An Integrated RNA-Seq And Network Study Reveals A Complex Regulation Process Of Rice Seed Germination

Generally, seed germination is a rapid transition process from dormancy to seedling growth, which starts a new round of life cycle for plants. Rice is one of the most important crops in the world and its whole genome sequence has been published, thus research of rice seed germination is benefit for agricultural production and further investigation. As genome sequencing technology and "omics" are developing fast, whole transcriptome sequencing become a new tool for seed germination study, however, there still lacks germination related study until now. Although several regulation mechanisms of seed germination have been reported, the whole regulation process by multiple factors is still unclear.To study rice seed germination in a whole transcriptome level, we selected Oryza sativa L. japonica. cv. Nipponbare seeds (embryos) at 0 (dry),12 and 48 (germination) hour post imbibition (HPI) and conducted RNA-Seq based on the next generation sequencing platform of SOLiD. On the basis of 83,273,725,71,036,373 and 90,085,278 generated reads, we detected 27,847,30,173 and 30,934 expressed genes respectively at 0,12 and 48 HPI, representing an increased transcriptional activity during germination. Differential gene expression analysis showed more significantly changed genes (6911) in early germination stage (0-12 HPI) than those (3083) in late germination stage (12-48 HPI), suggesting the importance of the first 12 HPI. Gene functional analysis revealed various molecular events during germination. For example, epigenetic regulation of gene expression, anatomical structure morphogenesis and carbohydrate binding were overrepresented at 0,12 and 48 HPI. Metabolism analysis showed that basical metabolisms including starch metabolism and cell wall modification related genes were significantly activated in the early germination to facilitate water-uptake, while lipid and amino acid metabolisms and light reaction related genes evidently increased in the late germination in preparation for seedling growth.Rice seed specific genes, especially those genes encoding late embryo abundant proteins (LEAs), heat shocked proteins (HSPs) and storage proteins showed a decreased expression pattern during germination. LEAs and HSPs accumulated in dry seeds to protect embryos and dropped significantly after imbibition in adaption to environment. Seed storage proteins were degraded in water-uptake to provide metabolic precursors and energy for seed germination.Phytohormones are critical in determining seed germination, but integrated regulations by all phytohormones and their interactions have not been reported in germination. In this study, we collected all biosynthesis and signaling related genes of eight phytohormones, including abscisic acid (ABA), gibberellin (GA), brassinosteroid (BR), ethylene (ET), cytokine (CK), auxin, jasmonate acid (JA) and salicylic acid (SA). Increased or decreased expression patterns of phytohormone related genes indicate positive or negative regulations of seed germination. ABA and GA is proved as a negative or positive regulator in germination, BR is considered as a positive factor, but roles of other phytohoremones can not be determined because their expression patterns were not consistently observed. To examine phytohormone interactions, an co-expression network of phytohormone related genes were constructed. Six among top ten core genes in the network encoded phosphorylation related proteins, implying the importance of phosphorylation in phytohormone interactions. For example, pathways of BR and ABA were connected by phosphorylation related proteins of BR insensitive 2s (BIN2s) and protein phosphatase 2Cs (PP2C), thus it is deduced that BR could repress ABA signaling through the phosphorylation of BIN2 on PP2C.Transcription factors (TFs) regulate gene transcription by binding to cis-elements. Hierarchical clustering of collected TF genes showed a similar expression pattern to total expressed genes in rice seed germination, indicating a regulation of germination. Based on statistical analysis of TF gene expression patterns, three groups of enriched TF families were determined for three stages. Based on gene co-expression network and gene ontolog (GO) analyses, the functional network of TFs was constructed, implying a relationship between enriched TF families and molecular events at certain time points. For example, enriched TF families of PHD, SET and SNF2 at 0 HPI were connected with epigenetic regulation in dry seeds, enriched families of HD-ZIP and M-type at 12 HPI were related with anatomical structure morphogenesis after imbibition, and enriched families of WRKY, AUX/IAA and NAC at 48 HPI were linked with lipid metabolism and light reaction.In combination of public datasets, we identified 35 rice seed germination specific TF genes in embryo. Genes with the highest expression at 12 HPI were considered most likely to be transcriptional regulators in germination, thus we randomly selected seven genes from those TFs to verify by qRT-PCR. Experimental results consisted with gene expression patterns in RNA-Seq, and this provides a gene reference for functional studies in future.Cis-elements, also known as TF binding sites, regulate gene transcription by binding to TFs. Enriched elements were searched for three gene classes with the highest expression at different time points, including ABRE (ABA responsive element), GARE (GA responsive element) and light reaction related elements enriched at 0,12 and 48 HPI. The results agreed with ABA and GA signaling gene expression patterns and physiological events during germination.Overall, this study analyzed transcriptional and functional changes during rice seed germination, and reveals a complex regulation process comprising of multiple factors such as phytohormones and TFs. Results also suggests the importance of phosphorylation in phytohormone interaction and implies a potential role of epigenetic regulation in seed dormancy.