- provides insight into gene expression dynamics during continuous salt stress. - However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. - Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h. - the number of DEGs was greatest within 12 h of salt stress. - Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation – reduction, transmembrane transport, transcription factor activity and ion channel activity. - The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq.. - In China, ephemeral plants are only distrib- uted in north Xinjiang, and mainly grow in the southern margin of the Gurbantunggut Desert based on the Flora Xinjiangensis [13]. - pumila in response to salt stress.. - Re- search on transcriptome-wide responses under salt stress have been performed in several salt-tolerant plant species using SGS: Populus pruinosa, Reaumuria trigyna, Suaeda fruticosa and Thellungiella salsuginea [16–19].. - These data provide a clear view of the tran- scriptomic dynamics for A. - pumila under salt stress Since salt stress causes active oxygen damage to plants, we investigated the physiological change in A. - pumila during the first 48 h of salt stress. - The activity of superoxide dismutase (SOD), gradually increased after the onset of salt stress, increased significantly by 6 h reached a peak at 12 h and then declined (Additional file 1: Figure S1D), in- dicating that the reactive oxygen species (ROS) scavenging system should begin to play a role in response to salt stress. - Leaf Na + concentration increased gradually after salt stress and continued to rise (Additional file 1: Figure S1E), but there were only minor changes in potassium (K. - concentration during the 48 h of salt stress (Add- itional file 1: Figure S1F). - pumila physiological changes in re- sponse to salt stress. - At the same time, plant cell accumulate osmoprotectant such as proline or upreg- ulate key enzyme for regulating reactive oxygen species such as SOD to balance the osmotic pressure of the ions to generate salt tolerance.. - pumila leaf tissues between control and salt-stress treat- ments, we employed joint the PacBio SMRT and SGS technologies for whole-transcriptome profiling. - To reduce the high error rates of the subreads, all 366,683 SMRT reads were corrected using the ap- proximately 1.15 billion Illumina clean reads as in- put data (Additional file 2: Table S1). - Transcription length distribution of SGS and TGS results showed that approximately 52.8% of the as- sembled transcripts from Illumina reads were <. - 600 bases, whereas only 0.75% of the transcripts from the SMRT were <. - Of the assembled transcripts from SMRT, 4.2% were >. - 5000 bases but only 1.4% of the assem- bled transcripts from Illumina were >. - Annotation and expression description of transcripts during salt stress. - This clustering pattern suggests that a set of genes was quickly activated during the early stage of salt stress (6 h), and other genes were activated with a longer period of stress. - 1 Clustering analysis of the differentially expressed genes (DEGs). - a, Hierarchical clustering graph of the 8075 DEGs based on the averaged log 10 (FPKM+ 1) values of all genes in each cluster. - salt stress than at other times. - Only 49 genes showing differential expression at 0.5 h of salt stress: 32 up-regulated and 17 down-regulated. - There were 488 DEGs at 3 h of salt stress: 283 up-regulated and 205 down-regulated. - Of the 3826 DEGs at 12 h of salt stress, 2121 were up-regulated, and 1705 were down-regulated. - and “single-organism metabolic process” were the most enriched terms of the biological process. - and “integral component of membrane” were the most enriched, suggesting that cell membrane proteins and sig- naling molecules play crucial roles in response to salt stress in A. - “catalytic activity” was the most enriched term of the molecular function, followed by “oxi- doreductase activity” and “transporter activity”, suggesting multiple molecular pathway during the processes of salt. - Osmotic stress and ion toxicity are considered to be the two major components of the plant salt-stress response [1, 6, 7]. - a, Differentially expressed genes were identified either by a comparison of each time point of salt stress to 0 h or comparing the adjacent stages. - b, Gene ontology classification of 2121 up-regulated DEGs at 12 h of salt stress. - We then analyzed the number of unique and shared DEGs between samples exposed to salt stress for and 48 h, and control using Venn diagrams. - Only three genes, Mitogen-activated protein kinase kinase kinase 18 (MAPKKK18), aminophospholipid ATPase 1 (ALA1) and CACTA-like transposase family (Tnp2/En/Spm) gene (Add- itional file 9: Table S5), were differentially co-expressed at all six time points of salt stress. - Only 11 genes were differentially co-expressed within 0–6 h of salt stress (Fig. - 4a), but more genes were differ- entially co-expressed with prolonged salt stress. - pumila transcriptome may facili- tate the deciphering of the salinity adaptation machinery and allele mining of salt tolerant genes.. - Data are represented as mean ± standard error of the mean (S.E.M). - The heat map of gene expression illustrated that some of these TF genes were extensively up-regulated in re- sponse to salt stress in A. - In addition to MAPKKK18, ALA1 and Tnp2, we selected another 21 genes from the top 10 DEGs at one or more time points under salt-stress conditions. - These analyses confirmed the reliability of the gene expression values gener- ated from SMRT sequencing results.. - Fur- thermore, correction of the SMRT long reads using Illumina short reads led to high-quality full-length transcripts, reducing mis-assemblies of genes and gene families with high sequence identity.. - pumila in response to continuous salt stress. - Several recent studies have reported transcript dynamics of different plant species in response to continuous salt stress but few studies addressed more than six time points of salt stress. - In our study, there were 8075 DEGs identified as responding to salt stress at all time points, but much fewer genes were differentially co-expressed for all time points. - The number of DEGs was most at 12 h of salt stress. - This information will be useful to explore salt-tolerance mechanisms and mine new salt stress-related genes spe- cific to A. - Most DEGs occurred during the first day of salt treatment, suggesting that we should pay more attention to the gene expression changes during the first 24 of salt stress in A. - Long-term exposure to continuous salt-stress condi- tion causes osmotic stress and ionic toxicity [1, 10]. - A number of studies have analyzed gene expression changes in response to salt stress and reported expressed change in a large number of genes associated with os- motic and ionic response. - Our study revealed that under salt stress conditions, more osmosis-related genes were up-regulated in A. - Identification of genes responsible for salt response Plant adaptation or tolerance to salt stress involves com- plex molecular or genetic networks. - Global analysis of stress-responsive genes facilitates understanding of the plant response to salt stress. - In this study, identification and molecular function analyses of the 8075 DEGs reflected general gene expression changes in response to continuous salinity stress in A. - Several enriched biological processes, metabolic path- ways and biochemical activities were identified based on GO and KEGG enrichment analysis of DEGs, providing gene expression overview underlying the salt-stress re- sponse in A. - osmotic balance play vital roles in salt-stress tolerance in A. - This information will be useful in elucidating salt-tolerance mechanisms and for mining new salt-stress related genes specific to A. - Proline plays a crucial role in oxidative and osmotic responses in higher plants, and proline accumulation is a well-known measure adopted for alleviation of salt stress. - Arginine and proline metabolism is one of the central pathways for the biosynthesis of the amino acids arginine and proline. - Arginine is a kind of free amino acids, and decreases when exposed to salt stress, whereas proline concentration rises in response to salt stress [1, 9, 30]. - Under salt stress conditions, osmoprotectants, such as proline and glycine betaine, must accumulate to balance the osmotic pressure of intracellular ions and provide tolerance towards stress.. - Expression of Arabidopsis MAPKKK20 was up-regulated with NaCl treatment, and transgenic plants overexpressing MAPKKK20 displayed tolerance to salt stress [54]. - Our results showed that MAPKKK18 was rapidly up-regulated within 0.5 h of salt stress and was also continuously and highly expressed during 2 days of salt stress (Fig. - Genes encoding TFs and TPs in response to salt stress In plants, many transcription factors have been identi- fied to confer salt tolerance using transcriptomic ap- proaches, including AP2-EREBP, bHLH, bZIP, C2H2, NAC and WRKY . - pumila to salt stress. - Gene expression analysis confirmed that many of them were up-regulated during the salt-stress treatment (Fig. - In the current study, we predicted that 1157 genes for TPs or regulating ion homeostasis were differentially expressed in response to salt stress (Additional file 13: Table S7). - pumila in response to salt stress. - The differential expression patterns of the TFs and TPs in our study are consistent with transcriptomic profiles of other plant species under salt stress . - However, the present study was the first to comprehen- sively study the transcriptomic responses using six time points of continuous salt stress by integrating short-read sequencing and long-read SMRT sequencing technologies.. - reduction process in response to salt stress of this species.. - Most DEGs were activated within 6 h of salt stress, and the number of DEGs was greatest at 12 h. - availability of full-length transcripts generated in this study provides a more accurate depiction of gene transcription, and facilitates understanding of the salt-adaptation mechan- ism for A. - pumila were collected in May 2012 from the southern margin (44°20’N and 87°46′E) of the Gurbantung- gut Desert in Xinjiang, China. - Tissues of young leaves of salt-stressed plants were separ- ately harvested at time points of and 48 h of salt stress. - The integrity of RNA samples was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). - Amplification was followed by size selection using the Blue- Pippin (Sage Science, Beverly, MA, USA) of the following bins for each sample and 2–6 kb. - After size se- lection, another amplification was performed using 12 PCR cycles of the above amplification conditions. - At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. - Considering the effect of the sequence depth and gene length on the fragments, the read-count values for each gene were converted into FPKM value. - The GO enrichment analysis of the DEGs was imple- mented using the GOseq R packages based on Wallenius non-central hyper-geometric distribution [68], which can adjust for gene length bias in DEGs. - (B) Species distribution of the result of NR annotation. - The number of DEGs in the pathways is indicated by the circle area, and the circle color represents the range of the corrected P values. - CLC-A: A member of the voltage-dependent chloride channel. - The funding bodies had no role in the design of the study, collection, the interpretation of data and in writing the manuscript. - Accession numbers for the Illumina cDNA libraries obtained from the controls and samples of leaves exposed to salt stress at and 48 h are SRR6320595, SRR6320596, SRR6320597, SRR6320598, SRR6320599, SRR6320600 and SRR6320601 under BioProject PRJNA417986.. - LY and YJ performed most of the experiments. - Salt stress or salt shock: which genes are we studying? J Exp Bot. - Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna. - Transcriptome assembly, profiling and differential gene expression analysis of the halophyte Suaeda fruticosa provides insights into salt tolerance. - Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. - A survey of the sorghum transcriptome using single-molecule long reads.. - Characterization of the human ESC transcriptome by hybrid sequencing.. - A single-molecule long-read survey of the human transcriptome. - Transcriptome analysis of canola (Brassica napus) under salt stress at the germination stage
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