Purification and characterization of a subtilisin‐like serine protease induced during the senescence of wheat leaves

Abstract

A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent K m and V max for the peptide were 1.18 mM and 2.27 mmol pNA mg À1 h À1 , respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60 C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65-75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process.

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum

“…Cysteine proteinases are also expressed during petal senescence in carnation (Jones et al 1995) and day-lily (Guerrero et al 1998). Although senescenceinduced serine proteases have been identified in wheat leaves (Roberts et al 2003), limited evidence is available for the role of serine proteinases other than the subtilisins (for review, see Beers et al 2000), or their inhibitors, in PCD. Interestingly, subtilisin-like serine proteases are expressed in Arabidopsis vascular bundles, pistils, ovules and anthers (Golldack et al 2003), similar tissues to which SaPIN2a and SaPIN2b have been localized in S. americanum.…”

Effect of different metals on protease activity in sunflower cotyledons

“…Five catalytic classes (serine, cysteine, aspartic, threonine and metallopeptidases) can been identified within the Arabidopsis genome sequence (Rawling et al 2004). In general, proteases are thought to be involved in a range of processes, including germination, senescence, defence responses and programmed cell death (Fontanini and Jones, 2002;Segarra et al 2002;Roberts et al 2003;Coffeen and Wolpert, 2004). In addition, proteases might also play a role in the plant adaptation to changing environmental conditions.…”

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants

“…Transcriptome study of Arabidopsis senescent leaves also revealed that cysteine and serine proteases are predominantly induced during natural and/or abiotic stress induced senescence (BuchananWollaston et al 2005). Roberts et al (2003) reported a serine protease activity in senescing wheat leaves for which rubisco was a target protein.…”