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This study aimed to reveal the potential genes and mechanisms involved in the biosynthesis of quercetin (QCT) in maize (Zea mays L.) through genome-wide association study (GWAS) and bioinformatics analysis, and provide a basis for further research on QCT. The content of QCT in 196 maize inbred lines was detected by LC-MS. The single-nucleotide polymorphism (SNPs) data from these samples were explored by Maize 55K SNP arrays, followed by population structure using fastStructure software and genome-wide association study (GWAS) analysis. Then, the SNP screening was performed with P < 1.00E-06 using Gramene software, followed by SNP annotation (150 KB up and downstream). The enrichment analysis was performed on all annotated genes. Finally, the protein-protein interaction (PPI) network analysis was performed to further reveal the relations among genes. A total of 12 SNPs were explored from original SNPs with P < 1.00E-06. The SNP annotation revealed several SNP-associated genes including Zm00001d036822 and Zm00001d007407. These genes were mainly enriched in metabolic pathway and functions like negative regulation of cell cycle phase transition. Finally, a PPI work was constructed by totally 34 annotated genes and 59 interactions. Zm00001d036822 and Zm00001d007407 might be novel candidate genes involved in the synthesis of QCT. Meanwhile, the metabolic pathway as well as negative regulation of cell cycle phase transition function might contribute to the biosynthesis of QCT.

Maize often exhibits asynchronous pollination under abiotic and biotic stress conditions; however, the molecular basis of this developmental deficiency has not been elucidated. Tassel development is a key process affecting the anthesis-silking interval (ASI) in maize. In this study, we showed that pollen shedding was delayed and ASI was significantly increased in B73 and Chang7-2 inbred lines under water deficit conditions, which resulted in longer barren tip length and decreased yields under both controlled and field conditions. Comparative transcriptome analysis performed on immature tassels derived from plants grown under well-watered and water deficit conditions identified 1931 and 1713 differentially expressed genes (DEGs) in B73 and Chang7-2, respectively. Further, 28 differentially co-expressed transcription factors were identified across both lines. Collectively, we demonstrated that the molecular regulation of tassel development is associated with water deficit stress at early vegetative stage in maize. This finding extends our understanding of the molecular basis of maize tassel development during abiotic stress.

Summary Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, pervasive environmental pollutants with mutagenic, teratogenic, and carcinogenic properties. There is interest in exploiting the nutritional capabilities of microbes to remove PAHs from various environments including those impacted by improper disposal or spills. Although there is a considerable body of literature on PAH degradation, the substrates and products for many of the enzymes have never been identified and many proposed activities have never been confirmed. This is particularly true for high molecular weight PAHs (e.g., phenanthrene, fluoranthene, and pyrene). As a result, pathways for the degradation of these compounds are proposed to follow one elucidated for naphthalene with limited experimental verification. In this pathway, ring fission produces a species that can undergo a non-enzymatic cyclization reaction. An isomerase opens the ring and catalyzes a cis to trans double bond isomerization. The resulting product is the substrate for a hydratase-aldolase, which catalyzes the addition of water to the double bond of an α,β-unsaturated ketone, followed by a retro-aldol cleavage. Initial kinetic and mechanistic studies of the hydratase-aldolase in the naphthalene pathway (designated NahE) and two hydratase-aldolases in the phenanthrene pathway (PhdG and PhdJ) have been completed. Crystallographic work on two of the enzymes (NahE and PhdJ) provides a rudimentary picture of the mechanism and a platform for future work to identify the structural basis for catalysis and the individual specificities of these hydratase-aldolases.

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.

5-Halo-2-hydroxy-2,4-pentadienoates (5-halo-HPDs) are reportedly generated in the bacterial catabolism of halogenated aromatic hydrocarbons by the meta-fission pathway. The 5-halo-HPDs, where the halogen can be bromide, chloride, or fluoride, result in the irreversible inactivation of 4-oxalocrotonate tautomerase (4-OT), which precedes the enzyme that generates them. The loss of activity is due to the covalent modification of the nucleophilic amino-terminal proline. Mass spectral and crystallographic analysis of the modified enzymes indicate that inactivation of 4-OT by 5-chloro- and 5-bromo-2-hydroxy-2,4-pentadienoate follows a different mechanism than that for the inactivation of 4-OT by 5-fluoro-2-hydroxy-2,4-pentadienoate. The 5-chloro- and 5-bromo derivatives undergo 4-OT-catalyzed tautomerization to their respective α-unsaturated ketones followed by attack at C-5 (by the prolyl nitrogen) with concomitant loss of the halide. For the 5-fluoro species, the presence of a small amount of the α,β-unsaturated ketone could result in a Michael addition of the prolyl nitrogen to C-4 followed by protonation at C-3. The fluoride is not eliminated. These observations suggest that the inactivation of 4-OT by a downstream metabolite could hamper the efficacy of the pathway, which is the first time that such a bottleneck has been reported for the meta-fission pathway.

Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of maize plant. However, few focused on seed development of maize. Here, we identified 753 lncRNA candidates in maize genome from six seed samples. Similar to the mRNAs, lncRNAs showed tissue developmental stage specific and differential expression, indicating their putative role in seed development. Increasing evidence shows that crosstalk among RNAs mediated by shared microRNAs (miRNAs) represents a novel layer of gene regulation, which plays important roles in plant development. Functional roles and regulatory mechanisms of lncRNAs as competing endogenous RNAs (ceRNA) in plants, particularly in maize seed development, are unclear. We combined analyses of consistently altered 17 lncRNAs, 840 mRNAs and known miRNA to genome-wide investigate potential lncRNA-mediated ceRNA based on “ceRNA hypothesis”. The results uncovered seven novel lncRNAs as potential functional ceRNAs. Functional analyses based on their competitive coding-gene partners by Gene Ontology (GO) and KEGG biological pathway demonstrated that combined effects of multiple ceRNAs can have major impacts on general developmental and metabolic processes in maize seed. These findings provided a useful platform for uncovering novel mechanisms of maize seed development and may provide opportunities for the functional characterization of individual lncRNA in future studies.

The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ-βα sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.