ZmGA3ox2, a candidate gene for a major QTL,qPH3.1, for plant height in maize

Maize plant height is closely associated with biomass, lodging resistance and grain yield. Determining the genetic basis of plant height by characterizing and cloning plant height genes will guide the genetic improvement of crops. In this study, a quantitative trait locus (QTL) for plant height, qPH3.1, was identified on chromosome 3 using populations derived from a cross between Zong3 and its chromosome segment substitution line, SL15. The plant height of the two lines was obviously different, and application of exogenous gibberellin A₃ removed this difference. QTL mapping placed qPH3.1 within a 4.0 cM interval, explaining 32.3% of the phenotypic variance. Furthermore, eight homozygous segmental isolines (SILs) developed from two larger F₂ populations further narrowed down qPH3.1 to within a 12.6 kb interval. ZmGA3ox2, an ortholog of OsGA3ox2, which encodes a GA3 β‐hydroxylase, was positionally cloned. Association mapping identified two polymorphisms in ZmGA3ox2 that were significantly associated with plant height across two experiments. Quantitative RT‐PCR showed that SL15 had higher ZmGA3ox2 expression relative to Zong3. The resultant higher GA₁ accumulation led to longer internodes in SL15 because of increased cell lengths. Moreover, a large deletion in the coding region of ZmGA3ox2 is responsible for the dwarf mutant d1‐6016. The successfully isolated qPH3.1 enriches our knowledge on the genetic basis of plant height in maize, and provides an opportunity for improvement of plant architecture in maize breeding.     

Sensory and Sympathetic Nerve Sprouting in the Rat Cornea Following Neonatal Administration of Capsaicin

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6–8 weeks both the CGRP-and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become “hyper-reinnervated”. The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 × 0.49 μm and 34.3 × 0.72 μm), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70–80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant “hyper-reinnervation” is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Microbial Resolution of Racemic Carvomenthols

By selected microorganisms dl-carvomenthyl acetate, dl-isocarvomenthyl acetate and dl-neo isocarvomenthyl acetate were asymmetrically hydrolyzed to l-carvomenthol with d-carvomenthyl acetate, l-isocarvomenthol with d-isocarvomenthyl acetate and d-neo isocarvomenthol with l-neo isocarvomenthyl acetate respectively; dl-neo carvomenthyl acetate was not hydrolyzed.     

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.