Salt Stress-Induced Structural Changes Are Mitigated in Transgenic Tomato Plants Over-Expressing Superoxide Dismutase

Abstract

Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na₂SO₄ solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na₂SO₄ treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.

Keywords: ROS; Solanum lycopersicum; cell structure; columella; cytoskeleton; root cap; salt stress; α-tubulin microtubule.

Figure 1

Transverse sections of root tips from wild-type (WT) (a–c) and transgenic tomato (d–f) plants grown without and supplemented with NaCl and Na₂SO₄. Modification of meristem and cap cells while the imitation of salinity effects in vitro culture. Responsiveness of the following parameters to salt stress is shown: the size and shape of the columella zone cells from the root cap (indicated by a vertical column with an average number of columella cell layers) and the initials extending from the meristematic zone (mz) from the root tip. Symbols: circle–stele cells, square–cortex cells, pentagon–columella cells. Scale bar: 50 μm. Transverse sections of root tips show the average number of columella cell layers. Values followed by the same letter significantly do not differ by Duncan’s test (α = 0.05) (50 transverse sections of root tips from five independent plants).

Figure 2

Fragments of transverse sections of root tips showing the zone of cap initials forming apex columella from WT (a–c) and transgenic tomato (d–f) plants grown without (a,d), and supplemented with the NaCl (b,e) and Na₂SO₄ (c,f). Modification of the cell walls of root apical meristem and cap cells of the tomato root tip is shown. The thickness and location of adjacent layers related to the cap initials, the transformation of the size and shape are visible as the most sensitive targets for salts treatments. Symbols: mz–meristem zone; cz–columella zone.

Figure 3

Distribution of reactive oxygen species (ROS) in the different zones of tomato roots. WT (a–c) and transgenic line 19 (d–f) plants were grown without, and with the addition of NaCl and Na₂SO₄. A change in the localization of ROS in the meristem and cap cells of the root tip is shown as a modification of fluorescence.

Figure 4

Tubuline cytoskeleton in cell cycle interphase of root cells from WT (a–c) and transgenic tomato (d–f) plants grown without, and with the addition of NaCl and Na₂SO₄. Nuclei were stained with DAPI (blue); microtubules were detected using antibodies to α-tubuline (green). While the bundles of microtubules are located in the cortical cytoplasm, microtubules are not visible in the perinuclear region in the interphase cells. Tomato root cells have obvious multiple lesions in the location of microtubules under the NaCl, Na₂SO₄. Cells form multiple chaotically arranged bundles and do not maintain peripheral position, which indicates violations in the process of cytoskeleton transformation, as a highly dynamic structure.

Figure 5

Ultrastructure of root cell from WT (a–c) and Fe-SOD-transgenic tomato (d–f) plants grown without and supplemented with NaCl and Na₂SO₄. Symbols: mt—microtubules, cw—cell wall, m–mitochondrion, v—vacuole, p—plastid, c—atypical cytoskeleton structure. Scale bars—250 nM.