V. Yu. Rakitin

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.

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

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

Inducible and constitutive mechanisms of salt stress resistance in Geum urbanum L.

The avens (Geum urbanum L.) seedlings were grown for 6 weeks until the expansion of five to six leaves and then exposed to salinity shock (300 mM NaCl in the nutrient medium) or to a gradual (within 4 days) increase in NaCl concentration from 100 to 400 mM. The dynamics of stress-dependent accumulation of Na+, Cl−, proline, and polyamines in leaves and roots was measured, together with activities of antioxidant enzymes, namely, superoxide dismutase (SOD) and guaiacol-dependent peroxidase occurring in soluble, ionically bound, and covalently bound forms. It is shown that avens plants can adapt to gradual salinization by mobilizing stressinducible protective mechanisms (accumulation of proline and spermine) and by activating constitutive enzyme systems (SOD and peroxidase).

Interaction between ethylene and ABA in the regulation of polyamine level in Arabidopsis thaliana during UV-B stress

The effects of treatment with ethylene (0.01–100 μl/l) on ABA and polyamine contents and treatment with ABA on ethylene synthesis, polyamines content, and the resistance to UV-B radiation of two-week-old Arabidopsis thaliana (L.) Heynh, Columbia ecotype plants grown u⊋er sterile conditions were studied. Ethylene stimulated the accumulation of polyamines only at concentrations of 0.1–10 μl/l, which could activate ABA synthesis. Treatment with ABA (50–5000 μM, 1 μl per plant) decreased the UV-B-induced ethylene synthesis and a spermine and spermidine loss, increasing the content of putrescine, the precursor of these polyamines. ABA inhibited fresh weight accumulation in irradiated and nonirradiated plants but prevented them from severe damage and death at the high (18 kJ/m2) and lethal (27 kJ/m2) UV-B dose, respectively. The data obtained demonstrated a mutual regulation of ethylene and ABA syntheses and the participation of these hormones in the control of the polyamine level during adaptation of A. thaliana to UV-B stress.

Ethylene evolution and ABA and polyamine contents in Arabidopsis thaliana during UV-B stress

Changes in plant growth, membrane integrity, ethylene evolution, ABA content, and the content of free polyamines were examined in 14-day-old Arabidopsis thaliana (L.) Heynh., strain Columbia (Col-0) plants after a single UV-B irradiation with low (3 kJ/m2), moderate (6–9 kJ/m2), high (18 kJ/m2), and lethal (27 kJ/m2) doses. The UV-B treatment caused dose-dependent suppression of plant growth. One hour after irradiation, the membrane damage was evident from the increased leakage of electrolytes. The low-dose and moderate-dose irradiation caused a transient increase in evolution of ethylene and in the content of putrescine (spermidine and spermine precursor) with the peaks of these parameters attained at 5 and 24 h, respectively. The high-and lethaldose irradiation induced a smaller rise in ethylene evolution, with a slight trend to its decrease, especially, after the exposure to the lethal dose. The high and lethal doses of UV-B suppressed putrescine accumulation, depleted spermidine and spermine pools, and caused severe injuries and plant death. During the first day after irradiation, the ABA content increased in proportion to the irradiation dose. On the second day, the accumulation of ABA was observed in plants irradiated with moderate doses. The accumulation was arrested after a high-dose irradiation and was diminished by 45% after a lethal dose treatment. The results provide evidence for the involvement of ethylene, ABA, and polyamines in plant responses induced by UV-B irradiation.

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 μl/l. BA (10–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.

Heat Shock Increases the Tolerance of Plants to UV-B Radiation: 2. Ethylene and Carbon Dioxide Evolution

The effects of heat shock (HS, 45°C) and UV-B radiation (280–320 nm, 18.3 kJ/(m2h)) and the consecutive action of the combination of these factors on ethylene production, gas exchange, and the growth of intact melon (Melo sativusSager.) seedlings were investigated. The changes in ethylene production and carbon dioxide exchange were described by a single-peaked curve. In the course of UV-B irradiation, the time of maximum ethylene and CO2evolution coincided (the first 5 min) and comprised 0.36 nl/(seedling h) for ethylene and 146.2 μl/(seedling h) for carbon dioxide. After HS, the maximum of ethylene production (0.37 nl/(seedling h)) was reached within 10 min, and that of carbon dioxide production (313 μl/(seedling h)), within 45 min. The rate of ethylene production (0.22 nl/(seedling h)), carbon dioxide production (97.7 μl/(seedling h)), and oxygen consumption (162.5 μl/(seedling h)) in the control seedlings did not change in the course of experiment. Throughout the experiment, the respiratory quotient of seedlings was ca. 0.6 regardless on the nature and duration of the acting factor. Preliminary heating at 45°C for 1 h increased the tolerance of seedlings to the subsequent UV-B radiation for 1 h. The protective effect of HS manifests itself in alleviating the inhibiting action of UV-B radiation on seedling growth and development, and this effect was preceded by an increase in ethylene production and respiration. The possible mechanisms of cross-tolerance of plants to overheating and UV-B radiation are discussed.

The Effect of Putrescine on the Apoplast Ultrastructure in the Leaf Mesophyll of Mesembryanthemum crystallinum under Salinity Stress

The effects of NaCl, putrescine (Put), and the combination of two agents on the contents of free polyamines (PA), peroxidase activity, and the ultrastructure of the mesophyll apoplast were studied in the third leaf pair of Mesembryanthemum crystallinum L. plants. The NaCl solution was added to soil daily for three days (100 ml of the 100 mM solution), and plants were sprayed with the 1 mM Put solution twice per day for a six-day period. The accumulation of Put and especially spermidine (Spd) by day 3 of salinization was followed by a dramatic drop in Put and Spd contents by day 6. In contrast, the activities of soluble and ion-bound peroxidases increased following a long lag-period. Treatments with Put and NaCl plus Put considerably enhanced this rise in two peroxidase activities. An electron microscopic examination of cell walls in the control and stressed plants demonstrated that a gap developed at the middle lamella with “pockets” filled with amorphous polysaccharides (AP), presumably pectins. At the maximum gap width, the pockets fused with the intercellular spaces, and, in this case, the intercellular spaces also contained AP. Following salinization, AP in the apoplast swelled and expanded. Apparently the genetic determination of high AP content in M. crystallinum plants is the basis for the ability of juvenile plants to bind Na+ and Cl– ions and excess PA and also to accumulate water. In the plants treated with NaCl plus Put, the number of pockets and their volume increased, and the surface of some cell walls became plated with suberin, thus providing an additional barrier for ion transport from the pockets and intercellular spaces into cells. The formation of suberin plates was correlated with the high activity of ion-bound peroxidase essential for suberin deposition. The authors presume that H2O2 results from PA oxidation and, in its turn, induces the activity of peroxidase involved in the suberin plate formation.

Ethylene-Induced Production of Cadaverine Is Mediated by Protein Phosphorylation and Dephosphorylation

Earlier, we discovered an unusual mode of interaction between cadaverine and ethylene in the facultative halophyte Mesembryanthemum crystallinum L. [1, 2], which was more likely synergistic rather than competitive (as in the case of spermidine). M. crystallinum responded to heat shock by transient release of ethylene, with subsequent interorgan translocation of cadaverine, which could be related to the adaptation and survival of plants under extremal conditions. To date, it is established that the regulation of physiological processes in plants by polyamines is closely related to ethylene [1–3]. However, the majority of studies in this field were performed using the polyamines of the putrescine family, because these polyamines and ethylene have a common precursor, S -adenosylmethionine (SAM). Under stress conditions, in addition to the competition for SAM, the relationship between polyamines and ethylene may be reciprocal, which is manifested in mutual inhibition of their biosyntheses) [3]. Although the processes of biosynthesis of ethylene and cadaverine are closely related (the latter is formed in a side branch of the aspartate pathway leading to the biosynthesis of methionine and SAM), the authors of the works cited practically did not pay attention for cadaverine. The nature of the interaction between ethylene and cadaverine has been poorly understood probably due to the fact that cadaverine is not widespread in plants (except for legumes) [4]. This is apparently due to the fact that storage proteins abound in lysine, which is used in the synthesis of diamine, catalyzed by lysine decarboxylase (LDC) [6]. Cadaverine is usually contained at low concentrations in leaves (10 –8 and 10 –9 M) and at higher concentrations in roots (as much as 10 − 6 M). Our previous studies showed that M. crystallinum also belongs to cadaverine-producing species [1, 2], because aspartate, a distant precursor of lysine, is one of the major metabolites produced from oxaloacetate in the course of assimilation of CO 2 by the CAM type [7]. M. crystallinum is an ideal model system for studying the mechanisms of stress-induced adaptation to abiotic stressors (including salinization) [8]. In view of this, it was important to determine whether NaCl-induced accumulation of cadaverine occurs in plants and, if it so, whether it is related to ethylene production.

Organ-specific changes in the content of free and conjugated polyamines in Mesembryanthemum crystallinum plants under salinity

The content of free and conjugated polyamines (PA) was studied in the leaves of secondary shoots and in roots of the facultative halophyte Mesembryanthemum crystallinum L. under salinity. Plants were grown in water culture and at the age of 10 weeks were subjected to salinity stress by a single introduction of NaCl into nutrient medium to a final concentration of 400 mM. In 0, 6, 24, and 48 h, the content of free, HClO4-soluble, and HClO4-insoluble conjugates of PA were measured in leaves and roots. The levels of free and conjugated PA in the roots of control plants and plants subjected to salinity stress were higher than in the leaves. In response to salinity shock, the content of all forms of spermine (Spm), particularly its HClO4-soluble conjugates, increased in roots and leaves. In contrast, the content of Spm precursors, putrescine (Put) and spermidine (Spd), as well as their HClO4-soluble conjugates, decreased. Salinity treatment elevated the content of free cadaverine both in roots and leaves, whereas the content of its conjugated forms decreased, which might suggest transition of conjugated forms of cadaverine into the free form. The product of oxidative degradation of Spm and Spd, 1,3-diaminopropane (Dap), was absent in leaves, whereas the content of free and conjugated forms of this diamine in roots increased under salinity conditions. The obtained data indicate organ specificity for the content of free and conjugated forms of PA, as well as their active role in adaptation of Mesembryanthemum crystallinum to salinity shock.