Regulation of arginine decarboxylase by spermine in osmotically-stressed oat leaves

Biosynthesis of polyamines in plants is controlled primarily by the enzymes ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (ADC: EC 4.1.1.19), which are responsible for the production of putrescine, and S -adenosyl-L-methionine (SAM) decarboxylase (EC 4.1.1.50) that is necessary for the formation of spermidine and spermine (Spm). Little is known about the metabolic or molecular mechanisms regulating the synthesis of these enzymes. We have studied the regulation of ADC synthesis by Spm in osmotically-stressed oat ( Avena sativa L. ev. Victory) leaves, using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic stress resulted in increased steady-state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation is explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a consequent decrease in the ADC-processed form. Spermine treatment leads to delayed loss of chlorophyll in dark-incubated and osmotically-treated oat leaves. Thus, post-translational regulation of ADC synthesis by Spm may be important in explaining its anti-senescence properties.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

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.     

Polyamine concentrations and arginine decarboxylase activity in wheat exposed to osmotic stress

The activity of L-arginine decarboxylase (ADC: EC 4.1.1.19)and polyamine content were examine in intact wheat plants ( Triticum aestivum L. cv. Sappo) exposed to osmotic stress (0.4 M mannitol) for 5 days. ADC activity was increased in first and second leaves and in roots of mannitol-stressed plants. Concentrations of putrescine, cadaverine and spermine were generally increased in leaves and roots of plants exposed to mannitol, whereas spermidine was reduced in first leaves and roots of these plants. In an attempt to determine the localization of mannitol in stressed wheat. ¹⁴C-mannitol was fed to plants grown in liquid culture. Most of the mannitol was detected in roots (84%), while small amounts were found in first (9%) and second (7%) leves.
Since it seemed possible that some of the effects on polyamine metabolism caused by exposure to mannitol could have been the result of water stress. polyamine metabolism was also studied in plants water stressed by exposure to 2% polyethylene glycol (PEG) 4000. ADC activity was not altered by exposure to PEG. but concentrations of putrescine, spermidine and spermine were generally reduced in leaves and roots of stressed plants. Cadaverine concentrations were not significantly affected by exposure to PEG. Spermidine and spermine concentrations were reduced in first and second leaves but remained unchanged in roots of plants exposed to PEG.     

Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Avena sativa L.) leaves

The uptake of [1-³H]geranylgeranyl diphosphate (GGPP) into protoplasts and intact etioplasts and the metabolic interconversion therein was studied after a 2 min pulse of white light. The chlorophyll synthetase reaction, Chlide+GGPPChl_GG, was taken as a natural probe for the etioplast compartment. This reaction yields labeled ChL_GG and, by hydrogenation, labeled Chl_P, when [1-³H]GGPP receives access to the etioplast stroma. It was found that penetration across the plastid envelope was rapid and that penetration across the plasma membrane of protoplasts, however, was slow. A cellular pool of soluble GGPP was detected. This pool was lost, in part, during preparation of the protoplasts and almost completely during preparation of the etioplasts. The membrane-bound phytol pool of etioplasts could not be replaced by exogenous [³H]GG. The endogenous GG and phytol pools of protoplasts, which were larger than those of etioplasts, could be replaced in part by exogenous [³H]GGPP. That part of this pool exists as soluble GGPP or as a direct precursor in the cytoplasm is discussed.Abbreviations GGPP geranylgeranyldiphosphate - Chl_GG geranylgeranyl chlorophyllide a - Chl_P phytyl chlorophyllide a - IPP isopentenyl diphosphate - Chlide chlorophyllide a     

外源亚精胺对盐胁迫下毕氏海蓬子体内多胺含量的影响

用含不同浓度(0、100、200、300、400和500 mmol·L 1)NaCl的Hoagland营养液及叶面喷施0.1 mmol·L-1外源亚精胺(Spd)处理毕氏海蓬子幼苗,研究外源Spd对NaCl胁迫下海蓬子体内游离态、结合态和束缚态3种形态多胺含量的影响,分析内源多胺含量的变化与植物耐盐性的关系.结果表明:(1)随盐胁迫浓度的升高,海蓬子幼苗叶片中3种形态腐胺(Put)含量均先降后升;同期的游离态Spd含量持续上升,结合态和束缚态Spd含量均先升后降;同期的游离和结合态精胺(Spm)含量均先升后降,而其束缚态Spm含量呈上升趋势;游离态和束缚态多胺(PAs)总量变化随盐浓度升高均呈上升趋势,而结合态PAs总量先升后降.(2)3种形态(Spd+ Spm) /Put比值均先升后降,而3种形态Put/PAs比值则均呈先降后升的相反趋势.(3)外源Spd处理提高了海蓬子幼苗叶片中结合态和束缚态PAs总量,也提高了游离态和束缚态(Spd+Spm)/Put比值.研究发现,外源Spd参与了NaCl胁迫下海蓬子内源PAs代谢的调节,可能通过促进盐胁迫植株中Put向Spd和Spm的转化,以及游离态多胺向结合态和束缚态多胺的转化来增强海蓬子耐盐性.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Polyamines inhibit lipid peroxidation in senescing oat leaves

Exogenous diaminopropane, spermidine, spermine and guazatine, an inhibitor of polyamine oxidase (EC 1.5.3.3) activity, were able to inhibit loss of chlorophyll and reduce the level of malondialdehyde in dark-incubated Avena sativa L. cv. Victory leaves, as well as in leaves subjected to osmotica used for protoplast isolation. Both lipoxygenase (EC 1.13.11.12) activity and enzyme-protein levels were reduced by incubation with spermine. The results provide support for the contention that the inhibition of lipid peroxidation by polyamines may be one of the mechanisms responsible for their anti-senescence effects.     

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.     

Spermidine and methylglyoxal bis(guanylhydrazone) affect maturation and endogenous polyamine content of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and methylglyoxal bis(guanylhydrazone) (MGBG), a putative inhibitor of Spd synthesis, improved somatic embryo formation of Scots pine (Pinus sylvestris L.). The induced maturation due to MGBG and Spd was accompanied by significantly retarded proliferation growth and by reduction in the concentration of free polyamines compared to the control cultures. The action of MGBG revealed that it has a non-specific effect on the whole polyamine metabolism of Scots pine. Furthermore, at certain concentrations it may induce plant differentiation as well.     

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.     

Plant Polyamines in Reproductive Activity and Response to Abiotic Stress*

In this review we will focus on two areas in which the accumulated evidence for a critical physiological role of polyamines is becoming compelling, i.e. reproductive activity and response to abiotic stress. Regarding reproductive behavior, it seems clear that polyamines are members of the array of internal compounds required for flower initiation, normal floral organ morphogenesis, fruit growth and fruit ripening in particular plant species. Abiotic stresses such as osmotic stress can “turn on” arginine decarboxylase (ADC) genes, resulting in a rapid increase in their mRNA levels. Localization of ADC enzyme in the chloroplast suggests a role of PAs in the maintenance of photosynthetic activity during senescence responses induced by osmotic stress. We envisage that the use of transgenic plants and improved molecular probes will resolve in the near future the physiological significance of stress-induced changes in PA metabolism as well as the role of these compounds in reproductive activity.     

Hydrogen peroxide pretreatment induces osmotic stress tolerance by influencing osmolyte and abscisic acid levels in maize leaves

Hydrogen peroxide (H₂O₂) functions as a signal molecule in plants under abiotic and biotic stresses. Leaves of detached maize (Zea mays L.) seedlings were used to study the function of H₂O₂ pretreatment in osmotic stress resistance. Low H₂O₂ concentration (10 mM) which did not cause a visual symptom of water deficit (leaf rolling) was applied to the seedlings. Exogenous H₂O₂ alone increased leaf water potential, endogenous H₂O₂ content, abscisic acid (ABA) concentration, and metabolite levels including soluble sugars, proline, and polyamines while it decreased lipid peroxidation and stomatal conductance. Osmotic stress induced by polyethylene glycol (PEG 6000) decreased leaf water potential and stomatal conductance but enhanced lipid peroxidation, endogenous H₂O₂ content, the metabolite levels, and ABA content. H₂O₂ pretreatment also induced the metabolite accumulation and improved water status, stomatal conductance, lipid peroxidation, ABA, and H₂O₂ levels under osmotic stress. These results indicated that H₂O₂ pretreatment may alleviate water loss and induce osmotic stress resistance by increasing the levels of soluble sugars, proline, and polyamines thus ABA and H₂O₂ production slightly decrease in maize seedlings under osmotic stress.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.     

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [³H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [³H]metabolites and the other for autoradiography. The³H-activity remaining in the leaf tissue after washing out the water-soluble³H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [³H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [³H]PE and [³H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [¹⁴C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [¹⁴C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.Abbreviations and symbols PE phosphatidylethanolamine - PC phosphatidylcholine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - TLC thin-layer chromatography - _leaf leaf water potential     

Growth and osmotic adjustment of two tomato cultivars during and after saline stress

The effect of a short period of saline stress was studied in two phenotypically different cultivars, one of normal fruit-size (L. esculentum cv. New Yorker) and one of cherry fruit-size (L. esculentum var.cerasiforme cv. PE-62). In both cultivars the relative growth rate (RGR) and the leaf area ratio (LAR) decreased following salinisation. The leaf turgor potential (_p) and the osmotic potential at full turgor (_os) decreased to the same extent in both cultivars. However, the contributions of organic and inorganic solutes to the osmotic adjustment was different between cultivars. New Yorker achieved the osmotic adjustment by means of the Cl^– and Na⁺ uptake from the substrate, and by synthesis of organic solutes. In the cherry cultivar organic solutes did not contribute to the osmotic adjustment, instead, their contribution decreased after salinisation. After the salt stress was removed, the water stress disappeared, the content of organic solutes decreased in plants of both cultivars and, therefore, their growth was not retarded by the diversion of resources for the synthesis of organic solutes. However, the toxic effects of the Cl^– and Na⁺ did not disappear after removal of the salt stress, and the net assimilation rate (NAR) and the rate of growth (RGR) did not recover.