Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

Changes in free polyamine levels induced by salt stress in leaves of cultivated and wild tomato species

The effects of N_aCl on endogenous free levels of the poluamines putrescine, spermi dine and spermine, and the relationships between polyamines, K⁺ levels and Na⁺ accumulation were determined in leaves of the cultivated tomato ( Lycopersicon esculentum Mill.) and its wild, salt-tolerant relative L. pennellii (Correll) D' Arcy at different exposure times during a 32-day period. Both stress treatments (100 and 200 m M NaCl) decreased the levels of putrescine and spermidine, although to a different degree for the cultivated and wild tomato species. The spermine levels did not decrease with salinity in L. pennellii over the salinization period, whereas they decreased in L. esculentum , except at the first application of the 100m M NaCl treatment. In both species, the changes induced by salinity in total polyamines and K⁺ were very similar, with the accumulation of Na⁺ in the leaf being concomitant with a decrease in both total polyamines and K⁺. This suggests that the main role of the polyamines in the leaf tissues. In this sense, a direct relationship between total polyamines and K⁺, and inverse relationship between polyamines and Na⁺ and between K⁺ and Na⁺ were found for both species. In the short term (up to 4 days) a peculiar physiological behavior was found in L. pennellii , as the total polyamine and K⁺ levels decreased at 100 m M but not at 200 m M NaCl, while after this time the latter plants had values lower than those of the 100 m M NaCl-treated plants at day 11.     

Priming of seeds with NaCl induces physiological changes in tomato plants grown under salt stress

The effects of seed priming with 6 M NaCl solution have been investigated with respect to growth and physiological responses of tomato plants ( Lycopersicon esculentum Mill. cv. Pera) exposed to 70 and 140 m M NaCl nutrient solutions from 11 to 60 days after sowing. Tomato seedlings from primed seeds emerged earlier than from non-primed seeds. At 70 m M , a lower shoot and root dry weight reduction was found in plants from primed seeds at the different harvests (30, 45 and 60 days after sowing), while at 140 m M the positive effect of seed priming was only shown in roots. Significant changes in Na⁺ and CI⁻ accumulation with seed priming were only found in roots at 60 days after sowing, with ion accumulation in roots being higher in plants grown at 70 and 140 m M from primed seeds. In leaves of salt-treated plants, significant increases in sugars and organic acids with seed priming were found from 30 days after sowing, and these increases were higher at longer treatment times. In roots, however, only the organic acids tended to increase in plants from primed seeds, although they increased less than in leaves, especially at 60 days after sowing. These results support the hypothesis that priming of seeds with NaCl induces physiological changes in the plants, changes which are shown more clearly at advanced growth stages.     

Fluxes of Na+, K+ and Cl-, and osmotic adjustment in Lycopersicon pimpinellifolium and L. esculentum during short- and long-term exposures to NaCl

NaCl (140 m M ) was applied to 14-day-old plants of salt-sensitive Lycopersicon esculentum Mill. cv. Volgogradskij and its wild relative L. pimpinellifolium Mill. accession PE-2. Changes in the relative growth rate of whole plant, and in the levels of inorganic and organic solutes in leaves, stems and roots were followed for 15 days after the application. Short-term salt exposure (4–6 days of salinization) resulted in enhanced relative growth rates for L. pimpinellifolium , but did not affect growth of L. esculentum , After 6 days of salinization, the relative growth rates of both species decreased significantly; leading to practically comparable growth rates for them by day 15. In all parts of both species, the contribution of organic solutes to the osmotic potential (Ψ_s) gradually decreased from 30% on day 0 to a value lower than 5% on day 4. In L. pimpinellifolium , compared to L. esculentum , short-term salt exposure resulted in (1) a higher percentage of adjustment of Ψ_s; and (2) increases in Na⁺ and K⁺ uptake rates, and in the levels of organic acids and proline (the level of which reached that of sugars, i.e., 10 μmol g^-1 dry weight. Conversely, in L. esculentum , drastic reductions of K⁺ uptake rates and organic acid levels occurred already on day 1. During long-term salt exposure, both species were able to adjust osmotically and both exhibited decreases in organic acid levels as well as in K⁺ uptake and accumulation rates in all parts. The results are discussed in an attempt to explain the adaptive responses during short-term salt exposure and the metabolic dysfunctions that lead to growth decrease after long-term exposure to salt.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

Altered levels of antioxidant enzymes associated with two mutations in tomato

Activity of peroxidase, superoxide dismutase and catalase were examined in leaves, stems and roots of olivacea ( oli ) and monstrosa ( mon ) mutants of Lycopersicon esculentum Mill. The extent of the difference between the pattern of oxidative enzyme activities of the wild type (wt) and the mutants was determined. The high peroxidase activity during the developmental stages of the leaves and stems of oli and mon phenotypes is associated with high levels of 4 anodic peroxidases in leaves and of two isozymes in the stem. Leaves of oli exhibit higher activity of the cathodic peroxidase C2, while both mutations have a marked increase of peroxidase C1 in stems. A positive relation between high peroxidase activity and oxidative stress damage was found: in chilling experiments at 5°C, peroxidase level in mutants and wt leaves was negatively correlated with electrolyte leakage. Superoxide dismutase (SOD) activity rises in oli stems around flowering time due to the high activity of the chloroplast forms SOD-1 and SOD-2. Catalases (CAT) were detectable only in early stages of plant development; CAT-2 was nearly absent in wild type tissues but well represented in mon and oli. The oli and mon mutations may affect critical steps of a regulatory pathway controlling various classes of oxidative enzymes in tomato.     

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L.

Calmodulin and NAD kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5–5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 mm. In-vitro NAD-kinase activity was found to increase 17-fold per cell over the apical 30 mm, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and NAD kinase were found to be highly dependent on extraction procedures.Abbreviation EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

等渗NaCl、干旱胁迫对番茄幼苗光合特性及叶绿体超微结构的影响

为分析等渗盐分和水分胁迫对番茄叶片光合功能的影响,选用耐盐性不同的4种基因型番茄(小果型的辽园红玛瑙、野生醋栗番茄、大果型的金田粉冠、超402)进行等渗的140 mmol·L^-1NaCl和15%PEG6000模拟盐分和水分胁迫.结果表明: 在光合特性方面,处理12 d后,两种胁迫导致4种不同基因型番茄的叶绿素含量(叶绿素a、叶绿素b、叶绿素总量)、光合速率、气孔导度、胞间CO₂浓度、蒸腾效率降低,气孔限制值升高.两种胁迫导致的光合下降由气孔性因素所致.等渗的盐分胁迫对番茄的光合系统损伤大于水分胁迫,这是因为盐分胁迫除了渗透胁迫还会导致离子伤害.4种不同基因型番茄中,耐盐型的辽园红玛瑙具有高光合特性,金田粉冠光合效率最差.在叶绿体超微结构方面,两种胁迫会造成番茄叶片的气孔密度增加,气孔张开率降低,叶绿体长度增加,宽度变小,长宽比增大,叶绿体内基粒数减少,嗜锇颗粒数增多.盐分胁迫下两种番茄叶绿体结构的影响大于水分胁迫,耐盐种醋栗番茄气孔变化小于盐敏感品种金田粉冠.     

Identification and quantitation of gibberellins in fruits of Lycopersicon esculentum, and their relationship to fruit size in L. esculentum and L. pimpinellfolium

GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were identified by combined gas chromatography-mass spectrometgry (GC-MS) in immature seeds and pericarp of Lycopersicon esculentum Mill. (tomato). Higher levels of these GAs were present in the seeds than in the pericarp; seeds in addition contained GA₁₅, GA₂₄, GA₂₅, and GA₄₄. Fruits of the Lycopersicon pimpinellifolium Mill. mutant I were smaller and contained lower GA₁ concentrations, but higher GA₂₀ concentrations, than those of mutants III and IV. In contrast, differences in fruit size in L. esculentum due to position on the truss did not correlate with GA₁ concentration in either the pericarp or seeds.     

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl₂ did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca²⁺ levels and ionic strength of the apoplast.     

The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt‐tolerant relative Lycopersicon pennellii

The possible involvement of the antioxidative system in the tolerance to salt stress was studied in the cultivated tomato Lycopersicon esculentum Mill. cv. M82 (M82) and its wild salt‐tolerant relative L. pennellii (Corn) D'Arcy accession Atico (Lpa). All analyses, except that of monodehydroascorbate reductase (MDHAR), were performed of the youngest fully‐expanded leaf of control and salt (100 m M NaCl) stressed plants, 4, 7, 10, 14, 18 and 22 days after completing the stress treatment. In Lpa, constitutive level of lipid peroxidation and activities of catalase (CAT) and glutathione reductase (GR) were lower while the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were inherently higher than in M82. Relative to M82, lipid peroxidation was much lower and the activities of SOD, CAT and APX were higher in Lpa at 100 m M NaCl. The activity of DHAR decreased more in Lpa than in M82 under salt stress, and the activity of MDHAR, which was lower in Lpa than in M82 under control conditions, increased much more and to a higher level in salt‐treated Lpa plants. GR activity decreased similarly in the two species under salt stress. The results of these analyses suggest that the wild salt‐tolerant Lpa plants are better protected against active oxygen species (AOS), inherently and under salt stress, than the relatively sensitive plants of the cultivated species.     

外源NO与蔗糖对盐胁迫下番茄(Lycopersicon esculentum Mill)幼苗氧化损伤的保护效应

选取长至6～8片真叶的健康番茄(Lycopersicon esculentum Mill)幼苗,分别进行蔗糖、硝普钠(sodium nitropresside, SNP, 作为外源NO供体)及其体积比例组合(1:1)处理;36h后施以NaCl胁迫,并分别于0h(胁迫前)、24h、48h和72h 取样,进行相关生理生化指标测定.具体5个实验处理如下:A. 蒸馏水(CK); B. 100 mmol/L NaCl;C. 0.1 mmol/L SNP 100 mmol/L NaCl;D. 0.1 mmol/L SNP 1.0mmol/L蔗糖 100 mmol/L NaCl; E. 1.0 mmol/L蔗糖 100 mmol/L NaCl.结果表明:与SNP和蔗糖单独处理相比,二者组合处理对缓解盐胁迫下番茄幼苗的氧化损伤存在正协同效应,主要表现在进一步增强了番茄幼苗超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)的活性;提高了脯氨酸(Pro)的含量,同时膜脂过氧化产物丙二醛(MDA)含量显著降低(P＜0.05).采用聚丙烯酰胺浓度梯度凝胶电泳对盐胁迫24 h和48 h材料的POD同功酶检测表明,当NaCl单独处理时,番茄幼苗叶片POD同功酶第V条带缺失,其它谱带酶量减少,抑制了POD同功酶的表达;SNP和蔗糖单独处理能够保护盐胁迫(24、48h)所导致的POD同功酶条带的完整;而组合处理既保证了POD同功酶条带的完整,又加强了酶量的表达.随着盐胁迫时间的延长,其氧化损伤程度愈烈,SNP和蔗糖组合处理能够更有效地缓解盐胁迫对番茄幼苗植株造成的氧化损伤.     

肉桂醛对NaCl胁迫下番茄幼苗生长特性的影响

为探明肉桂醛对番茄幼苗耐盐性的影响,该研究以番茄‘合作903’为试验材料,探究肉桂醛(100μg·L^-1)对处于100 mmol·L^-1 NaCl胁迫下番茄种子萌发与幼苗生长特性的影响。在试验中设置4组处理：对照(CK,蒸馏水)、NaCl (100 mmol·L^-1)、NaCl+CA(100 mmol·L^-1 NaCl+100μg·L^-1 CA)、CA(100μg·L^-1)。结果表明：(1)在100 mmol·L^-1 NaCl胁迫下,添加100μg·L^-1的肉桂醛,番茄种子的发芽势和发芽率均有所提升,番茄幼苗的根长和鲜重均显著提高,说明添加肉桂醛对NaCl胁迫下番茄种子的萌发与幼苗生长均有一定缓解作用。(2)肉桂醛能够有效降低NaCl胁迫导致的番茄幼苗根尖总活性氧(reactive oxygen species, ROS)的过量累积,缓解膜脂过氧化程度从而降低细胞死亡率。综上表明,肉桂醛处理能够通过缓解幼苗的氧化...