Transcriptome Analysis of Drought-Resistant and Drought-Sensitive Sorghum (Sorghum bicolor) Genotypes in Response to PEG-Induced Drought Stress

Abstract

Drought is a major limiting factor of crop yields. In response to drought, plants reprogram their gene expression, which ultimately regulates a multitude of biochemical and physiological processes. The timing of this reprogramming and the nature of the drought-regulated genes in different genotypes are thought to confer differential tolerance to drought stress. Sorghum is a highly drought-tolerant crop and has been increasingly used as a model cereal to identify genes that confer tolerance. Also, there is considerable natural variation in resistance to drought in different sorghum genotypes. Here, we evaluated drought resistance in four genotypes to polyethylene glycol (PEG)-induced drought stress at the seedling stage and performed transcriptome analysis in seedlings of sorghum genotypes that are either drought-resistant or drought-sensitive to identify drought-regulated changes in gene expression that are unique to drought-resistant genotypes of sorghum. Our analysis revealed that about 180 genes are differentially regulated in response to drought stress only in drought-resistant genotypes and most of these (over 70%) are up-regulated in response to drought. Among these, about 70 genes are novel with no known function and the remaining are transcription factors, signaling and stress-related proteins implicated in drought tolerance in other crops. This study revealed a set of drought-regulated genes, including many genes encoding uncharacterized proteins that are associated with drought tolerance at the seedling stage.

Keywords: drought resistance; gene expression; polyethylene glycol (PEG), drought; sorghum; transcriptome.

Figure 1

Evaluation of drought tolerance in four sorghum genotypes. (a) Seeds of four genotypes (BTx623, SC56, Tx-7000, PI-482662) were germinated and grown vertically on MS (control) and MS-containing polyethylene glycol (PEG) plates. (b) Quantification of reduction in root length in four genotypes under drought stress. The root length was measured after 48 h using ImageJ. Percent reduction in root length in the presence of PEG as compared to control is presented. Three biological replicates were used for each genotype. Tukey-Kramer HSD test was performed and significant differences (p < 0.05) between genotypes are labeled with different letters. The error bars represent +SD.

Figure 2

Genome-wide differential gene expression in the drought-resistant (DR1 and DR2) and sensitive (DS1 and DS2) lines of sorghum at 1 h (top) and 6 h (bottom). Each slice represents one of the 10 sorghum chromosomes. From the center, the first four concentric circles represent sorghum lines DR1, DR2, DS1, and DS2 respectively. The outermost circle shows the genomic coordinates with a step size of 10 million bp. The gene expression levels are shown by blue and purple coverage plots for the control and treated samples, respectively. Across the coverage plots, the up-regulated and down-regulated genes unique to resistant (DR1 and DR2) and sensitive (DS1 and DS2) genotypes are marked by green and red lines, respectively.

Figure 3

Differentially expressed (DE) genes in different genotypes in response to drought. (a) Total number of DE, up- and down-regulated genes that passed the cut-off Log₂ FC > 2 and q-value < 0.05 as compared to control condition. (b,c) Venn diagrams showing the unique and common DE genes among genotypes at 1 h (b) and 6 h (c).

Figure 4

Relative sequence read abundance (Integrated Genome Browser view) as histograms of differentially expressed genes only in the drought-resistant (DR1 and DR2) genotypes. Top two panels) One up-regulated (top) and one down-regulated gene (bottom) in resistant genotypes (DR1 and DR2) after 1-hr PEG-treatment. Replicates of each line are shown. Bottom two panels) One up-regulated (top) and one down-regulated gene (bottom) in resistant genotypes (DR1 and DR2) after 6-hr PEG-treatment. Replicates of each line are shown. The Y-axis indicates the read count. Gene structure and gene ID are shown below the read tracks. In the gene structure, lines represent introns and the boxes represent exons. The thinner boxes represent 5′ and 3′ UTRs.

Figure 5

Venn diagram showing the overlap between genes differentially expressed only in drought-resistant genotypes (DR1 and DR2) genotypes at 1 and 6 h.

Figure 6

Functional categories of DE genes expressed only in drought-resistant (DR1 and DR2) and drought-sensitive (DS1 and DS2) sorghum genotypes at 1 h (a,b) and 6 h (c,d) of post PEG-treatment. Blue represents up-regulated and red represents down-regulated genes. This figure was generated using MapMan and show DE genes that passed the cut-off value of Log₂ FC ≥2 and ≤−2 and q-value < 0.05. NA, Not Assigned.

Figure 7

Significant matches in the Arabidopsis TF database are shown for the motifs enriched in the promoter regions of differentially expressed genes in the sorghum drought-tolerant lines (DR1 and DR2) at T = 1 h (a–c) and T = 6 h (d–f). The enriched motifs were searched against the Arabidopsis TF database using the TomTom tool from the MEME suite. The transcription factors shown here are (a) AP2EREBP (b) REM, (c) C2C2, (d) HMG, (e) ABI3VP1, and (f) G2like.

Figure 8

The overlap between transcription factor families significantly matched to the enriched motifs in the promoter regions of the differentially expressed genes at T = 1 h and T= 6 h is shown.