Browsing by Author "Loubser, Johannes"

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    Analysis of the molecular and physiological effects following treatment with BC204 in Arabidopsis thaliana and Solanum lycopersicum
    (Stellenbosch : Stellenbosch University, 2020-12) Loubser, Johannes; Hills, Paul N.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology
    ENGLISH ABSTRACT: Plant biostimulants have been earmarked as one of the pivotal role players in the next much-needed agricultural revolution. Plant biostimulants are mostly from natural sources and they do not directly provide the plant with any nutrients. To date, many different biostimulants have been produced and tested on several different plant species. Although several reports indicate that they elicit an increase in overall plant growth, induce resistance to both abiotic and biotic stresses, increase crop yield and improve fruit/vegetable quality, the molecular data to back up these claims has generally been missing. One such plant biostimulant, BC204, is a citrus-based plant extract used on a variety of crop species in South Africa, China and Australia. There are internal reports from tests conducted by the producers of BC204 which show that it elicits physiological responses such as an increase in crop yield and fruit quality. One postgraduate research study reported that Croplife, a product closely related to BC204, has the potential to improve water utilisation efficiency in table grape cultivars (Van Zyl, 2007). However, no molecular data is available to explain the specific mechanisms associated with the increase in plant growth and tolerance to environmental stresses. Environmental stress is predicted to worsen due to climate change, but also due to irrigation practices on arable land areas, which can result in soil salinity. Although some progress has been made towards understanding plant mechanisms towards salt tolerance in efforts to combat the negative effects of salinity, these mechanisms are still a long way from being fully understood. BC204, like other plant biostimulants, could be a short-term alternative whilst salt tolerance and other abiotic stress mechanisms in plants are further unravelled. Such biostimulants can also be used to study salt tolerance, as the first part of this study provides preliminary evidence that BC204 significantly alleviates salt stress in Arabidopsis thaliana. BC204 treatment increased chlorophyll content, fresh and dry weights, whilst reducing proline, anthocyanin and malondialdehyde content in the presence of 10ds·m-1 EC salt stress. Stomatal conductivity was also reduced by BC204 in source leaves. In addition, BC204 had a significant effect on the expression of salinity-related genes, stimulating the expression of salinity-related genes RD29A and SOS1 independently of NaCl-stress, whilst suppressing the expression of SOT1 and P5CS1. In the second part of study, an RNA-seq approach was adopted to elucidate the effects of BC204 at the molecular level in the model plant species, Arabidopsis thaliana. BC204, applied via a soil drench at a low concentration of 0.01% (v/v), stimulated above-ground biomass production whilst eliciting a large change in gene expression levels across several biochemical pathways in Arabidopsis thaliana. Of the entire transcriptomic profile examined, a total of 8.212% of genes were significantly differentially expressed between the treated and control groups, of which 5.136% were upregulated and 3.076% downregulated. Most notably, genes involved photosynthesis, several aspects of cell wall metabolism, carbohydrate metabolism, signalling, stress and secondary metabolism were upregulated, which could explain the increase in plant growth. Genes related to transcription and RNA regulation were both strongly up- and downregulated, which suggests that BC204 plays a role in inducing and suppressing several pathways. In the third part of this study, the same RNA-seq approach was adopted to elucidate the effect of BC204 in Solanum lycopersicum, an important model crop species, at the molecular level under unstressed conditions. BC204, applied via foliar spray at a concentration of 0.05% (v/v), stimulated tomato root and shoot biomass production, root and shoot length and stem width compared to the untreated control plants. Of the 33308 transcripts analysed, a total of 18.059% genes were significantly differentially expressed between the control and treated groups, of which 8.776% were upregulated and 9.283% downregulated. Most notably, genes involved in signalling, stress and protein metabolism were upregulated, which could explain the increased growth that was observed. In both plant species, BC204 seemed to induce pathways involved in several environmental stresses. Together, the results of this study provide evidence that BC204 elicits a major change in a variety of metabolic processes which forms part of a complex network activating a broad priming response. These priming responses seem to start with enhanced photosynthesis, allowing additional energy to be channelled towards complex metabolic changes through RNA regulation and signalling. Very few metabolic plant processes seem to be unaffected by BC204 treatment.
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    The application of a commercially available citrus-based extract mitigates moderate NaCl-stress in arabidopsis thaliana plants
    (MDPI, 2020) Loubser, Johannes; Hills, Paul
    Aims: The aim of this study was to assess the effect of BC204 as a plant biostimulant on Arabidopsis thaliana plants under normal and NaCl-stressed conditions. Methods: For this study, ex vitro and in vitro growth experiments were conducted to assess the effect of both NaCl and BC204 on basic physiological parameters such as biomass, chlorophyll, proline, malondialdehyde, stomatal conductivity, Fv/Fm and the expression of four NaCl-responsive genes. Results: This study provides preliminary evidence that BC204 mitigates salt stress in Arabidopsis thaliana. BC204 treatment increased chlorophyll content, fresh and dry weights, whilst reducing proline, anthocyanin and malondialdehyde content in the presence of 10 dS·m−1 electroconductivity (EC) salt stress. Stomatal conductivity was also reduced by BC204 and NaCl in source leaves. In addition, BC204 had a significant effect on the expression of salinity-related genes, stimulating the expression of salinity-related genes RD29A and SOS1 independently of NaCl-stress. Conclusions: BC204 stimulated plant growth under normal growth conditions by increasing above-ground shoot tissue and root and shoot growth in vitro. BC204 also increased chlorophyll content while reducing stomatal conductivity. BC204 furthermore mitigated moderate to severe salt stress (10–20 dS·m−1) in A. thaliana. Under salt stress conditions, BC204 reduced the levels of proline, anthocyanin and malondialdehyde. The exact mechanism by which this occurs is unknown, but the results in this study suggest that BC204 may act as a priming agent, stimulating the expression of genes such as SOS1 and RD29A.
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    Tools for strigolactone research : Towards a strigolactone-responsive promoter and a strigolactone-activity inhibitor
    (Stellenbosch : Stellenbosch University, 2016-03) Loubser, Johannes; Hills, Paul N.; Kossmann, Jens; Stellenbosch University. Faculty of Agrisciences. Dept of Genetics. Institute for Plant Biotechnology (IPB).
    ENGLISH ABSTRACT: Strigolactones are a novel group of phytohormones reported to control branching in plants. Strigolactones also plays a pivotal role in the establishment of symbiotic relationships between plants and symbiotic fungi. Furthermore, its presence in the soil is responsible for the germination of the seeds of devastating plant parasitic plants known as broomrapes and witchweeds. They have also been implicated in playing roles in root development and architecture, secondary growth, adventitious root formation and leaf senescence. These phytohormones are derived from the carotenoid synthetic pathway, with β-carotene as the precursor. Several genes and the proteins (enzymes) they code for have been identified by reverse and forward genetics. A few components of the perception and signalling mechanism have been identified, but most of the pathway remains unknown. This highlights the need for more innovative scientific tools in order to fully elucidate the role of strigolactones in higher plants. A specific inhibitor of strigolactone signalling and strigolactone-responsive reporter are two tools that could aid in the further characterization of this poorly defined pathway. In this study, the effects of furanone-derivatives, with the main focus being on trimethylbutenolide (TMB), on the growth of Arabidopsis thaliana were investigated. The aim of this study was to determine if TMB is a competitive inhibitor of strigolactone signalling, because currently there are no known inhibitors of strigolactone signalling available. Having such an inhibitor would enable researchers to study the effects of strigolactones in non-model plant species where no mutants are available. Such an inhibitor would also aid in the further elucidation of the strigolactone signalling pathway. For the second part of this study, an attempt was made to create a strigolactone-response reporter construct in A. thaliana that is activated only in the presence of exogenously applied strigolactone. Having such a reporter-construct in A. thaliana would be valuable, as strigolactones are difficult to detect and quantify in these plants due to them being bioactive at picomolar concentrations in the plant. Such a reporter-construct would also aid in the further elucidation of the strigolactone pathway, the discovery of more functions and any interactions with other phytohormones and biochemical processes in the plant. During this study, it was found that the presence of TMB induced an increase in lateral root formation in wild-type A. thaliana seedlings, suggesting that it may act as a competitive inhibitor of strigolactone signalling, at least in terms of lateral rooting. It was also found that TMB affects the expression of a small group of strigolactone-responsive genes in an opposite way than GR24, a racemic mixture of strigolactone analogues. This effect on gene expression was observed after prolonged treatment of A. thaliana seedlings with TMB. For the second part of the project, we were unable to create a strigolactone-specific reporter construct, although the results suggest that the 990 bp region immediately upstream of AtBRC1 does respond more strongly to the presence of GR24 than the full, native promoter. It was also found that the 1480 bp and 990 bp regions immediately upstream of AtBRC1 is already severely deregulated version of the native AtBRC1 promoter. From these results, it was concluded that DNA binding motif/s for SMAX-like repressor proteins probably lie upstream of the 1480 bp promoter region of AtBRC1, while possible strigolactone-responsive motif/s lie downstream of the 990bp promoter region of AtBRC1.