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

Abstract

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.

A Soybean Dual-Specificity Kinase, GmSARK, and Its Arabidopsis Homolog, AtSARK, Regulate Leaf Senescence through Synergistic Actions of Auxin and Ethylene

“…During leaf senescence, the most prominent change in cell structure is the breakdown of the chloroplast, which has been widely used as a biomarker for leaf senescence (Thomson and Plat-Aloia, 1987;Otegui et al, 2005;Pruzinská et al, 2005;Prins et al, 2008). However, no definite conclusions have been reached as to whether and how the disintegration of chloroplasts affects the process of leaf senescence.…”

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

“…The subcellular location of these proteins is still unknown, with the exception of the multicystatins from potato (Solanum tuberosum) and tomato (Solanum lycopersicum) found in vacuoles and in cytoplasm and oryzacystatin OC-I from rice (Oryza sativa) detected in cytoplasm, vacuoles, and chloroplasts (Madureira et al, 2006;Prins et al, 2008;Nissen et al, 2009). Nevertheless, the signal peptide present in most of these inhibitors suggests a noncytosol location of these proteins (Martinez et al, 2005a;Abraham et al, 2006).…”

Autophagy Plays a Role in Chloroplast Degradation during Senescence in Individually Darkened Leaves

“…Using immunoelectron microscopy, we previously demonstrated in naturally senescing wheat (Triticum aestivum) leaves that Rubisco is released from chloroplasts into the cytoplasm and transported to the vacuole for subsequent degradation in small spherical bodies, named Rubisco-containing bodies (RCBs; Chiba et al, 2003). Similar chloroplast-derived structures were also subsequently confirmed in senescent leaves of soybean (Glycine max) and/or Arabidopsis (Arabidopsis thaliana) by electron microscopy (Otegui et al, 2005), and recently in tobacco (Nicotiana tabacum) leaves by immunoelectron microscopy, although the authors gave them a different name, Rubisco vesicular bodies (Prins et al, 2008). RCBs have double membranes, which seem to be derived from the chloroplast envelope; thus, the RCB-mediated degradation of stromal proteins represents a potential mechanism for chloroplast shrinkage during senescence.…”