Dissecting Heterosis During the Ear Inflorescence Development Stage in Maize via a Metabolomics-based Analysis

Abstract

Heterosis can increase the yield of many crops and has been extensively applied in agriculture. In maize, female inflorescence architecture directly determines grain yield. Thus, exploring the relationship between early maize ear inflorescence development and heterosis regarding yield-related traits may be helpful for characterizing the molecular mechanisms underlying heterotic performance. In this study, we fine mapped the overdominant heterotic locus (hlEW2b), associated with ear width, in an approximately 1.98-Mb region based on analyses of chromosome segment substitution lines and the corresponding testcross population. Maize ear inflorescences at the floral meristem stage were collected from two inbred lines, one chromosome segment substitution line that carried hlEW2b (sub-CSSL₁₆), the receptor parent lx9801, and the Zheng58 × sub-CSSL₁₆ and Zheng58 × lx9801 hybrid lines. A total of 256 metabolites were identified, including 31 and 24 metabolites that were differentially accumulated between the two hybrid lines and between the two inbred lines, respectively. Most of these metabolites are involved in complex regulatory mechanisms important for maize ear development. For example, nucleotides are basic metabolites affecting cell composition and carbohydrate synthesis. Additionally, nicotinate and nicotinamide metabolism is important for photosynthesis, plant stress responses, and cell expansion. Moreover, flavonoid and phenolic metabolites regulate auxin transport and cell apoptosis. Meanwhile, phytohormone biosynthesis and distribution influence the cell cycle and cell proliferation. Our results revealed that changes in metabolite contents may affect the heterotic performance related to ear width and yield in maize hybrid lines. This study provides new clues in heterosis at the metabolomics level and implies that differentially accumulated metabolites made distinct contributions to the heterosis at an early stage of ear inflorescences development.

Figure 1

Development of a CSSL testcross population and identification of heterotic loci. The Zheng58 × CSSL₁₂₅ population exhibited significant heterosis for maize ear width. The sub-CSSL population was constructed from 152 homozygous sub-CSSL F₂ plants, which were derived from a cross between CSSL₁₂₅ and lx9801. The corresponding test population was constructed by crossing the test parent Zheng58 with sub-CSSLs. Red, green, and blue represent the genomes of lx9801 (receptor parent), Chang7-2 (donor parent), and Zheng58 (test parent), respectively.

Figure 2

Principal component analysis score plots. The first two principal component (PCs) explained 42.4% of the variation. Additionally, PC1 separated the hybrid lines from the inbred lines, and explained 28.6% of the variation. Meanwhile, PC2 explained 13.8% of the variation, indicating the hybrid lines could be discriminated from the inbred lines. Three biological replicates of each sample were clustered in the same quadrant. HY: Zheng58 × sub-CSSL₁₆; CK: Zheng58 × lx9801.

Figure 3

Partial least squares discriminant scores plot. (A) Principal component 1 (PC1) and PC2 explained 31.4% and 16.4% of the variation, respectively, indicating Zheng58 × sub-CSSL₁₆ (HY) could be discriminated from Zheng58 × lx9801 (CK). (B) PC1 and PC2 explained 21% and 18.2% of the variation, illustrating the separation between sub-CSSL₁₆ and lx9801. R²X, R²Y, and Q² refer to the interpretation and predictability values of the partial least squares discriminant analysis models.

Figure 4

Differentially accumulated metabolites between hybrid and inbred lines. (A) Venn diagram of differentially accumulated metabolites in sub-CSSL₁₆ and lx9801 as well as in Zheng58 × sub-CSSL₁₆ (HY) and Zheng58 × lx9801 (CK). (B) Number of differentially accumulated metabolites in hybrids and inbred lines.

Figure 5

Integrated metabolite changes and differentially expressed genes for analyzing the secondary metabolite biosynthetic pathways. Differentially accumulated metabolites indicated in red and blue were upregulated and downregulated, respectively. The red-to-blue colors in squares indicate high-to-low gene expression levels, respectively. (A) surE, 5′-nucleotidase; IMPDH, inosine-5′-phosphate dehydrogenase; GMPS, guanosine 5′-monophosphate synthetase; APRT, adenine phosphoribosyl transferase; ADA, adenosine deaminase; ADK, adenosine kinase; CYP735A, cytokinin trans-hydroxylase; (B) NMNAT, nicotinamide mononucleotide adenylyltransferase; SDT1, pyrimidine and pyridine-specific 5′-nucleotidase; URH1, uridine nucleosidase; PNC1, nicotinamidase; NNM, nicotinamide N-methyltransferase.

Figure 6

Relative expression levels of genes in secondary metabolite biosynthetic pathways as determined by qRT-PCR. Relative expression levels are presented as the mean of three replicates ± standard error. HY: Zheng58 × sub-CSSL₁₆; CK: Zheng58 × lx9801. * and **Indicate significant differences at P < 0.05 and P < 0.01, respectively, as determined by a one-way ANOVA with Student’s t-test.