Ágnes Szepesi

Salicylic acid improves acclimation to salt stress by stimulating abscisic aldehyde oxidase activity and abscisic acid accumulation, and increases Na+ content in leaves without toxicity symptoms in Solanum lycopersicum L.

Pre-treatment with 10(-4)M salicylic acid (SA) in hydroponic culture medium provided protection against salinity stress in tomato plants (Solanum lycopersicum L. cv. Rio Fuego). The effect of 10(-7) or 10(-4)M SA on the water status of plants was examined in relation to the biosynthesis and accumulation of abscisic acid (ABA) in order to reveal the role of SA in the subsequent response to salt stress. Both pre-treatments inhibited the K+(86Rb+) uptake of plants, reduced the K+ content of leaves, and caused a decrease in leaf water potential (psi(w)). Due to the changes in the cellular water status, SA triggered the accumulation of ABA. Since the decrease in psi(w) proved to be transient, the effect of SA on ABA synthesis may also develop via other mechanisms. In spite of osmotic adaptation, the application of 10(-4)M, but not 10(-7)M SA, led to prolonged ABA accumulation and to enhanced activity of aldehyde oxidase (AO1, EC.1.2.3.1.), an enzyme responsible for the conversion of ABA-aldehyde to ABA, both in root and leaf tissues. AO2-AO4 isoforms from the root extracts also exhibited increased activities. The fact that the activities of AO are significantly enhanced both in the leaves and roots of plants exposed to 10(-4)M SA, may indicate a positive feedback regulation of ABA synthesis by ABA in this system. Moreover, during a 100mM NaCl treatment, higher levels of free putrescine or spermine were found in these leaves or roots, respectively, than in the salt-stressed controls, suggesting that polyamines may be implicated in the protection response of the cells. As a result, Na+ could be transported to the leaf mesophyll cells without known symptoms of salt toxicity.

Salicylic acid treatment via the rooting medium interferes with stomatal response, CO2 fixation rate and carbohydrate metabolism in tomato, and decreases harmful effects of subsequent salt stress.

Salicylic acid (SA) applied at 10(-3) m in hydroponic culture decreased stomatal conductance (g(s)), maximal CO(2) fixation rate (A(max) ) and initial slopes of the CO(2) (A/C(i)) and light response (A/PPFD) curves, carboxylation efficiency of Rubisco (CE) and photosynthetic quantum efficiency (Q), resulting in the death of tomato plants. However, plants could acclimate to lower concentrations of SA (10(-7) -10(-4) m) and, after 3 weeks, returned to control levels of g(s), photosynthetic performance and soluble sugar content. In response to high salinity (100 mm NaCl), the pre-treated plants exhibited higher A(max) as a function of internal CO(2) concentration (C(i) ) or photosynthetic photon flux density (PPFD), and higher CE and Q values than salt-treated controls, suggesting more effective photosynthesis after SA treatment. Growth in 10(-7) or 10(-4) m SA-containing solution led to accumulation of soluble sugars in both leaf and root tissues, which remained higher in both plant parts during salt stress at 10(-4) m SA. The activity of hexokinase (HXK) with glucose, but not fructose, as substrate was reduced by SA treatment in leaf and root samples, leading to accumulation of glucose and fructose in leaf tissues. HXK activity decreased further under high salinity in both plant organs. The accumulation of soluble sugars and sucrose in roots of plants growing in the presence of 10(-4) m SA contributed to osmotic adjustment and improved tolerance to subsequent salt stress. Apart from its putative role in delaying senescence, decreased HXK activity may divert hexoses from catabolic reactions to osmotic adaptation.

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross-tolerance to various stressors. SA-stimulated pre-adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole-plant level, SA-induced massive H₂O₂ accumulation only at high concentrations (10⁻³-10⁻² M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre-treatments. In the root tips, 10⁻³-10⁻² M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre-adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt-treated samples. This suggests that, the cross-talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1-aminocyclopropane-1-carboxylic acid, the compounds accumulating in pre-treated plants, enhanced the diphenylene iodonium-sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.

Physiological and morphological responses of the root system of Indian mustard (Brassica juncea L. Czern.) and rapeseed (Brassica napus L.) to copper stress.

Copper (Cu) is an essential microelement for growth and development, but in excess it can cause toxicity in plants. In this comparative study, the uptake and accumulation of Cu as well as the morphological and physiological responses of Indian mustard (Brassica juncea L. Czern.) and rapeseed (Brassica napus L.) roots to Cu treatment were investigated. The possible involvement of redox active molecules (reactive oxygen species and nitric oxide) and modification in cell wall structure associated with Cu-induced morphological responses were also studied. In short- and long-term treatments, B. juncea suffered more pronounced growth inhibition as compared with B. napus. In addition to the shortening of primary and lateral roots, the number and the density of the laterals were also decreased by Cu. Exposure to copper induced nitric oxide generation in the root tips and this event proved to be dependent on the duration of the exposure and on the plant species. In short- and long-term treatments, Indian mustard showed more significant activation of superoxide dismutase (SOD), inhibition of ascorbate peroxidase (APX) and oxidation of ascorbate (AsA) than B. napus. Moreover, H2O2-dependent lignification was also observed in the Cu-exposed plants. In longer term, significant AsA accumulation and callose deposition were observed, reflecting serious oxidative stress in B. juncea. Based on the morphological and physiological results, we conclude that rapeseed tolerates Cu excess better than Indian mustard.

Overexpression of the mitochondrial PPR40 gene improves salt tolerance in Arabidopsis.

Mitochondrial respiration is sensitive to environmental conditions and can be influenced by abiotic stress. Previously we described the Arabidopsis mitochondrial pentatricopeptide repeat domain protein PPR40, and showed that the stress hypersensitive ppr40-1 mutant is compromised in mitochondrial electron transport (Zsigmond et al., 2008) [20]. Overexpression of the PPR40 gene in Arabidopsis resulted in enhanced germination and superior plant growth in saline conditions. Respiration increased in PPR40 overexpressing plants during salt stress. Reduced amount of hydrogen peroxide, diminished lipid peroxidation, lower ascorbate peroxidase and superoxide dismutase activity accompanied salt tolerance. Proline accumulation was enhanced in the ppr40-1 mutant, but unaltered in the PPR40 overexpressing plants. Our data suggest that PPR40 can diminish the generation of reactive oxygen species by stabilizing the mitochondrial electron transport and protecting plants via reducing oxidative damage during stress.

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10−7 or 10−4 M salicylic acid (SA), and in the roots after the 10−4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH4)2SO4 revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.

CHANGES IN CHLOROPHYLL FLUORESCENCE PARAMETERS AND OXIDATIVE STRESS RESPONSES OF BUSH BEAN GENOTYPES FOR SELECTING CONTRASTING ACCLIMATION STRATEGIES UNDER WATER STRESS

Drought resistance of bean landraces was compared in order to select genotypes with either high morphological or high biochemical-physiological plasticity. The lines in the former group exhibited fast reduction in fresh and dry mass, decreased the water potential in primary leaves after irrigation withdrawal and the biomass mobilized from the senescent primary leaves was allocated into the roots. These genotypes had high frequency of primary leaf abscission under water stress. The genotypes with plasticity at the biochemical level maintained high water potential and photochemical efficiency, i.e. effective quantum yield, high photochemical (qP) and low non-photochemical (NPQ) quenching in primary leaves under drought stress. While superoxide dismutase activity was not influenced by the drought and the genotype, catalase activity increased significantly in the primary leaves of the genotypes with efficient biochemical adaptation. Lines with high morphological plasticity exhibited higher quaiacol peroxidase activity under drought. Proline may accumulate in both cases, thus it may be a symptom of protein degradation or a successful osmotic adaptation. On the basis of contrasting responses, the genetic material cannot be screened for a large-scale breeding program by a single physiological parameter but by a set of the methods presented in this work.

Opposite extremes in ethylene/nitric oxide ratio induce cell death in suspension culture and root apices of tomato exposed to salt stress

The plant hormone ethylene or the gaseous signalling molecule nitric oxide (NO) may enhance salt stress tolerance by maintaining ion homeostasis, first of all K+/Na+ ratio of tissues. Ethylene and NO accumulation increased in the root apices and suspension culture cells of tomato at sublethal salt stress caused by 100 mM NaCl, however, the induction phase of programmed cell death (PCD) was different at lethal salt concentration. The production of ethylene by root apices and the accumulation of NO in the cells of suspension culture did not increase during the initiation of PCD after 250 mM NaCl treatment. Moreover, cells in suspension culture accumulated higher amount of reactive oxygen species which, along with NO deficiency contributed to cell death induction. The absence of ethylene in the apical root segments and the absence of NO accumulation in the cell suspension resulted in similar ion disequilibrium, namely K+/Na+ ratio of 1.41 ± 0.1 and 1.68 ± 0.3 in intact plant tissues and suspension culture cells, respectively that was not tolerated by tomato.

Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants

The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

Modulation of Cu 2+ accumulation by (aminoethoxyvinyl)glycine and methylglyoxal bis (guanylhydrazone), the inhibitors of stress ethylene and polyamine synthesis in wheat genotypes

The effectiveness of Cu 2+ accumulation was investigated in three wheat cultivars with different copper sensitivity (Triticum aestivum cv. GK Tiszataj, GK Kata and GK Othalom). Supraoptimal Cu 2+ concentrations result in toxicity symptoms in the sensitive genotype and increase the production of the stress hormone, ethylene both in the leaves and root tissues of wheat seedlings. The sensitive cultivar, cv. Othalom produced less ethylene than the tolerant genotypes (cvs Tiszataj and Kata) in the roots whether the ethylene measurements were done over the 6-h period after Cu 2+ exposure. Levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, did not change characteristically during this period in either the tolerant or sensitive seedlings. The biosynthesis of ethylene has a common intermediate, S-adenosylmethionine (SAM) with the synthesis of the polyamine spermidine and 2-deoxymugineic acid, a Fe 3+ -solubilizing and transporting wheat phytosiderophore. These chelating substances also mediate the transport of different bivalent cations, such as Cu 2+. The biosynthetic pathways of ethylene and polyamines, spermidine and spermine may compete for SAM with the phytosiderophore synthesis. Simultaneous inhibition of SAM decarboxylase by 0.5 M methylglyoxal bis (guanylhydrazone) (MGBG), and 1-aminocyclopropane-1-carboxylic acid synthase by 10 μM (2-aminoethoxy-vinyl)glycine (AVG), significantly increased the Cu 2+ accumulation in root tissues of the wheat cultivars independently of their sensitivities. MGBG alone resulted in an enhanced copper content but AVG proved to be ineffective. This suggests that the amount of SAM allocated for polyamine formation may limit the phytosiderophore synthesis or spermidine (spermine) in itself may control the uptake of Cu 2+.