Physiological and biochemical responses induced by lead stress in Spirodela polyrhiza

The effects of increasing lead concentration on the activities of superoxide dismutase (SOD), peroxides (POD) and catalase (CAT), levels of ascorbate (AsA), reduced glutathione (GSH), Pb accumulation and its influence on nutrient elements, polyamines (PAs) content, as well as activities of polyamine oxidase (PAO) and ornithine decarboxylase (ODC), were investigated in Spirodela polyrhiza. POD and CAT activities increased progressively followed by a decline, while SOD activity gradually fell. The effect of Pb application on AsA content was similar to that seen for POD and CAT activities. GSH content initially rose but then declined. A significant enhancement in Pb accumulation was observed, except in the 25?μM Pb treatments. Nutrient elements were also affected. Moreover, Pb stress induced a considerable decrease in total spermidine (Spd), while the levels of total putrescine (Put) and spermine (Spm) initially increased at 25?μM Pb but then declined. Free and perchloric acid soluble conjugated (PS-conjugated) PAs contents changed in a similar way to total PAs. In addition, Pb stress induced a continuous accumulation of perchloric acid insoluble bound (PIS-bound) Spm and an initial accumulation of PIS-bound Put and Spd. The ratio of free (Spd?+?Spm)/Put significantly declined whereas the ratio of total (Spd?+?Spm)/Put rose at low Pb concentrations (25 and 50?μM). PAO activity rose gradually with an increase in Pb concentration, reaching peak values at 100?μM, while ODC activity first increased at 25?μM Pb and then declined. The results indicated that the tolerance of S. polyrhiza to Pb stress was enhanced by activating the antioxidant system, preventing the entry of the Pb ion and altering the content of polyamines.     

外源多胺对铜胁迫下水鳖叶片多胺代谢、抗氧化系统和矿质营养元素的影响

采用营养液水培的方法,研究了外源亚精胺(Spd)和精胺(Spm)对Cu胁迫下水鳖叶片3种形态多胺(PAs)、抗氧化系统及营养元素的影响。结果表明:(1)Cu胁迫使水鳖叶片腐胺(Put)急剧积累,Spd和Spm明显下降,从而使(Spd+Spm)/Put比值也随之下降。外源Spd和Spm显著或极显著逆转Cu诱导的PAs变化,抑制Put的积累,缓解Spd和Spm的下降,从而提高了(Spd+Spm)/Put比值。(2)外源Spd和Spm抑制了Cu胁迫诱导的多胺氧化酶(PAO)的增加,缓解了二胺氧化酶(DAO)的下降。(3)与单一Cu胁迫相比,Spd和Spm显著或极显著提高了超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和抗坏血酸(AsA)、谷胱甘肽(GSH)、游离脯氨酸(Pro)含量,从而降低了超氧阴离子(O2.-)产生速率和过氧化氢(H2O2)含量,极显著降低了丙二醛(MDA)含量,缓解了Cu诱导的氧化胁迫。(4)外源Spd和Spm显著或极显著缓解了Cu胁迫下矿质营养元素吸收平衡的紊乱。以上结果均说明了外施Spd和Spm可增加水鳖对Cu胁迫的耐受性。     

Putrescine overproduction does not affect the catabolism of spermidine and spermine in poplar and Arabidopsis

The effect of up-regulation of putrescine (Put) production by genetic manipulation on the turnover of spermidine (Spd) and spermine (Spm) was investigated in transgenic cells of poplar (Populus nigra × maximowiczii) and seedlings of Arabidopsis thaliana. Several-fold increase in Put production was achieved by expressing a mouse ornithine decarboxylase cDNA either under the control of a constitutive (in poplar) or an inducible (in Arabidopsis) promoter. The transgenic poplar cells produced and accumulated 8–10 times higher amounts of Put than the non-transgenic cells, whereas the Arabidopsis seedlings accumulated up to 40-fold higher amounts of Put; however, in neither case the cellular Spd or Spm increased consistently. The rate of Spd and Spm catabolism and the half-life of cellular Spd and Spm were measured by pulse-chase experiments using [¹⁴C]Spd or [¹⁴C]Spm. Spermidine half-life was calculated to be about 22–32 h in poplar and 52–56 h in Arabidopsis. The half-life of cellular Spm was calculated to be approximately 24 h in Arabidopsis and 36–48 h in poplar. Both species were able to convert Spd to Spm and Put, and Spm to Spd and Put. The rates of Spd and Spm catabolism in both species were several-fold slower than those of Put, and the overproduction of Put had only a small effect on the overall rates of turnover of Spd or Spm. There was little effect on the rates of Spd to Spm conversion as well as the conversion of Spm into lower polyamines. While Spm was mainly converted back to Spd and not terminally degraded, Spd was removed from the cells largely through terminal catabolism in both species.     

低氧胁迫下24-表油菜素内酯对黄瓜幼苗叶片光合特性及多胺含量的影响

采用1/2 Hoagland营养液培养,研究了低氧胁迫下24-表油菜素内酯(EBR)对黄瓜幼苗叶片光合特性及多胺含量的影响.结果表明：低氧胁迫下黄瓜幼苗的净光合速率(P_n)、气孔导度(g_s)、蒸腾速率(T_r)、胞间CO₂浓度(C_i)显著下降,而叶绿素含量显著提高,幼苗生长受抑;低氧胁迫显著提高了黄瓜幼苗叶片的腐胺(Put)、亚精胺(Spd)、精胺(Spm)、多胺(PAs)含量和Put/PAs,但降低了(Spd+Spm) /Put.低氧胁迫下,外源EBR不仅显著提高了黄瓜幼苗的P_n、g_s、T_r及叶绿素含量,也显著提高了黄瓜幼苗叶片的游离态Spm、结合态Spd、Spm及束缚态Put、Spd、Spm含量,促进了PAs的进一步积累,且降低了Put/PAs,提高了(Spd+Spm)/Put．可见,外源EBR调节了黄瓜幼苗内源多胺含量及形态的变化,维持了较高的光合性能,促进了叶面积和干物质量的显著增加,缓解了低氧胁迫对黄瓜幼苗的伤害.     

Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine

Distribution of biogenic amines—the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)—differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (α and β), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd–Spm are positive regulators of cellular amino acid metabolism.     

盐适应过程中香根草内源游离态、结合态、束缚态多胺含量的变化

研究了不同浓度NaCl胁迫下,香根草（Vetiteria zizanioides）根、叶中的游离态、结合态、束缚态多胺（PAs）[包括腐胺（Put）,尸胺（Cad）,亚精胺（Sod）和精胺（Spm）]含量的变化。在中度盐胁迫（100,200mmol L^-1NaCl）9天时,香根草基本能够正常生长,但在重度盐胁迫（300mmol L^-1NaCl）下,其生长受到严重抑制。在上述3个不同浓度的NaCl胁迫下,香根草根、叶中游离态Put,Cad,spd,Stma和总的游离态PAs含量明显下降,在高盐浓度下下降幅更大;结合态Put,Cad,Sod,Spm和总的结合态PAs含量显著上升,但在重度盐胁迫下升幅较小或与对照相当;束缚态Put,Cad和总的束缚态PAs含量均减少,而束缚态Spd和Spm含量在叶中是下降的,在根中则增加,且在中度盐胁迫下更明显。对根和叶片而言,除游离态（Spd＋Spm）,Put比值在重度盐胁迫下较对照显著下降外,其它游离态、结合态、束缚态和总的（Spd＋Spm）／Put比值在不同盐胁迫下均上升,在中度盐胁迫下更明显。这表明,维持多胺总量的稳态和较高的（Spd＋Spm）／Put比值是香根草适应中度盐胁迫的一个重要机制。     

Stress-dependent accumulation of spermidine and spermine in the halophyte Mesembryanthemum crystallinum under salinity conditions

We studied the effects of chloride salinity (300 and 500 mM NaCl) on the content of free polyamines (PAs) from putrescine (Put) family in Mesembryanthemum crystallinum L. leaves and roots. The contents of Put and spermidine (Spd) in leaves increased temporarily, achieving the highest values on the third day of salinity treatment; thereafter (by days 7–14), they dropped sharply. The content of spermine (Spm) increased gradually, and its high level was maintained until the end of experiment. The dynamics of Spm accumulation in leaves under salinity conditions resembled that of phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the water-saving CAM pathway of photosynthesis. This indicates indirectly the involvement of Spm in the common ice plant adaptation to salinity. A decrease in the molar ratios of Spd to Spm in the leaves under salinity conditions could point to the acceleration of Spm biosynthesis (accumulation) during plant adaptation, whereas the levels of Spm precursors, Put and Spd, did not increase. This phenomenon could be explained by an accelerated conversion of Spd into Spm, an active liberation of free Spm from its conjugates, or changes in the rates of studied PA biosynthesis and degradation under salinity. At the same time, the intracellular concentration of ethylene rose under these conditions. It was supposed and then demonstrated, that the pathway of ethylene biosynthesis and that of the synthesis of Put family PAs compete under severe salinity conditions. This competition might be based on the disturbances in sulfur metabolism and a decrease in the methionine content, an immediate precursor of S-adenosyl-L-methionine.     

Effect of root-applied spermidine on growth and respiratory metabolism in roots of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings under hypoxia

The effects of exogenous spermidine (Spd) application to hypoxic nutrient solution on the contents of endogenous polyamines (PAs) and respiratory metabolism in the roots of cucumber (Cucumis sativus L.) seedlings were investigated. Cucumber seedlings were grown hydroponically in control and hypoxic nutrient solutions with and without addition of Spd at a concentration of 0.05 mM. The activities of key enzymes involved in the tricarboxylic acid cycle (TCAC), such as succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH), were significantly inhibited under root-zone hypoxia with dissolved oxygen (DO) at 1 mg/l. In contrast, the activities of enzymes involved in the process of fermentation, such as pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and alanine aminotransferase (AlaAT), were significantly increased. Thus, aerobic respiration was inhibited and fermentation was enhanced in the roots of cucumber seedlings as a result of decreasing ATP content to inhibit the dry weight of seedlings under hypoxic stress. Moreover, the contents of free, soluble conjugated, and insoluble bound putrescine (Put), Spd, and spermine (Spm) in the roots of cucumber seedlings were significantly increased under hypoxia stress. Interestingly, application of Spd to hypoxic roots markedly suppressed the accumulation of free Put and, in contrast, promoted an increase in free Spd and Spm, as well as soluble conjugated and insoluble bound Put, Spd, and Spm contents. From these data, we deduced that exogenous Spd promotes the conversion of free Put into free Spd and Spm, and soluble conjugated and insoluble bound PAs under hypoxia stress. Furthermore, the activities of LDH, PDC, and ADH were suppressed and, in contrast, the activities of SDH and IDH were enhanced by application of exogenous Spd to hypoxic roots. As a result, aerobic respiration was enhanced but fermentation metabolism was inhibited in the roots of cucumber seedlings, leading to an increase in ATP content to alleviate the inhibited dry weight of seedlings due to hypoxia stress. These results suggest that application of Spd to hypoxic nutrient solution promoted conversion of free Put into free Spd and Spm as well as soluble conjugated and insoluble bound PAs, further enhanced IDH and SDH activities, and inhibited ethanol fermentation and lactate fermentation, resulting in increased ATP content and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

水分胁迫下山黧豆多胺代谢与β_N_草酰_L_α,β_二氨基丙酸积累相关性的研究

Seedlings of grass pea (Lathyrus sativus L.) were treated with polyethylene glycol (PEG), in addition with putrescine (Put) and α-difluoromethylarginine (DFMA) in order to study the relation between polyamine metabolism and accumulation of β-N-oxalyl-L-α, β-diaminopropionic acid (ODAP) under water stress. The results indicated that Put, spermidine (Spd) and spermine (Spm) contents of leaves gradually increased along with the extension of PEG-treatment time. Especially, the increase in Spm contents was significant together with a gradual increase of ODAP contents. Put and Spd contents of leaves markedly increased along with the extension of PEG+Put treatment time. There was little effect on Spm contents and ODAP contents in leaves with PEG+Put treatment. The accumulation of Put, Spd, Spm and ODAP in leaves was inhibited by PEG+DFMA treatment. The inhibition of DFMA for Put and Spd biosynthesis was partially overcome by adding exogenous Put, but little effect for the inhibition of DFMA on Spm biosynthesis by adding exogenous Put. The accumulation of ODAP was inhibited with PEG+DFMA+Put treatment in leaves. This indicated that there was a relationship between the increase of Spm contents and the accumulation of ODAP in leaves under water stress.     

Exogenous polyamines enhance copper tolerance of Nymphoides peltatum

The protective effects of polyamines (PAs) against copper (Cu) toxicity were investigated in the leaves of Nymphoides peltatum. Cu treatment increased the putrescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put ratio in leaves. Exogenous application of Spd or Spm markedly reversed these Cu-induced effects for all three PAs and partially restored the (Spd+Spm)/Put ratio in leaves. It also significantly enhanced the level of proline, retarded the loss of soluble protein, decreased the rate of O2*- generation and H2O2 content, and prevented Cu-induced lipid peroxidation. Furthermore, exogenous Spd and Spm reduced the accumulation of Cu and effectively maintained the balance of nutrient elements in plant leaves under Cu stress. These results suggest that exogenous application of Spd or Spm can enhance the tolerance of N. peltatum to Cu by increasing the levels of endogenous Spd and Spm as well as the (Spd+Spm)/Put ratio.     

水分胁迫对小麦幼苗叶片多胺含量的影响

水分胁迫下小麦幼苗叶片多胺含量变化的研究表明,聚乙二醇(PEG)的渗透胁迫明显提高了抗旱性强的周麦系列幼苗叶片游离态Put、Spd和Spm的含量和抗旱性弱的温麦6号的Put含量。外源Spd显著提高了水分胁迫下温麦6号的Spd的含量,对其抗性也有所改善。外源MGBG(Spd和Spm生物合成抑制剂)可提高水分胁迫下周麦12号Put的含量,但降低了Spd和Spm的含量和幼苗的抗性。     

Shoot-applied polyamines suppress nodule formation in soybean (Glycine max)

In legumes, the number of root nodules is controlled by a mechanism called autoregulation. Recently, we found that the foliar brassinosteroid (BR), a plant growth-regulating hormone, systemically regulates the nodule number in soybean plants. In the present study we report that such down-regulation of root nodule formation by a BR may occur through a change of the polyamine contents, with the experimental evidence as follows. The foliar contents of both spermidine (Spd) and spermine (Spm) in the super-nodulating soybean mutant, En6500, were always lower than those in its parent line, Enrei. This lower Spd and Spm content accompanied a striking accumulation of putrescine (Put) in the former plant. This finding indicates that Spd and Spm biosynthesis from their precursor Put is repressed in En6500. The foliar treatments with Spd or Spm of En6500 led to a reduction of both nodule number and root growth. On the other hand, foliar treatment with MDL74038, a specific inhibitor of Spd biosynthesis, apparently increased the root nodule number in Enrei. Foliar application of brassinolide (BL) of En6500 increased the leaf Spd level and reduced the nodule number. These results suggested that BL-induced Spd synthesis in shoots might suppress the root nodule formation.     

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.     

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (≥97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds.     

多胺及其合成抑制剂MGBC对稀脉萍成花反应的影响

腐胺、亚精胺和精胺对稀脉萍的成花均有一定的抑制作用,这种作用随多肢的浓度增加而增强。多胺合成抑制剂MGBG强烈抑制稀脉萍群体的增殖速率,并使稀脉萍群体在非诱导光周期下开花。这种由MGBG引起的增殖速率的降低及成花诱导作用均可被多胺逆转。稀脉萍成花诱导过程中,内源腐胺含量显著升高,亚精胺则下降。     

Effects of methylglyoxal-bis (guanylhydrazone) and abscisic acid on polyamine metabolism in embryonectomized barley seeds

Incorporation of L-[U-¹⁴C] arginine or L-[U-¹⁴C] ornithine into putrescine (Put), spermidine (Spd) and spermine (Spm) in embryonectomized barley seeds (Hordeum vulgare L. cv. Himalaya) was studied following imbition with methylglyoxal-bis (guanylhydrazone) (MGBG) and abscisic acid (ABA). Both radiolabeled amino acids were incorporated into the amines as a result of active polyamine biosynthesis in the seed during imbibition. In the aleurone layer, the Spd and Spn existed mainly in the free form (acid soluble). However about 50% of Put was recovered in conjugated form(s) (acid insoluble). Imbibition with 5 and 10M ABA for 3 days increased the accumulation of the free form of ¹⁴C-Put, probably as a result of inhibition (70%) of ¹⁴C-Spd accumulation. The ABA treatment showed no significant effect on levels of the conjugated form of Put and Spd. Imbibition with millimolar concentrations of MGBG resulted in (i) abnormal accumulation of the free form of Put and incorporation of ¹⁴C-amino acids into the diamine, (ii) progressive inhibition of the accumulation of the free forms of ¹⁴C-Spd and Spm, and (iii) reduction of the ¹⁴C incorporation into the conjugated forms of Put and Spd. Uptake of ¹⁴C-amino acids was not affected by MGBG treatment. The results indicate that MGBG may inhibit not only the synthesis of Spd and Spm, but the catabolism (e.g. oxidation) of Put in the aleurone layer.This paper is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Polyamines in marine macroalgae: Levels of putrescine, spermidine and spermine in the thalli and changes in their concentration during glycerol-induced cell growth in vitro

The levels of putrescine (Put), spermidine (Spd) and spermine (Spm) were analyzed in naturally collected samples of the marine macroalgae Dyctiota dichotoma, Gelidium canariensis and Grateloupia doryphora . Polyamines (PAs) appeared in free (35–134 μg g⁻¹ fresh weight) and bound TCA-insoluble form (1 667–2 624 μg g⁻¹ fresh weight). Axenic in vitro cultures of sporelings from G. doryphora were established in the medium containing glycerol. This medium promoted growth and morphogenesis and also increased the free and bound PA levels in the sporelings. Tracer experiments using 70 kBq [U-¹⁴C]-glycerol showed significant quantities of radioactivity in Put, Spd and Spm after 20 h of incubation. The effects of glycerol on growth were inhibited by the ornithine decarboxylase (EC 4.1.1.17) inhibitor α -difluoromethylornithine (DFMO). The presence of DFMO in the incubation medium with [U-¹⁴C]-glycerol also reduced the radioactivity in PAs.     

External spermine prevents UVA-induced damage of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 via increased catalase activity and decreased H<Subscript>2</Subscript>O<Subscript>2</Subscript> and malonaldehyde levels

Common polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), are cationic compounds known as beneficial factors for many cellular processes including cell division, proliferation, differentiation, and stress response in all living organisms. Effects of exogenous Spm on the protective responses of Synechocystis sp. PCC 6803 exposed to UVA were investigated. The presence of 0.5 mM Spm in the culture medium significantly reduced cell growth after 60 min under white light condition but protected the cells after growing for 60 min under UVA. The stress-tolerant response of Synechocystis cells represented by the ratio of putrescine/spermidine (Put/Spd) showed about a 6-fold increase after 60 min UVA in the presence of Spm. In addition, those levels of chlorophyll a, carotenoids, and photosynthetic oxygen evolution were increased by Spm supplementation in UVA-treated cells. Exogenous Spm induced the activity of catalase but not superoxide dismutase in cells under UVA treatment. On the other hand, Spm treatment enabled cells to apparently decrease the intracellular free radical H₂O₂ and malonaldehyde (MDA) levels. Overall results suggested that Spm supplementation could protect Synechocystis sp. PCC 6803 cells via the increase of Put/Spd ratio and the reduction of both H₂O₂ and MDA levels in conjunction with the induction of catalase activity. Interestingly, UVA-treated cells as compared to non-treated cells with exogenous Spm showed a decrease of Spm with an increase of Put and no change in Spd. This suggested the back conversion of Spm to Spd and finally to Put as cellular mechanism in response to UVA.     

Changes in free polyamine titers and expression of polyamine biosynthetic genes during growth of peach <Emphasis Type="Italic">in vitro</Emphasis> callus

In the present paper, correlation between free polyamines and growth of peach (Prunus persica cv. Yuzora) in vitro callus was investigated. Growth of the callus was divided into three phases based on measurement of fresh weight. Free polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), could be detected during peach callus growth. Changes in free Put titers followed the callus growth rate, as shown by low and stable levels in the first stage, quick increase at the beginning of the second phase, and slow increase in the last phase, whereas fluctuations of Spd and Spm titers were aberrant from that of Put at early stage. Expressions of five key genes involved in polyamine biosynthesis were characterized, in which only the genes leading to Put synthesis, ADC (arginine decarboxylase) and ODC (ornithine decarboxylase), agreed with callus growth and fluctuation of Put titers. Treatment of the callus with D-arginine, an inhibitor of ADC, led to significant growth inhibition and enormous reduction of endogenous Put, coupled with obvious decrease of mRNA levels of ADC and ODC. Exogenous application of Put partially restored the callus growth, along with resumption of endogenous Put and expression levels of ADC and ODC. Spd and Spm titers experienced minor change in comparison to Put. The data presented here suggested that free Put played an important part in peach callus growth. Putative mechanisms or mode of action underlying the role of Put in peach callus growth and different expression patterns of the genes responsible for polyamine biosynthesis are also discussed.