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

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Abstract

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 14C-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 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) 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 sub-fractions were isolated (thylakoid-post and stroma-post). 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.

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Abbreviations

bis-, mono-PU:

bis-, mono-(γ-glutamyl) putrescine

bis-, mono-SD:

bis-, mono-(γ-glutamyl) spermidine

bis-, mono-SM:

bis-, mono-(γ-glutamyl) spermine

PA:

polyamine

PU:

putrescine

RPG-TGase:

rat prostatic gland transglutaminase

Rubisco:

ribulose 1,5-bisphosphate carboxylase/oxygenase

SD:

spermidine

SM:

spermine

stroma-post:

stromal fraction isolated after the TGase assay of the entire chloroplast

stroma-pre:

stromal fraction isolated before the TGase assay

TGase:

transglutaminase

thylakoid-post:

thylakoids isolated after TGase assay of the entire chloroplast

thylakoid-pre:

thylakoids isolated before TGase assay

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Acknowledgements

This work was supported by a grant from Consiglio Nazionale delle Ricerche Contributo di Ricerca 98. 00514.CTO4 to D. Serafini-Fracassini and by the University of Bologna (Funds for Selected Topics: 'Segnali molecolari nel differenziamento cellulare'). The authors are grateful to Henkel S.p.A. (Fino Mornasco, Como), Prof. Carla Esposito (Salerno University), and Carlo Bergamini (Ferrara University) for their kind gifts of Deriphat C 160 KPC, TGase antibody, and erythrocyte purified TGase, respectively. We acknowledge N. Mele for technical assistance with photography and imaging, and the 'ESF protein Cross-linking-The ESF Transglutaminases Programme', for a concessional Travel Grant to Dr. I. Claparols to visit the Botany Laboratory of Bologna University.

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  1. L. Dondini

    Present address: Dipartimento di Colture Arboree, Università di Bologna, Via Fanin 46, 40126, Bologna, Italy

Authors and Affiliations

  1. Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Irnerio 42, 40126, Bologna, Italy

    L. Dondini, S. Del Duca, L. Dall'Agata, M. Della Mea, A. Di Sandro & D. Serafini-Fracassini

  2. Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, 37134, Verona, Italy

    R. Bassi & M. Gastaldelli

  3. Institut de Biologia Molecular, CID–CSIC, Jordi Girona 18, , 08034, Barcelona, Spain

    I. Claparols

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  1. L. Dondini
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Correspondence to D. Serafini-Fracassini.

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Dondini, L., Del Duca, S., Dall'Agata, L. et al. Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates. Planta 217, 84–95 (2003). https://doi.org/10.1007/s00425-003-0998-3

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