Senescence-Induced, Thylakoid-Bound Diisopropylfluorophosphate-Binding Protein in Spinach

Abstract

Changes in diisopropylfluorophosphate (DFP)-binding proteins during development and senescence of spinach (Spinacia oleracea) leaves were followed using [3H]DFP and sodium dodecylsulfate-polyacrylamide gel electrophoresis-fluorography. Experiments using a series of aging stages of leaves attached to plants and ones with detached leaves stored in the dark both showed that a protein of 38 kilodaltons was the only major DFP-binding protein in the membrane fraction and that its DFP-binding increased markedly as senescence proceeded, corresponding with the degradation of leaf protein. DFP binding to the 38-kilodalton protein was not affected by membrane solubilization with Triton X-100, and gradually decreased upon preservation of the membranes. The DFP binding was inhibited completely by phenylmethane-sulfonyl fluoride and slightly by p-chloromercuribenzoic acid, suggesting a serine protease-like character of the protein and a possible contribution of SH residues to the binding. Both differential and Percoll-gradient centrifugation indicated that the 38-kilodalton protein was localized in thylakoid membranes. The sedimentation behavior of the detergent-solubilized protein indicated that it belongs to a complex different from photosystem 1, photosystem 11, or coupling factor I of the ATP-synthesizing complex.(12, 27, 28), and the decrease in chloroplast content has been shown to be due to increased degradation, rather than to a decrease in synthesis (7, 10).Many attempts have been made to identify the degradative enzymes, in most cases proteases, functioning in the retrieval of nutrients (11,13,18,19), but little success has been achieved. One reason may be that the vacuole's large pool of degradative enzymes, which are not necessarily related to senescence (24), makes it difficult to detect other specific organellar enzymes in cell homogenates. Also, the substrate specificities of these enzymes are sometimes so narrow that a survey of enzymes with the small number of substrates hitherto used would seem to have limited prospects. Therefore, detection of proteases related to senescence may require alternative approaches.DFP3 is known to combine irreversibly with active serine residues of proteases and esterases, and proteins showing DFP binding activity can be analyzed simultaneously with high sensitivity using SDS-PAGE and fluorography (4, 21). In this study, aging-associated changes in DFP-binding proteins in spinach leaves were followed in an attempt to detect senescence-related enzymes. One senescence-induced protein was identified and analyzed further.

Senescence mechanisms

“…For example, PSII lated in senescing tissues (see Brady 1988), senescence and its components are removed synchronously, not one does not seem to be caused by tuming genes off component at a time (J. J. Guiamet, I. John, E. Pichersky (Nooden 1988d). Usually, the breakdown of some thylakoid proteins, e.g., LHCII, and RNA extracts are normalized on the basis of total RNA, some CP stromal enzymes (Kawasaki and Takeuchi 1989, which is predominantly rRNA, but massive degradation Yoshida and Minamikawa 1996). The influence necessary to prevent regeneration of cell components of Chi serves to illustrate this mutual stabilization be-broken down in the senescence process.…”

Senescence mechanisms

“…An important complication in the analyses of Exactly which proteases are responsible for disman-these changes lies in the method for normalizing the tling the protein components of the thylakoids is unclear, sample loads mn in the electrophoretic gels used to rebut there is some evidence implicating serine proteases in solve the mRNAs prior to northem blotting. Usually, the breakdown of some thylakoid proteins, e.g., LHCII, and RNA extracts are normalized on the basis of total RNA, some CP stromal enzymes (Kawasaki and Takeuchi 1989, which is predominantly rRNA, but massive degradation Yoshida and Minamikawa 1996). On the other hand, the of rRNAs occurs during senescence (see Brady 1988), thiol protease inhibitor E-64 retards the breakdown of and therefore, the baseline is changing.…”

Gene expression during leaf senescence

“…Kawasaki & Takeuchi (1989) have shown that a putative serine protease bound to the thylakoid membranes of spinach chloropiasts increases markedly in activity during senescence. They demonstrated this by the ability of a 38 kDa protein to bind H-diisopropylfluorophosphate, which is known to react irreversibly with the active serine residues of proteases and esterases.…”