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.     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

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.     

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.     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Exogenous spermidine enhances Hydrocharis dubia cadmium tolerance

The effects of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO), and diamine oxidase (DAO) activities, the rate of superoxide radical (O ₂ ^·? ) generation and polyamine (PA), malondialdehyde (MDA), and H₂O₂ contents in Hydrocharis dubia (Bl.) Backer leaves under cadmium (Cd) toxicity were studied after 6-day treatment. Cd stress increased putrescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels. In addition, the activities of ADC, DAO, and PAO were increased, while that of ODC was decreased. Exogenous application of Spd markedly reversed these Cd-induced effects. It also significantly reduced the generation of O ₂ ^·? and H₂O₂ and prevented lipid peroxidation. These results suggest that exogenous Spd can enhance the tolerance of H. dubia to Cd. The maintenance of PA homeostasis was necessary for plant metal tolerance.     

Polyamine metabolism and physiological responses of <Emphasis Type="Italic">Potamogeton crispus</Emphasis> leaves under lead stress

Aquatic macrophytes were found to be the potential scavengers of heavy metals from aquatic environment. In this study, influences of ladder concentration of lead (Pb) on the leaves of Potamogeton crispus Linn were studied after 7 days of treatment. The accumulation of Pb, nutrient element contents, the generation rate of superoxide radical (O₂^·−), MDA, proline, and polyamine (PAs) contents, as well as the activities of diamine oxidases (DAO), polyamine oxidases (PAO), arginine decarboxylase (ADC), and ornithine decarboxylase (ODC) in P. crispus leaves were investigated. The result indicated that Pb treatment decreased the activity of DAO, whereas the proline content, MDA content, the generation rate of O₂^·− and the activity of ODC increased in different degrees. Meantime, Pb treatment significantly increased the free putrescine (Put) level and made other PAs levels dynamic changes. The activities of PAO and ADC were declined firstly and then enhanced with the increase in the Pb concentration.     

盐胁迫对黄瓜幼苗根系生长和多胺代谢的影响

以两个不同抗盐性黄瓜品种为试材,采用营养液水培法,研究了NaCl胁迫对幼苗根系生长和多胺代谢的影响.结果表明:盐胁迫下黄瓜幼苗根系生长受抑制,膜脂过氧化和电解质渗漏升高,而弱抗盐品种‘津春2号'的变化幅度大于抗盐品种‘长春密刺';盐胁迫下‘长春密刺'根系精氨酸脱羧酶、鸟氨酸脱羧酶和S-腺苷蛋氨酸脱羧酶活性升高幅度均大于‘津春2号',其最高值分别比对照增加了149.3%、60.1%、69.4%和118.6%、56.2%、50.6%;'长春密刺'多胺氧化酶活性升高幅度小于‘津春2号',而二胺氧化酶活性仅在‘长春密刺'中增加.'长春密刺'根系游离态亚精胺和精胺、结合态和束缚态多胺含量均显著增加,而‘津春2号'根系游离态腐胺含量显著增加.表明黄瓜根系中较高的游离态亚精胺和精胺、结合态和束缚态多胺以及较低的游离态腐胺含量有利于提高幼苗对盐胁迫逆境的适应能力.     

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.     

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.     

盐胁迫下大麦幼苗多胺的种类和状态与多胺氧化酶活性的关系

200 mmol/L的NaCl胁迫8 d大麦幼苗叶片和根系中的三种形态多胺都有不同程度地下降,其中游离态多胺含量的下降幅度最大;高氯酸不溶性结合态多胺含量变化较小.根系中PAO的活性先上升后下降,而叶片中PAO的活性先下降后上升.游离态多胺中,亚精胺和精胺(Spd Spm)的含量变化与相应部位PAO的活性变化趋势相反,表明PAO在盐胁迫下可能调节了游离态多胺的含量从而影响高氯酸可溶结合态与高氯酸不溶结合态多胺的含量.     

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.     

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formationand polyamine metabolism was investigated in thin layer explantsof tobacco (Nicotiana tabacum L. cv. Samsun). A relatively lowconcentration of MJ (0.1 µM) enhanced explant fresh weight,but had no effect on the final number of shoots per explantwhile higher concentrations (1 and 10 µM) significantlyinhibited organogenesis. The histological study revealed that,with increasing concentrations of MJ, the formation of meristemoidsand shoot domes declined and the incidence of cell hypertrophyincreased. In explants cultured with 0.1, 1 or 10 µM MJ,the endogenous levels of free putrescine, spermidine and sperminegenerally declined compared with controls, after 7 and 15 d.Perchloric acid-soluble conjugated polyamines accumulated dramaticallyduring culture, but much more so in the presence of MJ thanin controls. Acid-insoluble conjugated spermidine alone increasedin response to the elicitor. Activities of the putrescine biosyntheticenzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithinedecarboxylase (ODC, EC 4.1.1.17) in the soluble fraction ofMJ-treated explants displayed up to 3-fold increases relativeto control explants. However, the most relevant increases inthese enzyme activities occurred in the particulate fraction.The activity of S-adenosylmethionine decarboxylase (SAMDC, EC4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis,was also stimulated by exposure to MJ. Northern analyses revealedMJ-induced, generally dose-dependent, increases in the mRNAlevels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6)activity was stimulated by MJ mainly in the cell wall fraction.The upregulation of polyamine metabolism is discussed in relationto the morphogenic behaviour of MJ-treated explants. Key words: Nicotiana tabacum, thin layers, shoot formation, methyl jasmonate, polyamine metabolism.     

Endogenous enzyme activities and polyamine levels in diverse rice cultivars depend on the genetic background and are not affected by the presence of the hygromycin phosphotransferase selectable marker

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.     

Polyamines,floral induction and floral development of strawberry (Fragaria ananassa Duch.)

In the short-day plant, strawberry (Fragaria ananassa Duch.), polyamines (putrescine, spermidine and spermine), conjugated spermidine (water-insoluble compounds) and bound amines (putrescine, spermidine, phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine) accumulated in the shoot tips during floral induction and before floral emergence. Different associations of free amines and conjugated amines were observed during floral induction, as compared with the reproductive phase. During the whole period of floral development, phenylethylamine (an aromatic amine) was the predominant amine, representing 80 to 90% of the total free amine pool. Phenylethylamine conjugates (water-insoluble compounds) were the predominant amides observed prior to fertilization. These substances decreased drastically after fertilization. In vegetative shoot tips from plants grown continously under long days, free polyamines (putrescine, spermidine) and bound polyamines (putrescine, spermidine) were low and no change was observed. Free amines (spermine and phenylethylamine), bound aromatic amines (phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine), conjugated spermidine and phenylethylamine did not appear. Male-sterile flowers were distinguished by their lack of conjugated spermidine and phenylethyalamine and by a decrease in free phenylethylamine. In normal and sterile strawberry plants -DL-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), caused inhibition of flowering and free and polyamine conjugates. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with -DL-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), did not affect flowering and polyamine titers. These results suggest that ornithine decarboxylase (ODC) and polyamines are involved in regulating floral initiation in strawberry. The relationship between polyamines, aromatic amines, conjugates, floral initiation and male sterility is discussed.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - Put putrescine - Spd spermidine - Spm spermine - Phen phenylethylamine - 3H4M Phen 3-hydroxy, 4-methoxyphenylethylamine     

Effects of Exogenous Manganese (Mn) on Mineral Elements,Polyamines and Antioxidants in Apple Rootstock <i>Malus robusta</i> Rehd.

Manganese (Mn) is one of the essential microelements in all organisms. However, high level of Mn is deleterious to plants. In this study, the effects of exogenous manganese application on mineral element, polyamine (PA) and antioxidant accumulation, as well as polyamine metabolic and antioxidant enzyme activities, were investigated in Malus robusta Rehd., a widely grown apple rootstock. High level of Mn treatments decreased endogenous Mg, Na, K and Ca contents, but increased Zn content, in a Mn-concentration-dependent manner. Polyamine metabolic assays revealed that, except the content of perchloric acid insoluble bound (PIS-bound) spermine, which increased significantly, the contents of putrescine (Put), spermidine (Spd) and spermine (Spm) all decreased progressively, accompanied with the decreased activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), and the increased activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.5.3.3). Further antioxidant capacity analyses demonstrated that contents of anthocyanin, non-protein thiols (NPT) and soluble sugar, and the activities of guaiacol peroxidase (POD, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1), also increased upon different concentrations of Mn treatments. Our results suggest that endogenous ion homeostasis is affected by high level of Mn application, and polyamine and antioxidant metabolism is involved in the responses of M. robusta Rehd. plants to high level of Mn stress.     

Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

Modified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots

ABSTRACT

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the D8 wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurred at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in the basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed.