Plant hormonal changes and differential expression profiling reveal seed dormancy removal process in double dormant plant-herbaceous peony

Herbaceous peony (Paeonia lactiflora Pall.) is a popular ornamental and medicinal plant. Taking approximately six to seven months, the seeds germination under natural conditions experiences dual dormancies, which seriously affects horticultural cultivation. Few studies have been conducted on exploring both biological and molecular mechanism that regulates dormancy removal process in hypocotyls double dormant plants. Here, we first measured ABA and GA3 content changes at four key dormancy break stages, and then performed transcriptomic analyses to identify the differentially expressed genes (DEGs) using RNA-seq. We subsequently carried out Quantitative real-time PCR (qRT-PCR) to validate RNA-seq data. ABA content decreased during the whole dormancy removal process and GA3 content exhibited decreasing slightly and then increasing trend. RNA sequencing de novo assembly generated a total of 99,577 unigenes. 20,344 unigenes were differentially expressed in the whole dormancy release process. The qPCR results of 54 selected unigenes were consistent with the FPKM values obtained from RNA-seq. Our results summarize a valuable collection of gene expression profiles characterizing the dormancy release process. The DEGs are candidates for functional analyses of genes affecting the dormancy release, which is a precious resource for the on-going physiological and molecular investigation of seeds dormancy removal in other perennial plants.

芍药（Paeonia lactiflora Pall.）是一种广受喜爱的观赏兼药用植物。自然条件下，其种子萌发需耗时约6至7个月，且会经历双重休眠现象，这严重制约了其园艺栽培推广。目前针对下胚轴双重休眠植物的休眠解除过程，同时探索其生物学与分子调控机制的研究尚少。本研究首先在4个关键休眠解除阶段测定了脱落酸（abscisic acid，ABA）与赤霉素3（Gibberellin A3，GA3）的含量变化，随后通过RNA测序（RNA-seq）开展转录组分析以筛选差异表达基因（differentially expressed genes，DEGs），并通过实时定量聚合酶链式反应（quantitative real-time PCR，qRT-PCR）对RNA测序结果进行验证。结果显示，在整个休眠解除过程中，脱落酸（ABA）含量持续下降，而赤霉素3（GA3）含量则先略微降低后呈现上升趋势。通过RNA测序从头组装（de novo assembly）共得到99577个单基因簇（unigenes），其中20344个单基因簇在整个休眠解除过程中呈现差异表达。我们选取54个单基因簇进行qPCR验证，其结果与RNA测序得到的FPKM值高度一致。本研究的结果总结了一套可用于表征芍药种子休眠解除过程的基因表达谱资源，具有重要的科研价值。这些差异表达基因可作为调控休眠解除相关基因功能研究的候选靶点，同时也为其他多年生植物种子休眠解除的生理与分子机制研究提供了宝贵的研究资源。