Seeds of Antirrhinum majus cv. Ribbon Yellow and Petunia hybridacv. Extra Blue were purchased from Known- You Seed Company, Taiwan. For gene cloning, seeds of A. majus were germinated, and young seedlings were transferred into pots and grown in a greenhouse until maturity. For plant transformation, seeds of P. hybrida were sterilized with 1% sodium hypochlorite for 20 min, washed thoroughly with sterile water, and germinated on MS basal medium containing MS salts [15] , 2% sucrose, 0.8% bacto-agar, pH 5.7. The cultures were then incubated in a 26˚C growth chamber under a 16-h illumination (100 mmol/m²/sec) and 8-h dark cycle. In vitro explants grown for 2 to 3 weeks were used to establish the plant transformation system.

To clone the aurone biosynthetic genes, RNA from different developmental stages of yellow flowers of A. majus was isolated by TRIzol protocol as previously described [16] . Primers SRY4'CGT-F1 (5'-ATGGGAGAAGAA- TACAAGAAA-3') and SRY4'CGT-R1 (5'-TTAACGAGTGACCGAGTTGAT-3') specific for A. majus chalcone 4'-O-glucosyltransferase (4'CGT), and primers SRYAS1-F1 (5'-ATGTTCAAAAATCCTAATATC-3') and SRYAS1-R1 (5'-TTAGCCATCAAGCTCAATCTT-3') specific for A. majus aureusidin synthase (AS1) were synthesized to amplify full-length reading frames of SRY4'CGT (1374 bp) and SRYAS1 (1689 bp), respectively. RT-PCR was carried out as previously described [16] . PCR products were analyzed by 1% agarose gel electrophoresis. After purification, PCR products were cloned into pGEM-T Easy vector (Promega), and then sequenced by an automatic DNA sequencer (ABI PRISM 3730 DNA sequencer, Perkin-Elmer). To construct the expression vectors, PCR products of snapdragon 4'CGT and AS1 were cloned into Gateway binary vectors pH2GW7 (carrying the hygromycin selection marker) and pK2WG7 (carrying the kanamycin selection marker), respectively, by using the pCR8/GW/TOPO TA cloning kit (Invitrogen). The resulting vectors pSRY4'CGT and pSRYAS1 were isolated, screened by PCR analysis, and confirmed by DNA sequencing analysis.

To introduce the vector DNA into Agrobacterium, 50 ml competent cells of Agrobacterium tumefaciens strain GV3101 were mixed with 100 ng plasmid DNA (pSRY4'CGT or pSRYAS1) and incubated on ice for 1 to 2 min. The mixture was then transferred into an ice-cold 0.1-cm electroporation cuvette. Electroporation was carried out in a Bio-Rad Gene Pulser II (USA) using the following parameters: 25 mF, 200 W, and 1.25 kV. After electroporation, 1 ml of YEP medium (10 g/l peptone; 10 g/l yeast extract; 5 g/l NaCl; pH 7.0) was added immediately into the mixture, incubated at 28˚C for 2 h, and 200 ml of bacterial mixture was transferred onto selective YEP plates containing 100 mg/l spectinomycin. The plates were incubated at 28˚C for 2 days. A single colony of plasmid-carrying Agrobacterium was picked up and incubated in 4 ml YEP medium containing 100 mg/l spectinomycin at 28˚C overnight. The value of OD₆₀₀ value should be 0.1 - 0.3. One ml of overnight culture was added to 50 ml AB medium (5 g/l glucose; 1 g/l NH₄Cl; 0.3 g/l MgSO₄∙7H₂O; 0.15 g/l KCl; 10 mg/l CaCl₂; 2.5 mg/l FeSO₄∙7H₂O; 3 g/l K₂HPO₄; 1.15 g/l NaH₂PO₄∙H₂O) supplemented with 100 mg/l spectinomycin, and incubated for 1 day at 28˚C. Acetosyringone (Fluka Chemika, Switerland) and glucose were added to the AB/ spectinomycin medium to yield a final concentration of 100 mM and 5%, respectively, and the culture was further incubated 4 h at 28˚C. The bacterial culture was then centrifuged at 5000 rpm for 5 min, and the pellet was dissolved in KCMS medium (MS salts; 0.9 mg/l thiamine; 0.2 mg/l 2,4-dichlorophenylacetic acid (2,4-D); 200 mg/l KH₂PO₄; pH 5.6) supplemented with 100 mM acetosyringone and 5% glucose. The suspension was then diluted such that the final OD₆₀₀ reading was 0.8 - 1.0.

Agrobacterium-mediated transformation was carried out as previously described [17] with modifications. For pre-culture prior to Agrobacterium infection, cotyledons or leaves were excised from 2- to 3-week-old in vitro plantlets of P. hybrida one day before infection, and incubated in KCMS solid medium supplemented with 100 mM acetosyringone for one day. Infection was carried out by adding 15 ml of diluted Agrobacterium suspension to the pre-cultured explants for 1 h. Excess Agrobacterium was blotted from the explants on sterile filter papers, then the infected explants were transferred onto KCMS solid medium containing 100 mM acetosyringone and 5% glucose, and left to grow for 2 days in a dark growth chamber at 26˚C. Explants were then washed 2-3 times with shaking (the first shaking was at 220 rpm, and subsequent shakings were at 100 rpm, 30 min each time) in KCMS liquid medium supplemented with 200 mg/l timentin. Explants were dried with sterile filter papers, and transferred to MSZ medium (MS salts; Nitsch vitamins; 1% sucrose; 2% mg/l zeatin; 0.7% bacto-agar; pH 6.0) supplemented with 200 mg/l timentin to inhibit Agrobacterium growth and 10 mg/l hygromycin (for selection in transforming vector pSRY4'CGT) or 100 mg/l kanamycin (for selection in transforming vector pSRYAS1), and incubated at 26˚C in a growth chamber with a 16-h light (100 - 200 mmol/m²/sec) and 8-h dark cycle. Explants were transferred to a new MSZ medium supplemented with 200 mg/l timentin and suitable antibiotic (10 mg/l hygromycin or 100 mg/l kanamycin) every two weeks. Formation of the initial calli could be seen on the wound site of explants after 3 weeks of transformation, and shoots were regenerated after 6 weeks. Single shoot was excised from the callus and transferred onto the MS basal medium supplemented with 100 mg/l timentin. The rooted plantlets were transferred into pots and grown in a greenhouse.

Total RNA was extracted from transgenic flowers, treated with DNase I, and 100 ng of treated RNA was used to perform reverse transcription-PCR (RT-PCR) analysis as previously described [12] . For amplification of gene- specific region, primers SRYAS1-F1 (5'-ATGTTCAAAAATCCTAATATC-3') and SRYAS1-R2 (5'-ACCAG- GTGAATCATAGTTCCA-3') were employed to amplify the 624-bp-long DNA fragment of snapdragon AS1 (GenBank accession no. EF650014), primers SRY4'CGT-F2 (5'-ATGGGAGAAGAATACAAGAAAAC-3') and SRY4'CGT-R3 (5'-CATGCTGGTGGATACTTC-3') were employed to amplify the 390-bp-long DNA fragment of snapdragon 4'CGT (GenBank accession no. EF650015), and primers CHS-C1 (5'-ACAACAAGGGCGCTC- GAG-3') and CHS-C2 (5'-CAAGCCCTTCACCAGTAG-3') were employed to amplify the 558-bp-long DNA fragment of Petunia chalcone synthase (GenBank accession No. AF233638).

T₁ seeds from self-fertilized transgenic plants were sterilized with 1% sodium hypochlorite as mentioned above. They were germinated in MS basal medium supplemented with 10 mg/l hygromycin (for transgenic SRY4'CGT lines) or 100 mg/l kanamycin (for transgenic SRYAS1 lines) for 3 weeks. Seedlings with white or pale green cotyledons, no true leaf development, inhibition of root extension and plant development were considered to be hygromycin or kanamycin sensitive, while seedlings with green cotyledons and healthy development of leaves and roots were considered to be hygromycin or kanamycin resistant.

Freeze-dried petals from transgenic Petunia flowers carrying SRY4'CGT or SRYAS1 transgene were homogenized by using liquid nitrogen with a pestle and mortar, transferred into a tube and dissolved in 50% acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid (TFA) to a final concentration of 0.1 g/ml (fresh weight/vol). The mixture was rotated for 3 h at room temperature, and then briefly centrifuged. The supernatant was transferred into a clean tube, and then vacuum-dried. The extracts were resuspended in 50% acetonitrile containing 0.1% (vol/vol) formic acid (FA), the final concentration was adjusted to 0.1 g/ml (fresh weight), and used for HPLC- DAD-MS/MS analysis.

The Agilent 1100 series liquid chromatograph system (Agilent, Waldbronn, Germany) coupled with a diode- array detector (DAD) and a Thermo Finnigan LCQ Advantage ion-trap mass spectrometer (San Jose, CA, USA) was employed for analysis at the Metabolomics Core Facility of the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan. The extracts were separated by HPLC with a RP-C18 column (Phenomenex Luna 3 μm C18, 150 mm × 2.0 mm) at a flow rate of 0.2 ml/min. The mobile phase consisted of water containing 0.1% (v/v) FA (mobile phase A) and acetonitrile containing 0.1% (v/v) FA (mobile phase B). The following conditions were used for the gradient elution: 10% - 23% B for 3 min, next with 23% B for 17 min, 23% - 80% B for 2 min and then 80% B for 3 min. The sample injection volume was 10 ml. Eluting peaks were monitored by DAD under the following conditions: Chalcones, aurones, anthocyanidins, and flavones were monitored respectively at 360 nm, at 400 nm, at 520 nm, and at 360 nm [11] . Both positive and negative ion polarity modes were used for compound ionization. The MS optimized parameters in positive ion mode were as follows: source voltage, 4.5 kV; sheath gas (N₂), 50 arbitrary units; capillary temperature, 250˚C; capillary voltage, 3 V; tube lens offset voltage, 30 V; in the negative ion mode, source voltage, −4 kV; capillary voltage, −47 V; tube lens offset voltage, −45 V. For full scan MS analysis, spectra were recorded in the range of m/z 100 - 2000.The data-de- pendent program was set so that the three most intense ions in each survey scan were selected and subjected to tandem mass spectrometry. The HPLC-DAD?MS/MS system was controlled by Xcalibur 2.0 SR2 software (Thermo Finnigan, San Jose, CA, USA).

Yellow flowers of snapdragon (Antirrhinum majus cv. Ribbon Yellow) at different stages from tiny flower bud to mature flower (Figure 1) were harvested, total RNA was isolated, and RT-PCR was carried out to clone the full-length cDNAs encoding chalcone 4'-O-glucosyltransferase (designated as SRY4'CGT) and aureusidin synthase (designated as SRYAS1) in aurone biosynthetic pathway. The SRY4'CGT clone is 1374 bp in length and contains a reading frame of 457 amino acids. Our clone is the same length as the published A. majus Am4'CGT nucleotide sequence ( [11] ; GenBank accession No. AB198665); however, it differed from the published clone by 16 nucleotides and 8 amino acids (Figure 2). To examine the accuracy of the sequence of our SRY4'CGT cDNA clone, the corresponding genomic DNA fragment was amplified by PCR analysis using genomic DNA isolated from A. majus cultivar Ribbon Yellow and primer set (SRY4'CGT-F1 and SRY4'CGT-R1) and then sequenced. As shown in Figure 2, nucleotide sequences of our cDNA and the genomic clones were identical, indicating that the genomic SRY4'CGT gene has no intron. This result is consistent with the finding that there is no intron in the Am4'CGT gene of A. majus [11] . The differences between the published clone and our clone reflect the two different cultivars (Ribbon Yellow versus Yellow Butterfly) examined. The full-length cDNA and genomic DNA encoding SRY4'CGT in cultivar Ribbon Yellow were submitted to GenBank database with accession numbers EF650015 and JQ234673, respectively. It was reported that chalcone 4'-O-glucosyltransferase catalyzes 2',4',6', 4-tetrahydroxychalcone (THC) to THC 4'-O-glucoside (colorless) or 2',4',6',3,4-pentahydrox- ychalcone (PHC) to PHC 4'-O-glucoside (colorless) in aurone biosynthetic pathway [11] .

The other clone, SRYAS1, is 1689 bp in length and contains a reading frame of 562 amino acids. Our SRY- AS1 differed from the published snapdragon AmAS1 nucleotide sequence ( [10] ; GenBank accession no. AB- 044884) by two bases in the coding region at nucleotide positions 285 and 892; however, the amino acids were identical to the corresponding published AmAS1 protein sequence (Figure 3). The full-length cDNA encoding SRYAS1 in snapdragon cultivar Ribbon Yellow was submitted to GenBank database with accession number EF650014. It was reported that aureusidin synthase catalyzes THC 4'-O-glucoside (colorless) or PHC 4'-O- glucoside (colorless) to produce aureusidin 6-O-glucoside (yellow pigment) or bracteatin 6-O-glucoside (yellow pigment), respectively, in the aurone biosynthetic pathway [11] . The deduced amino acid sequence of SRYAS1 protein showed high sequence similarity to the plant polyphenol oxidase family [10] , which has been demonstrated to be responsible for flower coloration [10] [11] [18] .

The relevant cDNA fragments were cloned separately into Gateway vectors, obtaining the expression vectors pSRY4'CGT and pSRYAS1 (Figure 4), which were confirmed by restriction enzyme digestion and DNA sequencing.

Agrobacterium-mediated transformation was carried out to transform pSRY4'CGT and pSRYAS1 separately into P. hybrida. Finally, 9 putative transgenic plants were obtained from a total of 186 cotyledon explants by

transforming the vector pSRY4'CGT and using 10 mg/l hygromycin as selection agent (Table 1); and 13 putative transgenic plants were obtained from a total of 205 cotyledon explants by transforming the vector pSR- YAS1 and using 100 mg/l kanamycin as selection agent (Table 2). Transformation efficiency for SRY'4CGT and SRYAS1 transgenes was 4.8% and 6.3%, respectively. All transgenic plants were grown in a greenhouse until

maturity.

Of the 9 transgenic Petunia plants carrying the SRY4'CGT transgene, only a few flowers (from transformants SRY4'CGT-1, -6, -7, -10) showed variegated blue-white sectors (Table 1). Those transformants that produced variegated blue-white flowers, at the late stage of flowering, more and more new-born flowers produced variegated blue-white flowers, and the areas of white sectors increased in the blue background of the petals. As time passed, most of the old flowers without alteration in pigmentation faded, and the number of new-born flowers carrying more white sectors/regions in the background blue color increased. Finally, petal color was changed to completely white in all the late-born flowers (Figure 5). In contrast, of the 13 transgenic Petunia plants carrying SRYAS1 transgene, three (SRYAS1-4, -6, -12) showed variegated blue-white flowers at the late stage of flowering (Table 2): a few variegated flowers carrying small portions of white sectors in the background blue petals were observed in these 3 transgenic lines. However, complete white flowers were not observed. The growth

Seeds were germinated on MS medium containing 10 mg/l hygromycin for 3 weeks.

pattern of a typical transgenic SRYAS1 plant carrying variegated flowers is shown in Figure 6. In addition, the amount and appearance of variegated blue-white flowers during the late stage of flowering (or plant senescence) in transgenic SRYAS1 plants were less than and later than, respectively, those of transgenic SRY4'CGT plants.

To examine the presence and expression of transgenes in transgenic plants, total RNA was isolated from variegated blue-white flowers in three transgenic SRYAS1 plants (SRYAS1-4, -6, -12) and four transgenic SRY4'CGT plants (SRY4'CGT-1, -6, -7, -10). In addition, total RNA was also isolated from blue flowers in wild-type P. hybrida. RT-PCR analysis was carried out to investigate the expression of transgene in transgenic plants (Figure 7). As expected, no transcripts were detected in wild-type Petunia flowers when gene-specific SRYAS1 or SRY4'CGT primers were employed. Transgenic SRYAS1 transcripts were detected in all three transgenic SRYAS1 plants, and transgenic SRY4'CGT transcripts were detected in all four transgenic SRY4'CGT plants (Figure 7). Thus, these results demonstrate clearly the presence and expression of transgenes in transgenic plants we examined.

Seeds were germinated on MS medium containing 100 mg/l kanamycin for 3 weeks.

The stable integration of the T-DNA region in two transformation vectors (Figure 4) into the petunia chromosomal genome was investigated by germinating T₁ seeds from self-fertilized T₀ transgenic plants on MS medium containing 10 mg/l hygromycin (Table 1) or 100 mg/l kanamycin sulfate (Table 2). In transgenic plants carrying SRY4'CGT transgene (Table 1), T₁ seeds were not available from three transformants (SRY4'CGT-1, -3, -5). Hygromycin-resistant and hygromycin-sensitive T₁ seedlings segregated in a 3:1 ratio in 2 transformants (SRY4'CGT-7 and SRY4'CGT-10), indicating the presence of single copy of the hygromycin-resistant transgene in the nuclear genomes of the 2 lines (Table 1). Since the transformation vector pSRY4'CGT contains expression cassettes for the selection marker gene (hygromycin resistant gene) and snapdragon SRY4'CGT transgene within the T-DNA region (Figure 4(a)), it is reasonable to predict that the same copy number of the hygromycin resistant transgene and SRY4'CGT transgene will be found in each transformant.

All transgenic plants carrying SRYAS1 transgene are shown in Table 2. No seeds were obtained from two transformants, SRYAS1-8 and SRYAS1-13. All T₁ seedlings from the two transformants SRYAS1-7 and SRYAS1-11 were sensitive to the selection agent kanamycin, suggesting that these two lines may not contain the foreign T-DNA fragment and can be considered to be false-positive clones. Thus, transformation efficiency in transgenic SRYAS1 plants was recalculated as 5.4%. Single insertion of the nptII transgene was determined in three transgenic plants SRYAS1-2, SRYAS1-4 and SRYAS1-12, as the segregation ratio of resistant and sensitive seedlings in these 3 transgenic plants was 3:1 (Table 2). Similarly, as transformation vector pSRYAS1 contains expression cassettes for the selection marker gene (nptII) and snapdragon SRYAS1 transgene within the T-DNA region (Figure 4(b)), it is reasonable to predict the same copy number of the nptII transgene and SRYAS1 transgene will be found in each transformant.

Five T₁ hygromycin-resistant seedlings were randomly selected in hygromycin-containing medium from T₀ selfed seeds of transgenic plant SRY4'CGT-7 that showed significant alteration in flower coloration, and grown in a greenhouse until maturity. Instead of no phenotypic change in 3 T₁ transgenic plants, obvious variegated blue-white flowers were still observed in 2 T₁ transgenic plants (Figure 8). However, no complete white flowers

were found even at the late stage of flowering in any lines. To examine the gene expression in these variegated flowers, white sectors and blue sectors were dissected from variegated flowers in 2 T₁ transgenic lines, RNA was isolated, and RT-PCR was performed using gene-specific primers (Figure 8(d)). As predicted, no SRY4'- CGT transcripts were detected in wild-type Petunia flowers. Transgenic SRY4'CGT transcripts were detected in both blue and white sectors from these two transgenic lines. When specific primers were employed for an upstream gene chalcone synthase (CHS) in the flavonoid/aurone biosynthetic pathway, CHS transcripts were only detected in blue sectors but not in white sectors of the transgenic plants examined (Figure 8(d)), suggesting that the expression of CHS gene was suppressed in the white sectors of transgenic plants.

To examine the pigment profile in transgenic flowers, and more importantly, to investigate whether the appearance of white sectors in blue background petals is due to the loss of anthocyanins, white sectors and blue sectors were dissected from variegated flowers in T₁ transgenic SRY4'CGT and SRYAS1 lines, and ground into a powder by liquid nitrogen. After extraction, crude extracts were analyzed by HPLC. For each sample, similar HPLC patterns were obtained when monitoring was performed at 360 nm (for measurement of chalcones, flavones), 400 nm (for measurement of aurones) or 520 nm (for measurement of anthocyanidins). As shown in Figure 9, an absorption peak was detected in all samples when retention time (RT) approached 2.51 to 2.59 min. However, two peaks at RT = 11.57 min and RT = 11.78 min were detected in blue sectors from transgenic SRY4'CGT- 7-T₁-1 flowers (designed as B1) but not in white sectors from transgenic SRY4'CGT-7-T₁-1 flowers (designed as W1). Again, two peaks at RT = 11.71 min and RT = 11.82 min were detected in blue sectors from transgenic SRY4'CGT-7-T₁-4 flowers (designed as B2) but not in white sectors from transgenic SRY4'CGT-7-T₁-4 flowers (designed as W2). Only one peak at RT = 11.85 min was detected in blue sectors from transgenic SRYAS1-3- T₁-5 flowers (designed as B3) but not in white sectors from transgenic SRYAS1-3-T₁-5 flowers (designed as W3); however, the HPLC pattern in B3 was distinguishable from the HPLC patterns in B1 and B2, which represented two individual T₁ plants from the same T₀ parent SRY4'CGT-7 plant. Besides the constitutive peak at RT = 2.51 - 2.59 min in all blue and white samples, almost no significant peaks were detected from white sectors (W1, W2, W3), suggesting that colored pigments may not exist or may accumulate at very low level in these white sectors. HPLC patterns monitored at 360 nm, 400 nm and 520 nm are shown in Figures 9(a)-(c), respectively.

To further identify the major compounds in these peaks, tandem mass spectrometry (MS/MS) was employed to identify fragmentation patterns of different anthocyanins. As shown in Figure 10 and Figure 11, several major peaks obtained from different retention times were chosen and then analyzed. The molecular weight of each fragment was measured as mass-to-charge ratio (m/z) in the ionization process, and the most abundant fragment in each RT was considered as 100%. Compound identification, mainly based on molecular ions and product ions, was performed with the aid of existing literatures spectral analysis regarding flavonoids/anthocyanins [19] - [22] . Cyanidin triglucoside was found as major compound in the RT = 2.62 min fraction (Figure 10; Figure 11). Fragmentation pattern of cyanidin 3,5,5'-triglucoside is shown in Figure 12(a).

Next, major compounds were identified in peaks around RT = 11.51 - 12.96 min (Figure 10; Figure 11). It is worth noting that these peaks can be detected only in the blue sectors of transgenic flowers but not in white sectors. The red color of cyanidin and its glucoside derivatives (cyanidin glucoside, cyanidin diglucoside, cyanidin malonyldiglucoside), and mauve (pale purple) color of malvidin and its derivative malvidin-3-(p-coumaroyl)- rutinoside-5-glucoside were identified in these tissues. Fragmentation patterns of these anothcyanins are shown in Figure 12(b), Figure 13(a) and Figure 13(b). These data demonstrated clearly that the major compounds of anthocyanins extracted from the blue sectors of transgenic SRY4'CGT and SRYAS1 flowers are cyanidin, malvidin and their derivatives. In contrast, cyanidin or malvidin was not detected or was accumulated at a very low level in the white sectors of transgenic flowers, as a result, white color was observed.

Finally, to evaluate whether aurones (the target yellow pigment) can be detected in our analysis system, yellow petals from snapdragon were excised, and sample preparation and HPLC-DAD-MS/MS were performed as previously described. As shown in Figure 14(a), several peaks including RT = 2.51 min were detected in HPLC analysis. Compound identification in the fraction at RT = 11.25 min revealed that aureusidin glucoside and its

dimer are the major compounds in that fraction (Figure 14(b)). It is worth pointing out that aureusidin is a yellow pigment belonging to the aurone, and aureusidin 6-O-glucoside (yellow pigment) occurs in the yellow petals of snapdragon. In addition, aureusidin 6-O-glucoside is the product of aureusidin synthase, one of our target

genes (SRYAS1) in this study. The fragmentation pattern aureusidin glucoside is shown in Figure 14(c). In conclusion, yellow pigment of aureusidin glucoside can be detected in our analysis system. However, aureusidin or aureusidin glucoside was not found in our transgenic Petunia flowers. In addition, genistin 7-O-glucuronide (an isoflavone derivative) was identified as a major compound in fraction at RT = 17.89 min (data not shown).

HPLC coupled with tandem mass spectrometry was employed to analyze the blue and white sectors from variegated flowers of transgenic SRY4'CGT and SRYAS1 plants, and the results clearly demonstrated that cyanidin, malvidin, and their derivatives are present in the blue sectors, but none of anthocyanins are present in white sectors, causing the white phenotype.