Genome wide transcriptome analysis reveals vital role of heat responsive genes in regulatory mechanisms of lentil (Lens culinaris Medikus)

Abstract

The present study reports the role of morphological, physiological and reproductive attributes viz. membrane stability index (MSI), osmolytes accumulations, antioxidants activities and pollen germination for heat stress tolerance in contrasting genotypes. Heat stress increased proline and glycine betaine (GPX) contents, induced superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities and resulted in higher MSI in PDL-2 (tolerant) compared to JL-3 (sensitive). In vitro pollen germination of tolerant genotype was higher than sensitive one under heat stress. In vivo stressed pollens of tolerant genotype germinated well on stressed stigma of sensitive genotype, while stressed pollens of sensitive genotype did not germinate on stressed stigma of tolerant genotype. De novo transcriptome analysis of both the genotypes showed that number of contigs ranged from 90,267 to 104,424 for all the samples with N₅₀ ranging from 1,755 to 1,844 bp under heat stress and control conditions. Based on assembled unigenes, 194,178 high-quality Single Nucleotide Polymorphisms (SNPs), 141,050 microsatellites and 7,388 Insertion-deletions (Indels) were detected. Expression of 10 genes was evaluated using quantitative Real Time Polymerase Chain Reaction (RT-qPCR). Comparison of differentially expressed genes (DEGs) under different combinations of heat stress has led to the identification of candidate DEGs and pathways. Changes in expression of physiological and pollen phenotyping related genes were also reaffirmed through transcriptome data. Cell wall and secondary metabolite pathways are found to be majorly affected under heat stress. The findings need further analysis to determine genetic mechanism involved in heat tolerance of lentil.

Figure 1

Plant exposed to heat stress (35/33 °C) and control (27/16 °C) in JL-3 (sensitive) and PDL- 2 (tolerant) genotypes at seedling stage in growth chamber under hydroponic conditions.

Figure 2

Changes in APX (µmol min⁻¹ g⁻¹fr.wt.) (a), GPX (µmol min⁻¹ g⁻¹fr.wt.) (b), CAT (µmol min⁻¹ g⁻¹fr.wt.) (c), SOD (unit min⁻¹ g⁻¹fr.wt.) (d), TBARS (µmol g⁻¹fr.wt.) (lipid peroxidation) (e), MSI (%) (f) Glycine betane content (µmol g⁻¹fr.wt.) (g), Proline content (µmol g⁻¹fr.wt.) (h), of heat tolerant (PDL-2) and sensitive (JL-3) lentil genotypes under control and heat stress.

Figure 3

Venn diagram showing overall expression of (A) down-regulated (B) up-regulated DEGs in combination tolerant treated vs. tolerant control (TT-TC), sensitive treated vs. sensitive control (ST-SC) and tolerant treated vs. sensitive treated (TT-ST).

Figure 4

HeatMap depicting 30 down-regulated DEGs with p value < 0.05 in combination tolerant treated vs sensitive treated.

Figure 5

HeatMap depicting 30 up-regulated DEGs with p value < 0.05 in combination tolerant treated vs sensitive treated.

Figure 6

Wego plot for upregulated and down regulated GO classification in accordance to GO groups: molecular function, biological process and cellular component in combination tolerant treated vs sensitive treated under heat stress.

Figure 7

Circular plot depicting DEGs involved in tolerant vs sensitive genotypes under heat stress was plotted using Circos version 0.62. Tabular data representing genotypes in columns & DEGs values in rows has been plotted in this circular plot. Rows are represented by circularly arranged segments (outermost ring), whose length is proportional to the total cell values in a row and columns are represented by circularly arranged segments (inner ring) whose length is proportional to the total cell values in a column. Ribbons represent row & column IDs. The 3 outer rings are stacked bar plots that represent relative contribution of a cell to row and column totals. Expression value is expressed in colours with red colour representing lowest value & purple colour representing highest values.

Figure 8

MapMan display for tolerant vs sensitive under heat stress. Up regulated genes are expressed in increased intense red while down regulated as blue at the amplitude of 4.5 to −4.5 (log2-value).

Figure 9

Genes relative expression profile using real time PCR. 2^-(ΔΔCT) method was used to obtain relative quantification using β-tubulin as reference gene. Data represents average from three biological replicates and error bars indicates standard deviation (±SD).

Figure 10

Correlation between RNA Seq and real time PCR. Log fold change is depicted through colour intensity as described in heat maps.