Regulation of arginine decarboxylase and putrescine levels in Cucumis sativus cotyledons

Arginine decarboxylase (arginine carboxy-lyase EC 4.1.1.19) of Cucumis sativus cotyledons, has a pH optimum of 8.3 and a temperature optimum of 40°. Among the various plant hormones administered to excised cotyledons in culture, benzyladenine and its riboside were most effective in increasing the arginine decarboxylase activity and putrescine content. The enzyme activity and putrescine content were significantly increased on acid feeding of the cotyledons and decreased by KCl treatment. The KCl effect could be only partially reversed by benzyladenine. Abscisic acid inhibited cotyledon growth and also reduced arginine decarboxylase and putrescine levels. This effect was overcome by cytokinins. The half life of the enzyme using cycloheximide was 3.7 hr. Dibutyryl cyclic AMP and 5′-AMP also marginally stimulated the enzyme and putrescine levels. Mixing experiments indicate that there is neither a non-dialysable activator nor inhibitor of the enzyme.     

Relationships among cytokinins, ethylene and poly amines during the stratification-germination process in seeds of Acer saccharum

Relatively high levels of dihydrozeatin and trans-zeatin were detected in unstratified seeds of Acer saccharum Marsh. Both cytokinins increased substantially over the first 20 days of stratification at 5°C and then fell rapidly to values well below original levels by first germination on day 55. In seeds held at 20°C, a non-afterripening temperature, cytokinin levels remained constant for the first 10 days and then declined to their lowest levels by day 20. Levels of putrescine, spermidine and spermine in the radicles and cotyledons did not change during the full course of the afterripening process, but large increments were noted during radicle emergence. A large increase in ethylene production at germination suggests that competition for S-adenosyl-methionine by the ethylene and polyamine biosynthetic pathways did not inhibit synthesis of ethylene or polyamines during seedling emergence and establishment. In seeds stratified at 20°C, ethylene showed an exceptionally large peak early in the stratification period, but polyamine contents remained low throughout the test. The present results are consistent with the hypothesis that cytokinins play a significant role in overcoming the metabolic block present in dormant seeds. This conclusion is supported by data showing that high levels of cytokinins develop concurrently with the start of tissue differentiation and at the time when abscisic acid and phenolic inhibitors decline in stratifying seeds. Changes in ethylene and polyamine contents did not correlate with any events in the afterripening process; however, large increases in levels of these substances were closely associated with the germinative process and, in the case of polyamines, specifically with the start of cell division.     

Putrescine uptake and translocation in higher plants

Putrescine uptake and translocation were studied by feeding [³H] putrescine to roots of tomato seedlings ( Lycopersicon esculentum Miller, cv. Earlypak 7) at the stage of expanded cotyledons, of maize seedlings ( Zea mais L.) at the coleoptile stage, and of one year old pines ( Pinus pinea L.). Putrescine translocation was rapid as radioactivity appeared in the upper part of the seedlings within 30 min, continuing to increase up to 24 h, while it decreased in roots. The putrescine supplied was partly metabolized to spermidine and spermine in the course of 24 h. The transport was temperature-dependent as it increased with increasing temperature from 4°C to 30°C. In plants kept in 100% relative humidity the transport decreased by 27% compared to controls kept in 50% relative humidity. The existence of basipetal transport was assessed by feeding labeled putrescine to cotyledons or to a primary leaf of tomato plants at different stages of growth. The influence of ringing at the hypocotyl level on polyamine translocation in pine plants was studied in order to exclude cortical parenchyma and phloem from transport. Radioactivity decreased in the hypocotyl just above the ring and in the upper parts (epicotyls with needles), but long-distance transport was low affected indicating xylem transport. It is suggested that polyamine transport is not polar, and that it occurs mainly through xylem vessels.     

外源亚精胺对盐胁迫下黄瓜(Cucumis sativus L.)叶绿体活性氧清除系统和结合态多胺含量的影响

采用营养液水培,研究了外源亚精胺(Spd)对NaCl胁迫下抗盐能力不同的两个黄瓜品种幼苗生长、叶绿体中活性氧清除系统、转谷酰胺酶(TGase)活性、结合态多胺含量及植株光合速率的影响.结果表明,外源Spd能提高NaCl胁迫下叶绿体中TGase活性、叶绿体结合态腐胺(Put)、Spd、精胺(Spm)及总多胺含量;提高超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性,提高抗坏血酸(AsA)、类胡萝卜素(Car)、还原型谷胱甘肽(GSH)含量及还原型谷胱甘肽/氧化型谷胱甘肽(GSH/ GSSG)比值,降低脱氢抗坏血酸/抗坏血酸(DAsA/AsA)比值;同时显著降低叶绿体过氧化氢(H2O2)和丙二醛(MDA)含量,提高植株净光合速率,缓解NaCl胁迫对幼苗生长的抑制.表明Spd对黄瓜盐害的缓解作用之一可能是通过提高叶绿体结合态多胺含量和叶绿体活性氧清除能力,从而缓解盐胁迫对叶绿体膜的伤害.     

Amine levels in Lathyrus sativus seedlings during development

In growing Lathyrus sativus seedlings, the levels of DNA, RNA and protein markedly decreased in the cotyledons and progressively increased in the embryo-axis. In cotyledons, spermidine and spermine contents were substantially reduced while those of agmatine and putrescine were sharply increased. By contrast the embryo-axis progressively accumulated relatively larger amounts of agmatine, homoagmatine. putrescine, cadaverine, spermidine and spermine in parallel with similar changes in its DNA, RNA and protein content. While the cotyledons contained ca 50% of the total agmatine and putrescine present in the plant embryo by day 10, the embryo-axis, though representing less than 20% of the dry wt, contained 90 and 75% of total cadaverine and homoagmatine respectively of the seedlings. Spermidine and spermine levels of this tissue were also comparatively higher, being of the order of 80 and 50% respectively of the total. The root and shoot portions of the embryo-axis also exhibited a similar relationship between changes in DNA, RNA and protein and all the above amines during development. However, the polyamine content of the shoots was relatively higher than those of the roots during the growth period.     

伏令夏橙愈伤组织体细胞胚发生中多胺水平的变化

以继代培养8年的伏令夏橙愈伤组织为材料,研究了不同类型愈伤组织体细胞胚发生能力的差异和多胺水平的变化及两者之间的关系.结果表明,胚性愈伤组织的多胺含量高于非胚性愈伤组织,体细胞胚发生能力与多胺水平呈正相关.体细胞胚发生早期Put含量的增加有利于体细胞胚发生.球形胚大量形成时,Spd达到最高值;球形胚发育后期并有少量心形胚形成时Spm达到峰值.随着倍性的增加,伏令夏橙体细胞胚发生能力降低.精氨酸脱羧酶的活性变化与Put水平呈正相关,表明它是调节伏令夏橙体细胞胚发生中多胺水平的重要因子.     

Differential effects of benzyladenine and potassium on DNA, RNA, protein and chlorophyll contents and on expansion growth of detached cucumber cotyledons in the dark and light

Benzyladenine (BA) and KCl were applied to detached cucumber ( Cucumis sativus L. cv. Ohio) cotyledons in continuous light or in the dark with subsequent light. BA brought about an increase in fresh weight and in DNA, RNA and carotenoid contents in both treatments. KCl did not cause an increase in fresh weight and cellular constituents in the dark, but it did result in an increased fresh weight and DNA content after illumination or in continuous light. BA + KCl treatment resulted in increased carotenoid and DNA contents in the dark, and in increases in fresh weight and all cellular constituents upon subsequent exposure to light. The effects of BA and BA + KCl on growth and chlorophyll synthesis decreased with cotyledon age.
BA pretreatment in the dark eliminated the lag phase in chlorophyll synthesis and increased the rate of synthesis. Treatment in continuous light had little effect. KCl did not shorten the lag phase in chlorophyll synthesis, but it stimulated the rate of synthesis in the light. Dark pretreatment with BA + KCl markedly increased the effect of BA on chlorophyll synthesis. Chlorophyll content and fresh weight were higher in cotyledons treated with BA followed by KCl than in cotyledons treated in the reverse order. These results suggest that growth and greening in cucumber cotyledons are primarily controlled by BA and that KCl intensifies the BA effect after irradiation.     

豌豆茎尖切除对茎和子叶中细胞分裂素含量的影响

豌豆植株去顶后６ｈ,茎中内源Ｚ／ＺＲ和ｉ－Ａｄｅ／Ａｄｏ的含量明显增加,１２ｈ后达到最高值,其后迅速下降、人工合成生长素ＮＡＡ可代替茎尖的作用抑制这些变化。经同样处理后子叶中的ＣＫｓ含量也表现出类似的变化。同时切去茎尖、子叶及根１２ｈ后茎中Ｚ／ＺＲ及ｉ－Ａｄｅ／Ａｄｏ的含量明显低于仅切去茎尖植株的ＣＫｓ含量,但高于ＮＡＡ处理。表明茎尖产生的生长素抑制了茎中ＣＫｓ含量的增加。     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Phytohormonal changes in greening and senescing intact cotyledons of oilseed rape and pumpkin: influence of the growth retardant BAS 111 ˙˙ W

The triazole growth retardant BAS 111‥W delayed senescence in cotyledons of pumpkin ( Cucurbita maxima L. cv. Gelbe genetzte Riesenmelone) and stimulated chlorophyll synthesis in greening cotyledons of oilseed rape ( Brassica napus L. cv. Petranova) seedlings. In both cases, changes of phytohormone-like substances in the cotyledons were analyzed on a fresh weight basis by immunoassay.
After soil treatment with increasing retardant concentrations, a close correlation was observed in senescing cotyledons of pumpkin between a reduced loss in total chlorophyll and increasing levels of dihydrozeatin riboside (DZR) and trans -zeatin riboside (ZR)-type cytokinins. In contrast, the levels of isopentenyladenosine (IPA)-type cytokinins, 3-indoleacetic acid (IAA) and gibberellin (GA) did not change significantly. The levels of abscisic acid (ABA) were slightly elevated at low retardant concentrations but dropped considerably below those of controls at higher doses. Consequently, the molar ratio of total cytokinin to ABA content changed from approximately 1:40 in controls (50% of initial chlorophyll) to 1:3 in cotyledons treated with 3 mg BAS 111‥W plant⁻¹ (85% of initial chlorophyll). These changes, together with the known reduction of ethylene production by plants treated with nitrogen-heterocyclic retardants, can explain the delayed senescence in pumpkin cotyledons. Likewise, when etiolated, BAS 111‥W-treated seedlings of oilseed rape were exposed to light, the stimulation of chlorophyll synthesis in the cotyledons was accompanied by an accumulation of DZR- and, particularly, ZR-type cytokinins and IAA. In contrast, GA and ABA contents decreased slightly. We conclude that the influence of BAS 111‥W on cytokinin levels might be involved in the stimulation of greening.     

Arabidopsis mutant plants with diverse defects in polyamine metabolism show unequal sensitivity to exogenous cadaverine probably based on their spermine content

Arabidopsis plants do not synthesize the polyamine cadaverine, a five carbon-chain diamine and structural analog of putrescine. Mutants defective in polyamine metabolic genes were exposed to exogenous cadaverine. Spermine-deficient spms mutant grew well while a T-DNA insertion mutant (pao4-1) of polyamine oxidase (PAO) 4 was severely inhibited in root growth compared to wild type (WT) or other pao loss-of-function mutants. To understand the molecular basis of this phenomenon, polyamine contents of WT, spms and pao4-1 plants treated with cadaverine were analyzed. Putrescine contents increased in all the three plants, and spermidine contents decreased in WT and pao4-1 but not in spms. Spermine contents increased in WT and pao4-1. As there were good correlations between putrescine (or spermine) contents and the degree of root growth inhibition, effects of exogenously added putrescine and spermine were examined. Spermine mimicked the original phenomenon, whereas high levels of putrescine evenly inhibited root growth, suggesting that cadaverine-induced spermine accumulation may explain the phenomenon. We also tested growth response of cadaverine-treated WT and pao4-1 plants to NaCl and found that spermine-accumulated pao4-1 plant was not NaCl tolerant. Based on the results, the effect of cadaverine on Arabidopsis growth and the role of PAO during NaCl stress are discussed.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

The process of abscisic acid-induced proline accumulation and the levels of polyamines and quaternary ammonium compounds in hydrated barley leaves

The contents of polyamines and quaternary ammonium compounds (QAC), substances involved in several kinds of stress phenomena, were tested in abscisk acid (ABA)-treated barley leaves (Hordeum vulgare L. cv. Georgie) accumulating pro-line. The characteristic parameters of the accumulation of proline induced by ABA [i.e. kinetics of accumulation, synergistic interaction hormone-K(Na)Cl, enhancing effect of Cl-, inhibiting effect of tetraethylammonium chloride (TEA), D-mannose and glucosamine] were tacking as far as polyamine and QAC content was concerned. Moreover: i) ABA slightly decreased the level of spermine and spermidine, slightly increased that of putrescine but did not influence the level of QAC; ii) the content of polyamines was reduced by KCl; iii) treatment with sorbitol increased the level of polyamines and prevented proline accumulation induced by ABA. These results indicate that there is no relationship between ABA-induced proline accumulation, polyamine level and QAC level; furthermore, accumulation of proline by ABA treatment is possible without increasing the levels of polyamines and QAC.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.     

Polyamine Anabolism in Germinating Glycine max (L.) Seeds : Dynamics of Cadaverine and Putrescine Formation in the Embryonic Axis

Active polyamine biosynthesis occurs in the embryonic axis, but not in the cotyledons, during germination of Glycine max (L.) cv Williams seeds and subsequent growth of the young seedlings. The hypocotyl and radicle synthesize and accumulate considerable amounts of cadaverine (Cad) and putrescine (Put) during the early stages of growth. Most of the amino acid precursors for the diamines are supplied from breakdown of the cotyledonary protein.     

Effect of putrescine on the conversion of protocorm-like bodies of <Emphasis Type="Italic">Dendrobium officinale</Emphasis> to shoots

The addition of 1.5 mM putrescine to the culture medium facilitated the conversion of protocorm-like bodies (PLBs) of Dendrobium officinale to shoots. The administration of 1.0 mM of the polyamine inhibitor, α-DL-difluoromethylarginine, decreased the conversion of PLBs to shoots. Compared to the control, the PLBs treated with 1.5 mM putrescine had higher levels of total intracellular free polyamines. The enhanced conversion of PLBs to shoots following the addition of putrescine was accompanied by an increase in the ratio of total cytokinins (CTKs) to indole-3-acetic acid (IAA). Analysis of enzyme activities indicated that the increased endogenous level of total CTKs driven by putrescine was associated with the inhibition of CTK decomposition by CTK oxidase, while the decreased endogenous level of IAA was associated with the promotion of IAA decomposition by IAA oxidase. In addition, putrescine increased the ratio of reduced glutathione to oxidized glutathione in the conversion process of PLBs to shoots.     

Role of polyamines in the cytokinin-dependent physiological processes II. Modulation of polyamine levels during cytokinin-stimulated expansion of cucumber cotyledons

The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyledons. Addition of K⁺ and Ca²⁺ ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity. The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons. The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.