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Thus, SA application triggers biotic or abiotic stress tolerance in plants through the regulation of these physiological or biochemical mechanisms [ 24 ]. SA induced systemic acquired resistance in response to phytopathogens attack. Upon infection, when the SA pathway is activated at the site of infection, the defense response is often activated in distal plant parts to protect undamaged tissues [ 25 ].

In addition, SA plays a role in the mitigation of abiotic stresses, such as toxic metals, heat, drought, and ultraviolet radiation stresses in plants, via the regulation of ions, antioxidant enzymes, endogenous hormones, and SA synthesis-related gene expression [ 25 — 29 ]. Particularly, accumulated SA in plants can induce saline tolerance by initiating a cascade of endogenous hormone signaling pathways. Conversely, findings suggest that high SA accumulation causes an oxidative burst and induces programmed cell death PCD in Arabidopsis under ozone stress [ 25 ].

Plants face multiple abiotic and biotic stress conditions. For example, elevated temperature or rainfall significantly influence the spread of pests and pathogens, thus inducing enormous yields [ 31 ]. To prevent crop yield loss, there is a need for studies investigating multiple stresses. Moreover, the role of exogenous SA application and its physiological response in plants under biotic stress is well reported. However, under abiotic stress—particularly salinity stress—it has not been fully explored.

Therefore, the current study was undertaken to examine the response of rice in exogenous SA application under salinity stress, particularly to investigate the scavengers or ROS and RNS regulation. Rice seeds Oryza sativa L. For consistency, we selected seedlings of similar lengths for transplant. Transplanted rice seedlings were cultivated in a hydroponic medium, Yoshida solution [ 32 ], for 1 week in the growth chamber. Two days following transplantation, Yoshida solution in designated pots was supplemented with 0.

Nineteen-day-old rice seedlings were divided into the following treatment groups: Treatments were conducted for 1, 2, 3, and 4 days. According to Munns and Tester [ 33 ], saline soil was defined as soil containing a high concentration of soluble salts, i. Thus, we used mM of NaCl to induce artificial abiotic stress salinity stress.

We previously showed that the application of 0. Similarly, the application of 1 mM SA has been reported to induce plant cell death in tomato [ 34 ]. Based on previous findings, we assumed that mM NaCl was sufficient to induce abiotic stress, and both concentrations of SA were able to induce biotic stress. All experiments were replicated three times. Sodium content in the shoots was analyzed. Sodium content in rice leaves was measured by inductively-coupled plasma mass spectrometry ICP-MS , as described by Jin and Zhu [ 35 ].

To assess SA accumulation in rice plants, we analyzed free SA content using a modified protocol [ 36 ]. Leaf samples were collected and placed directly into liquid nitrogen. Frozen samples were freeze-dried and ground into a very fine powder. A sample of freeze-dried leaf 0. Both methanol extracts were dried with a vacuum centrifuge, and dry pellets were re-dissolved in 2. More detailed information is listed in S1 Table. To investigate electrolytic leakage from cellular tissues, electrical conductivity EC was measured in rice leaves. After 24 h, 0.

EC data were used to calculate a ratio of the values obtained from the incubated and boiled samples.

Experimental Protocols for Reactive Oxygen and Nitrogen Species

To ensure the uniform collection of data, we used one-third of a leaf for the measurement. The method used to extract and quantify H 2 O 2 has been described by Cheeseman [ 38 ]. Rice plants shoots were homogenized in an extraction medium containing 0. The H 2 O 2 content was measured as described by Cheeseman [ 38 ]. The H 2 O 2 contents were measured to determine the difference in absorbance between — nm.

Lipid peroxidation activity was determined using the method described by Ohkawa et al. A mg fresh leaf sample was subjected to extraction using 10 mM phosphate pH 7. The mixture was heated in boiling water for 1 h and then cooled to room temperature. The lower layer was removed, and the upper organic layer was measured at nm with a spectrophotometer. Tetramethoxypropane was used as an external standard, and the level of lipid peroxidase was expressed as micromoles of malondialdehyde MDA formed per milligram of protein.

The total protein content was quantified using the Bradford assay [ 40 ]. Antioxidant activity was expressed as unit per milligram of protein. CAT was analyzed following the method described by Aebi, [ 41 ]. Crude enzyme extract was added to 0.


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CAT activity was calculated by the decrease in H 2 O 2 absorbance at nm, and was thus defined as micrograms of H 2 O 2 released per milligram of protein per minute. SOD activity was assayed using the protocol described by Marklund and Marklund [ 42 ]. A fresh leaf sample mg was homogenized with 0. After centrifuging, the supernatant was removed and used as a crude enzyme extract.

We prepared the reaction mixture, which included 2 ml of Tris—HCl buffer pH 8. This was mixed with 2 ml of Tris—HCl buffer pH 8. The prepared assay mixture was immediately analyzed at nm against a blank which did not contain tissue homogenate or pyrogallol at 3 min intervals in a spectrophotometer. APX activity was measured using the method described by Nakano and Asada [ 43 ]. Enzyme or hydrogen peroxide was added to the reaction mixture, which consisted of 50 mM potassium phosphate pH 7.

The collected data were used to define the reaction rate for H 2 O 2 independent of ascorbate oxidation. We followed the protocol described by Yun et al. Plant tissue mg was ground using a mortar and pestle with liquid nitrogen to produce a very fine powder. The supernatant was placed in a fresh tube and further centrifuged at 13, rpm for 10 min. To quantify the proteins, 1. All samples were measured with a spectrophotometer at nm following incubation for 10 min at room temperature.

Briefly, fresh rice leaf tissue was finely ground using liquid nitrogen, and 1 ml of TRIzol was added immediately after. The supernatant was transferred to a new 1. Centrifuge steps were conducted in between the aforementioned additions. OsUBI was used as the reference gene for data normalization, and all data were replicated three times. The experiment was repeated three times using the same external environment and treatments. Each replicate consisted of 10 plants.

The data collected from each repetition were pooled and analyzed statistically. Plant growth attributes were recorded daily 1—4 days after treatment, DAT. These growth attributes were significantly lower than those observed in the DW-applied control and NaCl-treated control plants.

Increasing concentrations of SA created a stressful situation for plants, which progressively reduced their shoot length at 1—4 DAT Table 1. The results showed that the increase in SA concentration had a further negative effect on plant growth.

Results of the ANOVA suggested that there were only significant differences in shoot length between treatments; however, there were significant differences in biomass among treatments and between treatment periods. Consequently, our data indicated that shoot length and fresh weight of rice plants were significantly affected by single and multi-stress conditions.

Therefore, our results show that application of different concentrations of SA, SA, and NaCl in combination, or NaCl only could induce physiological changes in rice plants. All data were collected from three consecutive repetitions of the same experiment in a time dependent manner.

Experimental protocols for reactive oxygen and nitrogen species - JH Libraries

The response to SA treatment suggests the presence of a negative feedback loop on growth with or without NaCl as compared to untreated rice plants. On other hand the phenotypic changes in rice plants following treatment with SA and NaCl alone or in combination are shown in Fig 1. Plant plasticity did not differ at 1 DAT among treatments; however, phenotype results differed significantly from 2—4 DAT.

Leaf tips started to undergo necrosis from 2 DAT; the greatest extent of leaf necrosis was observed in rice plants treated with combined SA and NaCl and increased with advancing exposure time Fig 1B, 1C and 1D. Arabic numbers indicate each treatment 1: The white vertical bar in the figure represents 10 cm. His book deserves to be widely read Would you like to tell us about a lower price? If you are a seller for this product, would you like to suggest updates through seller support?

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