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

CARRION CRISTIAN; COSTA LORENZA; MARTÍNEZ DANA; CHRISTINA MOHR; KLAUS HUMBECK; JUAN JOSÉ GUIAMET

JOURNAL OF EXPERIMENTAL BOTANY

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2013 vol. 64 p. 4967 - 4980

0022-0957

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process insenescing leaves. In spite of its importance in internal N recycling, the mechanism(s) andthe enzymes involved are largely unknown. Senescence Associated Vacuoles (SAV) aresmall, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteinsre-localize to SAVs during senescence, suggesting that SAVs might be involved inchloroplast protein degradation.SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance,visualized either with the acidotropic marker Lysotracker Red or by GFP fluorescence in aline expressing the senescence-associated cysteine protease SAG12 fused to GFP,increased during senescence induction in darkness, and peaked after 2-4 days, whenchloroplast dismantling was most intense. Increased abundance of SAVs correlated withhigher levels of SAG12 mRNA. Activity labeling with a biotinylated derivative of thecysteine protease inhibitor E64 was used to detect active cysteine proteases. The twoapparently most abundant cysteine proteases of senescing leaves, of 40 and 33 kDa weredetected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blockedby E64. Treatment of leaf disks with E64 in vivo substantially reduced degradation ofRubisco and leaf proteins. Overall, these results indicate that SAVs contain most of thecysteine protease activity of senescing cells, and that SAVs cysteine proteases are at leastpartly responsible for the degradation of stromal proteins of the chloroplast.