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. 2022 Sep 16;23(18):10792.
doi: 10.3390/ijms231810792.

Transcriptome Sequencing and Metabolome Analysis Reveals the Molecular Mechanism of Drought Stress in Millet

Xiaoning Cao  1   2 , Yulu Hu  1   2 , Jian Song  1   2 , Hui Feng  1   2 , Junjie Wang  1   2 , Ling Chen  1   2 , Lun Wang  1   2 , Xianmin Diao  3 , Yan Wan  4 , Sichen Liu  1   2 , Zhijun Qiao  1   2
Affiliations

Transcriptome Sequencing and Metabolome Analysis Reveals the Molecular Mechanism of Drought Stress in Millet

Xiaoning Cao et al. Int J Mol Sci. .

Abstract

As one of the oldest agricultural crops in China, millet (Panicum miliaceum) has powerful drought tolerance. In this study, transcriptome and metabolome analyses of 'Hequ Red millet' (HQ) and 'Yanshu No.10' (YS10) millet after 6 h of drought stress were performed. Transcriptome characteristics of drought stress in HQ and YS10 were characterized by Pacbio full-length transcriptome sequencing. The pathway analysis of the differentially expressed genes (DEGs) showed that the highly enriched categories were related to starch and sucrose metabolism, pyruvate metabolism, metabolic pathways, and the biosynthesis of secondary metabolites when the two millet varieties were subjected to drought stress. Under drought stress, 245 genes related to energy metabolism were found to show significant changes between the two strains. Further analysis showed that 219 genes related to plant hormone signal transduction also participated in the drought response. In addition, numerous genes involved in anthocyanin metabolism and photosynthesis were confirmed to be related to drought stress, and these genes showed significant differential expression and played an important role in anthocyanin metabolism and photosynthesis. Moreover, we identified 496 transcription factors related to drought stress, which came from 10 different transcription factor families, such as bHLH, C3H, MYB, and WRKY. Further analysis showed that many key genes related to energy metabolism, such as citrate synthase, isocitrate dehydrogenase, and ATP synthase, showed significant upregulation, and most of the structural genes involved in anthocyanin biosynthesis also showed significant upregulation in both strains. Most genes related to plant hormone signal transduction showed upregulated expression, while many JA and SA signaling pathway-related genes were downregulated. Metabolome analysis was performed on 'Hequ red millet' (HQ) and 'Yanshu 10' (YS10), a total of 2082 differential metabolites (DEMs) were identified. These findings indicate that energy metabolism, anthocyanins, photosynthesis, and plant hormones are closely related to the drought resistance of millet and adapt to adversity by precisely regulating the levels of various molecular pathways.

Keywords: Panicum miliaceum L.; drought stress; metabolome; plant hormone; transcription factors; transcriptome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure A1
Figure A1
Full-length transcriptome and differential genes overview of HQ and YS10. (A): The number of transcriptomes in the two strains. (B): The number of differentially expressed genes between each group in the pairwise comparison. (C): KEGG pathway analysis of DEGs.
Figure A2
Figure A2
Differential analysis and functional analysis of metabolites. (A): Venn diagram of the number of different metabolites. (B): Heatmap of the expression of metabolites that differ in positive ion mode and negative ion mode. (C): KEGG enrichment analysis of the DEMs in the HD vs. HC comparison. (D): KEGG enrichment analysis of the DEMs in the YD vs. YC comparison.
Figure A3
Figure A3
Molecular pathways shared by DEGs and DEMs. (A): KEGG pathways shared by the DEGs and DEMs in the HD vs. HC comparison. (B): KEGG pathways shared by the DEGs and DEMs in the YD vs. YC comparison.
Figure 1
Figure 1
The expression profile of genes involved in sugar metabolism. These genes include starch and sucrose metabolism, glycolysis, and the TCA cycle, involving 21 related enzymes and regulatory factors.
Figure 2
Figure 2
The expression profiles of the genes related to plant hormone signal transduction in HQ and YS10. These hormone pathways include the ABA signaling pathway, auxin signaling pathway, ethylene signaling pathway, cytokinin signaling pathway, BR signaling pathway, GA signaling pathway, and JA and SA signaling pathways.
Figure 3
Figure 3
Heat map diagrams of the relative expression levels of anthocyanin biosynthesis-related structural genes in response to DR. PAL: phenylalanine ammonia lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumaroyl-CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; DFR: dihydroflavonol-4-reductase; OMT: O-methyltransferase; and GST: glutathione S-transferase.
Figure 4
Figure 4
The expression profile of photosystem-related genes, including the PSA, PSB, LHC, ATPG, and ATPD genes.
Figure 5
Figure 5
Heat map diagrams of the relative expression levels of TFs annotated in anthocyanin biosynthesis in millet: bHLH, basic helix-loop-helix; bZIP, basic region/leucine zipper; C2H2, C2H2 zinc-finger proteins; C3H, Cys3His zinc finger; FAR1, far-red impaired response 1; TCP, TCP proteins; WD40, WD40 repeat proteins; MYB, MYB proteins; NAC, NAC proteins; and WRKY, WRKY proteins.
Figure 6
Figure 6
PCA of quantitative metabolite data and HMDB annotation of metabolites. (A,B): PCA of metabolites in positive ion mode and negative ion mode. (C,D): HMDB database annotations for metabolites in positive ion mode and negative ion mode.
Figure 7
Figure 7
qRT-PCR verification of 16 genes related to key metabolic pathways.
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