Transcriptome and co-expression network analyses of key genes and pathways associated with differential abscisic acid accumulation during maize seed maturation

Abstract

Background: Currently, mechanical maize kernel harvesting has not been fully utilized in developing countries including China, partly due to the absence of suitable cultivars capable of rapid desiccation during seed maturation. The initiation of rapid desiccation during seed maturation is regulated by abscisic acid (ABA). For further characterization of ABA-regulated key genes and cellular events, it is necessary to perform transcriptome analysis of maize developing embryos. The ABA synthesis-deficient mutant (vp5) and normal maize (Vp5) seeds are suitable materials for such purpose.

Results: In the present work, developing vp5 and Vp5 embryos were compared by ABA content and transcriptome analyses. Quantitative analysis revealed the significant difference in ABA synthesis between both genotypes. From 29 days after pollination (DAP), ABA content increased rapidly in Vp5 embryos, but decreased gradually in vp5 embryos. At 36 DAP, ABA level in vp5 decreased to 1/4 that of Vp5, suggesting that the differential ABA levels would affect seed maturation. Comparative transcriptomic analysis has found 1019 differentially expressed genes (DEGs) between both genotypes, with the most DEGs (818) at 36 DAP. Further, weighted correlation network analysis (WGCNA) revealed eight DEGs co-expression modules. Particularly, a module was negatively correlated with ABA content in vp5 embryos. The module was mainly involved in metabolic and cellular processes, and its hub genes encoded thiamine, NPF proteins, calmodulin, metallothionein etc. Moreover, the expression of a set of key genes regulated by ABA was further verified by RT-qPCR. The results of the present work suggested that because of ABA deficiency, the vp5 seeds maintained strong metabolic activities and lacked dormancy initiation during seed maturation.

Conclusion: Transcriptome and WGCNA analyses revealed significant ABA-related changes in metabolic pathways and DEGs between vp5 and Vp5 during seed maturation. The results would provide insights for elucidating the molecular mechanism of ABA signaling and developing high dehydration tolerance maize suitable for mechanical harvesting.

Keywords: Abscisic acid (ABA) synthesis deficiency; Differential expressed genes (DEGs); Seed maturation; Transcriptome; Weighted correlation network analysis (WGCNA); Zea mays.

Fig. 1

The change in ABA content during Vp5 and vp5 seed maturation. A, young embryos sampled at 15, 22, 29 and 36 DAP, respectively. B, comparison of ABA content between Vp5 and vp5 embryos

Fig. 2

Validation of expression levels of 12 randomly selected DEGs by RT-qPCR. Relative gene expression levels were represented in column. The inserted heatmap was drawn based on RNA-Seq results. Error bar represents standard deviation (n = 3). A, Vp5; B, vp5

Fig. 3

Principal component analysis (PCA) of RNA-seq data

Fig. 4

Summary of the genes detected in embryos during seed maturation in Venn diagram

Fig. 5

The number of DEGs identified between Vp5 and vp5 embryos. The changes in gene expression levels were calculated by Vp5 versus vp5

Fig. 6

GO enrichment analysis of DEGs in Vp5 versus vp5 at 36 DAP

Fig. 7

KEGG pathway analysis of DEGs in Vp5 versus vp5 at 36 DAP

Fig. 8

Network analysis of dendrogram showing modules identified using WGCNA. A, Hierarchical cluster tree showing co-expressing modules. B, Heatmap of module-ABA weight correlations and corresponding P-values

Fig. 9

Characterization of genes in midumpurple3 module. A, GO enrichment analysis. B, Heatmap of gene expression profile. C, Co-expression network analysis

Fig. 10

RT-qPCR verification of the expression of key genes regulated by ABA. Relative gene expression levels were represented in column. The inserted heatmap was drawn based on RNA-Seq results. Error bars represent standard deviation (n = 3). Asterisks indicate levels of significance of differential expression (Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.01)