Comparative transcriptomic analysis of rhizobial symbiosis effects on salinity stress responses in Vicia sativa shoots and roots under controlled nitrogen regimes
This study investigates how rhizobial symbiosis influences plant responses to salinity through a comparative transcriptomic framework, using Vicia sativa as the primary model. The experimental design integrates transcriptomics within a broader multi-omics approach, but is structured specifically to enable direct comparison of gene expression patterns as a function of nitrogen source and stress condition. Plants were grown under controlled conditions using a Leonard jar system with inert substrate and nitrogen-free nutrient solution. Two main nutritional regimes were established: (i) symbiotic plants inoculated with Rhizobium leguminosarum and allowed to develop active nodulation, and (ii) non-inoculated plants supplemented with mineral nitrogen (N-fed), which served as non-symbiotic controls. This design isolates the effect of biological nitrogen fixation from general nitrogen availability, ensuring that transcriptomic differences can be attributed to symbiosis rather than nutritional deficiency. [SalinitySy...al_Tracked | Word] To analyze the transcriptional response to salinity, a factorial design was implemented combining nitrogen source (nodulated vs. N-fed) and treatment (control vs. salt stress). Moderate salinity was applied by exposing four-week-old plants to 150 mM NaCl for 11 days. Tissue sampling was performed at a defined developmental stage (39-day-old plants), ensuring comparability across conditions and minimizing developmental variability. [SalinitySy...al_Tracked | Word] For transcriptomic analysis, both shoot and root tissues were collected, allowing the study to capture organ-specific responses. Samples were pooled from multiple plants to reduce biological noise, with three independent biological replicates per condition. This generated a total of 24 RNA-seq libraries (12 shoot and 12 root), representing the four experimental conditions across replicates. RNA quality was rigorously controlled prior to sequencing, and libraries were prepared using poly(A) selection followed by Illumina paired-end sequencing (2 × 151 bp), ensuring high coverage and sensitivity for differential expression analysis. [SalinitySy...al_Tracked | Word] Given the absence of a fully characterized reference genome for Vicia sativa, a de novo transcriptome assembly strategy was employed. Clean reads were assembled using Trinity, followed by redundancy reduction (Corset) and quality assessment (BUSCO), producing a high-confidence set of annotated unigenes. Functional annotation relied on multiple databases (NR, SwissProt, KEGG, GO, Pfam), providing comprehensive transcript classification and enabling downstream functional enrichment analyses. [SalinitySy...al_Tracked | Word] Gene expression quantification was performed by mapping reads back to the assembled transcriptome using Bowtie and estimating transcript abundance with RSEM. Differential expression analysis was conducted using statistical models based on negative binomial distributions (DESeq2/edgeR), with stringent thresholds (FDR-corrected p-value < 0.05 and |log2FC| > 1). This standardized pipeline ensures robust identification of differentially expressed genes across conditions. [SalinitySy...al_Tracked | Word] Critically, the design enables multiple layers of comparison: (i) salt vs. control within each nitrogen regime, (ii) nodulated vs. N-fed under the same stress condition, and (iii) shared versus condition-specific transcriptional responses. Additionally, parallel analysis of shoots and roots allows dissection of tissue-specific regulatory programs. Together, this experimental framework provides a comprehensive platform for assessing how symbiotic nitrogen fixation reshapes the transcriptional landscape under salinity stress.
This study investigates how rhizobial symbiosis influences plant responses to salinity through a comparative transcriptomic framework, using Vicia sativa as the primary model. The experimental design integrates transcriptomics within a broader multi-omics approach, but is structured specifically to enable direct comparison of gene expression patterns as a function of nitrogen source and stress condition. Plants were grown under controlled conditions using a Leonard jar system with inert substrate and nitrogen-free nutrient solution. Two main nutritional regimes were established: (i) symbiotic plants inoculated with Rhizobium leguminosarum and allowed to develop active nodulation, and (ii) non-inoculated plants supplemented with mineral nitrogen (N-fed), which served as non-symbiotic controls. This design isolates the effect of biological nitrogen fixation from general nitrogen availability, ensuring that transcriptomic differences can be attributed to symbiosis rather than nutritional deficiency. [SalinitySy...al_Tracked | Word] To analyze the transcriptional response to salinity, a factorial design was implemented combining nitrogen source (nodulated vs. N-fed) and treatment (control vs. salt stress). Moderate salinity was applied by exposing four-week-old plants to 150 mM NaCl for 11 days. Tissue sampling was performed at a defined developmental stage (39-day-old plants), ensuring comparability across conditions and minimizing developmental variability. [SalinitySy...al_Tracked | Word] For transcriptomic analysis, both shoot and root tissues were collected, allowing the study to capture organ-specific responses. Samples were pooled from multiple plants to reduce biological noise, with three independent biological replicates per condition. This generated a total of 24 RNA-seq libraries (12 shoot and 12 root), representing the four experimental conditions across replicates. RNA quality was rigorously controlled prior to sequencing, and libraries were prepared using poly(A) selection followed by Illumina paired-end sequencing (2 × 151 bp), ensuring high coverage and sensitivity for differential expression analysis. [SalinitySy...al_Tracked | Word] Given the absence of a fully characterized reference genome for Vicia sativa, a de novo transcriptome assembly strategy was employed. Clean reads were assembled using Trinity, followed by redundancy reduction (Corset) and quality assessment (BUSCO), producing a high-confidence set of annotated unigenes. Functional annotation relied on multiple databases (NR, SwissProt, KEGG, GO, Pfam), providing comprehensive transcript classification and enabling downstream functional enrichment analyses. [SalinitySy...al_Tracked | Word] Gene expression quantification was performed by mapping reads back to the assembled transcriptome using Bowtie and estimating transcript abundance with RSEM. Differential expression analysis was conducted using statistical models based on negative binomial distributions (DESeq2/edgeR), with stringent thresholds (FDR-corrected p-value < 0.05 and |log2FC| > 1). This standardized pipeline ensures robust identification of differentially expressed genes across conditions. [SalinitySy...al_Tracked | Word] Critically, the design enables multiple layers of comparison: (i) salt vs. control within each nitrogen regime, (ii) nodulated vs. N-fed under the same stress condition, and (iii) shared versus condition-specific transcriptional responses. Additionally, parallel analysis of shoots and roots allows dissection of tissue-specific regulatory programs. Together, this experimental framework provides a comprehensive platform for assessing how symbiotic nitrogen fixation reshapes the transcriptional landscape under salinity stress.
Centro de Biotecnologia y Genomica de Plantas