Xylem and Phloem Derived Polyamines during Flowering in Two Diverse Rose Species

Polyamine contents in xylem (root) and phloem (leaf) exudates in two diverse species of rose, viz. Rosa damascena Mill and Rosa bourboniana Desport, were analyzed before, during, and after flowering in the main flowering season, that is, April–May. Only free putrescine (Put) was detected in the xylem and phloem exudates at these time points, and it was high during the peak flowering period. In phloem, Put content was significantly higher in R. bourboniana than in R. damascena at all three stages; whereas in the xylem exudate it was relatively higher in R. damascena at the peak flowering period. A spray of α-difluoromethylornithine (DFMO), an irreversible inhibitor of the putrescine biosynthetic inhibitor ornithine decarboxylase (ODC), markedly decreased the flowering. This effect was reversed by application of Put alone or in combination with DFMO. The significance of this finding is discussed in light of polyamine translocation during flowering. *IHBT Communication: 0354     

Loading and translocation of various cytokinins in phloem and xylem of the seedlings of Ricinus communis L.

The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明：与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

Localization of ornithine decarboxylase and changes in polyamine content in root meristems of Zea mays

Polyamine content and the activity of arginine decarboxylase (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17) were studied with respect to meristematic activity in primary roots and in developing lateral roots of Zea mays L. (cv. Neve Ya'ar 170) seedlings. Comparative localization of active ornithine decarboxylase and of meristematic activity were determined by labelling roots either with α-[5-¹⁴C]-difluoromethyl ornithine or with [³H]-thymidine, respectively.
Lateral roots were formed during the 72 h post-decapitation period, accompanied by an initial decline in putrescine content and by a significant increase in spennidine con-tent at 48–72 h. High levels of spermidine and lower levels of putrescine were found in the primary root apex as well. A marked increase in ornithine and arginine decarboxylase activity, as measured by ¹⁴CO₂ release, was found during the 72 h post-decapitation period of lateral root development. This increase in ornithine decarboxylase activity was confirmed also by a parallel rise in the incorporation of α-[5-¹⁴C]-difluoromethyl ornithine into trichloroacetic acid-insoluble fractions. Microautoradiographs of longitudinal and cross sections of roots, labelled with α-[5-¹⁴C]-difluoromethyl ornithine, showed that ornithine decarboxylase is localized mainly in the meristematic zones, as evidenced by [³H]-thymidine incorporation. A close correlation between meristematic activity and polyamines was demonstrated in situ , suggesting that polyamine content and biosynthesis may have a role in meristematic activity in corn roots.     

Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (<Emphasis Type="Italic">Picea rubens</Emphasis> Sarg.)

Summary The major objective of this study was to determine if the observed changes in polyamines and their biosynthetic enzymes during somatic embryo development were specifically related to either the stage of the embryo development or to the duration of time spent on the maturation medium. Somatic embryos of red spruce (Picea rubens) at different developmental stages, grown in the embryo development and maturation media for various lengths of time, were separated from the associated subtending tissue (embryogenic and the suspensor cell masses) and analyzed for their polyamine content as well as for polyamine biosynthetic enzyme activities. Polyamine content was also analyzed in embryos representing different stages of developmentthat were collected from the sam culture plate at the same time and the subtending tissue surrouding them. Putrescine was the predominant polyamine in the pro-embryogenic tissue, while spermidine was predominant during embryo development. Significant changes in spermidine/putrescine and spermine/putrescine ratios were observed at all stages of embryo development as compared to the pro-embryogenic cell mass. Changes in the ratios of various polyamines were clearly correlated with the developmental stage of the embryo rather than the period of growth in the maturation medium. Whereas the activities of both ornithine decarboxylase and arginine decarboxylase increased by week 3 or 4 and stayed high during the subsequent 6 wk of growth, the activity of S-adenosylmethionine decarboxylase steadily declined during embryo development.     

嫁接对铜胁迫下黄瓜幼苗根系多胺代谢的影响

采用营养液栽培法,研究了嫁接(以黑籽南瓜为砧木)对铜胁迫下黄瓜幼苗根系活力及多胺代谢的影响.结果表明:铜胁迫下黄瓜幼苗根系活力下降,电解质渗漏率升高,而嫁接苗的变化幅度显著小于黄瓜自根苗;铜胁迫下黄瓜嫁接植株根系中除游离态腐胺(Put)含量显著低于自根苗外,结合态和束缚态Put、3种形态亚精胺(Spd)和精胺(Spm)含量均显著高于自根苗,嫁接苗根系中游离态Put含量及腐胺/多胺(Put/PAs)显著低于自根苗;铜胁迫下,嫁接苗根系精氨酸脱羧酶(ADC)、鸟氨酸脱羧酶(ODC)和S-腺苷蛋氨酸脱羧酶(SAMDC)活性高于自根苗,而二胺氧化酶(DAO)和多胺氧化酶(PAO)活性显著低于自根苗.表明嫁接黄瓜幼苗根系PAs的合成增加,降解减少,使PAs含量维持在较高水平,从而提高了黄瓜幼苗抗铜胁迫能力.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Involvement of polyamines in root development at low temperature in the subantarctic cruciferous species Pringlea antiscorbutica

Polyamine involvement in root development at low temperature was studied in seedlings of Pringlea antiscorbutica R. Br. This unique endemic cruciferous species from the subantarctic zone is subjected to strong environmental constraints and shows high polyamine contents. In the present study, free polyamine levels were modified by inhibitors of polyamine biosynthesis (D-arginine, difluoromethylornithine, cyclohexylammonium, and methylglyoxal-bis-guanylhydrazone) and variations of the endogenous pools were compared to changes in root growth. The arginine decarboxylase pathway, rather than that of ornithine decarboxylase, seemed to play a major role in polyamine synthesis in Pringlea antiscorbutica seedlings. Root, but not shoot, phenotypes were greatly affected by these treatments, which modified polyamine endogenous levels according to their expected effects. A positive correlation was found between agmatine level and growth rate of the primary root. Spermidine and spermine contents also showed positive correlations with primary root growth whereas the putrescine level showed neutral or negative effects on this trait. Free polyamines were therefore found to be differentially involved in the phenotypic plasticity of root architecture. A comparison of developmental effects and physiological concentrations suggested that agmatine and spermine in particular may play a significant role in the control of root development.     

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.     

Polyamine Metabolism in the Roots of Phaseolus vulgaris. Interaction of the Inhibitors of Polyamine Biosynthesis with Putrescine in Growth and Polyamine Biosynthesis

The effects of the inhibitors of polyamine biosynthesis, canavanineand -methyl ornithine on growth, the activities of argininedecarboxylase (EC 4.1.1.19 [EC] ) and ornithine decarboxylase (EC4.1.1.17 [EC] ) and on polyamine content were examined in two differentgrowth regions of Phaseolus vulgaris L. cv. Taylor's Horticulturalroots. Separately, in the same manner, in the same bean rootsystem exogenous putrescine effect and the interaction of canavaninewith putrescine were determined. The arginine and ornithine decarboxylase activities found inroot apex were high where cell division activity was highest.Polyamine (putrescine and spermine) content did not correlatewith these activities, but polyamine level was high in the rootbase where cell elongation is the main process. The arginineanalogue, canavanine, inhibited arginine decayboxylase activityand polymine liters. Putrescine partially reversed the canavanineinhibition of root growth as well as arginine decarboxylaseactivity and polyamine content. Similarly -methyl ornithineslightly inhibited the root length and ornithine decarboxylaseactivity in the root apex. Besides, exogenous putrescine didnot effect significantly the endogenous polyamine titers. Theseresults reinforce the growing connection between polyaminesand the rates of cell devision in the roots of bean plants.Separately, arginine decarboxylase is the main enzyme in thebean roots. (Received November 10, 1986; Accepted March 3, 1987)     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明:与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片乌氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.