Functional diversity inside the Arabidopsis polyamine oxidase gene family

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.     

Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion

Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.     

Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.     

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.     

干旱胁迫下小麦幼苗根、叶多胺含量和多胺氧化酶活性的变化

 以抗旱性不同的两个小麦品种（‘晋麦33’和‘温麦8’）（Triticum aestivum cv. Jinmai 33 and Wenmai 8）为材料,研究了干旱胁迫下多胺含量和多胺氧化酶活性的变化。结果表明：旱过程中,幼苗根、叶中腐胺（Put）、亚精胺（Spd）、精胺（Spm）3种多胺含量和多胺氧化酶（PAO）活性先迅速升高,而后下降。与抗旱性弱的‘晋麦33’相比,抗旱性强的品种‘温麦8’幼苗根、叶中Spd、Spm 含量初期升高幅度大,之后下降速率减慢;PAO活性的变化与之相反,‘晋麦33’的PAO活性提高的幅度大于‘温麦8号’。多胺含量和PAO活性在小麦幼苗的根与叶之间呈极显著正相关。干旱初期,小麦根、叶中多胺迅速积累可能是干旱胁迫反应的一个信号,随后较高的Spd、Spm 水平有利于增强小麦幼苗的抗旱性。     

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.     

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.     

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

The members of Arabidopsis thaliana PAO gene family exhibit distinct tissue- and organ-specific expression pattern during seedling growth and flower development

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis thaliana, five PAOs (AtPAO1-5) are present with cytosolic or peroxisomal localization. Here, we present a detailed study of the expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 during seedling and flower growth and development through analysis of promoter activity in AtPAO::β-glucuronidase (GUS) transgenic Arabidopsis plants. The results reveal distinct expression patterns for each studied member of the AtPAO gene family. AtPAO1 is mostly expressed in the transition region between the meristematic and the elongation zone of roots and anther tapetum, AtPAO2 in the quiescent center, columella initials and pollen, AtPAO3 in columella, guard cells and pollen, and AtPAO5 in the vascular system of roots and hypocotyls. Furthermore, treatment with the plant hormone abscisic acid induced expression of AtPAO1 in root tip and AtPAO2 in guard cells. These data suggest distinct physiological role(s) for each member of the AtPAO gene family.     

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。     

Nitric oxide enhances salt tolerance in cucumber seedlings by regulating free polyamine content

Nitric oxide (NO), an endogenous signaling molecule in plants and animals, mediates responses to abiotic and biotic stresses. This study was conducted in nutrient solution to investigate the effects of exogenous sodium nitroprusside (SNP), an NO donor, on plant growth and free polyamine content in cucumber leaves and roots under NaCl stress. The results showed that 100 μM SNP in solution significantly improved the growth of cucumber seedlings under NaCl stress for 8 days, as indicated by increased, plant height, stem thickness, fresh weight and increased dry matter accumulation. Further analysis demonstrated that the content of free polyamines and the activity of polyamine oxidase (PAO) in cucumber seedling leaves and roots initially increased dramatically under NaCl stress, although they decreased over a longer period of stress. Throughout the treatment period, the value of (spermine + spermidine)/putrescine [(Spd + Spm)/Put] also decreased under NaCl stress compared to the control. In contrast, the application of 100 μM SNP in the nutrient solution decreased the content of free Put, Spd, total free polyamines and PAO activity under NaCl stress. It also caused an increase in the content of Spm and the value of (Spd + Spm)/Put, adjusted the ratio of three kinds of free polyamines (Put, Spd, Spm) in cucumber seedlings. The high (Spd + Spm)/Put value and the accumulation of Spm were beneficial to improving the salt tolerance of plants. Therefore, NO alleviated the damage to cucumber seedlings caused by salt stress. NO enhanced the tolerance of cucumber seedlings to NaCl stress by regulating the content and proportions of the different types of free polyamines.     

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera . Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90–100 µmol m⁻² s⁻¹). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D- l -α-difluoromethylarginine (DFMA) and methylglyoxal bis (guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.     

氯化钠胁迫对嫁接黄瓜叶片多胺含量的影响

以日本耐盐品种‘帝王新土佐’南瓜为砧木,以’新泰密刺’黄瓜为接穗,在100 mmol·L^-1 NaCl胁迫下,对黄瓜嫁接和自根植株不同时期叶片中不同形态多胺含量的变化进行了研究.结果表明：NaCl胁迫下黄瓜嫁接植株游离态腐胺（Put）含量在胁迫2 d时与自根植株无显著差异,其余时间均显著高于自根植株;游离态亚精胺（Spd）和游离态精胺（Spm）含量在整个胁迫期间均显著高于自根植株;游离态多胺总量（PAs）在胁迫第4天出现峰值;嫁接植株游离态Put/PAs值在胁迫4 d时与自根植株无显著差异,其余胁迫时间均显著低于自根植株,而(Spd+Spm)/Put值在整个胁迫期间均显著高于自根植株;嫁接植株结合态和束缚态Put、Spd和Spm含量在整个胁迫期间均显著高于自根植株,结合态和束缚态PAs在胁迫第6天出现峰值;结合态多胺的Put/PAs值和(Spd+Spm)/Put值变化趋势与游离态多胺一致;嫁接植株束缚态Put/PAs值在胁迫6 d时与自根植株无显著差异,其余时间均显著低于自根植株,而(Spd+Spm)/Put值在整个胁迫期间均显著高于自根植株.表明黄瓜嫁接植株表现出较强的耐盐特征.     

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages – regenerating tissue and differentiated shoots. We observed significant (P<0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P<0.05), and lower levels of Spm (P<0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P<0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P<0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.     

Stress‐induced changes in polyamine and tyramine levels can regulate proline accumulation in tomato leaf discs treated with sodium chloride

The effect of salt stress on proline (Pro) accumulation and its relationship with the changes occurring at the level of polyamine (PA) metabolism and tyramine were investigated in leaf discs of tomato (Lycopersicon esculentum). The rate of accumulation of Pro, PA and tyramine was higher in the salt-sensitive than in the salt-tolerant cultivar. In the salt-sensitive cultivar, Pro started to accumulate 4 h after the onset of the NaCl treatment, its maximum level being reached 27 h later. The lag phase was associated with a rapid decrease in putrescine (Put) and spermidine (Spd) and some increase in 1,3-diaminopropane (Dap), a product of Spd and/or spermine (Spm) oxidation. This was followed by an increase in agmatine (Agm), cadaverine (Cad), Spm and tyramine. α-DL-difluoromethylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), induced a decrease in the Put level in both control and stressed discs, while α-DL-difluoromethylomithine (DFMO), an inhibitor of ornithine decarboxylase (ODC, EC 4.1.1.17), caused a decrease in Spd and Spm levels only in salinized discs. These data suggest that ADC is operating under both control and stress conditions, whereas ODC activity is promoted only in response to salt stress. DFMA also depressed the salt-induced Pro accumulation while DFMO did not inhibit this response. In salt-stressed leaf discs, the decrease in Spd level in response to methylglyoxal-bis-(guanylhydrazone) (MGBG) or cyclohexylammonium (CHA) treatment suggests that salt stress did not block SAM decarboxylase or Spd synthase activities. However, the increased level of Dap reflected a salt stress-promoted oxidation of PA. CHA and MGBG had no effect on Pro accumulation. Putrescine, Dap and especially tyramine supplied at low concentrations stimulated the Pro response which was, however, suppressed by application of Spm. Treatment with aminoguanidine, an inhibitor of diamine oxidases, also strongly inhibited Pro accumulation. These data suggest that salt-induced Pro accumulation in tomato leaf discs is closely related to changes in their PA metabolism, either via substrate-product relationships or regulatory effects at target(s) which remain to be characterized.     

水稻细胞质雄性不育系及其保持系幼穗发育过程中的多胺代谢

在幼穗发育过程中,不育系和保持系幼穗多胺含量先剧降后稳定或略回升,精氨酸脱羧酶活性快速下降,而二胶和多胺氧化酶活性缓慢下降。从雌雄蕊形成期到花粉母细胞形成期,不育系的多胺含量和精氨酸脱羧酶活性明显低于保持系;不过,两系二胺氧化酶和多胺氧化酶活性却差别不大。外施D-Arg抑制两系Put合成,也抑制以Put为前体的Spd的合成;外施MGBG抑制Spd和Spm的合成;同时,D-Arg或MGBG对不育系花粉育性影响不大,但明显降低保持系花粉育性,D-Arg＋MGBG对花粉育性的降低效应更强;Put和pd＋Spm可抵消（或部分抵消）D-Arg和MGBG的降低效应。且Put＋Spd＋Spm能使不育系花粉的育性得以轻度恢复。     

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.