Graduate School of Agriculture, Kyoto University, Kyoto, Kyoto, 606-8502, Japan.
Planta. 2024 Apr 8;259(5):114. doi: 10.1007/s00425-024-04395-1.
Two glycosyltransferase genes belonging to UGT88 family were identified to have 6'-deoxychalcone 4'-glucosyltransferase activity in dahlia. 6'-Deoxychalcones (isoliquiritigenin and butein) are important pigments for yellow and orange to red flower color. 6'-Deoxychalcones are glucosylated at the 4'-position in vivo, but the genes encoding 6'-deoxychalcone 4'-glucosyltransferase have not yet been identified. In our previous study, it was indicated that snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) has isoliquiritigenin 4'-glucosylation activity. Therefore, to identify genes encoding 6'-deoxychalcone 4'-glucosyltransferase in dahlia (Dahlia variabilis), genes expressed in ray florets that shared high homology with Am4'CGT were explored. As a result, c34671_g1_i1 and c35662_g1_i1 were selected as candidate genes for 6'-deoxychalcone 4'-glucosyltransferases in dahlia. We conducted transient co-overexpression of three genes (c34671_g1_i1 or c35662_g1_i1, dahlia aldo-keto reductase1 (DvAKR1) or soybean (Glycine max) chalcone reductase5 (GmCHR5), and chili pepper (Capsicum annuum) MYB transcription factor (CaMYBA)) in Nicotiana benthamiana by agroinfiltration. Transient overexpression of c34671_g1_i1, DvAKR1, and CaMYBA resulted in increase in the accumulation of isoliquiritigenin 4'-glucosides, isoliquiritigenin 4'-O-glucoside, and isoliquiritigenin 4'-O-[6-O-(malonyl)-glucoside]. However, transient overexpression of c35662_g1_i1, DvAKR1, and CaMYBA did not increase accumulation of isoliquiritigenin 4'-glucosides. Using GmCHR5 instead of DvAKR1 showed similar results suggesting that c34671_g1_i1 has isoliquiritigenin 4'-glucosyltransferase activity. In addition, we conducted co-overexpression of four genes (c34671_g1_i1, c35662_g1_i1 or Am4'CGT, DvAKR1 or GmCHR5, CaMYBA, and chalcone 3-hydroxylase from dahlia). Accumulation of butein 4'-O-glucoside and butein 4'-O-[6-O-(malonyl)-glucoside] was detected for c35662_g1_i1, suggesting that c35662_g1_i1 has butein 4'-glucosyltransferase activity. Recombinant enzyme analysis also supported butein 4'-glucosyltransferases activity of c35662_g1_i1. Therefore, our results suggested that both c34671_g1_i1 and c35662_g1_i1 are 6'-deoxychalcone 4'-glucosyltransferases but with different substrate preference.
两个属于 UGT88 家族的糖基转移酶基因在大丽花中被鉴定具有 6'-去氧查尔酮 4'-葡萄糖基转移酶活性。6'-去氧查尔酮（异甘草素和白杨素）是黄色到红色花颜色的重要色素。6'-去氧查尔酮在体内被 4'-位葡萄糖基化，但编码 6'-去氧查尔酮 4'-葡萄糖基转移酶的基因尚未被鉴定。在我们之前的研究中，表明金鱼草（Antirrhinum majus）查尔酮 4'-O-葡萄糖基转移酶（Am4'CGT）具有异甘草素 4'-葡萄糖基化活性。因此，为了鉴定大丽花（Dahlia variabilis）中编码 6'-去氧查尔酮 4'-葡萄糖基转移酶的基因，探索了与 Am4'CGT 具有高度同源性的射线小花中表达的基因。结果，选择 c34671_g1_i1 和 c35662_g1_i1 作为大丽花 6'-去氧查尔酮 4'-葡萄糖基转移酶的候选基因。我们通过农杆菌瞬时共表达三个基因（c34671_g1_i1 或 c35662_g1_i1、大丽花醛酮还原酶 1（DvAKR1）或大豆（Glycine max）查尔酮还原酶 5（GmCHR5）和辣椒（Capsicum annuum）MYB 转录因子（CaMYBA））在烟草原生质体中。c34671_g1_i1、DvAKR1 和 CaMYBA 的瞬时过表达导致异甘草素 4'-葡萄糖苷、异甘草素 4'-O-葡萄糖苷和异甘草素 4'-O-[6-O-（丙二酰基）-葡萄糖苷]的积累增加。然而，c35662_g1_i1、DvAKR1 和 CaMYBA 的瞬时过表达并没有增加异甘草素 4'-葡萄糖苷的积累。用 GmCHR5 代替 DvAKR1 显示出类似的结果，表明 c34671_g1_i1 具有异甘草素 4'-葡萄糖基转移酶活性。此外，我们还进行了四个基因（c34671_g1_i1、c35662_g1_i1 或 Am4'CGT、DvAKR1 或 GmCHR5、CaMYBA 和大丽花查尔酮 3-羟化酶）的共过表达。检测到 c35662_g1_i1 中白杨素 4'-O-葡萄糖苷和白杨素 4'-O-[6-O-（丙二酰基）-葡萄糖苷]的积累，表明 c35662_g1_i1 具有白杨素 4'-葡萄糖基转移酶活性。重组酶分析也支持 c35662_g1_i1 的白杨素 4'-葡萄糖基转移酶活性。因此，我们的结果表明，c34671_g1_i1 和 c35662_g1_i1 都是 6'-去氧查尔酮 4'-葡萄糖基转移酶，但具有不同的底物偏好。
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