Transcriptomic profile of Arabidopsis thaliana treated with exogenous Riboflavin. Transcriptomic profile of Arabidopsis thaliana treated with exogenous Riboflavin

Riboflavin, also known as vitamin B2, is crucial for cell function as it is the central component of FMN and FAD cofactors. These flavocoenzymes are essential for various cellular processes including the control of flavoproteins activity. Flavins are vital for growth and cellular metabolism of plants, supporting functions like photosynthesis, mitochondrial electron transport, and fatty acid oxidation. Further, previous studies have shown that exogenous application of riboflavin enhances plant defenses against abiotic and biotic stresses. In this study, to identify and implicate genes and signaling pathways involved in these processes, a holistic transcriptome analysis was conducted on Arabidopsis wild-type plants treated with Riboflavin and the mutant called “melin” which accumulates Riboflavin at exceptionally high levels. Overall design: Total RNA was extracted using the Direct-zol RNA Miniprep (Zymo Research, Irvine CA, USA) kit with an on-column DNase treatment according to the manufacturer’s instructions from six days-old Col-0 (wild-type) and fmn/fhy (mos) seedlings grown under standard conditions: 16 hours of light and 8 hours of darkness per day, with a light intensity of 100 μmol m–2 s–1. Additionally, RNA was extracted from Col -0 (wild-type) (CR) seedlings treated with 0.6 mM Riboflavin for 8 hours during the dark cycle. The quantity and integrity of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and agarose gel electrophoresis. RNA-seq libraries were generated using the polyA Strand-Specific Transcriptome Library method of BGI for DNBSEQ platform. Sequencing was performed on DNBSEQ platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample. Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (TAIR10). RNA-seq data were analyzed using SOAPnuke (version 1.5.2) with parameters “-l 15 -q 0.2 -n 0.05" and the HISAT2 pipeline (version 2.0.4) with parameters “--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8".

核黄素（Riboflavin）又称维生素B2，作为黄素单核苷酸（FMN）与黄素腺嘌呤二核苷酸（FAD）辅因子的核心组分，对细胞功能至关重要。这类黄素辅因子是调控黄素蛋白活性等多种细胞过程的必需物质。黄素类化合物对植物的生长与细胞代谢至关重要，参与光合作用、线粒体电子传递、脂肪酸氧化等生理功能。此外，既往研究表明，外源施加核黄素可增强植物对非生物与生物胁迫的防御能力。
本研究为鉴定并阐明参与上述过程的基因与信号通路，对经核黄素处理的拟南芥（Arabidopsis）野生型植株，以及核黄素积累量异常升高的“melin”突变体开展了全转录组分析。
实验整体设计如下：参照厂商说明书，采用Direct-zol RNA Miniprep（Zymo Research，美国加利福尼亚州欧文市）试剂盒并结合柱上DNase处理，从标准培养条件下生长6天的Col-0（野生型）与fmn/fhy（mos）幼苗中提取总RNA；培养条件为每日16小时光照、8小时黑暗，光照强度为100 μmol·m⁻²·s⁻¹。此外，还提取了黑暗周期中经0.6 mM核黄素处理8小时的Col-0（野生型，CR）幼苗的总RNA。
采用NanoDrop ND-1000分光光度计（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）与琼脂糖凝胶电泳，分别评估RNA的浓度与完整性。
采用BGI针对DNBSEQ测序平台开发的polyA链特异性转录组文库（polyA Strand-Specific Transcriptome Library）构建方法制备RNA-seq文库。所有样本均设置3个生物学重复，于北京基因组研究所（BGI）的DNBSEQ测序平台仪器上完成测序。
原始读段（raw reads）经过滤得到洁净读段（clean reads）后，比对至拟南芥基因组（TAIR10）。RNA-seq数据分析采用SOAPnuke（版本1.5.2），参数为"-l 15 -q 0.2 -n 0.05"，以及HISAT2流程（版本2.0.4），参数为"--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8"。