RESEARCH PAPER |
Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies
1School of Agriculture, Food and Rural Development, Agriculture Building, Newcastle University, Newcastle upon Tyne NE1 7RU, UK
2Forestry and Agricultural Biotechnology Institute, Botany Department, University of Pretoria, Pretoria 0002, South Africa
3School of Environmental Sciences and Development, Section Botany, North-West University, Potchefstroom 2520, South Africa
4CEBAS-CSIC, Department of Plant Physiology, PO Box 164, E-30080 Murcia, Spain
* To whom correspondence should be addressed. E-mail: christine.foyer{at}ncl.ac.uk
The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.
Key words: Chloroplast proteins, cysteine proteinase, photosynthesis, protein turnover, senescence, vesicle trafficking
Received 31 January 2008; Revised 26 February 2008 Accepted 27 February 2008
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