Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv 'Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25,23,20,16,12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pl = 8.9) and 60 kilodaltons (anionic, pI = 4.0).The observation that V5S1Na2SO4 was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.Ethylene is known to accelerate senescence in plants (1). While the loss of RNA, Chl, and protein during senescence is known (17,22), the enzymology of the degradation process is not understood. The original purpose of the work reported here was to characterize the enzymes involved in nucleic acid degradation. As an experimental system, we chose to measure enzyme changes during ethylene-enhanced senescence in cotyledons of 2-weekold cucumber seedlings since Lewington et al. (9) reported that yellowing of cucumber cotyledons was associated with a decrease in RNA, DNA, and protein and with an increase in RNase and DNase activity. While new proteins were detected in SDS-PAGE gels during senescence, none were associated with nuclease activity as measured in SDS-PAGE nuclease activity gels. However, two proteins with red chromaphores, which were subsequently found to be peroxidases, appeared during ethylene-induced senescence. This paper describes the isolation and purification of these enzymes.In spite of the fact that plant peroxidases have been studied by many workers, their physiological functions are only partially understood (7). One function ascribed to peroxidase is that of a Chl-degrading enzyme (12,23 Evidence has accumulated that peroxidase-based reactions may play a role in senescence. Lauriere (8) showed that peroxidase activity often increased during senescence. More specifically, Ford and Simon (6) have reported that peroxidase activity increased during senescence of cucumber plants. Thre...

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1-5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase

Ethylene enhanced the levels of peroxidases in the roots, stems, leaves, and cotyledons of 2-week-old cucumber Cucumis sativus cv Poinsett 76 seedlings. Antibodies to the isoelectric point (pi) 9 and pi 4 isoenzymes were used in a radial immunodiffusion assay to demonstrate that ethylene induced similar peroxidases in other cultivars of C. sativus, other species of Cucumis and other genera of Cucurbitaceae. Examination of ethylene-induced peroxidases, using isoelectric focusing gels, demonstrated the presence of a series of other peroxidases, mostly slightly acidic, whose isoelectric focusing pH was approximately 6. These pi 6 peroxidases were partially purified on a cation exchange column. Ouchterlony double diffusion gels indicated that these proteins cross-reacted with antibodies to both the pi 9 and pi 4 peroxidase. The data presented here suggest that the induction of peroxidase isoenzymes during ethyleneinduced senescence is a common response in this family of plants. In addition, antibody and isoelectric focusing studies indicate that both acidic and basic peroxidase are highly conserved in members of this family.