Relation of Polyamine Synthesis and Titer to Aging and Senescence in Oat Leaves

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Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chi breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. Exogenous Dap, like Spd, inhibits senescence, possibly by preventing the increases in protease activity and ethylene production that precede Chl breakdown. Inhibition of senescence is possibly effected through an initial interaction between Dap and membranes. MATERIALS AND METHODSPlant Material. The first leafof 14-d-old seedlings ofoat (Avena sativa L., var. Victory) was used for most experiments. The seeds (Swedish Seed Company, Ltd., Svalov, Sweden) were grown in vermiculite in controlled growth rooms maintained at 24 ± 1 C and with a 16-h photoperiod of about 12,000 lux.Incubation of leaf segments. For measurements of protease activity, leaves were sterilized as described earlier (19). The lower epidermis was peeled off and 45-mm-long leaf segments were rinsed in distilled H20 and floated in Petri dishes, stripped side down, on 5 ml of 1 mm phosphate buffer (pH 5.7). All manipulations were performed aseptically in a laminar flow hood. For measurements ofethylene production, leafsegments were similarly handled and floated on solutions in bottles sealed with rubber serum caps. For estimating senescence by measurements of residual Chl content, the following materials were used: leaf segments from oats, barley (Hordeum vulgare cv Himalaya), corn (Zea mays cv Golden cross bantam), and wheat (Triticum aestivum cv Yamhill) and leaf discs (10 mm) from mature leaves of 1-to 2-monthold plants of bean (Phaseolus vulgaris cv Taylor's Horticultural), radish (Early Scarlet Globe), and broccoli (Waltham). Leaves of these materials were floated on buffer in Petri dishes, with Dap, Spd (HCI salt, Sigma), and other effectors included as indicated in the appropriate tables. Incubation was carried out at 25°C in the dark or in the light in growth rooms as described above.Determination of Protease Activity. Twelve leaf segments (45 mm each) were homogenized in a prechilled mortar with 2 ml of cold 50 mm phosphate citrate buffer (pH 6.0). The homogenates were kept in the cold (4°C) for 0.5 h and then centrifuged at 12,000g for 15 min at 4°C. The clear supernatant fraction was assayed for protease activity using Azocoll (Calbiochem) as the substrate. The final l-ml reaction mixture contained 5 mg Azocoll, 0.8 ml of 50 mm phosphate-citrate buffer (pH 4.2 for acid protease and pH 6.6 for neutral protease), and 0.2 ml of the crude enzyme. For in vitro effects, the reaction mixture included 0.1 ml ofvarious concentrations of Dap and Spd. The tubes (1.5-ml microcentrifuge) wer...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves

Shih¹,

Kaur‐Sawhney²,

Fuhrer³

et al. 1982

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“…a-difluromethylarginine inhibited ethylene induced putrescine accumulation and coleoptile elongation. It seems that putrescine biosynthesis might be involved in the ethylene-induced elongation of rice coleoptiles.Polyamines, present ubiquitiously in a wide range of higher plants (8), are proposed to play important roles in the regulation of plant growth and development, including cell division, morphorgenesis, flower initiation, pollen tube growth and senescence (5,8,9,15). Stresses such as oxygen deficiency (19-21), chilling (11), high saline, low pH, and K+ and Mg2+ deficiency (5,8,9) increase polyamine contents and their biosynthetic enzyme activities (5,8,9).…”

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confidence: 99%

Ethylene-Induced Polyamine Accumulation in Rice (Oryza sativa L.) Coleoptiles

Lee

Cai²

1992

Effects of ethylene on free polyamine biosynthesis in rice (Oryza sativa L. cv Taichung Native 1) coleoptiles were investigated in sealed and aerobic conditions. In sealed conditions, putrescine increased significantly and coincided with ethylene accumulation. Application of ethylene in sealed containers promoted putrescine accumulation over that in sealed controls. This ethylene-enhanced putrescine accumulation was inhibited by the ethylene action inhibitor 2,5-norbornadiene at 4000 ML/L. In aerobic conditions, ethylene and 1 -aminocyclopropane-1 -carboxylic acid also induced putrescine accumulation. Activity of arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) increased on exposure to ethylene in aerobic conditions. Ornithine decarboxylase (EC 4.1.1.17) activity, however, remained unchanged. The ethylene-induced putrescine accumulation was inhibited by 5 x 10-4 M a-difluromethylarginine, but not by 5 x 10-4 M a-difluromethylornithine. Apparently, arginine decarboxylase, not ornithine decarboxylase, mediates the ethylene-induced putrescine accumulation. The increased S-adenosylmethioinine decarboxylase activity, however, did not result in a significant spermidine/spermine accumulation. In ethylene-treated coleoptiles, the accumulation of putrescine paralleled the increase of coleoptile length in both sealed and aerobic conditions. a-difluromethylarginine inhibited ethylene induced putrescine accumulation and coleoptile elongation. It seems that putrescine biosynthesis might be involved in the ethylene-induced elongation of rice coleoptiles.Polyamines, present ubiquitiously in a wide range of higher plants (8), are proposed to play important roles in the regulation of plant growth and development, including cell division, morphorgenesis, flower initiation, pollen tube growth and senescence (5,8,9,15). Stresses such as oxygen deficiency (19-21), chilling (11), high saline, low pH, and K+ and Mg2+ deficiency (5,8,9) increase polyamine contents and their biosynthetic enzyme activities (5,8,9). Ethylene was also found to influence polyamine biosynthesis (1,3,6,12,13,22,24); however, whether the effect was inhibition or stimulation depended on the plant system (5). In 3 Abbreviations: ADC, arginine decarboxylase; ACC, 1-aminocyclopropane-1-carboxylic acid; DFMA, a-difluromethylarginine; DFMO, a-difluromethylornithine; MeOH, methanol; ODC, omithine decarboxylase; PMSF, phenylmethylsulfonylfluride; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; PCA, perchloric acid.1.1.18) (12). The inhibition of the enzyme activity by ethylene was shown to be correlated with the ethylene-induced reduction of apical tissue activity. In contrast, ADC and SAMDC activities, and also polyamine contents, in deepwater rice stems increased in response to ethylene (3). The increased polyamines were suggested to be involved in the promotion of both cell division and elongation of rice stems (3). Thus, ethylene-mediated alterations in polyamine biosynthesis are parallel t...

“…It was worrisome that the activities of the two enzymes reported for comparable leaves of oat cv Victory seedlings, grown under controlled conditions, were much different from ours and at the same time showed significant variability. The reported ADC and ODC activities varied from 2.2 to about 32 and from 0.8 to 18 nmol/h x mg protein, respectively, with ODC/ADC ratios ranging from 0.1 to 6.0 (16,19,31,33,34). Thus, one might suppose that there is significant intraspecific variation in activity levels and/or substantial dependence on external uncontrolled factors; e.g.…”

Section: Resultsmentioning

confidence: 99%

Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

Birecka¹,

Birecki²,

Cohen³

et al. 1988

When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors-endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S.riddeliji and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence-with relatively very high levels of these compounds-in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ-or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence.Our previous study on PA '- the same borages were able to decarboxylate exogenous Orn to Put that in turn was converted into necines. Thus, in vivo transformation of an exogenous putative precursor does not always indicate the compound formed in situ that normally serves as a precursor. This is especially true in the case of Put, which can derive not only from Orn but also from Arg via Agm, the product of Arg decarboxylation by ADC. Our attempt to identify the in situ precursor(s) of the Put channeled into pyrrolizidines in Senecio vulgaris were unsuccessful due to the extremely low PA content of the shoots.We report here the effects of DFMO and DFMA on necine biosynthesis in S. riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants. These species occasionally produce extremely high levels of PAs, up to 9 to 17% of leaf dry weight (23). In previously analyzed samples of S. riddellii and S. longilobus plants from New Mexico the PA contents were about 1.5 and 2.8%, respectively (10). Since there is little information about polyamines, including Put, ADC, and ODC activities, or the site(s) of alkaloid biosynthesis in PA-bearing plants, and since such information is available only for Heliotropium (5, 7-9), a more detailed examination of PA-bearing plants seemed important. Flowering...