Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

Abstract

The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H₂O₂)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10⁻³ M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.

Several signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic^1–3 and abiotic stressors.^4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).⁷

The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms⁴ and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.⁸ NO is another important player that is required for the induction of defense mechanisms⁹ or for ROS-induced cell death.¹⁰

Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.⁸

Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.¹¹ The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O₃, an abiotic stressor which induces ROS-dependent cell death.¹² Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H₂O₂ accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H₂O₂ production and a functional ethylene signaling pathway.¹³ Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.¹⁴ It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.

It has been also shown that SA enhances NO synthesis in a dose-dependent manner.¹⁵ ROS, such as ^·O₂⁻ and H₂O₂ as well as NO can act together in the cell death regulation and propagation.^8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death.

Long-Term Effect of Salicylic Acid on the Production of ROS and NO in the Root Tip of Tomato

In our earlier work it was shown that SA, applied continuously in hydroponic culture through the root system of tomato plants, generated pre-adaptation responses which led to salinity tolerance.¹⁷ During the pre-adaptation period SA induced an extended H₂O₂ accumulation in tomato leaves at a high concentration (10⁻³ M), which later caused the death of the plants. However, sublethal concentrations of SA (10⁻⁷–10⁻⁴ M) induced only a transient raise in ROS accumulation, and no significant differences in the H₂O₂ content could be detected in the leaves and root tissues of control and SA-treated plants after three weeks. In the root tips, 10⁻³ M SA triggered the production of ROS and NO with a concomitant decline in cell viability but no significant enhancement of NO content could be detected at sublethal SA concentrations.¹⁸ It was concluded that those concentrations of SA which resulted in a significant increase in ROS and NO production during the pre-adaptation period led to PCD, while sublethal concentrations induced only a short and transient accumulation of ROS in root apices of tomato. Here we demonstrate that Nr mutation has a clear effect on the rate of SA-induced ROS and NO production in the root tips of tomato.

Salicylic Acid-Induced ROS and NO Production in the Root Apex of Never ripe Tomato Mutants

In tomato, six putative ethylene receptors have been found which bind ethylene with high affinity and transmit the signal to downstream Ser/Thre kinases (LeCTRs). These receptors have been classified in two sub-families on predicted protein structure and Never ripe (Nr, also called ETR3) mutant with two other members (LeETR1 and LeETR2), have been classified into sub-family 1.¹⁹ Dominant gain-of-function mutations in ethylene receptors such as Nr resulted in reduced sensitivity to ethylene and in contrast to other ripening mutants, the Nr mutants have lost the capacity to respond to ethylene in all tissues examined.

The Nr locus was mapped to the center of chromosome 9 in tomato²⁰ and the Nr gene proved to be partially dominant and to have pleiotropic effects on plant development. Nr mutants exhibit insensivity to ethylene via triple response, leaf petiole epinasty, senescence of petals and flower abscission and the severity of these responses were dependent on Nr gene dosage, suggesting its incomplete dominance in Solanum lycopersicum Ailsa Craig cultivar.²¹ The Nr/Nr plants were able to produce ethylene after pathogen attack indicating that the mutants are not impaired in ethylene biosynthesis.²¹

It has recently been found that ethylene receptors in Arabidopsis are capable of forming heteromer complexes, which has implications for signal amplification and fine-tuning.²² In spite of the complexity of the ethylene signal transduction, Nr mutants exhibited altered oxidative stress response compared with wild-type plants under the effect of abiotic stressors.²³

In order to clarify the role of functional ethylene signaling in the SA-induced ROS and NO production, shoot cuttings were prepared from wild-type tomato plants (S. lycopersicum L. cv Ailsa Craig) and from nearly isogenic lines homozygous for the Nr. Although ethylene may stimulate or reduce adventitious rooting, Nr mutants can produce adventitious roots.²⁴ The effect of SA was investigated on cuttings because three weeks after the preparation of cuttings the plants have well developed root system consisting of roots of equal priority, without the dominance of the taproot.

In wild-type plants, six hours of incubation in SA caused a very significant increase in ROS level in the apical 0.5 mm-long region of root tips (Fig. 1C) while Nr mutants displayed an attenuated level of ROS (Fig. 1D). In contrast, NO production remained at the level of untreated control at low SA concentrations and increased in the whole 1.5 mm long region of root apex at 10⁻³ M SA in wild-type plants (Fig. 1E). However, NO production of apical root tissues was about 80% higher in the Nr mutant than in the wild-type control and only modest increases were observed in the apical 1 mm. Interestingly, at 1.5 mm from the root apex the increase has already ranged from 60% to 140% in the function of SA concentration (Fig. 1F). Fluorescein diacetate staining indicated a considerable loss of cell viability in the root apex of wild-type plants exposed to 10⁻³ M SA. In these tissues SA-induced ROS and NO led to the activation of PCD and the disorganization of meristematic tissues in the root apex. In the absence of increased ROS production the loss of cell viability due to elevated NO level was significantly lower in Nr plants at 10⁻³ M SA (Fig. 1A and B). These results are in agreement with the data of Lehotai et al. who found that the cells of pea root meristem died when H₂O₂ and NO levels increased simultaneously after exposure to heavy metals, copper and cadmium. 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a scavenger of NO reversed the effect of heavy metal ions on the cell death detected by Evans-blue staining in the root apex.

Figure 1.

Shoot cuttings from wild-type and ethylene receptor (Nr) mutant of tomato (Solanum lycopersicum L.cv Ailsa Craig) were used in the experiments. After germination the seedlings were grown in soil until preparation of cuttings. The cuttings were then rooted in a hydroponic culture at pH 5.8 in a controlled environment under 300 µmol m⁻² s⁻¹ photon flux density (PPFD) (F36W/GRO lamps, Sylvania, Germany), with 12/12 h light/dark period, a day/night temperatures of 24/22°C and a relative humidity of 55–60% as described earlier in reference ²⁷. After three weeks the cuttings were treated with 10⁻⁷, 10⁻⁴ and 10⁻³ M SA (Sigma-Aldrich) for 6 h applied through the root system in the culture medium. ROS were detected by staining with 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA), NO with 4,5-diaminofluorescein diacetate (DAF2-DA) and the viability of cells with fluorescein diacetate (FDA) using a Zeiss Axiowert 200M fluorescence microscope (Carl Zeiss, Germany) equipped with a high-resolution digital camera (Axiocam HR). Wild-type controls are taken 100% and the data are means ± SD of four independent experiments. The least significant differences (LSD) at p ≤ 0.05 level for ROS, NO and viability were determined according to Fischer (SigmaPlot11.0 statistical software), and the bars represent the LSD values which refer to the data taken at 0.5, 1 and 1.5 mm distances from the root apex.

Our data clearly show that SA-induced ROS production requires a functional ethylene signaling pathway. In contrast, NO synthesis may be downregulated by ethylene in wild-type plants at sublethal SA concentrations because the mutant plants produce by ∼80% more NO in the absence of SA or at low SA concentrations (Fig. 2). In accordance with the results of Monteiro et al. who demonstrated reduced levels of H₂O₂ in the leaf tissues of Nr plants exposed to high salinity and heavy metal stress, these results indicate that ethylene signaling associated with the Nr receptor can modulate the oxidative stress response of apical root tissues in tomato. To the best of our knowledge, this is the first demonstration that at sublethal SA concentrations the ethylene signaling has been involved in the control of SA-induced ROS and NO production in the opposite direction. However, at PCD-inducing concentrations of SA NO production may be controlled predominantly by H₂O₂.²⁶ It can also be concluded that ethylene signal transduction determines the balance between ROS and NO production induced by SA.

Figure 2.

Model for the role of ethylene signaling in salicylic acid-induced control of ROS and NO production in the root apex of wild-type plants and Never ripe (Nr/Nr) mutants of tomato. PCD, programmed cell death.

Abbreviations

NO

nitric oxide

Nr

tomato mutant, Never ripe

ROS

reactive oxygen species

SA

salicylic acid