Glycerol 3-phosphate Dehydrogenase Gene Research Articles

Aspergillus nidulans gfdB, Encoding the Hyperosmotic Stress Protein Glycerol-3-phosphate Dehydrogenase, Disrupts Osmoadaptation in Aspergillus wentii.

The genome of the osmophilic Aspergillus wentii, unlike that of the osmotolerant Aspergillus nidulans, contains only the gfdA, but not the gfdB, glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and ΔgfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed a canonic hyperosmotic stress response characterized by the upregulation of the trehalose and glycerol metabolism genes (including gfdB), as well as the genes of the high-osmolarity glycerol (HOG) map kinase pathway. The deletion of gfdB caused only negligible alterations in the transcriptome, suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than did A. nidulans, e.g., the bulk upregulation of the glycerol and trehalose metabolism genes, along with the HOG pathway genes, was not detected. The expression of An-gfdB in A. wentii did not abolish osmophily, but it reduced growth and caused much bigger alterations in the transcriptome than did the missing gfdB gene in A. nidulans. Flexible glycerol metabolism and hence, two differently regulated gfd genes, may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

Identification of the glycerol-3-phosphate dehydrogenase (GPDH) gene family in wheat and its expression profiling analysis under different stress treatments

Glycerol-3-phosphate dehydrogenase (GPDH) catalyses the interconversion of glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), and plays key roles in different developmental processes and stress responses. GPDH family genes have been previously investigated in various plant species, such as Arabidopsis, maize, and soybean. However, very little is known in GPDH family genes in wheat. In this study, a total of 17 TaGPDH genes were identified from the wheat genome, including eight cytosolic GPDHs, six chloroplastic GPDHs and three mitochondrial GPDHs. Gene duplication analysis showed that segmental duplications contributed to the expansion of this gene family. Phylogenetic results showed that TaGPDHs were clustered into three groups with the same subcellular localization and domain distribution, and similar conserved motif arrangement and gene structure. Expression analysis based on the RNA-seq data showed that GPDH genes exhibited preferential expression in different tissues, and several genes displayed altered expression under various abiotic stresses. These findings provide the foundation for further research of wheat GPDH genes in plant growth, development and stress responses.

Genome-Wide Identification of Cotton (Gossypium spp.) Glycerol-3-Phosphate Dehydrogenase (GPDH) Family Members and the Role of GhGPDH5 in Response to Drought Stress.

Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme in plant glycerol synthesis and metabolism, and plays an important role in plant resistance to abiotic stress. Here, we identified 6, 7, 14 and 14 GPDH genes derived from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. Phylogenetic analysis assigned these genes into three classes, and most of the genes within the family were expanded by whole-genome duplication (WGD) and segmental duplications. Moreover, determination of the nonsynonymous substitution rate/synonymous substitution rate (Ka/Ks) ratio showed that the GPDH had an evolutionary preference for purifying selection. Transcriptome data revealed that GPDH genes were more active in the early stages of fiber development. Additionally, numerous stress-related cis-elements were identified in the potential promoter region. Then, a protein–protein-interaction (PPI) network of GPDH5 in G. hirsutum was constructed. In addition, we predicted 30 underlying miRNAs in G. hirsutum. Functional validation results indicated that silencing GhGPDH5 diminished drought tolerance in the upland cotton TM-1 line. In summary, this study provides a fundamental understanding of the GPDH gene family in cotton, GhGPDH5 exerts a positive effect during drought stress and is potentially involved in stomatal closure movements.

Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans

The genome of Aspergillus nidulans accommodates two glycerol 3-phosphate dehydrogenase genes, gfdA and gfdB. Previous studies confirmed that GfdA is involved in the osmotic stress defence of the fungus. In this work, the physiological role of GfdB was characterized via the construction and functional characterization of the gene deletion mutant ΔgfdB. Unexpectedly, ΔgfdB strains showed oxidative stress sensitivity in the presence of a series of well-known oxidants including tert-butyl-hydroperoxide (tBOOH), diamide as well as hydrogen peroxide. Moderate sensitivity of the mutant towards the cell wall stress inducing agent CongoRed was also observed. Hence, both Gfd isoenzymes contributed to the environmental stress defence of the fungus but their functions were stress-type-specific. Furthermore, the specific activities of certain antioxidant enzymes, like catalase and glutathione peroxidase, were lower in ΔgfdB hyphae than those recorded in the control strain. As a consequence, mycelia from ΔgfdB cultures accumulated reactive species at higher levels than the control. On the other hand, the specific glutathione reductase activity was higher in the mutant, most likely to compensate for the elevated intracellular oxidative species concentrations. Nevertheless, the efficient control of reactive species failed in ΔgfdB cultures, which resulted in reduced viability and, concomitantly, early onset of programmed cell death in mutant hyphae. Inactivation of gfdB brought about higher mannitol accumulation in mycelia meanwhile the erythritol production was not disturbed in unstressed cultures. After oxidative stress treatment with tBOOH, only mannitol was detected in both mutant and control mycelia and the accumulation of mannitol even intensified in the ΔgfdB strain.

Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Glycerol-3-phosphate (G3P) is a key intermediate of glycerol metabolism and is oxidized to dihydroxyacetone phosphate aerobically or anaerobically by appropriate G3P dehydrogenases. A hyperthermophilic archaeon Thermococcus kodakarensis KOD1 has a novel operon consisting of three genes encoding an anaerobic G3P dehydrogenase (G3PDH), an NADH oxidase (NOX), and a molybdopterin oxidoreductase (MOX). Typically, the G3PDH gene (glpA) is included in an operon with genes encoding essential subunits of the G3PDH complex, glpB and glpC. The three genes from T.kodakarensis were cloned and expressed in Escherichia coli, and their recombinant proteins, Tk-G3PDH, Tk-NOX and Tk-MOX, were characterized. The optimal temperature of Tk-G3PDH for activity was 80°C, indicating high thermal stability. Tk-G3PDH has flavin adenine dinucleotide as a prosthetic group and catalyzes oxidation of G3P with kcat/Km 1.93× 103M-1s-1at 80°C, compared with 9.83× 105M-1s-1 for the E.coli G3PDH complex at 37°C. Interestingly, Tk-G3PDH can catalyze this reaction even as a monomer, whereas GlpA must form a complex with GlpB and GlpC. Tk-G3PDH also forms a putative heteropentamer with Tk-NOX and Tk-MOX (G3PDH:NOX:MOX= 2:2:1). This complex may form an electron transfer pathway to a final electron acceptor in the cell membrane, as is the case for the typical G3PDH complex GlpABC.

Glycerol-3-phosphate dehydrogenase (GPDH) gene family in Zea mays L.: Identification, subcellular localization, and transcriptional responses to abiotic stresses.

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the formation of glycerol-3-phosphate, and plays an essential role in glycerolipid metabolism and in response to various stresses in different species. In this study, six ZmGPDH genes were obtained by a thorough search against maize genome, and designated as ZmGPDH1-6, respectively. The structural and evolutionary analyses showed that the ZmGPDHs family had typical conserved domains and similar protein structures as the known GPDHs from other plant species. ZmGPDHs were divided into NAD+-dependent type A form (ZmGPDH1-5) and FAD-dependent type B form (ZmGPDH6) based on their N-terminal sequences. Four full length ZmGPDHs were fused with GFP fusion proteins, and their subcellular localization was determined. ZmGPDH1 and ZmGPDH3 were located to the cytosol and mainly recruited to the surface of endoplasmic reticulum (ER), whereas ZmGPDH4 and ZmGPDH5 were located in the chloroplast. The transcriptional analysis of the ZmGPDHs in different maize tissues revealed relatively high level of transcripts accumulation of ZmGPDHs in roots and early stage developing seeds. Furthermore, we examined the transcriptional responses of the six GPDH genes in maize under various abiotic stresses, including salt, drought, alkali and cold, and significant induction of ZmGPDHs under osmotic stresses was observed. Together, this work will provide useful information for deciphering the roles of GPDHs in plant development and abiotic stress responses.

Predicted Glycerol 3-Phosphate Dehydrogenase Homologs and the Glycerol Kinase GlcA Coordinately Adapt to Various Carbon Sources and Osmotic Stress in Aspergillus fumigatus.

Glycerol plays an important role in the adaptation of fungi to various microenvironments and stressors, including heat shock, anoxic conditions and osmotic stress. Glycerol 3-phosphate dehydrogenase (G3PDH) is able to catalyze dihydroxyacetone phosphate to glycerol 3-phosphate (G3P), which is subsequently dephosphorylated into glycerol. However, current knowledge about the functions of G3PDH homologs in glycerol biosynthesis in Aspergillus fumigatus is limited. Here, we show that the A. fumigatus G3PDH gene, gfdA, is crucial for normal colony growth in glucose media under both normoxic and hypoxic conditions. In addition, failure of the overexpression of the gfdA homolog, gfdB, to rescue the phenotype of a gfdA null mutant suggests that gfdA plays a predominant role in the synthesis of G3P and glycerol. However, in a wild-type background, overexpressing either gfdA or gfdB is able to significantly enhance biomass production of mycelia, suggesting that gfdA and gfdB have similar functions in promoting the use of glucose. Interestingly, overexpression of the gene encoding the predicted glycerol kinase, GlcA, which is capable of phosphorylating glycerol to form G3P, significantly rescues the growth defects of gfdA null mutants in glucose media, indicating that the growth defects of gfdA null mutants might be due to the absence of G3P rather than glycerol. Moreover, Western blotting analysis revealed that gfdA is inducibly expressed by osmotic mediators. However, in the absence of gfdA, osmotic stress can rescue colony growth defects and allow colonies to partially bypass the gfdA requirement in a high osmolarity glycerol pathway-dependent manner. Therefore, the findings of this study elucidate how saprophytic filamentous fungi have developed pathways distinct from those of budding yeasts to adapt to varied carbon sources and survive environmental stresses.

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase.

Acetoin is widely used in food and cosmetic industry as taste and fragrance enhancer. For acetoin production in this study, Saccharomyces cerevisiae JHY605 was used as a host strain, where the production of ethanol and glycerol was largely eliminated by deleting five alcohol dehydrogenase genes (ADH1, ADH2, ADH3, ADH4, and ADH5) and two glycerol 3-phosphate dehydrogenase genes (GPD1 and GPD2). To improve acetoin production, acetoin biosynthetic genes from Bacillus subtilis encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) were overexpressed, and BDH1 encoding butanediol dehydrogenase, which converts acetoin to 2,3-butanediol, was deleted. Furthermore, by NAD+ regeneration through overexpression of water-forming NADH oxidase (NoxE) from Lactococcus lactis, the cofactor imbalance generated during the acetoin production from glucose was successfully relieved. As a result, in fed-batch fermentation, the engineered strain JHY617-SDN produced 100.1 g/L acetoin with a yield of 0.44 g/g glucose.

Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31%, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P < 0.05). mRNA of the peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P < 0.05). In conclusion, expression of the FABP3 gene enhances adipogenesis in 3T3-L1 preadipocytes primarily by upregulating lipogenic PPARγ, 422/aP2 and GPDH genes.

Association Between Cytosolic Glycerol 3-Phosphate Dehydrogenase Gene Expression in Human Subcutaneous Adipose Tissue and BMI

Background/Aims: Cytosolic glycerol 3-phosphate dehydrogenase (cGPDH) is a key enzyme providing glycerol 3-phosphate for triacylglycerol synthesis in adipose tissue and is regarded as a marker for adipocyte differentiation. The aim of this study was to test the hypothesis that an increase in cGPDH gene expression in subcutaneous adipose tissue is associated with obesity. Methods: mRNA levels in human subcutaneous adipose tissue were analysed by Real-Time PCR. Results: We found that human subcutaneous adipose tissue cGPDH activity and cGPDH mRNA level were greater in obese patients than in lean subjects and were positively correlated with BMI and fat mass. Moreover, a strong positive correlation between subcutaneous adipose tissue cGPDH mRNA level and cGPDH activity was found. The data presented here indicates also that PPARγ mRNA level is positively correlated with body mass index and fat mass as well as with adipose tissue cGPDH mRNA level. Moreover, the association between subcutaneous adipose tissue cGPDH mRNA level and fatty acid translocase (FAT/CD36) mRNA level was also observed. Conclusion: The obtained results suggest that in comparison to lean subjects the increase in subcutaneous adipose tissue cGPDH gene expression in the obese, is probably the result of adipose tissue expansion during obesity.

Molecular cloning and expression profile of snow trout GPDH gene in response to abiotic stress

Glycerol-3-phosphate dehydrogenase (GPDH) gene possibly plays a key role for cold acclimation process in snow trout during winter months when water temperature goes down to 4-5 °C. In this study, 1,012 bp nucleotide fragment of GPDH gene was obtained from two snow trout species (Schizothorax richardsonii and S. niger; family: Cyprinidae), distributed in several Himalayan rivers. The gene encoded a protein of 334 amino acids. The encoded protein sequence was very similar to GPDH of Danio rerio (94.36 %) using BLASTx searches. In S. richardsonii the qRT-PCR showed highest expression in muscle tissue followed by liver and also revealed 19 fold gene expression in liver tissue under cold (5 °C) in comparison with warm (15 °C) condition. The elevated expression levels of GPDH cDNA on cold treatment furthermore suggest that GPDH plays a role in stress related responses in S. richardsonii. The phylogenetic analysis showed that the two snow trout species GPDH share the same clade with characterized GPDHs from other teleost fishes suggesting a common evolutionary origin and a similar catalytic function. In addition, the Ka/Ks ratios of these sequences suggested that they are under purifying selection. Moreover, the expression profile of GPDH gene among co generic species of genus Schizothorax showed that GPDH cDNA expression was highest in S. richardsonii and lowest in S. esocinus which gives an indication of species specific adaptation in relation to different geographical areas.

In Silico Cloning and Characterization of the Glycerol-3-Phosphate Dehydrogenase (GPDH) Gene Family in the Green Microalga Chlamydomonas reinhardtii

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD(+). G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K(a)/K(s) ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production.

Development and characterisation of a recombinantSaccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties

The purpose of this study was to help lay the foundation for further development of xylose-fermenting Saccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant haploid strain that overexpressed only two genes of the xylose pathway. Previously, S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenous XKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADH1(p)-PsXYL1-ADH1(T) and PGK1(p)-PsXYL2-PGK1(T)) containing the xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2) genes from the xylose-fermenting yeast, Pichia stipitis, were integrated into the genome of a haploid S. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) overexpressing the P. stipitis XYL1 and XYL2 genes (but not the endogenous XKS1 gene) was subjected to ethyl methane sulfonate (EMS) mutagenesis. The resulting mutants were screened for faster growth rates on an agar medium containing xylose as the sole carbon source. A mutant strain (designated Y-X) that showed 20-fold faster growth in xylose medium in shake-flask cultures was isolated and characterised. In anaerobic batch fermentation, the Y-X mutant strain consumed 2.5-times more xylose than the YUSM 1001 parental strain and also produced more ethanol and glycerol. The xylitol yield from the mutant strain was lower than that from the parental strain, which did not produce glycerol and ethanol from xylose. The mutant also showed a 50% reduction in glucose consumption rate. Transcript levels of XYL1, XYL2 and XKS1 and the GPD2 glycerol 3-phosphate dehydrogenase gene from the two strains were compared with real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The mutant showed 10-40 times higher relative expression of these four genes, which corresponded with either the higher activities of their encoded enzymes or by-product formation during fermentation. Furthermore, no mutations were observed in the mutant's promoter sequences or the open reading frames of some of its key genes involved in carbon catabolite repression, glycerol production and redox balancing. The data suggest that the enhancement of the xylose fermentation properties of the Y-X mutant was made possible by increased expression of the xylose pathway genes, especially the XKS1 xylulokinase gene. (Less)

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.

Seasonal Freeze Resistance of Rainbow Smelt (Osmerus mordax) Is Generated by Differential Expression of Glycerol‐3‐Phosphate Dehydrogenase, Phosphoenolpyruvate Carboxykinase, and Antifreeze Protein Genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.

Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis.

Stearoyl-acyl-carrier-protein-desaturase-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is a key step, which regulates levels of unsaturated fatty acids in cells. We previously showed that stearoyl-acyl-carrier-protein-desaturase mutants ssi2/fab2 carrying a loss-of-function mutation in the plastidial glycerol-3-phosphate (G3P) acyltransferase (act1) have elevated 18:1 levels and are restored in their altered defense signaling. Because G3P is required for the acylation of 18:1 by G3P acyltransferase, it was predicted that reduction of G3P levels should increase 18:1 levels and thereby revert ssi2-triggered phenotypes. Here we show that a mutation in G3P dehydrogenase restores both salicylic acid- and jasmonic acid-mediated phenotypes of ssi2 plants. The G3P dehydrogenase gene was identified by map-based cloning of the ssi2 suppressor mutant rdc8 (gly1-3) and confirmed by epistatic analysis of ssi2 with gly1-1. Restoration of ssi2-triggered phenotypes by the gly1-3 mutation was age-dependent and correlated with the levels of 18:1. Regeneration of G3P pools by glycerol application in ssi2 and ssi2 gly1-3 plants caused a marked reduction in the 18:1 levels, which rendered these plants hypersensitive to glycerol. This hypersensitivity in ssi2 was rescued by the act1 mutation. Furthermore, overexpression of the ACT1 gene resulted in enhanced sensitivity to glycerol. Glycerol application also lowered the 18:1 content in SSI2 plants and converted these into ssi2-mimics. Our results show that 18:1 levels in plastids are regulated by means of acylation with G3P, and a balance between G3P and 18:1 is critical for the regulation of salicylic acid- and jasmonic acid-mediated signaling pathways.

Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae.

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene.

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.