Plant Mol Biol Rep (2011) 29:473–480 DOI 10.1007/s11105-010-0248-3

Isolation and Characterization of an AGAMOUS Homologue PmAG from the Japanese Apricot (Prunus mume Sieb. et Zucc.) Ji-Hua Hou & Zhi-Hong Gao & Zhen Zhang & Su-Mei Chen & Toshi Ando & Ji-Yu Zhang & Xin-Wei Wang

Published online: 14 September 2010 # Springer-Verlag 2010

Abstract An AGAMOUS homologue, named PmAG (GenBank accession number EU068730), was isolated from the Japanese apricot cultivar ‘Shirokaga’ (Prunus mume) via the homology cloning method. The cDNA was 812-base pairs long with an open reading frame of 732 base pairs, and encoded for a putative protein of 243 amino acid residues. Sequence alignment showed that the sequence of PmAG was 98% identical to that of the peach (Prunus persica). Genomic DNA analysis revealed an intron just before the stop codon of the ORF, and PmAG had AG motifs I and II in its C-terminal region, which suggested that PmAG was an AGAMOUS homologue. Real-time quantitative reverse transcription polymerase chain reaction and in situ hybridization showed that the PmAG mRNA was highly expressed in the sepals, carpel and stamens, and a weak signal was detected in the seed and nutlet. No expression was detected in the leaves or petals. Overexpression of PmAG in transgenic tobacco exhibited no effect on the phenotype of the flower organs, but changes in the floral color from pink to white. Keywords MADS-box gene . Japanese apricot . AGAMOUS . Expression

J.-H. Hou : Z.-H. Gao (*) : Z. Zhang : S.-M. Chen : J.-Y. Zhang : X.-W. Wang College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] T. Ando College of Horticulture, Chiba University, Matusdo 271-8510, Japan

Abbreviation qRT-PCR Quantitative reverse transcription polymerase chain reaction AG AGAMOUS gene Pm Prunus mume

Introduction The Japanese apricot (Prunus mume Sieb. et Zucc.) originated from China and is an important commercial woody plant owing to its flavorsome fruits and charming flowers. There are more than 200 cultivars in China (Chu 1999). Two types of flowers, namely perfect and imperfect flowers, occur in the Japanese apricot. The imperfect flowers are characterized by either pistils below the stamens, withered pistils or the absence of pistils, and hence fail to bear fruits. The percentage of imperfect flowers depends on the cultivar, the highest being 75.15% and the average being 35% (Gao et al. 2006). As a consequence, the yield of Japanese apricot may be greatly affected. We hypothesize that floral organogenesis controlling genes are involved in the differentiation of imperfect flowers. Studies have shown that the key gene that controls the development of the pistil is the AGAMOUS gene (AG) in Arabidopsis. This gene belongs to the C group of genes in the ABCDE model (Yanofsky et al. 1990; Theissen and Saedler 2001), and is also a key gene in flower development (Ito et al. 2007). To date, AG homologues have been cloned and their function and structure have been studied in more than 20 species (Zahn et al. 2006; Liu et al. 2010). AG homologues have been isolated from fruit trees such as apple (van der Linden et al. 2002), grapevine (Boss et al. 2001), and peach (Wu et al. 2004; Martin et al. 2006). To

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our knowledge, there are no reports of AG homologues in the Japanese apricot. In the present research, the AG homologue was isolated from the Japanese apricot via the homology cloning method and its expression pattern in different organs was also investigated.

Materials and Methods Plant Materials The Japanese apricot cultivar ‘Shirokaga’ (P. mume) was collected from the campus farm, Chiba University, Japan and the national germplasm nursery of the Japanese apricot, located at Nanjing Agricultural University, Nanjing, China. Total RNA Isolation Pistils were detached from the flower buds and ground into a fine powder in liquid nitrogen. Total RNA was extracted from the pistils by using the TRIzol® kit (Invitrogen™, Japan). The concentration of isolated RNA was determined from the absorbance at 260 nm with a Biophotometer (Eppendorf, Hamburg, Germany), purity was evaluated by the optical density (OD) absorption ratio OD260 nm/OD280 nm and integrity was estimated by electrophoresis on ethidium bromide-stained 1.0% agarose gels in 1× TAE buffer followed by visualization under UV light. Cloning of AGAMOUS Homologue in Japanese Apricot The first strand of cDNA synthesis was carried out with a 3′Full Race Core Set kit (Takara, Japan) according to the manufacturer's protocol. The second round PCR was conducted using primers AG1f and AG1r (Table 1) derived from the cDNA sequence of MADS4 in peach (AY705972). The cycling program for PCR consisted of an initial denaturation at 94°C for 4 min, 35 cycles at 94°C for 30 s, 52°C for 1 min, and 72°C for 2 min and a final extension at 72°C for 10 min. The amplified products were cloned into the pGEMT vector (Promega, USA) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer's protocol and analyzed using the ABI Prism 3700 DNA Analyzer (ABI 3730, USA). This gene, later named PmAG, was also independently cloned twice. Amino acid alignment protein sequences of AG homologues were downloaded from GenBank and the alignment of the amino acid sequences with that of PmAG was performed using PAUP 4b BETA 10 (Swofford 2003). A comparative phylogenetic tree was then constructed using

Plant Mol Biol Rep (2011) 29:473–480 Table 1 Primers used in this study Name

Primers sequences (5′-3′)

AG-1f AG-1r AG-2f AG-2r EF-1af EF-1ar tan-ag-f tan-ag-r

ATG GCC TAT GAA AAC AAA TCC A CCC GTA GCT ATC ATC ATC TGG T GAG TAT GCC AAC AAC AGT G CTC TCC AGA TTC TTC AGG TC CCC TCC GAC TAC CAC TTC AG GCA TCT CAA CAG ACT TAA CTT CAG GGA ATT CAC AAC AAC CAG CTC CTA C AAC TGC AGG TCT TTA CAT ACC CAC CAT

SP6 T7

CAT ACG ATT TAG GTG ACA CTA TAG TAA TAC GAC TCA CTA TAG GG

MEGA software version 4.0 by the p distance and neighbor-joining methods with 1,000 replication bootstrap tests (Tamura et al. 2007). The tree was then visualized using the Tree View package (Page 1996). Maize MADSbox ZAG1 was used as an outgroup. Quantitative Real-time PCR The tissue-specific and developmental expression patterns of PmAG were investigated by qRT-PCR. Total RNA was isolated from different organs including sepals, petals, stamens, carpel, seed, flesh, and leaves. Contaminating genomic DNA was removed with RNase-free DNase I (TaKaRa, Japan). The first strand of cDNA was synthesized by using 5 μg total RNA as the template with a cDNA synthesis kit ReverTraAce-α-™ (ToYoBo, Japan) in a 20μl reaction volume according to the manufacturer's instructions. The forward primer AG-2f and the reverse primer AG-2r (Table 1) designed from the cDNA of PmAG were used to amplify a 220-bp fragment. The EF-1a gene was used as a positive internal control with primers EF-1af and EF-1ar (Table 1), producing a 150-bp fragment. The specificity of the primers to the genes was tested by using a melting curve analysis of the PCR reaction and sequence analysis of the amplified PCR product. Real-time PCR amplification was carried out on a Rotor-Gene 3000 (Corbett Research, Australia) with a 20 μL reaction solution, containing 2 μL of 20-fold-diluted cDNA, 0.2 μL 10 pM of each primer, 10 μL SYBR Green Real time PCR Master Mix (×2; ToYoBo Japan) and 7.6 μL sterile double-distilled water. The following conditions were used: 30 min at 50°C for RT followed by denaturation at 94°C for 4 min and then 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 2 min followed by a final extension at 72°C for 10 min. Amplification was performed starting with initial denaturation for 1 min at 95°C and then 40 cycles of 95°C for

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20 s, 60°C for 20 s, and 72°C for 20 s. A melting temperature from 57°C to 95°C was used for each primer pair to verify the specificity of the RT-PCR reaction and the absence of primer dimers. Data were analyzed using the Rotor-Gene 6 Calculation Software (Corbett Research, Australia) for the relative expression compared to EF-1a in real-time PCR. Values for ΔΔCt were obtained from the software. Relative gene expression values were calculated using the log2-ΔΔCt method of Ramakers et al. (2003). Quantitative real-time RTPCR was conducted twice.

confirmed by PCR with T7 and SP6 primers and sequencing was conducted as described previously. The plasmid with the vector of pCAMBIA-S1300+ was finally transferred into Agrobacterium tumefaciens DH105 by freezing and thawing. The resulting plasmids were introduced into tobacco by Agrobacterium-mediated transformation with leaf discs as described (Wang et al. 2008) with slight modification. The leaf discs were incubated DH105 solution for 15 min and cultured in MS medium containing 250 mgl−1 carbenicillin and 10 mgl−1 hygromycin.

In situ Hybridization

PCR, Reverse Transcriptase PCR, and Phenotypic Analysis of PmAG Expression in Transgenic Plants

Floral buds (diameter: 2 mm) were collected from trees and fixed in a buffer containing 3.7% paraformaldehyde, 0.25% glutaraldehyde, and 50 mM sodium phosphate (pH 7.2) at 4°C for 24 h. They were then dehydrated through a graded series of ethanol and tert-butyl alcohol, embedded in Paraplast Plus (Sigma,USA) and sectioned to 8-μm thick with a rotary microtome and placed on aminopropylsilane-coated glass slides. Finally, the samples were baked at 42°C for 24 h on a slide warmer. The paraplast was removed from tissue sections with xylene and then rehydrated through an ethanol series, and then treated with proteinase K (5 μgml−1, Roche, Swiss) in Tris-HCl, pH 7.5 at 37°C for 30 min and prehybridized at room temperature for 2 h with a solution containing 50% formamide, ×5 SSC, ×5 Denhardt's solution, 100 μgml−1 tRNA and 500 μgml−1 herring sperm DNA. The labeled RNA probe was a fragment that corresponded to the 3′ terminal region of the PmAG cDNA between 522 and 814 bp. The fragment was amplified with the primer pair tanag-l and tan-ag-r (Table 1) and subcloned into pGEM-T vector. SP6 and T7 RNA polymerases were used to synthesize antisense and sense probes and labeled using the PCR DIG Probe Synthesis Kit (Roche, Swiss). Hybridization was performed at 42°C overnight with 0.5 μgml−1 of the labeled RNA probe in hybridization solution. After hybridization, slides were washed four times in ×4 SSC at 50°C for 10 min and the immunological signal was detected according to the Northern blotting kit protocol (Roche, Swiss). Photomicrography was performed using an Olympus Vanox-s microscope (Olympus, Tokyo, Japan). In situ hybridization was performed twice. Vector Construction and Tobacco Transformation The coding sequence of PmAG cDNA was fused between the 35S promoter and terminator in binary vector of pCAMBIAS1300+ to create the 35S: PmAG plasmid, then transferred into Escherichia coli. The E. coli cells were then cultured on Luria-Bertani media with 50 mgl−1 ampicilin. The insert was

PCR and RT-PCR were performed to confirm PmAG gene insertion in transgenic tobacco plants. Genomic DNA and RNA were isolated from the independent transformants by the modified CTAB method (Lassner et al. 1989) and the TRIzol® kit (Invitrogen™), respectively. The concentration of isolated DNA and RNA were determined as described previously. A primer pair of AG-PCRf and AG-PCRr was designed to target the sequence of PmAG cDNA as presented in Table 1. Using tobacco genomic DNA as template, PCR was performed with a pre-denaturing step at 94°C for 5 min, 35 cycles of denaturing at 94°C for 40 s, annealing at 65°C for 35 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. For RT-PCR analysis, the total RNA was reverse transcribed into cDNA using the ReverTrAce-α-™ kit (ToYoBo). The PCR cycling conditions consisted of 94°C for 4 min, 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by extension at 72°C for an additional 10 min. Positive transgenic plants were screened by PCR and RT-PCR, were grown in the greenhouse at 25°C under long-day conditions. The phenotypes of the flowers were subsequently observed.

Results cDNA Cloning of AGAMOUS Homologue from Japanese Apricot The nucleotide sequence of 812 bp was isolated by using primers based on the cDNA sequence of MADS4 from peach (AY705972). This sequence consisted of a 732-bp open reading frame and coded for a protein of 243 amino acids named PmAG (EU068730). The basic local alignment search tool (BLAST) was searched using the nucleotide sequence and deduced amino acid sequence, and revealed that this gene had high sequence similarity to GenBank

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peach (98% nucleotide identity and 100% amino acid identity; AY705972; Wu et al. 2004), followed by apple (AJ251118, 88% nucleotide identity) and Arabidopsis (AJ251118, 88% nucleotide identity). This suggested that PmAG is an AGAMOUS homologue in Japanese apricot. Sequence Analysis of PmAG The alignment and phylogenetic analysis of the deduced amino acid sequences with that of PmAG showed that the AG homologues isolated from Japanese apricot and peach were clustered in one group, which was clustered in a new group with PsAGAMOUS isolated from blackberry (Prunus serotina), whereas it had a longer distance from AGAMOUS from Arabidopsis. Monocotyledon maize (ZAG1, AAA02933) and lily (LLAG1, AAR98731) are two groups different from the dicotyledon plants including Japanese apricot (PmAG, ABU41518), peach (MADS4, AAU29531), apple (MdMADS15, CAC80858), rose (MASAKOC1, BAA90744 and RAG, AAD00025), hazel (MADS1, AAD03486), cucumber (CUM1, AAC08528), tomato (TAG1, Q40168), tobacco (NAG1, AAA17033), blackberry (PsAG, EU938540.1), and Arabidopsis (AGAMOUS, P17839; Fig. 1). The sequence of the MADS-domain of PmAG indicated high conservation and high similarity in region I and K. Moreover, PmAG bore the AGAMOUS motifs I and II, which are typical in AGAMOUS-like genes (Kramer et al. 2004; Zahn et al. 2006). Expression Patterns of PmAG Revealed by Quantitative Real Time RT-PCR and In situ Hybridization To gain further insight into the expression pattern of this gene, qRT-PCR experiments were conducted. PmAG was expressed specifically in the stamens and carpel but it was not

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detected in the petals or leaves. PmAG expression was also detected in the fruitlet and seed, but only weakly. Interestingly, PmAG was also highly expressed in sepals (Fig. 2). The results showed that PmAG is mainly expressed in reproductive organs and not in vegetative organs. This is consistent with the predicted functions of some genes homologueous to PmAG. Figure 2 shows that the PmAG transcripts were present not only in the carpel, stamens, and sepals of perfect flowers but also in the stamens and sepals of imperfect flowers without the carpel, which suggests that the expression pattern of PmAG mRNA is similar in perfect and imperfect flowers during the late stage of flower development. To confirm the expression pattern of PmAG mRNA in the different flower organs, in situ hybridization was performed. This result showed that PmAG transcripts were detected in the stamens and sepals in the late stage of flower development, but not in the petals (Fig. 3). Overexpression of PmAG in Transgenic Tobacco To investigate precise function of PmAG, we constructed the PmAG overexpression transgenic tobacco plants. From 123 independent transgenic lines, 11 were confirmed for the integration of the target gene into the tobacco genome by PCR. PmAG mRNA expression was detected by RT-PCR and transgenic plants were cultured in the greenhouse until flowering. We observed that overexpression of PmAG had no effect on floral organ development and the vegetative organs. However, the color of the flower was changed from pink, in control, to white, in transgenic plants (Fig. 4).

Discussion According to the BLAST and phylogenetic analysis, PmAG has high sequence homology to the AGAMOUS-like gene

Relative expression level

4.000 3.500 3.000 2.500 2.000 1.500 1.000 0.500 0.000

1

2

3

4

5

6

7

8

9

10

11

Different organs

Fig. 1 An alignment of the deduced amino acid sequence of PpAG with those deduced from the representative genes of the AG group and phylogenic tree of AGAMOUS homologues which were generated using PAUP 4b BETA 10 and MEGA software version 4.0, respectively. ZAG1 was used as an outgroup to root the tree

Fig. 2 Expression pattern of PmAG gene revealed by qRT-PCR analysis. Note: 1, sepals of perfect flower; 2, petals of perfect flower; 3, stamens of perfect flower; 4, carpel of perfect flower; 5, sepals of imperfect flower; 6, petals of imperfect flower; 7, stamens of imperfect flower; 8, leaves; 9, fruitlet; 10, seed, 11, flesh

Plant Mol Biol Rep (2011) 29:473–480 Fig. 3 Analysis of the spatial expression patterns of PmAG by in situ hybridization. Signals were brown. a hybridized with sense probe; b PmAG showed expression in the pollen, anthers, and carpel, but not in the corolla. se, sepal; pe, petal; st, stamen; po, pollen

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a

b

sta

se sta ca pe

po

pe ca

from peach (Wu et al. 2004; Martin et al. 2006). Furthermore, AGAMOUS group proteins were separated into C- and D-function clades based on the analysis of AGAMOUS-like genes from 15 angiosperm plants. Compared with D-function genes, most C-lineage AMAGOUSlike genes bear an intron before the last codon (Kramer et al. 2004; Skipper et al. 2006). To analyze whether the PmAG sequences possess an intron positioned in the last codon like some AGAMOUS sequences, we sequenced this region from genomic DNA. The results showed that a 136bp intron called intron-8 was present just before the stop codon of the PmAG sequence (data not shown), and that it had AG motifs I and II in its C-terminal region. Expression in stamens and carpels indicated that PmAG is a typical Clineage AGAMOUS-like gene. However, the related functions of this gene need to be studied further.

A

B

Fig. 4 Phenotypic characterization of transgenic tobacco plants expressing PmAG. a Whole plant morphologies of a non-transgenic plant (wildtype) as a control; b a transgenic plant with flowers in white

The AGAMOUS gene family contains important genes that regulate the identification and development of the carpel, and are normally expressed in reproductive organs such as the floral bud, pistil, and fruit but not in the vegetative organs (Yanofsky et al. 1990; Wu et al. 2004; Martin et al. 2006; Chaidamsari et al. 2006). However, the expression pattern of PmAG was more specific. It was expressed not only in the reproductive organs such as the pistil and fruit, but also in the sepals. Such results are similar to the expression pattern in apple (van der Linden et al. 2002). Based on an RT-PCR analysis, van der Linden et al. also found that the AG-like gene MdMADS15 from apple was not only expressed in reproductive organs but also in vegetative organs such as the leaf and shoot. However, Northern blot analysis revealed a low level of expression. A low level of expression of SAG1 was detected by RT-PCR in both mature needles and late vegetative buds. Furthermore, in situ hybridization analysis showed no hybridization signal in vegetative buds (Rutledge et al. 1998) Though Japanese apricot and peach are all stone fruit trees and have a close genetic relationship, the AG-like gene MADS4 isolated from peach was only expressed in reproductive organs, and not in vegetative organs such as the leaf (Wu et al. 2004; Martin et al. 2006). This suggests that the expression patterns of AG-like genes are different, and that other functions may exist that we have not yet found. Agrobacterium-mediated transformation is a wellaccepted method to study novel gene function. Since Japanese apricot is a perennial woody plant, no optimized genetic transformation systems have been established. Due to the Japanese apricot's long juvenile period before flowering, the functional analysis of PmAG was undertaken in the model species tobacco to investigate the function of PmAG overexpression driven by the 35S cauliflower mosaic virus promoter. However, we did not observe the typical ectopic expression of PmAG in tobacco as expected

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(Rutledge et al. 1998; Du and Pijut 2010). The exhibited phenotypes of the transgenic plants were normal, except for a change in petal color. The possible reasons for this phenomenon are that the AG homologue in the herbaceous plant tobacco could not be enhanced by overexpression or that the AG homologue was isolated from a woody plant and we did not use Arabidopsis as transgenic plant hosts as employed in studies investigating overexpression of a functional AG homologue from other plants (Chaidamsari et al. 2006; Martin et al. 2006). Moreover, Wang et al. (2009) found that misexpression of DBB1a resulted in floral development abnormity, while overexpression of DBB1a had no effect on floral development when they investigated the function of the DBB1a gene isolated from Arabidopsis. Further investigation is needed to determine whether the expression of PmAG antisense mRNA affects flower development. AG is the only C-functional gene that plays an important role in the determination of floral meristem

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and later in the development of stamens and carpels (Yanofsky et al. 1990; Martin et al. 2006). In the present research, we identified the function of PmAG by in situ hybridization, qRT-PCR and transgenic analysis in tobacco. The expression pattern was quite similar to that of other AG homologues isolated from woody plant, such as peach, blackberry, and apple (Martin et al. 2006; Liu et al. 2010; van der Linden et al. 2002). The expression pattern of PmAG was not significantly different between perfect and imperfect flowers in the late development stage, which suggests that AG genes contribute to flower and carpel development in perfect and imperfect flowers. However, the function of PmAG is not clear in the early stage of pistil determination necessitating additional extensive research. Acknowledgements This work was supported in part by the National Natural Science Foundation of China (30871681). We are grateful to Professor Cohara for providing plant materials.

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