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Evaluation the effects of ectopic expression of TaF5H1 in Arabidopsis driven by its native promoter on the the cell wall biosynthesis and salt tolerance by RNA-seq

ABSTRACT: Purpose: The goals of this study are to compare the transcriptome of Arabidopsis seedlings expressing pTaF5H1:TaF5H1(expression of TaF5H1 driven by its native promoter) with the wild-type control(Col-0) under/without salt treatment, which may give us clues to whether and how cell wall composition changes affect salt tolerance. Transcriptomes profiles of 14-day-old Arabidopsis seedlings of wild-type (Col-0) and seedlings harbour pTaF5H1:TaF5H1 without and under NaCl treatment for 6 hours were generated by RNA-seq, in triplicate, using Illumina a HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150 paired-end (PE) configuration; image analysis and base calling was conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format was processed by Cutadapt (V1.9.1) to be high quality clean data. For mapping, firstly, reference genome sequences and gene model annotation files of relative species were downloaded from ENSEMBL. Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1). For expression analysis, in the beginning, transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data. For differential expression analysis, DESeq2 Bioconductor package was used, a model based on the negative binomial distribution, the estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions, Padj of genes were setted <0.05 to detect differential expressed ones. Results: we mapped about 43.5 million sequence reads per sample to the Arabidopsis genome (TAIR10). Significant differential expressed genes(DEG) between samples was sreened for fold change≥2 and p-value (fdr, padj)≤0.05.Totally 7713 significant DEGs were discovered with 3430 up -regulated and 4483 down-regulated, no significant changes were detected as for the remaining 26505 genes among samples. Conclusions: Our study presents the detailed analysis of transcriptomes of Arabidopsis wild-type control(Col-0) and Arabidopsis seedlings expressing pTaF5H1:TaF5H1, with three biologic replicates, generated by RNA-seq technology. Our results show that ectopic expressing pTaF5H1:TaF5H1 in Arabidopsis changed the expression of some cell-wall related genes as well as genes related to salt stress response, inferring that changes in cell wall components may affect the salt tolerance of plants somehow.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE199116 | GEO | 2022/03/25

REPOSITORIES: GEO

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Project description:Transcriptional variation, also called expression level polymorphism (ELP), contributes to intra-specific phenotypic variation in many organisms. Differentially expressed transcripts are typically enriched for stress-related genes, suggesting that differences in response to the environment are a particularly common point of divergence among gentoypes. Analysis of ELPs also has been suggested as a way to assess unintended consequences of transgene introduction; however, it is important that interpretation of transcriptional changes be performed within the context of potential fitness effects. In these studies we sought to examine differential gene expression in response to salinity for two widely used Arabidopsis thaliana ecotypes, Wassilewskija (Ws) and Columbia (Col), and a single gene mutation (glabrous, gl1-1) in the Col background (Col(gl)), in relation to genetic, phenotypic, and fitness differences. Growth analyses were performed with seedlings germinated on culture media and growth chamber-grown plants carried through the full life cycle. Transcriptome analyses were performed with salt treated and control growth-chamber grown plants six days post initiation of salt stress. Ws plants had the least salt injury and highest dry matter accumulation and seed production in salt stressed conditions. ELPs among genoytypes and in response to 100 mM NaCl were enriched for genes associated with response to stress, including stress-associated transcription factors, heat shock and redox metabolism genes, and R genes. Application of salt resulted in many more transcripts up- or down-regulated in Col and Ws than in Col(gl). Many of the transcripts influenced by salt in Col were already altered in gl1-1 plants in the absence of salt, although Col(gl) plants did not show any detectable signs of stress, or effects on fecundity in the absence of salt treatment. The majority of salt-induced transcriptional changes that occurred in Ws also occurred in Col, suggesting common salt stress responses in these two ecotypes. Many more genes were affected by salt in Col than Ws, however, possibly reflecting the greater salt injury observed for Col. There was minimal overlap between the transcripts that differed for Ws and Col prior to salt treatment and those that were subsequently affected by salt stress. Thus, many genes conferring comparative salt stress tolerance in Ws likely differ from those whose expression levels are modified in response to salt stress. These studies demonstrate transcriptional variation among Arabidopsis genotypes in response to salt stress. Greater transcriptome differences did not necessarily correspond with greater genetic difference or phenotypic differences in morphology, fecundity, and resistance to salt stress. These results suggest that depending on circumstance, transcriptional changes can reflect response to injury, facilitate adaptive expression of fitness-associated traits, or allow for phenotypic buffering to minimize the impact of genetic changes.

Project description:The aim of this study was to analyze the impact of autotetraploidy on gene expression in Arabidopsis thaliana by comparing diploid versus tetraploid transcriptomes. In particular, this included the comparison of the transcriptome of different tetraploid A. thaliana ecotypes (Col-0 vs. Ler-0). The study was extended to address further aspects. One was the comparison of the transcriptomes in subsequent generations. This intended to obtain information on the genome wide stability of autotetraploid gene expression. Another line of work compared the transcriptomes of different diploid vs. tetraploid tissues. This aimed to investigate whether particular gene groups are specifically affected during the development of A. thaliana autotetraploids. Samples 1-8: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Col-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 9-12: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Ler-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 13-24: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Col-0 leaves (6th - 8th). The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 25-32: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Ler-0 leaves (6th - 8th). The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 33-36: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid vs. tetraploid Ler-0 seedlings from the second (F2) and third (F3) generation after induction, respectively. The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 37-40: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid vs. tetraploid Col-0 seedlings from the second (F2) and third (F3) generation after induction, respectively. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 41-44: Arabidopsis thaliana Col-0/Ler-0 diploid transcriptome. Transcriptional profiling and comparison of diploid Col-0 vs. diploid Ler-0 seedlings. The experiment was carried out with pedigree of esrablished lines. Samples 45-48: Arabidopsis thaliana Col-0/Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid Col-0 vs tetraploid Ler-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 and Ler-0 lines.

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Evaluation the effects of ectopic expression of TaF5H1 in Arabidopsis driven by its native promoter on the the cell wall biosynthesis and salt tolerance by RNA-seq

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