Integration of mRNA and microRNA analysis reveals the molecular mechanisms underlying drought stress tolerance in maize (Zea mays L.)

Abstract

Drought is among the most serious environmental issue globally, and seriously affects the development, growth, and yield of crops. Maize (Zea mays L.), an important crop and industrial raw material, is planted on a large scale worldwide and drought can lead to large-scale reductions in maize corn production; however, few studies have focused on the maize root system mechanisms underlying drought resistance. In this study, miRNA-mRNA analysis was performed to deeply analyze the molecular mechanisms involved in drought response in the maize root system under drought stress. Furthermore, preliminary investigation of the biological function of miR408a in the maize root system was also conducted. The morphological, physiological, and transcriptomic changes in the maize variety "M8186" at the seedling stage under 12% PEG 6000 drought treatment (0, 7, and 24 h) were analyzed. With prolonged drought stress, seedlings gradually withered, the root system grew significantly, and abscisic acid, brassinolide, lignin, glutathione, and trehalose content in the root system gradually increased. Furthermore, peroxidase activity increased, while gibberellic acid and jasmonic acid gradually decreased. Moreover, 32 differentially expressed miRNAs (DEMIRs), namely, 25 known miRNAs and 7 new miRNAs, and 3,765 differentially expressed mRNAs (DEMRs), were identified in maize root under drought stress by miRNA-seq and mRNA-seq analysis, respectively. Through combined miRNA-mRNA analysis, 16 miRNA-target gene pairs, comprising 9 DEMIRs and 15 DEMRs, were obtained. In addition, four metabolic pathways, namely, "plant hormone signal transduction", "phenylpropane biosynthesis", "glutathione metabolism", and "starch and sucrose metabolism", were predicted to have important roles in the response of the maize root system to drought. MiRNA and mRNA expression results were verified by real-time quantitative PCR. Finally, miR408a was selected for functional analysis and demonstrated to be a negative regulator of drought response, mainly through regulation of reactive oxygen species accumulation in the maize root system. This study helps to elaborate the regulatory response mechanisms of the maize root system under drought stress and predicts the biological functions of candidate miRNAs and mRNAs, providing strategies for subsequent mining for, and biological breeding to select for, drought-responsive genes in the maize root system.

Keywords: drought stress; mRNA; maize; miR408a; microRNA.

Figure 1

The morphology and physiochemical changes of maize variety “M8186” under drought stress. Values followed by different lowercase letters represent p ≤ 0.05.

Figure 2

The expression profile of drought stress-regulated DEMIRs and DEMRs in maize roots. (A, B) Column diagram representing the numbers of DEMIRs and DEMRs. (C, D) Venn diagrams representing the numbers of DEMIRs and DEMRs, and the overlaps of sets obtained across two comparisons. (E, F) Heat map of all miRNAs and all mRNAs expression profiles before and after drought stress.

Figure 3

GO and KEGG enrichment analysis across three time points during drought stress of maize plant roots. (A) Twenty representative profiles of different expression trends. The colored background parts showed significance, and the genes of the same type were gathered in the same cluster. (B, C) The most significantly enriched GO terms of DEMRs from the two comparison groups. (D, E) The most significantly enriched KEGG pathway of DEMRs from the two comparison groups.

Figure 4

miRNA and mRNA co-expression network diagram. Network analysis was performed using the Cytoscape network platform.

Figure 5

qRT-PCR analysis of DEMIs and DEMs in maize roots under drought stress. The 2^-ΔΔCt was used to calculate the fold change of expression in qRT-PCR analysis, with U6 and Actin as reference for miRNA and target genes, respectively. All experiments were repeated three times, and the expression data were log2 transformed before analysis. The error line is the standard error. The column charts represent qPCR data and heatmaps represent RNA-Seq data.

Figure 6

The key pathways of the drought stress response in maize. These pathways were “plant hormone signal transduction”, “phenylpropanoid biosynthesis”, “starch and sucrose metabolism”, and “glutathione metabolism”.

Figure 7

Analysis of phenotype and physiological and biochemical indexes of ZmmiR408a transgenic plants under drought stress. (A) Expression pattern analysis of ZmmiR408a in roots upon drought treatment. (B) Histochemical analysis of ZmmiR408a promoter: GUS transgenic maize roots were done under 0-h, 1-h, 3-h, and 5-h drought treatment. (C) Real-time quantitative PCR analysis of the expression level of ZmmiR408a gene in miR408a-OE and miR408a-KO plants. (D, E) Seed germination phenotypes of transgenic lines and wild-type plants under non-treatment and 12% PEG6000 treatment for 5 days. The scale bar represents 2 cm. (F, G) Analysis of wilting degree and recovery ability of transgenic lines and wild-type plants under drought treatment for 15 days and recovery for 5 days. All the plants were grown in a greenhouse at 25 ± 2°C under a 16-h light/8-h dark photoperiod. (H) Dry weight of wild-type, miR408a-KO, and miR408a-OE seedlings under normal and drought stress conditions. (I) Root length of wild-type, miR408a-KO, and miR408a-OE plants recorded after 15 days of non-treatment and drought treatment. (J) Phenotypes of plant height of wild-type, miR408a-KO and miR408a-OE plants under drought treatment in the field. (K, L) Grain phenotype and 100-seed weight analysis of wild-type, miR408a-KO, and miR408a-OE plants under drought treatment. (M) Seed vigor of wild-type, miR408a-KO and miR408a-OE plants under 12% PEG6000 treatment. A total of 0.4% TTC solution was used for staining observation of seed vigor. Control: maize kernels boiled in boiling water for 2 h; Untreatment: maize kernels without drought treatment. (N, O) Malondialdehyde (MDA) content and Proline (Pro) content in leaves of wild-type, miR408a-KO, and miR408a-OE plants under drought treatment. Data were expressed as the mean of triplicate values and error represented the SD, p < 0.05 (*) and p < 0.01 (**).

Figure 8

ZmmiR408a reduced drought tolerance by regulating ROS accumulation in maize. (A) Nitrotetrazolium Blue chloride (NBT) staining of wildtype, miR408a-KO, and miR408a-OE plants were conducted drought treatment for 15 days, which was used to monitor the ROS production in drought-treated leaves. (B, C) Hydrogen peroxide (H₂O₂) content and Superoxide radical (O₂ ⁻) content in leaves. (D–G) Analysis of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) activity in drought-treated leaves. (H–K) Analysis on expression patterns of ROS-related Marker genes in drought treated leaves. The expression level was normalized to that of Maize Actin. Data were expressed as the mean of triplicate values and error represented the SD, p < 0.05 (*) and p < 0.01 (**).