Proteomic analysis reveals that auxin homeostasis influences the eighth internode length heterosis in maize (Zea mays)

Abstract

Ear height is an important maize morphological trait that influences plant lodging resistance in the field, and is based on the number and length of internodes under the ear. To explore the effect of internodes on ear height, the internodes under the ear were analysed in four commercial hybrids (Jinsai6850, Zhengdan958, Xundan20, and Yuyu22) from different heterotic groups in China. The eighth internode, which is the third aboveground extended internode, exhibited high-parent or over high-parent heterosis and contributed considerably to ear height. Thus, the proteome of the eighth internode was examined. Sixty-six protein spots with >1.5-fold differences in accumulation (P < 0.05) among the four hybrids were identified by mass spectrometry and data analyses. Most of the differentially accumulated proteins exhibited additive accumulation patterns, but with epistatic effects on heterosis performance. Proteins involved in phenylpropanoid and benzoxazinoid metabolic pathways were observed to influence indole-3-acetic acid biosynthesis and polar auxin transport during internode development. Moreover, indole-3-acetic acid content was positively correlated with the eighth internode length, but negatively correlated with the extent of the heterosis of the eighth internode length.

Figure 1

Trait performance and mid-parent heterosis in four hybrids.

Figure 2

Comparison of internode length and plant height between the hybrids and parental inbred lines.

Figure 3

Two-dimensional maps of differentially accumulated proteins in the eighth internode of the hybrids and parental inbred lines. Proteins that accumulated differentially in hybrids and parental inbred lines are numbered.

Figure 4

Heat map and abundance patterns of differentially accumulated proteins in the eighth internode of the hybrids and parental lines. Note: Differences in accumulation are presented as a continuous range of colours. Red corresponds to the maximum accumulation (grey value: 449574); green corresponds to the minimum accumulation (grey value: 0); and yellow corresponds to the median accumulation (grey value: 224787).

Figure 5

Functional classification of all differentially accumulated proteins in the eighth internode of the hybrids and parental lines. Proteins were classified based on a maize protein database. Note: Proportions of different metabolic activities were calculated based on the number of identified proteins.

Figure 6

Summary of the phenylpropanoid metabolic pathway as well as the relative expression levels and patterns of key genes in the hybrids. Genes with the associated EC number are provided. Accessions encode the differentially accumulated proteins identified in this study. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl-alcohol dehydrogenase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; CHS, chalcone synthase; CHI, chalcone isomerase; and F3H, flavanone 3-hydroxylase.

Figure 7

Summary of the benzoxazinoid metabolic pathway as well as the relative expression levels and patterns of key genes in the hybrids. Genes with the associated EC number are provided. Accessions encode the differentially accumulated proteins identified in this study. BX1, benzoxazinless1; BX2 to BX5, cytochrome P450 enzymes; BX8 and BX9, UDP-glucosyltransferases; BX6, dioxygenase; BX7, O-methyltransferase; and DIBOA, 2,4-dihydroxy-2H-1,4-benzoxazin-3(4 H)-one.

Figure 8

Model of the development and heterosis of internodes. The development and heterosis of internodes are mainly regulated by an auxin gradient, which is the result of a balance between polar auxin transport (PAT) and auxin biosynthesis. In plant cells and organs, PAT is negatively regulated by flavonols, which are biosynthesised in the cytoplasm and stored in the vacuole. Transmembrane transport of flavonols to the vacuole is indirectly regulated by structural genes (e.g., BZ2) encoding glutathione S-transferases. Additionally, flavonol biosynthesis and BZ2 expression are regulated by a network of transcription factors. Once PAT is inhibited, auxin will accumulate in cells and organs to toxic levels. Moreover, excessive auxin concentrations induce ethylene production, which negatively regulates plant development. Thus, detoxification by benzoxazinoid metabolites and the competition for indole between the indole-3-acetic acid biosynthesis pathway and the benzoxazinoid metabolic pathway are required to protect cells and organs. Therefore, flavonols, which are synthesised in the phenylpropanoid metabolic pathway, and benzoxazinoids are important regulators of the development and heterosis of internodes via a complex network of key structural genes and transcription factors.