Subunit Of acetyl-CoA Carboxylase Research Articles

Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1–5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

Dynamic changes in the plastid and mitochondrial genomes of the angiosperm Corydalis pauciovulata (Papaveraceae)

BackgroundCorydalis DC., the largest genus in the family Papaveraceae, comprises > 465 species. Complete plastid genomes (plastomes) of Corydalis show evolutionary changes, including syntenic arrangements, gene losses and duplications, and IR boundary shifts. However, little is known about the evolution of the mitochondrial genome (mitogenome) in Corydalis. Both the organelle genomes and transcriptomes are needed to better understand the relationships between the patterns of evolution in mitochondrial and plastid genomes.ResultsWe obtained complete plastid and mitochondrial genomes from Corydalis pauciovulata using a hybrid assembly of Illumina and Oxford Nanopore Technologies reads to assess the evolutionary parallels between the organelle genomes. The mitogenome and plastome of C. pauciovulata had sizes of 675,483 bp and 185,814 bp, respectively. Three ancestral gene clusters were missing from the mitogenome, and expanded IR (46,060 bp) and miniaturized SSC (202 bp) regions were identified in the plastome. The mitogenome and plastome of C. pauciovulata contained 41 and 67 protein-coding genes, respectively; the loss of genes was a plastid-specific event. We also generated a draft genome and transcriptome for C. pauciovulata. A combination of genomic and transcriptomic data supported the functional replacement of acetyl-CoA carboxylase subunit β (accD) by intracellular transfer to the nucleus in C. pauciovulata. In contrast, our analyses suggested a concurrent loss of the NADH-plastoquinone oxidoreductase (ndh) complex in both the nuclear and plastid genomes. Finally, we performed genomic and transcriptomic analyses to characterize DNA replication, recombination, and repair (DNA-RRR) genes in C. pauciovulata as well as the transcriptomes of Liriodendron tulipifera and Nelumbo nuicifera. We obtained 25 DNA-RRR genes and identified their structure in C. pauciovulata. Pairwise comparisons of nonsynonymous (dN) and synonymous (dS) substitution rates revealed that several DNA-RRR genes in C. pauciovulata have higher dN and dS values than those in N. nuicifera.ConclusionsThe C. pauciovulata genomic data generated here provide a valuable resource for understanding the evolution of Corydalis organelle genomes. The first mitogenome of Papaveraceae provides an example that can be explored by other researchers sequencing the mitogenomes of related plants. Our results also provide fundamental information about DNA-RRR genes in Corydalis and their related rate variation, which elucidates the relationships between DNA-RRR genes and organelle genome stability.

High CO2 facilitates fatty acid biosynthesis and mitigates cellular oxidative stress caused by CAC2 dysfunction in Arabidopsis.

Increasing concentration of CO2 has significant impacts on many biological processes in plants, and its impact is closely associated with changes in the ratio of photosynthesis to photorespiration. Studies have reported that high CO2 can promote carbon fixing and alleviate plant oxidative damage in response to environmental stresses. However, the effect of high CO2 on fatty acid (FA) metabolism and cellular redox balance in FA-deficient plants is rarely reported. In this study, we identified a high-CO2 -requiring mutant cac2 through forward genetic screening. CAC2 encodes biotin carboxylase, which is one of the subunits of plastid acetyl-CoA carboxylase and participates in de novo FA biosynthesis. Null mutation of CAC2 is embryonic lethal. A point mutation of CAC2 in cac2 mutants produces severe defects in chloroplast development, plant growth and photosynthetic performance. These morphological and physiological defects were largely absent under high CO2 conditions. Metabolite analyses showed that FA contents in cac2-1 leaves were decreased, while photorespiratory metabolites, such as glycine and glycolate, did not significantly change. Meanwhile, cac2 exhibited higher reactive oxygen species (ROS) levels and mRNA expression of stress-responsive genes than the wild-type, indicating that cac2 plants may suffer oxidative stress under ambient CO2 conditions. Elevated CO2 significantly increased FA contents, especially C18:3-FA, and reduced ROS accumulation in cac2-1 leaves. We propose that stress mitigation by high CO2 in cac2 could be due to increased FA levels by promoting carbon assimilation, and the prevention of over-reduction due to decreased photorespiration.

Genome-wide identification of biotin carboxyl carrier subunits of acetyl-CoA carboxylase in Brassica and their role in stress tolerance in oilseed Brassica napus

BackgroundBiotin carboxyl carrier protein (BCCP) is a subunit of Acetyl CoA-carboxylase (ACCase) which catalyzes the conversion of acetyl-CoA to malonyl-CoA in a committed step during the de novo biosynthesis of fatty acids. Lipids, lipid metabolites, lipid-metabolizing and -modifying enzymes are known to play a role in biotic and abiotic stress tolerance in plants. In this regard, an understanding of the Brassica napus BCCP genes will aid in the improvement of biotic and abiotic stress tolerance in canola.ResultsIn this study, we identified 43 BCCP genes in five Brassica species based on published genome data. Among them, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus and Brassica juncea had six, seven, seven, 10 and 13 BCCP homologs, respectively. Phylogenetic analysis categorized them into five classes, each with unique conserved domains. The promoter regions of all BCCP genes contained stress-related cis-acting elements as determined by cis-element analysis. We identified four and three duplicated gene pairs (segmental) in B. napus and B. juncea respectively, indicating the role of segmental duplication in the expansion of this gene family. The Ka/Ks ratios of orthologous gene pairs between Arabidopsis thaliana and five Brassica species were mostly less than 1.0, implying that purifying selection, i.e., selective removal of deleterious alleles, played a role during the evolution of Brassica genomes. Analysis of 10 BnaBCCP genes using qRT-PCR showed a different pattern of expression because of exposure of the plants to biotic stresses, such as clubroot and sclerotinia diseases, and abiotic stresses such as drought, low temperature and salinity stresses.ConclusionsThe identification and functional analysis of the Brassica BCCPs demonstrated that some of these genes might play important roles in biotic and abiotic stress responses. Results from this study could lay the foundation for a better understanding of these genes for the improvement of Brassica crops for stress tolerance.

Overexpression of fatty acid synthesis genes in Synechocystis sp. PCC 6803 with disrupted glycogen synthesis increases lipid production with further enhancement under copper induced oxidative stress

In the present study, fatty acid synthesis genes such as alpha and beta subunits of acetyl CoA carboxylase (accA and accD) were overexpressed in the glgC (Glucose-1-phosphate adenylyltransferase) knockout Synechocystis sp. PCC 6803. The biomass and lipid contents were evaluated in both the wild type and the engineered strains after copper treatment. The maximum lipid production of 0.981 g/L with the productivity of 81.75 mg/L/d was obtained from the copper treated ΔglgC + A-OX strain, which showed a 3.3-fold increase compared to the untreated wild type with satisfactory biodiesel properties. After copper treatment the knockout strain improved the unsaturated fatty acids level contributing to the increase of the saturated and mono-unsaturated ratio with improvement of the fuel quality. Copper induced oxidative stress also improved the photosynthetic pigments in engineered strains leading to increased tolerance against oxidative stress in the engineered strains. The copper treatment increased the antioxidant enzyme activities in the engineered strains especially in ΔglgC + A-OX strain. The carbon flux to lipid synthesis was enhanced by the engineered strains particularly with the knockout-overexpression strains. The Synechocystis sp. PCC 6803 engineered with ΔglgC + A-OX showed high potential for fuel production after the copper treatment.

Increasing the Ascomycin Yield by Relieving the Inhibition of Acetyl/Propionyl-CoA Carboxylase by the Signal Transduction Protein GlnB.

Ascomycin (FK520) is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. In this study, we demonstrated that the inactivation of GlnB, a signal transduction protein belonging to the PII family, can increase the production of ascomycin by strengthening the supply of the precursors malonyl-CoA and methylmalonyl-CoA, which are produced by acetyl-CoA carboxylase and propionyl-CoA carboxylase, respectively. Bioinformatics analysis showed that Streptomyces hygroscopicus var. ascomyceticus contains two PII family signal transduction proteins, GlnB and GlnK. Protein co-precipitation experiments demonstrated that GlnB protein could bind to the α subunit of acetyl-CoA carboxylase, and this binding could be disassociated by a sufficient concentration of 2-oxoglutarate. Coupled enzyme activity assays further revealed that the interaction between GlnB protein and the α subunit inhibited both the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, and this inhibition could be relieved by 2-oxoglutarate in a concentration-dependent manner. Because GlnK protein can act redundantly to maintain metabolic homeostasis under the control of the global nitrogen regulator GlnR, the deletion of GlnB protein enhanced the supply of malonyl-CoA and methylmalonyl-CoA by restoring the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, thereby improving the production of ascomycin to 390 ± 10 mg/L. On this basis, the co-overexpression of the β and ε subunits of propionyl-CoA carboxylase further increased the ascomycin yield to 550 ± 20 mg/L, which was 1.9-fold higher than that of the parent strain FS35 (287 ± 9 mg/L). Taken together, this study provides a novel strategy to increase the production of ascomycin, providing a reference for improving the yield of other antibiotics.

Cinnamaldehyde inhibits the growth of Phytophthora capsici through disturbing metabolic homoeostasis.

BackgroundPhytophthora capsici Leonian (P. capsici) can cause wilting and roots rotting on pepper and other cash crops. The new fungicide cinnamaldehyde (CA) has high activity against this pathogen. However, its potential mechanism is still unknown.MethodsIn order to gain insights into the mechanism, isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics was used to analyze P. capsici treated with CA. The iTRAQ results were evaluated by parallel reaction monitoring (PRM) analysis and quantitative real-time PCR (qRT-PCR) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to speculate the biochemical pathways that the agent may act on.ResultsThe results showed that 1502 differentially expressed proteins were identified, annotated and classified into 209 different terms (like metabolic process, cellular process, single-organism process) based on Gene Ontology (GO) functional enrichment analysis and nine different pathways (glyoxylate and dicarboxylate metabolism, fatty acid metabolism and so on) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. This study suggested that CA disordered fatty acid metabolism, polysaccharide metabolism and leucine metabolism. Based on PRM analysis, five proteins including CAMK/CAMK1 protein kinase, glucan 1,3-beta-glucosidase, 1,3-beta-glucanosyltransferase, methylcrotonoyl-CoA carboxylase subunit alpha and isovaleryl-CoA dehydrogenase were down-regulated in P. capsici treated with CA. Furthermore, the qRT-PCR analysis showed that the gene expression level of the interested proteins was consistent with the protein expression level, except for CAMK/CAMK1 protein kinase, acetyl-CoA carboxylase and fatty acid synthase subunit alpha.ConclusionsCA destroyed the metabolic homoeostasisof P. capsici, which led to cell death. This is the first proteomic analysis of P. capsici treated with CA, which may provide an important information for exploring the mechanism of the fungicide CA against P. capsici.

Engineering of primary metabolic pathways for titer improvement of milbemycins in Streptomyces bingchenggensis.

Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1-based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing. KEY POINTS: • We compared the genomes of two strains with different titers of milbemycins. • We found three genes belonging to primary metabolism influence milbemycin production. • We improved titer of milbemycins by a combinatorial engineering of three targets.

The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii

Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites. The inherent genetic diversity of microalgae combined with their metabolic plasticity when grown using different trophic and illumination strategies necessitate the establishment of a reference knowledge base. In the present study we present a detailed characterisation of the combined effects of wavelength selection and trophic strategy on the growth kinetics and gene expression profile of the model microalgae Chlamydomonas reinhardtii grown under moderate to high light intensity (400 μmolph m−2·s−1). The aim is twofold: (a) to establish a list of reliable housekeeping genes valid for quantitative comparisons across several combinations of different wavelengths and trophic strategies and (b) to enable the investigation of the response of central carbon metabolic pathways under these process conditions. White, blue and red light emitting diodes (LEDs) were used to grow pH controlled photo- and mixo-trophic cultures over a period of 136 h in batch mode. A panel of 10 candidate genes, along with biomass growth rate and pigment content were dynamically monitored across all conditions. Statistical analysis identified genes (acetyl-CoA carboxylase subunit α and photosystem I reaction centre subunit II) with less variability observed in their expression levels across the entirety of conditions evaluated compared to housekeeping genes often referred to in literature (receptor of activated protein kinase C and ribosomal protein (large subunit) 19). Further analysis of gene expression profiles revealed substantial differences in response to changes in both wavelength selection (upregulation of ribulose bisphosphate carboxylase small subunit under red phototrophic growth) and trophic strategy (upregulation of malate synthase from phototrophic to mixotrophic conditions). The systematic approach used to establish reliable reference genes presented herein enables robust comparisons of cellular responses across different conditions to better understand algal metabolism and improve process performance.

Effect of Acetyl-CoA Carboxylase (ACC) on Lipid Production in Endophytic Fungi from Torreya grandis

Certain endophytic fungi isolated from Torreya grandis can produce similar unsaturated lipid of high economic value as its host plants. However, the lipid yield produced by endophytic fungi is very low. Acetyl-CoA carboxylase (ACC) is one of the key enzymes responsible for fatty acid synthesis. In this study, the impact of ACC on lipid yield of endophytic fungi were analyzed and the inhibitory effect of endophytic fungus metabolites on the ACC activity were investigated. Our results showed thatthe ACC activity was found to be positively correlated with the lipid content of endophytic fungi and the kernels of Torreya grandis. The ACC activity was inhibited by the metabolite, benzoic acid 2,2,6,6-tetramethyl-4-piperidinylester, which was extracted from the endophytic fungus Bionectria ochroleuca XF-38. Furthermore, the gene sequence of CT subunit of ACC in Bionectria ochroleuca XF-38 were cloned and successfully expressed in E. coli BL21 (DE3), and we revealed that the CT subunit of ACC is the target of benzoic acid 2,2,6,6-tetramethyl- 4-piperidinylester.

Specific diacylglycerols generated by hepatic lipogenesis stimulate the oncogenic androgen receptor activity in male hepatocytes.

Obesity-induced hepatocellular carcinoma (HCC) is more prevalent in males than in females, but the underlying mechanism remains unclear. The influence of hepatic androgen receptor (AR) pathway on the gender difference of HCC has been well documented. Here we investigated the role of hepatic lipogenesis, which is elevated in the livers of obese and nonalcoholic fatty liver disease (NAFLD) patients, in stimulating the AR pathway for the male preference of obesity induced HCC. Male C57BL/6J mice were fed a fructose-rich high carbohydrate diet (HCD) to induce hepatic lipogenesis. The effect of hepatic lipogenesis on AR was examined by the expression of hydrodynamically injected AR reporter and the endogenous AR target gene; the mechanism was delineated in hepatoma cell lines and validated in male mice. The hepatic lipogenesis induced by a fructose-rich HCD enhanced the transcriptional activity of hepatic AR in male mice, which did not happen when fed a high fat diet. This AR activation was blocked by sh-RNAs or inhibitors targeting key enzymes in lipogenesis, either acetyl-CoA carboxylase subunit alpha (ACCα), or fatty acid synthase (FASN), in vivo and in vitro. Further mechanistic study identified that specific unsaturated fatty acid, the oleic acid (C18:1 n-9), incorporated DAGs produced by hepatic lipogenesis are the key molecules to enhance the AR activity, through activation of Akt kinase, and this novel mechanism is targeted by metformin. Our study elucidates a novel mechanism underlying the higher risk of HCC in obese/NAFLD males, through specific DAGs enriched by hepatic lipogenesis to increase the transcriptional activity of hepatic AR, a confirmed risk factor for male HCC.

Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L.monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L.monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.

Allelic Diversity of Acetyl Coenzyme A Carboxylase accD/bccp Genes Implicated in Nuclear-Cytoplasmic Conflict in the Wild and Domesticated Pea (Pisum sp.).

Reproductive isolation is an important component of species differentiation. The plastid accD gene coding for the acetyl-CoA carboxylase subunit and the nuclear bccp gene coding for the biotin carboxyl carrier protein were identified as candidate genes governing nuclear-cytoplasmic incompatibility in peas. We examined the allelic diversity in a set of 195 geographically diverse samples of both cultivated (Pisum sativum, P. abyssinicum) and wild (P. fulvum and P. elatius) peas. Based on deduced protein sequences, we identified 34 accD and 31 bccp alleles that are partially geographically and genetically structured. The accD is highly variable due to insertions of tandem repeats. P. fulvum and P. abyssinicum have unique alleles and combinations of both genes. On the other hand, partial overlap was observed between P. sativum and P. elatius. Mapping of protein sequence polymorphisms to 3D structures revealed that most of the repeat and indel polymorphisms map to sequence regions that could not be modeled, consistent with this part of the protein being less constrained by requirements for precise folding than the enzymatically active domains. The results of this study are important not only from an evolutionary point of view but are also relevant for pea breeding when using more distant wild relatives.

Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies.

Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne’s disease research and diagnostics. Johne’s disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.

Design of a Seed-Specific Chimeric Promoter with a Modified Expression Profile to Improve Seed Oil Content.

Increasing the yield of plant oil is an important objective to meet the demand for sustainable resources and energy. Some attempts to enhance the expression of genes involved in oil synthesis in seeds have succeeded in increasing oil content. In many cases, the promoters of seed-storage protein genes have been used as seed-specific promoters. However, conventional promoters are developmentally regulated and their expression periods are limited. We constructed a chimeric promoter that starts to express in the early stage of seed development, and high-level expression is retained until the later stage by connecting the promoters of the biotin carboxyl carrier protein 2 (BCCP2) gene encoding the BCCP2 subunit of acetyl-CoA carboxylase and the fatty acid elongase 1 (FAE1) gene from Arabidopsis. The constructed promoter was ligated upstream of the TAG1 gene encoding diacylglycerol acyltransferase 1 and introduced into Arabidopsis. Seeds from transgenic plants carrying AtTAG1 under the control of the chimeric promoter showed increased oil content (up by 18–73%) compared with wild-type seeds. The novel expression profile of the chimeric promoter showed that this could be a promising strategy to manipulate the content of seed-storage oils and other compounds.

Transcriptional and post-translational regulation of AccD6 in Mycobacterium smegmatis

AccD6 is an important component of acetyl-CoA/propionyl-CoA carboxylase, which acts as a key role in mycolic acid synthesis and short chain fatty acyl-coenzyme A metabolism. In this study, we demonstrated that AccD6 of Mycobacterium smegmatis associates with AccA3 (α subunit of acetyl-CoA carboxylase, MSMEG_1807) and AccE (ε subunit, MSMEG_1812) to form the acetyl-CoA (propionyl-CoA) carboxylase. Results showed that the MSMEG_4331 subunit is a regulator that interacts with the promoter region of accD6 to inhibit its transcription. Transcription of accD6 was reduced by 50% in the mutant M. smegmatis strain overexpressing MSMEG_4331. Moreover, the activity of AccD6 was inhibited by acylation (such as acetylation and propionylation). These results demonstrate that AccD6 of M. smegmatis is regulated at both the transcriptional and post-translational levels. Our findings highlight the novel regulatory mechanism underlying mycolic acid biosynthesis in mycobacteria.

AccD nuclear transfer of Platycodon grandiflorum and the plastid of early Campanulaceae

BackgroundCampanulaceae species are known to have highly rearranged plastid genomes lacking the acetyl-CoA carboxylase (ACC) subunit D gene (accD), and instead have a nuclear (nr)-accD. Plastid genome information has been thought to depend on studies concerning Trachelium caeruleum and genome announcements for Adenophora remotiflora, Campanula takesimana, and Hanabusaya asiatica. RNA editing information for plastid genes is currently unavailable for Campanulaceae. To understand plastid genome evolution in Campanulaceae, we have sequenced and characterized the chloroplast (cp) genome and nr-accD of Platycodon grandiflorum, a basal member of Campanulaceae.ResultsWe sequenced the 171,818 bp cp genome containing a 79,061 bp large single-copy (LSC) region, a 42,433 bp inverted repeat (IR) and a 7840 bp small single-copy (SSC) region, which represents the cp genome with the largest IR among species of Campanulaceae. The genome contains 110 genes and 18 introns, comprising 77 protein-coding genes, four RNA genes, 29 tRNA genes, 17 group II introns, and one group I intron. RNA editing of genes was detected in 18 sites of 14 protein-coding genes. Platycodon has an IR containing a 3′ rps12 operon, which occurs in the middle of the LSC region in four other species of Campanulaceae (T. caeruleum, A. remotiflora, C. takesimana, and H. asiatica), but lacks accD, clpP, infA, and rpl23, as has been found in these four species. Platycodon nr-accD contains about 3.2 kb intron between nr-accD.e1 and nr-accD.e2 at the same insertion point as in other Campanulaceae. The phylogenies of the plastid genomes and accD show that Platycodon is basal in the Campanulaceae clade, indicating that IR disruption in Campanulaceae occurred after the loss of accD, clpP, infA, and rpl23 in the cp genome, which occurred during plastid evolution in Campanulaceae.ConclusionsThe plastid genome of P. grandiflorum lacks the rearrangement of the IR found in T. caeruleum, A. remotiflora, C. takesimana, and H. asiatica. The absence of accD, clpP, infA, and rpl23 in the plastid genome is a synapomorphic characteristic of Campanulaceae. The chloroplast genome phylogeny supports the hypothesis that chloroplast genomic arrangement occurred after accD nuclear transfer and loss of the four genes in the plastid of early Campanulaceae as a lineage of asterids.

On the brink: the highly reduced plastomes of nonphotosynthetic Ericaceae.

Ericaceae (the heather family) is a large and diverse group of plants that forms elaborate symbiotic relationships with mycorrhizal fungi, and includes several nonphotosynthetic lineages. Using an extensive sample of fully mycoheterotrophic (MH) species, we explored inter- and intraspecific variation as well as selective constraints acting on the plastomes of these unusual plants. The plastomes of seven MH genera were analysed in a phylogenetic context with two geographically disparate individuals sequenced for Allotropa, Monotropa, and Pityopus. The plastomes of nonphotosynthetic Ericaceae are highly reduced in size (c. 33-41kbp) and content, having lost all photosynthesis-related genes, and are reduced to encoding housekeeping genes as well as a protease subunit (clpP)-like and acetyl-CoA carboxylase subunit D (accD)-like open reading frames. Despite an increase in the rate of their nucleotide substitutions, the remaining protein-coding genes are typically under purifying selection in full MHs. We also identified ribosomal proteins under relaxed or neutral selection. These plastomes also exhibit striking structural rearrangements. Intraspecific variation within MH Ericaceae ranges from a few differences (Allotropa) to extensive population divergences (Monotropa, Hypopitys), which indicates that cryptic speciation may be occurring in several lineages. The pattern of gene loss within fully MH Ericaceae plastomes suggests an advanced state of degradation.

Whole-transcriptome RNA-seq, gene set enrichment pathway analysis, and exon coverage analysis of two plastid RNA editing mutants

ABSTRACTIn land plants, plastid and mitochondrial RNAs are subject to post-transcriptional C-to-U RNA editing. T-DNA insertions in the ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 gene resulted in reduced photosystem II (PSII) activity and smaller plant and leaf sizes. Exon coverage analysis of the ORRM6 gene showed that orrm6–1 and orrm6–2 are loss-of-function mutants. Compared to other ORRM proteins, ORRM6 affects a relative small number of RNA editing sites. Sanger sequencing of reverse transcription-PCR products of plastid transcripts revealed 2 plastid RNA editing sites that are substantially affected in the orrm6 mutants: psbF-C77 and accD-C794. The psbF gene encodes the β subunit of cytochrome b559, an essential component of PSII. The accD gene encodes the β subunit of acetyl-CoA carboxylase, a protein required in plastid fatty acid biosynthesis. Whole-transcriptome RNA-seq demonstrated that editing at psbF-C77 is nearly absent and the editing extent at accD-C794 was significantly reduced. Gene set enrichment pathway analysis showed that expression of multiple gene sets involved in photosynthesis, especially photosynthetic electron transport, is significantly upregulated in both orrm6 mutants. The upregulation could be a mechanism to compensate for the reduced PSII electron transport rate in the orrm6 mutants. These results further demonstrated that Organelle RNA Recognition Motif protein ORRM6 is required in editing of specific RNAs in the Arabidopsis (Arabidopsis thaliana) plastid.

Genome-Wide Identification and Expression Analysis of the Biotin Carboxyl Carrier Subunits of Heteromeric Acetyl-CoA Carboxylase in Gossypium.

Acetyl-CoA carboxylase is an important enzyme, which catalyzes acetyl-CoA’s carboxylation to produce malonyl-CoA and to serve as a committed step for de novo fatty acid biosynthesis in plastids. In this study, 24 putative cotton BCCP genes were identified based on the lately published genome data in Gossypium. Among them, 4, 4, 8, and 8 BCCP homologs were identified in Gossypium raimondii, G. arboreum, G. hirsutum, and G. barbadense, respectively. These genes were divided into two classes based on a phylogenetic analysis. In each class, these homologs were relatively conserved in gene structure and motifs. The chromosomal distribution pattern revealed that all the BCCP genes were distributed equally on corresponding chromosomes or scaffold in the four cotton species. Segmental duplication was a predominant duplication event in both of G. hirsutum and G. barbadense. The analysis of the expression profile showed that 8 GhBCCP genes expressed in all the tested tissues with changed expression levels, and GhBCCP genes belonging to class II were predominantly expressed in developing ovules. Meanwhile, the expression analysis for the 16 cotton BCCP genes from G. raimondii, G. arboreum and G. hirsutum showed that they were induced or suppressed by cold or salt stress, and their expression patterns varied among different tissues. These findings will help to determine the functional and evolutionary characteristics of the BCCP genes in Gossypium species.