The local and systemic accumulation of ethylene determines the rapid defence responses induced by flg22 in tomato (Solanum lycopersicum L.).

Plant defence responses induced by the bacterial elicitor flg22 are highly dependent on phytohormones, including gaseous ethylene (ET). While the regulatory role of ET in local defence responses to flg22 exposure has been demonstrated, its contribution to the induction of systemic responses is not clearly understood. For this consideration, we examined the effects of different ET modulators on the flg22-induced local and systemic defence progression. In our experiments, ET biosynthesis inhibitor aminoethoxyvinyl glycine (AVG) or ET receptor blocker silver thiosulphate (STS) were applied 1 h before flg22 treatments and 1 h later the rapid local and systemic responses were detected in the leaves of intact tomato plants (Solanum lycopersicum L.). Based on our results, AVG not only diminished the flg22-induced ET accumulation locally, but also in the younger leaves confirming the role of ET in the whole-plant expanding defence progression. This increase in ET emission was accompanied by increased local expression of SlACO1, which was reduced by AVG and STS. Local ET biosynthesis upon flg22 treatment was shown to positively regulate local and systemic superoxide (O2.-) and hydrogen peroxide (H2O2) production, which in turn could contribute to ET accumulation in younger leaves. Confirming the role of ET in flg22-induced rapid defence responses, application of AVG reduced local and systemic ET, O2.- and H2O2 production, whereas STS reduced it primarily in the younger leaves. Interestingly, in addition to flg22, AVG and STS induced stomatal closure alone at whole-plant level, however in the case of combined treatments together with flg22 both ET modulators reduced the rate of stomatal closure in the older- and younger leaves as well. These results demonstrate that both local and systemic ET production in sufficient amounts and active ET signalling are essential for the development of flg22-induced rapid local and systemic defence responses.

The role of ethylene signalling in the regulation of salt stress response in mature tomato fruits: Metabolism of antioxidants and polyamines.

Salt stress-induced ethylene (ET) can influence the defence responses of plants that can be dependent on plant organs. In this work, the effects of salt stress evoked by 75 mM NaCl treatment were measured in fruits of wild-type (WT) and ET receptor-mutant Never ripe (Nr) tomato. Salt stress reduced the weight and size of fruits both in WT and Nr, which proved to be more pronounced in mutants. In addition, significantly higher H2O2 levels and lipid peroxidation were measured after the salt treatment in Nr as compared to the untreated control than in WT. ET regulated the key antioxidant enzymes, especially ascorbate peroxidase (APX), in WT but in the mutant fruits the activity of APX did not change and the superoxide dismutase and catalase activities were downregulated compared to untreated controls after salt treatment contributing to a higher degree of oxidative stress in Nr fruits. The dependency of PA metabolism on the active ET signalling was investigated for the first time in fruits of Nr mutants under salt stress. 75 mM NaCl enhanced the accumulation of spermine in WT fruits, which was not observed in Nr, but levels of putrescine and spermidine were elevated by salt stress in these tissues. Moreover, the catabolism of PAs was much stronger under high salinity in Nr fruits contributing to higher oxidative stress, which was only partially alleviated by the increased total and reduced ascorbate and glutathione pool. We can conclude that ET-mediated signalling plays a crucial role in the regulation of salt-induced oxidative stress and PA levels in tomato fruits at the mature stage.

Role of ethylene in ER stress and the unfolded protein response in tomato (Solanum lycopersicum L.) plants.

The unfolded protein response (UPR) plays a significant role in the maintenance of cellular homeostasis under endoplasmic reticulum (ER) stress, which is highly dependent on the regulation of defense-related phytohormones. In this study, the role of ethylene (ET) in ER stress and UPR was investigated in the leaves of intact tomato (Solanum lycopersicum) plants. Exogenous application of the ET precursor 1-aminocyclopropane-1-carboxylic acid not only resulted in higher ET emission from leaves but also increased the expression of the UPR marker gene SlBiP and the transcript levels of the ER stress sensor SlIRE1, as well as the levels of SlbZIP60, after 24 h in tomato leaves. Using ET receptor Never ripe (Nr) mutants, a significant role of ET in tunicamycin (Tm)-induced ER stress sensing and signaling was confirmed based on the changes in the expression levels of SlIRE1b and SlBiP. Furthermore, the analysis of other defense-related phytohormones showed that the Tm-induced ET can affect positively the levels of and response to salicylic acid. Additionally, it was found that nitric oxide production and lipid peroxidation, as well as the electrolyte leakage induced by Tm, is regulated by ET, whereas the levels of H2O2 and proteolytic activity seemed to be independent of ET under ER stress in the leaves of tomato plants.

Interaction between polyamines and ethylene in the response to salicylic acid under normal photoperiod and prolonged darkness.

The impact of salicylic acid (SA) on ethylene (ET) production and polyamine (PA) metabolism was investigated in wild type (WT) and ET receptor mutant Never ripe (Nr) tomato leaves under normal photoperiod and prolonged darkness. Nr displayed higher ET emanation compared to WT under control conditions and after SA treatments, but the ET signalling was blocked in these tissues. The accumulation of PAs was induced by 1 mM but not by 0.1 mM SA and was higher in WT than in Nr leaves. Upon 1 mM SA treatment, which caused hypersensitive response, illuminated leaves of WT showed high spermine (Spm) content in parallel with an increased expression of S-adenosylmethionine decarboxylase and Spm synthase (SlSPMS) suggesting that this process depended on the light. In Nr, however, Spm content and the expression of the SlSPMS gene were very low independently of the light conditions and SA treatments. This suggests that Spm synthesis needs functional ET perception. In WT leaves 1 mM SA enhanced putrescine (Put) synthesis by increasing the expression of Put biosynthesis genes, arginine and ornithine decarboxylases under darkness, while they were down-regulated in Nr. The activities of diamine (DAO) and polyamine oxidases (PAO), however, were generally higher in Nr compared to the WT after SA treatments. In Nr both SA applications increased the expression of SlPAO1 under normal photoperiod, while SlPAO2 was down-regulated in the dark suggesting a diverse role of PAOs in PA catabolism. These results indicated that ET could modulate the SA-induced PA metabolism in light-dependent manner.

Activation of Local and Systemic Defence Responses by Flg22 Is Dependent on Daytime and Ethylene in Intact Tomato Plants.

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) mutants in the local and systemic defence responses of intact tomato plants after flg22 treatments. Flg22 was applied in the afternoon and at night and rapid reactions were detected. The production of hydrogen peroxide and nitric oxide was induced by flg22 locally, while superoxide was induced systemically, in wild type plants in the light period, but all remained lower at night and in Nr leaves. Flg22 elevated, locally, the ET, jasmonic acid (JA) and salicylic acid (SA) levels in the light period; these levels did not change significantly at night. Expression of Pathogenesis-related 1 (PR1), Ethylene response factor 1 (ERF1) and Defensin (DEF) showed also daytime- and ET-dependent changes. Enhanced ERF1 and DEF expression and stomatal closure were also observable in systemic leaves of wild type plants in the light. These data demonstrate that early biotic signalling in flg22-treated leaves and distal ones is an ET-dependent process and it is also determined by the time of day and inhibited in the early night phase.

Changes in physiological and photosynthetic parameters in tomato of different ethylene status under salt stress: Effects of exogenous 1-aminocyclopropane-1-carboxylic acid treatment and the inhibition of ethylene signalling.

Although ethylene (ET) is an important participant in plant responses to salt stress, its role in the early period of acclimation, especially in the case of photosynthesis has not been revealed in detail. In this study, the effects of tolerable (100 mM) or lethal (250 mM) NaCl concentrations were investigated in hydroponically grown tomato (Solanum lycopersicum L. cv. Ailsa Craig) plants of different ET status, in wild type (WT) plants, in WT plants pre-treated with the ET generator 1-aminocyclopropane-1-carboxylic acid (ACC) and in ET insensitive, Never ripe (Nr/Nr) mutants for 1-, 6- and 24 h. In the leaves ACC treatment reduced the osmotic effect of salt stress, while Nr mutation enhanced not only osmotic but ionic component of salt stress at 100 mM NaCl. ET insensitivity caused greater decline in stomatal conductance and photosynthetic CO2 assimilation rate than in the controls under tolerable salt stress, but both ACC treatment and Nr mutation helped to maintain positive carbon assimilation under lethal salt stress after 24 h. Nr mutant leaves showed highly enhanced regulated non-photochemical quenching (NPQ) and therefore lower quantum yield of photosystem II (PSII), due to more intensive cyclic electron flow around photosystem I (CEF-PSI), which was further increased under high salinity. Exogenous ACC treatment lowered CEF-PSI and enhanced PSII photochemistry after 6 h of lethal salt stress. Controlling PSI photoinhibition, ET is suggested to be an important regulator of CEF-PSI and photoprotection under salt stress. Furthermore, the altered ET status could cause contrasting effects under different stress severity.

Effects of Jasmonic Acid in ER Stress and Unfolded Protein Response in Tomato Plants.

Endoplasmic reticulum (ER) stress elicits a protective mechanism called unfolded protein response (UPR) to maintain cellular homeostasis, which can be regulated by defence hormones. In this study, the physiological role of jasmonic acid (JA) in ER stress and UPR signalling has been investigated in intact leaves of tomato plants. Exogenous JA treatments not only induced the transcript accumulation of UPR marker gene SlBiP but also elevated transcript levels of SlIRE1 and SlbZIP60. By the application of JA signalling mutant jai1 plants, the role of JA in ER stress sensing and signalling was further investigated. Treatment with tunicamycin (Tm), the inhibitor of N-glycosylation of secreted glycoproteins, increased the transcript levels of SlBiP. Interestingly, SlIRE1a and SlIRE1b were significantly lower in jai1. In contrast, the transcript accumulation of Bax Inhibitor-1 (SlBI1) and SlbZIP60 was higher in jai1. To evaluate how a chemical chaperone modulates Tm-induced ER stress, plants were treated with sodium 4-phenylbutyrate, which also decreased the Tm-induced increase in SlBiP, SlIRE1a, and SlBI1 transcripts. In addition, it was found that changes in hydrogen peroxide content, proteasomal activity, and lipid peroxidation induced by Tm is regulated by JA, while nitric oxide was not involved in ER stress and UPR signalling in leaves of tomato.

Effects of Light and Daytime on the Regulation of Chitosan-Induced Stomatal Responses and Defence in Tomato Plants.

Closure of stomata upon pathogenesis is among the earliest plant immune responses. However, our knowledge is very limited about the dependency of plant defence responses to chitosan (CHT) on external factors (e.g., time of the day, presence, or absence of light) in intact plants. CHT induced stomatal closure before dark/light transition in leaves treated at 17:00 hrs and stomata were closed at 09:00 hrs in plants treated at dawn and in the morning. CHT was able to induce generation of reactive oxygen species (ROS) in guard cells in the first part of the light phase, but significant nitric oxide production was observable only at 15:00 hrs. The actual quantum yield of PSII electron transport (ΦPSII) decreased upon CHT treatments at 09:00 hrs in guard cells but it declined only at dawn in mesophyll cells after the treatment at 17:00 hrs. Expression of Pathogenesis-related 1 (PR1) and Ethylene Response Factor 1 were already increased at dawn in the CHT-treated leaves but PR1 expression was inhibited in the dark. CHT-induced systemic response was also observed in the distal leaves of CHT-treated ones. Our results suggest a delayed and daytime-dependent defence response of tomato plants after CHT treatment at night and under darkness.

The Multifaceted Roles of Plant Hormone Salicylic Acid in Endoplasmic Reticulum Stress and Unfolded Protein Response.

Different abiotic and biotic stresses lead to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER), resulting in ER stress. In response to ER stress, cells activate various cytoprotective responses, enhancing chaperon synthesis, protein folding capacity, and degradation of misfolded proteins. These responses of plants are called the unfolded protein response (UPR). ER stress signaling and UPR can be regulated by salicylic acid (SA), but the mode of its action is not known in full detail. In this review, the current knowledge on the multifaceted role of SA in ER stress and UPR is summarized in model plants and crops to gain a better understanding of SA-regulated processes at the physiological, biochemical, and molecular levels.

Salicylic acid-induced ROS production by mitochondrial electron transport chain depends on the activity of mitochondrial hexokinases in tomato (Solanum lycopersicum L.).

The growth regulator, salicylic acid (SA) plays an important role in the induction of cell death in plants. Production of reactive oxygen species (ROS) by mitochondrial electron transport chain (mtETC), cytochrome c (cyt c) release from mitochondria and loss of mitochondrial integrity can be observed during cell death execution in plant tissues. The aim of this work was to study the putative role of hexokinases (HXKs) in the initiation of cell death using tomato (Solanum lycopersicum L.) leaves and mitochondria isolated from plants exposed to a sublethal, 0.1 mM and a cell death-inducing, 1 mM concentrations of SA. Both treatments enhanced ROS and nitric oxide (NO) production in the leaves, which contributed to a concentration-dependent loss of membrane integrity. Images prepared by transmission electron microscopy showed swelling and disorganisation of mitochondrial cristae and vacuolization of mitochondria after SA exposure. Using post-embedding immunohistochemistry, cyt c release from mitochondria was also detected after 1 mM SA treatment. Both SA treatments decreased the activity and transcript levels of HXKs in the leaves and the total mtHXK activity in the mitochondrial fraction. The role of mitochondrial hexokinases (mtHXKs) in ROS and NO production of isolated mitochondria was investigated by the addition of HXK substrate, glucose (Glc) and a specific HXK inhibitor, N-acetylglucosamine (NAG) to the mitochondrial suspension. Both SA treatments enhanced ROS production by mtETC in the presence of succinate and ADP, which was slightly inhibited by Glc and increased significantly by NAG in control and in 0.1 mM SA-treated mitochondria. These changes were not significant at 1 mM SA, which caused disorganisation of mitochondrial membranes. Thus the inhibition of mtHXK activity can contribute to the mitochondrial ROS production, but it is not involved in NO generation in SA-treated leaf mitochondria suggesting that SA can promote cell death by suppressing mtHXK transcription and activity.

Comparison of changes in water status and photosynthetic parameters in wild type and abscisic acid-deficient sitiens mutant of tomato (Solanum lycopersicum cv. Rheinlands Ruhm) exposed to sublethal and lethal salt stress.

Abscisic acid (ABA) regulates many salt stress-related processes of plants such as water balance, osmotic stress tolerance and photosynthesis. In this study we investigated the responses of wild type (WT) and the ABA-deficient sitiens mutant of tomato (Solanum lycopersicum cv. Rheinlands Ruhm) to sublethal and lethal salt stress elicited by 100 mM and 250 mM NaCl, respectively. Sitiens mutants displayed much higher decrease in water potential, stomatal conductance and net CO2 assimilation rate under high salinity, especially at lethal salt stress, than the WT. However, ABA deficiency in sitiens caused more severe osmotic stress and more moderate ionic stress, higher K+/Na+ ratio, in leaf tissues of plants exposed to salt stress. The higher salt concentration caused irreversible damage to Photosystem II (PSII) reaction centres, severe reduction in the linear photosynthetic electron transport rate and in the effective quantum yields of PSII and PSI in sitiens plants. The cyclic electron transport (CET) around PSI, which is an effective defence mechanism against the damage caused by photoinhibition in PSI, decreased in sitiens mutants, while WT plants were able to increase CET under salt stress. This suggests that the activation of CET needs active ABA synthesis and/or signalling. In spite of ABA deficiency, proline accumulation could alleviate the stress injury at sublethal salt stress in the mutants but its accumulation was not sufficient at lethal salt stress.

H2O2 homeostasis in wild-type and ethylene-insensitive Never ripe tomato in response to salicylic acid treatment in normal photoperiod and in prolonged darkness.

Ethylene proved to be an important modulator of salicylic acid (SA) signalling pathway. Since SA may regulate both the production and scavenging of hydrogen peroxide (H2O2), which show light-dependency, the aim of this study was to compare H2O2 metabolism in the leaves of SA-treated wild-type (WT) tomato (Solanum lycopersicum L. cv. Ailsa Craig) and in ethylene receptor Never-ripe (Nr) mutants grown in normal photoperiod or in prolonged darkness. H2O2 accumulation was higher in the WT than in the mutants in normal photoperiod after 1 mM SA treatment, while Nr leaves contained more H2O2 after light deprivation. The expression of certain superoxide dismutase (SOD) genes and activity of the enzyme followed the same tendency as H2O2, which was scavenged by different enzymes in the two genotypes. Catalase (CAT, EC 1.11.1.6) activity was inhibited by SA in WT, while the mutants maintained enhanced enzyme activity in the dark. Thus, in WT, CAT inhibition was the major component of the H2O2 accumulation elicited by 1 mM SA in a normal photoperiod, since the expression and/or activity of ascorbate (APX, EC 1.11.1.11) and guaiacol peroxidases (POD, EC 1.11.1.7) were induced in the leaves. The absence of APX and POD activation in mutant plants suggests that the regulation of these enzymes by SA needs functional ethylene signalling. While the block of ethylene perception in Nr mutants was overwritten in the transcription and activity of certain SOD and CAT isoenzymes during prolonged darkness, the low APX and POD activities led to H2O2 accumulation in these tissues.

Prolonged dark period modulates the oxidative burst and enzymatic antioxidant systems in the leaves of salicylic acid-treated tomato.

Salicylic acid (SA) is an important plant growth regulator playing a role in the hypersensitive reaction (HR) and the induction of systemic acquired resistance. Since the SA-mediated signalling pathways and the formation of reactive oxygen species (ROS) are light-dependent, the time- and concentration-specific induction of oxidative stress was investigated in leaves of tomato plants kept under light and dark conditions after treatments with 0.1mM and 1mM SA. The application of exogenous SA induced early superoxide- and H2O2 production in the leaves, which was different in the absence or presence of light and showed time- and concentration-dependent changes. 1mM SA, which induced HR-like cell death resulted in two peaks in the H2O2 production in the light but the first, priming peak was not detected in the dark. Unlike 0.1mM SA, 1mM SA application induced NADPH oxidase activity leading to increased superoxide production in the first hours of SA treatments in the light. Moreover, SA treatments inhibited catalase (CAT) activity and caused a transient decline in ascorbate peroxidase (APX), the two main enzymes responsible for H2O2 degradation, which led to a fast H2O2 burst in the light. Their activity as well as the expression of some isoenzymes of SOD and APX increased only from the 12th h in the illuminated samples. The activity of NADPH oxidase and expression SlRBOH1 gene encoding a NADPH oxidase subunit was much lower in the dark. In spite of low CAT and APX activity after SA treatments in the dark, the activation of guaiacol-dependent peroxidase (POD) could partially substitute H2O2 scavenging activity of these enzymes in the dark, which reduced the ROS burst and development of lesion formation in the leaves.

Proteasome Activity Profiling Uncovers Alteration of Catalytic ß2 and ß5 Subunits of the Stress-Induced Proteasome during Salinity Stress in Tomato Roots.

The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic ß subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic ß subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each ß subunit except for two genes encoding ß2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled ß2 and ß5 subunits, and an additional acidic pI shift for labeled ß5, whilst labeled ß1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress.

In vivo inhibition of polyamine oxidase by a spermine analogue, MDL-72527, in tomato exposed to sublethal and lethal salt stress.

The spermine analogue N1,N4-bis-(2,3-butadienyl)-1,4-butanediamine (MDL-72527), an effective inhibitor of polyamine oxidases (PAOs), triggers a systemic response in tomato (Solanum lycopersicum L.) exposed to sublethal (100mM) and lethal (250mM) NaCl concentrations. The accumulation of free polyamines (PAs), the terminal oxidation of PAs by diamine oxidases (DAOs) and PAOs, and the production of H2O2 by PA oxidases depends on the intensity of salt stress. Spermidine and spermine content increased significantly under sublethal salt concentrations, but remained low under lethal salt stress. Along with increased expression of the selected SlDAO1 and SlPAO1 genes in the leaves and roots, respectively, DAO and PAO activities and their product, H2O2, increased and initiated cell death by irreversible loss of electrolytes at 250mM NaCl. MDL-72527 significantly increased spermine, spermidine and/or putrescine contents as a result of reduced activity of PA oxidases; furthermore, it inhibited H2O2 and NO production during salt treatment. These results indicate that PAO contributed to H2O2 and NO production under salt stress, and the terminal activities of DAO and PAO play a role in cell death induction at 250mM NaCl. However, the inhibition of PAO by MDL-72527 does not increase the salt tolerance of plants, since electrolyte leakage increased significantly in the presence of the inhibitor.

Comparison of polyamine metabolism in tomato plants exposed to different concentrations of salicylic acid under light or dark conditions.

In this study the effect of exogenous 0.1 mM and 1 mM salicylic acid (SA) treatments were investigated on polyamine (PA) metabolism in tomato (Solanum lycopersicum L. cv. Ailsa Craig) leaves in illuminated or dark environments. The former proved to be sublethal and the latter lethal concentration for tomato leaf tissues. While PA biosynthetic genes, arginine- and ornitine decarboxylases or spermidine- and spermine synthases were highly up-regulated by 1 mM SA, the enzymes participating in PA catabolism, diamine- (DAOs, EC 1.4.3.6) and polyamine oxidases (PAOs, EC 1.5.3.3) displayed higher transcript abundance and enzyme activity at 0.1 mM SA. As a result, putrescine and spermine content but not that of spermidine increased after 1 mM SA application, which proved to be higher in the dark than in the light. H2O2 content produced on the effect of 1 mM SA was significantly higher than at 0.1 mM SA in the light. Since there was no coincidence between H2O2 accumulation and terminal PA catabolism, reactive oxygen species produced by photosynthesis and by other sources had more pronounced effect on H2O2 generation at tissue level than DAOs and PAOs. Accordingly, H2O2 in the absence of NO accumulation contributed to the initiation of defence reactions after 0.1 mM SA treatment, while high SA concentration generated simultaneous increase in H2O2 and NO production in the light, which induced cell death within 24 h in illuminated leaves. However, the appearance of necrotic lesions was delayed in the absence of NO if these plants were kept in darkness.

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.

The role of Arabidopsis glutathione transferase F9 gene under oxidative stress in seedlings.

Arabidopsis thaliana contains 54 soluble glutathione transferases (GSTs, EC 2.5.1.18), which are thought to play major roles in oxidative stress responses, but little is known about the function of individual isoenzymes. The role of AtGST phi 9 (GSTF9) in the salt- and salicylic acid response was investigated using 2-week-old Atgstf9 and wild type (Wt) plants. Atgstf9 mutants accumulated more ascorbic acid (AsA) and glutathione (GSH) and had decreased glutathione peroxidase (GPOX) activity under control conditions. Treatment of 2-week-old seedlings with 10⁻7 M salicylic acid (SA) for 48 h resulted in elevated H2O2level and enhanced GST activity in Atgstf9 plants, 10⁻5 M SA treatment enhanced the malondialdehyde and dehydroascorbate contents compared to Wt. 50 and 150 mM NaCl increased the GST activity, AsA and GSH accumulation in Atgstf9 seedlings more pronounced than in Wt plants. We found that the Atgstf9 mutants had altered redox homeostasis under control and stress conditions, in which elevated AsA and GSH levels and modified GST and GPOX activities may play significant role. The half-cell potential values calculated from the concentration of GSH and GSSG indicate that this GST isoenzyme has an important role in the salt stress response.

Salt stress-induced production of reactive oxygen- and nitrogen species and cell death in the ethylene receptor mutant Never ripe and wild type tomato roots.

The salt stress triggered by sublethal, 100 mM and lethal, 250 mM NaCl induced ethylene production as well as rapid accumulation of superoxide radical and H2O2 in the root tips of tomato (Solanum lycopersicum cv. Ailsa Craig) wild type and ethylene receptor mutant, Never ripe (Nr/Nr) plants. In the wild type plants superoxide accumulation confined to lethal salt concentration while H2O2 accumulated more efficiently under sublethal salt stress. However, in Nr roots the superoxide production was higher and unexpectedly, H2O2 level was lower than in the wild type under sublethal salt stress. Nitric oxide production increased significantly under sublethal and lethal salt stress in both genotypes especially in mutant plants, while peroxynitrite accumulated significantly under lethal salt stress. Thus, the nitro-oxidative stress may be stronger in Nr roots, which leads to the programmed death of tissues, characterized by the DNA and protein degradation and loss of cell viability under moderate salt stress. In Nr mutants the cell death was induced in the absence of ethylene perception. Although wild type roots could maintain their potassium content under moderate salt stress, K(+) level significantly declined leading to small K(+)/Na(+) ratio in Nr roots. Thus Nr mutants were more sensitive to salt stress than the wild type and the viability of root cells decreased significantly under moderate salt stress. These changes can be attributed to a stronger ionic stress due to the K(+) loss from the root tissues.

Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.