Candidate gene analysis of anthocyanin pigmentation loci in the Solanaceae.

Crop species in the Solanaceae, which includes tomato ( Lycopersicon esculentum), potato ( Solanum tuberosum), pepper ( Capsicum spp.), and eggplant ( S. melongena), exhibit natural variation in the types, levels, and tissue-specific expression patterns of anthocyanin pigments. While the identities of the genes underpinning natural variation in anthocyanin traits in these crops are largely unknown, many structural genes and regulators of anthocyanin biosynthesis have been isolated from the solanaceous ornamental species Petunia. To identify candidate genes that may correspond to loci controlling natural variation in the four crops, 13 anthocyanin-related genes were localized on a tomato F(2) genetic map. Gene map positions were then compared to mapped mutants in tomato and through comparative genetic maps to natural variants in potato, eggplant, and pepper. Similar map positions suggest that the tomato mutants anthocyaninless, entirely anthocyaninless, and anthocyanin gainer correspond to flavonoid 3'5'-hydroxylase ( f3'5'h), anthocyanidin synthase, and the Petunia Myb domain trancriptional regulatory gene an2, respectively. Similarly potato R, required for the production of red pelargonidin-based pigments, P, required for production of purple delphinidin-based pigments, and I, required for tissue-specific expression in tuber skin, appear to correspond to dihydroflavonol 4-reductase, f3'5'h and an2, respectively. The map location of an2 also overlaps pepper A and eggplant fap10.1, lla10.1, lra10.1, sa10.1, pa10.1 and ca10.1, suggesting that a homologous regulatory locus has been subjected to parallel selection in the domestication of many solanaceous crops. To test the hypothesis that tomato anthocyaninless corresponds to f3'5'h, a portion of the gene was sequenced. A premature stop codon was observed in an anthocyaninless mutant, but not in wild-type.

Organisation of the tomato polyphenol oxidase gene family.

We report the isolation and characterization of seven nuclear genes encoding polyphenol oxidase (PPO) in tomato (Lycopersicon esculentum cv. VFNT Cherry). The seven genes (PPOs A, A', B, C, D, E and F) fall into three structural classes (I, II, and III) based on Eco RI and Hind III restriction fragment length polymorphisms (RFLP). RFLP mapping and PFGE analysis demonstrated that the genes reside on chromosome 8, and may be clustered within a 165 kb region. Phage insert mapping demonstrated PPO E and PPO F (both class III), and PPOs B, D and A (classes I, II and I respectively) are grouped within separate 12.4 kb clusters. The complete nucleotide sequence was determined for each gene. Comparison to cDNAs revealed that the PPOs lack introns. A transcript of about 2 kb is expected for each PPO. Each PPO possesses a region encoding a transit peptide characteristic of polypeptides targeted to the thylakoid lumen. Predicted precursor polypeptides range in mass from 66 to 71 kDa and predicted mature polypeptides range from 57 to 62 kDa. All the PPOs encode two putative copper-binding sites characteristic of bacterial, fungal and mammalian tyrosinases. Five of the seven PPOs possess divergent DNA sequences in their 5' promoter regions. These flanking sequence differences may regulate the differential expression of PPO genes.

cDNA cloning and expression of potato polyphenol oxidase.

Polyphenol oxidases (PPOs) of plants are copper metalloproteins which catalyze the oxidation of mono- and o-diphenols to o-diquinones. Although PPOs are believed to be primarily responsible for the deleterious browning of many fruit and vegetable crops and are thought to be involved in plant-pest interactions, direct evidence for these roles is lacking. We report the cloning of two PPO cDNAs from Solanum tuberosum leaves. These cDNAs exhibit 97% and 98% sequence similarity at the DNA and deduced amino acid levels, respectively. Putative copper-binding regions of both cDNAs are very similar to those of mammalian, bacterial and Neurospora tyrosinases. Both leaf PPO cDNAs appear to encode polypeptides which are processed to a mature molecular weight of 57,000. In potato leaves, petioles, roots, and flowers, PPO is encoded by ca. 2 kb transcripts. Leaf PPO mRNA is developmentally regulated and only detectable in young foliage. In contrast, the protein profile of immunologically detectable PPO remains constant from the apical node through the eleventh leaf node.

Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii.

Type A glandular trichomes of the wild potato (Solanum berthaultii Hawkes) entrap insects by rapidly polymerizing the trichome contents after breakage by insect contact. Polymerization of trichome exudate appears to be driven by a soluble polyphenol oxidase (PPO). PPO constitutes up to 70% of the protein in individually collected trichomes and reaches a concentration approaching 200 mum in these organs. Trichome PPO has been purified and shown to be a monomeric copper metalloprotein with an isoelectric point of 5.5, possessing only o-diphenol oxygen oxido-reductase activity, and is larger than most other reported PPOs, with relative molecular weight of 59,000. Chlorogenic and caffeic acid were the most readily oxidized of 14 phenolic substrates tested. Polyclonal antibodies raised against the relative molecular weight 59,000 S. berthaultii trichome PPO were used to show that S. tuberosum L. trichomes express low levels of a cross-reactive protein that lacks detectable PPO activity.