Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

Similar enzymatic functions in distinct bioluminescence systems: Evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii , transfer sulfate in vivo to the luciferin substrate, vargulin. We find luciferin sulfotransferases of ostracods are not orthologous to known luciferin sulfotransferases of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Re-use of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

PanKmer: k-mer-based and reference-free pangenome analysis.

SUMMARY: Pangenomes are replacing single reference genomes as the definitive representation of DNA sequence within a species or clade. Pangenome analysis predominantly leverages graph-based methods that require computationally intensive multiple genome alignments, do not scale to highly complex eukaryotic genomes, limit their scope to identifying structural variants (SVs), or incur bias by relying on a reference genome. Here, we present PanKmer, a toolkit designed for reference-free analysis of pangenome datasets consisting of dozens to thousands of individual genomes. PanKmer decomposes a set of input genomes into a table of observed k-mers and their presence-absence values in each genome. These are stored in an efficient k-mer index data format that encodes SNPs, INDELs, and SVs. It also includes functions for downstream analysis of the k-mer index, such as calculating sequence similarity statistics between individuals at whole-genome or local scales. For example, k-mers can be "anchored" in any individual genome to quantify sequence variability or conservation at a specific locus. This facilitates workflows with various biological applications, e.g. identifying cases of hybridization between plant species. PanKmer provides researchers with a valuable and convenient means to explore the full scope of genetic variation in a population, without reference bias. AVAILABILITY AND IMPLEMENTATION: PanKmer is implemented as a Python package with components written in Rust, released under a BSD license. The source code is available from the Python Package Index (PyPI) at https://pypi.org/project/pankmer/ as well as Gitlab at https://gitlab.com/salk-tm/pankmer. Full documentation is available at https://salk-tm.gitlab.io/pankmer/.

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and re-emergence strategy.

Over 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high-quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high-quality transcripts and carried out RNA-seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza. Comparison of turion transcriptomes to that of fronds, the actively growing leaf-like tissue, were carried out. Bioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. We identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as those in pathways for starch and lipid utilization upon turion germination. Comparison of genome-wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. Similarities between turions and seeds provide evidence that key regulators for seed maturation and germination were retooled for their function in turion biology.

Neurodegeneration in a novel invertebrate model system: Failed microtubule-mediated cell adhesion and unraveling of macroglia.

Neurodegenerative diseases are among the main causes of death in the United States, leading to irreversible disintegration of neurons. Despite intense international research efforts, cellular mechanisms that initiate neurodegeneration remain elusive, thus inhibiting the development of effective preventative and early onset medical treatment. To identify underlying cellular mechanisms that initiate neuron degeneration, it is critical to identify histological and cellular hallmarks that can be linked to underlying biochemical processes. Due to the poor tissue preservation of degenerating mammalian brain tissue, our knowledge regarding histopathological hallmarks of early to late degenerative stages is only fragmentary. Here, we introduce a novel model organism to study histological hallmarks of neurodegeneration, the spider Cupiennius salei. We utilized toluidine blue-stained 0.9-µm serial semithin and 50-nm ultrathin sections of young and old spider nervous tissue. Our findings suggest that the initial stages of neurodegeneration in spiders may be triggered by (1) dissociation of neuron- and glia-derived microtubules, and (2) the weakening of microtubule-associated desmosomal junctions that lead to the unraveling of neuron-insulating macroglia, compromising the structural integrity of affected neurons. The involvement of macroglia in the disposal of neuronal debris described here-although different in the proposed transport mechanisms-shows resemblance to the mammalian glymphatic system. We propose that this model system is highly suitable to investigate invertebrate neurodegenerative processes from early onset to scar formation and that this knowledge may be useful for the study of neurodegeneration in mammalian tissue.

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding: insights from crystal structures and MD simulation.

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-ß-d-cellobioside (pNPC), p-nitrophenyl-ß-d-lactoside (pNPL) and methylumbelliferyl-ß-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-ß-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (kcat ) than TrCel7A, but higher Michaelis-Menten (KM ) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding.

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

The genome and preliminary single-nuclei transcriptome of Lemna minuta reveals mechanisms of invasiveness.

The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size, and developmental time makes this a challenge even in the diminutive model plant Arabidopsis (Arabidopsis thaliana). Duckweed, basal nongrass aquatic monocots, provide an opportunity to follow every cell of an entire plant due to their small size, reduced body plan, and fast clonal growth habit. Here we present a chromosome-resolved genome for the highly invasive Lesser Duckweed (Lemna minuta) and generate a preliminary cell atlas leveraging low cell coverage single-nuclei sequencing. We resolved the 360 megabase genome into 21 chromosomes, revealing a core nonredundant gene set with only the ancient tau whole-genome duplication shared with all monocots, and paralog expansion as a result of tandem duplications related to phytoremediation. Leveraging SMARTseq2 single-nuclei sequencing, which provided higher gene coverage yet lower cell count, we profiled 269 nuclei covering 36.9% (8,457) of the L. minuta transcriptome. Since molecular validation was not possible in this nonmodel plant, we leveraged gene orthology with model organism single-cell expression datasets, gene ontology, and cell trajectory analysis to define putative cell types. We found that the tissue that we computationally defined as mesophyll expressed high levels of elemental transport genes consistent with this tissue playing a role in L. minuta wastewater detoxification. The L. minuta genome and preliminary cell map provide a paradigm to decipher developmental genes and pathways for an entire plant.

Underwater CAM photosynthesis elucidated by Isoetes genome.

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known 'bacterial-type' PEPC, along with the 'plant-type' exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.

Streamlined variational inference for higher level group-specific curve models.

A two-level group-specific curve model is such that the mean response of each member of a group is a separate smooth function of a predictor of interest. The three-level extension is such that one grouping variable is nested within another one, and higher level extensions are analogous. Streamlined variational inference for higher level group-specific curve models is a challenging problem. We confront it by systematically working through two-level and then three-level cases and making use of the higher level sparse matrix infrastructure laid down in Nolan and Wand (2019). A motivation is analysis of data from ultrasound technology for which three-level group-specific curve models are appropriate. Whilst extension to the number of levels exceeding three is not covered explicitly, the pattern established by our systematic approach sheds light on what is required for even higher level group-specific curve models.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation.

Plants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24 h period, enabling the anticipation of these events. We used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. Approximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion than reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore, we identified sequence motifs enriched in the promoters of similarly phased genes, some potentially associated with transcription factors. When considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species.

Characterization of flavin binding in oxygen-independent fluorescent reporters.

Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen-free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn-on biosensor for vitamin B2.

Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging.

Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

Behavioral and Physiological Resistance to Desiccation in Spotted Wing Drosophila (Diptera: Drosophilidae).

With a high surface to volume ratio, small organisms must carefully regulate their internal water status. Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive frugivorous insect distributed across a wide range of geographical regions that can have periods of dry and hot weather, suggesting that this species has strategies to avoid stressful environments and reduce water loss. It also survives winter as an adult fly, indicating that it has adaptations to the low air humidity of this season. To determine the importance of water stress to D. suzukii, we studied their survival in environments of low humidity, which was manipulated using Drierite, and their survival and water loss in response to desiccation. Survival of both sexes was lower in drier conditions, and while female winter morph D. suzukii had higher mortality early on, remaining flies were able to survive longer in the drier conditions than the summer morphs. A bioassay method was adapted from Enjin et al. (2016) using 48-well plates to videotape the location of flies and quantify their behavioral responses to humidity. Male and female D. suzukii avoided dry conditions within the bioassay system, but only when there was at least 25% differential between humidity extremes. This response was observed for both summer and winter morphs of D. suzukii and our results provide guidance for attempts to manipulate crop environments to reduce the economic impact of this pest.

Aortic valve opening and closure: the clover dynamics.

BACKGROUND: Systolic aortic root expansion is reported to facilitate valve opening, but the precise dynamics remain unknown. A sonometric study with a high data sampling rate (200 to 800 Hz) was conducted in an acute ovine model to better understand the timing, mechanisms, and shape of aortic valve opening and closure. METHODS: Eighteen piezoelectric crystals were implanted in 8 sheep at each annular base, commissures, sinus of Valsalva, sinotubular junction, nodulus of Arantius, and ascending aorta (AA). Geometric changes were time related to pressures and flows. RESULTS: The aortic root was hemodynamically divided into left ventricular (LV) and aortic compartments situated, respectively, below and above the leaflets. During isovolumetric contraction (IVC), aortic root expansion started in the LV compartment, most likely due to volume redistribution in the LV outflow tract below the leaflets. This expansion initiated leaflet separation prior to ejection (2.1%±0.5% of total opening area). Aortic compartment expansion was delayed toward the end of IVC, likely related to volume redistribution above the leaflets due to accelerating aortic backflow toward the aortic valve and coronary flow reduction due to myocardial contraction. Maximum valve opening during the first third of ejection acquired a truncated cone shape [leaflet free edge area smaller than annular base area (-41.5%±5.5%)]. The distal orifice became clover shaped because the leaflet free edge area is larger than the commissural area by 16.3%±2.0%. CONCLUSIONS: Aortic valve opening is initiated prior to ejection related to delicate balance between LV, aortic root, and coronary dynamics. It is clover shaped at maximum opening in systole. A better understanding of these mechanisms should stimulate more physiological surgical approaches of valve repair and replacement.

Linking Endoplasmic Reticular Stress and Alternative Splicing.

RNA splicing patterns in antibody-secreting cells are shaped by endoplasmic reticulum stress, ELL2 (eleven-nineteen lysine-rich leukemia gene 2) induction, and changes in the levels of snRNAs. Endoplasmic reticulum stress induces the unfolded protein response comprising a highly conserved set of genes crucial for cell survival; among these is Ire1, whose auto-phosphorylation drives it to acquire a regulated mRNA decay activity. The mRNA-modifying function of phosphorylated Ire1 non-canonically splices Xbp1 mRNA and yet degrades other cellular mRNAs with related motifs. Naïve splenic B cells will activate Ire1 phosphorylation early on after lipopolysaccharide (LPS) stimulation, within 18 h; large-scale changes in mRNA content and splicing patterns result. Inhibition of the mRNA-degradation function of Ire1 is correlated with further differences in the splicing patterns and a reduction in the mRNA factors for snRNA transcription. Some of the >4000 splicing changes seen at 18 h after LPS stimulation persist into the late stages of antibody secretion, up to 72 h. Meanwhile some early splicing changes are supplanted by new splicing changes introduced by the up-regulation of ELL2, a transcription elongation factor. ELL2 is necessary for immunoglobulin secretion and does this by changing mRNA processing patterns of immunoglobulin heavy chain and >5000 other genes.

RNA Splicing in the Transition from B Cells to Antibody-Secreting Cells: The Influences of ELL2, Small Nuclear RNA, and Endoplasmic Reticulum Stress.

In the transition from B cells to Ab-secreting cells (ASCs) many genes are induced, such as ELL2, Irf4, Prdm1, Xbp1, whereas other mRNAs do not change in abundance. Nonetheless, using splicing array technology and mouse splenic B cells plus or minus LPS, we found that induced and "uninduced" genes can show large differences in splicing patterns between the cell stages, which could influence ASC development. We found that ∼55% of these splicing changes depend on ELL2, a transcription elongation factor that influences expression levels and splicing patterns of ASC signature genes, genes in the cell-cycle and N-glycan biosynthesis and processing pathways, and the secretory versus membrane forms of the IgH mRNA. Some of these changes occur when ELL2 binds directly to the genes encoding those mRNAs, whereas some of the changes are indirect. To attempt to account for the changes that occur in RNA splicing before or without ELL2 induction, we examined the amount of the small nuclear RNA molecules and found that they were significantly decreased within 18 h of LPS stimulation and stayed low until 72 h. Correlating with this, at 18 h after LPS, endoplasmic reticulum stress and Ire1 phosphorylation are induced. Inhibiting the regulated Ire1-dependent mRNA decay with 4u8C correlates with the reduction in small nuclear RNA and changes in the normal splicing patterns at 18 h. Thus, we conclude that the RNA splicing patterns in ASCs are shaped early by endoplasmic reticulum stress and Ire1 phosphorylation and later by ELL2 induction.

Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling.

RATIONALE: Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe3+), is desensitized to NO and limits cGMP production needed for downstream activation of protein kinase G-dependent signaling and blood vessel dilation. OBJECTIVE: Although reactive oxygen species are known to oxidize the sGC heme iron, the basic mechanism(s) governing sGC heme iron recycling to its NO-sensitive, reduced state remain poorly understood. METHODS AND RESULTS: Oxidant challenge studies show that vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein-protein complexes between cytochrome b5 reductase 3, also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in vascular smooth muscle cells reveal that cytochrome b5 reductase 3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show that cytochrome b5 reductase 3 directly reduces oxidized sGC required for NO sensitization as assessed by biochemical, cellular, and ex vivo assays. CONCLUSIONS: Together, these findings identify new insights into NO-sGC-cGMP signaling and reveal cytochrome b5 reductase 3 as the first identified physiological sGC heme iron reductase in vascular smooth muscle cells, serving as a critical regulator of cGMP production and protein kinase G-dependent signaling.