Browsing by Author "Kossmann, Jens"
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- ItemA comparative study of selected physical and biochemical traits of wild-type and transgenic sorghum to reveal differences relevant to grain quality(Frontiers Media, 2017) Ndimba, Roya J.; Kruger, Johanita; Mehlo, Luke; Barnabas, Alban; Kossmann, Jens; Ndimba, Bongani K.Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression.
- ItemDiverse exopolysaccharide producing bacteria isolated from milled sugarcane : implications for cane spoilage and sucrose yield(Public Library of Science, 2015) Hector, Stanton; Willard, Kyle; Bauer, Rolene; Mulako, Inonge; Slabbert, Etienne; Kossmann, Jens; George, Gavin M.Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.
- ItemImproving crops for a changing world(Frontiers Media S.A, 2021-09-06) Lloyd, James Richard; Kossmann, JensPlant biotechnology has been fundamental to the development of human civilisation. The domestication of plants helped increase food production, allowing the sustenance of populations in large settlements and they provide most calories in the human diet alongside being used as fodder for farm animals. They are also good sources of both therapeutic drugs and industrial feed stocks, while more recently they have been used to produce pharmaceutical proteins and biofuels. There are neverthelessmany areas where plants can be improved through geneticmanipulation and there are pressing reasons why this needs to be accomplished.
- ItemIn vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase(Springer, 2013-02-05) Van Der Vyver, Christell; Conradie, Tobie; Kossmann, Jens; Lloyd, JamesSelection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.
- ItemModification of cassava root starch phosphorylation enhances starch functional properties(Frontiers Media, 2018) Wang, Wuyan; Hostettler, Carmen E.; Damberger, Fred F.; Kossmann, Jens; Lloyd, James R.; Zeeman, Samuel C.Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.
- ItemThe molecular analysis of the effects of lumichrome as a plant growth promoting substance(Stellenbosch : University Stellenbosch, 2009-12) Gouws, Liezel Michelle; Kossmann, Jens; Kossmann, J. M.ENGLISH ABSTRACT: Through powerful signal molecules, rhizobacteria affect fundamental processes in plants. In recent years, a number of novel rhizobial molecules have been identified that positively affect plant growth and development. Previous studies have shown that Sinorhizobium meliloti, which form symbiotic relationships with leguminous plants, increases CO2 availability by enhancing root respiration in alfalfa. The active compound was identified as lumichrome, a previously unrecognized rhizosphere signal molecule that has been shown to promote plant growth in various studies. Lumichrome is a common breakdown product of riboflavin and produced by both chemical and biological factors. Various studies on lumichrome have proven its growth promoting effect in the interaction with plants. The mechanism through which lumichrome increases plant growth remains to be clarified. This study provides new insight into the molecular effects of the plant growth promoter lumichrome on the root metabolism of plants. The main aim of the work presented in this thesis was to investigate the molecular mechanism of the plant growth promoting substance lumichrome in the roots of the model plants Lotus japonicus and Solanum lycopersicon (tomato). To asses the impact of lumichrome on the root metabolism of Lotus japonicus and tomato and identify key genes involved in the growth stimulation, a comprehensive profile of differentially expressed genes, proteins and metabolites was compiled. As the effects of lumichrome as a plant growth promoter have not previously been tested on Lotus japonicus and tomato, basic growth studies were completed to determine if lumichrome indeed elicits plant growth at nanomolar concentrations, as was proven in numerous previous studies. Both Lotus japonicus and tomato showed significant increases in root biomass when treated with 5 nM of lumichrome. The treatment with lumichrome caused complex changes in gene expression. Generally, transcript profiling showed that the categories that were predominantly affected by lumichrome in both Lotus and tomato, were genes associated with RNA regulation of transcription and signaling, protein synthesis/degradation/modification and stress and defence. Proteomic studies revealed that the majority of the differentially expressed proteins were down-regulated. Lumichrome seems to largely influence proteins involved in protein folding and down-regulate proteins involved in glycolysis. Proteomics studies revealed that GS1 (Lotus) and GAPDH (Lotus and tomato) were present in lower abundance in lumichrome treated roots, therefore targeted analysis utilizing northern blots, western blots and the measurement of enzyme activities were completed to determine and verify their specific role in the lumichrome mediated growth promotion. The results indicated that GAPDH and GS1 seem to be under post-translational modification. The influence of lumichrome on the metabolome of Lotus roots was immense, however minute in tomato roots. The knowledge gained in the parallel analyses of both Lotus japonicus and tomato aided us in finding key genes involved in the growth stimulation. Overall, one of the most significant observations was that for the first time to our knowledge, six genes related to defence and pathogen responses were identified that are concurrently expressed in both Lotus and tomato. Through identifying a small number of genes involved in mediating the growth stimulation, these can be used for their functional analysis in the future, using reverse genetics to provide more insight into the molecular mechanisms that are triggered by lumichrome as a plant growth promoter.
- ItemMutations in glucan, water dikinase affect starch degradation and gametophore development in the moss physcomitrella patens(Nature Research, 2019) Mdodana, Ntombizanele T.; Jewell, Jonathan F.; Phiri, Ethel E.; Smith, Marthinus L.; Oberlander, Kenneth; Mahmoodi, Saire; Kossmann, Jens; Lloyd, James R.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.
- ItemRepression of sex4 and like sex four2 orthologs in potato increases tuber starch bound phosphate with concomitant alterations in starch properties physical(Frontiers Media, 2018-07-23) Samodien, Ebrahim; Jewell, Jonathan F.; Loedolff, Bianke; Oberlander, Kenneth; George, Gavin M.; Zeeman, Samuel C.; Damberger, Fred F.; Van Der Vyver, Christell; Kossmann, Jens; Lloyd, James R.To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 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 StSEX4 and in the C3 position in lines repressed in StLSF2 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 viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.
- ItemStarch trek : the search for yield(Frontiers Media, 2019) Lloyd, James R.; Kossmann, JensStarch is a plant storage polyglucan that accumulates in plastids. It is composed of two polymers, amylose and amylopectin, with different structures and plays several roles in helping to determine plant yield. In leaves, it acts as a buffer for night time carbon starvation. Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. Its degradation is also a source of sugars that can help alleviate abiotic stress. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed. Increasing starch in these organs can increase this as well as increasing yield. Enzymes involved in starch metabolism are well known, and there has been much research analyzing their functions in starch synthesis and degradation, as well as genetic and posttranslational regulatory mechanisms affecting them. In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield.