Chloroplast proteases from senescent and non-senescent wheat leaves

IRMA N. ROBERTS; SUSANA PASSERON; ATILIO J. BARNEIX

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión Anual SAIB; 2006

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

CHLOROPLAST PROTEASES FROM SENESCENT AND NON-SENESCENT WHEAT LEAVES Irma N. Roberts; Susana Passeron; Atilio J. Barneix IBYF-CONICET, Facultad de Agronomía, UBA. E-mail: iroberts@agro.uba.ar Higher plant chloroplasts contain large amounts of proteins, mainly involved in photosynthesis. Stromal proteins (including Rubisco) are the major source of nitrogen in leaves and the preferred point of attack for proteases at senescence. At present, there is a general consensus that at least the first steps in chloroplast protein degradation occur inside the organelle but the identity of the proteases involved remains unknown. The aim of this work was to search for chloroplast proteases with increased activity at senescence, as potential candidates for stromal protein degradation. Chloroplasts were isolated from non-senescent and senescent (72 h of incubation in darkness) wheat leaves by Percoll gradient, lysed by hypotonic shock and the stromal fraction recovered in the supernatant after centrifugation. In-gel protease assays were performed in polyacrylamide gels copolymerised with gelatin. Only one band of gelatin hydrolysis was visualised in the stromal fraction from non-senescent leaves, which was also observed in dark-induced senescent leaves but showing a weaker signal. The addition of different protease inhibitors to the incubation media indicated that it is a serine protease. Two other bands, detected only in dark-induced senescent leaves, were inhibited by o-phenantroline and EDTA indicating that they are metalloproteases. Supported by grants from ANPCyT and CONICET.