The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases

We have previously reported the occurrence of two serine endoproteases (referred to as P1 and P2) in dark-induced senescent wheat (Triticum aestivum L.) leaves. P1 enzyme was already purified and identified as a subtilisin-like serine endoprotease (Roberts et al. in Physiol Plant 118:483-490, 2003). In this paper, we demonstrate by Western blot analysis of extracts obtained from dark-induced senescent leaves that an antiserum raised against P1 was able to recognise a second protein band of 78 kDa which corresponded to P2 activity. This result suggested that both enzymes must be structurally related. Therefore, we purified and characterised P2 activity. According to its biochemical and physical properties (inhibition by chymostatin and PMSF, broad pH range of activity, thermostability and ability to hydrolyse Suc-AAPF-pNA) P2 was classified as a serine protease with chymotrypsin-like activity. In addition, P2 was identified by mass spectrometry as a subtilisin-like protease distinct from P1. Western blot analysis demonstrated that P1 appeared in extracts from non-detached dark-induced senescent leaves but was undetectable in leaves senescing after nitrogen (N) deprivation. In contrast, P2 was already present in non-senescent leaves and showed increased levels in leaves senescing after N starvation or incubation in darkness. P1 signal was detected at late stages of ethephon or methyl jasmonate-induced senescence but was undetectable in senescent leaves from plants treated with abscisic acid. None of the three hormones have any effect on P2 protein levels. These results indicate that despite their biochemical and structural similarities, both enzymes are probably involved in different physiological roles.