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. 2020 Nov 12;21(22):8520.
doi: 10.3390/ijms21228520.

Genome-Wide Gene Expression Profiles Analysis Reveal Novel Insights into Drought Stress in Foxtail Millet (Setaria italica L.)

Ling Qin  1   2 , Erying Chen  2 , Feifei Li  2 , Xiao Yu  3 , Zhenyu Liu  2 , Yanbing Yang  2 , Runfeng Wang  2 , Huawen Zhang  2 , Hailian Wang  2 , Bin Liu  2 , Yan'an Guan  2   3 , Ying Ruan  1
Affiliations

Genome-Wide Gene Expression Profiles Analysis Reveal Novel Insights into Drought Stress in Foxtail Millet (Setaria italica L.)

Ling Qin et al. Int J Mol Sci. .

Abstract

Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop because of its health benefits and adaptation to drought stress; however, reports of transcriptomic analysis of genes responding to re-watering after drought stress in foxtail millet are rare. The present study evaluated physiological parameters, such as proline content, p5cs enzyme activity, anti-oxidation enzyme activities, and investigated gene expression patterns using RNA sequencing of the drought-tolerant foxtail millet variety (Jigu 16) treated with drought stress and rehydration. The results indicated that drought stress-responsive genes were related to many multiple metabolic processes, such as photosynthesis, signal transduction, phenylpropanoid biosynthesis, starch and sucrose metabolism, and osmotic adjustment. Furthermore, the Δ1-pyrroline-5-carboxylate synthetase genes, SiP5CS1 and SiP5CS2, were remarkably upregulated in foxtail millet under drought stress conditions. Foxtail millet can also recover well on rehydration after drought stress through gene regulation. Our data demonstrate that recovery on rehydration primarily involves proline metabolism, sugar metabolism, hormone signal transduction, water transport, and detoxification, plus reversal of the expression direction of most drought-responsive genes. Our results provided a detailed description of the comparative transcriptome response of foxtail millet variety Jigu 16 under drought and rehydration environments. Furthermore, we identify SiP5CS2 as an important gene likely involved in the drought tolerance of foxtail millet.

Keywords: P5CS genes; RNA sequencing; drought stress; foxtail millet; gene expression.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effects of drought stress and re-watering on foxtail millet, variety Jigu 16. (A) Soil volumetric water content during the 9-day drought and 12-h re-watering treatments. Arrows indicate the time points when plants were sampled for RNA-seq. Each column represents the mean ± SD (n = 3). (B) Phenotypic alterations of foxtail millet seedlings under control conditions and after 9 days of drought stress and 12 h of re-watering. Bar = 5 cm. (C) Changes in leaf water content under 9-day drought and 12-h re-watering conditions. Each column represents the mean ± SD (n = 3 pools of x plants). Significance levels were determined by one-way ANOVA; Different letters above bars indicate significant differences, lowercase letter p < 0.05, uppercase letter p < 0.01.
Figure 2
Figure 2
Physiologic parameters in Jigu 16 foxtail millet leaves under drought stress and re-watering conditions. Total activity of P5CS (A) and the antioxidant enzymes, peroxidase (POD) (B), superoxide dismutase (SOD) (C), and catalase (CAT) (D) in Jigu 16 after 9 days drought stress and 12 h re-watering conditions. Malondialdehyde (MDA) (E) and proline (F) content in foxtail millet after 9 days drought stress and 12 h re-watering conditions. Each column represents the mean ± SD (n = 3 pools of x plants). Significance levels were determined by one-way ANOVA; Different letters above bars indicate significant differences, lowercase letter p < 0.05, uppercase letter p < 0.01.
Figure 3
Figure 3
Comparison of the expression profiles of selected DEGs determined by RT-qPCR and RNA-Seq analyses. (A,B) Expression levels of 12 DEGs in drought stress and re-watering conditions. Values are presented as log2(fold-change). The X-axis represents gene ID, according to the NCBI database. (C) Scatter plots of the expression levels of 12 DEGs in drought stress and re-watering conditions. X and Y axes represent log2 (fold-change) determined by RT-qPCR and RNA-seq experiments, respectively; ** p < 0.01.
Figure 4
Figure 4
Venn diagrams showing the numbers of differentially expressed genes (DEGs) co-modulated in leaves and roots following drought stress and re-watering treatment. (A) Venn diagrams showing DEGs between watered control (LCK), drought (LD) and re-watering (LR) treatments in leaves. (B) Venn diagrams showing DEGs between LCK-LD-up and LD-LR-down. (C) Venn diagrams showing DEGs between LCK-LD-down and LD-LR-up. (D) Venn diagrams showing DEGs between watered control (RCK), drought (RD) and re-watering (RR) treatments in roots. (E) Venn diagrams showing DEGs between RCK-RD-up and RD-RR-down. (F) Venn diagrams showing DEGs between RCK-RD-down and RD-RR-up.
Figure 5
Figure 5
Gene ontology (GO) enrichment of DEGs in response to drought and rehydration in Jigu 16 foxtail millet. (A) GO enrichment of DEGs in leaves between watered control (LCK) and drought (LD). (B) GO enrichment of DEGs in leaves between drought (LD) and re-watering (LR). (C) GO enrichment of DEGs in roots between watered control (RCK) and drought (RD). (D) GO enrichment of DEGs in roots between drought (RD) and re-watering (RR). Blue columns indicate the numbers of upregulated genes, while red columns indicate numbers of downregulated genes. The threshold for differential expression was set at log2 fold-change > 1 and FDR ≤ 0.05.
Figure 6
Figure 6
KEGG analysis of DEGs identified under drought and re-watering conditions. The “GeneRatio” shows the ratio of the number of DEGs to the total gene number in a specific pathway. Pathways are listed along the y-axis, while the x-axis indicates the enrichment factor. Red indicates a high q value while blue represents a low q value. The area of bubbles indicated the number of enriched DEGs. (A) KEGG analysis of DEGs identified between watered control (LCK) and drought (LD). (B) KEGG analysis of DEGs identified between drought (LD) and re-watering (LR). (C) KEGG analysis of DEGs identified between watered control (RCK) and drought (RD). (D) KEGG analysis of DEGs identified between drought (RD) and re-watering (RR).
Figure 7
Figure 7
Heatmap of primary drought-related genes. (A) Photosynthesis-related genes. (B) Signal transduction-related genes. (C) Phenylpropanoid biosynthesis-related genes. (D) Starch metabolism-related genes. (E) Amino acid synthesis-related genes. The X-axis represents gene ID, according to the NCBI database. The Y-axis represents different comparisons. Relative levels of genes expression are showed by a heatmap with color from blue to red representing the expression levels from low to high. The bar on the right side of the heatmap represents relative expression level of DEGs.
Figure 8
Figure 8
Schematic representation of the main processes involved in drought response in foxtail millet. The color scale represents increased (red) or decreased (blue) fold-change expression of DEGs in samples exposed to drought stress and re-watering.
Figure 9
Figure 9
Distribution of common transcription factor families differentially expressed in foxtail millet under drought conditions. (A) Leaves. (B) Roots.
Figure 10
Figure 10
Analysis of differential expression of SiP5CS genes under drought and rehydration conditions in leaves. (A) SiP5CS1. (B) SiP5CS2. Each column represents the mean ± SD (n = 3). Significance levels were determined by one-way ANOVA; Different letters above bars indicate significant differences, lowercase letter p < 0.05, uppercase letter p < 0.01.
Figure 11
Figure 11
Subcellular localization of SiP5CS2. SiP5CS2 is localized to the nucleus. The SiP5CS-GFP construct and the empty vector (GFP, green fluorescent protein) were co-transformed into Arabidopsis protoplasts with the pAN580-ECFP-Ghd7 vector (a nuclear marker). The fluorescent signal of SiP5CS-GFP (green, pseudo-color) was specifically detected in the nucleus and exclusively co-localized with pAN580-ECFP-Ghd7 (yellow). The free GFP signal was observed in both the nucleus and cytoplasm.
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