Aharoni Asaph, Giri Ashok P, Verstappen Francel W A, Bertea Cinzia M, Sevenier Robert, Sun Zhongkui, Jongsma Maarten A, Schwab Wilfried, Bouwmeester Harro J
Plant Research International, 6700 AA, Wageningen, The Netherlands.
Plant Cell. 2004 Nov;16(11):3110-31. doi: 10.1105/tpc.104.023895.
The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as alpha-pinene and beta-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of alpha-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of alpha-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.
水果产生的风味化合物混合物可作为生物香料，用于吸引包括人类在内的生物。它们包含数百种代谢产物，其特有的水果风味成分各不相同。在进化和驯化过程中，水果风味和香气化合物的增减机制在很大程度上尚不清楚。在此，我们报告了可能导致草莓（草莓属）果实风味成分多样性进化的过程。栽培草莓品种的萜类化合物谱以单萜芳樟醇和倍半萜橙花叔醇为主，而野生草莓品种的果实主要释放烯类单萜和乙酸桃金娘酯，这些在栽培品种中并不存在。我们利用cDNA微阵列分析在栽培草莓中鉴定出凤梨草莓橙花叔醇合酶1（FaNES1）基因，并表明在大肠杆菌细胞中产生的重组FaNES1酶，分别在供应香叶基二磷酸（GPP）或法呢基二磷酸（FPP）时，能够生成芳樟醇和橙花叔醇。对来自野生和栽培草莓品种中与FaNES1非常相似的其他基因（FaNES2和野草莓NES1）的表征表明，只有FaNES1专门存在于栽培（八倍体）品种的果实中并高度表达。它编码一个在其N端截短的蛋白质。绿色荧光蛋白定位实验表明，亚细胞定位的改变导致FaNES1酶同时接触到GPP和FPP，使其能够产生芳樟醇和橙花叔醇。相反，一个插入突变影响了一个与栽培品种不同的萜类合酶基因（称为凤梨草莓蒎烯合酶）的表达。它编码一种能够催化典型野生品种单萜如α-蒎烯和β-月桂烯生物合成的酶，并导致这些化合物在栽培草莓中的缺失。α-蒎烯的缺失也进一步影响了果实风味特征，因为它不再作为下游化合物桃金娘醇和乙酸桃金娘酯生产的底物。通过克隆和表征一个细胞色素P450基因（蒎烯羟化酶）证明了这一现象，该基因编码催化α-蒎烯C10羟基化生成桃金娘醇酶。这些发现揭示了导致不同风味特征的分子进化机制，这些风味特征最终在驯化物种中被选择。
