Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli

AbstractA DNA fragment containing genes for both large (A) and small (B) subunits ofribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-β-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures

Accumulation of Glycinebetaine in Rice Plants that Overexpress Choline Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic Stress

• Background and Aims Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice

Tissue Localization of the Glycine Betaine Biosynthetic Enzymes in Barley Leaves

Barley (Hordeum vulgare L.) plants accumulate glycine betaine (GB), a major compatible solute, in response to salt stress. In barley, GB is produced by a two-step oxidation of choline in a cooperative way in the cytosol and peroxisomes. In this study, we investigated the localization of two GB biosynthetic enzymes, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), in the tissues of barley plants (cv. Haruna-nijyo) grown under normal and saline conditions. Three-week-old barley plants grown hydroponically were treated with a hydroponic culture solution containing 200 mM NaCl for 72 h. Salt treatment resulted in increased expression of CMO and BADH proteins mainly in the leaves of barley but not in the roots. The expression of CMO protein was increased by the presence of NaCl in younger leaves but decreased in older leaves. The tissue localization of CMO and BADH proteins was analyzed by immunofluorescent labeling method using their primary antibodies and a fluorescein-conjugated secondary antibody. CMO and BADH proteins were constitutively co-localized in mesophyll and bundle sheath cells under both normal and saline conditions. A possible physiological function of GB in the salt tolerance of barley plants is discussed