Red coloration of apple (Malus x domestica) skin is an important determinant of consumer preference and marketability. Anthocyanins are responsible for this coloration, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. Regulation of expression of these genes is believed to be controlled by MYB transcription factors, and the MYB transcription factors involved in the activation of anthocyanin biosynthetic genes have been isolated in various plants. In the present study, we isolated and characterized a MYB transcription factor gene (MdMYBA) from apple skin. Characterization of MdMYBA demonstrated that (i) MdMYBA expression was specifically regulated depending on the tissue and cultivar/species; (ii) its expression level was much higher in a deep-red cultivar ('Jonathan') than in a pale-red cultivar ('Tsugaru'); (iii) when cauliflower mosaic virus 35S::MdMYBA was introduced into the cotyledons of apple seedlings by means of a transient assay, reddish-purple spots were induced, and MdMYBA also induced anthocyanin accumulation in reproductive tissues of transgenic tobacco; (iv) the expression of MdMYBA was induced by UV-B irradiation and low-temperature treatment, both of which are known to be important in the promotion of anthocyanin accumulation in apple skin; (v) MdMYBA bound specifically to an anthocyanidin synthase (MdANS) promoter region in a gel-shift assay; and (vi) MdMYBA was mapped to the near region of the BC226-STS (a1) marker for the red skin color locus (R(f)). These results suggest that MdMYBA is a key regulatory gene in anthocyanin biosynthesis in apple skin.
Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3' and 5' RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of a8FTQQYQa9 and a8EP-anti-IIWPNVa9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.
UDP-galactose:flavonoid 3-O-galactosyltransferase (UFGalT) is responsible for cyanidin 3-galactoside (cy3-gal) synthesis from cyanidin (cy) and UDP-galactose (UDP-gal) which are, respectively, catalyzed by anthocyanidin synthase (ANS) and UDP-glucose 4-epimerase (UGE). To clarify the contribution of UDP-galactose pathway to cy3-gal accumulation in apple skin, we analyzed the contents of UDP-gal and UDP-glucose (UDP-glu), cy, and, cy3-gal contents along with UGE activity. We confirmed that transcript levels for apple ANS and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) coincided with anthocyanin accumulation in three apple cultivars differing in their skin colors. During fruit development, changes in level of cy coincided with that of cy3-gal, whereas UDP-gal and UGE activity showed no similar trend with cy3-gal. Significant correlation was not observed between the changes in UGE activity and UDP-sugar contents. The effect of temperature and UV-B radiation (different environmental conditions) on the accumulation of UDP-sugars, cy and cy3-gal, and UGE activity were also investigated in a pale-red cultivar. High temperature tended to depress the accumulation of both UDP-sugars and cy concomitant with the decrease in cy3-gal content irrespective of UV-B radiation. Although there was no high inhibition of both cy and UDP-sugars at low-temperature without UV-B, cy3-gal accumulation was highly depressed. UGE activity was highest at low temperature with UV-B, but not much different under other conditions. Most of the parameters under different environmental conditions were significantly correlated with each other. Based on these results, contribution of UDP-sugar biosynthetic pathway to anthocyanin biosynthesis under different environmental conditions as well as during fruit development is discussed.
The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.
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