Expression of a Small Ubiquitin-Like Modifier Protease Increases Drought Tolerance in Wheat (Triticum aestivum L.)

Post-translation modification of proteins plays a critical role in cellular signaling processes. In recent years, the SUMO (Small Ubiquitin-Like Modifier) class of molecules has emerged as an influential mechanism for target protein management. SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responses. In the present study, the expression of an Arabidopsis thaliana cysteine protease (OVERLY TOLERANT TO SALT-1, OTS1) in wheat (Triticum aestivum L.) has led to improved plant growth under water stress conditions. Transformed wheat (pUBI-OTS1) displayed enhanced growth and delayed senescence under water deficit when compared with untransformed Gamtoos-R genotype or plants carrying an empty vector. Transformed pUBI-OTS1 plants also maintained a high relative moisture content (RMC), had a higher photosynthesis rate, and also had a higher total chlorophyll content when compared to untransformed plants or plants carrying an empty vector. SUMOylation of total protein also increased in untransformed plants but not in the AtOTS1 transformed plants. Our results suggest that SUMO-proteases may influence an array of mechanisms in wheat to the advantage of the crop to be more tolerant to water stress caused by drought. This is the first report to elucidate SUMOylation effects in the hexaploid crop wheat (T. aestivum L.)

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

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties

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

Stress-induced genome alterations in plants

Please read the abstract in the 00front part of this documentThesis (PhD (Botany))--University of Pretoria, 2006.Plant Scienceunrestricte

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase

CITATION: Van Der Vyver, C., et al. 2013. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase. In Vitro Cellular and Developmental Biology - Plant, 49(2):198–206, doi:10.1007/s11627-013-9493-0.The original publication is available at http://www.springer.com/life+sciences/plant+sciences/journal/11627Selection 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.Publisher's versio

Expression of a small ubiquitin-like modifier Protease increases drought tolerance in wheat (Triticum aestivum L.)

CITATION: Le Roux, M. L. et al. 2019. Expression of a small ubiquitin-like modifier Protease increases drought tolerance in wheat (Triticum aestivum L.). Frontiers in Plant Science, 10:266, doi:10.3389/fpls.2019.00266.The original publication is available at https://www.frontiersin.orgPost-translation modification of proteins plays a critical role in cellular signaling processes. In recent years, the SUMO (Small Ubiquitin-Like Modifier) class of molecules has emerged as an influential mechanism for target protein management. SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responses. In the present study, the expression of an Arabidopsis thaliana cysteine protease (OVERLY TOLERANT TO SALT-1, OTS1) in wheat (Triticum aestivum L.) has led to improved plant growth under water stress conditions. Transformed wheat (pUBI-OTS1) displayed enhanced growth and delayed senescence under water deficit when compared with untransformed Gamtoos-R genotype or plants carrying an empty vector. Transformed pUBI-OTS1 plants also maintained a high relative moisture content (RMC), had a higher photosynthesis rate, and also had a higher total chlorophyll content when compared to untransformed plants or plants carrying an empty vector. SUMOylation of total protein also increased in untransformed plants but not in the AtOTS1 transformed plants. Our results suggest that SUMO-proteases may influence an array of mechanisms in wheat to the advantage of the crop to be more tolerant to water stress caused by drought. This is the first report to elucidate SUMOylation effects in the hexaploid crop wheat (T. aestivum L.).https://www.frontiersin.org/articles/10.3389/fpls.2019.00266/fullPublisher's versio

Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin

Bacillus thuringiensis (Bt) crops require a high dosage of Bt toxin to delay development of insect resistance, in particular, when the refuge strategy is applied. This strategy is threatened by plant developmental and environmental factors that might reduce Bt toxin concentration and Bt efficacy in Bt crops. Growth of Bt (Cry1Ac) cotton under prolonged, moderate water deficit as a single stress factor was evaluated. Bt cotton plants were analysed for physiological performance, Bt toxin concentration and Bt efficacy. For performance analysis, leaf and total plant dry weight and leaf area were measured. Bt toxin concentration was determined by an immuno-assay. Effects of Bt toxin on growth and mortality of African cotton bollworm, Helicoverpa armigera, larvae were measured in different plant organs. Leaves from young plants exposed for 30 days to moderate water deficit had both higher Bt toxin concentrations and were more effective against larvae than leaves, flowers or bolls from mature flowering plants exposed to 60 days of moderate water deficit. Although growth of Bt cotton plants under moderate water-deficit conditions decreased Bt concentrations in leaves, flowers and bolls, this had no effect on efficacy against first-instar cotton bollworm larvae. No significant evidence was found that moderate water deficit, as a single stress factor, decreases Bt efficacy in Bt cotton

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of active cysteine proteases following inhibitor ingestion. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were first conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet bioassay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of young weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg- MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the diagnostic cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with these broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatininfiltrated banana stem disks. These promising results, which illustrate the natural susceptibility of C. sordidus to plant cystatins, are discussed in the light of current genomic data on coleopteran cysteine cathepsins and recent hypotheses suggesting a key role for digestive cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.Research Foundation of South Africa; Natural Science and Engineering Research Council of Canada; Rockefeller Foundation