Dynamic Patterns of Gene Expression and Regulatory Variation in seed coat of Maize

doi:10.21203/rs.3.rs-1768077/v1

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Dynamic Patterns of Gene Expression and Regulatory Variation in seed coat of Maize

https://doi.org/10.21203/rs.3.rs-1768077/v1

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Background：Seed size is an important factor contributing to maize yield, of which the molecular mechanism remains elusive. Seed coat, serves as one of the three components of maize grain, determines the seed size to a certain extent. The seed coat is a complete maternal genotype and is an ideal material for studying heterosis.

Results

In this study, the self-pollinated seeds of maize hybrid Yudan888 and its parental lines were continuously collected from 0DAP to 15DAP for phenotyping, cytological observation and RNA-seq. The phenotypic data showed that 3DAP and 8DAP are the best time points to study maize seed coat heterosis. Cytological observations indicated that heterosis in maize seed coat was dependent much on cell numbers other than cell size. Furthermore, RNA-seq indicats that the number of non-additive genes changed significantly. However, the number of genes expressed in heterozygotes and the number of genes expressed additively were not significantly different. Our findings suggest that seed coat heterosis, in hybrid Yudan888, is the result of non-additive expression caused by the dynamic changes of genes at different time points along seed expanding and seed coat development. This dynamic change suggests that a dose effect of gene expression results in Yudan888 seed coat heterosis. Go enrichment and KEGG analysis indicated that genes related to DNA replication, cell cycle regulation, circadian rhythms and metabolite accumulation contributed significantly to Yudan888 seed coat heterosis.

Conclusion

The maize seed coat area phenotyping allows us to infer that 3DAP and 8DAP are important time points in the study of seed coat heterosis. Our findings provide evidence for genes involved in DNA replication, cell cycle regulation, circadian rhythms and metabolite accumulation in hybrid with high or low parental expression as major contributors to Yudan888 seed coat heterosis.

Maize

Seed coat

Heterosis

RNA-seq

Seed size is an important component of grain yield and a key determinant trait for crop domestication [1]. Maize seeds consist three components: the embryo, which is the beginning of next generation; the endosperm, which provides nutrition for growth and germination of the embryo; and the seed coat, covering embryo and endosperm. The seed coat is developed from the integument with maternal genotype [2]. Ovules in angiosperms generally have two integuments, with the inner integument undergoes an additional periclinal division, resulting in a three-layered structure [3]. Seed size is co-ordinately determined by the development of triploid endosperm, diploid embryo, and seed coat [4, 5]. The seed coat protects the embryo and endosperm and grows co-ordinately with them. It plays an important role in the seed filling process [6]. Therefore, the seed coat determines the yield of cereal crops to a certain extent.

Heterosis showed that the hybrid F1 generation was superior to both of the parents in terms of viability, plant height, yield, stress and disease resistance, etc. [7]. Heterosis has been widely used in plant breeding, however, the genetic mechanism and molecular basis of heterosis remain enigmatic [8]. It is hypothesized that the difference in gene expression between hybrid and its parents is one of the main causes of heterosis [9, 10]. Genetic models to explain the increased yield of hybrid consider the interaction of alleles at many loci generating altered expression levels and patterns [11, 12]. Whole-genome transcriptomes of parents and hybrid have shown that genes with altered expression were over-represented in processes such as energy metabolism and transport [4]. In addition, changes in epigenetic states such as DNA methylation, small RNA production and histone modification have been found in hybrid genome [13, 14]. Although extensive research on the mechanism of heterosis have been conducted, it remains a study hot-spot.

Genetic mapping has been used to identify the detailed gene loci contributing to hybrid performances and investigate their mechanisms, in a number of plant species including Arabidopsis, rice, maize, sorghum, tomato, and etc. [15-19]. Rice GAI homologue ectopically expressed in F1 generation of Arabidopsis thaliana causes significant plant height changes [15]. The major quantitative trait gene Ghd7, which encodes a CCT domain protein in rice, was identified as a key factor for improving yield and adaptability of excellent hybrid Shanyou63 and other indica rice varieties [16]. The orthologue gene of flowering locus SFT for tomato [17] and Hd3a for rice [18], respectively, has been show as single-gene over-dominance in yield traits.

The final seed size is based on the coordinated development of embryo, endosperm and seed coat [5]. Of these three components, the development of seed coat and endosperm takes precedence over the embryo. In the early stage of seed development, from double fertilization to 6 days after pollination (DAP), the growth of seed coat is accompanied with the enlargement of endosperm. Within 72 hours after fertilization, the endosperm converts to the multinuclear stage [20]. 3-6 DAP, the endosperm of maize is in the stage of cellularization, during which the cytoskeleton recognizes the nuclei and under the traction of the microtubule system formed between the nuclei. All nuclei are separated and cytoplasmic division is carried out at the same time to form normal cells [21]. 8-12DAP is the stage of endosperm cell differentiation, during which nutrients and storage materials are accumulated along with cell proliferation and enlargement [22]. The morphological changes of the seed coat occurred only when the endosperm began to develop, and the development of the embryo did not promote the growth and differentiation of the seed coat [23, 24]. Thus, the seed coat is developed from the integument, and coincides with the enlargement of seed in the early stage of development. The seed coat regulates the accumulation of phloem assimilates in seeds during seed development [25]. At the beginning of filling stage of 12DAP, the seed coat thickness was continuously compressed until finally the seed coat thickness decreased to only a few layers of cells. At this stage, the seed coat cell wall thickened, providing physical protection for future seed dormancy. The consumption of seed coat during development may be to use its own starch for endosperm expansion and to provide more space for endosperm development. Seed coat is a complete maternal genotype. Due to its genetic stability and fixed heterosis, the seed coat of F2 seed obtained by self-pollination of F1 plant is in the stage of F1 heterozygosity, which is a good model for studying heterosis. Seed size determined by seed coat area is thus a typical heterosis trait. Whereas, the genetic basis of heterosis for seed coat has remained largely uncharacterized.

An impressive array of genomic and regulatory variations in kernel traits has been observed between maize inbred lines. However, how these variations affect gene expression in hybrid remains unknown. To this end, this study attempted to better define gene expression variations between seed coats of maize inbred lines, how these variations affect gene expression levels in F1 hybrid, and the relative contributions of cis- and trans-variantsto seed coat heterosis. Since the seed coat genotype is completely derived from the maternal genome, which is an important part of the seed. The parental selfing is a pure genotype and the hybrid after self-pollination is a heterozygous genotype. Together with the fact that seed size is a heterosis trait, the seed coat is an ideal material for studying heterosis. In this study, the self-pollinated kernels of Yudan888 hybrid and its parental lines were continuously collected from 0DAP to 15DAP for phenotyping, cytological observation and RNA-seq. The phenotypic data showed that 3DAP is the important time point for the expantion of the seed coat area of hybrid, and 8DAP is the key time point when the seed coat area of the hybrid significantly extending from its parents. 3DAP and 8DAP are the best time points to study maize seed coat heterosis. In addition, 8DAP cytological observations indicated that heterosis in maize seed coat area was dependent more on cell number other than cell size. Furthermore, results of 3DAP and 8DAP RNA-seq indicated that the number of genes expressed non-additively changed significantly in hybrid. However, the number of additive genes were not significantly different expressed. Our findings suggest that seed coat heterosis is the result of non-additive expression caused by the dynamic changes of genes at different time points. This dynamic change suggests that a dose effect of gene expression results in heterosis. Go enrichment and KEGG analysis indicated that at 3DAP, the early stage of seed coat development, genes related to DNA replication and cell cycle regulation contributed significantly to heterosis. While at 8DAP, hybrid weakened the cell cycle by enhancing metabolite accumulation combined with increased kernel weight and stabilize heterosis.We expect that this transcriptome comparison provides a new step towards understanding the causative mechanisms of altered gene expression in hybrid and the molecular mechanisms of maize seed coat heterosis.

The seed coat area of hybrid and its parents was significantly different on 8DAP.

Seed coat area and kernel weight from 0 to 15DAP of hybrid and parental lines were measured continually (Fig.1A). At 0DAP, seed coat area of hybrid was lower than both mid-parents value (MPV) and maternal line. On 6DAP, the hybrid values began to exceed the MPV but were still lower than maternal line. With the expansion of seed coat, hybrid value continued to be higher than MPV on 7DAP and slightly exceeded the maternal line. On 8DAP, there was a statistical difference (P value = 0.034) between the hybrid and the MPV of seed coat area (Fig.1B). From 9 to 15DAP, the seed area of the hybrid was more and more different from the MPV and paternal line values (Table S1). In conclusion, 8DAP is the inflection point of heterosis in maize seed coat, and also the entry point of heterosis in seed coat of maize. The kernel weight of hybrid was statistically different from that of mid-parents after 10DAP (Fig.1D). These results indicated that the heterosis of seed coat area was prior to seed weight.

In seed coat area, the relative growth rate of hybrid and paternal line peaked at 3DAP, and the maternal line grew most rapidly on 2DAP (Fig.1C). In terms of the relative increasing rate of kernel weight, the value of hybrid was the highest on 3DAP, 2DAP for maternal line and 4DAP for paternal line (Fig.1E). In general, seed coat area and kernel weight showed a large increase in 2-4DAP. More importantly, 3DAP is a critical time point for hybrid seed coat development.

Relative cell numbers in seed coat

In order to understand whether the increased seed coat area in hybrid was due to changes in cell number or cell size, we counted the seed coat cell number per unit area (0.1mm²) (Fig. 2). The middle longitudinal section of seed embryos collected on 8DAP were performed (Fig.2A-C). Surprisingly, the number of cells per unit area of the paternal line (319 cells per 0.1mm²) was higher than that of the hybrid (149.7 cells per 0.1mm²), the maternal line (138 cells per 0.1mm²) and the MPV (228.5 cells per 0.1mm²) on 8DAP (Fig. 2D). Although there was no difference in number of cells per unit area between hybrid and maternal line at 8DAP, there was a significant difference in seed coat surface area between them (Fig.2D, E). Thus, compared with maternal line, the cell number of hybrid is determined by its larger seed coat area. The difference in cell number between hybrid and MPV was significant, suggesting that the difference in seed coat size between hybrid and parents was mainly due to cell number rather than cell size.

Introduction of RNA-seq data

To investigate the expression of genes before and after the time points at which the seed coat of hybrid lines and parents showed statistical differences, we performed RNA-seq on 18 samples of 6 time points (2-4DAP and 8-10DAP) of hybrid and parental lines, with 3 biological replicates for each sample. 54 libraries in total, 2.4 billion high-quality reads were generated. The reads were then mapped to the maize B73 reference genome (RefGen_V4 [26]) by Hisat [27]. An average (87%) of reads were uniquely mapped (Table S2) to calculate the normalized gene expression level as fragments per kb of transcript per million mapped reads (FPKM). There was a high correlation among the three biological replicates in each time point (average R²=0.93) (Table S3). All the above results indicated that the sampling quality of this survey was exact, and RNA-seq sequencing data were accurate and reliable. Thus, we took the average FPKM value of the three replicates as the expression level for the sample at each time point.

Expressed genes

The number of expressed genes of Yudan888 hybrid and parental lines at 6 time points were investigated. Followed by the number of overlapping expressed genes of each material counted. The results showed that the total number of genes expressed by hybrid, maternal and paternal line at six time points were 23516, 23596 and 23247, respectively. 17691, 18328 and 17604 genes were overlapped at 6 time points, accounting for 75.23%, 77.67% and 75.73% of the total expressed genes (Fig.3A-C), respectively. Taken together, the results of overlapping expressed genes in hybrid and parental lines showed that the growth and development of seed coat was mainly due to gene expression quantity rather than the change of gene expression type and number.

Differentially expressed gene identification

It was suggested that the hybrid showed statistical differences with its parents on 8DAP, while the relative growth rate of hybrid seed coat area and kernel weight reached the peak at 3DAP. Therefore, we focused on RNA-seq data of 3DAP and 8DAP for subsequent analysis. In DEG analysis, the gene expression amounts in hybrid were compared one-by-one with their parental lines, setting the difference fold change (FC) ≥ 2 and (FC) ≤ -2 as up-regulated expression and down-regulated expression, respectively. Some DEGs were high or low expressed either in hybrid or parental lines. Among 3DAP, a total of 3915 DEGs varied between hybrid and maternal line (2134 (54.50%) up-expressed and 1781 (45.50%) down-expressed, 2647 DEGs varied between hybrid and paternal line (1248(47.15%) up- expressed and 1399 (52.85%) down-expressed). Compared with 3DAP, the proportion of differential genes up-regulated in 8DAP increased sharply. 2182 DEGs varied between hybrid and maternal line (1499(68.70%) up- expressed and 683(31.30%) down-expressed), and 2724 DEGs varied between hybrid and paternal line (1847(67.80%) up- expressed and 877(32.20%) down-expressed) (Fig.4A). The differences vary greatly in the reconciliation and/or down regulation of gene expression between hybrid and different parent combinations in the comparison of 3DAP and 8DAP. Some DEGs have genotype specific expression and thus identified as genotype specific uni-genes (Fig.4B). Venn diagram analysis indicated that 1156 genes showed DEGs (highlighted as black bold font) between hybrid and parents at both time points (Fig.4B). Out of the 1156 genes, 834 DEGs (sum of bold font numerals) were commonly observed between M vs H and P vs H at 3DAP. Whereas, 430 DEGs (sum of bold font numerals) were commonly observed between M vs H and P vs H at 8DAP (Fig.4B). In hybrids at both time points, 108 DEGs were found commonly differentially expressed (Fig.4B).

Expression patterns of DEGs

In order to explore and categorize the expression alteration trend, DEGs differentially expressed in 3DAP and 8DAP samples were divided into 13 possible hybridization and parental expression patterns (Table 1). Gene expression patterns were divided into additive expression and non-additive expression. We investigated that there were no significant changes in the number of additive expressed DEGs between the two time points. The expression pattern of most genes in hybrid at two time points was additive expressed (85.18% at 3DAP and 86.45% at 8DAP). However, the effects of non-additive DEGs are quite different, especially the dominantly expressed ones (classes 5, 7, 10 and 12). That is to say, the gene expression in hybrid is similar to that of one parent, which accounts for more than 13% of all DEGs at both time points. On 3DAP, the number of higher paternal dominantly expressed genes accounted for the highest proportion in non-additive pattern, while on 8DAP, the number of higher maternal dominance genes was the most abundant. In the number of over-dominantly expressed genes in transitional down expression (0.35% at 3DAP and 0.14% at 8DAP) is relatively higher than that in transitional up expression (0.10% at 3DAP and 0.08% at 8DAP).

We summarized 13 possible hybridization and parental expression patterns into mid-parent, high-parent and low-parent to analyse the expression changes at either time points. By comparing the number of DEGs in three expression patterns at two time points, it was found that the number of low-parents DEGs changed the most, followed by the high-parents DEGs, while the number of mid-parents expressed DEGs changed the least (Table S4). According to the analysis of the fold changes at two time points, it was suggested that the change of the number of DEGs expressed by low-parents and high-parents may play a role in Yudan888 seed coat heterosis.

Functional classification by gene ontology and KEGG pathway enrichment analysis

The DEGs with high and low parental expression at 3DAP and 8DAP were annotated by Gene Ontology (GO). The enrichment of GO with low-parental expression of 3DAP showed that a large number of DEG categories related to stress were significantly enriched in biological process, followed by cellular component, and categories related to plasma membrane (Fig.5A). 3DAP highly expressed GO enrichment results suggested that categories related to chromosome assembly and processing were enriched in cellular component, and categories related to cell cycle and DNA replication were enriched in biological process (Fig.5B). The enrichment results of GO with low-parental expression of 8DAP demonstrated that the categories related to cytoskeleton and microtubule were enriched in cellular component, and the categories enriched in biological process were mainly related to cell cycle, nuclear division and microtubule (Fig.5C). 8DAP highly expressed GO enrichment results indicated that categories related to metabolism were enriched (Fig.5D).

Leverage OmicShare Tools (https://www.omicshare.com/tools/Home/Report/ koenrich) Dynamic KEGG pathway enrichment analysis was carried out to further understanding of gene biological function and gene interactions. We performed KEGG enrichment analysis on DEGs with high and low-parental expression at 3DAP and 8DAP time points, respectively (Fig.6). Among 3DAP low-parental expressed DEGs, metabolic pathways, biosynthesis of secondary metabolites, protein processing in endoplasmic reticulum and glutathione metabolism were significantly enriched (Fig.6A). In the 3DAP high-parental expressed mode, biosynthesis of secondary metals, DNA replication and photosynthesis were significantly enriched (Fig.6B). Metabolic pathways, biosynthesis of secondary metabolites, alpha linolenic acid metabolism were significantly enriched in 8DAP low-parental expressed DEGs (Fig.6C). By contrast to 3DAP high parental expression, DNA replication was also enriched in 8DAP low-parental expression mode. In 8DAP high-parental expression pattern, among which the circadian rhythms, biosynthesis of secondary metals, glycolysis/gluconeogenesis, thiamine metabolism, metabolic pathways, galactose metabolism and other pathways were significantly enriched (Fig.6D).

Weighted gene co-expression network analysis (WGCNA)

In order to identify the specific genes highly related to seed coat area, we constructed co-expression networks by using the transcriptome data of 6 time points between hybrid and its parents, and associated the co-expression module with seed coat area traits. The results showed that these genes should to be divided into 41 modules (Fig.S1). By observing the correlation between modules and samples, it is found that the absolute value of the correlation coefficient between MEyellow (0.86) and MEsienna3(0.82) modules is the highest (Fig.S1). In other words, these two modules have the highest correlation with seed coat area. In these two modules, the genes with the top 100 weight values were screened to construct the regulation network diagram (Fig.7, Fig.S2). Firstly, the module MEyellow showed that Zm00001d019306 is a hub gene (Fig.7). However, in module MEsienna3, the regulatory network is not as simple and clear as that in MEyellow. We chose genes with a degree value of more than 10 as hub genes. Therefore, the hub genes in module MEsienna3 are Zm00001d006330, Zm00001d043289, Zm00001d007181, Zm00001d043290, Zm00001d033510, Zm00001d024732, Zm00001d015025, Zm00001d006331 and Zm00001d047796 (Fig.S2, Table S5).

Validtion of DEGs by Quantitative real-time PCR

The expression patterns of nine differentially expressed genes were further confirmed by quantitative real-time PCR (qRT-PCR). We compared the transcriptional profiles of each gene with the seed coat RNA samples from hybrid and parents at different time points. The data confirmed that the expression patterns of all 9 differentially expressed genes were consistent with the expression levels obtained by RNA-seq analysis (Fig.8, Fig.S3)

Heterosis has been discovered for centuries [28]. Maize seed is a character with heterosis, whose size is determined by maize seed coat area to a certain extent. Maize seed coat is developed from ovary wall with a complete maternal genotype. Due to its genetic stability and fixed heterozygosity, the seed coat of F2 seed obtained by self-pollination of F1 plant contained the genotype of heterozygosity, which is a good model for studying heterosis. Therefore, exploring the seed coat area obtained by self-pollination of maize F1 plant and its self-crossed parents can deepen our understanding of maize seed coat heterosis, and in turn seed size and yield heterosis.

The initiation of seed coat development depends on the fusion of the central cell with one of the sperm cells [29]. In our study, the relative growth rate of seed coat area of the hybrid and paternal line peaked at 3DAP, while maternal line grew most rapidly with the peak on 2DAP. This phenomenon may resulted from the fact that synthesis of auxin production in the endosperm after fertilization remove the block PRC2 on seed coat development, so as to promote the coordinated development of seed coat and endosperm [30]. There was a statistical difference between hybrid and mid-parental value on 8DAP in seed coat area, and it was concluded that the heterosis of seed coat area depended on number of cells rather than size of seed coat cells (Fig. 2). These results were consistent with the GO enrichment of the transcriptome comparison between hybrid and parents in the early stage of seed coat growth and development, suggesting that the heterosis of seed coat area was result from the enhanced cell cycle and cell division activity in the early stage. Previous studies have reported that plant growth heterosis is basically resulted from the change of cell number related to most plant characters [31]. Our results proved that maize seed coat developmental program of hybrid is not dramatically altered but greater cell proliferation is involved.

Transcriptome analysis is a well-established method to identify differential expression levels and regulatory mechanisms of genes from different samples at the transcriptional level [32]. Previous studies have identified heterosis genes associated with many traits between hybrid and parents of plants and still have proved that gene dose effect plays a role in heterosis [33–35]. RNA-seq was performed on the seed coats of 2-4DAP and 8-10DAP hybrid and parental lines. The result of overlapped expression indicated that the heterosis of seed coat extension mainly due to the dosage effect of gene expression rather than non-or-all expression.

The differential gene expression pattern of DEGs between hybrid and its parents plays a significant role in heterosis [13, 36]. In hybrid, where 2 different alleles of a gene are brought together, the combined allelic expression may deviate from that of either parent or the mid-parent predictions [37]. Concerning the relative gene expression level among a hybrid–parent triad, there are usually two scenarios about the relative gene expression level between the triples of hybrid parents. In the first case, the gene expression of hybrid showed a cumulative pattern, which was contributed by each allele of parents. This pattern was additive and mainly regulated by cis action. In another case, the expression deviates from the mid-parental level, and other trans acting factors may lead to the change of corresponding allele expression in hybrid [37]. In the present survey, majority (85.18% at 3DAP and 86.45% at 8DAP) of genes in hybrid are additively expressed, suggesting that cis-acting elements played a major role in regulation of gene expression. The non-additive genes expressed in our entire dataset were less than 15% in each time point but accounted for 54.35–56.24% of DEGs. This result can conclude that the difference of gene expression between hybrid and parental lines was mainly due to non-additive effects. Analysis of the expression of non-additive genes indicated that the allelic expression pattern of hybrid may not be a simple combination of alleles from both parents, but regulated by other genes or trans-action factors. By comparing the expression patterns of hybrids in 3DAP and 8DAP, the number and type of additive expressed genes changed slightly (19714 or 85.18% in 3DAP to 19460 or 86.45% in 8DAP DEGs), but the number and type of high-expressed and low-expressed genes of 3DAP and 8DAP varied greatly (Table S4, Fig. 5). In conclusion, it was suggested that the expression of non-additive genes in hybrid may be the main cause of heterosis in hybrid seed coats.

3DAP and 8DAP are the key stages of maize endosperm cellularization and differentiation, respectively [21, 22]. In this survey, the results of GO enrichment analysis and KEGG pathway analysis of different expression patterns in hybrid and parental seed coat on 3DAP and 8DAP could be matched with the endosperm development process. In other words, on 3DAP, compared with parents, hybrid can increase seed coat size mainly by enhancing cell cycle activity, weakening the response to stress and co-developing with their endosperm (Fig. 5, A-B). Although on 8DAP, the hybrid coat reduced the rate of cell division, instead by strengthened the metabolic process. At this stage, the seed coat changed mainly in coordination with the endosperm to enhance the synthesis and accumulation of nutrients in the seed, so as to enrich the grain and increase the kernel weight (Fig. 5, C-D). This result was supported by the statistical difference in kernel weight between hybrid and MPV at 10DAP (Fig. 1, D). In KEGG analysis of high parental expression of 8DAP, pathways related to circadian rhythm and metabolite accumulation were significantly enriched. Changing the circadian rhythm can improve the viability and biomass of hybrid [38]. In addition, evidence of changes in metabolic profiles has been recorded in hybrid [39, 40]. Moreover, Seed development is accompanied by metabolic activities for the synthesis and accumulation of stored products, including protein and carbohydrates [41]. Therefore, in addition to genetic control, the final size of seeds may also be affected by metabolic activities [42].

Zm00001d018415 and Zm00001d051995, two genes encoding proliferating cell nuclear antigen(PCNA)protein, shown high parental expression patterns in the GO and KEGG analysis of 3DAP. PCNA is a key protein in the mitotic DNA replication mechanism of all eukaryotes [43]. It acts as DNA polymerase δ co-factors thus necessary for the synthesis of new DNA strand by forms a homotrimeric complex [28, 44] that binds to various cyclin-CDK complexes [45–47]. In addition, as a sliding clamp, PCNA also plays a role in regulating DNA metabolism, processing DNA damage and controlling cell cycle [48]. Therefore, the high-parental expression of PCNA gene and its related proteins in 3DAP may play a role in enhancing DNA replication, repair and metabolism in the early stage of seed coat development, so as to increase the cell cycle and cell number of hybrid. Moreover, the excessive transcript abundance of these genes in hybrids in the early stage of seed coat development may eventually help to maintain heterosis through dominant gene effects to a great extent. Zm00001d039131, encoding the ADP-glucose pyrophosphorylase, which catalyze the key steps of starch synthesis pathway. This gene enriched in GO and the metabolic pathways and biosynthesis of secondary metabolites pathway in KEGG at 8DAP as a high-parental expression mode. This gene has been reported to be associated with seed development, filling and kernel weight [49–51]. 8-12DAP is the stage of endosperm cell differentiation, during which nutrients and storage materials accumulate with cell proliferation and enlargement [22]. Seed development is a coordinated process among seed coat, embryo and endosperm. The ADP-glucose pyrophosphorylase was high-parental expressed on 8DAP in hybrid, while the cell cycle related genes were low-parental expressed in the hybrid. This phenomenon may be the result of the rapid development and nutrient filling of endosperm by enhancing the accumulation of metabolites in hybrid. In other words, during this period, the gene expression pattern of hybrid changed, that is, it weakened the cell cycle activity and enhanced the metabolic process, so as to stabilize the Heterosis by cooperating with the development and filling of endosperm.

WGCNA can be used to analyze the relationship between gene set and sample phenotype, draw the regulatory network between genes in gene set and identify key regulatory genes [52]. Zm00001d019306, the hub gene of WGCNA in the highest correlation coefficient modules which was significantly positively correlated with the seed coat phenotype, coding indole-3-acetyl-leu hydrolase. It can catalyze hydrolysis of Indole-3-acetyl-L-leucine and release free Indole-3-acetic acid (IAA) [53]. Seed development is regulated by phytohormones [42]. Auxin, as a central plant hormone, regulates many biological processes, including maintenance of meristems, cell division and cell expansion [54–56]. In Arabidopsis, indole-3-acetyl-leu hydrolase regulate auxin response by regulating auxin homeostasis in endoplasmic reticulum [57]. Auxin is the key regulator of plant defense response and the main control factor of plant growth and development. The change of gene activity related to IAA pathway has potential importance in producing heterosis phenotype [58, 59]. Previous studies showed that in Arabidopsis, the utilization of IAA pathway caused the difference of auxin on cell proliferation control, resulting in different levels of heterosis [60]. Interestingly, these views are consistent with our results. On the one hand, at 3DAP, genes related to defense response in hybrids are significantly down-regulated. On the other hand, Heterosis of maize seed coat area is determined by the number of cells. To sum up, indole-3-acetyl-leu hydrolase may play a significant role in of IAA pathway and seed coat heterosis. It may regulate auxin response by regulating auxin homeostasis in different seed coat development processes, so as to effectively maintain the balance of hybrid immunity and growth and development on resource allocation at different development time points of seed coat.

Finally, the expression levels of 9 genes produced by RNA-seq analysis were verified by qRT-PCR, and the expression patterns of all tested gene were consistent with the results of RNA-seq, indicating the reliability of RNA-seq data in Yudan888 and its parents (Fig. 8). In conclusion, the genes identified in this paper may help to create genetic diversity with enhanced hybridization activity in maize. However, further validation is needed to confirm the heterosis association between gene expression patterns and maize target agronomic traits.

Materials

In summer 2020, maize hybrid Yudan888, a widely cultivated maize variety in Huang-Huai-Hai area of China, together with its parental lines 15S717 and T4691, were planted in farm of Henan Agricultural University (Yuanyang, China, 113°16’ E, 35°41’ N). The planting density was 75,000 plants per hectare, with 3 replicates each containing 600 seedlings. All the individual hybrid and parental seedlings were artificially self-pollinated.

Sampling

Seed coat sampling began on the very day of pollination and continues daily until 15DAP, which were guaranteed to be completed between 9am and 11am each day. During sampling, the seeds from upper and lower parts of ear were discarded with only the middle kernels sampled. At least six individual plants with no less than six kernels each were measured. Three biological replicates were collected for the same day samples. In addition, seeds from the centre of three different ears were fixed in FAA buffer (50% ethanol: formaldehyde: acetic acid = 90:5:5 volume) and then vacuumed at 4°C for further imaging.

RNA-seq samples were taken during the period when the parental and hybrid showed statistical differences in area of seed coats. The seed coat samples for RNA-seq were collected by manual dissection with scalpel. Three biological replicates were set for each samples of each time points. Each replicate samples were collected from at least three individual ears. The samples collected were frozen immediately in liquid nitrogen and stored at -80°C before RNA extraction.

Seed coat area Phenotyping

The kernel length, kernel width and kernel thickness were measured with vernier caliper immediately after sampling. The kernel weight was measured by an analytical balance (ME204E, METTLER TOLEDO) with accuracy of 0.0001. The measurement method of kernel coat area was initiated with peeling the whole seed coat with tweezers, following by photographed on a black background. Finally, the seed coat area were calculated by ImageJ software. The measured quantity of each material for phenotyping was at least six individual plants per DAP with no less than six kernels per plant.. Univariate ANOVA statistical analysis method in IBM SPSS statistics 24 was performed to test the difference significance of the measured the area of seed coat between the parental lines and the hybrid .

Microscope measurement of seed coat cells

Kernels from each samples were removed from the FAA solution and rinsed thoroughly in deionized water. Kernels were dissected at the longest part with a scalpel. Each dissected kernel was embedded in paraffin. Sections from kernels ranged from 8 to 10 μm in thickness. The maximum longitudinal cross section of intact kernels was dyed with 1% toluidine blue after dewaxing. Images of the sections were captured with a microscope (Axio Scope A1, Carl Zeiss) fitted with a digital camera (Axiocam 503 color, Carl Zeiss). ImageJ was supplied to measure the number of seed coat cells per unit area.

RNA extracting

Total RNA was extracted from Yudan888 and its parent seed coat samples at 2-4DAP and 8-10DAP using TRIZOL reagent (Invitrogen, United States). Every material at each DAPs was repeated in three biological replicates. The quality and concentration of each RNA sample was determined using both gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Only RNAs met the criterion OD260/280 ratio of 1.9-2.1 were stored at −80 °C freezer for further use.

RNA-seq and data analysis

A total amount of 1μg RNA was used as input material for construction of each RNA library, with NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (with RNase H). After second-strand cDNA synthesis, terminal repair, and poly(A) tail and sequencing oligonucleotide adaptors ligation, the fragments were purified and subsequently amplified by PCR. The PCR products were assessed with the Agilent Bioanalyzer 2100 system. Finally, the libraries of inserting cDNAs 150 bp in size were generated and sequenced by a commercial service company (Guoke Biotechnology, Beijing, China) on an Illumia Noveseq platform.

All data analyses were based on the clean data with high quality [61]. The feature Counts v1.5.0-p3 was used to count the read numbers mapped to each gene (ftp://ftp.ensemblgenomes.org/pub/release-40/plants/fasta/zea_mays/dna/). Multiple testing with the Benjamini-Hochberg approach for controlling the false discovery rate (FDR) was taken into account by using an adjusted P-value. Corrected P-value of 0.05 and absolute fold change of two times were set as the thresholds for significantly differential expression.

Classification of differential gene expression patterns

Based on previous studies on gene expression patterns [62-66], the fpkm value of genes were compared between hybrid and parents. The expression patterns were divided into additive and non-additive (dominance or over-dominance). The relative expression level of a hybrid gene located between parent values is classified as additive (whether there are DEGs between parents or not). The relative expression level of DEGs similar to that of dominant parents is classified as dominance. The relative expression level of DEGs is higher than that of parents (excessive up regulation) or lower than that of parents (excessive down regulation), which is classified as overdominance.

Weighted Gene Co-Expression Network Analysis (WGCNA)

The assignments of gene co-expression module were based on fpkm data, using WGCNA protocol (langfelder and Horvath, 2008). In order to determine the module related to seed coat surface area, we associated the module characteristic gene with seed coat area trait from 2-4DAP and 8-10DAP. The genes with an average fpkm＞1 in 54 samples were analyzed. The soft threshold power β of network construction was set to 11. The dynamic tree cutting algorithm with a minimum module size of 50 genes was used for hierarchical clustering cutting. 0.15 was used to merge similar modules. If the p-value of module trait association was less than 0.05, it was defined as a meaningful module. In R 4.0.1, WGCNA package was used to calculate the correlation weight of each gene and all other genes in the module, and the top 100 gene sets in the module were visualized using Cytoscape V3.9.1, and the gene with the highest degree was considered to be a hub gene.

qRT-PCR

9 differentially expressed genes were selected for qRT-PCR verification, the first strand of cDNA was synthesized by reverse transcription Using total RNA from seed coat of hybrid Yudan888, maternal line and paternal line as template. Primer 3 on-line toolbox was performed to design gene specific primers for qRT-PCR. The primers used for quantitative PCR are listed in Supplemental Table S6. Reverse transcription and qRT-PCR reaction system and reaction procedures were in accordance with PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa) and TB GreenTM Premix Ex TaqTM Ⅱ (TaKaRa) kit instructions. Each experiment has 3 biological replicates and 3 technical replicates. The relative expression level of these genes were analyzed by the 2^-^ΔΔ^Ctmethod with Zm-actin-1 serving as the internal reference [67].

DAP

days after pollination

MPV

mid-parental value

DEGs

Differentially expressed genes

Gene ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

WGCNA

Weighted Gene Co-Expression Network Analysis

qRT-PCR

Real-time quantitative PCR

Ethics approval and consent to participate

All experimental studies on plants were complied with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Availability of data and materials

The sequence datasets in fastq format of the current study are available in the NCBI Sequence Read Archive (SRA) database under Bioproject PRJNA828527 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA828527).

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by a grant from the National Nature Foundation of China (31971961, 31871641, 32060460) and the Science and Technique Foundation of Henan Province of China (202102110036).

Author' Contributions

DD, ZHC and JHT designed the research. JL and LFW performed the experiments. JL, LFW and JW analyzed data. JL wrote the manuscript, DD, ZHC and JHT revised the manuscript. All authors approved the final manuscript.

Acknowledgments

The authors would like to thank Huili Yang, Yanan Lin and Guoqiang Xu for assisting with this article.

Authors' information

¹College of Agronomy, Hunan Agricultural University, Changsha 410128, China

²National Key Laboratory of Wheat and Maize Crop Science; Collaborative Innovation Center of Henan Grain Crops; College of Agronomy; Henan Agricultural University, Zhengzhou 450002, China

³ Institute of Crop Germplasm Resources, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China

⁴ Hebi Academy of Agricultural Sciences, Hebi 458030, China

⁵The Shennong laboratory, Zhengzhou 450002, China

⁶Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China

Juan Li ^1,2,3#, Liangfa Wang^1,2,4#, Jiong Wan², Kuntai Dang², Yuan Lin², Shujun Meng², Xiaoqian Qiu², Qiyue Wang², Jiawen Zhao², Liqin Mu², Hongbing Luo¹, Dong Ding^2*, Zehui Chen^6*,and Jihua Tang^2,5*

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Dynamic Patterns of Gene Expression and Regulatory Variation in seed coat of Maize

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