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We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the fl owers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach ̒ Akatsuki ̓and the deep-red nectarine ̒ Flavortopʼ, expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach ̒ Mochizukiʼ, however, expression of the chalcone synthase gene (CHS) and the dihydrofl avonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the fl avanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-fl avonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of ̒ Mochizuki ̓peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine. The skin color of peach and nectarine fruit is an important factor in consumer appeal as well as in marketability. Anthocyanins are major pigments of fruit skins, and they are synthesized at an increasing rate during maturation, especially near maturity, reaching a maximum in the fully ripe fruit (Gross 1987). Six enzymes are generally involved in the anthocyanin biosynthesis pathway: chalcone synthase (CHS), chalcone isomerase (CHI), fl avanone 3-hydroxylase (F3H), dihydrofl avonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose-fl avonoid 3-O-glucosyltransferase (UFGT). The genes encoding these enzymes have been isolated and characterized in many plants (Holton and Cornish 1995). Some reports have described the relationship between the expression of these genes and anthocyanin accumulation (Boss et al., 1996a, 1996b; Honda et al., 2002; Kobayashi et al., 2001; Kondo et al., 2002; Li et al., 2001; Moyano et al., 1998). The key regulatory genes in the anthocyanin biosynthesis pathway vary with fruit species. In grape (Vitis vinifera L.), Boss et al. (1996a) and Kobayashi et al. (2001) demonstrated that UFGT was a key regulatory gene for anthocyanin accumulation. In strawberries (Fragaria ×ananassa Duch.), the putative DFR gene plays a main role in anthocyanin biosynthesis during color development (Li et al., 2001; Moyano et al. 1998). Kondo et al. (2002) reported that, in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] skin, expression of anthocyanin biosynthesis genes was positively related to anthocyanin concentration and that UFGT expression was closely related to anthocyanin expression at the ripening stage. Received for publication 21 Oct. 2003. Accepted for publication 14 May 2004. The authors thank Shozo Kobayashi, Hiroko Hayama, Toshiya Yamamoto, Masayuki Kita, Chungen Hu, and Zilian Zheng of the National Institute of Fruit Tree Science (NIFTS) for their technical advice. We also thank Yoshinori Ueda and Megumi Ishimaru of Osaka Prefecture Univ., Tadashi Baba of Tokyo Univ. of Agriculture, and Nobuko Mase of NIFTS for their helpful suggestions on earlier drafts. 1To whom reprint requests should be addressed: e-mail tomomi-t@ya2.so-net. ne.jp An understanding of the regulatory mechanisms of anthocyanin biosynthesis in peach skin will help effi cient management and breeding systems for improving coloration and thus marketability. Here, we compared the changes in expression of the six genes involved in the anthocyanin biosynthesis pathway during fruit development of the white peach ̒ Mochizukiʼ, the red peach ʻAkatsukiʼ, and the deep-red ʻFlavortop ̓nectarine. Materials and Methods PLANT MATERIALS. ʻMochizuki ̓and ʻAkatsuki ̓peaches and ʻFlavortop ̓nectarine, which were cultivated at the National Institute of Fruit Tree Science (Tsukuba, Ibaraki, Japan), were used as study materials. ̒ Mochizuki ̓is a white-skinned cultivar of peach, ʻAkatsuki ̓is a red-skinned cultivar of peach, and ʻFlavortop ̓is a deep-red cultivar of nectarine. Diameters along the equator of three fruit of each cultivar were measured with calipers on the tree at intervals of 3 to 8 d. Fruit were sampled at three developmental stages in 2002: 1) pre-endocarp-hardening stage, with rapid development and increase in pericarp size; 2) endocarp-hardening stage, with delayed size increase in the mesocarp; 3) mature fruit stage, with a rapid increase in mesocarp size (Tukey, 1933). Corresponding to these developmental stages, we set three sampling stages: Stage I, 49 DAFB (days after full bloom) (ʻMochizuki ̓and ̒ Akatsukiʼ) or 51 DAFB (ʻFlavortopʼ); Stage II, 81 DAFB (ʻMochizuki ̓ and ʻAkatsukiʼ) or 83 DAFB (ʻFlavortopʼ); and Stage III, 129 DAFB (ʻMochizukiʼ), 122 DAFB (ʻAkatsukiʼ), or 132 DAFB (ʻFlavortopʼ). We sampled fl owers of the three cultivars at the time of full bloom and fruit at the above three stages for RNA extraction, anthocyanin concentration, and coloration analysis. After sampling, fl owers and fruit skins were immediately frozen in liquid nitrogen and kept at –80 °C until preparation for RNA or anthocyanin extraction. Very occasionally, fruit of ʻMochizuki ̓had red pigment in their skins. These fruit were collected at Stage III, and the red parts of the skin were used for comparison with the normal white NovJournal.indb 857 10/26/04 11:32:14 AM 858 J. AMER. SOC. HORT. SCI. 129(6):857–862. 2004. skins. The coloration of the red parts of the skin was assessed only with a chromometer (as described below), because, unlike in the other peaches and the nectarine, there was not enough red skin to do the more complex analyses. DETERMINATION OF ANTHOCYANIN CONCENTRATION AND COLORATION IN SKIN. Anthocyanin was extracted from 0.2 g of fl owers (with calyces) or 1 g of fruit skins. We used 5 mL of HCl/MeOH 1:49 (v/v) in a slight modifi cation of the method of Sun and Francis (1967). The extracts were diluted with the same volume of HCl/MeOH 1:49 (v/v) and then the absorbance reading at a wavelength of 530 nm, as indicative of anthocyanin concentration, was determined with a spectrophotometer (UV-1600; Shimadzu, Kyoto, Japan) in fi ve replicates for each cultivar. Skin color was measured with a portable chromometer (CM-2002; Minolta, Osaka, Japan), and the fruit chromaticity was given an a* value as an indicator of redness. This a* value, based on spectrophotometric colorimetry, indicates a range from red to green (Voss, 1992) and is a useful fruit maturity index (Li et al., 2002; McGuire, 1992). CONSTRUCTION OF PRIMERS AND CLONING OF ANTHOCYANIN BIOSYNTHESIS GENES AND SEQUENCE ANALYSIS. Degenerate primers for the anthocyanin biosynthesis genes were designed from published sequences {CHS: Dianthus monspessulanus [DNA Data Bank of Japan accession no. AF267173], Equisetum arvense (AB030004), Hydrangea macrophylla (AB011467), Malus sp. (X68977), Persea americana (AD001672), Petunia ×hybrida (AF233638), Vitis vinifera (AF020709); CHI: Arabidopsis thaliana (M86358), Petunia ×hybrida (X14589), Zea mays (Z22760), Dianthus caryophyllus (Z67989); F3H: Daucus carota (AF184270), Juglans nigna (AJ278457), Malus ×domestica (AF117270), Vitis vinifera (X75965); DFR: Ipomoea batatas (AB019243), Malus ×domestica (AF117268), Petunia ×hybrida (AF233639); ANS: Daucus carota (AF184274), Vitis vinifera (X75966); UFGT: Forsythia ×intermedia (AF127218), Vitis vinifera (X75968), Scutellaria baicalensis (AB031274)}. Total RNA was isolated from mature skin of ̒ Akatsuki ̓by the CTAB (cetyltrimethylammonium bromide) method described by Kotoda et al. (2000). First-strand cDNA was synthesized from total RNA from the mature skin of ̒ Akatsuki ̓by using oligo (dT) primer (cDNA Synthesis System Plus kit; Amersham Pharmacia, Little Chalfont, U.K.). This was amplifi ed under the following conditions by using degenerate primers. pPpCHS was amplifi ed with AmpliTaq Gold (PE Applied Biosystems, Foster City, Calif.): after preheating of the sample at 95 °C for 12 min, a reaction cycle of 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 90 s was repeated 35 times, followed by 72 °C for 10 min. pPpCHI and pPpUFGT were amplifi ed by using Ex-Taq (TaKaRa, Otsu, Japan): after preheating of the sample at 95 °C for 3 min, a reaction cycle of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s was repeated 35 times, followed by 72 °C for 10 min. pPpF3H, pPpDFR, and pPpANS were amplifi ed with Z-Taq (TaKaRa): after preheating of the sample at 95 °C for 5 min, a reaction cycle of 95 °C for 10 s, 50 °C for 10 s, and 72 °C for 20 s was repeated 35 times, followed by 72 °C for 10 s. Amplifi ed fragments were cloned into pCR2.1 vector by using a TA cloning system (Invitrogen, San Diego, Calif.). The fragments were sequenced with a BigDye terminator cycle sequencing kit (PE Applied Biosystems) and an automated sequencer (model 377; PE Applied Biosystems). The cloned fragments were also PCR DIG-labeled (PCR DIG Probe Synthesis Kit; Roche Diagnostics, Mannheim, Germany) according to the method of Kobayashi et al. (2001) for RNA and DNA blot analyses. CHS-MF derived from the red parts of the skin of the ʻMochizuki ̓peach at Stage III and the deep-red skin of ʻFlavortop ̓ at Stage I was used for RNA blot analysis. RNA BLOT ANALYSIS FOR GENES OF THE ANTHOCYANIN BIOSYNTHESIS PATHWAY. To isolate total RNA, 2 g of fl owers or fruit skin at Stage I and 5.6 to 5.8 g at Stages II and III was extracted by the SDS (sodium dodecyl sulfate) method (Ikoma et al. 1996), and 5 μg of each RNA was separated in a 1.16% agarose gel in the presence of formaldehyde and transferred to a nylon membrane (Hybond N; Amersham Pharmacia). The membrane was prehybridized for more than 3 h and hybridized for 14 to 16 h at 50 °C with each probe in high-SDS buffer containin

Expression of Anthocyanin Biosynthesis Genes in the Skin of Peach and Nectarine Fruit