DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Identification and validation of DHCR7 as a diagnostic biomarker involved in the proliferation and mitochondrial function of breast cancer.

BACKGROUND: Energy metabolism has a complex intersection with pathogenesis and development of breast cancer (BC). This allows for the possibility of identifying energy-metabolism-related genes (EMRGs) as novel prognostic biomarkers for BC. 7-dehydrocholesterol reductase (DHCR7) is a key enzyme of cholesterol biosynthesis involved in many cancers, and in this paper, we investigate the effects of DHCR7 on the proliferation and mitochondrial function of BC. METHODS: EMRGs were identified from the Gene Expression Omnibus (GEO) and MSigDB databases using bioinformatics methods. Key EMRGs of BC were then identified and validated by functional enrichment analysis, interaction analysis, weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, Cox analysis, and immune infiltration. Western blot, qRT-PCR, immunohistochemistry (IHC), MTT assay, colony formation assay and flow cytometry assay were then used to analyze DHCR7 expression and its biological effects on BC cells. RESULTS: We identified 31 EMRGs in BC. These 31 EMRGs and related transcription factors (TFs), miRNAs, and drugs were enriched in glycerophospholipid metabolism, glycoprotein metabolic process, breast cancer, and cell cycle. Crucially, DHCR7 was a key EMRG in BC identified and validated by WGCNA, LASSO regression and receiver operating characteristic (ROC) curve analysis. High DHCR7 expression was significantly associated with tumor immune infiltration level, pathological M, and poor prognosis in BC. In addition, DHCR7 knockdown inhibited cell proliferation, induced apoptosis and affected mitochondrial function in BC cells. CONCLUSIONS: DHCR7 was found to be a key EMRG up-regulated in BC cells. This study is the first to our knowledge to report that DHCR7 acts as an oncogene in BC, which might become a novel therapeutic target for BC patients.

Fluorination of a conserved tyrosine in POR offers new clues for proton transfer.

Reduction of the 17,18-double bond in the D-ring during chlorophyll biosynthesis is catalyzed by the rare, naturally occurring photoenzyme protochlorophyllide oxidoreductase (POR). A conserved tyrosine residue has been suggested to donate a proton to C18 of the substrate in the past decades. Taylor and colleagues scrutinized the model with a powerful tool that utilized a modified genetic code to introduce fluorinated tyrosine analogues into POR. The presented results show that the suggested catalytically critical tyrosine is unlikely to participate in the reaction chemistry but is required for substrate binding, and instead, a cysteine residue preceding the lid helix is proposed to have the role of proton donor.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3ß-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Three linked variants have opposing regulatory effects on isovaleryl-CoA dehydrogenase gene expression.

While genome-wide association studies (GWAS) and positive selection scans identify genomic loci driving human phenotypic diversity, functional validation is required to discover the variant(s) responsible. We dissected the IVD gene locus-which encodes the isovaleryl-CoA dehydrogenase enzyme-implicated by selection statistics, multiple GWAS, and clinical genetics as important to function and fitness. We combined luciferase assays, CRISPR/Cas9 genome-editing, massively parallel reporter assays (MPRA), and a deletion tiling MPRA strategy across regulatory loci. We identified three regulatory variants, including an indel, that may underpin GWAS signals for pulmonary fibrosis and testosterone, and that are linked on a positively selected haplotype in the Japanese population. These regulatory variants exhibit synergistic and opposing effects on IVD expression experimentally. Alleles at these variants lie on a haplotype tagged by the variant most strongly associated with IVD expression and metabolites, but with no functional evidence itself. This work demonstrates how comprehensive functional investigation and multiple technologies are needed to discover the true genetic drivers of phenotypic diversity.

3ß-hydroxysteroid-Δ24 reductase dampens anti-viral innate immune responses by targeting K27 ubiquitination of MAVS and STING.

IMPORTANCE: The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

Malaria affects around 240 million people around the world every year. The microscopic parasite responsible for the disease are carried by certain mosquitoes and gets transmitted to humans through bites. These parasites are increasingly acquiring genetic mutations that make anti-malaria medication less effective, creating an urgent need for alternative treatment approaches. Several new malaria drugs being explored in preclinical research work by binding to an enzyme known as DHODH and preventing it from performing its usual role in the parasite. Previous work found that, in some cases, malaria parasites that evolved resistance to one type of DHODH inhibitor (by acquiring mutations in their DHODH enzyme) then became more vulnerable to another kind. It may be possible to leverage this 'collateral sensitivity' by designing treatments which combine two DHODH inhibitors and therefore make it harder for the parasites to evolve resistance. To investigate this possibility, Mandt et al. first tested several DHODH inhibitors to find the one that was most potent against drug-resistant parasites. In subsequent experiments, they combined TCMDC-125334, the best candidate that emerged from these tests, with a DHODH inhibitor that works well against vulnerable parasites. However, the parasites still rapidly evolved resistance. Further work identified a new DHODH mutation that allowed the parasites to evade both drugs simultaneously. Together, these findings suggest that the DHODH enzyme may not be the best target for new malaria drugs because many it can acquire many possible mutations that confer resistance. Such results may inform other studies that aim to harness collateral sensitivity to fight against a range of harmful agents.

Characterisation of putative class 1A DHODH-like proteins from Mucorales and dematiaceous mould species.

Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer.

DHODH: a promising target in the treatment of T-cell acute lymphoblastic leukemia.

Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) have a poor prognosis with few therapeutic options. With the goal of identifying novel therapeutic targets, we used data from the Dependency Map project to identify dihydroorotate dehydrogenase (DHODH) as one of the top metabolic dependencies in T-ALL. DHODH catalyzes the fourth step of de novo pyrimidine nucleotide synthesis. Small molecule inhibition of DHODH rapidly leads to the depletion of intracellular pyrimidine pools and forces cells to rely on extracellular salvage. In the absence of sufficient salvage, this intracellular nucleotide starvation results in the inhibition of DNA and RNA synthesis, cell cycle arrest, and, ultimately, death. T lymphoblasts appear to be specifically and exquisitely sensitive to nucleotide starvation after DHODH inhibition. We have confirmed this sensitivity in vitro and in vivo in 3 murine models of T-ALL. We identified that certain subsets of T-ALL seem to have an increased reliance on oxidative phosphorylation when treated with DHODH inhibitors. Through a series of metabolic assays, we show that leukemia cells, in the setting of nucleotide starvation, undergo changes in their mitochondrial membrane potential and may be more highly dependent on alternative fuel sources. The effect on normal T-cell development in young mice was also examined to show that DHODH inhibition does not permanently damage the developing thymus. These changes suggest a new metabolic vulnerability that may distinguish these cells from normal T cells and other normal hematopoietic cells and offer an exploitable therapeutic opportunity. The availability of clinical-grade DHODH inhibitors currently in human clinical trials suggests a potential for rapidly advancing this work into the clinic.

Theoretical and Experimental Studies on Plant Light-Dependent Protochlorophyllide Oxidoreductase as a Novel Target for Searching Potential Herbicides.

Herbicide resistance is a prevalent problem that has posed a foremost challenge to crop production worldwide. Light-dependent enzyme NADPH: protochlorophyllide oxidoreductase (LPOR) in plants is a metabolic target that could satisfy this unmet demand. Herein, for the first time, we embarked on proposing a new mode of action of herbicides by performing structure-based virtual screening targeting multiple LPOR binding sites, with the determination of further bioactivity on the lead series. The feasibility of exploiting high selectivity and safety herbicides targeting LPOR was discussed from the perspective of the origin and phylogeny. Besides, we revealed the structural rearrangement and the selection key for NADPH cofactor binding to LPOR. Based on these, multitarget virtual screening was performed and the result identified compounds 2 affording micromolar inhibition, in which the IC50 reached 4.74 µM. Transcriptome analysis revealed that compound 2 induced more genes related to chlorophyll synthesis in Arabidopsis thaliana, especially the LPOR genes. Additionally, we clarified that these compounds binding to the site enhanced the overall stability and local rigidity of the complex systems from molecular dynamics simulation. This study delivers a guideline on how to assess activity-determining features of inhibitors to LPOR and how to translate this knowledge into the design of novel and effective inhibitors against malignant weed that act by targeting LPOR.

Nucleotide Limitation Results in Impaired Photosynthesis, Reduced Growth and Seed Yield Together with Massively Altered Gene Expression.

Nucleotide limitation and imbalance is a well-described phenomenon in animal research but understudied in the plant field. A peculiarity of pyrimidine de novo synthesis in plants is the complex subcellular organization. Here, we studied two organellar localized enzymes in the pathway, with chloroplast aspartate transcarbamoylase (ATC) and mitochondrial dihydroorotate dehydrogenase (DHODH). ATC knock-downs were most severely affected, exhibiting low levels of pyrimidine nucleotides, a low energy state, reduced photosynthetic capacity and accumulation of reactive oxygen species. Furthermore, altered leaf morphology and chloroplast ultrastructure were observed in ATC mutants. Although less affected, DHODH knock-down mutants showed impaired seed germination and altered mitochondrial ultrastructure. Thus, DHODH might not only be regulated by respiration but also exert a regulatory function on this process. Transcriptome analysis of an ATC-amiRNA line revealed massive alterations in gene expression with central metabolic pathways being downregulated and stress response and RNA-related pathways being upregulated. In addition, genes involved in central carbon metabolism, intracellular transport and respiration were markedly downregulated in ATC mutants, being most likely responsible for the observed impaired growth. We conclude that impairment of the first committed step in pyrimidine metabolism, catalyzed by ATC, leads to nucleotide limitation and by this has far-reaching consequences on metabolism and gene expression. DHODH might closely interact with mitochondrial respiration, as seen in delayed germination, which is the reason for its localization in this organelle.

Effect of DHCR7 for the co-occurrence of hypercholesterolemia and vitamin D deficiency in type 2 diabetes: Perspective of health prevention.

Type 2 diabetes mellitus (T2DM) is a complex disease caused by multiple factors, which are often accompanied by the disorder of glucose and lipid metabolism and the lack of vitamin D.Over the years, researchers have conducted numerous studies into the pathogenesis and prevention strategies of diabetes. In this study, diabetic SD rats were randomly divided into type 2 diabetes group, vitamin D intervention group, 7-dehydrocholesterole reductase (DHCR7) inhibitor intervention group, simvastatin intervention group, and naive control group. Before and 12 weeks after intervention, liver tissue was extracted to isolate hepatocytes. Compared with naive control group, in the type 2 diabetic group without interference, the expression of DHCR7 increased, the level of 25(OH)D3 decreased, the level of cholesterol increased. In the primary cultured naive and type 2 diabetic hepatocytes, the expression of genes related to lipid metabolism and vitamin D metabolism were differently regulated in each of the 5 treatment groups. Overall, DHCR7 is an indicator for type 2 diabetic glycolipid metabolism disorder and vitamin D deficiency. Targeting DHCR7 will help with T2DM therapy.The management model of comprehensive health intervention can timely discover the disease problems of diabetes patients and high-risk groups and reduce the incidence of diabetes.

Cellular cholesterol loss by DHCR24 knockdown leads to Aß production by changing APP intracellular localization.

For the past 20 years, the majority of cell culture studies reported that increasing cholesterol level increases amyloid-ß (Aß) production. Conversely, other studies and genetic evidences support that cellular cholesterol loss leads to Aß generation. As a highly controversial issue in Alzheimer's disease pathogenesis, the apparent contradiction prompted us to again explore the role of cellular cholesterol in Aß production. Here, we adopted new neuronal and astrocytic cell models induced by 3ß-hydroxysterol-Δ24 reductase (DHCR24), which obviously differ from the widely used cell models with overexpressing amyloid precursor protein (APP) in the majority of previous studies. In neuronal and astrocytic cell model, we found that deficiency of cellular cholesterol by DHCR24 knockdown obviously increased intracellular and extracellular Aß generation. Importantly, in cell models with overexpressing APP, we found that APP overexpression could disrupt cellular cholesterol homeostasis and affect function of cells, coupled with the increase of APP ß-cleavage product, 99-residue transmembrane C-terminal domain. Therefore, we suppose the results derived from the APP knockin models will need to be re-evaluated. One rational explanation for the discrepancy between our outcomes and the previous studies could be attributed to the two different cell models. Mechanistically, we showed that cellular cholesterol loss obviously altered APP intracellular localization by affecting cholesterol-related trafficking protein of APP. Therefore, our outcomes strongly support cellular cholesterol loss by DHCR24 knockdown leads to Aß production.

"MiR-7 controls cholesterol biosynthesis through posttranscriptional regulation of DHCR24 expression".

Dysregulation of cholesterol homeostasis is associated with several pathologies including cardiovascular diseases and neurological disorders such as Alzheimer's disease (AD). MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of cholesterol metabolism. We previously established the role of miR-7 in regulating insulin resistance and amyloidosis, which represents a common pathological feature between type 2 diabetes and AD. We show here an additional metabolic function of miR-7 in cholesterol biosynthesis. We found that miR-7 blocks the last steps of the cholesterol biosynthetic pathway in vitro by targeting relevant genes including DHCR24 and SC5D posttranscriptionally. Intracranial infusion of miR-7 on an adeno-associated viral vector reduced the expression of DHCR24 in the brain of wild-type mice, supporting in vivo miR-7 targeting. We also found that cholesterol regulates endogenous levels of miR-7 in vitro, correlating with transcriptional regulation through SREBP2 binding to its promoter region. In parallel to SREBP2 inhibition, the levels of miR-7 and hnRNPK (the host gene of miR-7) were concomitantly reduced in brain in a mouse model of Niemann Pick type C1 disease and in murine fatty liver, which are both characterized by intracellular cholesterol accumulation. Taken together, the results establish a novel regulatory feedback loop by which miR-7 modulates cholesterol homeostasis at the posttranscriptional level, an effect that could be exploited for therapeutic interventions against prevalent human diseases.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts.

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

LncRNA ENST00000370438 promotes cell proliferation by upregulating DHCR24 in breast cancer.

Long noncoding RNAs (lncRNAs) are critically involved in the occurrence and development of breast cancer (BC). In this study, we performed RNA sequencing, and the results revealed an increase in the expression level of novel lncRNA ENST00000370438 in tissues of patients with BC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results showed an increase in lncRNA ENST00000370438 expression level in 23 pairs of BC tissues. Next, we determined the effect of ENST00000370438 on BC cell proliferation, and the results showed that ENST00000370438 promotes cell proliferation in BC. The proteomic analysis showed a decrease in DHCR24 expression level in BC cells transfected with ENST00000370438 small interfering RNA. Western blot and qRT-PCR assay results showed that ENST00000370438 regulated DHCR24 expression. Furthermore, the rescue experiment showed that the interference with ENST00000370438 expression could restore the effect of DHCR24 overexpression on BC cell proliferation, demonstrating that ENST00000370438 could promote cell proliferation by upregulating DHCR24. Finally, we showed that lncRNA ENST000000370438 could promote tumor growth by overexpressing DHCR24 in nude mice. Our results demonstrated that lncRNA ENST00000370438 promotes BC cell proliferation by upregulating DHCR24 expression.

Dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate wheat root growth.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

Regulation of chlorophyll biosynthesis by light-dependent acetylation of NADPH:protochlorophyll oxidoreductase A in Arabidopsis.

Chlorophylls are the major pigments that harvest light energy during photosynthesis in plants. Although reactions in chlorophyll biogenesis have been largely known, little attention has been paid to the post-translational regulation mechanism of this process. In this study, we found that four lysine sites (K128/340/350/390) of NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyzes the only light-triggered step in chlorophyll biosynthesis, were acetylated after dark-grown seedlings transferred to light via acetylomics analysis. Etiolated seedlings with K390 mutation of PORA had a lower greening rate and decreased PORA acetylation after illumination. Importantly, K390 of PORA was found extremely conserved in plants and cyanobacteria via bioinformatics analysis. We further demonstrated that the acetylation level of PORA was increased by exposing the dark-grown seedlings to the histone deacetylase (HDAC) inhibitor TSA. Thus, the HDACs probably regulate the acetylation of PORA, thereby controlling this non-histone substrate to catalyze the reduction of Pchlide to produce chlorophyllide, which provides a novel regulatory mechanism by which the plant actively tunes chlorophyll biosynthesis during the conversion from skotomorphogenesis to photomorphogenesis.