دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 2 2026 08 23 Effect of Iron Nanoparticles and Putrescine on Morphophysiological and Biochemical Traits of Medicinal Plant Salvia officinalis L. under Salinity Stress تأثیر نانوذره آهن و پوترسین بر صفات مورفوفیزیولوژیک و بیوشیمیایی گیاه دارویی مریم گلی (Salvia officinalis L.) تحت تنش شوری 1 22 30646 10.22108/ijpb.2026.149163.1455 FA اسحاق ساداتی گروه علوم باغبانی، مؤسسه آموزش عالی سنا، ساری، ایران مهیار گرامی گروه زیست شناسی، مؤسسه آموزش عالی سنا، ساری، ایران محدثه امیری گروه مرتع و آبخیزداری، دانشکده کشاورزی و منابع طبیعی، دانشگاه محقق اردبیلی، اردبیل، ایران Journal Article 2026 06 03 To investigate the effect of foliar application of iron nanoparticles and putrescine (Put) on morphological, physiological, and biochemical traits of the medicinal plant Salvia officinalis L. under salinity stress, a factorial experiment was conducted based on a completely randomized design. The treatments included salinity at three levels (0, 75, and 150 mM sodium chloride), iron nanoparticles at four levels (0, 50, 100, and 200 ppm), and putrescine at three levels (0, 0.5, and 1 mM). The results showed that salinity stress significantly reduced morphological traits (plant height, leaf number, shoot fresh and dry weight) and chlorophyll content, while increasing the content of carotenoids, soluble sugars, proline, flavonoids, and the activity of catalase (CAT) and peroxidase (POD) enzymes. Application of iron nanoparticles (particularly at 200 ppm) and putrescine (1 mM) significantly mitigated the negative effects of salinity and enhanced all studied traits compared to the control treatment. The highest increase in shoot fresh and dry weight, total chlorophyll content, and antioxidant enzyme activity was observed in the combined treatment of iron nanoparticles (200 ppm) plus putrescine (1 mM). Based on the findings, the application of iron nanoparticles and putrescine can ameliorate the adverse effects of salinity stress in sage plants by improving growth, physiological, and biochemical traits. Introduction Salvia officinalis L. is a valuable medicinal plant from the Lamiaceae family, widely used in pharmaceutical, food, and cosmetic industries due to its phenolic compounds, flavonoids, and essential oils. However, its cultivation faces salinity stress, which limits growth by inducing osmotic stress, causing ion imbalance, reducing photosynthesis, and increasing ROS production. The use of nanoparticles as nutritional elicitors and polyamines such as putrescine as growth regulators constitutes a novel strategy to enhance abiotic stress tolerance. Iron nanoparticles (Fe-NPs) reduce oxidative damage by enhancing the activity of antioxidant enzymes and chlorophyll synthesis. Putrescine contributes to osmotic adjustment, stabilizes membranes, and neutralizes free radicals. Despite previous studies, there is a lack of comprehensive data on their synergistic effects on Salvia officinalis under salinity. Thus, this study evaluated individual and combined effects of Fe-NPs and putrescine on improving salinity tolerance in Salvia officinalis. Materials and Methods A completely randomized factorial design with three replications was conducted in a greenhouse in 2025. Disinfected Salvia officinalis seeds were germinated, and uniform seedlings were transplanted into pots with perlite to peat moss (1:1 v/v) and irrigated with Hoagland solution (pH 5.5–5.7). Treatments included salinity (NaCl: 0, 75, 150 mM) applied via irrigation; Fe-NPs (0, 50, 100, 200 ppm); and putrescine (0, 0.5, 1 mM), with foliar sprays applied at five‑day intervals (three total applications). Sampling was performed four weeks after the last treatment. Morphological traits—height, leaf number, shoot fresh weight, and dry weight (48 h at 75°C)—were recorded. Photosynthetic pigments were extracted from 0.5 g fresh leaf in 80% acetone, measured at 663, 645, and 470 nm, and calculated via Arnon's formulas. Soluble sugars were determined using the anthrone method at 620 nm with a glucose standard curve. Proline content was determined using the Bates method with ninhydrin reagent, and absorbance was read at 520 nm after toluene extraction. Total flavonoids were measured by aluminum chloride colorimetry at 506 nm using a catechin standard curve. Antioxidant enzymes were extracted from 0.5 g tissue in phosphate buffer containing EDTA. Catalase activity was assayed by monitoring the decrease in H₂O₂ absorbance at 240 nm according to Luck's method. Peroxidase activity was measured by monitoring tetraguaiacol formation at 470 nm according to Tang & Newton's method. DPPH scavenging activity was measured at 517 nm. All assays were performed with three replicates. Data were subjected to analysis of variance (ANOVA) using SAS 9.2, and means were compared by Duncan's multiple range test at the 5% significance level. Normality and homogeneity of variances were confirmed by Kolmogorov–Smirnov and Levene's tests, respectively. Results and Discussion Salinity significantly reduced all morphological traits and photosynthetic pigment contents (p < 0.01). At 150 mM salinity, height decreased from 22.71 to 16.78 cm (26%), leaf number decreased from 9.67 to 6.25 (35%), fresh weight decreased from 1.01 to 0.70 g (31%), dry weight decreased from 0.21 to 0.13 g (38%), and total chlorophyll decreased from 2.72 to 2.20 mg/g FW (19%). These reductions resulted from impaired water and nutrient uptake, reduced turgor, chloroplast degradation, and decreased anabolic enzyme activity. Conversely, Fe-NPs at 200 ppm increased height by 36.26%, leaf number by 27.42%, fresh weight by 20.73%, and dry weight by 18.75%. Putrescine at 1 mM similarly increased height (20.92 cm), leaf number (8.30), fresh weight (0.94 g), and dry weight (0.19 g). The interaction between Fe-NPs and putrescine was significant for fresh and dry weight (p < 0.01), with the highest values in the NFe3Pu2 treatment (200 ppm Fe-NPs and 1 mM putrescine). For photosynthetic pigments, Fe-NPs at 100 and 200 ppm increased total chlorophyll by 20.45% and 20% over the control, respectively, while putrescine (1 mM) increased it by 12.28%. Salinity increased carotenoids by 20% (to 0.12 mg/g FW at 150 mM), indicating their protective role against oxidative stress. The Fe-NPs × salinity interaction showed that at 150 mM salinity, 200 ppm Fe-NPs raised total chlorophyll by 25% over the control, confirming the role of iron (supplied via Fe-NPs) in preserving chloroplast integrity. Biochemically, salinity elevated soluble sugars (11.67%), proline (12.68%), flavonoids (20.78%), catalase (28.33%), peroxidase (20.53%), and DPPH (10.04%), indicating activation of the plant's defense system. Fe-NPs and putrescine, individually or in combination, further enhanced these parameters. The NFe3Pu2 treatment exhibited the highest soluble sugars (172.91 mg/g FW), proline (38 μg/g FW), flavonoids (30.29 mg quercetin/g FW), catalase (18.09 ΔA/mg protein/min), and peroxidase (4.28 U/mg protein/min), corresponding to increases of 13.4%, 19.7%, 31.4%, 41%, and 24% over the control, respectively. The putrescine × salinity interaction was also significant for sugars, proline, and flavonoids; at 150 mM salinity, 1 mM putrescine increased soluble sugars by 13%. These increases can be attributed to iron‑mediated activation of catalase and peroxidase, which neutralizes ROS, and to putrescine‑induced membrane stabilization, which prevents lipid peroxidation and enhances osmotic regulation through proline and sugar accumulation. The synergistic effect is attributed to complementary mechanisms—enhanced antioxidant defense by iron and membrane protection by putrescine—leading to improved water uptake, enhanced photosynthesis, reduced oxidative damage, and increased secondary metabolite production. Conclusion Salvia officinalis showed relative tolerance to salinity up to 75 mM, whereas growth was severely restricted at 150 mM NaCl. The combined treatment of Fe-NPs (200 ppm) and putrescine (1 mM) was the most effective strategy to mitigate salinity stress, improving morphological traits, photosynthetic pigments, enzymatic and non‑enzymatic antioxidants, and compatible metabolites (proline and soluble sugars), which ultimately increased biomass. Considering the medicinal value of Salvia officinalis and the extensive saline lands in Iran, this combined treatment is recommended for integration into nutritional and stress management programs for cultivation in saline areas. Further field‑scale studies, along with investigations into essential oil quantity and quality, are recommended. جهت بررسی تأثیر محلول‌پاشی نانو ذره آهن و پوترسین بر صفات ریخت‌شناسی، فیزیولوژیک و بیوشیمیایی گیاه دارویی مریم گلی (Salvia officinalis L.) تحت تنش شوری، آزمایشی به صورت فاکتوریل در قالب طرح کامل تصادفی اجرا شد. تیمارهای مورد بررسی شامل شوری در سه سطح (صفر، 75 و 150 میلی‌مولار کلرید سدیم)، نانوذره آهن در چهار سطح (صفر، 50، 100 و 200 پی‌پی‌ام) و پوترسین در سه سطح (صفر، 5/0 و 1 میلی‌مولار) بودند. نتایج نشان دادند تنش شوری سبب کاهش معنی‌دار صفات ریخت‌شناسی (ارتفاع، تعداد برگ، وزن تر و خشک اندام هوایی) و محتوای کلروفیل شد، در حالی‌که میزان کاروتنوئید، قند محلول، پرولین، فلاونوئید و فعالیت آنزیم‌های کاتالاز و پراکسیداز افزایش یافت. کاربرد نانوذره آهن (به‌ویژه در غلظت 200 پی‌پی‌ام) و پوترسین (یک میلی‌مولار) به‌طور معنی‌داری اثرات منفی شوری را کاهش داده و مقادیر همه صفات مورد پژوهش را نسبت به تیمار شاهد افزایش دادند. بیشترین افزایش در وزن تر و خشک اندام هوایی، کلروفیل کل و فعالیت آنزیم‌های آنتی‌اکسیدانی در تیمار ترکیبی نانوذره آهن (200 پی‌پی‌ام) همراه با پوترسین (یک میلی‌مولار) مشاهده شد. بر اساس یافته‌ها، کاربرد نانوذرات آهن و پوترسین می‌تواند با بهبود فاکتورهای رشد، صفات فیزیولوژیک و بیوشیمیایی، اثرات سوء تنش شوری را در گیاه مریم گلی تعدیل نماید. آنزیم‌های آنتی‌اکسیدانی، پوترسین، رنگیزه‌های فتوسنتزی، شوری، مریم گلی، نانوذره آهن

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