Uncovering the Gene Regulatory Network of Maize Hybrid ZD309 under Heat Stress by Transcriptomic and Metabolomic Analysis

Abstract

Maize is an important cereal crop but is sensitive to heat stress, which significantly restricts its grain yield. To explore the molecular mechanism of maize heat tolerance, a heat-tolerant hybrid ZD309 and its parental lines (H39_1 and M189) were subjected to heat stress, followed by transcriptomic and metabolomic analyses. After six-day-heat treatment, the growth of ZD309 and its parental lines were suppressed, showing dwarf stature and rolled leaf compared with the control plants. ZD309 exhibited vigorous growth; however, M189 displayed superior heat tolerance. By transcriptomic and metabolomic analysis, hundreds to thousands of differentially expressed genes (DEGs) and metabolites (DEMs) were identified. Notably, the female parent H39 shares more DEGs and DEMs with the hybrid ZD309, indicating more genetic gain derived from the female instead of the male. A total of 299 heat shock genes detected among three genotypes were greatly aggregated in sugar transmembrane transporter activity, plasma membrane, photosynthesis, protein processing in the endoplasmic reticulum, cysteine, and methionine metabolism. A total of 150 heat-responsive metabolites detected among three genotypes were highly accumulated, including jasmonic acid, amino acids, sugar, flavonoids, coumarin, and organic acids. Integrating transcriptomic and metabolomic assays revealed that plant hormone signal transduction, cysteine, and methionine metabolism, and α-linolenic acid metabolism play crucial roles in heat tolerance in maize. Our research will be facilitated to identify essential heat tolerance genes in maize, thereby contributing to breeding heat resistance maize varieties.

Keywords: gene regulatory network; grain yield; heat tolerance; hybrid; maize (Zea mays L.); metabolome; physical alterations; transcriptome.

Figure 1

The effects of heat stress on maize growth and physical characters in ZD309 and its parental lines. (A–C) Performance of maize plants treated by 6 days heat stress in H39_1 ((A), maternal line), M189 ((B), paternal line), and ZD309 (C); (D–M) Physical alterations mediated by heat treatments, including proline content (D), malondialdehyde content (E), H₂O₂ (F), soluble sugar (G), BCA protein (H), chlorophyll a/b content (I,J), as well as SOD, POD, and CAT activity. For the content of proline, soluble sugar content, BCA protein, chlorophyll a/b, the measurement unit, mg g⁻¹, represents the respective content in 1 g of sample’s fresh weight; For the content of malondialdehyde and H₂O₂, the measurement unit, μmol mg⁻¹, represents the respective content in 1 mg of sample’s fresh weight; For the activity of SOD and POD, the measurement unit, U mg⁻¹, represents the respective activity units detected in 1 mg of sample’s fresh weight; For CAT activity, the measurement unit, U min⁻¹ mg⁻¹, 1 activity unit (U) represents the A240 absorbance decreased 0.1 OD in 1 min and 1 mg of sample’s fresh weight. * and ** represent that the corresponding physical character in heat-stressed maize plants are significantly and very significantly different from the control at p < 0.05 and p < 0.01 levels, respectively.

Figure 2

DEGs identifications and gene ontology analysis in transcriptomic data. (A,B) Numbers of the identified DEGs in the tested three genotypes under D3HS and D6HS treatments. (C,D) Gene ontology analysis for the conserved DEGs within ZD309 and its parental lines in H3HS (C) and H6HS (D) treatments. The heatmap presents statistical significance (log10-transformed corrected p-value) of GO terms over-representation.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for the conserved DEGs within ZD309 and its parental lines in H3HS (A) and H6HS (B) treatments.

Figure 4

The conserved DEGs identified between the two heat treatments. (A) Numbers of DEGs between the two heat treatments in the tested three genotypes; (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for between the two heat treatments in the tested three genotypes. The x-axis indicated the rich factor, and the y-axis indicated pathway; (C) expression patterns of the identified 299 conserved DEGs. The color scale at the right of the figure indicates the gene expression levels transformed by log₁₀ (FPKM + 1).

Figure 5

Verification the expression of DEGs by qRT-PCR. Bars show standard error of the relative expression levels. * and ** represent that the corresponding gene expression level in heat-stressed maize plants are significantly and very significantly different from the control at p < 0.05 and p < 0.01 levels, respectively.

Figure 6

Comparison of the identified DEGs in ZD309 and its parental lines. (A) Numbers of DEGs of ZD309 and its parental lines in D3HS or D6HS treatment. (B) Numbers of up- and down-regulated DEGs of ZD309 and its parental lines in D3HS or D6HS treatment. (C) KEGG enrichment analysis for the up- and down-regulated DEGs in each genotype. The heatmap presents statistical significance (p-value) of KEGG pathway term over-representation.

Figure 7

DEMs and expression pattern identifications within ZD309 and its parental lines. (A) DEMs numbers of ZD309 and its parental lines in D3HS treatment; (B) DEMs numbers of ZD309 and its parental lines in D6HS treatment. (C) Conserved DEGs numbers of ZD309 and its parental lines between D3HS and D6HS. (D) Expression patterns of the conserved DEMs between the two heat treatments.

Figure 8

The relative expression levels of 7 fructose and mannose metabolism-related genes in the tested three plant materials. Data are presented as heatmaps of a log₂ transformed fold change.

Figure 9

The alterations of JA levels and metabolism pathway-related genes expression levels in response to heat stress. (A) JA levels in three genotypes at both heat treatments; (B) The relative expression of the genes involved in JA biosynthesis. Data are presented as a heatmap by a log₂ transformed fold change.