Proteomics And Transcriptomics Analysis Of Desiccation Tolerance Of Mandshurica Ash Seeds In Response To Re-dehydration

Desiccation tolerance is the ability of orthodox seeds to achieve equilibrium with atmospheric relative humidity and to survive in this state. Understanding how orthodox seeds respond to dehydration is an important prerequisite for improving quality and long-term storage of seeds in low temperature and drought stress conditions. Long-term storage of seeds is an artificial situation because in most natural situations a seed that has been shed may not remain in a desiccated state for very long, and if dormant it may undergo repeated cycles of dehydration. Different types of seeds are differentially sensitive to desiccation and this directly affects long-term storage. For these reasons many researchers are investigating loss of desiccation tolerance during orthodox seed development to understand how desiccation tolerance is acquired. In this study, we investigate the orthodox seed proteome response of Fraxinus mandshurica Rupr. to dehydration (a relative water content of10%, which mimics seed dehydration) under different conditions (20℃;20℃with silica gel;1℃; and1℃after treated by Ca2+). The total proteins from the seeds dehydrated under different conditions were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). Then, a4800plus matrix-assisted laser desorption/ionization time of flight (TOF)/TOF (MALD1TOF/TOF) mass spectrometry was used to compare the proteomes of the seeds dehydrated under different conditions. A total of2919protein spots were detected on the gel, and high-resolution2D-DIGE revealed27differentially expressed proteins. Seven of these spots were identified using MALDI TOF/TOF mass spectrometry. Inferences from the bioinformatic annotations of these proteins revealed the possible involvement of detoxifying enzymes, transport proteins and nucleotide metabolism enzymes in response to dehydration. Of the seven differentially expressed proteins, six were down-regulated and one was up-regulated, while putative acyl-coenzyme A oxidase of the glyoxylate cycle also increased in abundance. In particular, kinesin-1was up-regulated in seeds exposed to low temperature stress, and this protein is important for regulation and cargo interaction. Though kinesin-1is present in all major lineages except for plants, a kinesin-1related protein has been predicted in the genome of Arabidopsis thaliana. These observations provide new insight into the proteome of seeds in deep dormancy towards different desiccation conditions.Desiccation tolerance in seeds is strictly developmentally controlled; however, it is hard to reach unifying mechanisms that determine desiccation tolerance in orthodox seeds. The causes for this are partly due to the complex aspects of seeds responses to water deficiency. Therefore, comparisons of gene associated with desiccation tolerance should be analysed on regulatory genes. Transcriptional regulation is mediated by transcription factor via interaction with specific elements. Nevertheless, the application of next-generation sequencing (NGS) technologies can be used to determine gene sequences and transcript abundance and permit large-scale analyses of gene expression patterns despite the lack of a reference genome. Here we use the Illumina Genome Analyzer sequencing platform to characterize transcript abundance data for dormancy-released seeds of Fraxinus mandshurica Rupr. under different conditions. This reveals that genes involved in several pathways, such as chaperones and folding catalysts, MAPK signalling pathway and protein processing in endoplasmic reticulum, and are tightly regulated at the level of gene expression.We identied17transcriptional factor families from the transcriptomes, including Zinc finger, NAC, Cytochrome P450, Protease, WRKY, MYB, bZIP, ERF, Lipase, AP2/ERF, ARF, RING finger, CIPK, DREB, bHLH, Pathogen-related protein, MADS-box. Expressional profiles analysis showed that stress response gene of Zinc finger were the most, followed by Protease, NAC, ERF, bZIP, Cytochrome P450, MYB. We further analysis indicated a few significant enrichment pathways, including chaperones and folding catalysts, mitogen-activated protein kinase signaling pathway and protein processing in endoplasmic reticulum. Several of these pathways were regulated in gene expression.In this study, we obtained approximately80million sequencing reads and assembled de novo, yielding52,992high quality sequences with average length of383bp from seed of F. mandshurica to dehydration (a relative water content of10%, which mimics seed dehydration) under different conditions (20℃;1℃; and1℃with Ca2+). Of these,44,251were identified as putative homologs of annotated sequences in the public protein databases, of which218were associated with the desiccation tolerance. Digital abundance analysis identified1,231transcripts differentially enriched among different conditions (20℃;1℃; and1℃with Ca2+). Unigenes found at each treatment were categorized according to their putative functional categories. These sequence and putative function data comprise a resource for future investigation of desiccation tolerance in seeds. Lastly, we predicted a total of1,683single nucleotide polymorphisms (SNP), among which649showed polymorphism between20℃and1℃,1034showed polymorphism between1℃and1℃with Ca2+. In addition, this study provides insight into the complex transcriptome of F. mandshurica in response to re-dehydration under different conditions and established a platform for future research.