Carboxylase Gene Research Articles

In Silico analysis of Phospho Enolpyrovate Carboxylase gene

The importance of sugar in many aspects of life, such as food, health and body care products, results inits great demand all around the world. From another hand, the annual production of sugar cannot meet theincreasing demand of the growing human population. Therefore, this study focused on one of the mostimportant genes that affect the quantity of sugar production, phosphoenolpyruvate carboxylase (PEPcase).The most important crop used to extract sugar is sugarcane where sugar is stored in the stalk internodes. Inthis study RNA extraction was performed to produce cDNA for cloning. Sequencing the main genes usingthe Basic Local Alignment Search Tool (BLAST).

Bovine pyruvate carboxylase gene proximal promoter activity is regulated by saturated and unsaturated fatty acids in Madin-Darby bovine kidney cells

An increase in bovine pyruvate carboxylase (PC; EC 6.4.1.1) at calving and during feed restriction corresponds with increased circulating nonesterified fatty acids as a consequence of negative energy balance. Regulation of PC mRNA and effect of specific combinations of saturated and unsaturated fatty acid profiles has yet to be explored. Our objective was to determine the effects of chain length, degree of saturation, and copresence of saturated and unsaturated fatty acids on activity of bovine PC promoter 1 (PCP1). For these experiments, Madin-Darby bovine kidney cells were transfected with a full-length bovine PCP1 construct from -1002 to +3 bp relative to the bovine PC gene transcription start site (bovine PCP1(-1002_+3)) ligated to a Firefly luciferase reporter, or with one of a series of nested 5' serial truncations (bovine PCP1(-773_+3), bovine PCP1(-494_+3), or bovine PCP1(-222_+3)). Cells were exposed for 23 h to either individual fatty acids (C16:0, C18:0, or C18:3n-3 cis) bound to BSA or to fatty acid mixtures in ratios of 90:10, 75:25, 50:50, or 25:75, corresponding to combinations of C16:0: C18:3n-3 cis or C18:0: C18:3n-3 cis. Total fatty acid concentration was 1.00 mM. Exposure to either C16:0 or C18:3n-3 cis alone elicited a significant increase in capacity to drive bovine PCP1(-1002_+3) activity compared with 1% BSA in Dulbecco's Modified Eagle's Medium control treatment (2.29, 2.89, and 1.00 ± 0.26 fold of promoter induction for C16:0, C18:3n-3 cis, and control, respectively). Treatment with C18:3n-3 cis alone caused a greater increase in promoter activity compared with C16:0 alone, indicating a lesser response to C16:0 alone for bovine PCP1(-1002_+3). Interestingly, inclusion of C18:3n-3 cis, at any level of fatty acid ratios examined, in combination with C16:0 increased promoter activity of bovine PCP1(-773_+3) or bovine PCP1(-222_+3) compared with treatment with C16:0 alone or control. Data from the bovine PCP1 truncation and fatty acid copresence experiments reveal the potential for response elements of unsaturated fatty acids or fatty acid ligands in several bovine PCP1 promoter regions. In silico analysis of bovine PCP1 identified putative peroxisome proliferator-activated receptor α and sterol regulatory element binding protein binding sites which may be implicated in fatty acid signaling to alter bovine PCP1 activity. Pyruvate carboxylase promoter 1 activity that is mediated by unsaturated fatty acids acting through elements within -1002 and -222 bp of bovine PCPI may determine PC response during periods of negative energy balance in dairy cows.

A powerful long metabarcoding method for the determination of complex diets from faecal analysis of the European pond turtle (Emys orbicularis, L. 1758).

High‐throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose‐1,5‐bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL‐trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items.

Reference genes for the study of herbicide stress responses in Leptochloa chinensis (L.) Nees and estimation of ACCase expression in cyhalofop-butyl resistant populations

Cyhalofop-butyl resistance in Leptochloa chinensis (L.) Nees is a threat to rice production. Qualitative changes to the acetyl-CoA carboxylase gene (ACCase) have been reported to induce cyhalofop-butyl resistance in some weed species, but the role of ACCase in cyhalofop-butyl resistance through quantitative changes remains uncertain. The accurate assessment of transcriptional changes in the functional genes associated with herbicide resistance in L. chinensis is challenging owing to the lack of available reference genes for expression normalization. Here, we selected nine candidate reference genes in L. chinensis and assessed their transcription stability in populations susceptible and resistant to cyhalofop-butyl. Transcription stability was compared under conditions of herbicide stress and control conditions using BestKeeper, NormFinder, and geNorm. Elongation factor 1 alpha, eukaryotic initiation factor 4A, and cap-binding protein CBP20 were the most stable reference genes under cyhalofop-butyl treatment. Transcription levels of ACCase were evaluated in seven resistant populations, one of which showed higher transcription than the susceptible population after 24 h cyhalofop-butyl treatment. However, the slight up-regulation of ACCase (approximately 2.0-fold) is unlikely to be responsible for the high resistance levels in these populations of L. chinensis.

Effects of ND vaccination combined LPS on growth performance, antioxidant performance and lipid metabolism of broiler

Newcastle Disease Virus (NDV) is the important pathogen of Newcastle Disease (ND) attacking chicken, turkey and other birds. Therefore, the purpose of this study was to assess the effects of immune stress induced by ND vaccination and lipopolysaccharide (LPS) on growth performance, antioxidant ability, and lipid metabolism of broilers. In total, 128 one-day-old broilers were randomly assigned to the following four groups and were treated as indicated: normal control (NC); vaccinated with live LaSota ND vaccine (CV); administered ND vaccine and 0.25 mg/kg body weight (BW) LPS (L-LPS); and administered ND vaccine and 0.5 mg/kg BW LPS (H-LPS). The results demonstrated that broiler feed conversion ratio (FCR) was increased in the groups CV, L-LPS and H-LPS from d 0 to 42 days compared with the group NC. The antioxidant function of broilers was decreased as indicated by the malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) levels in the serum of the treated groups. ND vaccination combined LPS increased the concentration of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), but decreased the concentration of high-density lipoprotein cholesterol (HDLC) compared with the group NC. The reverse transcription (RT)-PCR results revealed that the mRNA expression of acetyl-CoA carboxylase gene (ACC) and 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) in the liver were downregulated, whereas the mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) and peroxisome proliferator-activated receptor (PPAR)-α were upregulated compared with the group NC. These results suggest that ND vaccination combined LPS reduced broiler growth performance and antioxidant ability, whereas it activated AMPK-mediated lipid metabolism.

Genetic identification and hybridization in the seagrass genus Halophila (Hydrocharitaceae) in Sri Lankan waters.

Seagrasses, as marine angiosperms, play important roles in coastal ecosystems. With increasing anthropogenic impacts, they are facing dramatic declines on a global scale. Halophila is well-known as a complex taxonomic challenge mainly due to high morphological plasticity. By using only a morphological approach, the genus could be over-split or similar species could be erroneously lumped, thus masking its true biodiversity. In the present study, we incorporated genetic identification with morphological examination to reveal the identity of Halophila plants in southern and northwestern Sri Lankan waters. The nuclear ribosomal internal transcribed spacer (ITS) region and chloroplast ribulose-bisphosphate carboxylase gene (rbcL) were used to identify plants collected from the Gulf of Mannar, Puttalam Lagoon, and Matara, Sri Lanka. Based on genetic identification, H. major (Zoll.) Miquel is reported for the first time from Sri Lanka, which might have been misidentified as H. ovalis in previous literature based on morphology alone. We also observed a first hybridization case of Halophila cross between H. ovalis and H. major. Two potential cryptic species were found, herein designated Halophila sp. 1 (allied to H. minor) and Halophila sp. 2 (closely related to H. decipiens). In order to clarify taxonomic ambiguity caused by morphological plasticity and the low resolution of genetic markers, further comparative phylogenomic approaches might be needed to solve species boundary issues in this genus.

Morpho-physiological and molecular responses of two Libyan bread wheat cultivars to plant growth regulators under salt stress

To study the effects of salt stress and plant growth regulators (kinetin, gibberellic acid, potassium) on growth, yield, glycine betaine content, phosphoenolpyruvate carboxylase (PEPC) and ribulose biphosphate carboxylase (RBC) gene expression of two Libyan bread wheat varieties, a factorial design of greenhouse experiment with three replications was conducted. Results revealed that salt stress significantly reduced plant growth and productivity of both varieties. Moreover, the addition of kinetin + potassium and gibberellic acid + potassium had improved the performance of the morpho-metric parameters of both genotypes under salt stress; but the performance was more effective for kinetin treatment than for gibberellic acid. At the biochemical level, the results showed that salt stress increased glycine betaine contents in both varieties with different proportions. This increase is more elevated in the presence of kinetin + potassium than the treatment with gibberellic acid+ potassium, which showed an almost similar result as in only salt stress. At the molecular level, the effects of salt stress and plant growth regulators on the PEPC and RBC gene expression showed that the increase was significantly higher for kinetin, gibberellic acid, and salt stress when compared to the control.
 Highlights - Salt stress reduced plant growth and productivity of bread wheat varieties. - Growth regulator improved the performance of the morphometric parameters. - The performance was more effective for kinetin treatment than for gibberellic acid. - Kinetin improved the glycine betaine gene expression more than gibberellic acid. - Kinetin increased significantly the phosphoenolpyruvate carboxylase and ribulose biphosphate carboxylase gene expression.

Transcriptome Analysis of Bursaphelenchus xylophilus Uncovers the Impact of Stenotrophomonas maltophilia on Nematode and Pine Wilt Disease

Stenotrophomonas maltophilia influences the reproduction, pathogenicity, and gene expression of aseptic Bursaphelenchus xylophilus after inoculation of aseptic Pinus massoniana. Pine wilt disease is a destructive pine forest disease caused by B. xylophilus, and its pathogenesis is unclear. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. S. maltophilia is one of the most dominant bacteria in B. xylophilus, and its effect is ambiguous. This study aims to explore the role of S. maltophilia in pine wilt disease. The reproduction and virulence of aseptic B. xylophilus and B. xylophilus containing S. maltophilia were examined by inoculating aseptic P. massoniana seedlings. The gene expressions of two nematode treatments were identified by transcriptome sequencing. The reproduction and virulence of B. xylophilus containing S. maltophilia were stronger than that of aseptic nematodes. There were 4240 differentially expressed genes between aseptic B. xylophilus and B. xylophilus containing S. maltophilia after inoculation of aseptic P. massoniana, including 1147 upregulated genes and 2763 downregulated genes. These differentially expressed genes were significantly enriched in some immune-related gene ontology (GO) categories, such as membrane, transporter activity, metabolic processes, and many immune-related pathways, such as the wnt, rap1, PI3K-Akt, cAMP, cGMP-PKG, MAPK, ECM-receptor interaction, and calcium signaling pathways. The polyubiquitin-rich gene, leucine-rich repeat serine/threonine-protein kinase gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, acetyl-CoA carboxylase gene, and heat shock protein genes were the key genes associated with immune resistance. Moreover, there were four cell wall hydrolase genes, thirty-six detoxification- and pathogenesis-related protein genes, one effector gene and ten cathepsin L-like cysteine proteinase genes that were differentially expressed. After inoculation of the host pine, S. maltophilia could affect the virulence and reproduction of B. xylophilus by regulating the expression of parasitic, immune, and pathogenicity genes of B. xylophilus.

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

Post‐translational protein modifications derived from metabolic intermediates, such as acyl‐CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl‐CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl‐CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl‐CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl‐CoA accumulation in patient‐derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I‐driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl‐CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.

Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1

To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1). In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene. Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly. A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.

Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine.

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus.

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9°C) and treated for 4 weeks, with the 25°C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9°C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.).

C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

Effect of lignosulphonates on Vanilla planifolia shoot multiplication, regeneration and metabolism

Vanilla planifolia (V. planifolia) is a valuable orchidaceous plant, commonly grown for its pods that are used to produce the flavouring vanilla extract. Here, we evaluated the effect of calcium lignosulphonate (Ca-LIGN) and sodium lignosulphonate (Na-LIGN) on multiplication and regeneration of V. planifolia shoot tip culture. In 150 mg L−1 Ca-LIGN medium, the most number of shoots per explant (5.78 ± 0.63) was successfully obtained. Besides, Ca-LIGN also enhanced the shoot bud and primordial formation rate, as seen under scanning electron microscopy. In contrast, medium containing 150 mg L−1 Na-LIGN recorded the highest average of shoot length (4.72 ± 0.30 cm). Meanwhile, the best growth of root length (1.8 ± 0.32 cm) and root induction (96.67 ± 5.16%) were recorded on the explants treated with 150 mg L−1 Na-LIGN rooting medium. All rooted plantlets successfully acclimatized in the greenhouse (100.00% survival rate). Further biochemical analysis revealed that Ca-LIGN increased the total protein, chlorophyll, sugar, flavonoid and phenolic contents of V. planifolia. Notably, expression of both ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) genes were also elevated under the treatment of Ca-LIGN, implying a positive role in the photosynthetic process. Taken together, LIGN being an environmental friendly product could be used to enhance the growth and micropropagation of V. planifolia.

Isolation and genetic identification of Cylindrotheca closterium algae from Alexandria coastal region

Text Cylindrotheca closterium alga belongs to family Bacillariaceae, orderBacillariales, class Bacillariophyceae and phylum Bacillariophyta. In this studyCylindrotheca closterium alga was isolated from Alexandria coastal region(Eastern Harbor) using two methods: dilution method and streak and sprayplating technique. Isolated algal cells were cultured on Guillard F2 liquidmedium. Morphological identification was carried out by light microscope andscanning electric microscope. Algal cells were found to be wrapped by theraphe canal of valves, which is a typical characteristic of Cylindrothecaclosterium shape. The cells contained two plastids lying next to each otheralong the longitudinal axis of the cell and apprised to one side of the cell withlobes extending onto the other sides, and the nucleus centrally in between.However, previous studies indicated some morphological variations during thelife cycle of the alga.Genetic identification of Cylindrotheca closterium wascarried out using ribulose-bisphosphate carboxylase gene (rbcL) located in theplastids as molecular marker. Specific PCR primers were used to amplify rbcLgene fragment from total genomic DNA extracted from Cylindrothecaclosterium cells. Amplified rbcL (1177 bp) gene fragment was purified andsequenced. Nucleotide sequence was compared with Cylindrotheca sequencesavailable in the GenBank. The Genetic distances and multi alignments werecomputed by Pairwise Distance method using Clusteral W software analysis.The nucleotide sequence of amplified rbcL gene showed varied identitypercentages (81, 80 and 78%) with other related sequences in the GenBank.This could be correlated to the observed morphological variations. Previousstudies indicated high sequence divergences of rbcL gene amongCylindrotheca isolates, and numerous nucleotide variations of rbcL genecaused relatively few variation of deduced amino acid sequence. These resultsmay indicate that Cylindrotheca closterium is a species complex as waspreviously evidenced by the variations of rbcL gene.

Novel strategy to stimulate the food wastes anaerobic fermentation performance by eggshell wastes conditioning and the underlying mechanisms

A novel strategy of utilizing eggshell wastes to condition food wastes (FW) anaerobic fermentation for efficient volatile fatty acids (VFAs) production was proposed. The VFAs production, especially acetic and butyric acids, was significantly accelerated and enhanced by eggshell conditioning. The enhanced effects were dose-dependent and improved by approximate 13-folds with optimal dose when compared with that in control. Eggshell conditioning synchronously enhanced the FW solubilization, hydrolysis and acidification steps, resulting in high VFAs production. Further analysis found that the environmental conditions (i.e., pH, ORP, trace elements, etc.) were significantly improved by eggshell conditioning. Hence, the microbial activities (i.e., hydrolases, acidogenic enzymes and ATP) and functional fermentative bacteria related with VFAs generation were enhanced and enriched. The massive accumulation of H2 and CO2 in eggshell conditioning reactors stimulated the homoacetogenesis, which also contributed to the VFAs promotion. Metatranscriptomic analysis indicated that 3 carbohydrate-active enzymes (Glycoside hydrolases, carbohydrate esterases and carbohydrate binding modules), which participate in carbohydrates and energy metabolisms, were improved. Moreover, 10 kinds of synthetase, hydrolase and oxidoreductase genes, and 4 kinds of acetyl-CoA carboxylase genes that distributed throughout the fatty acid biosynthesis pathway (especially the acetic and butyric acids generation) were all enhanced. It was the highly expressed genes and metabolic enzymes that contributed to high microbial activities and subsequent efficient VFAs production in eggshell conditioning reactors. Besides, the improvement of dewaterability and phosphorus fixation in fermentation residues further increased the added-value of conditioning FW fermentation by eggshell wastes.

Effects of Cabbage-Apple Juice Fermented by Lactobacillus plantarum EM on Lipid Profile Improvement and Obesity Amelioration in Rats.

This study aimed to investigate the potential of cabbage-apple juice, fermented by Lactobacillus plantarum EM isolated from kimchi, to protect against obesity and dyslipidemia that are induced by a high-fat diet in a rat model. Male rats were fed a modified AIN-93M high-fat diet (HFD), the same diet supplemented with non-fermented cabbage-apple juice, or the same diet supplemented with fermented cabbage-apple juice for eight weeks. In the HFD-fermented cabbage- apple juice administered groups the following parameters decreased: body weight, liver and white fat pad weights, serum triglyceride (TG), total cholesterol (TC), LDL-cholesterol, insulin, glucose and leptin levels, TG levels, while HDL-C and adiponectin levels in serum increased as compared with the HFD group. The HFD-fed rats that were supplemented with fermented cabbage-apple juice exhibited significantly lower fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and malic enzyme gene expression levels when compared to the exclusively HFD-fed rats. The anti-obesity and hypolipidemic effects were marginally greater in the fermented juice administered group than in the non-fermented juice administered group. These results suggest that cabbage-apple juice—especially fermented cabbage-apple juice—might have beneficial effects on lipid metabolism dysfunction and obesity-related abnormalities. However, further studies are necessary for analyzing the biochemical regulatory mechanisms of fermented juice for obesity amelioration and lipid metabolic homeostasis.

Transcription Factor TaDof1 Improves Nitrogen and Carbon Assimilation Under Low-Nitrogen Conditions in Wheat

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits. Triticum aestivum Dof1 (TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressing TaDof1 transcription factor, under a constitutive promoter, was developed by Agrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T0 plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the complete TaDof1 cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T1 plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated by TaDof1 was observed after 4 weeks of nitrogen stress in T1 plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression of TaDof1 in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change in TaDof1 transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms.

Drought tolerance of transgenic rice overexpressing maize C<sub>4</sub>-PEPC gene related to increased anthocyanin synthesis regulated by sucrose and calcium

In order to reveal the role of sucrose (Suc) in early drought response in plants, transgenic rice (Oryza sativa L.) plants overexpressing the maize (Zea mays L.) C4-phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene (C4-pepc) (PC) and their untransformed wild type (WT) were used under 12 % (m/v) polyethylene glycol 6000 to simulate drought conditions. The results showed that PC has higher relative water content, the increased Suc content, and anthocyanin accumulation than WT during PEG treatment. By spraying 1 % Suc and 1 % Suc non-metabolic analog, turanose, on these plants, Suc in PC leaves increased anthocyanin content and Ca2+ content. Further experiments using the Ca2+ chelator (EGTA), Ca2+ channel antagonist (ruthenium red), and abscisic acid inhibitor (nordihydroguaiaretic acid), showed that, in PC plants, Suc content is closely related to the expression of sucrose nonfermenting-1-related protein kinases 2 (SnRK2s) such as SAPK8, SAPK9, and SAPK10 via abscisic acid, and the SnRK3 such as SnRK3.1, SnRK 3.4, and SnRK3.21 via Ca2+ and calcineurin B-like as well. Furthermore, the target genes associated with anthocyanin synthesis phenylalanine ammonia lyase, chalcone isomerase, chalcone synthase, flavonoid-3-hydrogenase, flavonoid-3'-hydrogenase, dihydroflavonone reductase, and anthocyanin synthase, their regulated genes basic helix-loop-helix (bHLH) proteins OsB1 and OsB2, R2R3-MYB transcription factor OsC1, and some transcription factors (constitutively photomorphogenic 1, elongated hypocotyl 5, and purple acid phosphatase gene 2) in PC plants also increased via Suc and Ca2+ during the PEG treatment. Some Suc tranporter genes OsSUT1 and OsSUT5 in PC lines during PEG treatment further showed an enhancement for the function of the signal of Suc. Thereby, increasing anthocyanin biosynthesis via Suc and Ca2+ signaling cascade is one of the important mechanisms on drought tolerance in the PC lines.

Confirmation and characterization of cyhalofop-butyl–resistant Chinese sprangletop (Leptochloa chinensis) populations from China

AbstractChinese sprangletop [Leptochloa chinensis (L.) Nees] is one grass weed severely affecting rice (Oryza sativa L.) growth in paddies in China. Cyhalofop-butyl is the main herbicide used to control grass weeds in Chinese paddy fields, especially for controlling L. chinensis; however, L. chinensis has evolved resistance to cyhalofop-butyl due to continuous and extensive application. To investigate cyhalofop-butyl resistance levels and mechanisms in L. chinensis in some of the Chinese rice areas, 66 field populations were collected and treated with cyhalofop-butyl. Of these tested populations, 10 showed a high level of resistance to cyhalofop-butyl; the 50% effective dose ranged within 108.4 to 1,443.5 g ai ha−1 with resistance index values of 9.1 to 121.8 when compared with the susceptible population. Acetyl-coenzyme A carboxylase genes (ACCase) of susceptible and all 10 resistant populations were amplified and sequenced. Among them, Ile-1781-Leu, Trp-2027-Cys, Trp-2027-Ser, and Ile-2041-Asn mutations were found in five resistant populations. No known resistance-related mutations were found in the other five resistant populations, indicating that resistance to cyhalofop-butyl in these populations was likely to be endowed by non–target site resistance mechanisms. Notably, the Ile-1781-Leu and Trp-2027-Cys substitutions have previously been reported, but this is the first report of Trp-2027-Ser and Ile-2041-Asn mutations in L. chinensis. Furthermore, three derived cleaved amplified polymorphic sequence methods were developed to rapidly detect these mutations in L. chinensis.