Table_8_RNAi silencing of wheat gliadins alters the network of transcription factors that regulate the synthesis of seed storage proteins toward maintaining grain protein levels.xlsx

Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.

谷蛋白（Gluten proteins）赋予小麦面团独特的黏弹性，但同时可诱发乳糜泻（celiac disease）患者的免疫应答。为降低小麦的免疫原性，研究人员获得了醇溶蛋白（gliadins）被高效沉默的RNA干扰（RNA interference, RNAi）小麦株系。其中E82株系的谷蛋白含量降幅最为显著，但籽粒中其余蛋白质的丰度有所上升，总氮含量仍与野生型（wild type）相当。为深入解析醇溶蛋白沉默后的调控机制，本研究对籽粒灌浆（grain filling）期E82株系的籽粒与叶片组织开展了转录组分析（transcriptomic analysis）。研究鉴定得到了调控籽粒中种子贮藏蛋白（seed storage proteins, SSPs）、α-淀粉酶/胰蛋白酶抑制剂（α-amylase/trypsin inhibitors）、脂质转运蛋白（lipid transfer proteins）、丝氨酸蛋白酶抑制剂（serpins）以及淀粉合成的候选转录因子（transcription factors, TFs）调控网络。此外，E82株系叶片中存在大量差异表达基因（differentially expressed genes），其富集的生物学过程涵盖营养有效性调控与物质转运等环节。叶片与籽粒间的源库互作（source-sink communication）分析显示，大量下调表达基因与蛋白酶活性、氨基酸及糖代谢及其转运过程密切相关。在叶片中，特定的脯氨酸转运蛋白（proline transporters）与赖氨酸-组氨酸转运蛋白（lysine-histidine transporters）分别呈现下调与上调表达模式。总体而言，该RNAi株系中醇溶蛋白的沉默主要通过富含赖氨酸的球蛋白（lysine-rich globulins）进行补偿，而这类蛋白并不属于上述候选转录因子调控网络，提示其调控机制与其他种子贮藏蛋白相互独立。本研究结果阐明了蛋白质补偿机制，有助于解析籽粒灌浆期复杂的转录因子调控网络。