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1.
Funct Integr Genomics ; 22(3): 407-421, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35286570

ABSTRACT

In this study, we characterized the fatty acid production in Neochloris aquatica at transcriptomics and biochemical levels under limiting, normal, and excess nitrate concentrations in different growth phases. At the stationary phase, N. aquatica mainly produced saturated fatty acids such as stearic acid under the limiting nitrate concentration, which is suitable for biodiesel production. However, it produced polyunsaturated fatty acids such as α-linolenic acid under the excess nitrate concentration, which has nutritional values as food supplements. In addition, RNA-seq was employed to identify genes and pathways that were being affected in N. aquatica for three growth phases in the presence of the different nitrate amounts. Genes that are responsible for the production of saturated fatty acids were upregulated in the cells grown under a limiting nitrogen amount while genes that are responsible for the production of polyunsaturated fatty acid were upregulated in the cells grown under excess nitrogen amount. Further analysis showed more genes differentially expressed (DEGs) at the logarithmic phase in all conditions while a relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase under limiting and excess nitrogen. Our results provide a foundation for identifying developmentally important genes and understanding the biological processes in the different growth phases of the N. aquatica in terms of biomass and lipid production.


Subject(s)
Fatty Acids , Transcriptome , Biomass , Fatty Acids/metabolism , Nitrates , Nitrogen/metabolism
2.
Funct Integr Genomics ; 19(5): 715-727, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31001704

ABSTRACT

Drought stress is one of the main environmental factors that affects growth and productivity of crop plants, including lentil. To gain insights into the genome-wide transcriptional regulation in lentil root and leaf under short- and long-term drought conditions, we performed RNA-seq on a drought-sensitive lentil cultivar (Lens culinaris Medik. cv. Sultan). After establishing drought conditions, lentil samples were subjected to de novo RNA-seq-based transcriptome analysis. The 207,076 gene transcripts were successfully constructed by de novo assembly from the sequences obtained from root, leaf, and stems. Differentially expressed gene (DEG) analysis on these transcripts indicated that period of drought stress had a greater impact on the transcriptional regulation in lentil root. The numbers of DEGs were 2915 under short-term drought stress while the numbers of DEGs were increased to 18,327 under long-term drought stress condition in the root. Further, Gene Ontology analysis revealed that the following biological processes were differentially regulated in response to long-term drought stress: protein phosphorylation, embryo development seed dormancy, DNA replication, and maintenance of root meristem identity. Additionally, DEGs, which play a role in circadian rhythm and photoreception, were downregulated suggesting that drought stress has a negative effect on the internal oscillators which may have detrimental consequences on plant growth and survival. Collectively, this study provides a detailed comparative transcriptome response of drought-sensitive lentil strain under short- and long-term drought conditions in root and leaf. Our finding suggests that not only the regulation of genes in leaves is important but also genes regulated in roots are important and need to be considered for improving drought tolerance in lentil.


Subject(s)
Dehydration/genetics , Droughts , Lens Plant/genetics , Plant Leaves/genetics , Plant Proteins/genetics , Plant Roots/genetics , Stress, Physiological , Gene Expression Regulation, Plant , Gene Ontology , High-Throughput Nucleotide Sequencing , Lens Plant/growth & development , Plant Leaves/growth & development , Plant Roots/growth & development , Transcriptome
3.
Bioresour Technol ; 250: 764-769, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29227826

ABSTRACT

In this study, we applied a second round of random mutagenesis using ethyl methanesulfonate to further increase the lipid productivity of a Chlorella vulgaris mutant strain. We generated a mutant (UV715-EMS25) with a lipid content and biomass that were respectively 67% and 35% higher than those of the wild type (WT). The highest achieved lipid productivity in UV715-EMS25 was 91 mg L-1 day-1. Gas chromatography-mass spectrophotometric analysis revealed that the fatty acid methyl ester content of the mutant was 3.9-fold higher compared with that of WT cells. Amounts of saturated and monounsaturated fatty acids were also higher in the mutant, while the total amounts of polyunsaturated fatty acids were lower. Finally, the mutant displayed superior lipid productivity compared with the WT during pilot-scale cultivation in a flat panel photobioreactor. All these results demonstrate that UV715-EMS25 is highly suitable for biodiesel production.


Subject(s)
Chlorella vulgaris , Fatty Acids , Biofuels , Biomass , Chlorella , Lipids , Mutagenesis
4.
Plant Sci ; 252: 125-132, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27717448

ABSTRACT

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.


Subject(s)
Glucose-1-Phosphate Adenylyltransferase/physiology , Plant Proteins/physiology , Solanum tuberosum/enzymology , Starch/biosynthesis , Binding Sites , Enzyme Stability , Glucose-1-Phosphate Adenylyltransferase/chemistry , Glycogen/biosynthesis , Hot Temperature , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Plant Proteins/chemistry , Protein Structure, Tertiary , Solanum tuberosum/genetics , Substrate Specificity
5.
Plant Cell Physiol ; 55(8): 1473-83, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24891561

ABSTRACT

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. Computational and experimental studies have revealed that the heterotetrameric assembly of AGPase is thermodynamically weak. Modeling studies followed by the mutagenesis of the LS of the potato AGPase identified a heterotetramer-deficient mutant, LS(R88A). To enhance heterotetrameric assembly, LS(R88A) cDNA was subjected to error-prone PCR, and second-site revertants were identified according to their ability to restore glycogen accumulation, as assessed with iodine staining. Selected mutations were introduced into the wild-type (WT) LS and co-expressed with the WT SS in Escherichia coli glgC(-). The biochemical characterization of revertants revealed that LS(I90V)SS(WT), LS(Y378C)SS(WT) and LS(D410G)SS(WT) mutants displayed enhanced heterotetrameric assembly with the WT SS. Among these mutants, LS(Y378C)SS(WT) AGPase displayed increased heat stability compared with the WT enzyme. Kinetic characterization of the mutants indicated that the LS(I90V)SS(WT) and LS(Y378C)SS(WT) AGPases have comparable allosteric and kinetic properties. However, the LS(D410G)SS(WT) mutant exhibited altered allosteric properties of being less responsive and more sensitive to 3-phosphoglyceric acid activation and inorganic phosphate inhibition. This study not only enhances our understanding of the interaction between the SS and the LS of AGPase but also enables protein engineering to obtain enhanced assembled heat-stable variants of AGPase, which can be used for the improvement of plant yields.


Subject(s)
Glucose-1-Phosphate Adenylyltransferase/metabolism , Protein Multimerization , Solanum tuberosum/enzymology , Allosteric Site , Amino Acid Sequence , Glucose-1-Phosphate Adenylyltransferase/genetics , Hot Temperature , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phenotype , Protein Stability , Protein Structure, Secondary , Reverse Genetics , Solanum tuberosum/genetics
6.
Plant Sci ; 205-206: 29-37, 2013 May.
Article in English | MEDLINE | ID: mdl-23498860

ABSTRACT

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme in plant starch biosynthesis. It contains large (LS) and small (SS) subunits encoded by two different genes. In this study, we explored the transcriptional regulation of both the LS and SS subunits of AGPase in stem and leaf under different photoperiods length in lentil. To this end, we first isolated and characterized different isoforms of the LS and SS of lentil AGPase and then we performed quantitative real time PCR (qPCR) to see the effect of photoperiod length on the transcription of the AGPase isforms under the different photoperiod regimes in lentil. Analysis of the qPCR results revealed that the transcription of different isoforms of the LSs and the SSs of lentil AGPase are differentially regulated when photoperiod shifted from long-day to short-day in stem and leaves. While transcript levels of LS1 and SS2 in leaf significantly decreased, overall transcript levels of SS1 increased in short-day regime. Our results indicated that day length affects the transcription of lentil AGPase isoforms differentially in stems and leaves most likely to supply carbon from the stem to other tissues to regulate carbon metabolism under short-day conditions.


Subject(s)
Gene Expression Regulation, Plant/genetics , Glucose-1-Phosphate Adenylyltransferase/genetics , Lens Plant/enzymology , Photoperiod , Base Sequence , Cloning, Molecular , Glucose-1-Phosphate Adenylyltransferase/isolation & purification , Glucose-1-Phosphate Adenylyltransferase/metabolism , Isoenzymes , Kinetics , Lens Plant/genetics , Lens Plant/radiation effects , Molecular Sequence Data , Organ Specificity , Phylogeny , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/radiation effects , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/radiation effects , Plant Stems/enzymology , Plant Stems/genetics , Plant Stems/radiation effects , Seeds/enzymology , Seeds/genetics , Seeds/radiation effects , Starch/metabolism
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