Summary Carotenoids are vital phytonutrients widely recognised for their health benefits. Therefore, it is vital to thoroughly investigate the metabolic regulatory network underlying carotenoid biosynthesis and accumulation to open new leads towards improving their contents in vegetables and crops. The outcome of our study defines SlWRKY35 as a positive regulator of carotenoid biosynthesis in tomato. SlWRKY35 can directly activate the expression of the 1‐deoxy‐d‐xylulose 5‐phosphate synthase (SlDXS1) gene to reprogramme metabolism towards the 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway, leading to enhanced carotenoid accumulation. We also show that the master regulator SlRIN directly regulates the expression of SlWRKY35 during tomato fruit ripening. Compared with the SlLCYE overexpression lines, coexpression of SlWRKY35 and SlLCYE can further enhance lutein production in transgenic tomato fruit, indicating that SlWRKY35 represents a potential target towards designing innovative metabolic engineering strategies for carotenoid derivatives. In addition to providing new insights into the metabolic regulatory network associated with tomato fruit ripening, our data define a new tool for improving fruit content in specific carotenoid compounds.

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The efficiency and high specificity of tobacco etch virus protease (TEVp) has made it widely used for cleavage of recombinant fusion proteins. However, TEVp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in E. coli and less soluble. So some mutants were designed to improve it, such as S219V, T17S/N68D/I77V and L56V/S135G etc. MD simulations for the WT TEVp and its mutants were performed to explore the underlying dynamic effects of mutations on TEVp. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of TEVp, including the elongation of β-sheet, conversion of loop to helix and the flexibility of active core. Our present study indicates that the three mutants for TEVp can change their secondary structure and tend to form more helixes and sheets to improve stability. The study also helps us to understand the effects of some mutations on TEVp, provides us insights into the change of them at the atomic level and gives a potential rational method to design an improved protein.

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Lutein is an oxygen-containing carotenoid synthesized in plant chloroplasts and chromoplasts. It plays an indispensable role in promoting plant growth and maintaining eye health in humans. The rate-limiting step of lutein biosynthesis is catalyzed by the lycopene ε-cyclase enzyme (LCYE). Although great progress has been made in the identification of transcription factors involved in the lutein biosynthetic pathway, many systematic molecular mechanisms remain to be elucidated. Here, using co-expression analysis, we identified a gene, G2-LIKE CAROTENOID REGULATOR (SlGCR), encoding a GARP G2-like transcription factor, as the potential regulator of SlLCYE in tomato. Silencing of SlGCR reduced the expression of carotenoid biosynthetic genes and the accumulation of carotenoids in tomato leaves. By contrast, overexpression of SlGCR in tomato fruit significantly increased the expression of relevant genes and enhanced the accumulation of carotenoids. SlGCR can directly bind to the SlLCYE promoter and activate its expression. In addition, we also discovered that expression of SlGCR was negatively regulated by the master regulator SlRIN, thereby inhibiting lutein synthesis during tomato fruit ripening. Taken together, we identified SlGCR as a novel regulator involved in tomato lutein biosynthesis, elucidated the regulatory mechanism, and provided a potential tool for tomato lutein metabolic engineering.