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Silk of maize (Zea mays L.) contains diverse metabolites with complicated structures and functions, making it a great challenge to explore the mechanisms of metabolic regulation. Genome‐wide identification of silk‐preferential genes and investigation of their expression regulation provide an opportunity to reveal the regulatory networks of metabolism. Here, we applied the expression quantitative trait locus (eQTL) mapping on a maize natural population to explore the regulation of gene expression in unpollinated silk of maize. We obtained 3,985 silk‐preferential genes that were specifically or preferentially expressed in silk using our population. Silk‐preferential genes showed more obvious expression variations compared with broadly expressed genes that were ubiquitously expressed in most tissues. We found that trans‐eQTL regulation played a more important role for silk‐preferential genes compared to the broadly expressed genes. The relationship between 38 transcription factors and 85 target genes, including silk‐preferential genes, were detected. Finally, we constructed a transcriptional regulatory network around the silk‐preferential gene Bx10, which was proposed to be associated with response to abiotic stress and biotic stress. Taken together, this study deepened our understanding of transcriptome variation in maize silk and the expression regulation of silk‐preferential genes, enhancing the investigation of regulatory networks on metabolic pathways. [ABSTRACT FROM AUTHOR]

SUMMARY: cis‐Regulatory variations contribute to trait evolution and adaptation during crop domestication and improvement. As the most important harvested organ in maize (Zea mays L.), kernel size has undergone intensive selection for size. However, the associations between maize kernel size and cis‐regulatory variations remain unclear. We chose two independent association populations to dissect the genetic architecture of maize kernel size together with transcriptomic and genotypic data. The resulting phenotypes reflected a strong influence of population structure on kernel size. Compared with genome‐wide association studies (GWASs), which accounted for population structure and relatedness, GWAS based on a naïve or simple linear model revealed additional associated single‐nucleotide polymorphisms significantly involved in the conserved pathways controlling seed size in plants. Regulation analyses through expression quantitative trait locus mapping revealed that cis‐regulatory variations likely control kernel size by fine‐tuning the expression of proximal genes, among which ZmKL1 (GRMZM2G098305) was transgenically validated. We also proved that the pyramiding of the favorable cis‐regulatory variations has contributed to the improvement of maize kernel size. Collectively, our results demonstrate that cis‐regulatory variations, together with their regulatory genes, provide excellent targets for future maize improvement. Significance Statement: GWAS associations confounded with population structure and relatedness were found to contribute to the genetic architecture of maize kernel size. Meanwhile, the favourable cis‐regulatory variations were found to involve in the improvement of maize kernel size. [ABSTRACT FROM AUTHOR]

Epialleles, the heritable epigenetic variants that are not caused by changes in DNA sequences, can broaden genetic and phenotypic diversity and benefit to crop breeding, but very few epialleles related to agricultural traits have been identified in maize. Here, we cloned a small kernel mutant, smk-wl10 , from maize, which encoded a tubulin-folding cofactor B (ZmTFCB) protein. Expression of the ZmTFCB gene decreased in the smk-wl10 mutant, which arrested embryo, endosperm and basal endosperm transfer layer developments. Overexpression of ZmTFCB could complement the defective phenotype of smk-wl10. No nucleotide sequence variation in ZmTFCB could be found between smk-wl10 and wild type (WT). Instead, we detected hypermethylation of nucleotide CHG (where H is A, C or T nucleotide) sequence contexts and increased level of histone H3K9me2 methylation in the upstream sequence of ZmTFCB in smk-wl10 compared with WT, which might respond to the attenuating transcription of ZmTFCB. In addition, yeast two-hybrid and bimolecular fluorescence complementation assays identified a strong interaction between ZmTFCB and its homolog ZmTFCE. Thus, our work identifies a novel epiallele of the maize ZmTFCB gene, which might represent a common phenomenon in the epigenetic regulation of important traits such as kernel development in maize. [ABSTRACT FROM AUTHOR]

Intron splicing is an essential event in post-transcriptional RNA processing in plant mitochondria, which requires the participation of diverse nuclear-encoded splicing factors. However, it is presently unclear how these proteins cooperatively take part in the splicing of specific introns. In this study, we characterized a nuclear-encoded mitochondrial P-type pentatricopeptide repeat (PPR) protein named EMP603. This protein is essential for splicing of intron 2 in the Nad1 gene and interacts with the mitochondria-localized DEAD-box RNA helicase PMH2-5140, the RAD52-like proteins ODB1-0814 and ODB1-5061, and the CRM domain-containing protein Zm-mCSF1. Further study revealed that the N-terminal region of EMP603 interacts with the DEAD-box of PMH2-5140, the CRM domain of Zm-mCSF1, and OBD1-5061, but not with OBD1-0814, whereas the PPR domain of EMP603 can interact with ODB1-0814, ODB1-5061, and PMH2-5140, but not with Zm-mCSF1. Defects in EMP603 severely disrupt the assembly and activity of mitochondrial complex I, leading to impaired mitochondrial function, and delayed seed development. The interactions revealed between EMP603 and PMH2-5140, ODB1-0814, ODB1-5061, and Zm-mCSF1 indicate a possible involvement of a dynamic 'spliceosome-like' complex in intron splicing, and may accelerate the elucidation of the intron splicing mechanism in plant mitochondria. [ABSTRACT FROM AUTHOR]

Summary: RUBylation plays essential roles in plant growth and development through regulating Cullin‐RING ubiquitin E3 ligase (CRL) activities and the CRL‐mediated protein degradations. However, the function of RUBylation in regulating kernel development remains unclear.Through genetic and molecular analyses of a small kernel 501 (smk501) mutant in maize (Zea mays), we cloned the smk501 gene, revealed its molecular function, and defined its roles in RUBylation pathway and seed development.Smk501 encodes a RUBylation activating enzyme E1 subunit ZmECR1 (E1 C‐TERMINAL RELATED 1) protein. Destruction in RUBylation by smk501 mutation resulted in less embryo and endosperm cell number and smaller kernel size. The transcriptome and proteome profiling, hormone evaluation and cell proliferation observation revealed that disturbing ZmECR1 expression mainly affects pathways on hormone signal transduction, cell cycle progression and starch accumulation during kernel development. In addition, mutant in zmaxr1 (Auxin resistant 1), another RUB E1 subunit, also showed similar defects in kernel development. Double mutation of zmecr1 and zmaxr1 lead to empty pericarp kernel phenotype.RUBylation is a novel regulatory pathway affecting maize kernel development, majorly through its functions in modifying multiple cellular progresses. [ABSTRACT FROM AUTHOR]

Splicing of plant organellar group II introns from precursor-RNA transcripts requires the assistance of nuclear-encoded splicing factors. Maturase (nMAT) is one such factor, as its three homologs (nMAT1, 2 and 4) have been identified as being required for the splicing of various mitochondrial introns in Arabidopsis. However, the function of nMAT in maize (Zea mays L.) is unknown. In this study, we identified a seed development mutant, empty pericarp 2441 (emp2441) from maize, which showed severely arrested embryogenesis and endosperm development. Positional cloning and transgenic complementation assays revealed that Emp2441 encodes a maturase-related protein, ZmnMAT3. ZmnMAT3 is highly expressed during seed development and its protein locates to the mitochondria. The loss of function of ZmnMAT3 resulted in the reduced splicing efficiency of various mitochondrial group II introns, particularly of the trans -splicing of nad1 introns 1, 3 and 4, which consequently abolished the transcript of nad1 and severely impaired the assembly and activity of mitochondrial complex I. Moreover, the Zmnmat3 mutant showed defective mitochondrial structure and exhibited expression and activity of alternative oxidases. These results indicate that ZmnMAT3 is essential for mitochondrial complex I assembly during kernel development in maize. [ABSTRACT FROM AUTHOR]

Leafy (Lfy1) is a typical late‐flowering mutant with locus being fine‐mapped, but its underlying molecular mechanism is unknown. In this study, two near‐isogenic lines, with and without the Lfy1 locus, were developed for phenotypic and transcriptional analysis of Lfy1‐mediated floral transition. Phenotypic observation showed that wild‐type (WT) plants transited into the tassel primordium at the V6 (6 expanded leaves) stage, whereas the Lfy1 mutant maintained the vegetative stage up to the V9 stage. Transcriptome analysis of shoot tips of both lines revealed more differentially expressed genes (DEGs) at the V5 (394) than the V3 stage (227). Functional enrichment analysis showed that circadian clock pathway genes exhibited lower expression levels in the Lfy1 mutant than in the WT at the V5 stage. A few integrator genes that functioned as downstream regulators of circadian clock genes also showed a lower transcript abundance in mutant lines, suggesting that the Lfy1‐mediated late‐flowering phenotype was partially caused by inefficient expression of circadian clock pathway genes. These results provide molecular clues for the late‐flowering phenotype of the Lfy1 mutant. [ABSTRACT FROM AUTHOR]

The "green revolution" gene gibberellin oxidase contributes to the semidwarf phenotype, improving product and lodging resistance. Dissecting the function of GA biosynthetic genes would be helpful for dwarf maize breeding. In this study, we edited the maize GA20ox3 gene and generated semidwarf maize plants using CRISPR/Cas9 technology. Application of exogenous gibberellin can recover the dwarf phenotype, indicating that the mutants are gibberellin deficient. The contents of GA12 and GA53 were elevated in the mutants due to the disruption of GA20 oxidase, whereas the contents of other GA precursors (GA15, GA24, GA9, GA44, and GA20) were decreased in the mutants, and the accumulation of bioactive GA1 and GA4 was also decreased, contributing to the semidwarf phenotype. Transgene-free dwarf maize was selected from T2-generation plants and might be useful for maize breeding in the future. [ABSTRACT FROM AUTHOR]

Callus induction in plants is similar to pluripotent stem cell induction in animals and can incite global changes in gene expression. Alternative splicing is considered a key factor underlying functional complexity and function in the induction of pluripotent stem cells in animals. However, the role of alternative splicing in plant callus induction remains unclear. We performed high-throughput RNA sequencing of maize callus induced on a callus induction medium (CIM) at different stages and recorded global alternative splicing changes. At every stage, over 20,000 alternative splicing events were detected, and the numbers slightly increased with callus induction. In total, 648 to 2580 genes were found to exhibit significant alternative splicing changes during callus induction—mainly those that belong to the spliceosome, metabolic pathways, and mRNA surveillance pathways. It was also found that alternative splicing can function alongside transcriptional regulation to contribute to callus induction. Especially, ZmWOX11, whose orthologous gene in Arabidopsis plays a vital role in the first-step cell fate transition of callus induction, was found to exhibit alternative splicing and expression level changes during callus induction. Our results establish a foundation from which researchers can further explore the molecular mechanism of callus induction in maize, especially at alternative splicing level. [ABSTRACT FROM AUTHOR]

Maize transformation is highly based on the formation of embryonic callus, which is mainly derived from scutellum cells of the immature maize embryo. However, only a few genes involved in callus induction have been identified in maize. To reveal the potential genes involved in the callus induction of maize, we carried out a high-throughput RNA sequencing on embryos that were cultured for 0, 1, 2, 4, 6, and 8 days, respectively, on a medium containing or lacking 2,4-dichlorophenoxyacetic acid. In total, 7,525 genes were found to be induced by 2,4-dichlorophenoxyacetic acid and categorized into eight clusters, with clusters 2 and 3 showing an increasing trend related to signal transmission, signal transduction, iron ion binding, and heme binding. Among the induced genes, 659 transcription factors belong to 51 families. An AP2 transcription factors, ZmBBM2 , was dramatically and rapidly induced by auxin and further characterization showed that overexpression of ZmBBM2 can promote callus induction and proliferation in three inbred maize lines. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus induction and are useful for further identification of the regulators governing callus formation. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins play crucial roles in intron splicing, which is important for RNA maturation. Identification of novel PPR protein with the function of intron splicing would help to understand the RNA splicing mechanism. In this study, we identified the maize empty pericarp602 (emp602) mutants, the mature kernels of which showed empty pericarp phenotype. We cloned the Emp602 gene from emp602 mutants and revealed that Emp602 encodes a mitochondrial-localized P-type PPR protein. We further revealed that Emp602 is specific for the cis -splicing of mitochondrial Nad4 intron 1 and intron 3, and mutation of Emp602 led to the loss of mature Nad4 transcripts. The loss of function of Emp602 nearly damaged the assembly and accumulation of complex I and arrested mitochondria formation, which arrested the seed development. The failed assembly of complex I triggers significant upregulation of Aox expression in emp602 mutants. Transcriptome analysis showed that the expression of mitochondrial-related genes, e.g. the genes associated with mitochondrial inner membrane presequence translocase complex and electron carrier activity, were extensively upregulated in emp602 mutant. These results demonstrate that EMP602 functions in the splicing of Nad4 intron 1 and intron 3, and the loss of function of Emp602 arrested maize seed development by disrupting the mitochondria complex I assembly. [ABSTRACT FROM AUTHOR]

Transgenic maize expressing the Cry1Ie gene was shown to efficiently kill susceptible and Cry1Ac-resistant insects, indicating that Cry1Ie might be a good candidate for the development of a new Bt-maize variety in China. As transgenic maize events with potential commercial usage are required to have one copy of a transgene, it is important to develop high-throughput methods to determine the transgene copy number initially and zygosity during subsequent breeding. In this study, a SYBR® Green quantitative real-time PCR (qRT-PCR) method was established and used to estimate the transgene copy number in Agrobacterium-derived transgenic maize lines expressing Cry1Ie gene. The amplification efficiencies of Cry1Ie, bar, and Adh1 with specific primers were calculated as 0.987, 1.194, and 0.992, respectively. The transgene copy number was calculated using the formula Ratio = 2, where a calibrator line with a single copy of the transgene was not required. Six homozygous transgenic maize lines were initially analyzed using this calculation method, and the results were as accurate as those obtained using 2, a common method for calculation of relative expression levels. More T generation transgene lines were compared using both the SYBR Green qRT-PCR and TaqMan qRT-PCR methods, and the results showed a high correlation ( R = 0.8942) between SYBR Green qRT-PCR and TaqMan qRT-PCR values, confirming the reliability of the SYBR Green qRT-PCR method. Using this method, we successfully determined the transgene copy number and zygosity in transgenic maize lines expressing Cry1Ie. [ABSTRACT FROM AUTHOR]

A bacterial protein delivery system for transient gene induction was used to study gene regulation in the cuticular wax biosynthesis pathway in maize. Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize. [ABSTRACT FROM AUTHOR]