Groszyk Jolanta, Kowalczyk Mariusz, Yanushevska Yuliya, Stochmal Anna, Rakoczy-Trojanowska Monika, Orczyk Waclaw
Department of Genetic Engineering, Plant Breeding and Acclimatization Institute - National Research Institute, Blonie, Poland.
Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation State Research Institute, Pulawy, Poland.
PLoS One. 2017 Feb 24;12(2):e0171506. doi: 10.1371/journal.pone.0171506. eCollection 2017.
The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye.
苯并恶嗪类化合物（BX）合成途径的第一步由一种具有吲哚 - 3 - 甘油磷酸裂解酶活性的酶催化，该酶由3个基因Bx1、TSA和Igl编码。在黑麦中已鉴定出一个与玉米和小麦Bx1高度同源的基因。本研究的目的是分析该基因，并通过实验验证其作为Bx1基因的黑麦直系同源基因在黑麦BX生物合成途径中的作用。该基因的表达在吸胀后3天（dai）出现峰值，在21 dai时无法检测到。直到21 dai，叶片中BX含量的变化与表达模式高度相关。在超过21 dai的植株中，尽管所分析基因的表达无法检测到，但仍有少量BX积累。通过将其天然表达和病毒诱导沉默与BX积累相关联，验证了该基因的功能。基于大麦条纹花叶病毒（BSMV）的载体用于诱导所分析基因的转录沉默（TGS）和转录后沉默（PTGS）。两种策略（PTGS和TGS）均显著降低了所分析基因的转录水平，且这与BX含量降低高度相关。用基于病毒的载体接种特异性诱导了所分析基因的表达，表明受到生物胁迫因子的上调。这是首次报道使用基于BSMV的系统对黑麦基因进行功能分析。研究结果证明所分析基因是Bx1基因的黑麦直系同源基因。其表达受发育调控，并受到生物胁迫的强烈诱导。在超过21 dai的植株中BX的稳定积累与ScBx1无法检测到的表达相关，这表明ScBx1在BX生物合成中的功能与另一个基因冗余。我们预计未知基因是Igl的假定直系同源基因，在黑麦中仍有待鉴定。
