Correction: Iron-promoted free radical cascade difunctionalization of unsaturated benzamides with silanes.

Correction for 'Iron-promoted free radical cascade difunctionalization of unsaturated benzamides with silanes' by Yaxin Ge et al., Chem. Commun., 2020, 56, 12656-12659, https://doi.org/10.1039/D0CC05213B.

Emission Characteristics of NOx and SO2 during the Combustion of Antibiotic Mycelial Residue.

The antibiotic mycelial residue (AMR) generated from cephalosporin C production is a hazardous organic waste, which is usually disposed of by landfilling that causes potential secondary environmental pollution. AMR combustion can be an effective method to treat AMR. In order to develop clean combustion technologies for safe disposal and energy recovery from various AMRs, the emission characteristics of NOx and SO2 from AMR combustion were studied experimentally in this work. It was found that the fuel-N is constituted by 85% protein nitrogen and 15% inorganic nitrogen, and the fuel-S by 78% inorganic sulfur and 22% organic sulfur. Nitrogen oxide emissions mainly occur at the volatile combustion stage when the temperature rises to 400 °C, while the primary sulfur oxide emission appears at the char combustion stage above 400 °C. Increasing the combustion temperature and airflow cause higher NOx emissions. High moisture content in AMR can significantly reduce the NOx emission by lowering the combustion temperature and generating more reducing gases such as CO. For the SO2 emission, the combustion temperature (700 to 900 °C), airflow and AMR water content do not seem to exhibit obvious effects. The presence of CaO significantly inhibits SO2 emission, especially for the SO2 produced during the AMR char combustion because of the good control effect on the direct emission of inorganic SO2. Employing air/fuel staging technologies in combination with in-situ desulfurization by calcium oxide/salts added in the combustor with operation temperatures lower than 900 °C should be a potential technology for the clean disposal of AMRs.

Free Radical-Promoted Monochloroalkylarylation of Alkenes with Chloralkanes.

Free radical-initiated cascade cyclization of unactivated alkenes with chloralkanes, which undergoes selective activation of the α-C(sp3)-H bond of chloralkanes, provides a protocol for the synthesis of chlorinated heterocycles or polycyclic compounds. A series of radical inhibition experiments, radical capture operations, and radical clock tests were studied in this system.

A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Iron-promoted free radical cascade difunctionalization of unsaturated benzamides with silanes.

Iron salt/peroxide promoted cascade difunctionalization of unsaturated benzamides with silanes has been reported. It provides a convenient, highly selective, and efficient protocol for the synthesis of various silylated dihydroisoquinolinones and 1,3-isoquinolinediones. In particular, the present methodology only gives cyclization products in good isolated yields under the typical conditions.

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa).

Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.

MAIN CONCLUSION: An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.

Silver or cerium-promoted free radical cascade difunctionalization of o-vinylanilides with sodium aryl- or alkylsulfinates.

An efficient cascade oxysulfonylation of o-vinylanilides with sodium aryl- or alkylsulfinates by a free radical mechanism has been developed, which provides a mild, facile and convenient method for the synthesis of various benzoxazines. Control experiments, including gram-level reactions and mechanistic studies, are involved in the reaction system.

Defined tetra-allelic gene disruption of the 4-coumarate:coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release.

BACKGROUND: The development of genome editing technologies offers new prospects in improving bioenergy crops like switchgrass (Panicum virgatum). Switchgrass is an outcrossing species with an allotetraploid genome (2n = 4x = 36), a complexity which forms an impediment to generating homozygous knock-out plants. Lignin, a major component of the plant cell wall and a contributor to cellulosic feedstock's recalcitrance to decomposition, stands as a barrier to efficient biofuel production by limiting enzyme access to cell wall polymers during the fermentation process. RESULTS: We developed a CRISPR/Cas9 genome editing system in switchgrass to target a key enzyme involved in the early steps of monolignol biosynthesis, 4-Coumarate:coenzyme A ligase (4CL). Three 4CL genes, Pv4CL1, Pv4CL2, and Pv4CL3, were identified in switchgrass. Expression analysis revealed that Pv4CL1 transcripts were more abundant in the stem than in the leaf, while Pv4CL2 transcripts were barely detectable and Pv4CL3 was mainly expressed in the leaf. Pv4CL1 was selected as the target for CRISPR/Cas9 editing because of its preferential expression in highly lignified stem tissues. Specific guide RNA was constructed to target Pv4CL1. After introducing the construct into switchgrass calli, 39 transgenic plants were regenerated. Using two rounds of PCR screening and sequencing, four plants were confirmed to have tetra-allelic mutations simultaneously. The Pv4CL1 knock-out plants had reduced cell wall thickness, an 8-30% reduction in total lignin content, a 7-11% increase in glucose release, and a 23-32% increase in xylose release. CONCLUSION: This study established a successful CRISPR/Cas9 system in switchgrass with mutation efficiency reaching 10%. The system allows the precise targeting of the selected Pv4CL1 gene to create switchgrass knock-out mutant plants with decreased lignin content and reduced recalcitrance.

The miR156-SPL4 module predominantly regulates aerial axillary bud formation and controls shoot architecture.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression. Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4. Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield.

Tall fescue (Festuca arundinacea Schreb.).

Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for the generation of a large number of transgenic tall fescue plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is used as the selectable marker, and hygromycin is used as the selection agent. Calli resistant to hygromycin are obtained after 4-6 weeks of selection. Soil-grown tall fescue plants can be regenerated 4-5 months after Agrobacterium tumefaciens-mediated transformation.

Transgenic expression of phytase and acid phosphatase genes in alfalfa (Medicagosativa) leads to improved phosphate uptake in natural soils.

Alfalfa (Medicagosativa L.) is one of the most widely grown crops in the USA. Phosphate (P) deficiency is common in areas where forage crops are grown. To improve the use of organic phosphate by alfalfa, two Medicagotruncatula genes, phytase (MtPHY1) and purple acid phosphatase (MtPAP1), were overexpressed in alfalfa under the control of the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Root enzyme activity analyses revealed that although both genes lead to similar levels of acid phosphatase activities, overexpression of the MtPHY1 gene usually results in a higher level of phytase activity than overexpression of the MtPAP1 gene. The MtPT1 promoter was more effective than the CaMV35S promoter in regulating gene expression and extracellular secretion under P-deficient conditions. Measurement of growth performance of the transgenic lines further proved that the best promoter-gene combination is the MtPHY1 gene driven by the MtPT1 promoter. Compared to the control, the plants with high levels of transgene expression showed improved growth. The biomass of several transgenic lines was three times that of the control when plants were grown in sand supplied with phytate as the sole P source. When the plants were grown in natural soils without additional P supplement, the best performing transgenic lines produced double the amount of biomass after 12 weeks (two cuts) of growth. Transgene effects were more obvious in soil with lower pH and lower natural P reserves than in soil with neutral pH and relatively higher P storage. The total P concentration in leaf tissues of the high-expressing transgenic lines was significantly higher than that of the control. The transgenes have great potential for improving plant P acquisition and biomass yield in P-deficient agricultural soils. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9628-0) contains supplementary material, which is available to authorized users.

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Genetic transformation of switchgrass.

Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes.

Phosphate is one of the least available macronutrients restricting crop production in many ecosystems. A phytase gene (MtPHY1) and a purple acid phosphatase gene (MtPAP1), both isolated from the model legume Medicago truncatula, were introduced into white clover (Trifolium repens L.) by Agrobacterium-mediated transformation. The transgenes were driven by the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Transcripts were detected in roots of the transgenic plants. Phytase or acid phosphatase (APase) activities in root apoplasts of the transgenic plants were increased up to three-fold compared to the wild type control. After the plants were grown 80 days in sand pots supplied with organic phosphorus (Po) as the sole P source, dry weights of shoot tissues of the best performing transgenic plants almost doubled that of the control and were comparable to the counterparts supplied with inorganic phosphorus (Pi). Relative biomass production of the transgenics under Po treatment was over 90% and 80% of that from the Pi treatment when the plants were grown in hydroponics (40 days) and sand pots (80 days), respectively. In contrast, biomass of the wild type controls under Po treatment was only about 50% of the Pi treatment in either hydroponic cultures or sand pots. In addition, shoot P concentrations of the transgenic plants were significantly increased compared to the control. Transgenic plants accumulated much higher amounts of total P (up to 2.6-fold after 80 days of growth) than the control in Po supplied sand pots. The results showed that transgenic expression of MtPHY1 or MtPAP1 in white clover plants increased their abilities of utilizing organic phosphorus in response to P deficiency.

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation.

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4-6 weeks of selection and transgenic plants were produced in 10-13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.

Bermudagrass (Cynodon spp.).

Bermudagrass is an important warm-season forage and turf species widely grown in the southern United States. This chapter describes a rapid and efficient protocol that allows for the generation of a large number of transgenic bermudagrass plants, bypassing the callus formation phase. Stolon nodes are infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA binary vectors. Hygromycin phosphotransferase gene (hph) is used as the selectable marker and hygromycin is used as the selection agent. Green shoots are directly produced from infected stolon nodes 4 to 5 wk after hygromycin selection. Without callus formation and with minimum tissue culture, this procedure allowed us to obtain well-rooted transgenic plantlets in only 7 wk and greenhouse-grown plants in only 9 wk.

Tall Fescue (Festuca arundinacea Schreb.).

Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for the generation of large number of transgenic tall fescue plants by Agrobacterium-mediated transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is used as the selectable marker and hygromycin is used as the selection agent. Calli resistant to hygromycin are obtained after 4-6 wk of selection. Soil-grown tall fescue plants can be regenerated 4-5 mo after Agrobacterium-mediated transformation.

Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation.

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4-5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10-12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea).

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.