Biochemical and structural characterization of quizalofop-resistant wheat acetyl-CoA carboxylase

A novel nucleotide mutation in ACC1 resulting in an alanine to valine amino acid substitution in acetyl-CoA carboxylase (ACCase) at position 2004 of the Alopecurus myosuroides reference sequence (A2004V) imparts quizalofop resistance in wheat. Genotypes endowed with the homozygous mutation in one or two ACC1 homoeologs are seven- and 68-fold more resistant to quizalofop than a wildtype winter wheat in greenhouse experiments, respectively. In vitro ACCase activities in soluble protein extracts from these varieties are 3.8- and 39.4-fold more resistant to quizalofop with the homozygous mutation in either one or two genomes, relative to the wildtype. The A2004V mutation does not alter the specific activity of wheat ACCase, suggesting that this resistance trait does not affect the catalytic functions of ACCase. Modeling of wildtype and quizalofop-resistant wheat ACCase demonstrates that the A2004V amino acid substitution causes a reduction in the volume of the binding pocket that hinders quizalofop’s interaction with ACCase. Docking studies confirm that the mutation reduces the binding affinity of quizalofop. Interestingly, the models suggest that the A2004V mutation does not affect haloxyfop binding. Follow up in vivo and in vitro experiments reveal that the mutation, in fact, imparts negative cross-resistance to haloxyfop, with quizalofop-resistant varieties exhibiting higher sensitivity to haloxyfop than the wildtype winter wheat line.

First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland

Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Differential expression of acetyl-CoA carboxylase beta in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding acetyl-CoA carboxylase beta, ACACB, when comparing primary tumors of the breast to the tissue of origin, the normal breast. ACACB mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of ACACB in primary tumors of the breast was correlated with recurrence-free survival in patients with luminal B subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. ACACB may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Impact of a Novel W2027L Mutation and Non-Target Site Resistance on Acetyl-CoA Carboxylase-Inhibiting Herbicides in a French Lolium multiflorum Population

Herbicides that inhibit acetyl-CoA carboxylase (ACCase) are among the few remaining options for the post-emergence control of Lolium species in small grain cereal crops. Here, we determined the mechanism of resistance to ACCase herbicides in a Lolium multiflorum population (HGR) from France. A combined biological and molecular approach detected a novel W2027L ACCase mutation that affects aryloxyphenoxypropionate (FOP) but not cyclohexanedione (DIM) or phenylpyraxoline (DEN) subclasses of ACCase herbicides. Both the wild-type tryptophan and mutant leucine 2027-ACCase alleles could be positively detected in a single DNA-based-derived polymorphic amplified cleaved sequence (dPACS) assay that contained the targeted PCR product and a cocktail of two discriminating restriction enzymes. Additionally, we identified three well-characterised I1781L, I2041T, and D2078G ACCase target site resistance mutations as well as non-target site resistance in HGR. The non-target site component endowed high levels of resistance to FOP herbicides whilst partially impacting on the efficacy of pinoxaden and cycloxydim. This study adequately assessed the contribution of the W2027L mutation and non-target site mechanism in conferring resistance to ACCase herbicides in HGR. It also highlights the versatility and robustness of the dPACS method to simultaneously identify different resistance-causing alleles at a single ACCase codon.

Acetyl-CoA Carboxylase 1 Is Essential for Hematopoietic Stem Cell Homeostasis

Though fatty acid oxidation has been shown to be essential for hematopoietic stem cell maintenance, the importance of fatty acid uptake from the bone marrow microenvironment vs. de novo lipogenesis has not been elucidated. The process of de novo lipogenesis begins with the generation of malonyl-CoA from acetyl-CoA and is catalyzed by the enzyme acetyl-CoA carboxylase 1 (ACC1), encoded by Acaca. In leukemia, both ACC1 activation and degradation of ACC1 have been shown to enhance disease progression, suggesting an important role in leukemogenesis. The function of ACC1 in normal hematopoiesis remains unknown. To characterize the role of ACC1 in normal hematopoiesis, we bred mice that harbored an Acaca allele in which exons 22 through 26 were flanked by loxp sites. We crossed these animals to a mouse line harboring the Mx1-Cre transgene, generating Acaca fl/fl; Mx1-Cre + (ACC1 KO) and Acaca fl/fl; Mx1-Cre -(ACC1 WT) mice. Animals were then treated with intraperitoneal poly(I:C) injection to drive Mx1-Cre expression and Acaca excision. By 4-6 weeks after poly(I:C) injection, ACC1 KO animals developed expansion of hematopoietic stem cells and progenitors, including Lineage - Sca-1 +cKit hi (LSK) and the LSK CD48 -CD150 + long-term hematopoietic stem cell (LT-HSC) populations. In addition, these animals developed splenomegaly with extramedullary LSK expansion and enhanced myelopoiesis. ACC1 KO LSKs showed increased cell cycle activity and LT-HSCs demonstrated a reduced quiescent fraction compared to ACC1 WT controls. These data suggest that ACC1 deficiency results in increased cell cycle activity among primitive hematopoietic progenitors and biases cell fate to the myeloid lineage. To further test the impact of ACC1 deletion on LT-HSC function, we established competitive chimerism maintenance assays in which equal numbers of CD45.2 +Acaca fl/fl; Mx1-Cre +or CD45.2 +Acaca fl/fl; Mx1-Cre - bone marrow cells were mixed with CD45.1 + competitor bone marrow and transplanted into lethally irradiated recipient mice. Transplants were then given 8 weeks to establish stable engraftment. After documenting comparable engraftment, 5-poly(I:C) injections were administered every other day. Over the course of 16 weeks of tracking, the ACC1 KO graft showed a significant reduction in trilineage hematopoietic output as indicated by a reduction in B cell, T cell, and myeloid populations in the peripheral blood. After 16 weeks, animals were sacrificed and the LT-HSC compartment was analyzed, showing a significant reduction in ACC1 KO LT-HSCs. Together, these data suggest that the loss of ACC1 significantly impairs the ability of LT-HSCs to maintain long term hematopoiesis in competitive transplants. Collectively, these data are the first report of a critical role for ACC1 in hematopoiesis and LT-HSC function. Mechanistically, in the absence of ACC1, FACS sorted LSK demonstrated increased ATP content compared to WT controls, suggesting an altered metabolic state in these progenitor cells. Further studies are ongoing to determine if these findings are due to an essential function for ACC1 within the context of de novo lipogenesis, or if ACC1 participates in an as of yet uncharacterized tumor suppressive role. Disclosures No relevant conflicts of interest to declare.