James Lloyd - Academia.edu (original) (raw)
Papers by James Lloyd
bioRxiv, 2020
The availability of nutrients impacts cell size and growth rate in many organisms. Research in E.... more The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ΔmalP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was i...
South African Journal of Botany, 2009
Biotechnology Journal, 2008
Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product becaus... more Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.
Frontiers in Plant Science
Journal Of Experimental Botany
This article comments on: Chia T, Chirico M, King R et al. 2019. A carbohydrate-binding protein, ... more This article comments on: Chia T, Chirico M, King R et al. 2019. A carbohydrate-binding protein, B-granule content 1 influences starch granule-size distribution in a dose dependent manner in polyploid wheat. Journal of Experimental Botany 70, 105–115.
Scientific Reports
The role of starch degradation in non-vascular plants is poorly understood. To expand our knowled... more The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.
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](Frontiers in plant science, 2018
To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where ort... more To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of and () were repressed using RNAi constructs. Although repression of either or inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in and in the C3 position in lines repressed in expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid...
Planta, 2003
For quantification of a-glucan, water dikinase (GWD) activity in crude extracts of plant tissues ... more For quantification of a-glucan, water dikinase (GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and 33 P-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP applied was labeled at the b position. In wild-type extracts from leaves of Arabidopsis thaliana (L.) Heynh. the maximum rate of starch phosphorylation was approximately 27 pmol min-1 (mg protein)-1. Leaf extracts from the GWDdeficient sex1 mutants of Arabidopsis showed no significant incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude extracts. Keywords Arabidopsis AE Dikinase reaction AE a-Glucan, water dikinase protein AE sex1 mutant AE Solanum AE Starch phosphorylation Abbreviation GWD: a-glucan, water dikinase
Planta, Jan 17, 2018
Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plan... more Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement....
Methods in Molecular Biology, 2015
Defining metabolite abundance and resulting fractional isotope enrichments, within and between ce... more Defining metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane filters) or visualization of metabolites/metabolic states (e.g., fluorescence resonance energy transfer (FRET) or redox-sensitive fluorescent markers (roGFP)) have significantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defined. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.
South African Journal of Botany, 2009
Planta, 1999
In order to examine whether alterations in the supply of precursor molecules into the starch bios... more In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway aected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.
Planta, 2010
Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (St... more Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the Wrst detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking
Current Opinion in Plant Biology, 1998
Current Opinion in Biotechnology, 1999
Critical Reviews in Plant Sciences, 2000
Starch is one of the most important products synthesized by plants that is used in industrial pro... more Starch is one of the most important products synthesized by plants that is used in industrial processes. If it were possible to increase production or modify starches in vivo, using combinations or either genetically altered or mutant plants, it may make them cheaper for use by industry, or open up new markets for the modified starches. The conversion of sucrose to starch in storage organs, therefore, is, discussed. In particular the roles of the different enzymes directly involved in synthesizing the starch molecules on altering starch structure are reviewed, as well as the different models for the production of the fine structure of amylopectin. In addition, the process of starch phosphorylation, which is also important in determining the physical properties of starches, is reviewed. It is hoped that detailed knowledge of these processes will lead to the rational design of tailored starches. Starch degradation is also an important process, for example, in the cold-sweetening of potato tubers, but outside of cereal endosperm little is known about the processes involved. The enzymes thought to be involved and the evidence for this are discussed.
Biochemical Journal, 1999
A chimaeric antisense construct was used to reduce the activities of the two major starch-synthas... more A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90 % inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the
PLANT PHYSIOLOGY, 2004
A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was i... more A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 48C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anionexchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.
South African Journal of Botany
In the next couple of decades, nanotechnology-enabled healthcare applications will significantly ... more In the next couple of decades, nanotechnology-enabled healthcare applications will significantly influence the diagnosis, prevention and treatment of human diseases. Since pharmaceutical products (PPs) have been detected in various environmental compartments, and low-level chronic exposure to PPs has induced adverse and sometimes unexpected effects on non-target organisms, the question of potential environmental risks from increased usage of nanomedical products arises. The risks and benefits to patients from nanomedicines are the focus of exhaustive evaluation by regulatory agencies; by contrast, risks to the environment from nanomedicines are only briefly considered by regulators and are rarely discussed by nanoscientists. To start to fill this gap, 66 experts from nanomedicine R&D, representatives of research funding agencies and of institutions involved in safeguarding public health and the environment were interviewed using a semi-structured questionnaire regarding possible hazards and risks from nanomedicine and on the adequacy of current the environmental risk assessment (ERA) framework for medicines. The interview recordings were transcribed verbatim and analysed via qualitative content analysis. Experts interviewed commented that hazards were possible but risks were unlikely from nanomedicines due to expected minimal exposure. They qualified their statements by comparing risks from nanomedicine with risks from nanomaterials in other industries, conventional pollutants and larger global issues like climate change. Regarding adequacy of the current risk framework for assessment of environmental risks from nanomedicines, perceptions of experts were more varied; some argued that complete overhaul of the ERA framework was required including changes in toxicity endpoints, whereas others suggested that the framework was adequate, though some adjustments were needed.
FEMS microbiology letters, Feb 24, 2017
E. coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phospho... more E. coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen.
bioRxiv, 2020
The availability of nutrients impacts cell size and growth rate in many organisms. Research in E.... more The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ΔmalP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was i...
South African Journal of Botany, 2009
Biotechnology Journal, 2008
Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product becaus... more Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.
Frontiers in Plant Science
Journal Of Experimental Botany
This article comments on: Chia T, Chirico M, King R et al. 2019. A carbohydrate-binding protein, ... more This article comments on: Chia T, Chirico M, King R et al. 2019. A carbohydrate-binding protein, B-granule content 1 influences starch granule-size distribution in a dose dependent manner in polyploid wheat. Journal of Experimental Botany 70, 105–115.
Scientific Reports
The role of starch degradation in non-vascular plants is poorly understood. To expand our knowled... more The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.
Frontiers in plant science, 2018
To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where ort... more To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of and () were repressed using RNAi constructs. Although repression of either or inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in and in the C3 position in lines repressed in expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid...
Planta, 2003
For quantification of a-glucan, water dikinase (GWD) activity in crude extracts of plant tissues ... more For quantification of a-glucan, water dikinase (GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and 33 P-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP applied was labeled at the b position. In wild-type extracts from leaves of Arabidopsis thaliana (L.) Heynh. the maximum rate of starch phosphorylation was approximately 27 pmol min-1 (mg protein)-1. Leaf extracts from the GWDdeficient sex1 mutants of Arabidopsis showed no significant incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude extracts. Keywords Arabidopsis AE Dikinase reaction AE a-Glucan, water dikinase protein AE sex1 mutant AE Solanum AE Starch phosphorylation Abbreviation GWD: a-glucan, water dikinase
Planta, Jan 17, 2018
Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plan... more Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement....
Methods in Molecular Biology, 2015
Defining metabolite abundance and resulting fractional isotope enrichments, within and between ce... more Defining metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane filters) or visualization of metabolites/metabolic states (e.g., fluorescence resonance energy transfer (FRET) or redox-sensitive fluorescent markers (roGFP)) have significantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defined. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.
South African Journal of Botany, 2009
Planta, 1999
In order to examine whether alterations in the supply of precursor molecules into the starch bios... more In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway aected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.
Planta, 2010
Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (St... more Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the Wrst detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking
Current Opinion in Plant Biology, 1998
Current Opinion in Biotechnology, 1999
Critical Reviews in Plant Sciences, 2000
Starch is one of the most important products synthesized by plants that is used in industrial pro... more Starch is one of the most important products synthesized by plants that is used in industrial processes. If it were possible to increase production or modify starches in vivo, using combinations or either genetically altered or mutant plants, it may make them cheaper for use by industry, or open up new markets for the modified starches. The conversion of sucrose to starch in storage organs, therefore, is, discussed. In particular the roles of the different enzymes directly involved in synthesizing the starch molecules on altering starch structure are reviewed, as well as the different models for the production of the fine structure of amylopectin. In addition, the process of starch phosphorylation, which is also important in determining the physical properties of starches, is reviewed. It is hoped that detailed knowledge of these processes will lead to the rational design of tailored starches. Starch degradation is also an important process, for example, in the cold-sweetening of potato tubers, but outside of cereal endosperm little is known about the processes involved. The enzymes thought to be involved and the evidence for this are discussed.
Biochemical Journal, 1999
A chimaeric antisense construct was used to reduce the activities of the two major starch-synthas... more A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90 % inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the
PLANT PHYSIOLOGY, 2004
A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was i... more A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 48C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anionexchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.
South African Journal of Botany
In the next couple of decades, nanotechnology-enabled healthcare applications will significantly ... more In the next couple of decades, nanotechnology-enabled healthcare applications will significantly influence the diagnosis, prevention and treatment of human diseases. Since pharmaceutical products (PPs) have been detected in various environmental compartments, and low-level chronic exposure to PPs has induced adverse and sometimes unexpected effects on non-target organisms, the question of potential environmental risks from increased usage of nanomedical products arises. The risks and benefits to patients from nanomedicines are the focus of exhaustive evaluation by regulatory agencies; by contrast, risks to the environment from nanomedicines are only briefly considered by regulators and are rarely discussed by nanoscientists. To start to fill this gap, 66 experts from nanomedicine R&D, representatives of research funding agencies and of institutions involved in safeguarding public health and the environment were interviewed using a semi-structured questionnaire regarding possible hazards and risks from nanomedicine and on the adequacy of current the environmental risk assessment (ERA) framework for medicines. The interview recordings were transcribed verbatim and analysed via qualitative content analysis. Experts interviewed commented that hazards were possible but risks were unlikely from nanomedicines due to expected minimal exposure. They qualified their statements by comparing risks from nanomedicine with risks from nanomaterials in other industries, conventional pollutants and larger global issues like climate change. Regarding adequacy of the current risk framework for assessment of environmental risks from nanomedicines, perceptions of experts were more varied; some argued that complete overhaul of the ERA framework was required including changes in toxicity endpoints, whereas others suggested that the framework was adequate, though some adjustments were needed.
FEMS microbiology letters, Feb 24, 2017
E. coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phospho... more E. coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen.