Role of nitrogen metabolism in the development of salt tolerance in barley plants

The 7- to 8-day-old barley (Hordeum vulgare L.) seedlings grown in KNO3 solutions (1-40 mM) were characterized by the substrate activation of nitrate reductase (NR) in the apical leaf segments (1–2 cm in length), as well as by stimulated growth, broadened leaf blades, and by vigorously developed system of shortened roots. When the seedlings were grown in the presence of 20 mM KNO₃, the ability of leaf segments to generate superoxide anion radical remained at the level typical of control plants grown in water. The content of 5-aminolevulinic acid (ALA) in plants grown in the presence of 20 mM KNO₃ was 2.2–2.4 times higher than in control plants. The plants grown in the presence of nitrate had an elevated content of chlorophylls a and b, heme, and protein (by 42%). At the same time, the proline content was almost twofold lower than in control plants, which was due to substantial reduction (by 40%) in activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS). It is concluded that the substrate activation of NR by KNO₃ under normal growth conditions results in predominant utilization of glutamic acid (the primary product of inorganic nitrogen assimilation) for biosynthesis of tetrapyrroles and protein amino acids at the expense of inhibition of proline synthesis. When barley seedlings were grown in 150 mM NaCl solution, the plant growth and the root system development were suppressed to the levels of 63 ± 6% and 61 ± 11% of the control values, respectively. In the apical leaf tissues of plants adapted to NaCl, there was a slight decrease in the total NR activity (by 10%), a significant reduction in protein content (by 32%), and a parallel increase in the content of ALA (by a factor of 4.3), chlorophylls, heme, carotenoids, proline (2.2-fold) and P5CS (1.6-fold) with respect to the control values. It is proposed that the accumulation of ALA and proline under salinity-induced suppression of nitrogen assimilation results from the predominant allocation of glutamate for biosyntheses of ALA and proline at the expense of inhibition of growth-related processes requiring intense protein synthesis. The substrate activation of NR by KNO₃ under salinity conditions was associated with prevailing allocation of the assimilated nitrogen for synthesis of proline and protein amino acids, which reinforced plant cell protection against salinity and stimulated plant growth.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

Changes in water relations,photosynthetic activity and proline accumulation in one-year-old olive trees (<Emphasis Type="Italic">Olea europaea</Emphasis> L. cv. Chemlali) in response to NaCl salinity

The comparative responses of young olive trees (Olea europaea L. cv “Chemlali”) to different NaCl salinity levels were investigated over 11 months. One-year-old own rooted plants were grown in 10-L pots containing sand and perlite mixture (1:3 v/v). Trees were subjected to three irrigation treatments: CP (control plants that were irrigated with fresh water); SS1 (salt stressed plants irrigated with water containing 100 mM NaCl) and SS2 plants (salt stressed plants irrigated with water containing 200 mM NaCl). Shoot elongation rate, relative water content, leaf water potential and net carbon dioxide exchange rates decreased significantly with increased NaCl salinity level. Under stressed conditions, the increase of Na⁺ and Cl⁻ ions in both leaves and roots was accompanied with that of proline and soluble sugars. The above results show that the accumulation of proline and sugars under stressed conditions could play a role in salt tolerance. The absence of toxicity symptoms under both stress treatments and the superior photosynthetic activity recorded in SS1-treated plants suggest that cv Chemlali is better able to acclimatize to 100 mM NaCl than at 200 mM NaCl. Our findings indicate that saline water containing 100 mM NaCl, the most available water in arid region in Tunisia, can be recommended for the irrigation of cv Chemlali in the arid south of Tunisia.     

Light-Dark Changes in Proline Content of Barley Leaves under Salt Stress

Proline accumulation in leaves of barley (Hordeum vulgare L. cv. Alfa) seedlings treated with 150 mM NaCl was promoted in the light and suppressed in the dark. The light/dark changes of proline content was enhanced with each 12 h light/12 h dark cycle and the proline content increased steadily. Root and shoot concentrations of Na⁺ and Cl^– in salt treated plants increased about 10 to 25 times as compared to the control. The content of these ions and the content of malondialdehyde were higher in the shoot of seedlings exposed to salt stress for 4 d in the light in comparison with the seedlings exposed to NaCl for 4 d in darkness. Light stimulated both ions and proline accumulation in the leaves and has no effect in the roots. Oxygen uptake was higher in the seedlings kept 4 d in the light which have higher endogenous free proline content. Chlorophyll fluorescence measurements showed that the photochemical activity of PS 2 slightly decreased as a result of salt stress and was not influenced by light regimes during plant growth.     

外源5-氨基乙酰丙酸对NaCl胁迫下黑果枸杞幼苗生理及生长的影响

5-氨基乙酰丙酸(ALA)是植物血红素、叶绿素等四吡咯化合物的关键生物合成前体,对植物适应非生物胁迫至关重要。为验证外源ALA对黑果枸杞幼苗生理生长的影响,该研究用300 mmol·L^-1 NaCl和不同浓度(0、5、10、15、20、25 mg·L^-1)的ALA共同处理黑果枸杞幼苗,并测定其相关的生理指标和生长指标,综合评价各处理幼苗的耐盐性。结果表明：(1)NaCl胁迫使黑果枸杞幼苗总生物量和叶片总叶绿素、类胡萝卜素、可溶性糖含量以及过氧化物酶(POD)活性较CK分别显著降低了33.39%、19.06%、24.38%、39.57%和47.91%(P<0.05),使黑果枸杞幼苗脯氨酸和丙二醛的含量较CK分别显著增加了165.74%和49.16%。(2)当外源ALA和NaCl同时处理时,黑果枸杞幼苗叶片类胡萝卜素和丙二醛含量、POD和过氧化氢酶(CAT)活性以及株高、总生物量均恢复至对照水平,叶片总叶绿素和脯氨酸含量以及SOD活性较CK显著增加。(3)黑果枸杞幼苗叶片叶绿素和脯氨酸含量以及抗氧化酶活性、生物量等指标随ALA浓度增加均呈先...     

Salt‐stress induced modulation of chlorophyll biosynthesis during de‐etiolation of rice seedlings

Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100–200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (–58%) and Chl accumulation (–66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41–45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5‐aminolevulinic acid (ALA) dehydratase (EC‐2.4.1.24), porphobilinogen deaminase (EC‐4.3.1.8), coproporphyrinogen III oxidase (EC‐1.3.3.3), protoporphyrinogen IX oxidase (EC‐1.3.3.4), Mg‐protoporphyrin IX chelatase (EC‐6.6.1.1) and protochlorophyllide oxidoreductase (EC‐1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt‐stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl‐tRNA reductase (EC‐1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt‐stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.     

Sodium,potassium, chloride and proline concentrations of chloroplasts isolated from a halophyte,Mesembryanthemum crystallinum L.

Concentrations of four major solutes (Na⁺, K⁺, Cl^-, proline) were determined in isolated, intact chloroplasts from the halophyte Mesembryanthemum crystallinum L. following long-term exposure of plants to three levels of NaCl salinity in the rooting medium. Chloroplasts were obtained by gentle rupture of leaf protoplasts. There was either no or only small leakage of inorganic ions from the chloroplasts to the medium during three rapidly performed washing steps involving precipitation and re-suspension of chloroplast pellets. Increasing NaCl salinity of the rooting medium resulted in a rise of Na⁺ und Cl^- in the total leaf sap, up to approximately 500 and 400 mM, respectively, for plants grown at 400 mM NaCl. However, chloroplast levels of Na⁺ und Cl^- did not exceed 160–230 and 40–60 mM, respectively, based upon a chloroplast osmotic volume of 20–30 l per mg chlorophyll. At 20 mM NaCl in the rooting medium, the Na⁺/K⁺ ratio of the chloroplasts was about 1; at 400 mM NaCl the ratio was about 5. Growth at 400 mM NaCl led to markedly increased concentrations of proline in the leaf sap (8 mM) compared with the leaf sap of plants grown in culture solution without added NaCl (proline 0.25 mM). Although proline was fivefold more concentrated in the chloroplasts than in the total leaf sap of plants treated with 400 mM NaCl, the overall contribution of proline to the osmotic adjustment of chloroplasts was small. The capacity to limit chloroplast Cl^- concentrations under conditions of high external salinity was in contrast to an apparent affinity of chloroplasts for Cl^- under conditions of low Cl^- availability.Abbreviation Chl chlorophyll     

Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea)

ALA is a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, and was found to induce temporary elevations in the photosynthesis rate, APX, and CAT; furthermore, treatment with ALA at a low concentration might be correlated to the increase of NaCl tolerance of spinach plants. The photosynthetic rate and the levels of active oxygen-scavenging system in the 3rd leaf of spinach (Spinacia oleracea) plants grown by foliar treatment with 0, 0.18, 0.60 and 1.80 mmol/L 5-aminolevulinic acid under 50 and 100 mmol/L NaCl were analyzed. Plants treated with 0.60 and 1.80 mmol/L ALA showed significant increases in the photosynthetic rate at 50 and 100 mmol/L NaCl, while that of 0.18 mmol/L ALA did not show any changes at 50 mmol/L NaCl and a gradual decrease at 100 mmol/L NaCl. In contrast, the rate with 0 mmol/L ALA showed reduction at both concentrations of NaCl. The increase of hydrogen peroxide content by treatment with 0.60 and 1.80 mmol/L ALA were more controlled than that of 0 mmol/L ALA under both NaCl conditions. These ALA-treated spinach leaves also exhibited a lower oxidized/reduced ascorbate acid ratio and a higher reduced/oxidized glutathione ratio than the 0 mmol/L-treated spinach leaves when grown at both NaCl conditions. With regard to the antioxidant enzyme activities in the leaves, ascorbate peroxidase, catalase, and glutathione reductase activities were enhanced remarkably, most notably at day 3, by treatment with 0.60 and 1.80 mmol/L ALA under both NaCl conditions in comparison to that of 0 and 0.18 mmol/L ALA. These data indicate that the protection against oxidative damage by higher levels of antioxidants and enzyme activities, and by a more active ascorbate-glutathione cycle related to the increase of the photosynthesis rate, could be involved in the increased salt tolerance observed in spinach by treatment with 0.60 to 1.80 mmol/L ALA with NaCl.     

Effect of salinity on chlorophyll and proline contents in three aquatic macrophytes

The role of proline in imparting tolerance to salinity was investigated in Hydrilla verticillata, Najas indica and Najas gramenia. The plants were exposed to different concentrations of NaCl and artificial sea water (SWS) separately. The chlorophyll (Chl) a/b ratio decreased significantly in all the three plant species in both NaCl and SWS treatments, comparatively more in former than the latter. NaCl resulted in drastic decrease in this ratio in salt sensitive H. verticillata and N. indica, but in somewhat lesser decrease in salt resistant N. gramenia. Proline content increased at both NaCl and SWS treatments, especially at the latter. However, in H. verticillata proline content at 1.5 and 2.5 % NaCl decreased. It was concluded that proline cannot be used as a biochemical marker of salt tolerance in aquatic plants, however, the decrease in Chl a/b ratio in response to NaCl may be used as an index of salt sensitivity in this ecological group of plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of NaCl and Proline on Polyphenol Oxidase Activity in Bean Seedlings

In this work, the effects of NaCl (0, 50, 100, and 150 mM), proline (0, 5 and 10 mM) and NaCl + proline in combinations on activity of polyphenol oxidase (PPO; E.C. 1.10.3.1) and soluble protein content have been investigated in the root, stem and leaf tissues of bean (Phaseolus vulgaris L.) seedlings grown in embryo culture. PPO activities were higher in all the tissues treated with NaCl, proline and NaCl + proline combinations those that of the control tissues. The protein content was very high in tissues exposed to proline and NaCl + proline combination, but NaCl alone decreased protein contents in root and leaf tissues. The results suggest that proline may play a role as an enzyme-stabilizing agent in salt stress.     

Physiological effects of exogenous nitric oxide on Brassica juncea seedlings under NaCl stress

The study was conducted to investigate the physiological effects of exogenous NO on potherb mustard (Brassica juncea Coss.) seedlings under salt stress. The plants were grown in Hogland nutrient solution for 15 d and treated with 150 mM NaCl, NO donor sodium nitropruside (SNP) and NO scavenger methylene blue (MB-1) for 4 d. The NaCl stress increased superoxide dismutase, peroxidase and ascorbate peroxidase activities and malondialdehyde (MDA) and free proline contents, and decreased soluble protein content. However, the application of exogenous NO limited the production of MDA and free proline, while markedly promoted SOD, POD and APX activity.     

Response of barley grains to the interactive e.ect of salinity and salicylic acid

Effect of grain soaking presowing in 1 mM salicylic acid (SA) and NaCl (0, 50, 100, 150 and 200 mM) on barley (Hordeum vulgare cv Gerbel) was studied. Increasing of NaCl level reduced the germination percentage, the growth parameters (fresh and dry weight), potassium, calcium, phosphorus and insoluble sugars content in both shoots and roots of 15-day old seedlings. Leaf relative water content (RWC) and the photosynthetic pigments (Chl a, b and carotenoids) contents also decreased with increasing NaCl concentration. On the other hand, Na, soluble sugars, soluble proteins, free amino acids including proline content and lipid peroxidation level and peroxidase activity were increased in the two plant organs with increasing of NaCl level. Electrolyte leakage from plant leaves was found to increase with salinity level. SA-pretreatment increased the RWC, fresh and dry weights, water, photosynthetic pigments, insolube saccharides, phosphorus content and peroxidase activity in the stressed seedlings. On the contrary, Na⁺, soluble proteins content, lipid peroxidation level, electrolyte leakage were markedly reduced under salt stress with SA than without. Under stress conditions, SA-pretreated plants exhibited less Ca²⁺ and more accumulation of K⁺, and soluble sugars in roots at the expense of these contents in the plant shoots. Exogenous application (Grain soaking presowing) of SA appeared to induce preadaptive response to salt stress leading to promoting protective reactions to the photosynthetic pigments and maintain the membranes integrity in barley plants, which reflected in improving the plant growth.     

Salt stress responses of a halophytic grass <Emphasis Type="Italic">Aeluropus lagopoides</Emphasis> and subsequent recovery

To investigate the salt tolerance mechanisms, Aeluropus lagopoides as a halophytic plant was used. Plants were treated with 0, 150, 450, 600, and 750 mM NaCl and harvested at 0, 4, 8, and 10 days after treatment and 1 day and 1 week after recovery. Optimal growth, measured as fresh and dry weights, occurred at 150 mM NaCl, but it was suppressed by 450, 600, and 750 mM NaCl. Recovery significantly increased fresh and dry weights only in 750 mM NaCl-treated plants. Water content was decreased after NaCl treatment and increased after recovery. Na⁺ and proline contents and activity of superoxide dismutase (SOD) were increased after NaCl treatment and decreased after recovery in all treated plants. In contrast, K⁺ content and ascorbate peroxidase activity decreased after NaCl treatment and increased after recovery in all treated plants. Catalase (CAT) was activated only in 750 mM NaCl-treated plants. Total content of soluble protein was slightly changed after NaCl treatment. It was concluded that proline accumulation for osmotic adjustment, SOD activation for O₂^·− scavenging, and CAT activation at the higher level of salt stress to detoxify produced H₂O₂ were main A. lagopoides strategies under salt stress. A. lagopoides salt tolerance was not based on the restriction of Na⁺ uptake.     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

Gamma radiation and hormone treatment as tools to reduce salt stress in rice (Oryza sativa L.)

We investigated the effects of jasmonic acid (JA) and gamma irradiation on the growth and metabolic responses to salt stress in rice (Oryza sativa L.) plants. The relative growth rate (RGR), relative water content (RWC), and chlorophyll (Chl) content were lower in NaCI-treated plants than in the control, whereas the malondialdehyde content (MDA), electrolyte leakage (EL), and contents of proline and abscisic acid (ABA) were higher in the treated plants. When induced by the salt stress, those effects, however, were somewhat alleviated by the application of JA or gamma irradiation. The most significant response was manifested by the proline content, with relatively lower values for alleviation being recorded for the contents of RGR, RWC, Chl, and MDA, as well as EL. Moreover, although total Chl content was not significantly influenced by JA or gamma irradiation in plants under salt stress, an increase in the level of Chl a resulted in a markedly changed ratio of Chl a/b. The degree of alleviation, in terms of growth and metabolic responses, was more extensive for JA-treated plants than for those exposed to gamma irradiation.     

Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity

Proline accumulation was studied in the leaves of Glycine max (L.) Merr. subjected to salt stress in the presence of aminoguanidine (AG, a specific inhibitor of diamine oxidase, DAO) and exogenous putrescine (Put). Both DAO activity and proline content were increased while endogenous Put content was decreased in soybean leaves under 50 to 150 mM NaCl. There was a negative correlation between proline accumulation and endogenous Put content. The addition of AG during NaCl stress inhibited DAO activity, caused Put accumulation and a 15 to 20 % decrease in proline content. Application of 1 mM Put to NaCl solution markedly increased proline content. The promotive effect of Put application could be alleviated by the treatment with Put plus AG. Moreover an application of AG had no effect on proline accumulation in soybean seedlings grown under normal condition. These results indicate that the quantitative contribution of Put degradation to proline formation is 15 to 20 %.     

Photodynamic processes and the synthesis of 5-aminolevulinic acid in chlorella cells treated with amino acids and 1,10-phenanthroline

Treatment of chlorella (Chlorella sp.) cells for 2 h in darkness with tetrapyrrole-dependent photodynamic herbicides (TDPH) derived on the basis of 0.3 mM 1,10-phenanthroline (Ph) combined with 0.6 mM Glu or 0.6 mM Gln induced the accumulation of sensitizers of photodynamic processes: magnesium protoporphyrin IX (MgPP) and MgPP monomethyl ester (MgPPE). Within the first day after chlorella cells treated with TDPH were illuminated, photodestruction of MgPP(E) was observed, and production of the first specific precursor of chlorophyll (Chl), 5-aminolevulinic acid (ALA), in the cells declined. Then the accumulation of ALA was stimulated, and the level of heme, which is a retroinhibitor of ALA synthesis, simultaneously fell. During the first two days of illumination, the content of Chl and carotenoids in the algae treated with TDPH did not differ from their levels in control culture, which suggests a high resistance of photosynthetic pigments to photodynamic process induced by porphyrins. Subsequently, a slight but rising in time accumulation of pheophytin (Pheo) was observed, as well as photodestruction of Chl and carotenoids. After five days of illumination, the difference in the content of Chl between the culture treated with TDPH and control material was 10–30% depending on the illuminance. Chlorella cells treated with TDPH remained capable of producing Chl from exogenous ALA in the dark for at least eight days. In the experiments simultaneously conducted with a higher plant, cucumber (Cucumis sativa L.), which accumulated in the dark essentially the same content of porphyrins in response to TDPH as algae did, the residual level of Chl after five days of illumination was only 10–20% of control plants. It was assumed that a high tolerance of the chlorella pigment pool to photooxidative stress induced by the accumulation of MgPP(E) and Pheo depended on a highly active state of the antioxidant protective system and the ability of ALA molecules additionally formed under the influence of TDPH to be converted into Chl, thereby participating in its de novo synthesis.     

Effect of ABA on the contents of proline, polyamines, and cytokinins in the common ice plants under salt stress

The effects of ABA treatment on the contents of proline, polyamines (PA), and cytokinins (CK) in the facultative halophyte the common ice plant (Mesembryanthemum crystallinum L.) subjected to salt stress were studied. Plants grown in the phytotron chamber on Jonson nutrient medium for 6 weeks were subjected to 6-day-long salinity by a single NaCl adding to medium. During first three days of salinity, half plants of each treatment were placed for 30 min on nutrient medium containing 0, 100, or 300 mM NaCl plus ABA in the final concentration of 1 μM. Salinity reduced biomass accumulation and water and chlorophyll contents in plants. This was accompanied by the increase in the levels of MDA, proline, and sodium ions. ABA treatment of salt-stressed plants favored biomass accumulation and photosynthetic pigment protection, reduced the intensity of oxidative stress and the level of NaCl-induced proline accumulation. ABA treatment increased the contents of putrescine (Put) and spermidine (Spd) in the leaves and roots of control plants (not subjected to salt stress), reduced the losses of Put in the leaves and roots and Spd in the roots in the presence of 100 mM NaCl, and suppressed cadaverine (Cad) accumulation in the roots in the presence of 300 mM NaCl. In the presence of NaCl, ABA reduced the contents of zeatin and zeatin riboside and increased the level of zeatin-O-glucoside in the roots and isopentenyladenosine and isopentenyladenine in the leaves. Thus, ABA protective action under salinity can be realized through the weakening of oxidative stress (a decrease in MDA content) and the regulation of PA, proline, and CK metabolism, which has a great significance in plant adaptation to injurious factors.     

Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro

The use of in vitro shoot cultures to evaluate osmotic and salt tolerance and the effects of salt and mannitol in the medium on proline and sugar accumulation were investigated in two poplar species, P. euphratica and P. alba cv. Pyramidalis × P. tomentosa. Shoot length, leaf number, whole plant dry weight, and the accumulation of proline and total soluble sugars in leaves were quantified after 2 weeks. All P. euphratica plantlets survived at all levels of mannitol and NaCl, while the mortality of P. alba cv. Pyramidalis × P. tomentosa increased both at the mannitol and the NaCl treatments. A significant increase in proline accumulation was observed in both young and mature P. euphratica leaves at 200 mM mannitol and above, and at 150 mM NaCl and above. The total soluble sugar content increased in young P. euphratica leaves at 250 mM NaCl; however, it decreased in the mature leaves. Similar increases of the total soluble sugar content were not seen in P. alba cv. Pyramidalis × P. tomentosa plants in response to either mannitol or NaCl treatment. Our results suggest that accumulated proline and sugars promote osmotic and salt tolerance. The effects of accumulated proline and total soluble sugars on leaves are discussed in relation to growth and osmotic adjustment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Accumulation of photosynthetic pigments in Larix decidua Mill. and Picea abies (L.) Karst. cotyledons treated with 5-aminolevulinic acid under different irradiation

European larch (Larix decidua Mill.) and Norway spruce [Picea abies (L.) Karst.] synthesize chlorophyll (Chl) in darkness. This paper compares Chl accumulation in 14-d-old dark-grown seedlings of L. decidua and P. abies after shortterm (24 h) feeding with 5-aminolevulinic acid (ALA). We used two ALA concentrations (1 and 10 mM) fed to cotyledons of both species in darkness and in continuous light. The dark-grown seedlings of L. decidua accumulated Chl only in trace amounts and the seedlings remained etiolated. In contrast, P. abies seedlings grown in darkness were green and had significantly higher Chl content. After ALA feeding, higher protochlorophyllide (Pchlide) content was observed in L. decidua than in P. abies cotyledons incubated in darkness. Although short-term ALA feeding stimulated the synthesis of Pchlide, Chl content did not change significantly in cotyledons incubated in darkness. The Chl accumulation in cotyledons fed with ALA was similar to the rate of Chl accumulation in the controls. Higher Chl accumulation was reported in control samples after illumination: 86.9% in L. decidua cotyledons and 46.4% in P. abies cotyledons. The Chl content decreased and bleaching occurred in cotyledons incubated with ALA in light due to photooxidation. Analyses of Chlbinding proteins (D1 and LHCIIb) by Western blotting proved differences between Chl biosynthesis in L. decidua and P. abies seedlings in the dark and in the light. No remarkable increase was found in protein accumulation (D1 and LHCIIb) after ALA application. Our results showed interspecific difference in Chl synthesis between two gymnosperms. Shortterm ALA feeding did not stimulate Chl synthesis, thus ALA synthesis was not the rate-limiting step in Chl synthesis in the dark.