Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Due to global climate change, drought is emerging as a major threat to plant growth and agricultural productivity. Abscisic acid (ABA) has been implicated in plant drought tolerance, however, its retarding effects on plant growth cannot be ignored. The reactions catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) proteins are critical steps within the isoprenoid biosynthesis in plants. Here, five DXS (CtDXS1-5) and two DXR (CtDXR1-2) genes were identified from Cassia tora genome. Based on multiple assays including the phylogeny, cis-acting element, expression pattern, and subcellular localization, CtDXS1 and CtDXR1 genes might be potential candidates controlling the isoprenoid biosynthesis. Intriguingly, CtDXS1 transgenic plants resulted in drought tolerance but retardant growth, while CtDXR1 transgenic plants exhibited both enhanced drought tolerance and increased growth. By comparison of β-carotene, chlorophyll, abscisic acid (ABA) and gibberellin 3 (GA3) contents in wild-type and transgenic plants, the absolute contents and (or) altered GA3/ABA levels were suggested to be responsible for the balance between drought tolerance and plant growth. The transcriptome of CtDXR1 transgenic plants suggested that the transcript levels of key genes, such as DXS, 9-cis-epoxycarotenoid dioxygenases (NCED), ent-kaurene synthase (KS) and etc, involved with chlorophyll, β-carotene, ABA and GA3 biosynthesis were induced and their contents increased accordingly. Collectively, the trade-off effect induced by CtDXR1 was associated with redesigning architecture in phytohormone homeostasis and thus was highlighted for future breeding purposes.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

The methylerythritol 4-phosphate (MEP) pathway is of paramount importance for generating plastidial isoprenoids. The first enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), catalyzes a flux-controlling step. In plants the DXS gene family is composed of three distinct classes with non-redundant functions. Although the DXS1 and DXS2 subfamilies have been well characterized, the DXS3 subfamily has been considerably understudied. Here, we carried out in silico and functional analyses to better understand the DXS3 class. Our phylogenetic analysis showed high variation in copy number among the different DXS classes, with the apparent absence of DXS1 class in some species. We found that DXS3 subfamily emerged later than DXS1 and DXS2 and it is under less intense purifying selection. Furthermore, in the DXS3 subfamily critical amino acids positions in the thiamine pyrophosphate binding pocket are not conserved. We demonstrated that the DXS3 proteins from Arabidopsis, Maize, and Rice lack functional DXS activity. Moreover, the Arabidopsis DXS3 protein displayed distinctive sub-organellar chloroplast localization not observed in any DXS1 or DXS2 proteins. Co-expression analysis of the DXS3 from Arabidopsis showed that, unlike DXS1 and DXS2 proteins, it co-expresses with genes related to post-embryonic development and reproduction and not with primary metabolism and isoprenoid synthesis.

Nannochloropsis spp. are promising industrial microalgae for scalable oil production and the lipid production can be boosted by nutrient starvation and high irradiance. However, these stimuli halt growth, thereby decreasing overall productivity. In this study, we created transgenic N. oceanica where AtDXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) derived from Arabidopsis thaliana was overexpressed in vivo. Compared with the wild type (WT), engineered Nannochloropsis showed a higher CO2 absorption capacity and produced more biomass, lipids, and carbohydrates with more robust growth in either preferred conditions or various stressed conditions (low light, high light, nitrogen starvation, and trace element depletion). Specifically, relative to the WT, lipid production increased by ~68.6% in nitrogen depletion (~1.08 ​g ​L-1) and ~110.6% in high light (~1.15 ​g ​L-1) in the transgenic strains. As for neutral lipid (triacylglycerol, TAG), the engineered strains produced ~93.2% more in nitrogen depletion (~0.77 ​g ​L-1) and ~148.6% more in high light (~0.80 ​g ​L-1) than the WT. These values exceed available records in engineered industrial microalgae. Therefore, engineering control-knob genes could modify multiple biological processes simultaneously and enable efficient carbon partitioning to lipid biosynthesis with elevated biomass productivity. It could be further exploited for simultaneous enhancement of growth property and oil productivity in more industrial microalgae.

To fight the growing threat of antibiotic resistance, new antibiotics are required that target essential bacterial processes other than protein, DNA/RNA, and cell wall synthesis, which constitute the majority of currently used antibiotics. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase is a vital enzyme in bacterial central metabolism, feeding into the de novo synthesis of thiamine diphosphate, pyridoxal phosphate, and essential isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. While potent and selective inhibitors of DXP synthase in vitro activity have been discovered, their antibacterial activity is modest. To improve the antibacterial activity of selective alkyl acetylphosphonate (alkylAP) inhibitors of DXP synthase, we synthesized peptidic enamide prodrugs of alkylAPs inspired by the natural product dehydrophos, a prodrug of methyl acetylphosphonate. This prodrug strategy achieves dramatic increases in activity against Gram-negative pathogens for two alkylAPs, butyl acetylphosphonate and homopropargyl acetylphosphonate, decreasing minimum inhibitory concentrations against Escherichia coli by 33- and nearly 2000-fold, respectively. Antimicrobial studies and LC-MS/MS analysis of alkylAP-treated E. coli establish that the increased potency of prodrugs is due to increased accumulation of alkylAP inhibitors of DXP synthase via transport of the prodrug through the OppA peptide permease and subsequent amide hydrolysis. This work demonstrates the promise of targeting DXP synthase for the development of novel antibacterial agents.

Malaria remains one of the most serious infectious diseases in the world. There are five human species of the Plasmodium genus, of which Plasmodium falciparum is the most virulent and responsible for the vast majority of malaria related deaths. The unique biochemical processes that exist in Plasmodium falciparum provide a useful way to develop novel inhibitors. One such biochemical pathway is the methyl erythritol phosphate pathway (MEP), required to synthesize isoprenoid precursors. In the present study, a detailed computational analysis has been performed for 1-deoxy-d-xylulose-5-phosphate synthase, a key enzyme in MEP. The protein is found to be stable and residues from 825 to 971 are highly conserved across species. The homology model of the enzyme is developed using three web-based servers and Modeller software. It has twelve disordered regions indicating its druggability. Virtual screening of ZINC database identifies ten potential compounds in thiamine diphosphate binding region of the enzyme.

We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.