A method for in vivo determination of subtilase activity in germinating seeds.

Abstract Plant proteolytic enzymes belonging to the S8 family of serine proteases are known as subtilases or subtilisin-like proteases. Subtilases compose the largest family of plant serine peptidases and have been associated to a wide range of physiological responses and developmental stages. We here describe a simple and fast method to reveal subtilase activity in vivo in germinating seeds of different crops. The method is based on the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA), a synthetic peptide highly specific for subtilases, and subsequent diazotization of liberated p-nitroaniline giving an intense pink dye. The method was tested in germinating seeds of four cereals (wheat, maize, sorghum and rice). In order to verify subtilase activity in such materials, soluble extracts were prepared and analyzed by a standard protocol for in vitro measurement of Suc-AAPF-pNA degradation. Results revealed subtilase activity in vivo in all the cereal grains tested, showing strong correlation with spectrophotometric measurement of subtilase activity in soluble extracts. The present work not only describes for the first time a method to asses for subtilase activity in vivo , but also reveals the ubiquity of active subtilases during the germination of different plant species. Highlights • Subtilase activity was visualized in germinating wheat grains after diazotization. • Using phenylmethylsulfonyl fluoride (PMSF) and chymostatin it was demonstrated the specificity of the method. • Subtilase activity could be detected in situ in rice, maize and sorghum. • Subtilase activity was confirmed by a standard protocol for in vitro measurement. [ABSTRACT FROM AUTHOR]