Pergamon 0031-9422(95)00635-4 Phytochemistry, Vol. 41, No. 2, pp. 377-384, 1996 Copyright © 1996ElsevierScienceLtd Printe, d in Great Britain. All rights reserved 0031-9422/96$15.00+ 0.00 ENDOPEPTIDASES DURING THE DEVELOPMENT AND SENESCENCE OF LOLIUM TEMULENTUM LEAVES KARL MORRIS, HOWARD THOMAS and LYNDON J. ROGERS*~' Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB, U.K.; *Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed SY23 3DD, U.K. (Received 24 May 1995) Key Word Index--Lolium temulentum; Gramineae; leaf development; senescence; cysteine endopep- tidases. Abstract--The endopeptidase activity of Lolium temulentum leaf tissue was measured using azocasein as a substrate. The enzyme increased with leaf age, and also during senescence of excised leaf tissue. There were at least two distinct endopeptidase activities, characterized by different pH optima. The predominant form in leaves of intact plants was maximally active at pH 5. In detached leaves during the later stages of senescence this activity was replaced by an enzyme with an optimum at pH 8. An antibody raised against the non-glycosylated cysteine endopeptidase papain cross-reacted with polypeptides in protein preparations of L. temulentum leaf tissue. The correlation between enzyme activity and the pattern of immunoreactive polypeptides suggested that the polyclonal antibody was able to recognize the Lolium homologues of papain. The switch from pH 5 to pH 8 enzyme in detached leaves was associated with an evident decrease in the M, values of papain-like antigens detected on western immunoblots, from ca 60 000 to 30 000. It is possible that the alkaline activity is derived from the acid form, perhaps by limited autolysis in protein-depleted tissue at an advanced stage of senescence. On the other hand, the response of protease activation to treating leaf tissue with inhibitors of protein biosynthesis is more consistent with de novo appearance of a different form of the enzyme in late senescence. INTRODUCTION The leaves of many plant species are rich sources of proteolytic enzymes [1-3], but the physiological func- tions of the major proteases are not clear. Although there are exceptions, protease activities in most leaves increase with age, and particularly when foliar senescence is pro- moted by stress or excision. Protein mobilization is char- acteristic of senescing tissues, but it has proved difficult to establish a direct connection with increased levels of protease. In cereals, grasses and many other species, most of the measurable proteolytic activity of leaves is accounted for by a small number of acid endopeptidases [1,4-6]. Consistent with the low pH optimum is the observation that the acid endopeptidase activity of me- sophyll cells is located predominantly in the vacuole [7-11]. For these enzymes to play a direct part in pro- teolysis during senescence there must be some kind of interchange between the vacuole and the chloroplast, which is the site of most of the mobilizable protein of leaf cells [12, 13]. Such contacts between the two organelles have never been convincingly demonstrated. tAuthor to whom correspondence should be addressed. As well as acid endopeptidases, activities at neu- tral-alkaline pH values have been detected [3, 14-16]. The alkaline protease of oat leaves is located outside the vacuole [ 11, 16]. Experiments with inhibitors indicate that senescence-related increases in protease activity require protein synthesis [2]. Recently, cDNAs encod- ing proteases with homology to cysteine endopeptidases have been isolated from Arabidopsis [17] and maize [18] and used to show increased abundance of the corresponding mRNAs during senescence. It seems likely, therefore, that both transcription and transl- ation are necessary for protease activation in ageing leaf tissue. Lolium temulentura has been used extensively for studies of leaf growth and senescence [ 19-21]. Detached leaves of this species [15] initially accumulated acid protease activity and subsequently replaced it with a neutral-alkaline protease, the dominant enzyme in late senescence. We have re-examined L. temulentura leaf proteases with the object of characterizing the pH response in more detail, applying an immunolog- ical approach to visualizing qualitative changes in pro- tease complement, and establishing the degree to which these changes depend on current protein synthesis. 377