Red Anthocyanins and Yellow Carotenoids Form the Color of Orange-Flower Gentian (Gentiana lutea L. var. aurantiaca)

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.

花色是决定观赏植物商业价值的关键性状。龙胆花作为切花的栽培应用范围较窄，其自然花色范围较为有限。黄龙胆（Gentiana lutea L.）变种aurantiaca（缩写为aurantiaca）以其橙色花为典型特征，但其介导该花色形成的具体色素组分尚未明确。为此，本研究针对橙色花变种aurantiaca与黄花变种黄龙胆原变种（Gentiana lutea L. var. lutea，缩写为lutea）的花瓣，在花发育过程中的类胡萝卜素（carotenoid）与类黄酮（flavonoid）组成展开了系统分析。研究发现，两个变种在花发育早期与终花期的类胡萝卜素含量仅存在细微差异；仅aurantiaca的花瓣能够积累天竺葵素糖苷（pelargonidin glycosides）类物质，而lutea花瓣中未检测到此类化合物。本研究从两变种的花瓣中克隆并测序了花青素生物合成基因（anthocyanin biosynthetic gene）片段，并对这些基因在花瓣中的表达模式进行了分析，以解析二者花瓣花色差异形成的分子机制。对分离得到的花青素cDNA片段所编码的推定氨基酸序列进行比对后发现，查尔酮合酶（chalcone synthase, CHS）、查尔酮异构酶（chalcone isomerase, CHI）、花青素合酶1（anthocyanidin synthase 1, ANS1）与ANS2在两个变种中完全一致；而二者的推定黄烷酮3-羟化酶（flavonone 3-hydroxylase, F3H）与二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase, DFR）则存在少量氨基酸序列差异。与lutea花瓣相比，aurantiaca花瓣中CHS、F3H、DFR、ANS以及UDP-葡萄糖：类黄酮3-O-葡萄糖基转移酶（UDP-glucose:flavonoid-3-O-glucosyltransferase）基因的转录本表达水平显著更高。因此，aurantiaca花瓣中天竺葵素糖苷的合成，似乎与其较高的天竺葵素合成相关基因稳态转录水平密切相关。此外，DFR酶底物特异性的潜在改变，可能是aurantiaca花瓣中生成红色天竺葵素糖苷的另一潜在调控机制。本研究揭示了aurantiaca花瓣特异性积累天竺葵素糖苷的现象，该发现可为通过调控红色花青素的合成来改造龙胆花色提供重要的理论参考。