Developing Maize Kernels Research Articles

N6-methyladenosine transcriptome-wide profiles of maize kernel development.

Maize (Zea mays L.) kernel development is a complex and dynamic process involving cell division and differentiation, into a variety of cell types. Epigenetic modifications, including DNA methylation, play a pivotal role in regulating this process. N6-methyladenosine modification is a universal and dynamic post-transcriptional epigenetic modification that is involved in the regulation of plant development. However, the role of N6-methyladenosine in maize kernel development remains unknown. In this study, we have constructed transcriptome-wide profiles for maize kernels at various stages of early development. Utilizing a combination of MeRIP-seq and RNA-seq analysis, we identified a total of 11,170, 10,973, 11,094, 11,990, 12,203 and 10,893 N6-methyladenosine peaks in maize kernels at 0, 2, 4, 6, 8, and 12 days after pollination, respectively. These N6-methyladenosine modifications were primarily deposited at the 3'-UTRs and were associated with the conserved motif-UGUACA. Additionally, we found that conserved N6-methyladenosine modification are involved in the regulation of genes that are ubiquitously expressed during kernel development. Further analysis revealed that N6-methyladenosine peak intensity was negatively correlated with the mRNA abundance of these ubiquitously expressed genes. Meanwhile, we employed phylogenetic analysis to predict potential regulatory proteins involved in maize kernels development and identified several that participate in the regulation of N6-methyladenosine modifications. Collectively, our results suggest the existence of a novel post-transcriptional epigenetic modification mechanism involved in the regulation of maize kernels development, thereby providing a novel perspective for maize molecular breeding.

PPR21 is involved in the splicing of nad2 introns via interacting with PPR–small MutS-related 1 and small PPR protein 2 and is essential to maize seed development

Pentatricopeptide repeat (PPR) proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression. Here, we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development. PPR21 is a typical P-type PPR protein targeted to mitochondria. The ppr21 mutants are arrested in embryogenesis and endosperm development, leading to embryo lethality. Null mutations of PPR21 reduce the splicing efficiency of nad2 intron 1, 2, and 4 and impair the assembly and activity of mitochondrial complex I. Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns. However, our protein interaction analyses reveal that PPR21 does not interact with EMP12. Instead, both PPR21 and EMP12 interact with the small MutS-related (SMR) domain–containing PPR protein 1 (PPR-SMR1) and the short P-type PPR protein 2 (SPR2). PPR-SMR1 interacts with SPR2, and both proteins are required for the splicing of many introns in mitochondria, including nad2 intron 1, 2, and 4. These results suggest that a PPR21–(PPR-SMR1/SPR2)–EMP12 complex is involved in the splicing of nad2 introns in maize mitochondria.

Insight into the composition and differentiation of endophytic microbial communities in kernels via 368 maize transcriptomes

IntroductionKernels are important reproductive organs in maize, yet there is a lack of systematic investigation on the differences in the composition of endophytic microorganisms in plants from a population perspective. ObjectivesWe aimed to elucidate the composition of endophytic microorganisms in developing maize kernels, emphasizing differences among various inbred lines. MethodsThe transcriptomic data of 368 maize inbred lines were used to explore the composition and diversity of endophytic microorganisms. ResultsThe findings revealed a higher abundance of fungi than bacteria in developing maize kernels, followed by protozoa, while viruses were less abundant. There were significant differences in the composition and relative abundance of endophytic microorganisms among different maize lines. Diversity analysis revealed overall similarity in the community composition structure between tropical/subtropical (TST) and temperate (NSS) maize germplasm with apparent variations in community richness and abundance. The endophytic microorganisms network in the kernels from TST genotypes exhibited higher connectivity and stability compared to NSS kernels. Bacteria dominated the highly connected species in the networks, and different core species showed microbial phylum specificity. Some low-abundance species acted as core species, contributing to network stability. Beneficial bacteria were predominant in the core species of networks in TST kernels, while pathogenic bacteria were more abundant in the core species of networks in NSS kernels. ConclusionTropical maize germplasm may have advantages in resisting the invasion of pathogenic microorganisms, providing excellent genetic resources for disease-resistant breeding.

Toward unveiling transcriptome dynamics and regulatory modules at the maternal/filial interface of developing maize kernel.

The basal region of maize (Zea mays) kernels, which includes the pedicel, placenta-chalazal, and basal endosperm transfer layers, serves as the maternal/filial interface for nutrient transfer from the mother plant to the developing seed. However, transcriptome dynamics of this maternal/filial interface remain largely unexplored. To address this gap, we conducted high-temporal-resolution RNA sequencing of the basal and upper kernel regions between 4 and 32 days after pollination and deeply analyzed transcriptome dynamics of the maternal/filial interface. Utilizing 790 specifically and highly expressed genes in the basal region, we performed the gene ontology (GO) term and weighted gene co-expression network analyses. In the early-stage basal region, we identified five MADS-box transcription factors (TFs) as hubs. Their homologs have been demonstrated as pivotal regulators at the maternal/filial interface of rice or Arabidopsis, suggesting their potential roles in maize kernel development. In the filling-stage basal region, numerous GO terms associated with transcriptional regulation and transporters are significantly enriched. Furthermore, we investigated the molecular function of three hub TFs. Through genome-wide DNA affinity purification sequencing combined with promoter transactivation assays, we suggested that these three TFs act as regulators of 10 basal-specific transporter genes involved in the transfer of sugars, amino acids, and ions. This study provides insights into transcriptomic dynamic and regulatory modules of the maternal/filial interface. In the future, genetic investigation of these hub regulators must advance our understanding of maternal/filial interface development and function.

Drought stress during maize flowering may cause kernel abortion by inhibition of plasma membrane H+‐ATPase activity

AbstractBackgroundDrought stress during flowering of maize (Zea mays L.) frequently results in decreased kernel setting, leading to grain yield depressions. Plasma membrane (PM) H+‐ATPase was identified as a key enzyme responsible for supply of assimilates to the developing maize kernels shortly after pollination. The activity of this enzyme was strongly inhibited under salt stress, pointing to an involvement in kernel abortion.AimsThis study aimed to determine whether also drought stress causes inhibition of PM H+‐ATPase in developing maize kernels shortly after pollination, leading to diminished hexose uptake and finally kernel abortion. The key questions are as follows: What are the limiting factors for grain yield production of maize plants facing drought? Are physiologically relevant parameters, quantified at flowering, reflected by yield determinants at maturity?MethodsMaize plants were cultivated using the container technique, and drought stress was imposed during 3 weeks bracketing flowering compared to well‐watered conditions throughout the entire growth period. The developing kernels were harvested 2 days after pollination, and PM vesicles were isolated and purified using two‐phase partitioning.ResultsWater deficit caused a significant decrease in grain yield at maturity (−35%), which was determined by a reduced kernel number (−42%). Source limitation in the developing kernels under stress could be excluded. Acid invertase activity was unaffected by water deficit. Hexose availability was also no limiting factor for kernel setting and development. However, Vmax of in vitro hydrolytic activity of PM H+‐ATPase was significantly decreased in the developing maize kernels under drought stress and the maximal pH gradient at the PM was also significantly reduced. The observed inhibiting effects on PM H+‐ATPase were mainly of quantitative nature, as a lower number of proton pumps was present in the kernel PM. Qualitative changes of the enzyme (activation energy Ea, Michaelis constant Km) due to drought were not observed.ConclusionsThe lower pH gradient probably decreased the proton‐driven transport of hexoses by carriers into the cytosol of the kernel cells, leading to kernel starvation and eventually contributing to kernel abortion.

The cytosolic isoform of triosephosphate isomerase, ZmTPI4, is required for kernel development and starch synthesis in maize (Zea mays L.)

Triosephosphate isomerase (TPI) is an enzyme that functions in plant energy production, accumulation, and conversion. To understand its function in maize, we characterized a maize TPI mutant, zmtpi4. In comparison to the wild type, zmtpi4 mutants showed altered ear development, reduced kernel weight and starch content, modified starch granule morphology, and altered amylose and amylopectin content. Protein, ATP, and pyruvate contents were reduced, indicating ZmTPI4 was involved in glycolysis. Although subcellular localization confirmed ZmTPI4 as a cytosolic rather than a plastid isoform of TPI, the zmtpi4 mutant showed reduced leaf size and chlorophyll content. Overexpression of ZmTPI4 in Arabidopsis led to enlarged leaves and increased seed weight, suggesting a positive regulatory role of ZmTPI4 in kernel weight and starch content. We conclude that ZmTPI4 functions in maize kernel development, starch synthesis, glycolysis, and photosynthesis.

Insights into ZmWAKL in maize kernel development: genome-wide investigation and GA-mediated transcription

BackgroundThe functional roles of the Wall Associated Kinase (WAK) and Wall Associated Kinase Like (WAKL) families in cellular expansion and developmental processes have been well-established. However, the molecular regulation of these kinases in maize development is limited due to the absence of comprehensive genome-wide studies.ResultsThrough an in-depth analysis, we identified 58 maize WAKL genes, and classified them into three distinct phylogenetic clusters. Moreover, structural prediction analysis showed functional conservation among WAKLs across maize. Promoter analysis uncovered the existence of cis-acting elements associated with the transcriptional regulation of ZmWAKL genes by Gibberellic acid (GA). To further elucidate the role of WAKL genes in maize kernels, we focused on three highly expressed genes, viz ZmWAKL38, ZmWAKL42 and ZmWAKL52. Co-expression analyses revealed that their expression patterns exhibited a remarkable correlation with GA-responsive transcription factors (TF) TF5, TF6, and TF8, which displayed preferential expression in kernels. RT-qPCR analysis validated the upregulation of ZmWAKL38, ZmWAKL42, ZmWAKL52, TF5, TF6, and TF8 following GA treatment. Additionally, ZmWAKL52 showed significant increase of transcription in the present of TF8, with ZmWAKL52 localizing in both the plasma membrane and cell wall. TF5 positively regulated ZmWAKL38, while TF6 positively regulated ZmWAKL42.ConclusionsCollectively, these findings provide novel insights into the characterization and regulatory mechanisms of specific ZmWAKL genes involved in maize kernel development, offering prospects for their utilization in maize breeding programs.

RNA m6A modification facilitates DNA methylation during maize kernel development.

N6-methyladenosine (m6A) in mRNA and 5-methylcytosine (5mC) in DNA have critical functions for regulating gene expression and modulating plant growth and development. However, the interplay between m6A and 5mC is an elusive territory and remains unclear mechanistically in plants. We reported an occurrence of crosstalk between m6A and 5mC in maize (Zea mays) via the interaction between mRNA adenosine methylase (ZmMTA), the core component of the m6A methyltransferase complex, and decrease in DNA methylation 1 (ZmDDM1), a key chromatin-remodeling factor that regulates DNA methylation. Genes with m6A modification were coordinated with a much higher level of DNA methylation than genes without m6A modification. Dysfunction of ZmMTA caused severe arrest during maize embryogenesis and endosperm development, leading to a significant decrease in CHH methylation in the 5' region of m6A-modified genes. Instead, loss of function of ZmDDM1 had no noteworthy effects on ZmMTA-related activity. This study establishes a direct link between m6A and 5mC during maize kernel development and provides insights into the interplay between RNA modification and DNA methylation.

Genome-wide analysis of sugar transporter genes in maize (Zea mays L.): identification, characterization and their expression profiles during kernel development.

Sugar transporters (STs) play a crucial role in the development of maize kernels. However, very limited information about STs in maize is known. In this study, sixty-eight ZmST genes were identified from the maize genome and classified into eight major groups based on phylogenetic relationship. Gene structure analysis revealed that members within the same group shared similar exon numbers. Synteny analysis indicated that ZmSTs underwent 15 segmental duplication events under purifying selection. Three-dimensional structure of ZmSTs demonstrated the formation of a compact helix bundle composed of 8-13 trans-membrane domains. Various development-related cis-acting elements, enriched in promoter regions, were correlated with the transcriptional response of ZmSTs during kernel development. Transcriptional expression profiles exhibited expression diversity of various ZmST genes in roots, stems, leaves, tassels, cobs, embryos, endosperms and seeds tissues. During kernel development, the expression of 24 ZmST genes was significantly upregulated in the early stage of grain filling. This upregulation coincided with the sharply increased grain-filling rate observed in the early stage. Overall, our findings shed light on the characteristics of ZmST genes in maize and provide a foundation for further functional studies.

DEFECTIVE KERNEL 56 functions in mitochondrial RNA editing and maize seed development.

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.

A peptide chain release factor 2a gene regulates maize kernel development by modulating mitochondrial function

Mitochondrial protein translation that is essential for aerobic energy production includes four essential steps of the mitochondrial ribosome cycle, namely, initiation, elongation, termination of the polypeptide, and ribosome recycling. Translation termination initiates when a stop codon enters the A site of the mitochondrial ribosome where it is recognized by a dedicated peptide release factor (RF). However, RFs and mechanisms involved in translation in plant mitochondria, especially in monocotyledons, remain largely unknown. Here, we identified a crumpled kernel (crk5 allele) mutant, with significantly decreased kernel size, 100-kernel weight, and an embryo-lethal phenotype. The Crk5 allele was isolated using map-based cloning and found to encode a mitochondrial localization RF2a. As it is an ortholog of Arabidopsis mitochondrial RF2a, we named the gene ZmmtRF2a. ZmmtRF2a is missing the 5th–7th exons in the crk5 resulting in deletion of domains containing motifs GGQ and SPF that are essential for release activity of RF, mitochondrial ribosome binding, and stop codon recognition. Western blot and qRT-PCR analyses indicate that the crk5 mutation results in abnormal mitochondrion structure and function. Intriguingly, we observed a feedback loop in the crk5 with up-regulated transcript levels detected for several mitochondrial ribosome and mitochondrial-related components, in particular mitochondrial complexes CI, CIV, and a ribosome assembly related PPR. Together, our data support a crucial role for ZmmtRF2a in regulation of mitochondrial structure and function in maize.

Spatial transcriptomics uncover sucrose post-phloem transport during maize kernel development

Maize kernels are complex biological systems composed of three genetic sources, namely maternal tissues, progeny embryos, and progeny endosperms. The lack of gene expression profiles with spatial information has limited the understanding of the specific functions of each cell population, and hindered the exploration of superior genes in kernels. In our study, we conduct microscopic sectioning and spatial transcriptomics analysis during the grain filling stage of maize kernels. This enables us to visualize the expression patterns of all genes through electronical RNA in situ hybridization, and identify 11 cell populations and 332 molecular marker genes. Furthermore, we systematically elucidate the spatial storage mechanisms of the three major substances in maize kernels: starch, protein, and oil. These findings provide valuable insights into the functional genes that control agronomic traits in maize kernels.

ZmDRR206 functions in maintaining cell wall integrity during maize seedling growth and defense response to external stresses

Plants adaptively change their cell wall composition and structure during their growth, development, and interactions with environmental stresses. Dirigent proteins (DIRs) contribute to environmental adaptations by dynamically reorganizing the cell wall and/or by generating defense compounds. A maize DIR, ZmDRR206, was previously reported to play a dominant role in regulation of storage nutrient accumulation in endosperm during maize kernel development. Here we show that ZmDRR206 mediates maize seedling growth and disease resistance by coordinately regulating biosynthesis of cell wall components for cell-wall integrity (CWI) maintenance. Expression of ZmDRR206 was induced in maize seedlings upon pathogen infection. ZmDRR206 overexpression in maize resulted in reduced seedling growth and photosynthetic activity but increased disease resistance and drought tolerance, revealing a tradeoff between growth and defense. Consistently, ZmDRR206 overexpression reduced the contents of primary metabolites and down-regulated genes involved in photosynthesis, while increasing the contents of major cell wall components, defense phytohormones, and defense metabolites, and up-regulated genes involved in defense and cell-wall biosynthesis in seedlings. ZmDRR206-overexpressing seedlings were resistant to cell-wall stress imposed by isoxaben, and ZmDRR206 physically interacted with ZmCesA10, which is a cellulose synthase unit. Our findings suggest a mechanism by which ZmDRR206 coordinately regulates biosynthesis of cell-wall components for CWI maintenance during maize seedling growth, and might be exploited for breeding strong disease resistance in maize.

Comprehensive Identification of the Pum Gene Family and Its Involvement in Kernel Development in Maize.

The Pumilio (Pum) RNA-binding protein family regulates post-transcription and plays crucial roles in stress response and growth. However, little is known about Pum in plants. In this study, a total of 19 ZmPum genes were identified and classified into two groups in maize. Although each ZmPum contains the conserved Pum domain, the ZmPum members show diversity in the gene and protein architectures, physicochemical properties, chromosomal location, collinearity, cis-elements, and expression patterns. The typical ZmPum proteins have eight α-helices repeats, except for ZmPum2, 3, 5, 7, and 14, which have fewer α-helices. Moreover, we examined the expression profiles of ZmPum genes and found their involvement in kernel development. Except for ZmPum2, ZmPum genes are expressed in maize embryos, endosperms, or whole seeds. Notably, ZmPum4, 7, and 13 exhibited dramatically high expression levels during seed development. The study not only contributes valuable information for further validating the functions of ZmPum genes but also provides insights for improvement and enhancing maize yield.

Recessive waxy1 and opaque2 genes synergistically regulate accumulation of amylopectin, lysine and tryptophan in maize

Traditional maize is poor in amylopectin (75%), lysine (0.200%), and tryptophan (0.045%). While the recessive waxy1 (wx1) gene enhances amylopectin and the recessive opaque2 (o2) gene increases lysine and tryptophan. Here, we analyzed transcripts of wx1 and o2 genes in (i) three wild-type (Wx1Wx1/O2O2), (ii) six waxy (wx1wx1/O2O2), (iii) four QPM (Wx1Wx1/o2o2), and (iv) eight waxy-QPM (wx1wx1/o2o2) inbreds at 20-, 30- and 40-days after pollination (DAP). The waxy inbreds possessed ∼1.4-fold higher amylopectin, while QPM inbreds showed 1.5- and 1.7-fold more accumulation of lysine and tryptophan over wild types. Interestingly, double mutant (wx1wx1/o2o2) had 2.1% higher amylopectin, and 15.6% and 16.6% more lysine and tryptophan, respectively over single mutants. Amylopectin showed an increase from 20-DAP (94.66%) to 30-DAP (96.56%) with a peak at 40-DAP (97.32%). On contrary, lysine and tryptophan peaked at 20-DAP (lysine: 0.552%, tryptophan: 0.267%), which gradually reduced through 30-DAP (lysine: 0.448%, tryptophan: 0.166%) and 40-DAP (lysine: 0.325%, tryptophan: 0.081%). Waxy and QPM inbreds possessed significantly lower expression of wx1 (∼3.0-fold) and o2 (∼1.1-fold) alleles over wild-type alleles (Wx1 and O2). Expression of wx1 and o2 in double mutant was 57.8% and 80.7% lower over single mutants, respectively. This is the first report on the expression of wx1 and o2 genes during maize kernel development.

Defective kernel 66 encodes a GTPase essential for kernel development in maize.

The mitochondrion is a semi-autonomous organelle that provides energy for cell activities through oxidative phosphorylation. In this study, we identified a defective kernel 66 (dek66)-mutant maize with defective kernels. We characterized a candidate gene, DEK66, encoding a ribosomal assembly factor located in mitochondria and possessing GTPase activity (which belongs to the ribosome biogenesis GTPase A family). In the dek66 mutant, impairment of mitochondrial structure and function led to the accumulation of reactive oxygen species and promoted programmed cell death in endosperm cells. Furthermore, the transcript levels of most of the key genes associated with nutrient storage, mitochondrial respiratory chain complex, and mitochondrial ribosomes in the dek66 mutant were significantly altered. Collectively, the results suggest that DEK66 is essential for the development of maize kernels by affecting mitochondrial function. This study provides a reference for understanding the impact of a mitochondrial ribosomal assembly factor in maize kernel development.

ZmDRR206 Regulates Nutrient Accumulation in Endosperm through Its Role in Cell Wall Biogenesis during Maize Kernel Development.

Dirigent proteins (DIRs) contribute to plant fitness by dynamically reorganizing the cell wall and/or by generating defense compounds during plant growth, development, and interactions with environmental stresses. ZmDRR206 is a maize DIR, it plays a role in maintaining cell wall integrity during seedling growth and defense response in maize, but its role in regulating maize kernel development is unclear. Association analysis of candidate genes indicated that the natural variations of ZmDRR206 were significantly associated with maize hundred-kernel weight (HKW). ZmDRR206 plays a dominant role in storage nutrient accumulation in endosperm during maize kernel development, ZmDRR206 overexpression resulted in small and shrunken maize kernel with significantly reduced starch content and significantly decreased HKW. Cytological characterization of the developing maize kernels revealed that ZmDRR206 overexpression induced dysfunctional basal endosperm transfer layer (BETL) cells, which were shorter with less wall ingrowth, and defense response was constitutively activated in developing maize kernel at 15 and 18 DAP by ZmDRR206 overexpression. The BETL-development-related genes and auxin signal-related genes were down-regulated, while cell wall biogenesis-related genes were up-regulated in developing BETL of the ZmDRR206-overexpressing kernel. Moreover, the developing ZmDRR206-overexpressing kernel had significantly reduced contents of the cell wall components such as cellulose and acid soluble lignin. These results suggest that ZmDRR206 may play a regulatory role in coordinating cell development, storage nutrient metabolism, and stress responses during maize kernel development through its role in cell wall biogenesis and defense response, and provides new insights into understanding the mechanisms of kernel development in maize.

Is hypoxia good or bad? Role of oxygen levels in maize kernel development.

Is hypoxia good or bad? Role of oxygen levels in maize kernel development.

Dek219 encodes the DICER-LIKE1 protein that affects chromatin accessibility and kernel development in maize

Chromatin accessibility plays a vital role in gene transcriptional regulation. However, the regulatory mechanism of chromatin accessibility, as well as its role in regulating crucial gene expression and kernel development in maize (Zea mays) are poorly understood. In this study, we isolated a maize kernel mutant designated as defective kernel219 (dek219), which displays opaque endosperm and embryo abortion. Dek219 encodes the DICER-LIKE1 (DCL1) protein, an essential enzyme in miRNA biogenesis. Loss of function of Dek219 results in significant reductions in the expression levels of most miRNAs and histone genes. Further research showed that the Heat shock transcription factor17 (Hsf17)-Zm00001d016571 module may be one of the factors affecting the expression of histone genes. Assay results for transposase-accessible chromatin sequencing (ATAC-seq) indicated that the chromatin accessibility of dek219 is altered compared with that of wild type (WT), which may regulate the expression of crucial genes in kernel development. By analyzing differentially expressed genes (DEGs) and differentially accessible chromatin regions (ACRs) between WT and dek219, we identified 119 candidate genes that are regulated by chromatin accessibility, including some reported to be crucial genes for kernel development. Taken together, these results suggest that Dek219 affects chromatin accessibility and the expression of crucial genes that are required for maize kernel development.

Integrating microRNAs and mRNAs reveals the hormones synthesis and signal transduction of maize under different N rates

The effect of nitrogen (N) fertilizer on the development of maize kernels has yet to be fully explored. MicroRNA-mRNA analyses could help advance our understanding of how kernels respond to N. This study analyzed the morphological, physiological, and transcriptomic changes in maize kernels under different N rates (0, 100, 200, and 300 kg ha–1). The result showed that increasing N application significantly increased maize grains’ fresh and dry weight until N reached 200 kg ha–1. Higher levels of indole-3-acetic acid, cytokinin, gibberellin, and a lower level of ethylene were associated with increased N applications. We obtained 31 differentially expressed genes (DEGs) in hormone synthesis and transduction, and 9 DEGs were regulated by 14 differentially expressed microRNAs (DEMIs) in 26 pairs. The candidate DEGs and DEMIs provide valuable insight for manipulating grain filling under different N rates.