Browsing by Author "Mdodana, Ntombizanele Thobela"

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    Analysis of aspects of starch metabolism in Physcomitrella patens
    (Stellenbosch : Stellenbosch University, 2021-12) Mdodana, Ntombizanele Thobela; Lloyd, James Richard; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is an important polysaccharide produced by plants and is widely used in industry mainly as a food thickener, but also in other important processes, such as the development of textiles and p aper manufacture. This polyglucan consists of two glucose polymers, amylose and amylopectin. As the major storage carbohydrate, starch is synthesised during the day before being catabolised at night to sustain plant growth and metabolism. Starch metabolism is well studied in vascular plants such as Arabidopsis thaliana and Solanum tuberosum, however, information about these processes are less well understood in non-vascular plants. The bryophyte Physcomitrella patens is an excellent plant model system for many reasons; for example its fully sequenced genome and the ability to produce knockout mutants using homologous recombination. Attempts to gain a better understanding of the function and regulation of some of the key metabolic enzymes involved in starch metabolism in non-vascular plants have recently emerged (Stander, 2015; Jacobs, 2018; Mdodana et., al 2019). This project focused on two aspects of starch metabolism to determine whether some of the pathways and mechanisms involved during these processes are conserved between Physcomitrella patens and vascular plants. The first part of this dissertation examines the roles of glucan water dikinase enzymes (GWD) in P. patens. In angiosperms these polypeptides are involved in starch degradation through catalysing starch phosphorylation. Five isoforms, PpGWDa-e, were identified in and phylogenetic analysis demonstrated the two (PpGWDa and PpGWDb) were most similar to Arabidopsis GWD1 or GWD2, while another two (PpGWDd and PpGWDe) were most similar to GWD3/PWD. The final isoform (PpGWDc) was likely to be inactive as it lacks the essential catalytic histidine. Both PpGWDa and PpGWDb targeted to chloroplasts. Using homologous recombination, knockout mutant lines were successfully generated for PpGWDa and PpGWDb each isoform both as individually and together in double mutants. Inserts in either gene resulted in reduced amounts of starch phosphate compared to the control, with Ppgwda mutant lines interestingly containing less glucose 6-phosphate in starch than Ppgwdb lines. Double mutant (Ppgwda/Ppgwdb; DM) lines contained even less glucose 6-phosphate in starch than Ppgwda single mutants. When plants were grown over a diurnal cycle it was shown that, like vascular plants, starch accumulated in the light period and was degraded at night. Both Ppgwd1a and DM lines accumulated significantly higher amounts of starch compared to Ppgwd1b and the control lines at almost all time points. Soluble sugars on the other hand were significantly reduced in Ppgwd1a and the DM lines compared with Ppgwd1b and the control lines. The Ppgwd1a and DM mutant lines also demonstrated a n observable morphological phenotypic alteration characterized by lack of gametophore development which could be reversed by growing the plant on media supplemented with glucose. In the second experimental chapter, two isoamylase (ISA) like starch debranching enzymes, ISA1 and ISA2, were examined. Mutations in these genes in vascular plants can result in the production of the water-soluble polysaccharide (WSP), phytoglycogen at the expense of starch. Both single and double mutants showed loss of a debranching enzyme activity band in activity gels indicating that ISA1 and ISA2 exist as a heterocomplex in Physcomitrella patens. Analysis of purified recombinant protein demonstrated, surprisingly that both PpISA1 and PpISA2 peptides were catalytically active. Analysis of single and double mutant plants demonstrated that all contained increased amounts of WSP.
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    The effects of the synthetic strigolactone GR24 on Arabidopsis thaliana callus culture
    (Stellenbosch : Stellenbosch University, 2012-12) Mdodana, Ntombizanele Thobela; Hills, Paul N.; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Plant growth promoting substances (PGPS) are emerging as useful tools in the investigation of important plant growth traits. Two PGPS, smoke-water derived from burning plant material and a synthetic strigolactone analogue, GR24, have been reported to regulate a wide variety of developmental and growth processes in plants. These PGPS are beginning to receive considerable attention in the area of improving plant biomass yield and production. Variation in growth between plants is a major impediment towards the complete understanding of the intrinsic processes that control biomass production. Callus cultures of the model plant Arabidopsis thaliana could overcome some of these hindrances. However, the suitability of these callus cultures as a model system for plant biomass production must be established first. This study aimed at using A. thaliana callus cultures as a platform to study the plant growth promoting activities of smoke-water and GR24. The first part of this study was conducted to develop an optimal protocol for inducing A. thaliana callus formation. Wild-type A. thaliana Col-O, as well as strigolactone deficient and insensitive mutants (max1-1, max2-1, max2-2, max3-9 and max4-1) were cultured for callus induction. Hypocotyl and leaf explants were cultured onto MS media supplemented with different hormone concentrations of 2,4-D and kinetin (2:2 mg/L 2,4-D:kinetin and 0.5:0.05 mg/L 2,4-D:kinetin). Both media proved suitable for callus induction of all genotypes, with max1-1 showing the highest efficiency (83.33% and 92.22%) of callus induction. Calli were then used as a platform for future investigations into the effects of smoke-water and GR24. Secondly, this study examined the effects of smoke-water and GR24 on wild-type A. thaliana Col-O callus. Basic physiological studies were conducted to determine if these two compounds would positively affect callus growth, as was shown in previous studies using whole plants. Calli cultivated on MS media containing the two different hormone concentrations were transferred onto the same fresh MS medium, supplemented with either smoke-water or GR24. Growth promotion by smoke-water and GR24 in calli was characterized by a significantly increased mass (biomass). Calli were additionally transferred onto MS medium containing either auxin only or kinetin only and supplemented with GR24 or smoke-water. In the auxin only system, increased mass was recorded for both GR24 and smoke-water treatments, while these two compounds seemed to reduce growth in the kinetin only system. The positive growth stimulatory effect observed for the auxin only system could be attributed to the synergistic relationship between auxin and strigolactones, whilst the reduced mass in the latter system could be due to the antagonistic interaction between strigolactones and cytokinins. Finally, this study has discovered a dual role of strigolactones in biomass accumulation and adventitious root formation for Arabidopsis thaliana callus. On an auxin- and cytokinin-free MS medium supplemented with GR24, calli of Arabidopsis thaliana strigolactone deficient mutants (max1-1 and max4-1) and the wild-type Col- O, but not the strigolactone response mutant (max2-2), showed enhanced biomass accumulation. In addition to this, the max4-1 mutant and wild-type Col-O demonstrated enhanced adventitious rooting, which was not apparent in max2-2. Together these data suggested that the biomass accumulation and the adventitious rooting activities of GR24 in Arabidopsis thaliana calli are controlled in a MAX2- dependent manner. The interaction between strigolactone, auxin and cytokinin signalling pathways in regulating these responses appears to be complex. Gene expression profiling showed regulation of stress-related genes such as B-box transcription factors, CALCINEURIN B-LIKE and RAP4.2 Genes encoding hormones associated with stress (ABA, ethylene) and defence mechanisms (JA) were upregulated. Expression of stress related genes indicated clues on some kind of stress mediation that might be involved during the regulation of the rhizogenic response. Conversely, smoke-water treatment could not enhance the biomass of the calli and nor could it induce adventitious rooting in the absence of auxin and cytokinin. This observation strongly emphasized the distinct roles of these two compounds, as well as the importance of the interaction and ratio of auxin and cytokinin in callus growth. This study has demonstrated a novel role of strigolactones in plant growth and development, i.e. enhancement of biomass production in callus cultures. Secondly the enhanced adventitious rooting ability is in agreement with recently published literature on the role of strigolactones in regulating root architecture. In vitro callus production is advantageous to plant sciences. It creates an opportunity for increasing plant material for cultivation and offers the use of cell cultures that accurately mimic specific growth responses. It could greatly contribute to the study of intricate regulatory and signalling pathways responsible for growth and development in plants. Because the regulation of plant biomass production is very complex and the molecular mechanisms underlying the process remain elusive, it is of paramount importance that further work be done in order to gain more in-depth insights and understanding of this aspect and subsequently improve efficiency and returns when applying biotechnology tools on commercially important crop plants.