Oryzacystatin I expression in transformed tobacco produces a conditional growth phenotype and enhances chilling tolerance

Abstract

SummaryA recent strategy for pest control in plants has involved transformation with genes encoding cysteine proteinase inhibitors (cystatins). Little is known, however, about the effects of constitutive cystatin expression on whole plant physiology. The present study using oryzacystatin I (OC-I) expression in transformed tobacco was designed to resolve this issue and also to test the effects on abiotic stress tolerance. All transformed plants expressing OC-I showed a conditional phenotype. A marked effect on stem elongation was observed in plants grown under low light intensities. After 7 weeks of growth at low light, the plants expressing OC-I were smaller with fewer expanded leaves and a slightly lower total biomass than empty vector controls or wild type plants. Maximal rates of photosynthesis ( A max ) were also decreased, the inhibitory effect being greatest in the plants with highest OC-I expression. After 12 weeks of growth at low light, however, the plants expressing OC-I performed better in terms of shoot biomass production, which was nearly double that of the empty vector or wild type controls. All plants showed similar responses to drought, however photosynthesis was better protected against chilling injury in plants constitutively expressing OC-I. Photosynthetic CO 2 assimilation was decreased in all plants following exposure to 5 ° C, but the inhibition was significantly less in the OC-I expressing plants than in controls. The transformed tobacco plants expressing OC-I therefore show a phenotypeenvironment interaction with important implications for biotechnological applications.

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

“…Several transgenic plant lines expressing exogenous cystatins were successfully produced over the last 15 years (e.g. Masoud et al, 1993;Benchekroun et al, 1995;Leplé et al, 1995;GutierrezCampos et al, 1999;Van der Vyver et al, 2003;Outchkourov et al, 2004), but recent studies reported pleiotropic effects of these proteins affecting major biological processes such as flowering, programmed cell death, tolerance to biotic stresses, and defense protein induction (Gutierrez-Campos et al, 2001;Belenghi et al, 2003;Van der Vyver et al, 2003;Rojo et al, 2004;Vaillancourt, 2005). Little is still known about the exact regulatory functions of Cys proteases in plant cells, but their obvious importance at the genome level (Beers et al, 2004) and their broad distribution in plant tissues and cells (Grudkowska and Zagdanska, 2004;Schaller, 2004) suggest a significant role for these enzymes in various cellular and developmental processes.…”

“…Several studies used protein engineering approaches to improve the effectiveness of protease inhibitors toward herbivorous insect or nematode proteases (Urwin et al, 1995;Inanaga et al, 2001;Koiwa et al, 2001;Ceci et al, 2003;Martinez et al, 2003;Melo et al, 2003), but little attention has been paid to the impact of such modifications in a multitrophic context. The ability of recombinant plant cystatins to alter digestive protease activities in insect predatory arthropods via their herbivorous prey fed the modified plant has been documented in recent years (Bouchard et al, 2003a(Bouchard et al, , 2003bFerry et al, 2003;Alvarez-Alfageme et al, 2007), as well as some unintended pleiotropic effects of these inhibitors in planta significantly altering developmental processes and stress responses in the modified plant (Gutierrez-Campos et al, 2001;Belenghi et al, 2003;Van der Vyver et al, 2003;Vaillancourt, 2005). Using a collection of 29 cystatin variants derived from the eighth inhibitory unit of tomato (Solanum lycopersicum) multicystatin, SlCYS8 (formerly LeCYS8; Girard et al, 2007), we assessed here the usefulness of site-directed mutagenesis at hypervariable (positively selected) amino acid sites for the improvement of both the inhibitory potency and specificity of plant cystatins toward herbivorous insect digestive Cys proteases.…”

Ectopic phytocystatin expression increases nodule numbers and influences the responses of soybean (Glycine max) to nitrogen deficiency

“…The coordinated expression of transcripts encoding cysteine protease and cystatin interacting partners has been reported in senescent spinach leaves (Tajima et al, 2011). Ectopic OCI expression not only altered the growth and development of tobacco (Van der Vyver et al, 2003;Prins et al, 2008), soybean and Arabidopsis thaliana plants (Quain et al, 2014) but also enhanced tolerance to abiotic stresses, such as low temperatures and drought (Prins et al, 2008;Quain et al, 2014).…”

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…Our bioassays with OsCYS1 and CpCYS1 were conducted with excess concentrations of inhibitor, estimated at ~0.6 mg/g.fresh weight of infiltrated stem disk after re-extraction (not shown). Such concentrations would represent accumulation rates reaching ~2% of leaf soluble proteins in tissues of transgenic banana lines, compared to lower rates of 0.1-1.0% typically observed in cystatinexpressing transgenic plants (Cloutier et al, 2000;Van der Vyver et al, 2003). In practice, a strong inhibitory potency of the protease inhibitor selected is critical for an effective inhibition effect, as it directly determines the minimal amount of recombinant inhibitor required in the modified host plant to provide sufficient pesticidal effects (De Leo et al, 1998;Goulet et al, 2008).…”