Department of Agricultural Sciences, Viikki Plant Science Centre, University of Helsinki, P.O. Box 27, Helsinki, FIN-00014, Finland.
Plant J. 2016 Sep;87(6):548-58. doi: 10.1111/tpj.13216. Epub 2016 Jul 19.
Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4-hydroxy-5-methylcoumarin (HMC). Unlike most coumarins, 5-methylcoumarins have been suggested to be derived through the acetate-malonate pathway. All of these polyketide-derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2-pyrone synthases (G2PS1-3). The enzymes are chalcone synthase-like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7-dihydroxy-5-methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site-directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.
大丁草（Gerbera hybrida）是一种经济上重要的观赏植物，也是菊科植物花发育和次生代谢的模式植物。在几乎所有大丁草组织中都大量产生两种苦味的糖苷类内酯，即大丁苷和副伞形花内酯。大丁草及其近缘种还产生一种罕见的香豆素，即 4-羟基-5-甲基香豆素（HMC）。与大多数香豆素不同，5-甲基香豆素被认为是通过乙酸-丙二酸途径衍生而来的。所有这些聚酮衍生的糖基化分子都被认为在大丁草防御草食动物和植物病原体方面发挥作用。大丁草表达三个编码 2-吡喃酮合酶（G2PS1-3）的基因。这些酶是查尔酮合酶样聚酮合酶，具有改变的起始底物特异性。我们之前已经表明，G2PS1 负责合成 4-羟基-6-甲基-2-吡喃酮（三乙酰醇内酯），这是大丁苷和副伞形花内酯的假定前体。在这里，我们表明聚酮合酶 G2PS2 和 G2PS3 是大丁草中 HMC 生物合成所必需的，并且可能需要一种还原酶来完成 HMC 的途径。G2PS2 在大丁草的叶片和花序中表达，而 G2PS3 则严格是根特异性的。G2PS2 或 G2PS3 在烟草中的异源表达导致 4,7-二羟基-5-甲基香豆素的形成，显然是 HMC 的未还原前体，而在大丁草中的异位表达导致在非转基因组织不表达基因且不积累化合物的组织中形成 HMC。通过蛋白质建模和定点突变，我们确定 G2PS2 和 G2PS3 中的残基 I203 和 T344 对五酮合酶活性至关重要。
