This document summarizes a study that manipulated ascorbic acid (vitamin C) biosynthesis pathways in tomato (Solanum lycopersicum) plants by expressing three genes involved in ascorbic acid production. GDP-mannose pyrophosphorylase (GMPase) from yeast, arabinono-1,4-lactone oxidase (ALO) from yeast, and myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana were independently expressed in tomato under the control of a constitutive promoter. Expression of GMPase increased ascorbic acid levels up to 70% in leaves, 50% in green fruit, and 35% in red fruit.
Cyclotides are small globular proteins found in plants that have a unique circular backbone stabilized by three disulfide bonds forming a cystine knot motif. This structure makes them exceptionally stable and able to penetrate cells. They can be produced through chemical synthesis or recombinant expression. Cyclotides show potential as scaffolds for therapeutic peptides due to their stability and ability to cross cell membranes and target protein-protein interactions. One example is an engineered cyclotide that targets the p53-Hdm2/HdmX interaction for cancer treatment. Overall, cyclotides' unique circular topology and stability make them promising molecular scaffolds for novel peptide-based therapeutics.
1) Researchers engineered E. coli strains SZ63 and SZ85 to produce optically pure D(-)-lactate and L(+)-lactate respectively from various sugars. However, these strains lacked the ability to metabolize sucrose.
2) Sucrose utilization genes (cscR cscA cscKB) were cloned from E. coli KO11 and expressed in plasmid pLOI3501. This plasmid was stable and functionally expressed in SZ63 but not SZ85.
3) Strain SZ63 containing pLOI3501 was able to produce over 500 mM D(-)-lactate from sucrose and molasses substrates. This demonstrates the ability to utilize inexpensive sucrose and molasses for bi
The document describes the cloning of a large modular polyketide synthase (PKS) gene complex from "Candidatus Endobugula sertula", an uncultivated bacterial symbiont of the marine bryozoan Bugula neritina. The PKS is proposed to be responsible for biosynthesis of the bryostatin family of anticancer compounds found in B. neritina. One gene in the complex, called bryA, was characterized in more detail. bryA contains domains typical of PKSs but lacks acyltransferase domains. The authors propose that bryA synthesizes part of the pharmacologically active portion of bryostatin and could be useful for semisynthesis
Crimson publishers-5-MethylcytosineDNA Methylation Patterns among Gut Predomi...CrimsonpublishersMedical
5-MethylcytosineDNA Methylation Patterns among Gut Predominate Commensal Escherichia coli and Lactobacilli from the Balbas and Mazekh Domestic Sheep Breeds by Pepoyan AZ* in Research in Medical &Engineering Sciences
Each chapter begins with an overview of the key concepts to be discussed, in this case the cellular basis of inheritance. The chapter then presents a series of figures illustrating these concepts. It explores how organisms develop from fertilized eggs through cell division, the life cycles of animals, fungi and plants, the processes of crossing over and independent assortment during meiosis, the stages and outcomes of meiosis and mitosis, examples of genetic disorders caused by nondisjunction during meiosis, and different types of chromosomal rearrangements like inversions and translocations.
This document provides figures and captions describing cell structure and function. It includes figures of different cell types viewed under light and electron microscopes. It also includes figures and descriptions of cellular components like the plasma membrane, nucleus, mitochondria, chloroplasts and their structures and functions. Transport mechanisms across membranes like diffusion, osmosis, and active transport are depicted. Endocytosis and exocytosis are also summarized. The document serves as a visual guide to key concepts in cell biology.
Synthetic Biology Of Plant Specialised Metabolism Using NGS Information Of No...Fabio Caligaris
Presented at Plant Genomics and Gene Editing Congress: Asia. For more information visit: www.global-engage.com
Synthetic biology is powerful strategy to reconstruct biosynthetic
pathways when molecular tools are available. Here, we report
the potentials of this strategy as a case study in isoquinoline
alkaloid biosynthesis. So far, we have characterized biosynthetic
enzyme genes of specialized metabolism using a combination of
transcriptome and metabolome. In this presentation, identification of several biosynthetic enzyme genes and production of some metabolites are discussed
This document contains captions for 17 figures related to the key concepts of biotechnology discussed in Chapter 10. The figures cover topics such as:
1) Tools used for DNA analysis like thermal cyclers and gel electrophoresis.
2) Processes involved in biotechnology like polymerase chain reaction (PCR), molecular cloning, gene therapy, and transgenic organisms.
3) Applications of biotechnology like cloning animals, curing genetic diseases, genetically modifying crops, and developing renewable fuels.
Cyclotides are small globular proteins found in plants that have a unique circular backbone stabilized by three disulfide bonds forming a cystine knot motif. This structure makes them exceptionally stable and able to penetrate cells. They can be produced through chemical synthesis or recombinant expression. Cyclotides show potential as scaffolds for therapeutic peptides due to their stability and ability to cross cell membranes and target protein-protein interactions. One example is an engineered cyclotide that targets the p53-Hdm2/HdmX interaction for cancer treatment. Overall, cyclotides' unique circular topology and stability make them promising molecular scaffolds for novel peptide-based therapeutics.
1) Researchers engineered E. coli strains SZ63 and SZ85 to produce optically pure D(-)-lactate and L(+)-lactate respectively from various sugars. However, these strains lacked the ability to metabolize sucrose.
2) Sucrose utilization genes (cscR cscA cscKB) were cloned from E. coli KO11 and expressed in plasmid pLOI3501. This plasmid was stable and functionally expressed in SZ63 but not SZ85.
3) Strain SZ63 containing pLOI3501 was able to produce over 500 mM D(-)-lactate from sucrose and molasses substrates. This demonstrates the ability to utilize inexpensive sucrose and molasses for bi
The document describes the cloning of a large modular polyketide synthase (PKS) gene complex from "Candidatus Endobugula sertula", an uncultivated bacterial symbiont of the marine bryozoan Bugula neritina. The PKS is proposed to be responsible for biosynthesis of the bryostatin family of anticancer compounds found in B. neritina. One gene in the complex, called bryA, was characterized in more detail. bryA contains domains typical of PKSs but lacks acyltransferase domains. The authors propose that bryA synthesizes part of the pharmacologically active portion of bryostatin and could be useful for semisynthesis
Crimson publishers-5-MethylcytosineDNA Methylation Patterns among Gut Predomi...CrimsonpublishersMedical
5-MethylcytosineDNA Methylation Patterns among Gut Predominate Commensal Escherichia coli and Lactobacilli from the Balbas and Mazekh Domestic Sheep Breeds by Pepoyan AZ* in Research in Medical &Engineering Sciences
Each chapter begins with an overview of the key concepts to be discussed, in this case the cellular basis of inheritance. The chapter then presents a series of figures illustrating these concepts. It explores how organisms develop from fertilized eggs through cell division, the life cycles of animals, fungi and plants, the processes of crossing over and independent assortment during meiosis, the stages and outcomes of meiosis and mitosis, examples of genetic disorders caused by nondisjunction during meiosis, and different types of chromosomal rearrangements like inversions and translocations.
This document provides figures and captions describing cell structure and function. It includes figures of different cell types viewed under light and electron microscopes. It also includes figures and descriptions of cellular components like the plasma membrane, nucleus, mitochondria, chloroplasts and their structures and functions. Transport mechanisms across membranes like diffusion, osmosis, and active transport are depicted. Endocytosis and exocytosis are also summarized. The document serves as a visual guide to key concepts in cell biology.
Synthetic Biology Of Plant Specialised Metabolism Using NGS Information Of No...Fabio Caligaris
Presented at Plant Genomics and Gene Editing Congress: Asia. For more information visit: www.global-engage.com
Synthetic biology is powerful strategy to reconstruct biosynthetic
pathways when molecular tools are available. Here, we report
the potentials of this strategy as a case study in isoquinoline
alkaloid biosynthesis. So far, we have characterized biosynthetic
enzyme genes of specialized metabolism using a combination of
transcriptome and metabolome. In this presentation, identification of several biosynthetic enzyme genes and production of some metabolites are discussed
This document contains captions for 17 figures related to the key concepts of biotechnology discussed in Chapter 10. The figures cover topics such as:
1) Tools used for DNA analysis like thermal cyclers and gel electrophoresis.
2) Processes involved in biotechnology like polymerase chain reaction (PCR), molecular cloning, gene therapy, and transgenic organisms.
3) Applications of biotechnology like cloning animals, curing genetic diseases, genetically modifying crops, and developing renewable fuels.
The document describes a novel three-step synthesis of the N(10)-C(17) fragment of cyclotheonamides, potent serine protease inhibitors. The key steps are:
1) A Passerini reaction between protected argininal, dipeptide isonitrile, and proline derivatives that produces adduct 13 in good yield.
2) Orthogonal deprotection of 13 leads to an O- to N-acyl migration, providing compound 14.
3) Compound 14 constitutes the desired N(10)-C(17) fragment and is produced in each step in good yield under mild conditions, representing an atom-economical synthesis.
This document contains figures and captions describing cell reproduction and the cell cycle. Figure 6.1 shows a sea urchin undergoing cell division from a single cell to a multicellular organism. Figure 6.2 shows the chromosomes in a human cell. Figure 6.3 describes the phases of the cell cycle, including interphase and the mitotic phase where the cell divides. Figures 6.4-6.9 provide more details on mitosis, cytokinesis, checkpoints in the cell cycle, DNA damage response, and bacterial binary fission.
This document provides an overview of key concepts in molecular biology through a series of figures. It discusses DNA replication through the semiconservative model and repair through proofreading, mismatch repair, and nucleotide excision. It also examines transcription, from initiation to elongation, and translation, from the genetic code and recruitment of tRNAs to termination. Finally, it notes that eukaryotic gene expression is regulated at transcription, RNA processing, translation, and through post-translational modifications.
This document summarizes a study that analyzed a knockout mutant (Mtp5cs3) of the MtP5CS3 gene in Medicago truncatula. The study found that the Mtp5cs3 mutant, which cannot produce the MtP5CS3 protein, showed decreased proline accumulation and increased sensitivity to salt, drought, and low water potential stresses. Metabolomic analysis revealed that some osmoprotective metabolites accumulated more in Mtp5cs3 roots, but this was insufficient to compensate for proline deficiency. Overexpression of MtP5CS3 increased stress tolerance, suggesting it plays a critical role in proline accumulation and stress tolerance in M. truncatula.
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
Omar Quintero-Monzon has over 20 years of experience in biochemistry, protein purification and characterization, antibody drug development, and assay development. He has worked at multiple biotech companies leading projects and developing novel techniques. His experience includes developing assays for glycoprotein analysis, antibody and protein purification methods, and biomarker assays with improved sensitivity.
This study compared the enzyme activity of ASAT2, which catalyzes the biosynthesis of acyl sugars, natural pesticides produced by tomato plants, in different wild tomato species. The ASAT2 enzyme from two wild accessions, S. chilense and S. arcanum, was found to add C12, aiC5, or iC5 chains to monacyl sucrose, unlike the cultivated S. lycopersicum allele which does not add iC5. Comparing amino acid sequences identified four substitutions in the wild alleles that may alter acyl-CoA binding affinity and account for the additional activity. This research helps understand how small changes impact acyl sugar biosynthesis, with implications for manipulating this pathway
Gene expression and protein activity are controlled through modifications of genetic material and proteins. At the gene level, DNA can be methylated to suppress gene expression. Histone proteins can also be methylated or acetylated to control transcriptional activation or silencing. At the protein level, post-translational modifications like phosphorylation, glycosylation, and cleavage can regulate protein activity, localization, and function. These modifications allow for versatile control over biological processes.
This document summarizes research on sortase enzymes. Sortases are bacterial transpeptidase enzymes that covalently attach secreted proteins to the bacterial cell wall. They play important roles in virulence and pathogenesis. The document discusses various classes of sortases, their mechanisms of action, roles in antibiotic resistance and biofilm formation. It also outlines applications of sortases in areas like vaccine development, drug targeting, and protein immobilization. Overall, the document provides an overview of the sortase enzyme family and their significance in microbial physiology and disease.
Ribosomal Proteins and their Extra Ribosomal Functions in Abiotic Stress Tole...CrimsonpublishersMCDA
Ribosomal proteins (RPs) that include both small (RPS) and large subunit (RPL) proteins have been known to be involved in several very important functions in ribosome assembly, protein synthesis and other cellular functions in association with several other components [1]. The composition of ribosomal protein subunits that are involved in ribosome assembly is heterogeneous [2] indicating clearly that individual subunit protein components have functions also in phenomena like stress tolerance. Although each RP gene has multiple paralogs, the expression of all of them is differentially required for normal development with some of them functioning in spatio-temporal and signal-induced manner while others exhibit binding properties. The expression of ribosomal proteins has been shown to be regulated by various environmental cues and treatments with signaling molecules [3,4]. The involvement of ribosomal proteins in extra ribosomal functions in animal systems has been well documented [5].
https://crimsonpublishers.com/mcda/fulltext/MCDA.000591.php
For more open access journals in Crimson Publishers please click on link: https://crimsonpublishers.com
For more articles on journal of agronomy and crop science please click on below link: https://crimsonpublishers.com/mcda/
This document summarizes research on variation in the acylsugar biosynthetic pathway between wild tomato species. Acylsugars are specialized metabolites produced in tomato trichomes that serve as an insect defense. The pathway diverged between cultivated tomato (Solanum lycopersicum) and wild relative S. pennellii, producing different acylsugar structures. Through site-directed mutagenesis of acylsugar acyltransferase genes (ASAT) and analysis of resulting acylsugar products, specific amino acids were found to control acyl donor/acceptor specificity and pathway divergence between species. Mutating residues in S. pennellii ASAT2 and S. lycopersicum
This document describes a project to develop an in vitro lipoylation reaction to screen chemicals hypothesized to trigger primary biliary cirrhosis (PBC) through molecular mimicry. The student conducted a series of optimization reactions altering amounts of lipoic acid, GTP/ATP, or lipoate activating enzyme and lipoyltransferase. Little success was achieved and time did not allow screening of chemicals, requiring further work. In conclusion, both enzymes are required for lipoylation but varying amounts did not greatly affect the reaction, prompting questions about enzyme purification for future studies.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
The document summarizes research characterizing alcohol/aldehyde dehydrogenase (ADHE) enzymes from six Entamoeba varieties. Key findings include:
1) ADHE genes were isolated, sequenced, and expressed from E. dispar, E. invadens, E. moshkovskii, and E. terrapinae.
2) The ADHE proteins showed similar molecular weights (2.7 kDa) and consistency in amino acid structure, indicating evolutionary convergence.
3) Biochemical analysis of the ADHE enzymes' alcohol and aldehyde dehydrogenase activities and kinetics will provide insight into their structure-function and evolution.
4) Inhibiting the EhADH2 enzyme in E
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
This document reports the discovery and characterization of three new polyene macrolactams called lobosamides A-C produced by the marine actinobacterium Micromonospora sp. Lobosamide A exhibited submicromolar activity against Trypanosoma brucei, the causative agent of human African trypanosomiasis. The biosynthetic gene cluster of the lobosamides was sequenced and used to deduce the complete absolute configurations of the compounds. A "molecules-to-genes-to-molecules" approach identified a related gene cluster in Actinosynnema mirum, leading to the discovery of two additional related macrolactams called mirilactams A-B
This document summarizes research analyzing relationships between lysine ubiquitylation and acetylation sites from proteomic datasets in Arabidopsis thaliana proteins. The research compared ubiquitylation and acetylation sites identified in previous publications and found that 9 non-nuclear proteins experienced both post-translational modifications. Most of these proteins were located in energy-associated organelles. While this provides support for potential cross-talk between ubiquitylation and acetylation, more acetylation and ubiquitylation data is still needed to further investigate these relationships and whether acetylation directly or indirectly influences ubiquitylation.
This study identified and characterized a new collagenase, ColAh, produced by Aeromonas piscicola AH-3. The researchers identified a gene in A. hydrophila ATCC 7966 that encodes a putative collagenase. They constructed a knockout mutant of this gene (colAh) in A. piscicola AH-3. Tests showed the mutant had significantly lower collagenolytic activity and cytotoxic effects compared to the wild type, demonstrating the colAh gene encodes an active collagenase. Further analysis found ColAh is a ~100kDa metalloprotease that can cleave and bind collagen. This contributes to A. piscicola's collagen degradation and cytotoxicity. ColAh contains the conserved HEXX
1. The study examines how progesterone receptor activity is maintained in decidualizing human endometrial stromal cells exposed to oxidative stress.
2. It finds that modest oxidative stress activates the JNK pathway in stromal cells, leading to enhanced sumoylation and inhibition of the progesterone receptor.
3. However, JNK pathway signaling and its effects on sumoylation are disabled during decidualization, maintaining progesterone receptor activity even under oxidative stress. This is mediated by increased expression of the JNK pathway regulator MKP1 upon decidualization.
The SKA (Square Kilometre Array) will be the world's largest radio telescope, consisting of thousands of antennas linked together across thousands of kilometers. It will be used to study the formation and evolution of galaxies, dark matter, magnetic fields, and to search for signs of life in the universe. The SKA will be constructed in phases, with Phase 1 beginning construction in 2016 and Phase 2 from 2019-2024. It will be built through international collaboration between over a dozen countries, including South Africa which is contributing the 64-dish MeerKAT telescope and has several scientists and engineers working on the project.
DevOpsDays Baltimore 2017.
Product owners are under pressure from Marketing and Leadership to focus on features, while operability (availability, performance, monitoring, etc) are an afterthought to be bolted on later. Deployments fail, customers complain, and work isn't fun. How can DevOps reach out to Product?
People from a "Product background" often have zero technical experience, but find themselves needing to dictate the deliverables. Product owners are under great pressure from Marketing and Leadership to focus on "features" from a customer perspective; the so-called "non-functional requirements" often fall by the wayside. Operability - monitorabilty, recoverability, availability, performance, among other aspects - is difficult to bake into an application that was developed without such consideration.
This talk will present practical approaches to bridge-building between Ops and Product. Focusing especially on cross-functional Agile teams with leadership with little or no Ops background, we will explore whether "planning the work will result in the planned work being the work that is done." When working with a mixed team, doing development, deployment, incident response, and everything in support of that, such plans go off the rails. Methods of championing Ops needs while avoiding "the sky is falling" perceptions will be presented. What kinds of unplanned work exist? Are there steps we can take to convert unplanned work into planned work? How does work flow through the team? How does unplanned work disrupt the flow?
The document describes a novel three-step synthesis of the N(10)-C(17) fragment of cyclotheonamides, potent serine protease inhibitors. The key steps are:
1) A Passerini reaction between protected argininal, dipeptide isonitrile, and proline derivatives that produces adduct 13 in good yield.
2) Orthogonal deprotection of 13 leads to an O- to N-acyl migration, providing compound 14.
3) Compound 14 constitutes the desired N(10)-C(17) fragment and is produced in each step in good yield under mild conditions, representing an atom-economical synthesis.
This document contains figures and captions describing cell reproduction and the cell cycle. Figure 6.1 shows a sea urchin undergoing cell division from a single cell to a multicellular organism. Figure 6.2 shows the chromosomes in a human cell. Figure 6.3 describes the phases of the cell cycle, including interphase and the mitotic phase where the cell divides. Figures 6.4-6.9 provide more details on mitosis, cytokinesis, checkpoints in the cell cycle, DNA damage response, and bacterial binary fission.
This document provides an overview of key concepts in molecular biology through a series of figures. It discusses DNA replication through the semiconservative model and repair through proofreading, mismatch repair, and nucleotide excision. It also examines transcription, from initiation to elongation, and translation, from the genetic code and recruitment of tRNAs to termination. Finally, it notes that eukaryotic gene expression is regulated at transcription, RNA processing, translation, and through post-translational modifications.
This document summarizes a study that analyzed a knockout mutant (Mtp5cs3) of the MtP5CS3 gene in Medicago truncatula. The study found that the Mtp5cs3 mutant, which cannot produce the MtP5CS3 protein, showed decreased proline accumulation and increased sensitivity to salt, drought, and low water potential stresses. Metabolomic analysis revealed that some osmoprotective metabolites accumulated more in Mtp5cs3 roots, but this was insufficient to compensate for proline deficiency. Overexpression of MtP5CS3 increased stress tolerance, suggesting it plays a critical role in proline accumulation and stress tolerance in M. truncatula.
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
Omar Quintero-Monzon has over 20 years of experience in biochemistry, protein purification and characterization, antibody drug development, and assay development. He has worked at multiple biotech companies leading projects and developing novel techniques. His experience includes developing assays for glycoprotein analysis, antibody and protein purification methods, and biomarker assays with improved sensitivity.
This study compared the enzyme activity of ASAT2, which catalyzes the biosynthesis of acyl sugars, natural pesticides produced by tomato plants, in different wild tomato species. The ASAT2 enzyme from two wild accessions, S. chilense and S. arcanum, was found to add C12, aiC5, or iC5 chains to monacyl sucrose, unlike the cultivated S. lycopersicum allele which does not add iC5. Comparing amino acid sequences identified four substitutions in the wild alleles that may alter acyl-CoA binding affinity and account for the additional activity. This research helps understand how small changes impact acyl sugar biosynthesis, with implications for manipulating this pathway
Gene expression and protein activity are controlled through modifications of genetic material and proteins. At the gene level, DNA can be methylated to suppress gene expression. Histone proteins can also be methylated or acetylated to control transcriptional activation or silencing. At the protein level, post-translational modifications like phosphorylation, glycosylation, and cleavage can regulate protein activity, localization, and function. These modifications allow for versatile control over biological processes.
This document summarizes research on sortase enzymes. Sortases are bacterial transpeptidase enzymes that covalently attach secreted proteins to the bacterial cell wall. They play important roles in virulence and pathogenesis. The document discusses various classes of sortases, their mechanisms of action, roles in antibiotic resistance and biofilm formation. It also outlines applications of sortases in areas like vaccine development, drug targeting, and protein immobilization. Overall, the document provides an overview of the sortase enzyme family and their significance in microbial physiology and disease.
Ribosomal Proteins and their Extra Ribosomal Functions in Abiotic Stress Tole...CrimsonpublishersMCDA
Ribosomal proteins (RPs) that include both small (RPS) and large subunit (RPL) proteins have been known to be involved in several very important functions in ribosome assembly, protein synthesis and other cellular functions in association with several other components [1]. The composition of ribosomal protein subunits that are involved in ribosome assembly is heterogeneous [2] indicating clearly that individual subunit protein components have functions also in phenomena like stress tolerance. Although each RP gene has multiple paralogs, the expression of all of them is differentially required for normal development with some of them functioning in spatio-temporal and signal-induced manner while others exhibit binding properties. The expression of ribosomal proteins has been shown to be regulated by various environmental cues and treatments with signaling molecules [3,4]. The involvement of ribosomal proteins in extra ribosomal functions in animal systems has been well documented [5].
https://crimsonpublishers.com/mcda/fulltext/MCDA.000591.php
For more open access journals in Crimson Publishers please click on link: https://crimsonpublishers.com
For more articles on journal of agronomy and crop science please click on below link: https://crimsonpublishers.com/mcda/
This document summarizes research on variation in the acylsugar biosynthetic pathway between wild tomato species. Acylsugars are specialized metabolites produced in tomato trichomes that serve as an insect defense. The pathway diverged between cultivated tomato (Solanum lycopersicum) and wild relative S. pennellii, producing different acylsugar structures. Through site-directed mutagenesis of acylsugar acyltransferase genes (ASAT) and analysis of resulting acylsugar products, specific amino acids were found to control acyl donor/acceptor specificity and pathway divergence between species. Mutating residues in S. pennellii ASAT2 and S. lycopersicum
This document describes a project to develop an in vitro lipoylation reaction to screen chemicals hypothesized to trigger primary biliary cirrhosis (PBC) through molecular mimicry. The student conducted a series of optimization reactions altering amounts of lipoic acid, GTP/ATP, or lipoate activating enzyme and lipoyltransferase. Little success was achieved and time did not allow screening of chemicals, requiring further work. In conclusion, both enzymes are required for lipoylation but varying amounts did not greatly affect the reaction, prompting questions about enzyme purification for future studies.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
The document summarizes research characterizing alcohol/aldehyde dehydrogenase (ADHE) enzymes from six Entamoeba varieties. Key findings include:
1) ADHE genes were isolated, sequenced, and expressed from E. dispar, E. invadens, E. moshkovskii, and E. terrapinae.
2) The ADHE proteins showed similar molecular weights (2.7 kDa) and consistency in amino acid structure, indicating evolutionary convergence.
3) Biochemical analysis of the ADHE enzymes' alcohol and aldehyde dehydrogenase activities and kinetics will provide insight into their structure-function and evolution.
4) Inhibiting the EhADH2 enzyme in E
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
This document reports the discovery and characterization of three new polyene macrolactams called lobosamides A-C produced by the marine actinobacterium Micromonospora sp. Lobosamide A exhibited submicromolar activity against Trypanosoma brucei, the causative agent of human African trypanosomiasis. The biosynthetic gene cluster of the lobosamides was sequenced and used to deduce the complete absolute configurations of the compounds. A "molecules-to-genes-to-molecules" approach identified a related gene cluster in Actinosynnema mirum, leading to the discovery of two additional related macrolactams called mirilactams A-B
This document summarizes research analyzing relationships between lysine ubiquitylation and acetylation sites from proteomic datasets in Arabidopsis thaliana proteins. The research compared ubiquitylation and acetylation sites identified in previous publications and found that 9 non-nuclear proteins experienced both post-translational modifications. Most of these proteins were located in energy-associated organelles. While this provides support for potential cross-talk between ubiquitylation and acetylation, more acetylation and ubiquitylation data is still needed to further investigate these relationships and whether acetylation directly or indirectly influences ubiquitylation.
This study identified and characterized a new collagenase, ColAh, produced by Aeromonas piscicola AH-3. The researchers identified a gene in A. hydrophila ATCC 7966 that encodes a putative collagenase. They constructed a knockout mutant of this gene (colAh) in A. piscicola AH-3. Tests showed the mutant had significantly lower collagenolytic activity and cytotoxic effects compared to the wild type, demonstrating the colAh gene encodes an active collagenase. Further analysis found ColAh is a ~100kDa metalloprotease that can cleave and bind collagen. This contributes to A. piscicola's collagen degradation and cytotoxicity. ColAh contains the conserved HEXX
1. The study examines how progesterone receptor activity is maintained in decidualizing human endometrial stromal cells exposed to oxidative stress.
2. It finds that modest oxidative stress activates the JNK pathway in stromal cells, leading to enhanced sumoylation and inhibition of the progesterone receptor.
3. However, JNK pathway signaling and its effects on sumoylation are disabled during decidualization, maintaining progesterone receptor activity even under oxidative stress. This is mediated by increased expression of the JNK pathway regulator MKP1 upon decidualization.
The SKA (Square Kilometre Array) will be the world's largest radio telescope, consisting of thousands of antennas linked together across thousands of kilometers. It will be used to study the formation and evolution of galaxies, dark matter, magnetic fields, and to search for signs of life in the universe. The SKA will be constructed in phases, with Phase 1 beginning construction in 2016 and Phase 2 from 2019-2024. It will be built through international collaboration between over a dozen countries, including South Africa which is contributing the 64-dish MeerKAT telescope and has several scientists and engineers working on the project.
DevOpsDays Baltimore 2017.
Product owners are under pressure from Marketing and Leadership to focus on features, while operability (availability, performance, monitoring, etc) are an afterthought to be bolted on later. Deployments fail, customers complain, and work isn't fun. How can DevOps reach out to Product?
People from a "Product background" often have zero technical experience, but find themselves needing to dictate the deliverables. Product owners are under great pressure from Marketing and Leadership to focus on "features" from a customer perspective; the so-called "non-functional requirements" often fall by the wayside. Operability - monitorabilty, recoverability, availability, performance, among other aspects - is difficult to bake into an application that was developed without such consideration.
This talk will present practical approaches to bridge-building between Ops and Product. Focusing especially on cross-functional Agile teams with leadership with little or no Ops background, we will explore whether "planning the work will result in the planned work being the work that is done." When working with a mixed team, doing development, deployment, incident response, and everything in support of that, such plans go off the rails. Methods of championing Ops needs while avoiding "the sky is falling" perceptions will be presented. What kinds of unplanned work exist? Are there steps we can take to convert unplanned work into planned work? How does work flow through the team? How does unplanned work disrupt the flow?
DevOpsDays Baltimore - 2017.
What can an Italian plumber, falling Russian blocks, and sword wielding adventurers teach us about DevOps? Well, quite a bit actually if we stay awhile and listen.
In this lighthearted session I'll share a few lessons learned from a lifetime playing and developing video games, and how the same mindset that we naturally assume when we play games can be used to create a successful DevOps culture.
See mom, I wasn't just wasting my time all those years...
HealthSeeker - Gamification in healthcare - Manu Melwin Joymanumelwin
This document discusses Healthseeker, a Facebook game developed by the Diabetes Hands Foundation and Joslin Diabetes Center to gamify healthcare. Players choose missions, take small daily actions, complete levels, earn points by interacting with friends, and help their friends reach goals. The game shows that social networks can help people complete health challenges at higher rates than if trying alone.
Generalidades e Incisivo Central Superiorcaiqueacm
O documento fornece detalhes técnicos sobre a anatomia dental, incluindo a nomenclatura de estruturas como arcadas, quadrantes, dentes individuais e suas partes. Há também descrições da morfologia de dentes específicos como incisivos e molares.
Developing New Services in Science LibrariesAPLICwebmaster
This document discusses how science libraries can provide new services to better serve scientists. It suggests that libraries provide free bibliographic and publication services like maintaining staff bibliographies, populating institutional repositories, and helping integrate publications into the broader scientific publishing ecosystem. These services allow libraries to showcase the work of their scientists while also reusing bibliographic information to serve multiple audiences.
El documento define Web 1.0, Web 2.0, SlideShare, wikis y blogs. Web 1.0 se refiere a páginas web estáticas donde los usuarios solo pueden leer, mientras que Web 2.0 permite una mayor interacción entre usuarios a través de herramientas como blogs y redes sociales. SlideShare permite compartir presentaciones en línea, wikis son espacios de escritura colaborativa, y los blogs son sitios web con contenido actualizado regularmente donde los lectores pueden comentar.
Singaporean jewelry designer Choo Yilin crafts pieces from reclaimed metals and discarded gems, believing in "imperfect beauty". A former political analyst, she launched her sustainable luxury label in 2008 in Bangkok and focuses on ethical designs, working with hill tribes in Thailand and bringing attention to ocean destruction. Her latest collection features tree-inspired pieces honoring 2011 as the International Year of Forests.
This document summarizes work on developing a data processor for analyzing atomic force microscopy (AFM) scans of polymer brush samples. It describes an algorithm created to automatically analyze AFM data by determining slope, derivatives, borders, height, width, and other metrics of patterned lines. Testing showed the program could accurately process data faster than manual methods. The work involved operating AFM and SEM equipment to collect data, developing data processing skills, and redesigning a lab website. It concluded by acknowledging those who contributed to the project.
This study aimed to 1) develop an LC-MS/MS method to quantify ionophores (MON, SAL, NAR) in poultry litter using a modified QuEChERS sample preparation, 2) quantify the levels of ionophores in poultry litter before and after three pilot-scale composting processes (aeration, turning, combination), and 3) identify transformation products of ionophores formed during composting using high-resolution LC-QToF/MS. The validation results showed good accuracy (71-119% recovery) and precision (19% RSD). Composting reduced ionophore levels by 13-68% depending on the conditions. Three transformation products and one
The document summarizes a study that examined the effects of different concentrations of tropospheric ozone exposure on soybean plants through proteomic analysis. It found that ozone exposure significantly altered the expression and oxidation states of proteins involved in important metabolic pathways like carbon metabolism and nitrogen homeostasis. In particular, proteins related to photosynthesis like RuBisCO showed increased oxidation and expression with higher ozone levels, which could reduce their catalytic activity and photosynthetic capacity.
Effect of nitrogen and phosphorus amendment on the yield of a Chlorella sp. s...Agriculture Journal IJOEAR
Abstract— A strain of microalgae was isolated from phytoplankton samples collected from the sea coast of Amsheet, North Lebanon. Molecular diagnosis based on ribosomal RNA genes showed it to be most closely related to Chlorella sp. (GenBank accession KC188335.1) with over 90 % nucleotide identity. It was then evaluated whether N and P amendments of seawater fertilized with Guillard’s f/2 medium would improve algal growth and production. Addition of nitrogen (30 ppm) and/or phosphorus (2 ppm) to microalgae grown under laboratory conditions in 3L bioreactors resulted in improved biomass yield (mg dry matter/ L) by approximately 48%, and increased protein yield by approximately 56%, from 19.5% to 30.6% of DM content. Total protein yield/L of culture medium was therefore increased by approximately 83%. Total lipid content and carotenoid levels of the microalgal culture were not affected by the N+P amendement, whereas chlorophyll content was almost doubled. When lower levels of N+P supplementations, 10 and 20 ppm N, were tried, the biomass yield was also improved. The experiment was repeated in 20 L bioreactors in a plastic greenhouse, under normal environmental conditions, with an average temperature of 28°C and a maximum temperature of 36°C. At these relatively high temperatures, the growth rate was slowed down, but N supplementations at 10 and 20 ppm resulted in improved dry matter yield by 25 and 45% respectively, and protein content by 17 and 35%, respectively. Knowledge of the optimal culturing conditions of this local Chlorella strain is essential for its efficient production and is expected to serve future environmental and biotechnological purposes.
This document summarizes the results of a study that used metagenomics to elucidate the cytochrome P450 (CYPome) of prokaryotic and eukaryotic communities in a chronically polluted soil. The CYPome comprised 94 CYP proteins across bacteria, archaea, and eukaryotes. Bacteria dominated, with 72 CYP families affiliated with Bacillus, Streptomyces, and Mycobacterium. Streptomyces CYPs were mainly involved in secondary metabolite biosynthesis. Mycobacterial CYPs included those related to virulence. Eukaryotic CYPs spanned animal, plant, and fungal taxa. The study provides insight into CYP family
Quantitative PCR (qPCR), also known as real-time PCR, is a laboratory technique used to quantify the amount of a specific DNA sequence in a sample. Some key points about how qPCR is used to determine the amount of DNA:
- qPCR works by amplifying a target DNA sequence over multiple cycles. It monitors the amplification in real-time using fluorescent dyes or probes.
- The fluorescent signal increases as more DNA is amplified. The point at which the fluorescence crosses a defined threshold is called the cycle threshold (Ct).
- Samples with more DNA copies of the target sequence will reach the threshold earlier in the amplification process (lower Ct value). Samples with fewer copies will reach it later (higher Ct value).
The study investigated the effects of methoxychlor (MXC), an organochlorine pesticide, on liver and kidney function in rats and the potential protective effects of propolis. Rats were exposed to MXC, propolis, or both for 6 or 12 months. MXC exposure significantly increased liver enzymes and oxidative stress markers in the liver and caused histological damage. It also increased kidney dysfunction biomarkers and caused tubular degeneration. Co-administration of propolis with MXC ameliorated many of the toxic effects of MXC on the liver and kidney, decreasing oxidative stress and normalizing biomarker levels. The study suggests that propolis has protective effects against MXC-induced toxicity in
Effects of organic and mineral fertilizers on total antioxidant, polyphenolic...Alexander Decker
This study examined the effects of organic and mineral fertilizers on antioxidant, polyphenol, and carotenoid content in orange-fleshed sweet potato tubers. Organic fertilizer significantly increased all three phytochemicals, with annual applications having the highest levels. Mineral fertilizers also significantly affected levels, with combinations of nitrogen, phosphorus, and potassium influencing different compounds. The combination of minimal mineral doses with annual or biennial organic fertilizer produced the highest antioxidant, polyphenol, and carotenoid concentrations in the tubers.
Science 2013-schuenemann-179-83 leprosy önemliHazal Sav
This study obtained near-complete genome sequences of Mycobacterium leprae from skeletal remains dating from the 11th to 14th centuries in Europe, as well as from recent patient biopsies. Genome comparisons revealed remarkable genomic conservation of M. leprae over the past 1000 years. The ancient genomes suggest a European origin for leprosy in the Americas and the presence of a genotype in medieval Europe now commonly associated with the Middle East. Exceptional DNA and mycolic acid preservation in the ancient skeletal remains provides insights into pathogen evolution and the disappearance of leprosy in Europe.
E-screen assay validation: evaluation of estrogenic activity by MCF7 cell cul...Agriculture Journal IJOEAR
— Natural and synthetic estrogens have been detected in rivers, lakes and estuaries in many parts of the world. Primary sources of these compounds are domestic and industrial effluents, which are not deleted after the water treatment. Estrogen has been the endocrine disruptor most researched to be very active biologically and be the etiologic agent of diverse types of cancer and other conditions such as endometriosis, precocious puberty, feminization, masculinization, sterility. In this context, we use water of 36 natural reservoirs or dams, in a bioassay to characterize their estrogenicity in culture of MCF7 cells and obtained high concentration of estrogen in samples taken in Ibiúna and Equestrian Santo Amaro / SP. However, certain concentration in our samples for most water samples from different regions was very close to the limit of quantification by bioassay and estrogen was in fmol. It has been shown that e-screen assay with MCF7 cells is a sensitive and stable tool for quantitative analysis of estrogenicity of water and can easily be developed and implemented for routine for estrogen quantification also in animal food and man, aqueous and plastics etc. Keywords— endocrine disrupters, estrogen, breast cancer cells, (MCF7) bioassay: E-screen assay
The document summarizes recent research on the role of the phytohormone indole-3-acetic acid (IAA) in microbial and microorganism-plant signaling. It discusses that diverse bacterial species can produce IAA through different biosynthesis pathways, with redundancy widespread. Interactions between IAA-producing bacteria and plants can result in outcomes ranging from pathogenesis to phytostimulation. The review highlights that bacteria use IAA to interact with plants during colonization, including phytostimulation and circumventing plant defenses. Additionally, recent evidence indicates IAA can act as a signaling molecule in bacteria and directly impact bacterial physiology.
Growth Pattern, Molecular Identification and Bio molecules Analysis of FOMITO...journal ijrtem
Abstract : Fomitopsis feei, a brown rot fungus is identified tentatively using morphological characteristics and confirmed phylogenetically by 28S rDNA analysis and sequence was submitted in EMBL Nucleotide Sequence Database. Its growth pattern was studied on eight different solid media and found to be good on Malt extract agar medium. Biomolecules such as proteins and lipid were screened qualitatively and estimated quantitatively. Aminoacid analysis by chromatography and fatty acid analysis by FAME were also done and revealed that tryptophan (20.53%), valine (20.51%) and cis-linoleic acid (43.38%) and palmetic acid (17.88%) were in high percentage.
Key words : Fomitopsis feei, growth, molecular identification and biomolecules
Plant Defence inducing molecules against pathogens - Lessons learned and path...Ashajyothi Mushineni
This document summarizes a seminar presentation on defence inducing molecules against plant pathogens. It discusses various molecules that can induce plant defenses, such as BABA, probenazole, SA analogues, jasmonic acid, chitosan, oligogalacturonides, harpin proteins, and vitamins. Case studies demonstrate the effects of these molecules in enhancing disease resistance in plants like tomato and pepper. While these defense activators provide advantages over fungicides, challenges include potential environmental and health issues. Further research is needed to develop more economical and effective defense inducing strategies.
1) Abscisic acid (ABA) induces stomatal closure in pea plants by raising both the cytosolic pH and nitric oxide (NO) levels in guard cells.
2) The rise in cytosolic pH occurs earlier than the increase in NO, suggesting that pH increases are upstream of NO production during ABA-induced stomatal closure.
3) Modulators that raise cytosolic pH like methylamine enhance stomatal closure and NO production by ABA, while agents that lower pH like butyrate prevent the effects of ABA.
The document discusses epigenetics and epigenomics in plants, describing the main epigenetic modifications including DNA methylation, histone modifications, and non-coding RNAs. It reviews several ongoing research projects applying epigenomics to improve crop traits and stress resistance in important crops like wheat, rice, and maize. Going forward, further research is needed to better understand how epigenetic changes influence plant development and physiology, their degree of heritability, and how epigenomics can be used to enhance crop breeding.
This study analyzed the occurrence and diversity of integrons in bacteria isolated from an urban wastewater treatment plant. A total of 697 isolates of Enterobacteriaceae and Aeromonas were screened for integrons. Three new gene cassettes were identified, including a novel aadA variant and genes involved in cell signaling and unknown functions. Thirteen different gene cassette arrays were detected, with four representing novel integrons. Approximately 80% of isolates were resistant to at least 3 antibiotic classes. The presence of novel integron structures in treated effluent suggests wastewater treatment plants may facilitate the formation and spread of antibiotic resistance genes.
This document describes the construction of a physical and genetic map of the Gluconacetobacter diazotrophicus PAL5 chromosome using pulsed-field gel electrophoresis and DNA hybridization. Key findings include:
1) G. diazotrophicus has a circular chromosome approximately 4,240 kb in size containing 4 rRNA operons.
2) Hybridization results allowed positioning of 42 genetic markers on the chromosome, including 39 single-copy genes and 3 repeated elements.
3) One rRNA operon was found to have an inverted orientation compared to the others.
Methods for detection of aflatoxins in agricultural food cropsfstdesk.com
This review article summarizes various methods used for detecting aflatoxins in agricultural food crops. Aflatoxins are toxic carcinogenic metabolites produced by fungi that contaminate foods. The review examines detection methods including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), enzyme-linked immune-sorbent assay (ELISA), and electrochemical immunosensors. Each method has advantages and limitations in sensitivity, specificity, and ease of use. The review provides details on the metabolism and toxicity of aflatoxins and critically analyzes the strengths and weaknesses of each detection technique.
This document describes a study that characterized two glycolytic enzymes - glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) - found on the outer surface of the probiotic bacterium Lactobacillus plantarum 299v. The enzymes were found to specifically bind plasminogen, fibronectin, and intestinal epithelial cells. GAPDH also weakly bound mucin. Both enzymes bound to intestinal epithelial cells in a pH-dependent manner. Several probiotic and non-probiotic Lactobacillus strains were also analyzed but no correlation was found between the presence of these surface enzymes and probiotic status.
This document describes a study probing the catalytic mechanism of the insulin receptor kinase (IRK) using a tetrafluorotyrosine-containing peptide substrate. Key findings include:
1) pH-rate profiles indicate the neutral phenol, not the phenoxide ion, is required for phosphorylation, supporting a dissociative transition state.
2) The pKa of the tetrafluorotyrosyl hydroxyl is elevated on the enzyme, and the phenoxide anion behaves as a weak competitive inhibitor.
3) Crystal structures show hydrogen bonding of the tetrafluorotyrosyl group to enzyme residues, consistent with its elevated pKa.
The document summarizes research on variation in the ability of plant-colonizing Pseudomonas bacteria to utilize histidine and urocanate. The researchers analyzed 164 Pseudomonas strains isolated from sugar beets and found that most could use both histidine and urocanate, but some could only use one or the other. Analysis of transporter genes in Pseudomonas genomes showed variation but metabolic genes were conserved. Experiments with Pseudomonas fluorescens SBW25 identified two histidine transport systems and one dedicated to urocanate transport. Transfer of these transporter genes complemented defects in strains unable to use histidine or urocanate. Thus, the variation in phenotypes is attributable to genetic differences in
Similar to Cronje, 2012, Planta article - Copy (20)
1. ORIGINAL ARTICLE
Manipulation of L-ascorbic acid biosynthesis pathways in Solanum
lycopersicum: elevated GDP-mannose pyrophosphorylase activity
enhances L-ascorbate levels in red fruit
Christelle Cronje • Gavin M. George •
Alisdair R. Fernie • Jan Bekker •
Jens Kossmann • Rolene Bauer
Received: 6 June 2011 / Accepted: 12 September 2011 / Published online: 7 October 2011
Ó Springer-Verlag 2011
Abstract Ascorbate (AsA) plays a fundamental role in
redox homeostasis in plants and animals, primarily by
scavenging reactive oxygen species. Three genes, repre-
senting diverse steps putatively involved in plant AsA
biosynthesis pathways, were cloned and independently
expressed in Solanum lycopersicum (tomato) under the
control of the CaMV 35S promoter. Yeast-derived GDP-
mannose pyrophosphorylase (GMPase) and arabinono-1,4-
lactone oxidase (ALO), as well as myo-inositol oxygenase 2
(MIOX2) from Arabidopsis thaliana, were targeted.
Increases in GMPase activity were concomitant with
increased AsA levels of up to 70% in leaves, 50% in green
fruit, and 35% in red fruit. Expression of ALO significantly
pulled biosynthetic flux towards AsA in leaves and green
fruit by up to 54 and 25%, respectively. Changes in AsA
content in plants transcribing the MIOX2 gene were
inconsistent in different tissue. On the other hand, MIOX
activity was strongly correlated with cell wall uronic acid
levels, suggesting that MIOX may be a useful tool for the
manipulation of cell wall composition. In conclusion, the
Smirnoff–Wheeler pathway showed great promise as a
target for biotechnological manipulation of ascorbate levels
in tomato.
Keywords Arabinono-1,4-lactone oxidase Á Ascorbate Á
GDP-mannose pyrophosphorylase Á Myo-inositol
oxygenase Á Solanum
Abbreviations
GMPase Guanidine-diphosphate mannose
pyrophosphorylase
ALO Arabinono-1,4-lactone oxidase
MIOX Myo-inositol oxygenase
MI Myo-inositol
L-GulL L-Gulono-1,4-lactone
GlucA D-Glucuronic acid
DHA Dehydroascorbate
L-Asc L-Ascorbate
AsA Total ascorbate
GalUR Galacturonic acid reductase
L-GalLDH L-Galactono-1,4-lactone dehydrogenase
GME GDP-D-mannose 3,5-epimerase
O/N Over night
GDP Guanidine-diphosphate
Introduction
The L-enantiomer of ascorbate (AsA), or vitamin C, acts as
a scavenger of the free radicals generated by photosyn-
thesis, cellular respiration, and abiotic stresses such as
Electronic supplementary material The online version of this
article (doi:10.1007/s00425-011-1525-6) contains supplementary
material, which is available to authorized users.
C. Cronje Á G. M. George Á J. Bekker Á J. Kossmann
Genetics Department, Institute for Plant Biotechnology,
Stellenbosch University, Private Bag X1, Matieland 7602,
South Africa
A. R. Fernie
Max Planck Institute for Molecular Plant Physiology,
Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany
Present Address:
R. Bauer (&)
Department of Biotechnology, Institute for Microbial
Biotechnology and Metagenomics, University of the Western
Cape, Bellville, Private Bag X17, Cape Town 7535, South Africa
e-mail: rbauer@uwc.ac.za
123
Planta (2012) 235:553–564
DOI 10.1007/s00425-011-1525-6
2. ozone and UV radiation (Levine 1986; Conklin et al. 1996;
Smirnoff and Pallanca 1996; Noctor and Foyer 1998;
Smirnoff and Wheeler 2000). AsA has additionally been
shown to play an important role as an enzyme cofactor
while participating in defense, cellular elongation, division,
and fruit ripening (Arrigoni and De Tullio 2000, 2002;
Pastori et al. 2003; Green and Fry 2005). In animals, AsA
is synthesized from D-glucose which is converted into
L-gulono-1,4-lactone (L-GulL) via the intermediates D-glu-
curonic acid (GlucA) and L-gulonate (Fig. 1; Electronic
Supplementary Material Fig. A). L-GulL is oxidized to AsA
by L-gulono-1,4-lactone oxidase (Burns and Mosbach
1956). Humans cannot synthesize AsA due to a mutation in
the L-gulono-1,4-lactone oxidase gene and have to acquire
Vitamin C through the regular ingestion of fruit and veg-
etables (Nishikimi et al. 1994). Vitamin C micronutrient
deficiency is associated with conditions such as scurvy and
low immunity because of its integral role as enzyme
cofactor and in the biosynthesis of carnitine and collagen
(reviewed by Padayatty et al. 2003). The biofortification of
crops has become a major focus in developing countries
where poverty and micronutrient deficiencies are synony-
mous and are largely responsible for poor health and
fatalities (reviewed by Mu¨ller and Krawinkel 2005).
Several AsA biosynthetic pathways have been identified
and characterized in plants (Fig. 1; Electronic Supplemen-
tary Material Fig. A). The ‘‘Smirnoff–Wheeler’’ pathway is
considered the principal route for de novo synthesis of AsA
and involves the conversion of D-mannose into AsA via a
series of L-galactose containing intermediates (Barber 1979;
Wheeler et al. 1998; Conklin et al. 1999, 2000, 2006;
Bartoli et al. 2000; Wolucka and Van Montagu 2003;
Smirnoff et al. 2004; Dowdle et al. 2007; Laing et al. 2007;
Loannidi et al. 2009). Conklin et al. (1997) has demon-
strated that ascorbate deficient Arabidopsis thaliana
mutants display reduced GDP-mannose pyrophosphorylase
(GMPase) activity, an enzyme that catalyzes one of the first
steps of the ‘‘Smirnoff–Wheeler’’ pathway. Expression of
an Acerola GMPase in tobacco resulted in up to 100%
increased levels of AsA (Badejo et al. 2007). Loannidi et al.
(2009) has shown that galactose-1-phosphate phosphatase
expression is up regulated during fruit development, sug-
gesting an important control point in ascorbate biosynthesis.
The final biosynthetic step, oxidation of L-galactono-1,4-
lactone (L-GalL) into AsA is catalyzed by galactono-
1,4-lactone dehydrogenase (L-GalLDH), the only
membrane-bound enzyme of this pathway (Hancock et al.
2003). A yeast homologue, arabinono-1,4-lactone oxidase
(ALO), has been shown to promiscuously convert L-GalL,
as well as L-guluno-1,4-lactone (L-GulL) into AsA (Huh
et al. 1994; Lee et al. 1999; Hancock et al. 2000; Sauer et al.
2004; Hancock 2009). The ‘‘Smirnoff–Wheeler’’ pathway
can, furthermore, be augmented through a ‘‘pectin scav-
enging’’ pathway whose products are directly utilized by L-
GalLDH (Agius et al. 2003). Support for this alternative
route to AsA stem from radiotracer, transcription, and
expression studies of various pathway intermediates (Lo-
ewus 1999; Agius et al. 2003; Cruz-Rus et al. 2010).
Fig. 1 A schematic
representation of proposed
ascorbic acidbiosynthesis
pathways: the Smirnoff–
Wheeler pathway (Wheeler
et al. 1998) the pectin
scavenging pathway (Agius
et al. 2003) and the animal and
animal-like AsA biosynthetic
pathways (Wolucka and Van
Montagu 2003; Lorence et al.
2004). GMPase GDP-mannose
pyrophosphorylase; MIOX myo-
inositol oxygenase; ALO
arabinono-1,4-lactone oxidase;
L-GulLDH L-gulono-1,4-lactone
dehydrogenase; L-GalLDH
L-galactono-1,4-lactone
dehydrogenase
554 Planta (2012) 235:553–564
123
3. Overexpression of a MIOX gene in Arabidopsis was
shown to increase AsA levels two- to threefold (Lorence
et al. 2004). A de novo ‘‘MIOX’’ or ‘‘animal-like’’ path-
way, involving the ring cleavage of myo-inositol (MI) by
myo-inositol oxygenase (MIOX) into D-glucuronic acid,
was proposed (Fig. 1). Labeling experiments revealed that
myo-inositol was incorporated not only into cell wall
components but also into L-gulonate, which in turn may be
converted into L-GulL (Lorence et al. 2004; Zhang et al.
2008). L-GulL was shown to serve a direct precursor of
L-ascorbic acid in plant cells (Wolucka and Van Montagu
2003).
Our current study was initiated with the intent of
increasing total AsA in tomato. Temporal analyses of
changes in the levels of AsA, as well as precursors and
breakdown products, have suggested that ascorbate
metabolism is highly complex in tomato (Carrari and
Fernie 2006; Wang et al. 2009; Garcia et al. 2009). Here
we report on the heterologous expression of GMPase,
ALO, and MIOX under the control of a constitutive
promoter and the corresponding effect on AsA content
within leaf and fruit tissue. GMPase has been shown to
affect ascorbate biosynthesis in several Solanaceous
species (Conklin et al. 1999; Keller et al. 1999; Badejo
et al. 2007), ALO effectively metabolizes a range of
substrates towards ascorbate production in situ (Huh
et al. 1994), and MIOX is thought to play a central role
in an ‘‘animal like’’ AsA biosynthetic pathway (Lorence
et al. 2004).
Materials and methods
Constructs and transformations
GMPase (GenBank accession number NM_001180114)
and ALO (accession number AY693120.1) were PCR
amplified from Saccharomyces cerevisiae strain FY23
(S288C) (Winston et al. 1995) genomic DNA. The coding
region of the Arabidopsis thaliana L. MIOX2 gene
(accession number NM_127538) was amplified from A.
thaliana Columbia-O cDNA [NASC (http://arabidopsis.
info/)]. Appropriate PCR primer pairs are given in Table 1.
Amplification, using pfu polymerase (Fermentas, Glen
Burnie, MD, USA), introduced XhoI and HindIII restriction
sites. PCR products were independently cloned into the
pGEMÒ
-T Easy vector (Promega, Madison, WI, USA) and
sub-cloned into the pART7 vector (Gleave 1992) under
control of the constitutive CaMV 35S promoter. Expres-
sion cartridges were transferred into the pART27 plant
transformation vector as NotI fragments as described by
Basson et al. (2010b). The constructs, i.e. pART27::
GMPase, pART27::ALO, and pART27::MIOX2, were
mobilized into Agrobacterium tumefaciens EHA 105 cells
using the freeze–thaw method (Ho¨fgen and Willmitzer
1988). The Solanum lycopersicum ‘Money maker’ cultivar
was infiltrated as described by Obiadalla-Ali et al. (2004).
Plant material
Stem cuttings representing different transformation events
were transferred onto MS agar (4.4 g/L Murashige and
Skoog, 15 g/L sucrose and 3 g/L, agar, pH 7) and grown in
tissue culture at 22°C under continuous light conditions.
After 2 weeks, plants were transferred to the glass house
and progressively hardened off in soil (Double Grow,
Durbanville, South Africa) at 22°C in a 16/8 h day night
cycle. Seeds were harvested from ripe fruit and germinated
in the glasshouse. At 4 weeks, plantlets were moved to a
greenhouse (summer between the months of November and
March) and grown under controlled irrigation. Every
4 days, plants were supplied with 1 g/L calcium nitrate and
1.5 g/L carbon-free hydroponic nutrient supplement
(Hygrotech Hygroponic Nutrients, Pretoria, South Africa
Reg No. K5709). Leaf samples were collected at 8 weeks
and whole fruit samples were harvested during green and
red stages of maturity at 25 days and 60 ± 5 days,
respectively, post anthesis (Basson et al. 2010a). The
pericarp was not separated from the locular tissue as this
would initiate a wound response thereby affecting ascor-
bate levels (Loannidi et al. 2009). Care was taken to har-
vest all samples at noon on days with non-overcast skies. In
each case, five replicates were sampled for each line.
Samples were immediately frozen, ground in liquid nitro-
gen, and stored at -80°C.
Selection of transformants by polymerase chain
reaction
Plant material was ground in liquid nitrogen and genomic
DNA extracted from 50 mg of tissue according to the
method of McGarvey and Kaper (1991) and in the presence
of 0.5 g/L spermidine. DNA concentration and quality
were determined spectrophotometrically (Basson et al.
2010a, b). GMPase, ALO, and MIOX transgenic lines were
screened using forward primer 10 and reverse primers 7, 8,
and 9, respectively (Table 1). PCR screening reactions
were performed with PromegaGoTaqÒ
PCR (Promega,
Madison, WI, USA). Amplicons were visualized in a 1%
agarose gel containing ethidium bromide (4 lL/100 mL).
WT plants and plasmids containing the cloned genes
of interest were used as negative and positive controls,
respectively.
Planta (2012) 235:553–564 555
123
4. RNA extraction and RT-PCR
RNA was extracted from frozen leaf and fruit material
according to Burgos et al. (1995) with the following
modifications. The extraction buffer contained 5% b-merca-
ptoethanol and RNA was precipitated with one-quarter
volume 8 M lithium chloride. The dried RNA pellet was
reconstituted in *50 lL MQ water and RNA concen-
trations were normalized to 100 ng/lL. All samples were
DNase-treated using DNase I (Fermentas). First strand
cDNA synthesis was performed with 5 lg RNA using
RevertAid H Minus Reverse Transcriptase (Fermentas).
Gene-specific forward primers (Table 1, numbers 1, 3,
and 5) and reverse primers (Table 1, numbers 2, 4, and
6) were used to amplify expressed sequences. TIP41, a
reference gene for quantitative transcriptomics in Sola-
num lycopersicum (Expo´sito-Rodrı´guez et al. 2008) was
used as a constitutively expressed gene control (Table 1,
number 11 and 12). All RT-PCR reaction conditions
were as follows: 3 min at 94°C; (25 cycles of: 30 s at
94°C, 30 s at 55°C, 30 s at 72°C); 7 min at 72°C.
Protein extraction
Total protein from GMPase expressing plants was extrac-
ted from frozen tissue in 10 volumes of ice cold buffer
containing 50 mM Tris–HCl (pH 7.5), 0.05% Triton
X-100, 5 mM EDTA, 5 mM DTT, 0.01% b-mercap-
toethanol and 1 mM PMSF. Samples were centrifuged
(18,000 g, 5 min, 4°C), one volume 50% PEG 6000
was added to the supernatant, and protein precipitated
for 30 min on ice. Samples were centrifuged (14,000 g,
10 min, 4°C) and pellets resuspended in 100 mM Tris pH
7.5. MIOX protein was extracted in 10 volumes of ice-
cold buffer containing 100 mM Tris–HCl pH 7.6, 2 mM
L-cysteine, 1 mM ammonium ferrous sulfate hexahydrate,
1 mM EDTA, and 1% PVPP. Protein was precipitated as
described above and resuspended in 100 mM KPO4 buffer
(pH 7.2) containing 2 mM L-cysteine and 1 mM ammo-
nium ferrous sulfate hexahydrate.
Activity assays
GMPase activity was measured using a stopped radio-
assay as described by Keller et al. (1999) with the fol-
lowing modifications. The assay was started by adding
400 lL crude protein extract to 400 lL assay mix
(100 mM Tris pH 7.5, 4 mM MgCl2, 5 mM sodium
pyrophosphate, 0.1 mM cold GDP-mannose, and 0.04 Cu
14
C GDP-mannose) and stopped after 1 h with the
addition of 2 mg activated charcoal. Scintillation fluid
(5 mL) was added and 14
C D-mannose-1-P determined
using the Tri-Carb 2100 TR Liquid Scintillation Ana-
lyzer (Packard Instrument Company, Meriden, CT,
USA).
MIOX activity was determined within the linear range
of an endpoint assay (Reddy et al. 1981) modified as
follows: Protein (500 lg per sample) was incubated for
30 min at 30°C in a buffer containing 100 mM KPO4
(pH 7.2), 2 mM L-cysteine, 1 mM ammonium ferrous
sulfate hexahydrate, and 60 mM myo-inositol (Electronic
Supplementary Material Fig. B). The reaction was stop-
ped by boiling for 10 min and denatured protein removed
by centrifugation (18,000 g, 10 min). Glucuronic acid
was measured as described by Van den Hoogen et al.
(1998).
Table 1 Primers used for this study: GDP-mannose pyrophosphorylase (GMPase); arabinono-1,4-lactone oxidase (ALO); myo-inositol
oxygenase (MIOX); cauliflower mosaic virus 35S promoter (CaMV 35S); TIP41-like protein (TIP41) (Expo´sito-Rodrı´guez et al. 2008)
Prime number Name Bp Oligo sequence Accession no.
1 GMPase F 30 50
GGCTCGAGCATATATAATTGAAAAATGAAAGG 30
NM_001180114
2 GMPase R 29 50
GGAAGCTTAGTTCGTTTTCCTAACTCACA 30
3 ALO F 28 50
GGCTCGAGTCAGGTTTTTCACCCCATGT 30
AY693120
4 ALO R 30 50
CCAAGCTTACAAAAAGAGACTAGTCGGACA 30
5 MIOX F 29 50
GGCTCGAGTCAAATTCCGAGCAAGATGAC 30
NM_127538
6 MIOX R 31 50
GGAAGCTTTGACTCGTAGCTTTATCTCACCA 30
7 GMPase R 21 50
AACAATGTTGGCACCTGTAGC 30
8 ALO R 21 50
ATCCCATTGCTTCAAAAGGTT 30
9 MIOX R 20 50
GGGTCGTGCCATTCTTCTTA 30
10 CaMV 35S 21 50
TCCACTGACGTAAGGGATGAC 30
11 TIP41 F 22 50
ATGGAGTTTTTGAGTCTTCTGC 30
SGN-U321250
12 TIP41 R 19 50
GCTGCGTTTCTGGCTTAGG 30
Bp base pairs, F forward primer, R Reverse primer
556 Planta (2012) 235:553–564
123
5. Ascorbic acid measurement
Frozen plant tissue was ground in five volumes of 6% (w/v)
meta-phosphoric acid and total AsA quantified with the aid
of ascorbic acid oxidase (EC 1.10.3.3) and the reductant
tris[2-carboxyethyl]phosphine hydrochloride (TCEP) as
described by Basson et al. (2010a). Content was calculated
against a standard curve of 0–80 lM ascorbic acid. Total
AsA is given as the sum of oxidized AsA (L-ascorbic acid)
and reduced AsA (DHA).
GC–MS for metabolite profiling
Extraction and derivatization of plant tissue was done
according to the method of Roessner et al. (2000) with
modifications. The polar fraction was extracted from
60 mg frozen leaf tissue homogenized in 1,400 lL 100%
methanol and with 60 lL ribitol (0.2 mg/mL water) as
internal standard. Samples were extracted at 70°C for
15 min, vortexed and centrifuged (18,000 g, 10 min). The
supernatant was added to one volume chloroform and two
volumes water, vortexed and centrifuged (5,500 g,
15 min), and the upper phase vacuum dried for derivati-
zation. Dried samples were reconstituted in 40 lL meth-
oxyamine hydrochloride (20 mg/mL in pyridine),
derivatized for 2 h at 37°C, and incubated for a further
30 min (37°C) in the presence of 70 lL MSTFA and 40 lL
internal retention time standard.
Analysis was performed using a 6890-N gas chro-
matograph and 5975 inert mass selective inhibitor mass
spectrometer (Agilent Technologies; Santa Clara, CA,
USA). 1-lL Volumes of were injected with a 7683B
Series splitless injector (Agilent Technologies) and gas
chromatography was performed on a 30-m RtxÒ
-5Sil MS
Integra Guard column with 0.25 mm internal diameter
and 0.25 lm film thickness (Restek, Bellefonte, PA,
USA). Injection- and ion source temperatures were set at
230°C and 200°C, respectively, and the program was set
to 5 min at 70°C, a first ramp of 1°C/min to 76°C, and a
second ramp of 6°C/min to 350°C. Temperature was
equilibrated to 70°C prior to injection of each sample and
mass spectra recorded (2 scans per s in range of
50–600 m/z). Data were analyzed using the Automated
Mass Spectral Deconvolution and Identification System
(AMDIS, http://www.amdis.net/index.html, National
Institute of Standards and Technology, Gaithersburg,
MD, USA) (Stein 1999) and compared with a custom RI-
annotated supervised plant metabolite mass spectral
database (http://gmd.mpimp-golm.mpg.de/) (Schauer et al.
2005) and the NIST/EPA/NIH Mass Spectral Library
(NIST 05) using the NIST Mass Spectral Search Program
Version 2.0d.
Preparation of alcohol insoluble residues (AIR)
and measurement of cell wall uronic acids
Ethanol was added to ground plant tissue (125 ± 10 mg)
and incubated for 20 min at 70°C. Samples were centri-
fuged at 8,500 g for 10 min and supernatants discarded.
Ethanol extraction was repeated four times. Samples were
washed in acetone and vacuum dried. Cell wall uronic acids
were measured using an adaptation of methods previously
described (Blumenkrantz and Asboe-Hansen 1973; Van den
Hoogen et al. 1998). Dried AIR samples (10 mg) were
reconstituted in 200 lL 12 M sulfuric acid and incubated
for 2 h at 4°C. The sulfuric acid was diluted to 2 M and cell
wall polysaccharides hydrolyzed for 2 h at 80°C. Concen-
trated sulfuric acid containing 120 mM sodium tetraborate
was added to 40-lL aliquots of AIR sample (200 lL per
aliquot), incubated at room temperature for 30 min, and
background OD measured at 540 nm. Uronic acids were
measured as described by Van den Hoogen et al. (1998)
against a galacturonic acid standard of 0–8 lg.
Results
Constructs, transformations, and selection
Regenerated plant transformants were screened by PCR for
the presence of pART27::GMPase, pART27::ALO, and
pART27::MIOX2 constructs, respectively. GMPase positive
line G2 was not selected for further analyses due to the
high probability that it exhibited somaclonal variation
(Electronic Supplementary Material Fig. C), while ALO
line A16 was rejected due to an uncharacteristically low
fruit yield. Tomato seeds were collected and at least five
biological replicates established per line.
GMPase activity
Lines positive for the presence of the yeast-derived GMPase
gene were assayed for protein activity using a radiolabel
incorporation assay. In comparison with untransformed
controls, GMPase activity in leaves of transgenic lines
increased between 26 and 31 times (Fig. 2). Similarly, in
green fruit tissue activity increased 13–17 times. Despite the
fact that the baseline activity in different wild-type tissues
was very similar, transgenic leaf material displayed up to
100% more activity than transgenic green fruit.
ALO transcription
Arabinono-1,4-lactone oxidase (ALO) activity could not be
reliably measured because the protein is embedded within
Planta (2012) 235:553–564 557
123
6. the mitochondrial membrane. Membrane fractions con-
tained varying amounts of active protein, complicating
measurements, and standardization of enzymatic assays.
Therefore, transcript levels of ALO were measured semi-
quantitatively and compared with the expression level of
the constitutively expressed TIP41 gene. RT-PCR con-
firmed the unique transcription of the heterologous gene in
transgenic lines (Fig. 3).
MIOX activity
Transgenic lines displayed approximately three- to fourfold
increased MIOX activity in leaves compared with wild-
type controls (Fig. 4). In green fruit, activity in line M8
was not significantly higher than in wild-type plants,
whereas lines M2 and M4 exhibited twofold increases
(P 0.1).
Ascorbate
Total ascorbate, measured as the sum of L-AsA and DHA,
was determined in leaves, green fruit and red fruit to
study the effect of introduced transgenes on ascorbate
biosynthesis or its steady-state levels. Due to the direct
link between ascorbate levels and the wounding response,
fruits were frozen and analyzed whole (Loannidi et al.
2009). During senescence, the locule becomes filled with
water and soluble sugars. In red fruit, DHA concentra-
tions per fresh weight were below the limits of detection,
and ascorbate content was therefore represented by L-AsA
alone. Increase in GMPase activity was concomitant with
increased ascorbate levels in all tissues measured
(Table 2). Ascorbate content in leaves was increased up
to 66% compared with 50 and 35% in green and red fruit,
respectively. Most transgenic ALO lines displayed
increased ascorbate levels (P 0.05) in leaf tissue, typi-
cally between 21 and 54% (Table 3). Levels in green fruit
were increased up to 25% (P 0.1), while red fruit
contained levels invariant from the wild type. In leaf
material, increased MIOX activity was associated with up
to 30% reduction in ascorbate content (Table 4). Con-
versely, transgenic green fruit with increased MIOX
activity displayed up to 35% increased ascorbate levels
(P 0.1).
0
1
2
3
WT G5 G6 G21
GMPaseActivity
(µMoles.g.min)-1-1
Green fruit
Leaf
*
*
*
*
*
*
Fig. 2 GDP-mannose pyrophosphorylase (GMPase) activity mea-
surements in plants expressing GMPase from Saccharomyces cere-
visiae using [14
C]GDP-mannose, cold GDP-mannose and PPi as
substrates. Activity was measured as the amount of radio label
incorporated into the product, mannose-1-phosphate. Values calcu-
lated as average ± standard deviation; n = 3; P 0.05
ALO
ALO
TIP41
TIP41
+C WT A8 A13 A16 A21 A22 A23
+C WT A8 A13 A16 A21 A22 A23
Leaves
Green
fruit
Fig. 3 Agarose gel of RT-PCR products showing transcription of the arabinono-1,4-lactone oxidase (ALO) gene in leaves (top) and green fruit
(bottom) of transformed plants using TIP41 as a constitutively expressed control gene
0
10
20
30
40
50
WT M2 M4 M8
MIOX2Activity
(µMolesGlucA.g.min)-1-1
Green fruit
Leaf
*
*
* *
*
Fig. 4 Myo-inositol oxygenase (MIOX) activity measurements in
leaves of MIOX lines and wild-type controls. Myo-inositol was
provided as substrate and MIOX activity measured relative to the
amount of glucuronic acid produced. Optical density was determined
at 540 nm before and after samples developed a pink color with
addition of a 3-hydroxybiphenylphenol color reagent. Values calcu-
lated as average ± standard deviation; n = 3; P 0.1 (green fruit);
P 0.05 (leaves)
558 Planta (2012) 235:553–564
123
7. Metabolite profiling
In order to determine whether precursor molecules within
the various pathways of AsA biosynthesis were affected,
GC–MS metabolite profiling was performed on leaf tissue.
Comparison of the GC–MS chromatograms with plant
metabolite and NIST mass spectral libraries revealed
numerous metabolites consistently present in all samples
and several significant deviations in the metabolite profiles
of the transgenic plants (Table 5). GMPase transgenic lines
showed an increase in galactono-1,4-lactone and galacto-
nate, and a concomitant decrease in glucuronic acid. Major
increases in citric acid cycle components, fumarate, and
succinate were also observed. Principal component analy-
sis (PCA) (Electronic Supplementary Material Fig. D) of
the GC–MS data (Electronic Supplementary Material
Table 1) revealed increases in threonate (P 0.1). Galac-
tonate, galactose, myo-inositol, and sucrose decreased
significantly in most ALO lines. Decreases in myo-inositol
content were most evident in MIOX lines, by between 72
and 90% (P 0.05), with concomitant increases in
gulonate.
Cell-wall analysis
Cell wall uronic acids were determined in leaf and green
fruit tissue of MIOX lines (Fig. 5). In leaf tissue, all three
transgenic lines displayed small increases in cell wall
uronic acids (P 0.1). In green fruit, levels were increased
by more than 100% in lines M2 and M4.
Discussion
Three different genes, GMPase, MIOX, and ALO, were
targeted for heterologous expression with the aim of
(re)directing carbon flux toward AsA biosynthesis in
plants. These genes were ectopically expressed in tomato in
an attempt to overcome rate-limiting steps in production, or
to increase the contribution of secondary pathways.
Expression of GDP-mannose pyrophosphorylase
A yeast-derived GMPase, catalyzing the conversion of
D-mannose-1-P to GDP-D-mannose (Hashimoto et al.
1997) was expressed in an attempt to accelerate the flux
of carbon through the Smirnoff–Wheeler AsA pathway
(Fig. 1). Transgenic tomato lines exhibited up to 31 and
17-fold increased GMPase activity in leaves and green
fruit, respectively. Total ascorbate levels increased up to
70%, most apparent in photosynthesizing tissues as
reported earlier (Yabuta et al. 2008). Heterologous
expression of a plant GMPase in tobacco leaves has
previously resulted in about 100% increased AsA content
(Badejo et al. 2007). In the current study, an increase in
GMPase activity was accompanied by up to 375% more
galactono-1,4-lactone, a downstream intermediate in the
Smirnoff–Wheeler pathway, and a significant increase in
galactonate, an intermediate in the cell wall scavenging
pathway. DHA (the reduced form of ASA) was signifi-
cantly increased in leaf tissue of all transgenic lines. Both
the rate of AsA synthesis and recycling via DHA, and
monodehydroascorbate reductase are critical in the
maintenance of a high AsA redox state (Conklin and
Barth 2004). Statistical principal component analysis
(PCA) of metabolic profiles in leaves revealed an overall
increase in threonate production in transgenic plants
(Electronic Supplementary Material Fig. D). Pallanca and
Smirnoff (2000) suggested that the rate at which AsA is
recycled and catabolized can be inferred from the levels
of DHA, glutathione or the breakdown products tartrate
and threonate. Significant increases in the citric acid
cycle components, fumarate and succinate, were mea-
sured in leaves. It has been shown that AsA biosynthetic
Table 2 L-Ascorbate (L-asc), dehydroascorbate (DHA) and total ascorbate (AsA) levels measured in leaf, green fruit and red fruit material from
plants with increased GDP-mannose pyrophosphorylase (GMPase) activity
Leaf Green fruit Red fruit
L-asc DHA AsA L-asc DHA AsA L-asc
WT 1.17 ± 0.29 0.43 ± 0.07 1.6 ± 0.36 0.76 ± 0.06 0.11 ± 0.06 0.87 ± 0.1 0.55 ± 0.08
G5 1.71 ± 0.08** 0.53 ± 0.01** 2.29 ± 0.07** 1.14 ± 0.06** 0.13 ± 0.01 1.27 ± 0.02** 0.66 ± 0.03**
G6 1.63 ± 0.35* 0.5 ± 0.12* 2.19 ± 0.46* 1.02 ± 0.03** 0.11 ± 0.004 1.13 ± 0.03* 0.73 ± 0.02**
G21 1.98 ± 0.45** 0.64 ± 0.14* 2.67 ± 0.59** 1.12 ± 0.05** 0.16 ± 0.01 1.28 ± 0.1* 0.74 ± 0.11**
DHA could not be detected in red fruit using the methods described. Values calculated as average ± standard deviation and measured in
lMoles/g FW
n = 3
* P 0.1
** P 0.05
Planta (2012) 235:553–564 559
123
8. rates are affected by the flow of electrons through the
respiratory electron transport chain (Millar et al. 2003;
Alhagdow et al. 2007). Increased flux through the Smirnoff–
Wheeler pathway creates an increased demand for oxi-
dized cytochrome c, which is diverted from ATP
synthase. A resulting demand for citric acid cycle derived
NADH could plausibly lead to increased turnover and
intermediates such as succinate and fumarate. While
GMPase may not exert majority metabolic control over
this pathway, the study suggests that increased substrate
supply from early steps of the L-galactose pathway pos-
itively affects vitamin C production, especially in pho-
tosynthesizing tissue.
Expression of arabinono-1,4-lactone oxidase
D-Arabinono-1,4-lactone oxidase (ALO), the yeast analog
of galactono-1,4-lactone dehydrogenase (L-GalLDH),
converts D-arabinono-1,4-lactone to erythroascorbate,
while promiscuously converting L-galactono-1,4-lactone
and L-guluno-1,4-lactone to AsA (Huh et al. 1994; Lee
et al. 1999; Hancock et al. 2000; Sauer et al. 2004;
Hancock 2009). ALO was expressed in order to assess if
increased turnover of the terminal step in the ascorbate
biosynthetic pathway would increase carbon flux towards
AsA biosynthesis. L-GalLDH is sensitive to irradiance,
ascorbate oxidase activity, cytochrome c activity, and
respiration (Millar et al. 2003; Tamaoki et al. 2003;
Nunes-Nesi et al. 2005; Bartoli et al. 2006, 2009; Bulley
et al. 2009). By contrast, ALO has not shown sensitivity
to light or reductant availability. ALO activity in tomato
extracts could not be reliably quantified due to its pre-
sumed interaction with the inner mitochondrial membrane
as demonstrated for its plant homologue L-GalLDH
(Hancock et al. 2003). Transcription of the ALO transgene
was, however, confirmed (Fig. 4) and has resulted in
significantly higher AsA levels in leaves (up to 54%) and
green fruit (up to 25%). DHA levels in transgenic green
Table 3 L-Ascorbate (L-asc), dehydroascorbate (DHA) and total ascorbate (AsA) levels measured in leaf, green fruit and red fruit material from
plants transcribing the yeast arabinono-1,4-lactone oxidase (ALO) gene
Leaf Green fruit Red fruit
L-asc DHA AsA L-asc DHA AsA L-asc
WT 1.1 ± 0.16 0.13 ± 0.03 1.18 ± 0.11 1 ± 0.07 0.16 ± 0.02 1.12 ± 0.05 0.97 ± 0.04
A8 1.2 ± 0.11 0.17 ± 0.02* 1.43 ± 0.16** 1.15 ± 0.08** 0.2 ± 0.05* 1.41 ± 0.01** 0.88 ± 0.08
A13 1.11 ± 0.03 0.07 ± 0.02 1.2 ± 0.02 1.2 ± 0.09** 0.27 ± 0.08** 1.47 ± 0.01** 1.01 ± 0.11
A21 1.6 ± 0.2** 0.17 ± 0.02* 1.64 ± 0.19** 1.1 ± 0.09* 0.27 ± 0.07** 1.34 ± 0.04** 0.91 ± 0.05
A22 1.7 ± 0.08** 0.15 ± 0.03 1.82 ± 0.07** 1.26 ± 0.1** 0.15 ± 0.03 1.4 ± 0.08** 1.02 ± 0.07
A23 1.52 ± 0.13** 0.14 ± 0.12 1.51 ± 0.07** 1.11 ± 0.15* 0.25 ± 0.03** 1.35 ± 0.19* 0.88 ± 0.11
DHA could not be detected in red fruit using the methods described. Values calculated as average ± standard deviation and measured in
lMoles/g FW
n = 3
* P 0.1
** P 0.05
Table 4 L-Ascorbate (L-asc), dehydroascorbate (DHA) and total ascorbate (AsA) levels measured in leaf, green fruit and red fruit material from
plants containing the myo-inositol oxygenase2 (MIOX2) gene
Leaves Green fruit Red fruit
L-asc DHA AsA L-asc DHA AsA L-asc
WT 1.61 ± 0.09 0.09 ± 0.06 1.65 ± 0.07 0.65 ± 0.02 0.03 ± 0.003 0.69 ± 0.003 0.55 ± 0.08
M2 1.34 ± 0.1** 0.06 ± 0.02 1.4 ± 0.1** 0.82 ± 0.16* 0.07 ± 0.007** 0.89 ± 0.06** 0.69 ± 0.07**
M4 1.2 ± 0.24** n/d 1.14 ± 0.08** 0.9 ± 0.16** 0.04 ± 0.008 0.93 ± 0.08** 0.63 ± 0.15
M8 1.33 ± 0.12** n/d 1.29 ± 0.07** 0.49 ± 0.16 0.05 ± 0.02 0.55 ± 0.09 0.37 ± 0.22
DHA could not be detected in any of the red fruits because the assay is not sensitive enough. DHA could not be detected in red fruit using the
methods described. Values calculated as average ± standard deviation and measured in lMoles/g FW
n/d not detected
n = 3
* P 0.1
** P 0.05
560 Planta (2012) 235:553–564
123
9. fruit also increased, suggesting an increase in AsA turn-
over. AsA feeding experiments have shown that AsA pool
size is directly proportionate to turnover rate (Pallanca
and Smirnoff 2000). Metabolite profiling of leaf tissue
revealed up to 42% reduction in galactose (an
intermediate in the Smirnoff–Wheeler pathway), up to
45% reduction of (galactonate an intermediate in the
pectin degradation pathway) and up to 90% reduction of
myo-inositol. GC–MS did not allow discrimination
between D- and L-galactose. The yeast isoform (ALO)
appears to pull carbon flux towards AsA biosynthesis. To
our knowledge, this is the first report on the successful
expression of ALO in planta.
Expression of myo-inositol oxygenase
Myo-inositol is converted into GlucA by the activity of
MIOX. However, whether GlucA acts as a precursor to
AsA in an ‘‘animal like’’ pathway in plants has not been
established with certainty (Lorence et al. 2004; Zhang
et al. 2008; Endres and Tenhaken 2009). The gene family
for the MIOX enzyme from Arabidopsis was shown to be
represented by four members (Kanter et al. 2005). The
current study investigated expression of the MIOX2 iso-
form in tomato. Transcription of the transgene resulted in
increased MIOX activity in leaf material without a
concomitant increase in AsA content. In contrast, a
Table 5 Metabolite profiling of leaf material from GDP-mannose pyrophosphorylase (GMPase), arabinono-1,4-lactone oxidase (ALO) and
myo-inositol oxygenase (MIOX) lines, together with wild-type controls
Galactonate Galactono-1,4-lactone Glucuronic acid Fumarate Succinate
Wild type 0.044 ± 0.008 0.012 ± 0.004 0.064 ± 0.013 0.065 ± 0.004 0.013 ± 0.002
G5 0.089 ± 0.016** 0.045 ± 0.005** 0.036 ± 0.004* 0.135 ± 0.028** 0.034 ± 0.004**
G6 0.068 ± 0.009** 0.028 ± 0.004** 0.038 ± 0.006* 0.085 ± 0.010** 0.026 ± 0.001**
G21 0.089 ± 0.003** 0.028 ± 0.005** 0.027 ± 0.008* 0.213 ± 0.056** 0.024 ± 0.001**
Galactonate Galactose Myo-inositol Sucrose
Wild type 0.244 ± 0.033 0.036 ± 0.008 11.548 ± 0.895 6.439 ± 1.246
A8 0.135 ± 0.033** 0.022 ± 0.005* 6.636 ± 2.627* 2.450 ± 0.146**
A13 0.142 ± 0.021** 0.021 ± 0.001* n/d 3.863 ± 0.577*
A16 0.173 ± 0.017* 0.027 ± 0.004 5.512 ± 3.300* 3.932 ± 0.705*
A21 0.169 ± 0.012** 0.023 ± 0.002* 4.407 ± 1.707** 4.220 ± 0.205*
A22 0.211 ± 0.026 0.023 ± 0.001* 3.920 ± 2.102** 4.154 ± 0.195*
A23 0.147 ± 0.016** 0.030 ± 0.008 6.325 ± 3.180* 3.193 ± 0.561**
Galactonate Gulonate Myo-inositol
Wild type 0.244 ± 0.033 0.027 ± 0.011 11.548 ± 0.895
M2 0.145 ± 0.020** 0.315 ± 0.024** 1.155 ± 0.611**
M4 0.228 ± 0.036 0.602 ± 0.252** 3.208 ± 0.657**
M8 0.174 ± 0.025* 0.386 ± 0.085** 1.357 ± 0.667**
GC–MS analysis was used to identify compounds affected by increased GMPase, ALO and MIOX expression. Values calculated as average peak
area ± standard deviation
n/d not detected
n = 3
* P 0.1
** P 0.05
0
2.5
5
7.5
10
WT M2 M4 M8
%UronicacidsofAIR
Green fruit
Leaves
*
**
*
*
Fig. 5 Uronic acid measurements in myo-inositol oxygenase
(MIOX) lines representative of cell wall biosynthesis. Measurements
were performed on leaf and green fruit material with wild-type
controls and expressed as a weight percentage of total alcohol
insoluble residues (AIR) extracted from the cell wall. Values
calculated as average ± standard deviation; n = 3; P 0.1 (leaves);
P 0.05 (green fruit)
Planta (2012) 235:553–564 561
123
10. significant decrease in AsA in leaf tissue, inversely pro-
portionate to the level of MIOX activity, was apparent.
Previously, expression of the MIOX4 gene in Arabidopsis
was shown to increase AsA levels two- to threefold
(Lorence et al. 2004; Zhang et al. 2008). In contrast,
MIOX4 overexpressing Arabidopsis lines were recently
shown to be largely invariant from the wild type (Endres
and Tenhaken 2009).
Steady-state myo-inositol levels in lines with increased
MIOX activity were decreased to as low as 10% of levels
in wild type controls, while a tenfold increase in gulonate
was observed (Table 5). Gulonate resides downstream of
myo-inositol and is converted to L-gulono-1,4-lactone, the
terminal substrate in the ‘animal-like’ AsA biosynthesis
pathway (Fig. 1). While increased MIOX activity plays an
ambiguous role in AsA biosynthesis, the enzyme clearly
controls the metabolite level of myo-inositol and deriva-
tives in plants as suggested previously (Endres and
Tenhaken 2009). The authors have reported on increased
incorporation of MIOX-derived sugars into cell wall
polymers, while overexpressors exhibited a lower steady-
state level of myo-inositol due to an enhanced turnover
rate.
D-Glucuronic acid is a major precursor in cell wall
biosynthesis (Kanter et al. 2005). Expressed as a per-
centage of the AIR of the cell wall, uronic acid content
was significantly higher in the leaves of all MIOX lines.
Increased uronic acid levels were also observed in green
fruits with significantly higher MIOX activity, indicative
of a shunt of glucuronic acid into the cell wall (Fig. 5).
Green fruit with measurably higher MIOX activity levels
and uronic acids also showed significant increases in AsA.
Either carbon is being directed towards AsA biosynthesis
through an ‘animal-like’ pathway, or increases in cell wall
components provide more substrate for AsA biosynthesis
via the pectin scavenging pathway. The strong correlation
between MIOX activity and cell wall uronic acid levels
suggests that MIOX may be a useful tool for the
manipulation of cell wall composition. Downregulation of
GDP-D-mannose 3,5-epimerase (GME) isoforms in tomato
was recently shown to result in significant changes in cell
wall composition (Gilbert et al. 2009). Garcia et al.
(2009) showed direct correlations between intermediates
of ascorbate and cell wall biosynthetic pathways. Such
studies strengthen the concept of a cell wall-ascorbate
nexus.
Acknowledgments Technical support from Ilse Balbo and scien-
tific discussions with Prof. Adriano Nunes-Nesi (Max Planck Insti-
tute for Molecular Plant Physiology, Golm, Germany) are much
appreciated. Dr Be´ne´dicte A Lebouteiller (Institute for Plant Bio-
technology; Stellenbosch University; South Africa) is thanked for
her assistance as is funding from the National Research Foundation;
South Africa.
References
Agius F, Gonzalez-Lamothe R, Caballero JL, Munoz-Blanco J,
Botella MA, Valpuesta V (2003) Engineering increased vitamin
C levels in plants by overexpression of a D-galacturonic acid
reductase. Nat Biotechnol 21:177–181
Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D,
Petit J, Beauvoit B, Fernie AR, Rothan C, Baldet P (2007)
Silencing of the mitochondrial ascorbate synthesizing enzyme
L-galactono-1, 4-lactone dehydrogenase affects plant and fruit
development in tomato. Plant Physiol 145:1408–1422
Arrigoni O, De Tullio MC (2000) The role of ascorbic acid in cell
metabolism: between gene-directed functions and unpredictable
chemical reactions. J Plant Physiol 157:481–488
Arrigoni O, De Tullio MC (2002) Ascorbic acid: much more than just
an antioxidant. Biochim Biophys Acta 1569:1–9
Badejo AA, Jeong ST, Goto-Jamamoto N, Esaka M (2007) Cloning
and expression of GDP-D-mannose pyrophosphorylase gene and
ascorbic acid content of acerola (Malphighia glabra L.) fruit at
ripening stages. Plant Physiol Biochem 45:665–672
Barber GA (1979) Observations on the mechanism of the reversible
epimerization of GDP-mannose to GDP-L-galactose by an
enzyme from Chlorella pyrenoidosa. J Biol Chem 254:7600–
7603
Bartoli CG, Pastori GM, Foyer CH (2000) Ascorbate biosynthesis in
mitochondria is linked to the electron transport chain between
complexes III and IV. Plant Physiol 123:335–343
Bartoli CG, Yu J, Go´mez F, Ferna´ndez L, McIntosh L, Foyer CH
(2006) Inter-relationships between light and respiration in the
control of ascorbic acid synthesis and accumulation in Arabid-
opsis thaliana leaves. J Exp Bot 57:1621–1631
Bartoli CG, Tambussi EA, Diego F, Foyer CH (2009) Control of
ascorbic acid synthesis and accumulation and glutathione by the
incident light red/far red ratio in Phaseolus vulgaris leaves.
FEBS Lett 583:118–122
Basson CE, Groenewald JH, Kossmann J, Cronje C, Bauer R (2010a)
Sugar and acid-related quality attributes and enzyme activities in
strawberry fruits: invertase is the main sucrose hydrolyzing
enzyme. Food Chem 121:1156–1162
Basson CE, Groenewald JH, Kossmann J, Cronje´ C, Bauer R (2010b)
Upregulation of pyrophosphate: fructose 6-phosphate 1-phos-
photransferase (PFP) activity in strawberry. Transgenic Res
20:925–931
Blumenkrantz N, Asboe-Hansen G (1973) New method for quanti-
tative determination of uronic acids. Anal Biochem 54:484–489
Bulley SM, Rassam M, Hoser D, Otto W, Schu¨nemann N, Wright M,
MacRae E, Gleave A, Laing W (2009) Gene expression studies
in kiwifruit and gene over-expression in Arabidopsis indicates
that GDP-L-galactose guanyltransferase is a major control point
of vitamin C biosynthesis. J Exp Bot 60:765–778
Burgos RC, Chiang VL, Zhang XH, Campbell ER, Podila GK,
Campbell WH (1995) RNA isolation from plant tissues
recalcitrant to extraction in guanidine. Biotechniques 19:734–
737
Burns JJ, Mosbach EH (1956) Further observations in the biosynthe-
sis of L-ascorbic acid from D-glucose in the rat. J Biol Chem
221:107–111
Carrari F, Fernie AR (2006) Metabolic regulation underlying tomato
fruit development. J Exp Bot 57:1883–1897
Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule
intertwined in the response of plants to ozone, pathogens, and the
onset of senescence. Plant Cell Environ 27:959–970
Conklin PL, Williams EH, Last RL (1996) Environmental stress
sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc
Natl Acad Sci USA 93:9970–9974
562 Planta (2012) 235:553–564
123
11. Conklin PL, Pallanca JE, Last RL, Smirnoff N (1997) L-Ascorbic acid
metabolism in the ascorbate-deficient Arabidopsis mutant vtc1.
Plant Physiol 115:1277–1285
Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last
RL (1999) Genetic evidence for the role of GDP-mannose in
plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci
USA 96:4198–4203
Conklin PL, Saracco SA, Norris SR, Last RL (2000) Identification of
ascorbic acid-deficient Arabidopsis thaliana mutants. Genetics
154:847–856
Conklin PL, Gatzek S, Wheeler GL, Dowdle J et al (2006)
Arabidopsis thaliana VTC4 encodes L-galactose 1-P phospha-
tase, a plant ascorbic acid biosynthetic enzyme. J Biol Chem
281:15662–15670
Cruz-Rus E, Botella MA, Valpuesta V, Gomez-Jimenez MC (2010)
Analysis of genes involved in L-ascorbic acid biosynthesis
during growth and ripening of grape berries. J Plant Physiol
167:739–748
Dowdle J, Ishikawa T, Gatzek S, Rolinski S, Smirnoff N (2007) Two
genes in Arabidopsis thaliana encoding GDP-L-galactose phos-
phorylase are required for ascorbate biosynthesis and seedling
viability. Plant J 52:673–689
Endres S, Tenhaken R (2009) Myoinositol oxygenase controls the
level of myoinositol in Arabidopsis, but does not increase
ascorbic acid. Plant Physiol 149:1042–1049
Expo´sito-Rodrı´guez M, Borges AA, Borges-Pe´rez A, Pe´rez JA (2008)
Selection of internal control genes for quantitative real-time RT-
PCR studies during tomato development process. BMC Plant
Biol 8:131
Garcia V, Stevens R, Gil L, Gibert L, Gest N, Petit J, Faurobert M
et al (2009) An integrative genomics approach for deciphering
the complex interactions between ascorbate metabolism and fruit
growth and composition in tomato. C R Biol 332:1007–1021
Gilbert L, Alhagdow M, Nunes-Nesi A, Quemener B, Guillon F,
Bouchet B, Faurobert M et al (2009) GDP-D-mannose 3,5-
epimerase (GME) plays a key role at the intersection of
ascorbate and non-cellulosic cell-wall biosynthesis in tomato.
Plant J 60:499–508
Gleave AP (1992) A versatile binary vector system with a T-DNA
organizational structure conducive to efficient integration of
cloned DNA into the plant genome. Plant Mol Biol 20:1203–
1207
Green MA, Fry SC (2005) Apoplastic degradation of ascorbate: novel
enzymes and metabolites permeating the plant cell wall. Plant
Biosyst 139:2–7
Hancock RD (2009) Recent patents on vitamin C: opportunities for
crop improvement and single-step biological manufacture.
Recent Pat Food Nutr Agric 1:39–49
Hancock RD, Galpin JR, Viola R (2000) Biosynthesis of L-ascorbic
acid (vitamin C) by Saccharomyces cerevisiae. FEMS Microbiol
Lett 186:245–250
Hancock RD, McRae D, Haupt S, Viola R (2003) Synthesis of
L-ascorbic acid in the phloem. BMC Plant Bio 3:7
Hashimoto H, Sakakibara A, Yamasaki M, Yoda K (1997) Saccha-
romyces cerevisiae VIG9 encodes GDP-mannose pyrophosphor-
ylase, which is essential for protein glycosylation. J Biol Chem
272:16308–16314
Ho¨fgen R, Willmitzer L (1988) Storage of competent cells for
Agrobacterium transformation. Nucleic Acids Res 16:9877
Huh W, Kim S, Yang K, Seok Y, Hah YC, Kang S (1994)
Characterisation of D-arabinono-1, 4-lactone oxidase from
Candida albicans ATCC 10231. Eur Biochem 225:1073–1079
Kanter U, Usadel B, Guerineau F, Li Y, Pauly M, Tenhanken R
(2005) The inositol oxygenase gene family of Arabidopsis is
involved in the biosynthesis of nucleotide sugar precursor for
cell-wall matrix polysaccharides. Planta 221:243–254
Keller R, Springer F, Renz A, Kossmann J (1999) Antisense
inhibition of the GDP-mannose pyrophosphorylase reduces the
ascorbate content in transgenic plants leading to developmental
changes during senescence. Plant J 19:131–141
Laing WA, Wright MA, Cooney J, Bulley SM (2007) The missing
step of the L-galactose pathway of ascorbate biosynthesis in
plants, an L-galactose guanylyltransferase, increases leaf ascor-
bate content. Proc Natl Acad Sci USA 104:9534–9539
Lee B, Huh W, Kim S, Lee J, Kang S (1999) Bacterial production of
D-erythroascorbic acid and L-ascorbic acid through functional
expression of Saccharomyces cerevisiae D-arabinono-1, 4-lac-
tone oxidase in Escherichia coli. Appl Environ Microbiol
65:4685–4687
Levine M (1986) New concepts in the biology and biochemistry of
ascorbic acid. N Eng J Med 314:892–902
Loannidi E, Kalamaki MS, Engineer C, Pateraki I, Alexandrou D,
Mellidou I, Giovannonni J, Kanellis AK (2009) Expression
profiling of ascorbic acid-related genes during tomato fruit
development and ripening and in response to stress conditions.
J Exp Bot 60:663–678
Loewus FA (1999) Biosynthesis and metabolism of ascorbic acid in
plants and analogs of ascorbic acid in fungi. Phytochemistry
52:193–210
Lorence A, Chevone BI, Mendes P, Nessler CL (2004) Myo-inositol
oxygenase offers a possible entry point into plant ascorbate
biosynthesis. Plant Physiol 134:1200–1205
McGarvey P, Kaper JM (1991) A simple and rapid method for
screening transgenic plants using the PCR. Biotechniques
11:428–432
Millar AH, Mittova V, Kiddle G, Haezlewood JL, Bartoli CG,
Theodoulou FL, Foyer CH (2003) Control of ascorbate synthesis
by respiration and its implications for stress response. Plant
Physiol 133:443–447
Mu¨ller O, Krawinkel M (2005) Malnutrition and health in developing
countries. Can Med Assoc J 173:279–286
Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K (1994)
Cloning and chromosomal mapping of the human nonfunctional
gene for L-gulono-gamma-lactone oxidase, the enzyme for
L-ascorbic acid biosynthesis missing in man. J Biol Chem
269:13685–13688
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping
active oxygen under control. Annu Rev Plant Physiol Plant Mol
Biol 49:249–279
Nunes-Nesi A, Lytovchenko A, Smith AM, Loueiro ME, Ratcliffe
RG, Sweetlove LJ, Fernie AR (2005) Enhanced photosynthetic
performance and growth as a consequence of decreasing
mitochondrial malate dehydrogenase activity in transgenic
tomato plants. Plant Physiol 137:611–622
Obiadalla-Ali H, Fernie AR, Lytovchenko A, Kossmann J, Lloyd JR
(2004) Inhibition of chloroplastic fructose 1,6-bisphosphatase in
tomato fruits leads to decreased fruit size, but only small changes
in carbohydrate metabolism. Planta 219:533–540
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee J, Chen S, Corpe
C, Dutta A, Dutta SK, Levine M (2003) Vitamin C as an
antioxidant: evaluation of its role in disease prevention. J Am
Coll Nutr 22:18–35
Pallanca JE, Smirnoff N (2000) The control of ascorbic acid synthesis
and turnover in pea seedlings. J Exp Bot 51:669–674
Pastori GM, Kiddle G, Antoniw J, Bernard S, Veljovic-Jovanovic S,
Verrier PJ, Noctor G, Foyer CH (2003) Leaf vitamin C contents
modulate plant defense transcripts and regulate genes that control
development through hormone signaling. Plant Cell 15:939–951
Reddy CC, Swan JS, Hamilton GA (1981) Myo-inositol oxygenase
from hog kidney. Purification and characterization of the
oxygenase and of an enzyme complex containing the oxygenase
and D-glucuronate reductase. J Biol Chem 256:8510–8518
Planta (2012) 235:553–564 563
123
12. Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L (2000)
Simultaneous analysis of metabolites in potato tuber by gas
chromatography–mass spectrometry. Plant J 23:131–142
Sauer M, Branduardi P, Valli M, Porro D (2004) Production of
L-ascorbic acid by metabolically engineered Saccharaomyces
cerevisiae and Zygosaccharomyces bailii. Appl Environ Micro-
biol 70:6086–6091
Schauer N, Steinhauser D, Strelkov S, Schomburg D, Allison G et al
(2005) GC-MS libraries for the rapid identification of metabo-
lites in complex biological samples. FEBS Lett 579:1332–1337
Smirnoff N, Pallanca JE (1996) Ascorbate metabolism in relation to
oxidative stress. Biochem Soc Trans 24:472–478
Smirnoff N, Wheeler GL (2000) Ascorbic acid in plants: biosynthesis
and function. Crit Rev Biochem Mol Biol 35:291–314
Smirnoff N, Running JA, Gaztek S (2004) Ascorbate biosynthesis: a
diversity of pathways. In: Asard H, May JM, Smirnoff N (eds)
Vitamin C: functions and biochemistry in animals and plants.
BIOS Scientific Publishers, London, pp 7–29
Stein SE (1999) An integrated method for spectrum extraction and
compound identification from gas chromatography/mass spec-
trometry data. J Am Soc Mass Spectrom 10:770–781
Tamaoki M, Mukai F, Asai N, Nakajima N, Kubo A, Aono M, Saji H
(2003) Light-controlled expression of a gene encoding L-galac-
tono-1, 4-lactone dehydrogenase which affects ascorbate pool
size in Arabidopsis thaliana. Plant Sci 164:1111–1117
Van den Hoogen BM, Van Weeren RP, Lopes-Cardozo M, Van Golde
LMG, Barneveld A, Van de Lest CHA (1998) A microtiter plate
assay for the determination of uronic acids. Anal Biochem
257:107–111
Wang H, Schauer N, Usadel B, Frasse P, Zouine M, Hernould M,
Latche A, Pech JC, Fernie AR, Bouzyen M (2009) Regulatory
features underlying pollination-dependent and independent
tomato fruit set revealed by transcript and primary metabolite
profiling. Plant Cell 21:1428–1452
Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway
of vitamin C in higher plants. Nature 393:365–369
Winston F, Dollard C, Ricupero-Hovasse SL (1995) Construction of a
set of convenient Saccharomyces cerevisiae strains that are
isogenic to S288C. Yeast 11:53
Wolucka BA, Van Montagu M (2003) GDP-mannose 30
, 50
-epimerase
forms GDP-L-gulose, a putative intermediate for the de novo
biosynthesis of vitamin C in plants. J Biol Chem 278:47483–
47490
Yabuta Y, Maruta T, Nakamura A, Mieda T, Yoshimura K, Ishikawa T,
Shigeoka S (2008) Conversion of the L-galactono-1, 4-lactone to
L-ascorbate is regulated by the photosynthetic electron transport
chain in Arabidopsis. Biosci Biotechnol Biochem 72:2598–2607
Zhang W, Gruszewski HA, Chevone BI, Nessler CL (2008) An
Arabidopsis purple acid phosphatase with phytase activity
increases foliar ascorbate. Plant Physiol 146:431–440
564 Planta (2012) 235:553–564
123