Grain size and grain weight
To elucidate the consequence of mutations on grain size development, we performed morphological analysis using tc19 and Chang7-2 in two locations for 2 years. We found that the length, width, thickness, and 100-kernel weight of the mature seeds of tc19 were significantly greater than in Chang7-2 (Table 1). Grain length in tc19 increased by 3.57%, grain width increased by 8.8%, and grain thickness increased by 3.88% compared with Chang7-2. The grain volume and 100-kernel weight of tc19 increased by 18.75 and 16.92%, respectively. However, ear length and ear weight in tc19 were significantly lower than in Chang7-2 (Table 1).
Environmental factors have a great influence on plant growth and development. In this study, the grain length, grain width, grain thickness, and 100-kernel weight of Chang7-2 and tc19 were influenced significantly by the environment. However, the grain length, grain width, grain thickness, and 100-kernel weight of tc19 were significantly greater than those of Chang7-2 in every environment (Fig. 1), indicating that grain size is mainly controlled by genetic factors.
Grain width changed most obviously between the mature seeds of tc19 and Chang7-2. To determine the stage at which this difference occurred, we measured the grain width from 14 to 28 days after pollination (DAP) every 7 days. Before 21 DAP, the grain width of tc19 was significantly smaller than that of Chang7-2. However, after 28 DAP, the grain width of tc19 was significantly larger than that of Chang7-2. The grain width of tc19 increased rapidly from 14 to 28 DAP, which ultimately contributed to the difference between tc19 and Chang7-2 (Fig. 2).
Plant endogenous hormones, indole-3-acetic acid (Auxin), gibberellins (GAs), cytokinin (CTK) and brassinosteroids (BR), play important roles in the regulation of seed size. To elucidate the effects of hormones between tc19 and Chang7-2, we detected the concentrations of mixed hormones at five stages after pollination (Table S1). The concentrations of auxin and BR in tc19 were higher than those in Chang7-2 at all five stages (Fig. 3). The concentration of auxin in tc19 peaked at 21 DAP, following which it decreased slightly at 28 and 35 DAP. The concentration of auxin in Chang7-2 also peaked at 21 DAP, reaching its lowest level at 28 DAP and then increasing slightly at 35 DAP (Fig. 3A). The concentration of GA decreased in tc19 but increased in Chang7-2 from 7 to 35 DAP. The concentration of GA in tc19 was significantly higher than that in Chang7-2 from 7 to 21 DAP and was no longer significant after 28 DAP (Fig. 3B). The concentration of CTK in tc19 was higher than that in Chang7-2 at 21 DAP, but it was not significantly different at 7, 14, 28, and 35 DAP (Fig. 3C). The concentration pattern of BR was similar to that of auxin (Fig. 3D).
Sequencing data quality assessment
First, all of the parameters for RNA quality met the library construction standards. After Illumina sequencing, we generated 20-35 M reads for the samples (Table 2). Q30 for all the samples ranged from 93.8 to 94.7%, the GC content ranged from 52.76 to 56.22% (Table S2). After alignment, more than 70% of the reads in each sample were aligned to the reference B73 genome sequence (Zm-B73-REFERENCE-NAM-5.0) (Table 2). PCA analysis successfully classified samples into different groups (Fig. S1). The pearson correlation among different groups were higher than 0.97 (Fig. S2). These results confirmed that the data were reliable.
Differential gene statistics
We then screened differentially expressed genes (DEGs) between Chang7-2 and tc19 at three stages after pollination. There were 2987, 2647, and 3209 DEGs at 14, 21, and 28 DAP, respectively. Compared with Chang7-2, 1201 genes increased and 1786 genes decreased in tc19 at 14 DAP. A total of 1647 genes increased and 1000 genes decreased in tc19 at 21 DAP, and 1995 genes increased and 1214 genes decreased in tc19 at 28 DAP (Fig. 4).
Gene ontology (GO) analysis
To know the function of the DEGs, we performed GO analysis (Fig. 5). The cellular components involved at 14 DAP include lipid particle, extracellular region, nucleus, cell wall, proteasome core complex, microtubule associated complex, external encapsulating structure. The molecular functions at 14 DAP are hydrolase activity, tetrapyrrole binding, DNA binding, transcription cofactor activity. The biological processes at 14 DAP include DNA duplex unwinding, carbohydrate metabolic process, axis specification and others (Fig. 5A).
The cellular components at 21 DAP include chromatin, extracellular region, chromosomal part, cell periphery et al. The molecular function of the DEGs at 21 DAP are related to oxidoreductase activity, protein dimerization activity, oxidoreductase activity, endopeptidase inhibitor activity et al. The biological processes of the DEGs at 21 DAP include transcription, nucleic acid-templated transcription, response to heat, regulation of hydrolase activity et al. (Fig. 5B).
The cellular components of the DEGs at 28 DAP include extracellular region, cell periphery, proteasome core complex, plasma membrane et al. The molecular function of the DEGs at 28 DAP are involved in oxidoreductase activity, tetrapyrrole binding, catalytic activity, peptidase regulator activity et al. The biological processes of the DEGs at 28 DAP include peptidase activity, proteolysis, hydrolase activity, catalytic activity et al. (Fig. 5C).
Kyoto encyclopedia of genes and genomes enrichment (KEGG) analysis
To determine the biochemical metabolic pathways and signal transduction pathways associated with the DEGs, we performed KEGG analysis. 556, 500 and 633 DEGs at 14, 21 and 28 DAP were pathway annotated, respectively. The DEGs at 14 DAP were enriched mainly in the phenylpropane biosynthesis pathway, plant hormone signal transduction, phenylalanine metabolism, and starch sucrose metabolism pathway (Fig. 6A). The DEGs at 21 DAP were enriched mainly in endoplasmic reticulum protein processing, plant hormone signal transduction, phenylpropanoid biosynthesis, and α-linolenic acid metabolic pathways (Fig. 6B). The DEGs at 28 DAP were enriched mainly in phenylpropanoid biosynthesis, plant hormone signal transduction, brassinosteroid synthesis, and α-linolenic acid metabolism (Fig. 6C). Above all, the DEGs in the hormone signal transduction pathway were significantly enriched. This indicated that the signal transduction pathway may play an important role in seed development.
Genes enriched in hormone signal transduction
We found a total of 77 DEGs related to the hormone signal transduction pathway (Fig. 7A). Among them, 27 genes were involved in the IAA signal transduction pathway; 5 genes were involved in the BR signal transduction pathway; 7 genes were involved in the CTK signal transduction pathway; 2 genes were involved in the GA signal transduction pathway; 6 genes were involved the abscisic acid (ABA) signal transduction pathway; 9 genes were involved in the ethylene (ET) signal transduction pathway; 11 genes were involved in the jasmonic acid (JA) signal transduction pathway; and 10 genes were involved in the SA signal transduction pathway.
We detected 27 DEGs involved in the IAA signal transduction pathway. ARF3 (Zm00001d012731) and IAA15 (Zm00001d039624) showed high expression levels. The expression level of ARF3 in tc19 was higher than that in Chang7-2. From 14 to 28 DAP, the expression of ARF3 was significantly increased in tc19, whereas it increased only slightly in Chang7-2 (Fig. 7B). The expression of IAA15 in Chang7-2 was higher than that of tc19 (Fig. 7C). AO2 (Zm00001d034388) in tc19 was higher than that in Chang7-2 (Fig. 7D). Endogenous hormone analysis showed that the BR concentrations of Chang7-2 and tc19 differed significantly. Analysis of the BR biosynthesis pathway indicated that DWF4 (ZM00001d003349) and XTH (Zm00001d014617) were highly expressed in tc19 than in Chang7-2 (Fig. 7E and F).
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.