Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.Plants are estimated to produce more than 500,000 secondary metabolites (1). These compounds have many functions, including attracting pollinators, communicating with neighboring plants, and defending against pathogens and herbivores (2, 3). The importance of secondary metabolites is highlighted by the extensive resources that plants invest in producing these compounds. Although once thought to be metabolically simple, more than 170 secondary metabolites have been identified in Arabidopsis thaliana (reviewed in Ref. 4).Triterpenoids are the 30-carbon subset of terpenoids, the largest class of secondary metabolites. Triterpenoid biosynthesis is diagramed in Fig. 1. Isopentenyl diphosphate and dimethylallyl diphosphate are synthesized from mevalonate and oligomerized to farnesyl diphosphate by farnesyl diphosphate synthase (FPS).5 Farnesyl diphosphate is dimerized to squalene by squalene synthase. Squalene epoxidase (SQE)-mediated oxidation then produces oxidosqualene, which triterpene synthases cyclize to Ͼ80 triterpene skeletons (5, 6). Further metabolism of these compounds produces membrane sterols, brassinosteroid phytohormones, saponins, other defense compounds, cuticular waxes, and numerous triterpenoids that have not been functionally characterized.The yeasts and mammals that have been investigated each encode a single squalene epoxidase. In contrast, several plants have multiple genes predicted to encode squalene epoxidases, a diversity suggesting that this step may be subject to additional or unique regulation in plants. Two Medicago truncatula SQE enzymes have been biochemically characterized (7). The Brassica napus (8), Populus trichocarpa, and Oryza sativa genomes each have multiple predicted SQE enzymes. Despite the likely importance of SQE to plant growth and development, no plant mutants with defects in these enzymes have been reported.In this work, we heterologously expressed the six Arabidopsis putative SQE enzymes in Saccharomyces cerevisiae lacking squalene epoxidase to determine which have squalene epoxidase activity. We isolated Arabidopsis sqe1 loss-of-function mutants and found t...

Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium.

The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50-mediated resistance to stem rust (Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.

Rust diseases are among the major constraints for wheat production worldwide due to the emergence and spread of highly destructive races of Puccinia. The most common approach to minimise yield losses due to rust is to use cultivars that are genetically resistant. Modern wheat cultivars, landraces, and wild relatives can contain undiscovered resistance genes, which typically encode kinase or nucleotide-binding site leucine rich repeat domain containing receptor proteins. Recent research has shown these genes can provide either resistance in all growth stages (all-stage resistance; ASR) or specially in later growth stages (adult-plant resistance; APR). ASR genes are pathogen and race-specific, meaning they can function against selected races of the rust fungus due to the necessity to recognise specific avirulence molecules in the pathogen. APR genes are either pathogen specific or multipathogen resistant but often race-nonspecific. Prediction of resistance genes through rust infection screening alone remains complex when more than one resistance gene is present. However, breakthroughs during the past half century such as single nucleotide polymorphism (SNP) based genotyping techniques and resistance gene isolation strategies such as MutRenSeq (Mutagenesis, Resistance gene enrichment and Sequencing), MutChromSeq (Mutagenesis and Chromosome Sequencing), and AgRenSeq (Association genetics combined with RenSeq) enable rapid transfer of resistance from source to modern cultivars. There is a strong need for combining multiple genes for better efficacy and longer-lasting resistance. Hence techniques such gene cassette creation speed up the gene combination process, but their widespread adoption and commercial use is limited due to their transgenic nature.

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