Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/ dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by C-14-polyamines under light and light/ dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of C-14-polyamines to Rubisco. In both fractions (thylakoid-pre and stromapre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their subfractions were isolated (thylakoid-post and stromapost). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.