Spermidine application enhances tomato seedling tolerance to salinity-alkalinity stress by modifying chloroplast antioxidant systems

The purpose of this study was to elucidate whether exogenous spermidine (Spd) protection of tomato (Solanum lycopersicum L.) seedlings under salinity-alkalinity stress is associated with antioxidant enzymes in the chloroplast. The effects of exogenous Spd on antioxidant enzyme activity and antioxidant content in the chloroplast were evaluated in seedlings of salt-sensitive ecotype (Zhongza 9) grown in a 75 mM salinity-alkalinity solution, with or without 0.25 mM Spd foliar spraying. Results showed that salinity-alkalinity stress increased MDA content, superoxide anion O₂^?- generation rate, superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) activities and ratio of AsA/DHA and reduced contents of ascorbate (AsA), dehydroascorbate (DHA), AsA+DHA, glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, dehydroascorbate reductase (DHAR) activity and ratio of GSH/GSSG in chloroplasts. The exogenous Spd application combined with salinity-alkalinity stress decreased the O₂^?- generation rate and MDA content compared to salinity-alkalinity stress alone. The exogenous Spd also increased AsA-GSH cycle components and increased all antioxidant enzyme activities in most cases. Therefore, exogenous Spd alleviates salinity-alkalinity stress damage using antioxidant enzymes and non-enzymatic systems in chloroplasts.     

Exogenous spermidine alleviates fruit granulation in a <Emphasis Type="Italic">Citrus</Emphasis> cultivar (<Emphasis Type="Italic">Huangguogan</Emphasis>) through the antioxidant pathway

The role of exogenous spermidine (Spd) in alleviating fruit granulation in the grafted seedlings of a Citrus cultivar (Huangguogan) was investigated. Granulation resulted in increased electrical conductivity, cell membrane permeability, and total pectin, soluble pectin, cellulose, and lignin contents. However, it decreased the activities of superoxide dismutase, peroxidase, and catalase, as well as the (Spd + Spm):Put ratio. The application of exogenous Spd onto Huangguogan seedlings significantly increased proline and ascorbate contents, but decreased the H₂O₂ and O ₂ ^?· levels, which suggested that exogenous Spd provided some protection from oxidative damage. In addition, exogenous Spd decreased cell membrane permeability and MDA content, and increased the (Spd + Spm):Put ratio. The activities of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase, were increased in Spd-treated seedlings affected by fruit granulation, resulting in a decrease in oxidative stress levels. The protective effects of Spd were reflected by a decrease in superoxide levels through osmoregulation, increased proline and ascorbate contents, and increased antioxidant activities. Our observations reveal the importance of exogenous Spd in alleviating citrus fruit granulation.     

Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO^?) and the level of O ₂ ^?? or H₂O₂ in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H₂O₂ and O ₂ ^?? in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO^? production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.     

Involvement of phosphoenolpyruvate carboxylase and antioxydants enzymes in nitrogen nutrition tolerance in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> plants

Plants of Sorghum bicolor (C₄ species) were grown at different nitrate or ammonium concentrations (0.5, 5, 20 and 50 mM) in order to examine the effect of nitrogen nutrition on growth, phosphoenolpyruvate carboxylase (PEPC) and antioxidant enzymes activities in both roots and leaves of 30-day-old plants. At high NO₃^? levels (20 and 50 mM) the fresh weight was significantly higher. When the nitrogen source was in ammonium form, the leaf and root mass increased drastically at low concentration 5 mM and significantly at 20 mM, however similar fresh weight was found at high level of ammonium (50 mM). The leaves catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), and glutathione S-transferase (GST) activities and the roots glutathione reductase and glutathione S-transferase activities were significantly higher in the NH₄⁺-fed plants than those grown in the nitrate medium. Activity and proteins levels of phosphoenolpyruvate carboxylase in both leaves and roots of sorghum plants were increased progressively with increasing external nitrogen concentration. This increase was more pronounced at high level of ammonium (50 mM), being 2-fold at 50 mM of NO₃^? and 3-fold at 50 mM of NH₄⁺. Our results suggested that antioxidant enzymes activities and PEPC play a key role in ammonium detoxification and tolerance in sorghum plants.     

Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber <Emphasis Type="Italic">in vitro</Emphasis>

The relationship between polyamines (PAs) metabolism and adventitious shoot morphogenesis from cotyledons of cucumber was investigated in vitro. The endogenous levels of free putrescine (Put) and spermidine (Spd) in the explants decreased sharply, whereas endogenous spermine (Spm) increased during adventitious shoot morphogenesis. The presence of 1–15 mM Put, 1–2 mM Spd, 0.05–1 mM Spm, 5–10 M aminoethoxyvinylglycine (AVG) or 5 M AVG together with 50 M 1-aminocyclopropane-1-carboxylic acid (ACC) in the regeneration medium could promote adventitious shoot formation. Conversely, 1–5 mM D-arginine (D-Arg) or 0.01–0.1 mM methylglyoxal bis-guganylhydrazone (MGBG) inhibited regeneration; and 0.005–0.05 mM ACC displayed little or no evident effects. The explants growing on medium containing 5 M AVG produced higher levels of free Put and Spm, and on medium containing 5 mM Put the explants responded similarly to the AVG-treated explants. However, the exogenous use of 1 mM D-Arg reduced the levels of Put, Spd and Spm, and 0.1 mM MGBG reduced the levels of free Spd and Spm. Moreover, although the explants cultured on medium containing Put and MGBG enhanced ethylene production, AVG and D-Arg inhibited ethylene biosynthesis. This study shows the PAs requirement for the formation of adventitious shoot from cotyledons of cucumber in vitro and the enhanced adventitious shoot morphogenesis may be associated with the elevated level of endogenous free Spm, albeit the promotive effect of PAs on adventitious shoot morphogenesis may not be related to ethylene metabolism.     

The effect of total inorganic nitrogen and the balance between its ionic forms on adventitious bud formation and callus growth of ‘Węgierka Zwykła’ plum (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)

The effect of different ammonium NH ₄ ⁺ and nitrate NO ₃ ^? ratios (4:1, 2:1, 1:1, 1:2, 1:4, 1:6) on organogenesis of ‘W?gierka Zwyk?a’ leaf explants cultivated on media with nitrogen levels equalling full- or half-MS was investigated. On media with total nitrogen equal to ½ MS, explant regeneration increased significantly and was highest on media with 1:2 or 1:4 NH ₄ ⁺ :NO ₃ ^? ratio. An excess of ammonium versus nitrate ions had a negative effect on both regeneration and biomass. Addition of potassium to the medium increased the fresh weight of explants and the number of adventitious buds.     

Chemical composition of the Tatra Mountain lakes: Recovery from acidification

Ninety-one lakes distributed along the Tatra Mountains (most of lakes > 1 ha and 65% of lakes > 0.01 ha) were sampled and analysed for ionic and nutrient composition in September 2004 (15 years after reduction in acid deposition). Eighty-one lakes were in alpine zone and ten lakes in Norway spruce forest. The results were compared to similar lake surveys from 1994 (the beginning of water recovery from acidification) and 1984 (maximum acidification). Atmospheric deposition of SO ₄ ^2? and inorganic N decreased 57% and 35%, respectively, in this region from the late 1980s to 2000. Lake water concentrations of SO ₄ ^2? and NO ₃ ^? have decreased both by ～50% on average (to 23 and 19 μmol L^?1, respectively, in 2004) since 1984. While the decrease in SO ₄ ^2? concentrations was stable throughout 1984–2004, most of the NO ₃ ^? decrease occurred from 1994 to 2004. The declines in SO ₄ ^2? and NO ₃ ^? concentrations depended on catchment coverage with vegetation, being most rapid for SO ₄ ^2? in forest lakes and for NO ₃ ^? in rocky lakes. Concentrations of the sum of base cations (dominated by Ca²⁺) significantly decreased between 1984 and 2004, with the highest change in rocky lakes. Most of this decline occurred between 1994 and 2004. Acid neutralising capacity (ANC) did not change in the 1984–1994 period, but increased on average by 29 μmol L^?1 between 1994 and 2004, with the highest change in rocky lakes. Over the last decade, the proportion of lakes with ANC > 150 μmol L^?1 increased from 15% to 21% and that of ANC < 20 μmol L^?1 decreased from 37% to 20%. The highest decline in H⁺ and Al concentrations occurred in the most acid lakes. On a regional basis, no significant change was observed for total phosphorus, total organic nitrogen, and dissolved organic carbon (DOC) in the 1994–2004 period. However, these parameters increased in forest lakes, which exhibited an increasing trend in DOC concentrations, inversely related (P < 0.001) to their decreasing ionic strength (30% on average in 1994–2004).     

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.     

Characterization of ammonium and nitrate uptake and assimilation in roots of tea plants

It has been pointed out that tea (Camellia sinensis (L.) O. Kuntze) prefers ammonium (NH ₄ ⁺ ) over nitrate (NO ₃ ^? ) as an inorganic nitrogen (N) source. ¹⁵N studies were conducted using hydroponically grown tea plants to clarify the characteristics of uptake and assimilation of NH ₄ ⁺ and NO ₃ ^? by tea roots. The total ¹⁵N was detected, and kinetic parameters were calculated after feeding ¹⁵NH ₄ ⁺ or ¹⁵NO ₃ ^? to tea plants. The process of N assimilation was studied by monitoring the dynamic ¹⁵N abundance in the free amino acids of tea plant roots by GC-MS. Tea plants supplied with ¹⁵NH ₄ ⁺ absorbed significantly more ¹⁵N than those supplied with ¹⁵NO ₃ ^? . The kinetics of ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^? influx into tea plants followed a classic biphasic pattern, demonstrating the action of a high affinity transport system (HATS) and a low affinity transport system (LATS). The V _max value for NH ₄ ⁺ uptake was 54.5 nmol/(g dry wt min), which was higher than that observed for NO ₃ ^? (39.3 nmol/(g dry wt min)). K_M estimates were approximately 0.06 mM for NH ₄ ⁺ and 0.16 mM for NO ₃ ^? , indicating a higher rate of NH ₄ ⁺ absorption by tea plant roots. Tea plants fed with ¹⁵NH ₄ ⁺ accumulated larger amounts of assimilated N, especially glutamine (Gln), compared with those fed with ¹⁵NO ₃ ^? . Gln, Glu, theanine (Thea), Ser, and Asp were the main free amino acids that were labeled with ¹⁵N under both conditions. The rate of N assimilation into Thea in the roots of NO ₃ ^? -supplied tea plants was quicker than in NH ₄ ⁺ -supplied tea plants. NO ₃ ^? uptake by roots, rather than reduction or transport within the plant, seems to be the main factor limiting the growth of tea plants supplied with NO ₃ ^? as the sole N source. The NH ₄ ⁺ absorbed by tea plants directly, as well as that produced by NO ₃ ^? reduction, was assimilated through the glutamine synthetase-glutamine oxoglutarate aminotransferase pathway in tea plant roots. The ¹⁵N labeling experiments showed that there was no direct relationship between the Thea synthesis and the preference of tea plants for NH ₄ ⁺ .     

Interaction between NO 3 − and abscisic acid in the regulation of lateral root elongation in Zea mays L.

Lateral root (LR) elongation rate of 7–8-day maize seedlings depends on the availability of NO ₃ ^? , NO ₂ ^? , and abscisic acid (ABA) in an environment. Four-hour exposure to 0.01–1.5 mM NO ₂ ^? increases the relative LR elongation rate; in the case of NO ₂ ^? , the stimulation occurs only at an NO ₂ ^? concentration equal to 0.01 mM. Exogenous ABA (10^?6 M) inhibits the LR elongation process. In the case of a combined influence of NO ₃ ^? and ABA or NO ₂ ^? and ABA, the character of the response elongation reaction is different. The NO role in the regulation of LR elongation is discussed.     

Carbon and nitrogen in the ground waters of the Tellerman forest (European southern forest steppe)

Under the upland forests growing on the Dnieper loamy soils at 150–160 m above sea level, sandy aquifers are found at ~120 and ~140 m above sea level. The general mineralization of groundwaters (GWs) and the concentration of SO ₄ ^-2 increase upon approaching the foundation of fluvial glacial sediments. The concentration of C supplied to the local GWs is 10–12 mg L^–1 per year^–1. The concentration of C(CO ₃ ^-2 ; HCO ₃ ^-1 ; NO ₃ ^-1 ; NO ₂ ^-1 ) in the DSW is consistent with the C balance of upper biogeocenoses and the rate of groundwater migration. The concentrations of N(nh ₄ ⁺¹ ) are unrelated to the filtration of biogeocenosis moisture. They are identical both under sodic grassy glades and highly productive tree stands. The main part of N-containing ions in situ is synthesized and destroyed by anaerobic microorganisms living in the GW.     

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.     

Reactive oxygen and nitrogen species’ effect on <Emphasis Type="Italic">lux</Emphasis>-biosensors based on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H₂O₂ > OCl^– > NO^? > RОO^? > ONOO^–> O₂^?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO^? > H₂O₂ > ONOO^– > RОO^? > OCl^– > O₂^?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H₂O₂ and OCl^–, along with O₂^?- and NO^?. Among the used reactive oxygen and nitrogen species, H₂O₂ showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors.     

Partitioning of previously-accumulated nitrate to translocation,reduction, and efflux in corn roots

The effect of nitrate uptake, or its absence, on the utilization of nitrate previously accumulated by dark-grown, decpitated maize (Zea mays L., cv. DeKalb XL-45) seedlings was examined. Five-d-old plants that had been pretreated with 50 mM ¹⁴NO ₃ ^? for 20 h were exposed for 8 h to nutrient solutions containing either no nitrate or 50 mM ¹⁵NO ₃ ^? , 98.7 atom % ¹⁵N. The ambient solution, xylem exudate, and plant tissue were analyzed to determine the quantities of previously-accumulated (endogenous) ¹⁴NO ₃ ^? that were translocated to the xylem, lost to the solution, or reduced within the tissue during the 8-h period. Energy was continuously available to the roots from the attached endosperm. In the absence of incoming nitrate, appreciable reduction and translocation of the endogenous ¹⁴NO ₃ ^? occurred, but efflux of ¹⁴NO ₃ ^? to the external solution was minimal. In contrast, during ¹⁵NO ₃ ^? uptake, there was considerable efflux of ¹⁴NO ₃ ^? as well as translocation of ¹⁴NO ₃ ^? to the xylem, but little ¹⁴NO ₃ ^? was reduced. Thus there appeared to be an inverse relationship between ¹⁴NO ₃ ^? efflux and reduction. The data are tentatively interpreted on the basis of a model which envisages (a) two storage locations within roots, one of which primarily supplies nitrate for translocation and the other of which primarily supplies nitrate for outward passage through plasmalemma, and (b) the majority of nitrate reduction as occurring during or immediately following influx across the plasmalemma, with endogenous ¹⁴NO ₃ ^? initially moving outward being recycled inward and thereby being reduced.     

Influence of different mineral nitrogen sources (NO 3 − -N vs. NH 4 + -N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices–cowpea symbiosis

Labeled nitrogen (¹⁵?N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO ₃ ^? vs. NH ₄ ⁺ ) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO ₃ ^? and NH ₄ ⁺ . However, the amount of N transferred from the FC to the plant was higher when NO ₃ ^? was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher ¹⁵?N enrichment when the FC was supplied with ¹⁵NH ₄ ⁺ compared with ¹⁵NO ₃ ^? . The ¹⁵?N shoot/root ratio of plants supplied with ¹⁵NO ₃ ^? was much higher than that of plants supplied with ¹⁵NH ₄ ⁺ , indicative of a faster transfer of ¹⁵NO ₃ ^? from the root to the shoot and a higher accumulation of ¹⁵NH ₄ ⁺ in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH ₄ ⁺ preferentially over NO ₃ ^? but that export of N from the hyphae to the root and shoot may be greater following NO ₃ ^? uptake. The need for NH ₄ ⁺ to be assimilated into organically bound N prior to transport into the plant is discussed.     

Dominance of biologically produced nitrate in upland waters of Great Britain indicated by stable isotopes

Atmospheric deposition of nitrogen (N) compounds is the major source of anthropogenic N to most upland ecosystems, where leaching of nitrate (NO ₃ ^? ) into surface waters contributes to eutrophication and acidification as well as indicating an excess of N in the terrestrial catchment ecosystems. Natural abundance stable isotopes ratios, ¹⁵N/¹⁴N and ¹⁸O/¹⁶O (the “dual isotope” technique) have previously been used in biogeochemical studies of alpine and forested ecosystems to demonstrate that most of the NO ₃ ^? in upland surface waters has been microbially produced. Here we present an application of the technique to four moorland catchments in the British uplands including a comparison of lakes and their stream inflows at two sites. The NO ₃ ^? concentrations of bulk deposition and surface waters at three sites are very similar. While noting the constraints imposed by uncertainty in the precise δ¹⁸O value for microbial NO ₃ ^? , however, we estimate that 79–98% of the annual mean NO ₃ ^? has been microbially produced. Direct leaching of atmospheric NO ₃ ^? is a minor component of catchment NO ₃ ^? export, although greater than in many similar studies in forested watersheds. A greater proportion of atmospheric NO ₃ ^? is seen in the two lake sites relative to their inflow streams, demonstrating the importance of direct NO ₃ ^? deposition to lake surfaces in catchments where terrestrial ecosystems intercept a large proportion of deposited N. The dominance of microbial sources of NO ₃ ^? in upland waters suggests that reduced and oxidised N deposition may have similar implications in terms of contributing to NO ₃ ^? leaching.     

Element fluxes in watershed-lake ecosystems recovering from acidification: Plešné Lake,the Bohemian Forest, 2001–2005

Fluxes of major ions and nutrients were measured in the watershed-lake ecosystem of a strongly acidified lake, Ple?né jezero (Ple?né Lake), in the Czech Republic in hydrological years from 2001 through 2005. The lake is situated in a Norway spruce forest and has a steep watershed between elevations of 1090 and 1378 m. The average water input and output from the ecosystem was 1372 mm and 1157 mm (37 L km^?2 s^?1), respectively, and the water residence time averaged 306 days. Despite ecosystem recovery from acidification occurring since the late 1980s, the Ple?né watershed was an average net source of 25 mmol SO ₄ ^2? m^?2 yr^?1. Nitrogen saturation of the watershed caused low retention of the deposited inorganic N (< 44% on average) before 2004. Then, the watershed became a net source of 28–32 mmol m^?2 yr^?1 of inorganic N in the form of NO ₃ ^? due to climatic effects (a dry summer in 2003 and a cold winter in 2004) and forest dieback caused by a bark beetle attack in 2004. Nitrogen transformations and SO ₄ ^2? release were the dominant terrestrial sources of H⁺ (72 and 49 mmol m^?2 yr^?1, respectively) and the watershed was a net source of 24 mmol H⁺ m^?2 yr^?1. Ionic composition of surface inlets showed seasonal variations, with the most pronounced changes in NO ₃ ^? , ionic Al (Al_i), and DOC concentrations, while the composition of subsurface inlets was more stable. The in-lake biogeochemical processes reduced on average 59% of the incoming H⁺ (251 mmol H⁺ m^?2 yr^?1 on a lake-area basis). NO ₃ ^? assimilation and denitrification, photochemical and microbial decomposition of allochthonous organic acids, and SO ₄ ^2? reduction in the sediments were the most important aquatic H⁺ consuming processes (358, 121, and 59 mmol H⁺ m^?2 yr^?1, respectively), while hydrolysis of Al_i was the dominant in-lake H⁺ generating process (233 mmol H⁺ m^?2 yr^?1). Photochemical liberation from organic complexes was an additional in-lake source of Al_i. The net in-lake retention or removal of total phosphorus, total nitrogen, and silica were on average 50%, 27%, and 23%, respectively. The lake was a net source of NH ₄ ⁺ due to a cease in nitrification (pH < 5) and from NH ₄ ⁺ production by dissimilation exceeding its removal by assimilation.     

Polyamines and jasmonic acid induce plasma membrane potential variations in lima bean

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.⁵ JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.⁵ Here, we report that a plasma membrane potential (V_m) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V_m but the perfusion of JA and the polyamines significantly increased Cad and Put V_m depolarization. When JA was perfused with polyamines, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Key words: polyamines, lima bean, herbivore-induced volatile organic compounds, calcium and ROS signalling, jasmonic acid, quantitative gene expression, transmembrane potentialPolyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.^1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.^1–4 We recently reported that exogenously applied polyamines ∼diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)]∽ induce volatile emission in Lima bean leaves.⁵ Membrane potentials (V_m) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in V_m.⁶ The plasma membrane potential (V_m), which lies in the range of −50 to −200 mV in Lima bean leaves,⁷ may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.Measurement of V_m were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).⁷ Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different V_m depolarization, depending on the polyamine. In general, a V_m depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd V_m depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad V_m depolarization (p = 0.435). In all cases, V_m depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the V_m was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min⁻¹; R = 0.96), equal for Put and Cad (4.8 mV min⁻¹; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min⁻¹; R = 0.96).Open in a separate windowFigure 1Effect of 1 mM polyamines (arrow) on the V_m of Lima bean palisade cells. Spermine (Spm) caused the lowest V_m depolarization, whereas spermidine (Spd) showed the highest values of V_m depolarization. intermediate values were found when putrescine (Put) and cadaverine (cad) were perfused. after washing the tissues with fresh buffer (double arrow) V_m was always hyperpolarized, however the initial potential was recovered only for Put, while for all other polyamines the V_m never reached the initial values. Metric bars indicate standard deviation.Perfusion with JA caused a slight and not significant (p = 0.332) V_m depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in V_m depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min⁻¹; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min⁻¹, R = 0.99; Spd: 13.49 mV min⁻¹, R = 0.99) and lower for Spm (1.34 mV min⁻¹; R = 0.93). For JA the rate of V_m depolarization was 0.19 mV min⁻¹ (R = 0.96). With the addition of JA, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Open in a separate windowFigure 2Effect of 1 mM polyamines + 0.1 mMJA (arrow) on the V_m of Lima bean palisade cells. the perfusion with Ja did not cause any variation in the V_m. addition of JA to Spm and Spd caused the same V_m depolarization observed in the absence of JA, whereas when JA was added to Put and Cad a stronger and significantly different V_m depolarization was observed. even in this case washing the tissues with fresh buffer (double arrow) caused a V_m hyperpolarized, however in this case Spd reached V_m values significantly more negative that the initial V_m. Metric bars indicate standard deviation. For abbreviations see Figure 1.Since ion fluxes through channels directly influence V_m, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.⁸ In the present study, rapid and reversible V_m depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the V_m may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.⁹ Liu et al.¹⁰ showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.^1,11 Both mechanisms can be correlated to the observed V_m depolarization, and the positive correlation between intracellular Ca²⁺ concentration⁵ and V_m depolarizing activity of polyamines confirms the involvement of Ca²⁺ during polyamine uptake.¹¹     

Role of spermidine and spermine in alleviation of drought-induced oxidative stress and photosynthetic inhibition in Chinese dwarf cherry (Cerasus humilis) seedlings

To investigate the effect of exogenous Spermidine (Spd) and Spermine (Spm) on drought-induced damage to seedlings of Cerasus humili, relative water content (RWC), malondialdehyde content, relative electrolyte leakage, superoxide (O₂ ^?, SOD) generation rate, hydrogen peroxide (H₂O₂), endogenous polyamines (PAs), antioxidant enzymes [SOD and peroxidase (POD)] activities, PA-biosynthetic enzymes [arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC)] activities, as well as photosynthetic parameters, were measured in greenhouse cultured seedlings of C. humili. The results showed that either exogenous Spd or Spm (0.2 mM) significantly enhanced the level of RWC and prevented drought-induced lipid peroxidation. They also significantly enhanced photosynthetic capability and decreased O₂ ^? generation rate and H₂O₂ content. In addition, Spd and Spm helped to maintain SOD and POD activities in C. humili seedlings subjected to water stress, suggesting that they exerted a positive effect on antioxidant systems. The contents of endogenous free putrescine, Spd and Spm were increased to different extents in water-stressed C. humili seedlings. By the end of drought treatment (21 days) with exogenous Spd or Spm, the contents of free Spd increased by 30 and 38 %, respectively, and endogenous Spm increased by 41 and 26 %, respectively, compared with water-stressed plants. Furthermore, exogenous Spd or Spm enhanced the activities of ADC, ODC, and SAMDC. The pretreatment with Spd or Spm prevents oxidative damage induced by drought, and the protective effect of Spd was found to be greater than that of Spm.