The Zea mays mutants opaque2 and opaque16 disclose lysine change in waxy maize as revealed by RNA-Seq

Abstract

In maize, opaque2 (o2) and opaque16 (o16) alleles can increase lysine content, while the waxy (wx) gene can enhance the amylopectin content of grains. In our study, o2 and o16 alleles were backcrossed into waxy maize line (wxwx). The o2o2o16o16wxwx lines had amylopectin contents similar to those of waxy line. Their nutritional value was better than waxy line, but the mechanism by which the o2 and o16 alleles increased the lysine content of waxy maize remained unclear. The o2o2o16o16wxwx lines and their parents on kernels (18th day after pollination) were subjected to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed 272 differentially expressed genes (DEGs). Functional analyses revealed that these DEGs were mainly related to biomass metabolism. Among them, in o2o2o16o16wxwx lines, 15 genes encoding α-zein were down-regulated, which resulted in the reduction of α-zein synthesis and increased lysine content; lkr/sdh1 and Zm00001d020984.1 genes involved in the lysine degradation pathway were down-regulated, thereby inhibited lysine degradation; sh2, bt2 and ae1 genes involved in starch metabolism were upregulated, leaded to wrinkling kernel and farinaceous endosperm. Our transcriptional-level identification of key genes responsible for increased grain lysine content and farinaceous endosperm formation following introgression of o2 and o16 alleles should promote molecular breeding for maize quality.

Figure 1

Phenotypic features of o2o2o16o16wxwx lines and their parents. (A) Photographs of intact ears taken under normal light. (B) Light transmission of mature kernels on a light box. (C) Cross-sections of mature kernels on a light box, Bars = 1 cm.

Figure 2

(A) Scanning electron micrograph for endosperms of QCL3024, Taixi19, QCL5019, QCL8006_1 and QCL8006_2 at 700× magnification, respectively. (B) Scanning electron micrograph for endosperms of QCL3024, Taixi19, QCL5019, QCL8006_1 and QCL8006_2 at 1500× magnification. SG, starch granules; Blue arrows, matrix protein.

Figure 3

The contents of protein (A), starch (B) and 17 FAAs (C) in mature kernels of QCL5019, QCL8006_1 and QCL8006_2. *P < 0.05 and **P < 0.01.

Figure 4

(A) The column diagram of DEGs for QCL5019 vs. QCL8006_1 and QCL5019 vs. QCL8006_2. X axis represents pairwise and Y axis means number of screened DEGs. Blue bar denotes down-regulated genes and red bar for the up-regulated. (B) The intersection heatmap of DEGs for QCL5019 vs. QCL8006_1 and QCL5019 vs. QCL8006_2. Gradient color barcode at the right top indicates log2 (FC) value (FC, Fold change of expression in triple recessive mutant vs waxy parent). Each row represents a DEG and each column represents one condition pairwise. DEGs with similar fold change value are clustered both at row and column level.

Figure 5

The GO analysis of differentially expressed genes. X axis represents GO terms. Y axis means number of DEGs (the number is presented by its square root value). All GO terms are grouped into three ontologies: brown is for biological process, orange is for cellular component and blue is for molecular function.

Figure 6

Pathway analysis of differentially expressed genes. X axis means number of DEGs. Y axis represents second KEGG pathway terms. All second pathway terms are grouped in top pathway terms indicated in different color.

Figure 7

(A) The qRT-PCR log2 ratio of 17 DEGs for QCL5019 vs. QCL8006_1 and QCL5019 vs. QCL8006_2. (B) The RNA-Seq log2 ratio of 17 DGEs for QCL5019 vs. QCL8006_1 and QCL5019 vs. QCL8006_2. (C) The qRT-PCR validation of 17 DGEs for QCL5019 vs. QCL8006_1 identified by RNA-seq. Pearson’s r = 0.8409. (D) The qRT-PCR validation of 17 DGEs for QCL5019 vs. QCL8006_2 identified by RNA-seq. Pearson’s r = 0.8377. P1-P17, 17 DEGs.

Figure 8

A proposed model of the regulatory network of waxy corn following the introgression of the o2 and o16 alleles. MAS, Marker-assisted selection; FS, Foreground selection; BS, Background selection; RNA-seq, RNA sequencing; ↑, -up-regualted; ↓, down-regulated.