Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Abstract

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [14C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [14C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminasedirected modifications of ribulose 1,5-bisphosphate carboxylase/ oxygenase.

Effects of Exogenous Putrescine on Mycorrhiza, Root System Architecture, and Physiological Traits of Glomus mosseae-Colonized Trifoliate Orange Seedlings

“…PAs are involved in photosynthetic functions (Margosiak et al, 1990). In the present work, supplied exogenous Put significantly increased contents of chlorophyll a, carotenoid, and total chlorophyll, compared with the non-Put treatment (Tab.…”

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotiana tabacum) flowers

“…Chloroplast TGases (ChlTGases) are different from those of the cytoplasm and are active in chloroplasts of both higher and lower plants. 20,27,51,57,58 Having as substrates the antenna complexes and Rubisco, 26,27 they are possibly involved in photosynthesis. The decrease in total TGase activity during petal senescence could thus be partly related to the breakdown of chloroplasts.…”

“…actin and tubulin 24 or cell wall proteins, 21 and through its involvement in the polymerisation of Rubisco. 26 Different roles for TGase have been proposed in chloroplasts 27 and mitochondria. 28 In contrast to the considerable number of reports concerning TGase in dying animal cells, no information is available regarding this enzyme family during PCD in plants.…”

The Acropetal Wave of Developmental Cell Death of Tobacco Corolla Is Preceded by Activation of Transglutaminase in Different Cell Compartments

“…In plant cells, the roles of TGases are similar to those in animal cells, in terms of several biochemical features, however, their presence in particular plant compartments and their substrates suggest that they may fulfil additional roles. TGases were found in the chloroplasts of several higher plants and some algae, where they possibly stabilize and protect antenna complexes and Rubisco, thus possibly being involved in the regulation of the photosynthetic process (Margosiak et al, 1990;Della Mea et al, 2004b;for review, see Serafini-Fracassini and Del Duca, 2002). In some lower organisms, like Chlamydomonas (Waffenschmidt et al, 1999) and some fungi, TGase activity has been found in the cell walls, where the enzyme has a structural role or acts as the defense elicitors of higher plants (for review, see Del Duca and Serafini-Fracassini, 2005).…”