In vivo inhibition of cysteine proteases provides evidence for the involvement of ‘senescence-associated vacuoles’ in chloroplast protein degradation during dark-induced senescence of tobacco leaves

Abstract

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

Roles of C1A peptidases during barley leaf senescence mediated by abiotic stresses

“…This extraplastidial pathway of degradation is dependent on ATG genes and implies a complex trafficking of proteins from the chloroplast to the central vacuole, not yet studied in depth. The detection of Rubiscocontaining bodies (RCBs) carrying stromal proteins to the central vacuole in senescing leaves of several plant species corroborate this dynamic protein traffic (Chiba et al, 2003;Ishida et al, 2008;Prins et al, 2008;Carrion et al, 2013) All these data confirm the importance of C1A CysProt as the main proteases located in different cell compartments during the leaf senescence process, as well as the important regulatory effect of cystatins in the leaf senescence physiology.…”

“…Outside the chloroplast, plastid proteins including Rubisco, occur in the vesicular transport system mediated by SAVs where proteolysis may continue due to the presence of active CysProt (Carrion et al, 2013). Besides, RCBs derived from chloroplast and carrying stromal proteins or their hydrolytic products, are redirected to the central vacuole.…”

“…Intra-plastid proteolysis involves multiple isomeric forms of FtsH-, Clp- higher levels of Rubisco in older leaves (Kato et al, 2004;2005). In addition, the nonchloroplastic proteolysis is produced by the action of vacuolar proteases and by the independent Senescent-Associated Vacuoles (SAVs) enriched in CysProt activities (Otegui et al, 2005, Martinez et al, 2008Carrion et al, 2013). The lytic central vacuoles also contain C1A CysProt among other enzymes (Thoenen et al, 2007;Ishida et al, 2008;van Doorn et al, 2011).…”

“…More recently, the same research group has shown in a very elegant way that active CysProt are responsible for the degradation of some plastid proteins in SAVs during dark-induced senescence of tobacco leaves (Carrion et al, 2013). Studies with potential orthologs of Arabidopsis SAG12 gene in other dicot species, such as BnSAG12-1 and BnSAG12-2 in Brassica napus (Noh and Amasino, 1999;Desclos et al, 2008;Gombert et al, 2006), SPG31 in sweet potato (Chen et al, 2002) and NtCP1 and NtSAG12 in tobacco (Beyene et al, 2006;Carrion et al, 2013) et al, 2010). XCP1 and XCP2 are other cathepsin Llike CysProt involved in Arabidopsis senescence.…”

“…As an outcome, a massive degradation of macromolecules, dismantling of cellular structures, mainly chloroplasts, and the subsequent mobilization of mineral nutrients and N-containing molecules to sustain further growth and development are observed (Hörtensteiner, 2007;Diaz-Mendoza et al, 2014). Up-regulation of proteases, from plastidial and nuclear genomes, are needed for protein breakdown throughout stress responses, implying a complex traffic of proteins, peptides and amino acids among subcellular compartments (Roberts et al, 2012;Carrion et al, 2013;Diaz-Mendoza et al, 2014). In particular, Cysteine Proteases (CysProt) of the C1A papain family are the predominantly up-regulated plant proteases (Roberts et al, 2012;Diaz and Martinez, 2013;Diaz-Mendoza et al, 2014).…”