Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance.

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes.

All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. Mitochondrial localization of PPO2 in Arabidopsis was ruled out. Since Arabidopsis PPO2 does not encode a cleavable transit peptide, integration of the protein into the chloroplast envelope must make use of a noncanonical import route. However, when a chloroplast transit peptide was fused to the N-terminus of PPO2, the enzyme was detected predominantly in thylakoid membranes and was able to fully complement ppo1. Thus, the 2 PPO isoforms in Arabidopsis are functionally equivalent but spatially separated. Their distinctive localizations within plastids thus enable the synthesis of discrete subpools of the PPO product protoporphyrin IX, which may serve different cellular needs.

Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome.

SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

Myelodysplastic syndromes with ring sideroblasts.

Myelodysplastic syndromes (MDS) are acquired bone marrow malignant disorders characterized by ineffective hematopoiesis, resulting from a complex interaction between genetic and epigenetic mutations, alterations of the marrow microenvironment, and the immune system. In 2001, the World Health Organization (WHO) proposed a classification that integrates morphologic and genetic information, considering the MDS with ring sideroblasts (MDS-RS) as a distinct entity. Considering the strong association between MDS-RS and SF3B1 mutation and its importance in the development of MDS, the last WHO classification replaced the prior entity of MDS-RS with MDS with SF3B1 mutation. Several studies were performed to explore this genotype-phenotype correlation. Mutant SF3B1 protein deregulates the expression of genes implicated in developing hematopoietic stem and progenitor cells. Of paramount importance are PPOX and ABCB7 involved in iron metabolism. Another essential role in hemopoiesis is played by the transforming growth factor-beta (TGF-ß) receptor. This gene exerts its effects on SMAD pathways, regulating hematopoiesis through effects on balancing proliferation and apoptosis cell inactivity, differentiation, and migration. Luspatercept (ACE-536) is a soluble fusion protein that inhibits molecules in the TGF-ß superfamily. Since its structure resembles the TGF-ß family receptor, it catches TGF-ß superfamily ligands before binding to the receptor, resulting in reduced activation of SMAD signaling, thus enabling erythroid maturation. Luspatercept was investigated in the phase III trial MEDALIST, showing promising efficacy in treating anemia compared to placebo. Nowadays, further studies are needed to explore the real potential of luspatercept, investigating the biological features likely associated with treatment response, the potential use in combination treatments, and its role in the treatment of naïve MDS.

Heterologous complementation systems verify the mosaic distribution of three distinct protoporphyrinogen IX oxidase in the cyanobacterial phylum.

The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.

Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX.

Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.

Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection.

Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. This inhibition can be enhanced by 15- to 25-fold with two oxadiazon derivatives, lending therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans, such as Plasmodium spp., Cystoisospora, Eimeria, Neospora, and Sarcocystis.

Mutation of Protoporphyrinogen IX Oxidase Gene Causes Spotted and Rolled Leaf and Its Overexpression Generates Herbicide Resistance in Rice.

Protoporphyrinogen IX (Protogen IX) oxidase (PPO) catalyzes the oxidation of Protogen IX to Proto IX. PPO is also the target site for diphenyl ether-type herbicides. In plants, there are two PPO encoding genes, PPO1 and PPO2. To date, no PPO gene or mutant has been characterized in monocotyledonous plants. In this study, we isolated a spotted and rolled leaf (sprl1) mutant in rice (Oryza sativa). The spotted leaf phenotype was sensitive to high light intensity and low temperature, but the rolled leaf phenotype was insensitive. We confirmed that the sprl1 phenotypes were caused by a single nucleotide substitution in the OsPPO1 (LOC_Os01g18320) gene. This gene is constitutively expressed, and its encoded product is localized to the chloroplast. The sprl1 mutant accumulated excess Proto(gen) IX and reactive oxygen species (ROS), resulting in necrotic lesions. The expressions of 26 genes associated with tetrapyrrole biosynthesis, photosynthesis, ROS accumulation, and rolled leaf were significantly altered in sprl1, demonstrating that these expression changes were coincident with the mutant phenotypes. Importantly, OsPPO1-overexpression transgenic plants were resistant to the herbicides oxyfluorfen and acifluorfen under field conditions, while having no distinct influence on plant growth and grain yield. These finding indicate that the OsPPO1 gene has the potential to engineer herbicide resistance in rice.

The hydrogen bonding network involved Arg59 in human protoporphyrinogen IX oxidase is essential for enzyme activity.

Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.

Gene co-expression and alternative splicing analysis of key metabolic tissues to unravel the regulatory signatures of fatty acid composition in cattle.

Increasing the healthy/unhealthy fatty acid (FA) ratio in meat is one of the urgent tasks required to address consumer concerns. However, the regulatory mechanisms ultimately resulting in FA profiles vary among animals and remain largely unknown. In this study, using ~1.2 Tb high-quality RNA-Seq-based transcriptomic data of 188 samples from four key metabolic tissues (rumen, liver, muscle, and backfat) together with the contents of 49 FAs in backfat, the molecular regulatory mechanisms of these tissues contributing to FA formation in cattle were explored. Using this large dataset, the alternative splicing (AS) events, one of the transcriptional regulatory mechanisms in four tissues were identified. The highly conserved and absent AS events were detected in rumen tissue, which may contribute to its functional differences compared with the other three tissues. In addition, the healthy/unhealthy FA ratio related AS events, differential expressed (DE) genes, co-expressed genes, and their functions in four tissues were analysed. Eight key genes were identified from the integrated analysis of DE, co-expressed, and AS genes between animals with high and low healthy/unhealthy FA ratios. This study provides an applicable pipeline for AS events based on comprehensive RNA-Seq analysis and improves our understanding of the regulatory mechanism of FAs in beef cattle.

Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis.

KEY MESSAGE: We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides. We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52-54 kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

Acute hepatic porphyrias: Identification of 46 hydroxymethylbilane synthase, 11 coproporphyrinogen oxidase, and 20 protoporphyrinogen oxidase novel mutations.

The acute hepatic porphyrias (AHPs) are inborn errors of heme biosynthesis, which include three autosomal dominant porphyrias, Acute Intermittent Porphyria (AIP), Hereditary Coproporphyria (HCP), and Variegate Porphyria (VP), and the ultra-rare autosomal recessive porphyria, δ-Aminolevulinic Acid Dehydratase Deficiency Porphyria (ADP). AIP, HCP, VP, and ADP each results from loss-of-function (LOF) mutations in their disease-causing genes: hydroxymethylbilane synthase (HMBS); coproporphyrinogen oxidase (CPOX); protoporphyrinogen oxidase (PPOX), and δ-aminolevulinic acid dehydratase (ALAD), respectively. During the 11-year period from January 1, 2007 through December 31, 2017, the Mount Sinai Porphyrias Diagnostic Laboratory diagnosed 315 unrelated AIP individuals with HMBS mutations, including 46 previously unreported mutations, 29 unrelated HCP individuals with CPOX mutations, including 11 previously unreported mutations, and 54 unrelated VP individuals with PPOX mutations, including 20 previously unreported mutations. Overall, of the 1692 unrelated individuals referred for AHP molecular diagnostic testing, 398 (23.5%) had an AHP mutation. Of the 650 family members of mutation-positive individuals tested for an autosomal dominant AHP, 304 (46.8%) had their respective family mutation. These data expand the molecular genetic heterogeneity of the AHPs and document the usefulness of molecular testing to confirm the positive biochemical findings in symptomatic patients and identify at-risk asymptomatic family members.

Tetrapyrrole biosynthetic enzyme protoporphyrinogen IX oxidase 1 is required for plastid RNA editing.

RNA editing is a posttranscriptional process that covalently alters the sequence of RNA molecules and plays important biological roles in both animals and land plants. In flowering plants, RNA editing converts specific cytidine residues to uridine in both plastid and mitochondrial transcripts. Previous studies identified pentatricopeptide repeat (PPR) motif-containing proteins as site-specific recognition factors for cytidine targets in RNA sequences. However, the regulatory mechanism underlying RNA editing was largely unknown. Here, we report that protoporphyrinogen IX oxidase 1 (PPO1), an enzyme that catalyzes protoporphyrinogen IX into protoporphyrin IX in the tetrapyrrole biosynthetic pathway, plays an unexpected role in editing multiple sites of plastid RNA transcripts, most of which encode subunits of the NADH dehydrogenase-like complex (NDH), in the reference plant Arabidopsis thaliana. We identified multiple organellar RNA editing factors (MORFs), including MORF2, MORF8, and MORF9, that interact with PPO1. We found that two conserved motifs within the 22-aa region at the N terminus of PPO1 are essential for its interaction with MORFs, its RNA editing function, and subsequently, its effect on NDH activity. However, transgenic plants lacking key domains for the tetrapyrrole biosynthetic activity of PPO1 exhibit normal RNA editing. Furthermore, MORF2 and MORF9 interact with three PPRs or related proteins required for editing of ndhB and ndhD sites. These results reveal that the tetrapyrrole biosynthetic enzyme PPO1 is required for plastid RNA editing, acting as a regulator that promotes the stability of MORF proteins through physical interaction.

An Unusual Cause of Headache and Fatigue in a Division 1 Collegiate Athlete.

Variegate porphyria (VP) is an autosomal dominant disorder of porphyrin metabolism. We report a case of a 21-year-old male collegiate athlete who complained of recurrent headache and fatigue. Extensive testing after initial presentation failed to identify a cause. Months later, his grandmother was diagnosed with VP after being hospitalized; hence, he was tested. He was positive for a heterozygous missense mutation, R168H, in one protoporphyrinogen oxidase allele. This case highlights a rare disorder of heme synthesis that should be considered in the differential diagnosis of exertional fatigue and headaches in athletes. When other more common causes of fatigue and/or headache are unable to be identified, a more focused history and examination may lead to a more unusual but crucial diagnosis. To our knowledge, there are no reported cases of this condition in Division I collegiate athletes.

The assessment of noncoding variant of PPOX gene in variegate porphyria reveals post-transcriptional role of the 5' untranslated exon 1.

The PPOX gene encodes for the protoporphyrinogen oxidase, which is involved in heme production. The partial deficiency of protoporphyrinogen oxidase causes variegate porphyria. The tissue-specific regulation of other heme biosynthetic enzymes is extensively studied, but the information concerning transcriptional and post-transcriptional regulation of PPOX gene expression is scarcely available. In this study, we characterized functions of three variants identified in the regulatory regions of the PPOX gene, which show a novel role for the 5' untranslated exon 1. Using luciferase assays and RNA analysis, we demonstrated that only c.1-883G>C promoter variant causes a significant loss in the transcriptional activity of PPOX gene whereas c.1-413G>T 5' UTR variant inhibits translation of PPOX mRNA and c.1-176G>A splicing variant causes 4bp deletion in 5' UTR of PPOX mRNA variant 2. These observations indicate that the regulation of PPOX gene expression can also occur through a post-transcriptional modulation of the amount of gene product and that this modulation can be mediated by 5' untranslated exon 1. Moreover this study confirms that these regulatory regions represent an important molecular target for the pathogenesis of variegate porphyria.

Biallelic inactivation of protoporphyrinogen oxidase and hydroxymethylbilane synthase is associated with liver cancer in acute porphyrias.

Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals.

Quantitative structural insight into human variegate porphyria disease.

Defects in the human protoporphyrinogen oxidase (hPPO) gene, resulting in ~50% decreased activity of hPPO, is responsible for the dominantly inherited disorder variegate porphyria (VP). To understand the molecular mechanism of VP, we employed the site-directed mutagenesis, biochemical assays, structural biology, and molecular dynamics simulation studies to investigate VP-causing hPPO mutants. We report here the crystal structures of R59Q and R59G mutants in complex with acifluorfen at a resolution of 2.6 and 2.8 Å. The r.m.s.d. of the Cα atoms of the active site structure of R59G and R59Q with respect to the wild-type was 0.20 and 0.15 Å, respectively. However, these highly similar static crystal structures of mutants with the wild-type could not quantitatively explain the observed large differences in their enzymatic activity. To understand how the hPPO mutations affect their catalytic activities, we combined molecular dynamics simulation and statistical analysis to quantitatively understand the molecular mechanism of VP-causing mutants. We have found that the probability of the privileged conformations of hPPO can be correlated very well with the k(cat)/K(m) of PPO (correlation coefficient, R(2) > 0.9), and the catalytic activity of 44 clinically reported VP-causing mutants can be accurately predicted. These results indicated that the VP-causing mutation affect the catalytic activity of hPPO by affecting the ability of hPPO to sample the privileged conformations. The current work, together with our previous crystal structure study on the wild-type hPPO, provided the quantitative structural insight into human variegate porphyria disease.

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 µm and ~0.1 µm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.

Metabolic Resistance to Protoporphyrinogen Oxidase-Inhibitor Herbicides in a Palmer amaranth Population from Kansas.

Palmer amaranth has evolved target and nontarget site resistance to protoporphyrinogen oxidase-inhibitor herbicides in the United States. Recently, a population (KCTR) from a long-term conservation tillage study in Kansas was found to be resistant to herbicides from six sites of action, including to PPO-inhibitors, even with this herbicide group being minimally used in this field. This research investigated the level of resistance to postemergence PPO-inhibitors, target- and nontarget-site resistance mechanism(s), and efficacy of pre-emergence chemistries. The greenhouse experiments confirmed 6.1- to 78.9-fold resistance to lactofen in KCTR, with the level of resistance increasing when KCTR was purified for the resistance trait. PPO2 sequences alignment revealed the absence of known mutations conferring resistance to PPO-inhibitors in KCTR Palmer amaranth, and differential expression of the PPO2 gene did not occur. KCTR metabolized fomesafen faster than the susceptible population, indicating that herbicide detoxification is the mechanism conferring resistance in this population. Further, treatment with the cytochrome P450-inhibitor malathion followed by lactofen restored the sensitivity of KCTR to this herbicide. Despite being resistant to POST applied PPO-inhibitors, KCTR Palmer amaranth was completely controlled by the labeled rate of the PRE applied PPO-inhibitors fomesafen, flumioxazin, saflufenacil, sulfentrazone, and oxadiazon. The overall results suggest that P450-mediated metabolism confers resistance to PPO-inhibitors in KCTR, rather than alterations in the PPO2, which were more commonly found in other Palmer amaranth populations. Future work will focus on identifying the fomesafen metabolites and on unravelling the genetic basis of metabolic resistance to PPO-inhibitor herbicides in KCTR Palmer amaranth.