Identification of a cysteine protease in senescent barley leaves.

HOLLMANN J; CARRIÓN C.A; MATROS A.; GREGERSEN P.L; MOCK H.-P; GUIAMET J.J.; KRUPINSKA K

Hemavan

Conferencia; The 1st International Conference on Plant Proteases; 2011

Rubisco is the most abundant protein in chloroplasts and its degradation during leaf senescence is of pivotal importance for nitrogen remobilization and crop yield (Gregersen et al. 2008). So far, no plastid enzymes responsible for degradation of Rubisco have been identified in barley. Rather it seems that Rubisco might be degraded outside of chloroplasts by cysteine proteases in lytic compartments called senescence associated vacuoles (Martínez et al. 2008). To identify cysteine proteases potentially involved in degradation of Rubisco during leaf senescence an affinitity chromatography approach was undertaken. For this purpose E64 a specific inhibitor for cysteine proteases was fused to biotin to allow binding to streptavidin-Sepharose beads (Greenbaum et al. 2000). The tagged inhibitor was incubated with protein extracts from either nonsenescent or senescent leaves. Bound proteases obtained by incubation of senescent leaves and not detectable in control leaves were identified by mass spectrometry. Amino acid sequences information allowed the identification of three cysteine proteases potentially involved in senescence associated protein degradation. Quantitative real time PCR with RNA prepared from barley flag leaves collected in field plots at different times of development revealed that one of the proteases is highly upregulated during leaf senescence. The cDNA will be used for overexpression in E. coli. Immunological analyses will be done to elucidate the subcellular localization of the enzyme.