved by Afl II to produce a 327 bp fragment; the amplified fragment from an heterozygote ag-1 Arabidopsis plant was cleaved by Afl II to produce a 355 bp and a 327 bp fragments; the wild-type amplicon was cleaved by Afl II to produce a fragment of 355 bp fragment.
sion of CpAG1 and CpAG2 induced rose-like flowers with multiple petals [30]. On the other hand, alternative splicing of AG orthologous gene resulted in mutant proteins with key domain deletions that enable production of double flowers. For example, exon skipping in the AG orthologous gene PrseAG from P. lannesiana resulted in a mutant prseag-1 protein with the C-terminal AG motifs I and II deletions and production of double flowers, in which stamens were converted into petals or petaloid anthers, and carpels changed into leaf-like organs [15]. Alternative splicing of AG orthologous
gene ThtAG1 from T. thalictroides resulted in mutant proteins with K-domain deletions, and production of double flower in ‘Double White’ cultivar [14]. In M. stellata, three alternative splicing transcripts of AG orthologue were simultaneously expressed in stamens and carpels. Ectopic expression of MastAG rescued carpel development without complementation of stamen development in the Arabidopsis ag-1 mutant (Fig. 9A-c), and converted the first whorl sepals of wildtype Arabidopsis into carpeloid organs bearing ovules (Fig. 9B-c). These results suggested that the functional conservation between AG and MastAG in specifying carpel identity and repression of Aclass gene expression. However, a 144 bp exon skipping in MastAG resulted in a truncated mastag 2 protein lacking the completely I domain and 18 aa of the K1 subdomain. In addition, a 165 bp exon skipping in MastAG produced a truncated mastag 3 protein with a 6 aa deficiency of the K3 subdomain and completely C terminal deletion. Previous studies revealed that the I domain in MADS-box proteins was responsible for dimerization and protein functional specificity, and the K-domain was essential for protein–protein interactions, and the C-terminal domain was involved in both transcriptional activation and the formation of higher order MADS protein complexes [25,31,32]. These domain omissions or amino acid truncations in key subdomain resulted in the C-function loss or changes in AG orthologous proteins [14,15,21]. In Arabidopsis, the ag-4 mutation with partial loss of the C terminus of the K domain resulted in flowers with normal stamens, but with carpels converted into four sepals [21]. In our study, ectopic expression of mastag 2 and mastag 3 failed to rescued stamen and carpel development in the Arabidopsis ag-1 mutant, respectively (Fig. 9A-e,f). However, 35S::mastag 2 transgenic Arabidopsis in wild-type background produced an interesting flower phenotype with carpeloid sepals bearing ovules, but without petals and stamens (Fig. 9B-e). In addition, 35S::mastag 3 transgenic Arabidopsis in wild-type background produced an interesting flower phenotype with homeotic transformation of stamens into petals and carpels into another
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Fig. 9. Comparison of phenotypes of the wild-type, homozygous ag-1 mutant, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in a homozygous ag1 mutant and wild-type background, respectively. A: Phenotypic comparison of the homozygous ag-1 mutant, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in the homozygous ag-1 mutant background. Flowers of: (A-a) homozygous ag-1 mutant A. thaliana; (A-b) transgenic ag-1 mutant Arabidopsis transformed with the pBI121 vector only (negative control); (A-c) 35S::MastAG transgenic lines with strong complementation phenotypes of ag Arabidopsis, showing 4 sepals in the first whorl, 8 petals in the second and one fat gynoecium in the third; (A-d) 35S::MastAG transgenic lines with medium rescue phenotype, showing a terminator of unlimited number of perianth by a central fat gynoecium; (A-e) 35S::mastag 2 transgenic ag Arabidopsis; (A-f) 35S::mastag 3 transgenic ag Arabidopsis. B: Phenotypic comparison of the wild-type Arabidopsis, 35S::MastAG, 35S::mastag 2 and 35S::mastag 3 transgenic Arabidopsis in wild-type background. Flower of: (B-a) wild-type Arabidopsis; (B-b) transgenic Arabidopsis transformed with the pBI121 vector only (negative control); (B-c) 35S::MastAG transgenic Arabidopsis showing strong phenotype with the first whorl sepals converted into carpeloid organs; (B-d) 35S::MastAG transgenic Arabidopsis showing weaker phenotype with smaller petals and shorter filament; (B-e) 35S::mastag 2 transgenic Arabidopsis showing strong phenotype with carpeloid sepals, but without petal and stamen; (B-f) 35S::mastag 3 transgenic Arabidopsis showing strong phenotype with transformation of stamens into petals and carpels into another ag flower. sep, sepal; pet, petal; sta, stamen; sti, stigma; ov, ovule. Bars: 1000 m.
ag-like mutant flower (Fig. 9B-f). A similar ag-like flower phenotype was also observed in transformants with a 35S::AG construct encoding a truncated AG protein lacking the C-terminal region in Arabidopsis, indicating that this truncated AG protein inhibited normal AG function; however, the transformants with a 35S::AG construct encoding a truncated AG protein lacking both the K domain and C-terminal region produced determinate flowers with more stamens and carpels, indicating that the truncated AG protein slightly reduced normal AG function [33]. These results provided strong evidence to suggest that both the K domain and the Cterminal region have important and distinct in vivo functions. In M. stellata, mastag 2 and mastag 3 proteins may work together to partially inhibit normal MastAG function, resulting in an increased numbers of tepals and normal reproductive organs. However, the AG orthologous gene from M. wufengensis, a closely related species with fewer tepals, has been identified but no abnormal alternatively spliced transcripts were isolated [34]. Previous studies suggested that AS events resulted in truncated protein isoforms lacking certain functional domains. However, most truncated transcriptional factors lacking certain functional modules may still perform part of their functions and hence may act as dominant-negative regulators [35]. In our study, the flowers phenotypes of MastAG overexpres-
sion in ag-1 and wild-type Arabidopsis provided strong evidence for the C-functional conservation of MastAG. However, the flowers phenotypes of mastag 2 and mastag 3 overexpression in ag-1 and wild-type backgrounds indicated that both the AS isoforms lost their ability to develop stamens and carpels, but may dominant negatively regulate Arabidopsis AG. All these results suggested that the key domain or subdomain deletion resulted in C-function loss of mastag 2 and mastag 3, whereas the expression of both the abnormal splicing transcripts could dominant-negatively regulate MastAG and affect flower phenotype. Acknowledgements We thank Robert McIntosh (Plant Breeding Institute, The University of Sydney, Australia) for critical reading of the manuscript. This research was supported by grants from the Natural Science Foundation of China (Grant No. 31170625 and No. 31370256). References [1] F. Birzele, G. Csaba, R. Zimmer, Alternative splicing and protein structure evolution, Nucleic Acids Res. 36 (2008) 550–558.
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