N. I. Shevyakova

Polyamines and stress: Biological role, metabolism, and regulation

In this review, we consider recent advances in the study of the multifaceted biological role of polyamines, primarily under stress conditions, discuss molecular mechanisms controlling their anabolism, catabolism, and transport, and also the regulation of gene expression for key enzymes of their biosynthesis and degradation. To understand the place and role of polyamines in plant adaptation, we focus the data concerning gene expression obtained by modern physicochemical methods on mutant and transgenic plants and also on natural stress-tolerant species manifesting a high tolerance to salinity, drought, and other abiotic factors.

Proline antioxidant role in the common ice plant subjected to salinity and paraquat treatment inducing oxidative stress

Leaves of 4-week-old (juvenile) and 9-week-old (adult) plants of the halophyte Mesembryanthemum crystallinum L. (the common ice plant), cultured under controlled conditions in the phytotron, were treated with paraquat (0.1 μM), which produces superoxide radical, and (or) paraquat combined with introduction of NaCl (100 mM) or proline (5 mM) into nutrient medium. After a 20-h dark period (23°C), plants were transferred into light (4 h at 54.1 W/m2 of photosynthetically active radiation) for stimulation of O°2− formation in plastids. Activities of antioxidant enzymes, the contents of MDA, H2O2, chlorophyll, and free proline were measured in leaves. Plant responses in two age groups, which differed in the type of photosynthesis (juvenile plants had C3 type of photosynthesis, whereas adult plants were at the transition stage to Crassulacean Acid Metabolism (CAM) photosynthesis), differed in the levels of constitutive proline and proline, induced by NaCl and paraquat, as well as in activities of superoxide dismutase (SOD) and catalase. Changes in SOD activity and proline accumulation in response to paraquat treatment combined with NaCl revealed opposite dependence to accumulation of proline: the more proline accumulated in leaves, the lower activity of the enzyme. In response to paraquat treatment, the content of chlorophylls a and b most drastically declined in juvenile plants. Negative effect of salinity on the content of chlorophylls was lower than that of paraquat and was almost the same in plants of both age groups. Protective effect of exogenous proline was most profound in the case of paraquat treatment. Exogenous proline decreased the rate of lipid peroxidation, the content of superoxide radical and, consequently, SOD activity (almost fivefold), and increased the content of chlorophylls (a and b) in leaves of adult plants. The obtained data suggest that stress-induced accumulation of proline in the common ice plant has both osmoprotectory and antioxidant functions.

Compartmentation of Cadmium and Iron in Mesembryanthemum crystallinum Plants during the Adaptation to Cadmium Stress

The common ice plants (Mesembryanthemum crystallinum) at the stage of five leaf pairs were exposed to cadmium chloride solutions (1, 0.1, and 0.01 mM) under the conditions of water culture. After five days, the partition of cadmium and iron in the plant organs and in the cell structures of the apical root region were investigated. Plant adaptation to excess cadmium in the environment was assessed by an increase in the leaf and root weight, a change in peroxidase activity, and an accumulation of proline. The common ice plant accumulated cadmium mainly in the root system. At a high concentration of cadmium in the nutrient solution (1 mM), its content in the root exceeded 2 g/kg fr wt, while at a concentration of 0.01 mM, it was as low as 10 mg/kg. Dithizone staining of transverse sections of the root apical region showed that, after a 48-h-long exposure of plants to 0.1 mM cadmium chloride, cadmium was localized in the cell walls of endodermis and metaxylem. The level of cadmium in leaves varied from 0.5 to 18 mg/kg fr wt. However, there was only a weak correlation between cadmium accumulation and the extent of a biomass decrease in the leaves of various stories, when cadmium concentration in the medium (1 mM cadmium chloride) was toxic. This fact could be related to a marked efflux of endogenous iron from old leaves into the young ones and to a change in the cadmium/iron ratio in the tissues. Proline accumulation in the third leaf pair and in the roots occurred at a relatively low cadmium content (10–12 mg/kg fr wt) in these organs. Maxima of activity of all three forms of peroxidase, viz., soluble, ionically-bound, and covalently-bound peroxidases, in roots were found at a high accumulation of cadmium in these organs (45 mg/kg fr wt). These maxima exceeded 3–4-fold the activity in aging leaves containing 5 mg cadmium/kg fr wt. A decrease in peroxidase activity in leaves was accompanied by a 3.3-fold decrease in iron content; thus, it could be caused by a deficiency of available iron necessary for the enzyme functioning. It was concluded that the resistance of Mesembryanthemum crystallinum, a halophyte, to excess cadmium content in the medium was achieved by its predominant accumulation in roots, where excess cadmium is compartmentalized in the apoplast and seems to be subjected to detoxification through pectate formation. Moreover, the leaves and, particularly, the roots are characterized by a high activity of the antioxidant systems, such as guaiacol-dependent peroxidases, and an occurrence of proline at modest cadmium concentrations.

Ultrastructure of Chloroplasts and Their Storage Inclusions in the Primary Leaves of Mesembryanthemum crystallinum Affected by Putrescine and NaCl

The effects of salinity (300 mM NaCl), putrescine (Put), and the combination of two agents on the structure of chloroplasts and storage deposits were studied in the third leaf pair of a facultative halophyte Mesembryanthemum crystallinum. Within 6 days, the common ice plants responded to NaCl and Put treatments by diminished chloroplast volumes and swollen grana. Different effects of the experimental treatments were primarily manifested in the chloroplast storage inclusions. Under the salinity conditions, the starch content dropped down almost threefold as compared to untreated plants (control), whereas the number of plastoglobules did not change. Put and Put + NaCl treatments further decreased the starch content per unit section area; in contrast, the plastoglobule area per chloroplast section increased eightfold and tenfold in Put and Put + NaCl treatments, respectively. The morphology and electronic density of plastoglobules changed in all treatments. In both Put treatments there ware no destructive changes in the chloroplasts, and therefore the authors presume that the increase in the numbers plastoglobules was related to the redirection of cell metabolism towards the products of the higher reduction potential. The ferritin deposits in the chloroplasts were observed in all treatments they were more abundant in the vascular parenchyma cells, especially under salinity. The ability of the common ice plants to accumulate large Fe quantities in their chloroplasts and the characteristic pectin-filled “pockets”, which were observed earlier, and intercellular spaces are probably related to the genetically determined traits of plant adaptation to salinity and water deficit.

Role of antioxidant systems in wild plant adaptation to salt stress

Wild plants differing in the strategies of adaptation to salinity were grown for six weeks in the phytotron and then subjected to salt stress (100 mM NaCl, 24 h). The activities of principal antioxidant enzymes and the accumulation of sodium ions and proline were studied. Independently of the level of constitutive salt tolerance, plants of all species tested accumulated sodium ions under salinity conditions but differed in their capability of stress-dependent proline accumulation and superoxide dismutase (SOD) and guaiacol-dependent peroxidase activities. Proline-accumulating species were found among both halophytes (Artemisia lerchiana and Thellungiella halophila) and glycophytes (Plantago major and Mycelis muralis). The high activities of ionically-bound and covalently bound peroxidases were characteristic of Th. halophila plants. High constitutive and stress-induced SOD activities were, as a rule, characteristic of glycophytes with the low constitutive proline level: Geum urbanum and Thalictrum aquilegifolium. Thus, a negative correlation was found between proline content and SOD activity in wild species tested; it was especially bright in the halophyte Th. halophila and glycophyte G. urbanum. An extremely high constitutive and stress-induced levels of proline and peroxidase activity in Th. halophila maybe compensate SOD low activity in this plant, and this contributed substantially into its salt resistance. Thus, monitoring of stress-dependent activities of some antioxidant enzymes and proline accumulation in wild plant species allowed a supposition of reciprocal interrelations between SOD activity and proline accumulation. It was also established that the high SOD activity is not obligatory trait of species salt tolerance. Moreover, plants with the high activity of peroxidase and active proline accumulation could acclimate to salts stress (100 mM NaCl, 24 h) independently of SOD activity.

Functioning of Defense Systems in Halophytes and Glycophytes under Progressing Salinity

Six-week-old Plantago major L. and Thellungiella halophila Mey. plants were subjected to progressing salinity by a daily increase in the NaCl concentration by 100 mM until the final concentration of 400 mM. A dynamics of stress-dependent accumulation of Na+ and Cl− ions, proline, and free polyamines and also activities of antioxidant enzymes, superoxide oxidase (SOD) and free, ion-bound, and covalently bound guaiacol-dependent peroxidases was studied. We also examined the intensity of gene expression encoding enzymes of proline metabolism and polyamine biosynthesis. It was shown that the high salt-resistance of the halophyte T. halophila was determined by plant capability of ion accumulation and stress-dependent proline accumulation. An important role in the maintenance of this plant homeostasis under salinity plays a high constitutive levels of activities of three types of peroxidases tested and also of proline manifesting a polyfunctional protective action. In contrast, P. major plants characterized by a lower tolerance to salt excess did not display a high constitutive level of proline or the activity of guaiacol-dependent peroxidases; they also were not capable of stress-induced accumulation of compatible osmolytes and did not accumulate the salt. However, this glycophyte contained relatively much spermidine and active SOD, which provided for a decrease in the damaging effects of reactive oxygen species under salt shock. In both plant species, it was established that salinity changed the intracellular content of polyamines, which was not dependent on the activity of gene transcription encoding the enzymes of their biosynthesis. The results obtained support a hypothesis that halophytes and glycophytes have some common mechanisms of tolerance to salinity, but the control of these mechanisms differs substantially.

Proline involvement in the common sage antioxidant system in the presence of NaCl and paraquat

To elucidate proline antioxidant properties in common sage (Salvia officinalis L.) plants, they were treated with paraquat (a producer of superoxide radical) and/or NaCl and also with paraquat and proline at the stage of 4–5 true leaves. The paraquat solution (1 ml containing 0.1 μmol of the agent) was applied to the leaf surface; NaCl (200 mM) and proline (the final concentration of 5 mM) were added to nutrient medium. Experimental plants were firstly kept in darkness for 12 h, then illuminated, and in 3, 6, and 12 h, leaves and roots were fixed for biochemical analyses. The results obtained are in agreement with the supposition of proline antioxidant properties. In particular, it was established that paraquat induced a slight increase in the proline level in the leaves during dark period of plant growth and also during subsequent 3 h after light switching on. This transient proline accumulation in the leaves was accompanied by its level decrease in the roots. Proline addition to the nutrient medium of paraquat-treated plants neutralized paraquat damaging action on the leaves. In the presence of paraquat, proline treatment reduced the accumulation in the roots of hydrogen peroxide and malondialdehyde, the product of membrane lipid peroxidation. It also affected indirectly the activities of superoxide dismutase (SOD) and free, covalently bound, and ionically bound peroxidases. Keeping in mind that, in the presence of paraquat, superoxide-induced changes in SOD activity in the roots were negatively correlated with the level of proline, which content was the highest during the last hours of experiments, we can conclude that proline antioxidant effects are manifested only after 12 h of stressor action, whereas antioxidant enzymes are involved in ROS scavenging during the earlier stage of damaging factor action.

Do Polyamines Participate in the Long-Distance Translocation of Stress Signals in Plants?

Accumulation and ethylene-dependent translocation of free polyamines was studied in various organs, the phloem and xylem exudates of common ice plants (Mesembryanthemum crystallinum L.). Under normal conditions (23–25°C), spermidine predominated among polyamines. Cadaverine was found in old leaves, stems, and, in large quantities, in roots. The heat shock treatment (HS; 47°C, 2 h) of intact plant shoots induced intense evolution of ethylene from leaves but reduced the leaf content of polyamines. Under these conditions, the concentration of polyamines in roots, particularly of cadaverine, increased many times. The HS treatment of roots (40°C, 2 h) induced translocation of cadaverine to stems and putrescine to leaves. An enhanced polyamine content after HS treatment was also found in the xylem and phloem exudates. The exposure of detached leaves to ethylene led to a reduction in their putrescine and spermidine and accumulation of cadaverine, which implies the ethylene-dependent formation of cadaverine and a possible relation between the HS-induced translocation of this diamine to roots and the transient ethylene evolution by leaves. To validate this hypothesis, we compared the ethylene evolution rate and interorgan partitioning of cadaverine and other polyamines for two lines of Arabidopsis thaliana: the wild type (Col-0) and ein4 mutant with impaired ethylene reception. In plants grown in light at 20–21°C, the rate of ethylene evolution by rosetted leaves was higher in the mutant than in the wild type. The content of putrescine and spermidine was reduced in mutant leaves, whereas cadaverine concentration increased almost threefold compared with the wild type. In roots, cadaverine was found only in the wild type and not in the mutant line. Our data indicate the ethylene-dependent formation of cadaverine in leaves and possible involvement of cadaverine and ethylene in the long-distance translocation of stress (HS) signal in plants.

Phytoremediation potential of Amaranthus hybrids: Antagonism between nickel and iron and chelating role of polyamines

Three amaranth hybrids (Amaranthus paniculatus f. cruentus (Vishnevyi dzhem), A. paniculatus (Bronzovyi vek), and A. caudatus f. iridis (Izumrud) were grown in the climate-controlled chamber on Jonson nutrient medium supplemented with 2 μM Fe3+-EDTA. When plants developed 5–6 true leaves (six-week-old plants), NiCl2 was added to medium to final concentrations of 0 (control), 50, 100, 150, 200, and 250 μM. In 6 days, the increment in biomass of young and mature leaves, stems, and roots, and also the contents of Ni and Fe in them were measured. The red leaf amaranth hybrid Vishnevyi dzhem manifested the highest phytoremediation potential. i.e., the highest capacity for Ni accumulation in the shoots and the most pronounced symptoms of Fe deficit. In the presence of 150 and 250 μM NiCl2 in medium, the shoots of these plants contained about 2 and 4 mg Ni/g dry wt, respectively. In experiments with Fe deficit in plants grown for a week in the presence of NiCl2 (0, 25, 50, 75, and 100 μM), it was established that all tested nickel concentrations suppressed iron reduction in intact roots, which is catalyzed by ferric-chelate reductase, and this may underlie the antagonism between the two metals. In the presence of 50 μM NiCl2 in medium and 2 μM Fe3+ (Fe deficit) and especially 100 μM Fe3+ (Fe excess), the content of MDA and proline in leaves increased and superoxide dismutase was activated; this indicates a development of oxidative stress. Leaf treatment with polyamines (putrescine or spermidine) with aminoguanidine (the inhibitor of H2O2 generation at polyamine oxidation) and with 1,3-diaminopropane led to the increase in nickel accumulation in leaves but did not result in the appearance of any signs of injury. This confirms our previous suggestion that polyamines manifest their protectory action as Ni chelators and detoxicants.

Cadaverine-Induced Induction of Superoxide Dismutase Gene Expression in Mesembryanthemum crystallinum L.

Polyamines—putrescine, spermidine, spermine, and cadaverine—are the components of cellular antioxidant systems that are conventionally regarded as scavengers of oxy radicals [4]. However, the mechanisms of their antioxidative action are poorly understood. It was shown that the involvement of polyamines in ROS quenching is based on their ability to bind to proteins to form conjugates with various phenol derivatives rather than on the fact that their amino groups readily undergo autooxidation and oxygen-dependent and enzymatic oxidation [5]. It was also shown that, in salt stress, the maximal accumulation of cadaverine in Mesembryanthemum crystallinum plants was observed upon their transition from the ë 3 type photosynthesis to the crassulacean acid metabolism (CAM), which enables intense ROS generation [6]. Cadaverine, capable of a rapid stress-dependent accumulation and interorgan transport [7], does not function in this case as a signal factor inducing the expression of gene Ppc1 , which encodes phosphoenolpyruvate carboxylase, the key enzyme of CAM-type photosynthesis, and triggering the development of water-saving adaptation mechanism [6]. It cannot be also ruled out that the biological role of cadaverine in oxidative stress is mediated by the signal role of H 2 O 2 produced during its oxidative degradation [8]. To date, the prooxidative role of polyamines was considered primarily in connection O2 .– H . with the generation of H 2 O 2 in the apoplast, which is required for the formation of suberin, lignin, and oxyproline proteins [3]. Eventually, cadaverine on its own may function as an inducer of the expression of genes encoding antioxidant enzymes.