Dokument

Summary:
Raspberry (Rubus idaeus L.) is a nutrient-rich fruit crop containing high yields of natural bioactive compounds, such as flavonoids and carotenoids, which are known to have potential health benefits in humans. Various colored raspberry fruits offer a unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of colorless leucoanthocyanidins to colored anthocyanidins, a key step in biosynthesis of anthocyanins. The current study revealed that reduced anthocyanins in yellow raspberry (“Anne”) were due to loss of function mutation or inactive ans allele. A 5-bp insertion (ans+5) in the coding region creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids of wild type ANS of red raspberry “Tulameen”. Apparently, the mutated ans gene transcripts are suppressed as a secondary effect because of nonsense-mRNA mediated decay (NMD). Functional characterization and complementation of Ans/ans alleles in planta provide strong proof of inactive ANS protein of “Anne” as compared to the functional protein of “Tulameen”. Further, molecular screening of various colored raspberries for Ans/ans alleles indicated that most of the yellow and orange fruiting raspberries contain various types of ans mutations that cause frameshifts and initiate premature stop codons leading to loss of function of the ANS proteins. In anthocyanin-free varieties, yellow/orange fruit pigmentation seems to exist as a net result of accumulation/degradation of specific carotenoids at ripe stage. The putative carotenoid pathway genes from Rubus “Anne” inserted in standard expression cassettes along with plasmids capable of generating different carotenoid precursors resulted in the successful characterization of the pathway genes via complementation in a bacterial host. It suggests that accumulation of β-branch carotenoids like β-carotene and xanthophylls (lutein) are the principal components that provide yellow coloration to anthocyanin-free raspberry fruits. Taken together, molecular and functional characterization of the carotenoid pathway genes helped to predict a preliminary pathway map for pigmentation in non-red (yellow, orange) fruiting raspberries.

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