Navarro Gallón Sandra M, Elejalde-Palmett Carolina, Daudu Dimitri, Liesecke Franziska, Jullien Frédéric, Papon Nicolas, Dugé de Bernonville Thomas, Courdavault Vincent, Lanoue Arnaud, Oudin Audrey, Glévarec Gaëlle, Pichon Olivier, Clastre Marc, St-Pierre Benoit, Atehortùa Lucia, Yoshikawa Nobuyuki, Giglioli-Guivarc'h Nathalie, Besseau Sébastien
EA2106 Biomolécules et Biotechnologies Végétales, Université François Rabelais de Tours, Tours, France.
Laboratorio de Biotecnologıa, Sede de Investigacion Universitaria, Universidad de Antioquia, Medellin, Colombia.
Planta. 2017 Jul;246(1):45-60. doi: 10.1007/s00425-017-2681-0. Epub 2017 Mar 27.
The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.
使用病毒诱导基因沉默（VIGS）方法使新鉴定的苹果树鲨烯合酶（SQS）亚型沉默，揭示了植物甾醇在质体色素沉着和叶片发育中的生物学功能。三萜类化合物是苹果中大量积累的有益健康的化合物；然而，它们的代谢调控却知之甚少。鲨烯合酶（SQS）是参与植物甾醇和三萜生物合成的关键分支点酶。在本研究中，在苹果基因组中鉴定出两种SQS亚型。两种亚型都位于内质网表面，并通过体外活性测定证明是有功能的SQS酶。MdSQS1和MdSQS2在其表达谱上对植物器官和环境限制具有特异性。这表明每个亚型可能优先参与植物甾醇和/或三萜代谢途径，正如使用RNAseq元转录组分析进一步论证的那样。最后，使用病毒诱导基因沉默（VIGS）方法使MdSQS1和MdSQS2沉默。两种MdSQS亚型的同时下调强烈影响植物甾醇合成，而三萜积累没有改变，因为发现三萜特异性氧化鲨烯合酶被上调以补偿代谢通量的减少。沉默植物中的植物甾醇缺乏明显扰乱了叶绿体色素沉着，并导致影响叶片分裂而非伸长或分化的异常发育。总之，除了对苹果树中两种SQS亚型的表征外，这项工作为每个亚型在植物甾醇和三萜途径中的特定参与提供了线索，并强调了植物甾醇在发育和叶绿体完整性中的生物学功能。我们的报告还为利用基于苹果潜隐球形病毒的VIGS方法在苹果属植物中进行代谢研究打开了大门。
