Planta (1993, 189:201 206 P l a n t a 9 Springer-Verlag1993 Effect of polyamines on stabilization of molecular complexes in thylakoid membranes of osmotically stressed oat leaves R.T. Besford 1., C.M. Richardson 1, J.L. Campos 2, and A.F. Tiburcio 2 1 Horticulture Research International, Littlehampton,West Sussex BN17 6LP, UK 2 FisiologiaVegetal, Facultat de Farmficia,Universitat de Barcelona, Spain Received 6 May; accepted 27 August 1992 Abstract. Monocotyledonous leaves subjected to osmot- ica used for protoplast isolation accumulate a massive amount of putrescine (Put), lose chlorophyll and senesce rapidly. Treatment with spermidine (Spd) or spermine (Spm) prevents the loss of chlorophyll, indicating preservation of the thylakoid membranes at the site of the chlorophyll-protein complexes. Using several recent- ly produced antibody probes, the effects on the stabiliza- tion of thylakoid membranes of applying either difluoro- methylarginine (DFMA), a specific inhibitor of putres- cine synthesis via arginine decarboxylase, or the poly- amines Spd, Spm, or diaminopropane (Dap) to osmoti- cally shocked oat leaves (Arena sativa L.) have been investigated. High protein levels were maintained in thy- lakoid membranes of leaf tissue incubated in the dark in the presence of 0.6 M sorbitol when pretreated with DFMA. After 48 h incubation, the level of the thylakoid protein D 1, at the core of photosystem II, was higher in the DFMA-pretreated leaves as was the stromal protein ribulose-l,5-bisphosphate carboxylase-oxygenase (Rub- isco; as indicated by the level of large subunits). Applica- tions of Spd, Spm or Dap were effective in retarding the loss of D1, D2 and cytochrome f from the thylakoid membranes as well as Rubisco large subunits and chlorophyll from the leaf tissue. The effects of polyamine applications may be mediated through Dap since most of the added Spd or Spm was converted to Dap within 6 h. The possible mechanisms of action of polyamine application s and DFMA-pretreatment on stabilizing the composition of the thylakoid membrane are also discuss- ed. * To whom correspondence should be addressed; FAX: 44 (903) 72 67 80 Abbreviations : Cyt= cytochrorne; Dap = diaminopropane; DFMA = DL-cc-difluoromethylarginine ; LSU = large subunit (of Rubisco); Put=putrescine; Rubisco=ribulose-l,5-bisphosphate carboxylase-oxygenase; Spd = spermidine; Spin= spermine; SDS-PAGE = sodiumdodecylsulphate-polyacrylamide gel electro- phoresis Key words: Polyamine - Ribulose-l,5-bisphosphate car- boxylase/oxygenase (large subunit) - Thylakoid mem- brane (stabilization) - Thylakoid protein (D1, D2, cytochrome J) Introduction It is difficult to retain viability of the protoplasts from monocotyledonous species compared with those derived from dicotyledons and this may be related to their differ- ing polyamine metabolism in the osmotica used for protoplast work. During loss of viability either of cereal protoplasts due to osmotic shock or during dark-induced senescence in leaves, there is a rapid increase in arginine decarboxylase and a massive accumulation of putrescine (Put) (Flores and Galston 1984). This is in sharp con- trast to dicotyledons where osmotic treatments lead to increases in spermidine (Spd) and spermine (Spin) with a decline in Put and in the activities of ornithine decar- boxylase and arginine decarboxylase (Tiburcio et al. 1986c). These authors speculated that a high ratio of Put to polyamines (Spd+Spm), as occurs in osmotically shocked cereai leaf tissue, is detrimental. Also, in such oat leaves there is an accompanying rise in the activities of RNase and protease, and a loss of chlorophyll (Alt- man et al. 1977; Kaur-Sawhney and Galston 1979). Treatment with the polyamines Spd or Spm inhibits or retards these processes (Kaur-Sawhney and Galston 1979; Galston et al. 1978) and induces DNA synthesis and limited protoplast mitosis (Kaur-Sawhney et al. 1980). A similar effect is produced by pretreating oat leaves with DL-a-difluoromethylarginine (DFMA) (which inhibits synthesis of Put via arginine decar- boxylase) before osmotic treatment (Tiburcio et al. 1986a). Levels of Spd and Spm increase relative to Put levels and osmotically induced inhibition of Spd-syn- thase activity which catalyses conversion of Put to Spd is prevented by DFMA pretreatment (Tiburcio et al. 1986a, b).