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Introduction Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Artemisia annuais an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression ofProduction of Anthocyanin Pigment 1(PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences ofA. annuaand obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-Box cis-element. Eight promoters were cloned for activity analysis. Duel luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 μM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per petri dish. HPLC-qTOF-MS/MS based profiling showed that these concentrations did not alter anthocyanin profiles. Real time RT-PCR was completed to characterize the expressionPAP1and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutivePAP1transgene and three pathway genes significantly and eliminated chalcone synthase gene in expression either significantly or slightly. These data indicate that the constitutivePAP1expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells ofA. annua.ConclusionPromoters of eight anthocyanin pathway genes were cloned. Four types of AC cis-elements were identified from six promoters and G-Box elements were also determined from six promoters. PAP1 enhanced the activity of eight promoters. 2,4-D downregulated the expression of the constitutive PAP1 transgene leading to the decrease the biosynthesis of anthocyanins.

Artemisia annuais the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs),; however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated fromA. annua. In this study, our goals were to engineer anthocyanins inA. annuacells and to understand the artemisinin biosynthesis in anthocyanin-producing cells. ArabidopsisPAP1(AtPAP1) was used to engineer four types of transgenic anthocyanin-producingA. annua(TAPA1 to 4) cells. Three wild type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from DHAA in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells ofA. annua.

Purpose The purpose of this paper is to generate quantitative managerial insights for the improvement of the energy-saving potential and the coordinated development between economic growth and environmental protection. Design/methodology/approach A novel data envelopment analysis (DEA) model, based on the classical DEA theory, is developed from the perspective of emission reduction. Findings The empirical results indicate that China’s overall environmental efficiency is low and that there is huge improvement space for energy saving. Under the concerns of emission reduction, the energy-saving potential of the central region exceeds that of both the eastern and western regions. With regard to water, electricity and gas consumption, the electricity-saving potential exceeds the potential for both water saving and gas saving. Originality/value Previous studies rarely focused on the energy-saving potential, while considering environmental pollution. This paper applies a novel DEA method to evaluate the energy-saving potential of 30 Chinese provinces for 2015 with a focus on emission reduction concerns. Furthermore, both regional differences and energy type differences of the saving potential were analyzed.

Rice blast is one of the most serious rice diseases. It is caused by Magnaporthe oryzae resulting in rice shortages. Biological control has become a new measure in the control of plant diseases. In this study, we isolated antagonistic bacteria from the traditional Chinese medicinal plant Baizhi (Angelica dahurica) against M. oryzae. Biochemical, physiological and 16S rDNA sequence analysis proved that it was Bacillus amyloliquefaciens and we named the strain Rdx5. The experiment proved that Rdx5 can produce cellulase, protease, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. The sterilized culture filtrate of Rdx5 had strong antifungal activity after treatment at 100 °C for 30 min. In addition, its antifungal activity did not change in strong acid, neutral and weak alkaline environment. However, under strong alkaline conditions, the antifungal activity decreased significantly. In vitro and in vivo experiments showed that Rdx5 could prevent and treat rice blast, since the prevention group performed better than the treatment group. B. amyloliquefaciens Rdx5 might provide an alternative resource for the biocontrol of rice blast.

Transcription factors (TFs) andWe selected two Arabidopsis TF types and two tobacco CRE types to design aG-box and MYB recognition elements (MREs) were identified in fourPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activatedG-boxes, MREs, and two TF types are appropriate molecular tools for a

IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P+T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P+T and PAP1 genotypes.ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Background: WRKY transcription factor (TF) is one of the largest TF families in plants and plays an important role in plant development and stress protection. However, the function of individual WRKY gene in plants are still under investigation. Here, we identified a new member of WRKY TF family, OsWRKY97 , and analyze its role in stress resistance by using a series of transgenic plant lines.Results: OsWRKY97 , which positively regulates drought tolerance in rice. OsWRKY97 was expressed in all examined tissues and could be induced by various abiotic stresses. OsWRKY97 is located in the nucleus. Compared with the wild-type (WT), OsWRKY97 -overexpressing (OE) plants enhanced the tolerance of rice to drought and osmotic stress. In addition, OE plants also showed higher sensitivity to exogenous abscisic acid (ABA). When exposed to drought stress, up-regulation OsWRKY97 increased the accumulation of ABA and reduced water loss. Furthermore, OE plants achieved higher proline content and reduced levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The homeostasis of ROS was maintained via the activity of active oxygen scavenging enzyme, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), activated by OsWRKY97Conclusions: These results indicate that OsWRKY97 plays a crucial role in the response to drought stress and may possess high potential value in improving drought tolerance in rice.

Rice blast is one of the most destructive diseases of rice and is caused by the infection with rice blast fungus, Magnaporthe oryzae. In the present study, we obtained 12 bacterial isolates from Sichuan basin neutral purplish soil. Among them, the strain named ZW-10 showed significant activity against M. oryzae. Morphological and biochemical studies, along with 16S rDNA sequencing analysis, proved that the strain belongs to Bacillus velezensis. The cell-free culture filtrate of ZW-10 inhibited the growth of M. oryzae, suggesting the presence of active enzymes and secondary metabolites. The experimental results showed that ZW-10 produced cellulase, protease, and peroxidase. The temperature experiment indicated that the secondary metabolisms of ZW-10 remained active at high temperature. In addition, this strain had good acidity resistance. As shown in leaf experiments in vitro as well as field trials, the liquid culture and the cell-free culture filtrate of ZW-10 had significant inhibitory effects on rice blast. In conclusion, ZW-10 can be used as a potential biological agent in the control of rice blast.

PurposeThe existing literature has a lack of modeling of procedural fairness concerns in the supply chain level. This paper aims to investigate how procedural fairness concerns affect channel decisions, performance and coordination.Design/methodology/approachThis paper considers a supply chain consisting of one supplier and one retailer who have procedural fairness concerns in a classic Stackelberg game setting. The model is set in sales promotional environment. According to the existing literature, engagement is used to depict fair process. Some findings are made through analyzing respective decisions of the supplier and the retailer under the influence of procedural fairness concerns.FindingsThe results show that the channel efficiency can be improved when the retailer exhibits procedural fairness concerns, but if the aversion to unfair process exceeds a certain threshold, the retailer cannot benefit from it. Besides, the retailer profits more when he cares about distributional fairness, although the whole channel surplus can be improved by procedural fairness concerns.Originality/valueThis is the first paper to study the influences of procedural fairness concerns on supply chain decisions and channel performance. Finally, a mechanism combining a wholesale price contract with slotting allowances is proposed to coordinate the supply chain.

In some traditional formative and additive manufacturing processes widely used in the fabrication of steel products, stress conditions and metallurgy typically result in a strong texture and mechanical anisotropy in both rolling high-strength low-alloy steel and additive manufacturing stainless steel products. To resolve this problem, we have investigated the effectiveness of a novel wire-arc additive manufacturing method in which the heat source is substituted by high-efficiency submerged arc plasma i.e., submerged arc additive manufacturing. An isotropic microstructure can be obtained in the bulk of large-scale low-carbon steel components by ensuring a complete columnar-to-equiaxed transition. We found that the top zone of the as-deposited component comprised columnar α-Fe grains with a typical preferential α orientation. Multiple allotropic transformations were triggered by an in-situ intrinsic heat treatment (mainly the combination of multi-layer-penetration normalizing, full-layer-penetration inter-critical annealing, and long-duration tempering—i.e., NIT). Consequently, when the net bead height was no greater than the width of the fine grain zone affected by the previous layer of deposition, the microstructures in each new layer could be progressively refined and homogenized. Therefore, the intermediate zone was predominantly composed of fully equiaxed α-Fe and displayed homogeneous characteristics along the build direction. During full-layer NIT treatment, the pearlite was spheroidized slightly, reduced both in size (from 4 µm to 2 µm) and in area fraction (from ~5% to ~1%), thereby mitigating cracking susceptibility. Moreover, not only were the intracrystalline dislocations significantly reduced, but also their morphology evolved from tangled to movable lines. These changes, along with finer grains and the formation of high-angle grain boundaries, led to lower and more dispersed internal strain in the α-Fe matrix. Consequently, excellent Charpy toughness (over 300 J at − 60 °C) with minimal deterioration in strength was achieved. Impact, tensile, and hardness tests revealed nearly isotropic mechanical characteristics. We believe that this novel method shows tremendous promise in large-scale additive manufacturing.

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the three major diseases affecting rice production and quality; it reduces rice grain yield by nearly 30%. In the early stage of this study, a strain of Bacillus velezensis with strong inhibition of M. oryzae was isolated and named ZW10. In vitro assays indicated prolonged germination time of conidia of M. oryzae treated with the antifungal substances of ZW10, 78% of the conidia could not form appressorium, and the conidial tubes expanded to form vacuolar structure and then shrank. The results of FDA-PI composite dyes showed that the antifungal substances of ZW10 inhibited the normal activity of M. oryzae hyphae that were rarely able to infect the epidermal cells of rice leaf sheath in vivo tests. In addition, rice treated with the antifungal substances of ZW10 showed a variety of defense responses, including activation of defense-related enzymes, increased expression of the salicylic acid pathway genes, and accumulation of hydrogen peroxide (H2O2), which might function directly or indirectly in resistance to pathogen attack. The field experiment with rice blast infection in different periods showed that the antifungal substances of ZW10 had the same control effect as carbendazim. The significant biological control activity of ZW10 and its capacity to stimulate host defenses suggest that this B. velezensis strain has the potential to be developed into a biopesticide for the biocontrol of rice blast.

Biological control as a promising method to combat plant disease has gained public attention in recent years. In the present study, we isolated 12 strains resistant to Magnaporthe oryzae from western Sichuan subalpine soil. Among them, CQ07 exhibited remarkable activity against M. oryzae. The result of 16S rRNA sequence analysis revealed that CQ07 is approximately 99% similar to Bacillus australimaris. The sterilized culture filtrate of CQ07 inhibited the growth of M. oryzae, which motivated us to deduce the influence of CQ07 on the pathogenicity of M. oryzae. As shown by experimentation, sterilized culture filtrate (10 μl/ml) of CQ07 can delay and even suppress the germination of conidia and prevent the formation of appressorium in vitro and in vivo. In addition, by simulative field tests, the spraying of conidia suspension diluted with sterilized culture filtrate of CQ07 reduced infection of rice blast. To better control rice blasts, understanding the infection mechanism of M. oryzae and inhibiting the mechanism of the antagonistic strain is of great importance.

Sulfonated polyether ether ketone (SPEEK)/polyvinylidene fluoride (PVDF) blends are employed to prepare the ion-exchange membranes for vanadium redox flow battery (VRB) application. The addition of the highly crystalline and hydrophobic PVDF effectively confines the swelling behavior of SPEEK. The vanadium ion permeability of SPEEK/PVDF membranes is one order of magnitude lower than that of Nafion117 membrane and SPEEK membrane. In VRB single-cell test, the SPEEK/PVDF binary membranes exhibit higher energy efficiency (EE) than recast SPEEK and Nafion117 at 30 mA cm−2. The blend membrane with 30 wt% of PVDF (S0.7P0.3) shows EE of 83.2% at 30 mA cm−2, which is superior to that of the Nafion117 and recast SPEEK membranes. In the self-discharge test, S0.7P0.3 membrane also possesses twice longer duration in open circuit decay test. These results indicate that the addition of PVDF is a simple and efficient way to improve the ion selectivity of SPEEK. With all the good properties and low cost, the SPEEK/PVDF membranes are expected to have excellent commercial prospects as ion-exchange membranes for VRB system.

Biological control is a promising measure in the control of plant disease. In the present study, we isolated 13 endophytic strains from Angelica dahurica. Among them, an endophytic strain which was named GYLH001 exhibited remarkable activity against Magnaporthe oryzae. 16S rRNA sequence analysis, biochemical and physiological proved that it is Bacillus tequilensis. The sterilized culture filtrate of GYLH001 can inhibit the growth of M.oryzae, which suggests the presence of secondary metabolites. Proved by experiment, GYLH001 can produce cellulase, protease, gelatinase, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. In addition, the temperature experiment showed that secondary metabolites produced by GYLH001 had good thermal stability. They can remain activity even heated at 100°C for 30 min. They also had good acid-resistance in heavily acidic condition. But under alkaline condition, the antifungal effect decreased significantly. By simulative field tests, the spraying of GYLH001 spore solution could prevent and treat rice blast. Through continuous separation and purification of sterilized culture filtrate and identification by mass spectrometry, the molecular weight of an active substance is 364.26. In the control of rice blast, B. tequilensis GYLH001 has potential as a biological control agent in agriculture.