بررسي نقش حفاظتي نيتريك اكسيد در كاهش صدمات ناشي از تنش شوري در گياه هميشه بهار (L. cv. Gitan Orange (Calendula officinalis

Investigation on the Protective Role of Nitric Oxide in Reducing Damages Induced by Salinity Stress in Calendula officinalis L.

شوری یكی از فاكتورهای مهم محیطی است كه رشد و نمو گیاهان را تحت تاثیر قرار داده و تولید گیاهان را محدود می‌كند. سدیم نیترو پروسید (SNP) به طور معمول به عنوان تركیب رها كننده نیتریك اكسید (NO) در گیاهان استفاده می‌شود. نیتریك اكسید رادیكال گازی نسبتاً پایداری است كه در غلظت‌های پایین با ممانعت از تولید رادیكال‌های فعال اكسیژن مانع خسارت آن‌ها می‌گردد. این پژوهش به صورت فاكتوریل با طرح كاملاً تصادفی در سه تكرار، تیمار كلرید سدیم در پنج سطح صفر (شاهد)، 25، 50، 75 و 100 میلی‌مولار و سدیم نیتروپروسید به صورت محلول پاشی برگی درچهار غلظت صفر (شاهد)، 25/0، 5/0 و 75/0 میلی‌مولار انجام شد. بنابر نتایج بدست آمده در این پژوهش، تنش شوری باعث كاهش رشد رویشی گردید و با افزایش غلظت نمك، بیشتر ویژگی‌های مورفولوژیك گیاه تحت تاثیر منفی تنش قرار گرفتند. تنش شوری، نیز دارای اثرات منفی قابل توجهی بر ویژگی‌های فیزیولوژیكی بود به گونه‌ای كه باعث كاهش كلروفیل شد و از طرفی باعث افزایش ظرفیت آنتی‌اكسیدانی، پرولین، قند محلول و نشت الكترولیت در گیاه گردید. تیمارهای مختلف سدیم نیتروپروسید توانست به طور معنی‌داری اثر منفی شوری به ویژه در سطوح پایین شوری بر گیاه را كاهش دهد. در بین غلظت‌های مختلف سدیم نیتروپروسید، غلظت 25/0 و 5/0 میلی‌مولار بر روی بهبود صفات مورفولوژیكی، غلظت های 25/0، 5/0 و 75/0 میلی‌مولار بر روی بهبود صفات فیزیولوژیكی و غلظت‌های 5/0 و 75/0 میلی مولار بر روی بهبود صفات بیوشیمیایی موثر واقع شدند.

Introduction: Salinity is one of the most important environmental factors that regulates plant growth and development, and limits plant production. Researchers have shown that some plant growth regulators such as nitric oxide improve the plants resistance to environmental stresses such as heat, cold, drought and salinity. Sodium nitroprusside (SNP) commonly has been used as nitric oxide (NO) donor in plants. NO is a diffusible gaseous free radical. Low concentrations of NO inhibit the production of reactive oxygen species and protect plants against ROS damages. The aim of this study was to evaluate the role of SNP as NO donor on salt tolerance of Calendula officinalis and its effects on some morphological, physiological and biochemical characteristics of this plant. Materials and Methods: In this study, the effects of salinity (0, 25, 50, 75 and 100 mM) and sodium nitroprusside (0.0, 0.25, 0.50 and 0.75 mM) on morphological and physiological characteristics of Calendula officinalis L. were investigated. Total leaf area and number of leaves were determined in the end of the experiment. Electrolyte leakage was used to asses’ membrane permeability. This procedure was based on Lutts et al.,1995. Soluble sugars were extracted and estimated by the method of Irigoyen et al., 1992. Chlorophyll a, b and carotenoid content were calculated from the absorbance of extract at 653, 666 and 470 nm using the formula of Dere et al., 1998. Proline was extracted by the method of Bates et al., 1973. DPPH radical- scavenging activity of sample was performed as described previously of Cleep et al., 2012. The SAS software was used for the analysis of variance (ANOVA), comparisons with P <0.05 were considered significantly different. Results and Discussion: In this research NO can reduce the adverse effects of salt stress. When Calendula plants were grown under 100 mM NaCl without SNP application, total leaf area and number of leaves were at least compared to control and other treatments. The concentration of 0.25 and 0.5 mM SNP with 50 mM NaCl caused to increase the total leaf area and the number of leaves compared to control. This is possibly a cumulative impact generated by SNP through the regulation of exo- and endo-β-D-glucanase in the cell wall. These promoting effects of NO were evidenced by using NO-deficient mutants, where enzyme activity decreased and plant growth remained restricted. The exo- and endo-β-Dglucanase enzymes hydrolyze glycosidic linkages between glucose units within the cell wall to favor loosening and increase cell extensibility. Electrolyte leakage was increased by salt treatment, however 0.25 and 0.5 mM nitric oxide significantly decreased electrolyte leakage in 50 mM NaCl concentration under salt condition. We found that applied antioxidants, SNP (NO donor) had a protective effect on salt induced membrane damage. Soluble sugar content was increased by salt treatment, thus application of 0.5 and 0.75 mM SNP increased soluble sugar content compared to control. The mechanism of NO induced sugar synthesis still needs to be further explored. Chlorophyll a and b content were decreased with increasing NaCl concentration but cartotenoid content was increased with increasing salt stress. Foliar application of SNP could increase chlorophyll a and b content in 50 mM NaCl. Salt stress significantly induced accumulation of proline in Calendula leaves and application of 0.25 and 0.5 mM of SNP significantly increased proline concentration under salt stress. High levels of accumulated proline enabled the plant to maintain turgor and thus water potential. The role of proline has been established as a compatible solute under stressful conditions. Our experimental data showed that the DPPH radical- scavenging activity was significantly increased after NaCl treatment. Treatment with 0.5 and 0.75 mM SNP resulted in a remarkable increasing in the activity of DPPH radical- scavenging until 50 mM NaCl. Increasing of DPPH radical scavenging activity in plants usually depends on the antioxidant metabolites. The stimulation of antioxidant capacity of NO might be attributed to induction of antioxidant metabolites synthesis. However, the effectiveness of NO is dependent on its time of application, stage of development at which NO application or stress is applied to plants, type of variety and environmental conditions. Conclusions: Our study demonstrated that NO can effectively protect Calendula seedlings from salt stress damage by enhancing DPPH radical scavenging activity, proline, chlorophyll content, soluble sugar and decreasing the electrolyte leakage. In morphological traits, the concentrations of 0.25 and 0.5mM SNP, in physiological traits 0.25, 0.5 and 0.75 mM and in biochemical traits 0.5 and 0.75 mM were effective under salt stress conditions.