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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.

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.