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PhD student

Department

Plant Sciences
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Primary Supervisor
Juan Vorster

Co Supervisor
Chris Cullis

Agriculture is an important aspect of the South African economy and biotechnology will play a crucial part in the development of the strategic knowledge based economy for South Africa. Soybean is one of the most important sources for vegetable protein for food and animal feed world-wide and is also an important South African legume crop. In soybean the symbiotic association with rhizobia allows the fixation of atmospheric nitrogen by the bacteria inside specialized organs called root nodules. Nodulation of the soybean root system occurs after infection of the root-hair cells by Bradyrhizobium japonicum. The plant uses the products from this bacterial nitrogen assimilation to synthesise essential macromolecules, such as proteins. The symbiosis drives plant development and ultimately determines the plant’s yield. Nitrogen fixation therefore offers an important advantage in soybean when compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of high-protein seed, soybean has about twice the amount of seed protein than wheat or maize. The nitrogen fixation symbiosis further reduces the cost of production drastically by minimizing the need for nitrogen supplementation with fertilizers. However, the life of a nodule in an annual crop species, such as soybean, is remarkably short (11-13 weeks), nitrogen fixation declines rapidly as the nodules age and has almost ceased by the time pod-filling starts. Such early losses in nitrogen fixation capacity lead to nitrogen limitation within the plant, which has major impact on seed production, crop quality and yield. It is known that plant cysteine proteases as well as their inhibitors (cystatins) play an important role during this plant development processes and in the proposed project the cysteine protease-cystatin system, particularly during soybean nodule development, will be studied. Although there are published data available for the expression of cysteine proteases during nodule senescence, there is very limited information currently available about the expression of cysteine proteases during early nodule development (Oh et al., 2004), but no information about expression of any cystatins in soybean nodules. With the release of the draft assembly of the soybean genome it is now possible to identify all the different cysteine proteases, as well as the cystatins present in the soybean genome. A search carried out of the genome revealed that there are up to 52 putative cysteine protease and 19 putative cystatin sequences in the soybean genome. However, it is not known whether all of these protease and inhibitor sequences are expressed and functional in the soybean proteome and where and when they are expressed. Our interest in studying the cysteine protease-cystatin system in greater detail is based on our current EU-IRSES “LEGIM” project together with Leeds and Ghent University which is aimed to extend our knowledge and concepts of nodule development and sustainability. Through characterization of the individual components of the protease–protease inhibitor system we might be able, as a possible outcome, either silence a particular cysteine protease or recombinantly express a specific natural or engineered cysteine protease inhibitor(s) in soybean nodules which might possibly delay either natural or stress-induced nodule senescence. In previous research we have already established a protease activity profile during nodule development and we found that protease activity including cysteine protease activity increases dramatically during early developmental stages as well as during senescence. Interestingly, detectable cysteine protease activity, which was completely inhibited by the commonly used cysteine protease inhibitor E64, could not be blocked by a natural purified plant cystatins from either rice or papaya, which raises the question if this interaction with a natural inhibitor is prevented due to the structure of the inhibitor.  In proposed project, we would like to identify those cysteine proteases and also their inhibitors (cystatins) that are specifically expressed during the different stages of nodule development and in particular during senescence. The development of new ecologically safe technologies for agricultural production of high value crops depend on a better understanding of these metabolic pathways and enzyme functioning within these pathways.The research will contribute to ultimately provide a detailed understanding of the cysteine protease-cystatin system regulating the senescence process in legumes. Expression profiling of proteases have been done, but no work on cystatins thus will be of novel interest. Through characterization of the individual components of the protease–protease inhibitor system we might be able, as a possible outcome, either silence a particular cysteine protease or recombinantly express a specific natural or engineered cysteine protease inhibitor(s) in soybean nodules which might possibly delay either natural or stress-induced nodule senescence. The created knowledge will also reveal ways in which this system can be manipulated for beneficial use, e.g. potentially enhancing the yield of a major economic crops in future applications.


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Publication
Pillay P, Kunert KJ, van Wyk S, Makgopa ME, Cullis CA, Vorster BJ. (2016) Agroinfiltration contributes to VP1 recombinant protein degradation. Bioengineered 7(6):459-477. 10.1080/21655979.2016.1208868
Van Wyk SG, Kunert KJ, Cullis CA, Pillay P, Makgopa ME, Schluter U , Vorster BJ. (2016) The future of cystatin engineering. Plant Science 246:119-127. 10.1016/j.plantsci.2016.02.016 PDF
van Wyk SG, Du Plessis M, Cullis CA, Kunert KJ, Vorster BJ. (2014) Cysteine protease and cystatin expression and activity during soybean nodule development and senescence. BMC Plant Biology 14(294) 10.1186/s12870-014-0294-3
Pillay P, Schlüter U, van Wyk S, Kunert KJ, Vorster BJ. (2014) Proteolysis of recombinant proteins in bioengineered plant cells. Bioengineered 5(1):15-20. 10.4161/bioe.25158
Vorster BJ, Schlüter U, du Plessis M, van Wyk S, Makgopa ME, Ncube I, Quain MD, Kunert K, Foyer CH. (2013) The Cysteine Protease – Cysteine Protease Inhibitor System Explored in Soybean Nodule Development. Agronomy 3(3):550-570. 10.3390/agronomy3030550
Du Plessis J, Vanheel H, Janssen CEI, Roos L, Slavik T, Stivaktas PI, Nieuwoudt M, van Wyk SG, Vieira W, Pretorius E, Beukes M, Farré R, Tack J, Laleman W, Fevery J, Nevens F, Roskams T, Van der Merwe SW. (2013) Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function. Journal of Hepatology 58(6):1125-1132. 10.1016/j.jhep.2013.01.038