Protection of recombinant glutathione reductase by Oryzacystatin-I in transgenic tobacco

Protein degradation poses a significant challenge for the efficient production of recombinant proteins in plants, affecting the stability and yield of the recombinant protein. In this study the E. coli-derived enzyme glutathione reductase (GR) was transiently expressed in transgenic tobacco plants constitutively expressing the cysteine protease inhibitor OC-I and non-transgenic plants. A protein resembling the GR was detected in infiltrated leaves. Transiently expressing GR in transgenic N tabacum plants resulted in almost two fold significant increases in GR activity. Transgenic tobacco plants expressing the rice cysteine protease inhibitor OC-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher GR activity and also higher GR amounts in transgenic plants. The study has demonstrated that OC-I is an effective companion protease inhibitor candidate with the potential to protect other high value proteins such as GR, from cysteine protease degradation.Dissertation (MSc)--University of Pretoria, 2012.Plant Scienceunrestricte

Improvement of rhizobium-soybean symbiosis and nitrogen fixation under drought

The symbiotic interaction between soybean plants and rhizobacteria can be severely affected by drought, which results in a reduction in symbiotic nitrogen fixation and ultimately decreased yields. The aim of our research was to determine whether symbiotically efficient rhizobia that can better tolerate soil water deficits can improve nodule performance in plants subjected to drought. Firstly, rhizobial strains were selected that exhibited differences in tolerance to salt (NaCl) or water deficit (PEG 6000). Sinorhizobium fredii strain SMH12 showed the highest tolerance to these treatments while Bradyrhizobium diazoefficiens strain WB74‐1 showed the lowest tolerance. Greenhouse‐grown Prima 2000 soybean plants were then inoculated with either SMH12 or WB74‐1 and subjected to two water deficit regimes. Different nodule and plant growth traits were determined, including nodule number, dry weight, water potential, and the accumulation of malondialdehyde and ureide. Plants inoculated with SMH12 had significantly more nodules under water deficit conditions than those inoculated WB74‐1, despite having lower root and shoot biomass. SMH12‐inoculated plants had higher nodule water potentials and lower malondialdehyde contents than the WB74‐1‐inoculated plants. These results demonstrate that inoculation of soybean plants with the more water deficit‐tolerant S. fredii strain improved nodule characteristics when plants were grown under water deficit conditions. However, these improved nodule characteristics do not always directly translate into better plant growth.University of Pretoria; NRF/DST; Oil and Protein Development Trust (OPOT); Sasol Agriculture trust in South Africa; NRF Young researcher development grant (112144) (BJV).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2048-3694https://wileyonlinelibrary.com/journal/fes3pm2021Forestry and Agricultural Biotechnology Institute (FABI)Plant Production and Soil Scienc

Use of transgenic Oryzacystatin-I expressing plants enhances recombinant protein production

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter–GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.This work was supported by a grant from the National Research Foundation in South Africa.http://link.springer.com/journal/12010hb201