Global analysis of switchgrass (Panicum virgatum L.) transcriptomes in response to interactive effects of drought and heat stresses

Abstract

Background: Sustainable production of high-quality feedstock has been of great interest in bioenergy research. Despite the economic importance, high temperatures and water deficit are limiting factors for the successful cultivation of switchgrass in semi-arid areas. There are limited reports on the molecular basis of combined abiotic stress tolerance in switchgrass, particularly the combination of drought and heat stress. We used transcriptomic approaches to elucidate the changes in the response of switchgrass to drought and high temperature simultaneously.

Results: We conducted solely drought treatment in switchgrass plant Alamo AP13 by withholding water after 45 days of growing. For the combination of drought and heat effect, heat treatment (35 °C/25 °C day/night) was imposed after 72 h of the initiation of drought. Samples were collected at 0 h, 72 h, 96 h, 120 h, 144 h, and 168 h after treatment imposition, total RNA was extracted, and RNA-Seq conducted. Out of a total of 32,190 genes, we identified 3912, as drought (DT) responsive genes, 2339 and 4635 as, heat (HT) and drought and heat (DTHT) responsive genes, respectively. There were 209, 106, and 220 transcription factors (TFs) differentially expressed under DT, HT and DTHT respectively. Gene ontology annotation identified the metabolic process as the significant term enriched in DTHT genes. Other biological processes identified in DTHT responsive genes included: response to water, photosynthesis, oxidation-reduction processes, and response to stress. KEGG pathway enrichment analysis on DT and DTHT responsive genes revealed that TFs and genes controlling phenylpropanoid pathways were important for individual as well as combined stress response. For example, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) from the phenylpropanoid pathway was induced by single DT and combinations of DTHT stress.

Conclusion: Through RNA-Seq analysis, we have identified unique and overlapping genes in response to DT and combined DTHT stress in switchgrass. The combination of DT and HT stress may affect the photosynthetic machinery and phenylpropanoid pathway of switchgrass which negatively impacts lignin synthesis and biomass production of switchgrass. The biological function of genes identified particularly in response to DTHT stress could further be confirmed by techniques such as single point mutation or RNAi.

Keywords: Differential gene expression; Drought stress; Gene ontology; Heat stress; Panicum virgatum; Transcription factors; Transcriptome.

Fig. 1

Hierarchical clustering analysis of Control, DT, and DTHT treated samples

Fig. 2

The number of common and specific up-regulated (A), and down-regulated (B) genes among switchgrass during DT and DTHT stress in the Venn diagram. The genes were significantly differentially expressed (DE) in more than one comparison of the time point, 0 h, 72 h, 96 h, 120 h, 144 h, and 168 h. DE genes for each comparison were quantified at log2 fold changes and P-value < 0.05

Fig. 3

a. The Gene Ontology (GO) terms enriched by responsive genes to DT stress. The DEGs were annotated against the GO database. The GO terms are in the three GO domains (biological process, molecular function and cellular compartment). These terms were significantly enriched (p < 0.05) in combined DT and HT treated samples compared to control plants. The number of genes enriched in each term were plotted against the GO term. b. The Gene Ontology (GO) terms enriched by responsive genes to DTHT stress. The DEGs were annotated against the GO database. The GO terms are in the three GO domains ( biological process, molecular function, and cellular compartment). These terms were significantly enriched (p < 0.05) in combined DT and HT treated samples compared to control plants. The number of genes enriched in each term were plotted against the GO term

Fig. 4

Heat map with clusters based on FPKM values for A) DT vs Control, B) DTHT vs control and C) DTHT vs DT TFs. The Heat map shows a grouping of control samples and stress samples. Extended periods of DTHT to stress samples showed abundant up-regulated TFs (A and B) and down-regulated TFs (C) compared to their control samples. For example, there were more responsive TFs which were up-regulated at time 144/72 h compared to its control sample at Control 144/72 h (A)

Fig. 5

Metabolism overview in MapMan showing the DEGs between DT vs Control (A) and DTHT vs control (B) switchgrass samples. The log-fold ratio is indicated as a gradient with red color (down-regulated) and blue color (up-regulation)

Fig. 6

Heat map indicating genes enriched in module 1 from the WGCNA analysis. DTHT and HT responsive genes were enriched in module 1

Fig. 7

a. Validation of the relative expression levels of five selected genes responsive to combined DTHT stress from RNA-Seq analysis by quantitative real-time PCR (qPCR). The genes selected were differentially expressed, and the time point at which these genes showed high expression from the RNA-Seq data were selected with its control for qPCR validation. b. Validation of relative expression of DT-responsive gene UDP-glucosyl transferase 85A3. UDP-glucosyl transferase 85A3 was up-regulated and down-regulated at different time points during DT stress from the RNA-Seq data. The expression pattern of the qPCR analysis is like results from the RNA-Seq analysis. The different alphabets in the Figure show that the samples collected from the different time point of DT are significantly different from the control at p-value< 0.05. qPCR results from two technical replicates and three biological replicates were analyzed using ANOVA from Minitab 18 software. The x-axis shows the treatment imposed on switchgrass. The y-axis shows the relative expression of the genes

Fig. 8

Control chamber: Regular watering (80% FC) and optimum temperature (30°/23 °C day/night temperature); DT chamber: withhold watering at 45 days after transplanting the ramets and kept at optimum temperature (30°/23 °C day/night temperature); DT + HT chamber: imposed HT after 72 h of DT (35°/25 °C day/night temperature); Leaf tissue samples were collected at 0 h-DT (dt), 72 h-dt/0 h-HT (ht), 96 h-dt/24 h-ht, 120 h-dt, 48 h-ht, and 144 h-dt/72 h-ht impositions