Association of the molecular regulation of ear leaf senescence/stress response and photosynthesis/metabolism with heterosis at the reproductive stage in maize

Abstract

Maize exhibits a wide range of heterotic traits, but the molecular basis of heterosis at the reproductive stage has seldom been exploited. Leaf senescence is a degenerative process which affects crop yield and quality. In this study, we observed significantly delayed ear leaf senescence in the reciprocal hybrids of B73/Mo17 and Zheng58/Chang7-2 after silking, and all the hybrids displayed larger leaf areas and higher stems with higher yields. Our time-course transcriptome analysis identified 2,826 differentially expressed genes (DEGs) between two parental lines (PP-DEGs) and 2,328 DEGs between parental lines and the hybrid (PH-DEGs) after silking. Notably, several senescence promoting genes (ZmNYE1, ZmORE1, ZmWRKY53 and ZmPIFs) exhibited underdominant expression patterns in the hybrid, whereas putative photosynthesis and carbon-fixation (ZmPEPC)-associated, starch biosynthetic (ZmAPS1, ZmAPL), gibberellin biosynthetic genes (ZmGA20OX, ZmGA3OX) expressed overdominantly. We also identified 86 transcription factors from PH-DEGs, some of which were known to regulate senescence, stress and metabolic processes. Collectively, we demonstrate a molecular association of the regulations of both ear leaf senescence/stress response and photosynthesis/metabolism with heterosis at the late developmental stage. This finding not only extends our understanding to the molecular basis of maize heterosis but also provides basic information for molecular breeding.

Figure 1. Heterotic phenotypes of maize hybrids at the reproductive stage.

(a) The photograph shows four inbred lines and their reciprocal hybrid lines at silking stage. Z58 and C7-2 represent Zheng58 and Chang7-2, respectively. (b) Phenotypes of ear leaves from different lines at the indicated time points after silking (weeks after silking, WAS). (c) Chlorophyll contents of ear leaves from different genotypes at the indicated time points. (d,e) Leaf widths and lengths of different genotypes. “Third down” and “Third up” refer to the third leaves down and up from the ear leaves, respectively. (f) Grain yields per plant and thousand-kernel weights of different genotypes. All the analyses involve three biological replicates and error bars represent the standard deviation.

Figure 2. Gene ontology enrichment analysis of differentially expressed genes (DEGs) between B73 and Mo17.

(a) 1205 genes showed higher expressions in Mo17 than B73, 147 of them were shared by all three stages. Top 5 enriched GO categories were listed in the figure, while 147 genes in (a) only showed enrichment of three GO categories. (b) 1576 genes showed higher expression in B73 than Mo17, 231 of them were shared by three stages. Top 5 enriched GO categories are listed in the figure.

Figure 3. Clustering and functional category enrichment analysis of DEGs between parental lines and the hybrid (PH-DEGs).

(a) The percentages (%) in the graph represent the ratios of additive genes and non-additive genes during S0 to S2. (b) Six clusters were identified along the three developmental stages using the K-Means clustering algorithm. (c) Functional categories enrichment (modified MapMan bins) among six clusters according to the MapMan annotations, the color key represents (−log₁₀ P).

Figure 4. Expression levels of Chlorophyll biosynthesis- and degradation- associated genes.

(a) Chlorophyll synthetic pathway in Arabidopsis and putative maize homologous genes. (b) Expression patterns of several putative maize chlorophyll synthetic genes. (c) A diagram illustrating the chlorophyll degradation pathway in Arabidopsis. NYE1: Non-Yellowing 1; PPH: pheophytin pheophorbide hydrolase; PAO: pheophorbide a oxygenase; RCCR: red chlorophyll catabolite reductase; pFCC: primiary fluorescent catabolites. (d) Expression patterns of putative ZmNYE1, ZmPAO, and ZmPPH.

Figure 5. Expression levels of photosynthesis-associated enzyme genes.

(a) A diagram shows the intermediates and key enzymes involved in calvin cycle in maize. (b) Overdominant expression patterns of six genes encoding carbon fixation- and calvin cycle-associated enzymes. PEP: phosphoenolpyruvate; OAA: oxaloacetate; Asp: aspartic acid; Asn: Asparagine; RuBP: ribulose-1,5-bisphosphate; PGA: phosphoglycerate; G-3-P: glyceraldehyde-3-phosphate; DHAP: Dihydroxyacetone phosphate; F-1,6-P: fructose-1,6- phosphate; F-6-P: fructose-6-phosphate; Xu5P: Xylulose 5-phosphate; AST: aspartate transaminase; Rubisco: Ribulose bisphosphate carboxylase oxygenase; PGK: 3-phosphoglycerate kinase; RPE: ribulose-5-phosphate epimerase; PEPC: phosphoenolpyruvate carboxylase; TRK: Transketolase; FBA: fructose-bisphospate aldolase; PRK: phosphoribulokinase.

Figure 6. Expression levels of senescence-associated genes.

(a) Expression levels of six putative senescence-associated positive regulators in maize. (b) Expression levels of putative cytokinin-related negative regulators of senescence. (c) Expression levels of ethylene biosynthesis- and signaling-related genes.

Figure 7. Dynamic changing inventories of differentially expressed transcription factors.

(a) Distribution of differentially expressed transcription factor families at S0, S1 and S2. Fisher’s exact test was used to compare the ratios of TF families in our list to their ratios of total identified TF families, *p < 0.05. (b) Representative transcription factors with non-additive expression patterns in the hybrid.