دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 1 2026 05 22 Combined effect of methyl jasmonate, yeast extract and salicylic acid on flavonoids, rosmarinic acid and other phenolic acids accumulation in Moldavian balm (Dracocephalum moldavica L.) اثر ترکیبی متیل جاسمونات، عصاره مخمر و سالیسیلیک اسید بر تولید فلاونوئیدها، رزمارینیک اسید و سایر اسیدهای فنلی بادرشبو (Dracocephalum moldavica L.) 1 22 30507 10.22108/ijpb.2026.147859.1429 FA نساء انصاری گروه تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ملایر، ملایر، ایران محمد عبدلی گروه تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ملایر، ملایر، ایران محمد رضا طالبیان گروه تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ملایر، ملایر، ایران Journal Article 2025 12 21 Elicitation is an effective approach to increase the accumulation of plant secondary metabolites. This study aimed to increase the production of rosmarinic acid and other phenolic compounds in Dracocephalum moldavica L. by combining different concentrations of methyl jasmonate, yeast extract, and salicylic acid. The experiment was conducted in a completely randomized design with 13 treatments and three replications. The treatments included spraying with distilled water (control) and the combined effects of methyl jasmonate (50 and 100 μM), salicylic acid (80 and 160 mg/L), and yeast extract (1 and 1.5 g/L). The contents of phenolic and flavonoid compounds, including rosmarinic acid, chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid, apigenin, quercetin and luteolin, was measured using HPLC. The results showed that the treatments significantly increased the levels of phenolic acids and flavonoids. Foliar application of 160 mg/L salicylic acid + 100 μM methyl jasmonate resulted in the greatest increase in rosmarinic acid, chlorogenic acid, p-coumaric acid, and quercetin, with increases of 4.2-, 3.5-, 10.3-, and 6.2-fold, respectively, compared to the control. The results of the correlation analysis among the traits showed that the amount of rosmarinic acid had a positive and significant correlation with chlorogenic acid (r=0.819**), ferulic acid (r=0.773**), quercetin (r=0.987**), and p-coumaric acid (r=0.986**) and a negative and significant correlation with caffeic acid (r=-0.920**). The heatmap of the studied traits showed distinct clustering between the control and some combined treatments of these elicitors. Overall, the results highlighted the synergistic effect of methyl jasmonate and salicylic acid on increasing polyphenolic compound production in D. moldavica. Introduction Moldavian balm or dragonhead (Dracocephalum moldavica L.) is an annual herbaceous plant belonging to the Lamiaceae family, widely used in the food and pharmaceutical industries. Its extract exhibits various therapeutic effects, including anticancer, antirheumatic, anti-inflammatory, antibacterial, analgesic, sedative, and antioxidant activities. The quality of medicinal plants intended for pharmacological use is often assessed by the content of biologically active compounds. Phytochemical analyses have revealed that phenolic compound production in D. moldavica is often limited. Elicitation represents an effective approach to enhance the accumulation of plant secondary metabolites. This study aimed to increase the production of flavonoids, rosmarinic acid, and other phenolic compounds in D. moldavica by applying combinations of methyl jasmonate (MeJA), yeast extract (YE), and salicylic acid (SA) at different concentrations. Materials and Methods The experiment was conducted in a completely randomized design with 13 treatments and three replications under greenhouse conditions. Treatments included foliar application of distilled water (control) and the combinations: salicylic acid (80 mg/L)+yeast extract (1 g/L), salicylic acid (80 mg/L)+yeast extract (1.5 g/L), salicylic acid (80 mg/L)+methyl jasmonate (50 μM), salicylic acid (80 mg/L)+methyl jasmonate (100 μM), salicylic acid (160 mg/L)+yeast extract (1 g/L), salicylic acid (160 mg/L)+yeast extract (1.5 g/L), salicylic acid (160 mg/L)+methyl jasmonate (50 μM), salicylic acid (160 mg/L)+methyl jasmonate (100 μM), methyl jasmonate (50 μM)+yeast extract (1 g/L), methyl jasmonate (50 μM)+yeast extract (1.5 g/L), methyl jasmonate (100 μM)+yeast extract (1 g/L), and methyl jasmonate (100 μM)+yeast extract (1.5 g/L). For each treatment, three pots were used, each containing five plants (one replicate). Elicitors were applied as foliar sprays to the aerial parts at the 50% flowering stage. Six days after spraying, all aerial parts were harvested. The harvested material was shade-dried for seven days at ambient temperature (25-30 °C) with adequate ventilation. The content of phenolic compounds, including rosmarinic acid, chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid, apigenin, quercetin, and luteolin, was determined using high-performance liquid chromatography (HPLC; UNICAM, Crystal 200 model, England). Retention times were as follows: chlorogenic acid (9.8 min), caffeic acid (16.2 min), p-coumaric acid (22.5 min), ferulic acid (24.8 min), rosmarinic acid (34.1 min), luteolin (40.9 min), quercetin (41.7 min), and apigenin (42.3 min). Concentration of rosmarinic acid and other compounds was expressed as mg/g dry weight. Statistical analysis was performed using SPSS software (version 26). Means were compared using Duncan's multiple range test at P ≤ 0.01. A heatmap was generated using ClustVis, and Pearson correlation coefficients were calculated with SPSS. Principal component analysis (PCA) was performed using PAST software (version 4.17) Results and Discussion The results of analysis of variance (ANOVA) revealed that the combined elicitor treatments significantly (P ≤ 0.01) increased the phenolic compounds contents in D. moldavica compared to the control. The results showed that the type, combination, and concentration of elicitors markedly influenced the accumulation of flavonoids, rosmarinic acid, and other phenolic acids in D. moldavica compared to the control. The most effective treatment was a foliar application of 160 mg/L salicylic acid combined with 100 μM methyl jasmonate, which increased rosmarinic acid, chlorogenic acid, p-coumaric acid, and quercetin by 4.2-, 3.5-, 10.3-, and 6.2-fold, respectively, relative to the control. The results showed that within salicylic acid 160 mg/L treatments, the order of efficacy for rosmarinic acid accumulation was: 100 μM methyl jasmonate>50 μM methyl jasmonate>1.5 g/L yeast extract>1 g/L yeast extract. The results of the correlation analysis indicated that rosmarinic acid content was positively and significantly correlated with chlorogenic acid (r=0.819**), ferulic acid (r=0.773**), quercetin (r=0.987**), and p-coumaric acid (r=0.986**), but negatively correlated with caffeic acid (r=-0.920**). The heatmap clustering clearly distinguished the control group from the combined elicitor treatments, and principal component analysis further supported these groupings. Conclusion Foliar application of 160 mg/L salicylic acid combined with 100 μM methyl jasmonate proved most effective in enhancing the accumulation of phenolic compounds in D. moldavica. Rosmarinic acid, the major phenolic compound in this species, showed strong positive correlations with chlorogenic acid, ferulic acid, quercetin, and p-coumaric acid. These findings suggest that the combined use of salicylic acid and methyl jasmonate is a viable strategy to significantly increase the levels of valuable phenolic and flavonoid metabolites in D. moldavica, with potential applications in the pharmaceutical and food industries. یکی از رویکردهای مؤثر برای افزایش تولید متابولیتهای ثانویه گیاهی، استفاده از محرکها است. این پژوهش، با هدف افزایش تولید رزمارینیک اسید و سایر ترکیبات فنلی بادرشبو (Dracocephalum moldavica L.) توسط اثر ترکیبی غلظتهای مختلف متیل جاسمونات، عصاره مخمر و سالیسیلیک اسید انجام شد. آزمایش در قالب طرح کامل تصادفی با 13 تیمار و در سه تکرار انجام شد. تیمارها شامل محلولپاشی با آب مقطر (شاهد)، اثر توام متیل جاسمونات (50 و 100 میکرومولار)، سالیسیلیک اسید (80 و 160 میلیگرم در لیتر) و عصاره مخمر (1 و 5/1 گرم در لیتر) بود. میزان ترکیبات فنلی و فلاونوئیدی شامل رزمارینیک اسید، کلروژنیک اسید، فرولیک اسید، کافئیک اسید، پیکوماریک اسید، آپیژنین، کوئرسیتین و لوتئولین توسط دستگاه HPLC اندازهگیری شدند. نتایج نشان داد تیمارها بر افزایش مقدار اسیدهای فنلی و فلاونوئیدی بادرشبو تأثیر معنیدار داشتند. محلول‌پاشی با 160 میلیگرم در لیتر سالیسیلیک اسید+100 میکرومولار متیل جاسمونات بهترین نتیجه را در افزایش میزان رزمارینیک اسید، کلروژنیک اسید، پیکوماریک اسید و کوئرسیتین داشت و بهترتیب 2/4، 5/3، 3/10 و 2/6 برابر نسبت به شاهد افزایش داشتند. همچنین نتایج همبستگی بین صفات نشان داد که میزان رزمارینیک اسید همبستگی مثبت و معنیدار با کلروژنیک اسید (**819/0r=)، فرولیک اسید (**773/0r=)، کوئرسیتین (**987/0r=) و پیکوماریک اسید (**986/0r=) و منفی و معنیدار با کافئیک اسید (**920/0-r=) دارد. نتایج نقشه حرارتی برای صفات مورد پژوهش، بین شاهد و برخی تیمارهای توام این محرکها خوشهبندی متمایزی نشان داد. در مجموع، نتایج بر اثر همافزایی سالیسیلیک اسید با متیل جاسمونات در افزایش تولید ترکیبات پلیفنلی بادرشبو دلالت داشت.

https://ijpb.ui.ac.ir/article_30507_d8f4e109b221716768e39ac244dcb3f8.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 1 2026 05 22 The Effect of Delayed Sowing Date (as a Cold Stress Inducer) and Seed Pre-treatment on Leaf Nutrient Concentration, Physiological Indices, and Wheat Yield تأثیر تأخیر در تاریخ کاشت (به عنوان القاکننده تنش سرما) و پیش‌تیمار بذر بر غلظت عناصر غذایی برگ، شاخص‌های فیزیولوژیک و عملکرد گندم 23 40 30391 10.22108/ijpb.2026.148213.1435 FA اسماعیل قلی نژاد گروه علوم کشاورزی، دانشکده فنی و مهندسی، دانشگاه پیام نور، تهران، ایران سعیده صلواتی گروه علوم کشاورزی، دانشکده فنی و مهندسی، دانشگاه پیام نور، تهران، ایران علیرضا عیوضی بخش تحقیقات نهال و بذر، مرکز تحقیقات کشاورزی و منابع طبیعی استان آذربایجان غربی، سازمان تحقیقات، آموزش و ترویج کشاورزی، ارومیه، ایران بختیار لله گانی گروه علوم کشاورزی، دانشکده فنی و مهندسی، دانشگاه پیام نور، تهران، ایران Journal Article 2026 01 31 Late planting dates, due to the induction of cold stress, represent one of the most critical limiting factors for wheat production in cold and temperate regions, causing significant yield losses by disrupting physiological and nutritional processes. The use of seed pre-treatment with bio- and chemical stimulants has emerged as a promising strategy to mitigate the effects of this stress. This study aimed to evaluate the effects of planting date and various pre-treatment treatments on leaf nutrient concentrations, physiological indices, and grain yield of the Simin wheat cultivar. A factorial experiment was conducted within a randomized complete block design with three replications during the 2024–2025 growing season at the Urmia Agricultural Research Center. The first factor consisted of two planting dates (October 22 and December 22), and the second factor comprised seven pre-treatment treatments (control, salicylic acid, gibberellic acid, GABA, potassium nitrate, zinc sulfate, and melatonin). Measured traits included leaf nutrient concentrations, membrane stability index, calcium nutritional index, stress index, nutrient use efficiency, grain filling rate, and grain yield. Results showed that the second planting date reduced magnesium, calcium, and iron concentrations by 28%, 27%, and 19%, respectively. Furthermore, the interaction of planting date and seed pre-treatment with melatonin increased grain yield by 37% on the first planting date and 42% on the second planting date. Melatonin, GABA, and potassium nitrate treatments were significantly superior in improving physiological indices compared to other treatments. Overall, the first planting date combined with seed pre-treatment using melatonin, GABA, or potassium nitrate is recommended as the optimal management strategy to improve nutritional status, enhance cold tolerance, and achieve optimal yield of the Simin wheat cultivar under conditions similar to the study region. The choice of pre-treatment should be based on planting date and management objectives (improving nutrition or increasing stress tolerance). Introduction Delayed sowing date is a major limiting factor for wheat production in cold and cold semi-arid regions, causing significant yield loss by disrupting physiological and nutritional processes. Cold stress, particularly during early growth stages, impairs root activity, reduces nutrient uptake and translocation, damages membrane integrity, and limits photosynthetic efficiency. In Iran, where large areas of wheat fields are located in cold semi-arid zones, autumn and early winter frosts annually cause substantial economic damage. Seed priming with biological and chemical stimulants is a pre-treatment approach to mitigate these adverse effects by enhancing germination, improving seedling vigor, and activating stress-responsive mechanisms. This study aimed to evaluate the impact of sowing date (optimal vs. delayed) and different seed pre-treatments on leaf nutrient concentration (magnesium, calcium, iron, zinc, potassium), physiological indices (membrane stability index, calcium nutrition index, stress index, element use efficiency, grain filling rate), and grain yield of Simin wheat, a cold-adapted bread wheat cultivar widely grown in Iran. Materials and Methods A factorial experiment was conducted based on a randomized complete block design with three replications during the 2024-2025 growing season at the Urmia Agricultural Research Center, West Azerbaijan Province, Iran (latitude 37°44’ N, longitude 45°02’ E, altitude 1352 m). The region has a cold semi-arid climate with average annual rainfall of 390 mm and a mean temperature of 11.3°C. Factors included two sowing dates (November 1 as optimal sowing, and December 1 as delayed sowing to induce cold stress) and seven seed pre-treatments: control (dry seed), salicylic acid (1 mM), gibberellic acid (1500 µM), GABA (10 mM), potassium nitrate (200 mM), zinc sulfate (50 mM), and melatonin (100 µM). Seeds were soaked in priming solutions for 15 hours, air-dried for 48 hours, and then sown by drill using a broadcast method at a density of 500 seeds m⁻². Standard agronomic practices, including four spring irrigations, were applied. Measured traits included leaf concentrations of Mg, Ca, Fe, Zn, and K; total leaf element concentration; membrane stability index (MSI); calcium nutrition index (NIC); stress index (SI); element use efficiency (EUE); grain filling rate (GFR); and grain yield (GY). Data were analyzed using SAS software (version 9.1), and means were compared using Tukey’s HSD test at p ≤ 0.05. Results and Discussion Results indicated that the interaction between sowing date and seed pre-treatments was significant (p ≤ 0.01) for leaf nutrient concentration, nutrient use efficiency, and grain filling rate. Delayed sowing (December 1) significantly reduced leaf concentrations of magnesium, calcium, and iron by 28%, 27%, and 19%, respectively, compared to optimal sowing (November 1). This reduction is primarily attributed to impaired root function, reduced transpiration-driven nutrient transport, and decreased metabolic activity under cold stress conditions experienced by late-sown plants during early growth stages. Furthermore, delayed sowing decreased the membrane stability index by approximately 9.5%, indicating greater electrolyte leakage and membrane damage. Regarding seed pre-treatments, GABA, melatonin, and potassium nitrate were consistently superior in improving physiological indicators. Specifically, GABA increased leaf Mg by 30%, while melatonin and KNO₃ raised leaf Ca by 30% and 29%, respectively, compared to the control. All priming treatments significantly enhanced leaf Fe concentration, with GABA, salicylic acid, and melatonin showing up to 31% improvement. The interaction between sowing date and priming was significant for leaf Zn and K. Under optimal sowing, KNO₃ increased leaf Zn by 81%, whereas under delayed sowing, GABA was most effective (60% increase). For leaf K, KNO₃ (53% increase) and melatonin (26%) performed best in the first sowing date, while GABA (42%) and melatonin (33%) excelled under late sowing. Compared to the unprimed control within each sowing date, melatonin increased grain yield by 37% in the first planting date and 42% in the second planting date compared to the unprimed control. GABA and KNO₃ also significantly enhanced grain yield, particularly under optimal and late sowing conditions, respectively. Melatonin consistently improved the calcium nutrition index (NIC), grain filling rate, and stress index (SI), highlighting its role as a broad-spectrum stress protectant. In general, early sowing combined with seed pre-treatment using melatonin, GABA, or potassium nitrate is recommended as the best management strategy to improve nutritional status, enhance cold tolerance, and achieve optimal yield in Simin wheat under similar regional conditions. The superior performance of melatonin is attributed to its dual role as a direct antioxidant and a signaling molecule that regulates stress-responsive genes, stabilizes membranes, and improves nutrient redistribution. GABA’s effectiveness is linked to its role in reducing oxidative stress, maintaining osmotic balance, and protecting photosynthetic pigments. Potassium nitrate likely acts through nutritional and osmotic regulation, improving K⁺/Na⁺ homeostasis and activating antioxidant enzymes. The choice of seed pre-treatments should be aligned with the sowing date and management objectives (nutrition improvement vs. stress tolerance enhancement). For instance, KNO₃ is more suitable under near-optimal conditions to boost nutrient content, while GABA and melatonin are preferable under cold stress conditions to enhance physiological resilience and yield stability. Conclusion Early sowing combined with seed pre-treatment using melatonin, GABA, or potassium nitrate is recommended as the optimal management strategy to improve nutritional status, enhance cold tolerance, and achieve maximum yield in Simin wheat under similar regional conditions. تاریخ کاشت دیرهنگام به علّت القای تنش سرما یکی از مهمترین عوامل محدودکننده تولید گندم در مناطق سرد و معتدله است که با اختلال در فرآیندهای فیزیولوژیک و تغذیه‌ای، خسارت قابل توجهی به عملکرد وارد می‌کند. استفاده از روش پیشتیمار بذر با محرک‌های زیستی و شیمیایی، به‌عنوان راهکاری امیدبخش برای تعدیل اثرات این تنش مطرح است. هدف از این پژوهش، ارزیابی تأثیر تاریخ کاشت و تیمارهای مختلف پیشتیمار بر غلظت عناصر غذایی برگ، شاخص‌های فیزیولوژیک و عملکرد دانه گندم رقم سیمین بود. این آزمایش به‌صورت فاکتوریل در قالب طرح بلوک‌های کامل تصادفی در سه تکرار در سال زراعی 1404-1403 در مرکز تحقیقات کشاورزی ارومیه اجرا شد. فاکتور اول شامل دو تاریخ کاشت (اول آبان و اول دی) و فاکتور دوم هفت تیمار پیشتیمار (شاهد، سالیسیلیک اسید، جیبرلیک اسید، گابا، نیترات پتاسیم، سولفات روی و ملاتونین) بود. صفات مورد اندازه‌گیری شامل غلظت عناصر غذایی برگ، شاخص پایداری غشاء، شاخص تغذیه‌ای کلسیم، شاخص تنش، کارایی مصرف عناصر، نرخ پر شدن دانه و عملکرد دانه بود. نتایج نشان داد تاریخ کاشت دوم، غلظت منیزیم، کلسیم و آهن را بهترتیب بهمیزان 28، 27 و 19 درصد کاهش داد. همچنین بر همکنش تاریخ کاشت و پیشتیمار بذر با ملاتونین، عملکرد دانه را بهترتیب 37 درصد در تاریخ کاشت اول و 42 درصد در تاریخ کاشت دوم افزایش داد. تیمارهای ملاتونین، گابا و نیترات پتاسیم در بهبود شاخص‌های فیزیولوژیک در مقایسه با سایر تیمارها برتری معنیداری داشتند. به‌طور کلی، تاریخ کاشت اول همراه با پیشتیمار بذر با ملاتونین، گابا یا نیترات پتاسیم، به‌عنوان بهترین راهکار مدیریتی برای بهبود وضعیت تغذیه‌ای، افزایش تحمل به سرما و دستیابی به عملکرد بهینه در گندم رقم سیمین در شرایط مشابه منطقه‌ای توصیه می‌شود. انتخاب تیمار پیشتیمار باید با توجه به تاریخ کاشت و اهداف مدیریتی (بهبود تغذیه یا افزایش تحمل به تنش) صورت گیرد.

https://ijpb.ui.ac.ir/article_30391_7761a2f990ea4c22d472e63b64081c88.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 1 2026 05 22 Micromorphology, numerical taxonomy, and ancestral character reconstruction of petals in Cleome and Rorida from Iran ریزریخت‌شناسی، تاکسونومی عددی و بازسازی صفات اجدادی گلبرگ‌ها در سرده‌های Cleome و Rorida در ایران 41 68 30442 10.22108/ijpb.2026.148253.1436 FA آتنا اسلامی فاروجی بخش زیست شناسی، دانشکده علوم، دانشگاه شیراز، شیراز، ایران Journal Article 2026 02 03 In this study, for the first time, the micromorphology of the dorsal and ventral surfaces of petals in species of the genera Cleome and Rorida (family Cleomaceae) was investigated in Iran using a scanning electron microscope (SEM). Epidermal cell traits of petals, including cell shape (conical papillose or tabular-rugose), anticlinal wall shape (straight or curved), cuticular ornamentation (striate or rugose), presence or absence of trichomes and their type, and the presence or absence of stomata, were examined. After character coding and scoring, the data were analyzed using cluster analysis, Principal Coordinate Analysis (PCoA), and Detrended Correspondence Analysis (DCA) in PAST software. Ancestral state construction of petal characters was also performed based on molecular data. The results showed that the dorsal and ventral surfaces of petals in Rorida and some Cleome species (C. heratensis, C. foliolosa, and C. turkmena) exhibited tabular-rugose epidermal cells. In contrast, other species demonstrated conical papillose cells. Anticlinal wall shapes were straight or curved, or exclusively curved, and trichomes were observed in simple or glandular forms (short or long); stomata were reported only in some species. The inferred ancestral states included tabular-rugose cells, striate cuticular ornamentation, and the absence of trichomes and stomata. The obtained results provided valuable information on petal traits at the microscopic scale and successfully distinguished the two genera under study. Introduction The spider-flower family, Cleomaceae, comprises annual and perennial herbs, shrubs, and small trees. It is mainly distributed in tropical, desert, and temperate regions and encompasses around 27 genera and 270 species worldwide (Bayat et al., 2018). Taxonomically, this family was treated as a subfamily of Capparaceae (Pax & Hoffmann, 1936; Messina, 2020), while some taxonomists reassigned Cleomaceae as a distinct family (Heywood et al., 2007; Iltis & Cochrane, 2007). In a phylogenetic context, Cleomaceae is closely related to Brassicaceae (Nikolov et al., 2019; Mabry et al., 2023). Some taxa within this family possess medicinal, nutritional, and ornamental value (Hassan et al., 2022; Khuntia et al., 2022). The genera Cleome and Rorida belong to the family Cleomaceae, and comprise approximately 203 and 15 species worldwide, respectively (POWO, 2026), and ca. 10 and 6 species in Iran, respectively. Although Rorida is taxonomically assigned as a synonym of Cleome (POWO, 2026), recent taxonomic and phylogenetic investigations supported its recognition as a distinct genus (Thulin & Roalson, 2017; Eslami-Farouji et al., 2025). While the petal micromorphology has been investigated in several plant families have been studied previously, the micromorphology of petals within Cleomaceae has remained unexplored. Therefore, the present study specifically aims to investigate the micromorphology, numerical taxonomy, and ancestral character reconstruction of petals of Cleome and Rorida. Materials and Methods In this study, a total of 13 species from the genera Cleome and Rorida were investigated. The studied specimens are preserved in the Herbarium of Shiraz University (HSHU). The information regarding the collected specimens, including locality, date, altitude, geographical coordinates, and collector’s name, was recorded. The mature flowers of each species were selected. Specimens were transferred to the Central Laboratory of Shiraz University for further analyses using scanning electron microscopy (SEM). The dorsal and ventral surfaces of the petals were placed on the aluminum stubs and fixed with double-sided carbon tape. The stubs were then coated with a thin layer of gold for ten minutes. Microscopic examination was performed using a TESCAN Vega-3 SEM at an accelerating voltage of 20 kV. Epidermal cell traits of petals, comprising cell shape (conical papillose or tabular-rugose), anticlinal wall shape (straight or curved), cuticular ornamentation (striate or rugose), presence or absence of trichomes and their type, and the presence or absence of stomata, were recorded. Qualitative and quantitative micromorphological traits were examined. The latter characteristics were measured using Digimizer software. After character state determination and scoring, the data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA), Principal Coordinate Analysis (PCoA), and Detrended Correspondence Analysis (DCA) in PAST software. Ancestral state reconstruction of petal characters was also conducted using Bayesian Binary MCMC in the RASP software. Arivela viscosa was selected as the outgroup. Results and Discussion In this study, various images were captured and used to examine qualitative and quantitative characteristics. One-way ANOVA and Welch F test show that all measured quantitative traits (petal length, width, epidermal cell area) differed significantly among the studied species of two genera within Iran (p<0.05). Multivariate statistical analyses of 13 Cleome and Rorida species using the Gower coefficient revealed three distinct phenons. The first phenon comprises species related to Rorida, while the remaining two phenons belong to the genus Cleome. Based on DCA analyses, the most diagnostic traits were identified in all three phenons. Box plots were used to illustrate differences in cell area of the dorsal and ventral surfaces of each species. Subsequently, the maximum and minimum variations were recorded. According to the Bayesian Binary MCMC analyses, the graphical output of the petal character analyses showed the ancestral and derived traits, respectively (Tabular-rugose with striation cells vs. Papillose conical cells with striation/ striate vs. non-striate/ glabrous vs. glandular trichome/ without stomata vs. with stomata). Overall, numerous morphological and micromorphological investigations have been conducted by researchers to establish the taxonomic delimitation of taxa. Indeed, selecting apomorphic traits within numerical taxonomy plays an important role in delineating taxa. Therefore, understanding the evolutionary trends of morphological characters is crucial. Conclusion Due to the limited information on petal micromorphology in the two genera Rorida and Cleome, detailed investigations are necessary. In this study, the micromorphology of the dorsal and ventral petal surfaces was investigated for the first time. The results provided valuable information on petal characteristics and led to the interpretation of ancestral and derived characters. Moreover, these traits enabled us to distinguish the two genera based on the specimens studied. However, since the present findings are limited to Iran, comprehensive worldwide plant sampling is necessary. These results support future research and highlight the utility of petal traits in generic and specific delimitation, as well as in understanding the evolutionary history of this group. Acknowledgement The author expresses her gratitude to Shiraz University for financial support and to the Central Laboratory of Shiraz University for providing SEM images. در این پژوهش، برای نخستین بار ریز ریخت‌شناسی سطوح شکمی و پشتی گلبرگ در گونه‌های دو سرده Cleome و Rorida از تیره Cleomaceae در ایران توسط میکروسکوپ الکترونی روبشی (Scanning Electron Microscope: SEM) بررسی شد. صفات گلبرگ شامل شکل سلول‌ها (مخروطی پاپیلوز یا تخت چین‌دار)، شکل دیواره آنتی‌کلینال (صاف یا موج‌دار)، تزئینات کوتیکول (خط‌دار یا چروکیده)، حضور یا عدم حضور کرک و نوع آن، و وجود یا عدم وجود روزنه مورد بررسی قرار گرفت. پس از تعیین حالات صفات و امتیازدهی، داده‌ها با بهره‌گیری از آنالیز‌های خوشه‌بندی، مختصات اصلی و آنالیز تطبیقی قوس‌گیری‌شده در نرم‌افزار PAST واکاوی شدند. همچنین، بازسازی حالات صفات اجدادی گلبرگ‌ها براساس داده‌های مولکولی انجام گرفت. نتایج نشان داد سطح شکمی و پشتی گلبرگ‌ها در سرده Rorida و برخی گونه‌هایCleome (C. heratensis, C. foliolosa, & C. turkmena) دارای سلول‌های اپیدرمی تخت و چین‌دار است، با این وجود، در سایر گونه‌ها حالت مخروطی پاپیلوز مشاهده شد. شکل دیواره آنتی‌کلینال به صورت صاف یا موج‌دار یا صرفاً موج‌دار بود و کرک‌ها به شکل ساده یا غدّه‌ای (کوتاه و بلند) مشاهده شد؛ روزنه تنها در برخی از گونه‌ها گزارش شد. حالت‌های صفات اجدادی شامل سلول‌های صاف و چین‌خورده، تزئینات کوتیکول شیاردار و فقدان کرک و روزنه تشخیص داده شد. نتایج حاصل، اطّلاعات ارزشمندی درباره صفات گلبرگ‌ها در مقیاس میکروسکوپی فرآهم آورد و در جداسازی دو سرده مورد بررسی موفق عمل نمود.

https://ijpb.ui.ac.ir/article_30442_b686f0a375ae2f1a24423c51f9faabe5.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 1 2026 05 22 Comparison of morphological and physiological traits of three ecotypes of Teucrium polium L. under drought stress and evaluation of drought-tolerant genes MYB and LEA in the tolerant ecotype مقایسه صفات ریخت شناسی و فیزیولوژیکی سه اکوتیپ گیاه دارویی کلپوره Teucrium polium L. در شرایط تنش خشکی و بررسی بیان ژنهای متحمل به تنش خشکی MYB و LEA در اکوتیپ متحمل 69 86 30396 10.22108/ijpb.2026.147569.1426 FA زهرا سادات توحیدی گروه زیست شناسی، دانشگاه پیام نور، تهران، ایران. حمید سبحانیان گروه زیست شناسی، دانشگاه پیام نور، تهران، ایران. امین باقی زاده گروه بیوتکنولوژی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی ، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان-ایران Journal Article 2025 11 24 Drought stress is one of the most important limiting factors for plant growth and performance, affecting a wide range of plant responses from metabolic alterations to changes in growth and productivity. The severity of this stress varies depending on water deficit level, growth stage, and plant species. Considering Iran’s location in arid and semi-arid climates and the continuous decline in water resources, identifying drought-tolerant plants and ecotypes is essential. In recent years, research has increasingly focused on understanding the molecular mechanisms of drought tolerance and the key genes involved in this process. In this study, morphological and physiological traits of three ecotypes of the medicinal plant Kalpooreh Teucrium polium from Kerman province were evaluated under different drought stress levels (40%, 70%, and 100% field capacity), along with the activity of antioxidant enzymes (SOD, CAT, POD) and lipid peroxidation (MDA). Additionally, expression of the MYB and LEA genes was assessed in the tolerant ecotype using Real-time PCR. The results showed that increasing drought stress intensity reduced stem height and total chlorophyll content, while root length, proline content, and protein content increased. Antioxidant enzyme activity and MDA levels also rose significantly, indicating intensified oxidative stress. Moreover, expression of the MYB and LEA genes showed a marked increase in the tolerant ecotype under drought stress. These findings confirm the combined roles of physiological, biochemical, and molecular responses in drought tolerance and suggest that these genes could serve as useful indicators for genetic improvement and selection of drought-resistant germplasms in Teucrium polium Introduction Drought stress is one of the most critical abiotic constraints limiting plant growth, productivity, and distribution, affecting plants at multiple levels from cellular metabolism to whole-plant morphology. The severity of drought damage depends on stress intensity and duration, developmental stage, and the genetic characteristics of each species or ecotype. Given Iran's location in arid and semi-arid regions and the continuous decline in water resources, identifying drought-tolerant plants and ecotypes is a major priority. Medicinal plants, despite their economic and therapeutic value, remain underexplored in terms of stress physiology. Teucrium polium L., widely distributed across Iran, possesses strong antioxidant and pharmacological properties, yet its drought-response mechanisms are poorly documented. In this study, morphological and physiological traits of three ecotypes of T. polium from Kerman province were evaluated under different drought stress levels (40%, 70%, and 100% field capacity), alongside antioxidant enzyme activities (SOD, CAT, POD) and lipid peroxidation (MDA). Additionally, expression of the MYB and LEA genes was assessed in the tolerant ecotype using Real-time PCR. The results showed that increasing drought stress reduced stem height and total chlorophyll content, while root length, proline, and protein content increased. Antioxidant enzyme activity and MDA levels also rose significantly, indicating intensified oxidative stress. Furthermore, MYB and LEA gene expression showed a marked increase in the tolerant ecotype, confirming the combined roles of physiological, biochemical, and molecular responses in drought tolerance. Materials and Methods A factorial experiment based on a completely randomized design with three replications was conducted in a greenhouse in Kerman, Iran. Three ecotypes (Sarcheshmeh, Rafsanjan, Sirjan) were subjected to 100%, 70%, and 40% field capacity. Morphological traits measured included stem length and root length. Physiological and biochemical traits included total chlorophyll, proline, soluble proteins, and MDA content. Antioxidant enzyme activities (SOD, CAT, POD) were quantified by standard spectrophotometric assays. For molecular analysis, RNA was extracted from the tolerant ecotype, cDNA was synthesized, and expression of MYB and LEA genes was analyzed using Real-time PCR. Relative expression was calculated by the 2^-ΔΔCt method. ANOVA analyzed the data, and means were compared using Duncan's test at the 5% probability level. Results Drought stress significantly affected almost all measured traits. Increasing drought intensity reduced stem length and total chlorophyll content across all ecotypes, indicating impaired photosynthesis and inhibited aerial growth. In contrast, root length increased under moderate and severe drought, representing an adaptive strategy to improve water acquisition. Biochemically, proline concentration rose sharply under water deficit, likely contributing to osmotic adjustment and cellular protection. Protein content also increased, suggesting the production of stress-related proteins. Antioxidant enzyme activities (SOD, CAT, POD) increased significantly with increasing drought severity, reflecting activation of the antioxidant defense system to detoxify reactive oxygen species. MDA content, indicative of lipid peroxidation, also increased, though tolerant ecotypes showed lower MDA accumulation, suggesting reduced membrane damage. At the molecular level, MYB and LEA genes were strongly upregulated in the tolerant ecotype, especially under 40% field capacity, corresponding with higher antioxidant activity, increased osmolyte accumulation, and improved root performance. Discussion Drought tolerance in T. polium is driven by a combination of structural, metabolic, and molecular mechanisms. Reduced shoot growth minimizes transpiration, while increased root length supports deeper soil exploration under water deficit. Chlorophyll reduction and increased ROS under drought are well-known physiological outcomes, but tolerant ecotypes demonstrated better chlorophyll retention and stronger oxidative defense. Accumulation of osmoprotectants such as proline and proteins contributes to maintaining cell turgor and stabilizing membranes. The marked increase in antioxidant enzyme activity reveals effective ROS scavenging, limiting oxidative damage. The strong upregulation of MYB and LEA genes further confirms their roles in regulating transcriptional and protective responses under drought. These genes may therefore serve as functional biomarkers for screening drought-tolerant germplasm. Conclusion This study highlights substantial ecotypic variation in drought responses of T. polium. Drought tolerance is associated with improved root development, higher osmolyte and protein accumulation, strengthened antioxidant capacity, and significant upregulation of the MYB and LEA genes. The tolerant ecotype identified here, along with its molecular markers, holds promise for breeding programs, cultivation under limited water conditions, and future molecular studies aimed at enhancing drought resistance in medicinal plants. تنش خشکی یکی از مهم‌ترین عوامل محدودکننده رشد و عملکرد گیاهان است و طیف وسیعی از واکنش‌های گیاهی، شامل تغییرات متابولیکی تا دگرگونی در رشد و تولید را تحت تأثیر قرار می‌دهد. شدت این تنش بسته به میزان کم‌آبی، مرحله رشد و گونه گیاهی متفاوت است. با توجه به قرارگیری ایران در اقلیم خشک و نیمه‌خشک و کاهش منابع آب، شناسایی گیاهان و اکوتیپ‌های سازگار با کم‌آبی اهمیت زیادی دارد. در سال‌های اخیر، پژوهش‌ها به سمت شناخت سازوکارهای مولکولی مقاومت به خشکی و ژن‌های مؤثر در این فرایند هدایت شده‌اند. در این مطالعه، صفات ریخت شناسی و فیزیولوژیکی سه اکوتیپ گیاه دارویی کلپوره Teucrium polium استان کرمان تحت سطوح مختلف تنش خشکی (۴۰، ۷۰ و ۱۰۰ درصد ظرفیت زراعی)، همراه با فعالیت آنزیم‌های آنتی‌اکسیدانی سوپراکسید دیسموتاز، کاتالاز، پراکسیداز و میزان پراکسیداسیون لیپیدها (MDA) ارزیابی شد. همچنین در اکوتیپ مقاوم، بیان دو ژن MYB و LEA با روش Real-time PCR بررسی شد. نتایج پژوهش نشان داد افزایش شدت تنش خشکی سبب کاهش ارتفاع ساقه و محتوای کلروفیل کل و در مقابل افزایش طول ریشه، مقدار پرولین و میزان پروتئین شد. همچنین فعالیت آنزیم‌های آنتی‌اکسیدانی و میزان MDA نیز به طور معنی‌داری افزایش یافت که بیانگر تشدید تنش اکسیداتیو است. علاوه بر این، بیان ژن‌های MYB و LEA در اکوتیپ مقاوم تحت تأثیر تنش افزایش قابل‌توجهی داشت. این یافته‌ها نقش همزمان واکنش‌های فیزیولوژیکی، بیوشیمیایی و مولکولی را در تحمل خشکی تأیید کردند و نشان دادند ژن‌های مذکور می‌توانند به‌عنوان شاخص‌هایی کاربردی برای اصلاح ژنتیکی و انتخاب ژرم‌پلاسم‌های مقاوم در کلپوره مورد استفاده قرار گیرند.

https://ijpb.ui.ac.ir/article_30396_80963e015e0ab3349dd72509f0018a7a.pdf

دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 1 2026 05 22 Metabolic responses induced by silver nanoparticles in the green microalga Dunaliella salina پاسخ‌های متابولیکی ناشی از نانو ذرات نقره در ریز جلبک سبز Dunaliella salina 87 108 30567 10.22108/ijpb.2026.148814.1445 FA علیرضا عینعلی گروه زیست شناسی، دانشکده علوم، دانشگاه سیستان و بلوچستان، زاهدان، ایران سمیه شعلی بر گروه زیست شناسی، دانشکده علوم، دانشگاه زابل، زابل، ایران امید عزیزیان شرمه گروه زیست شناسی، دانشکده علوم، دانشگاه سیستان و بلوچستان، زاهدان، ایران شهلا نجفی گروه زیست شناسی، دانشکده علوم، دانشگاه زابل، زابل، ایران Journal Article 2026 05 05 In the present study, the biochemical responses and antioxidant enzyme activities of the unicellular green alga Dunaliella salina were investigated following exposure to phytosynthesized silver nanoparticles synthesized using aqueous extracts of German chamomile (Matricaria chamomilla) leaves and flowers. Algal cultures grown in 1.5 M NaCl and harvested during the exponential growth phase were exposed to 0, 5, 15, and 25 picomolar (pM) concentrations of silver nanoparticles. Cell number, reducing sugars, non-reducing sugars, and starch content either increased or remained unchanged following exposure to 5 pM silver nanoparticles. However, at higher nanoparticle concentrations, cell number and soluble sugar contents decreased markedly. In addition, the contents of chlorophyll, β-carotene, starch, proteins, and amino acids, as well as the activities of catalase and ascorbate peroxidase, declined significantly at all nanoparticle concentrations, whereas cell fresh weight increased. These results indicate that silver nanoparticle-induced stress in algal cells is tolerated through changes in carbon partitioning rather than through enhanced antioxidant enzyme activity or β-carotene accumulation. Therefore, tolerance of D. salina to silver nanoparticles appears to be associated with physiological and metabolic reprogramming, particularly through the redistribution of carbon toward alternative metabolic pathways, such as glycerol and lipid accumulation, which may contribute to nanoparticle tolerance. Introduction Silver nanoparticles (AgNPs) are among the most extensively utilized engineered nanomaterials because of their unique physicochemical properties and broad applications in medicine, agriculture, environmental remediation, and industrial processes. The increasing release of these nanoparticles into aquatic environments has raised concerns regarding their potential impacts on aquatic organisms, particularly primary producers such as microalgae. Microalgae constitute the foundation of aquatic food webs and play a vital role in global carbon cycling. Therefore, understanding their physiological and biochemical responses to nanoparticle exposure is essential for evaluating the ecological consequences of nanomaterial contamination. Dunaliella salina is a unicellular halotolerant green microalga widely recognized as a model organism for investigating stress physiology, carotenoid biosynthesis, and metabolic adaptation. The remarkable ability of this microalga to survive under extreme environmental conditions makes it a valuable system for studying cellular responses to abiotic stress factors. In recent years, increasing attention has been directed toward understanding how nanoparticles influence the metabolism and stress-response mechanisms of D. salina. The present study aimed to investigate the metabolic and biochemical responses of D. salina exposed to phytosynthesized silver nanoparticles produced using aqueous extracts of German chamomile (Matricaria chamomilla). Materials and methods Silver nanoparticles were synthesized biologically and characterized using UV–Visible spectroscopy and transmission electron microscopy. The synthesized nanoparticles exhibited a characteristic surface plasmon resonance peak at approximately 410 nm and were predominantly spherical with an average particle size of approximately 13 nm. Exponentially growing cultures of D. salina were exposed to different concentrations of silver nanoparticles (0, 5, 15, and 25 pM). After 48 h of treatment, various physiological and biochemical parameters were evaluated, including cell number, fresh biomass, photosynthetic pigments, soluble sugars, starch, proteins, free amino acids, and antioxidant enzyme activities. Results and discussion The results revealed that exposure to silver nanoparticles significantly influenced algal growth and metabolism. Cell number decreased progressively with increasing nanoparticle concentration, particularly at 15 and 25 pM. This reduction suggests that silver nanoparticles negatively affect cell division and cellular proliferation. In contrast, fresh biomass increased significantly under nanoparticle treatments, indicating that nanoparticle exposure induced substantial physiological and metabolic modifications. The observed inverse relationship between cell number and fresh biomass suggests that factors other than cell proliferation contribute to biomass accumulation under nanoparticle stress. Photosynthetic pigments were highly sensitive to nanoparticle treatment. Chlorophyll a, chlorophyll b, total chlorophyll, and β-carotene contents decreased significantly at higher nanoparticle concentrations. The reduction in chlorophyll content indicates impairment of the photosynthetic apparatus and decreased photosynthetic efficiency. Moreover, the decline in β-carotene accumulation suggests a reduction in photoprotective capacity because β-carotene functions as a major antioxidant pigment in Dunaliella species. The decrease in the β-carotene-to-total chlorophyll ratio further confirms the greater sensitivity of carotenoid metabolism to nanoparticle-induced stress. Silver nanoparticles also altered carbon metabolism. Low nanoparticle concentration (5 pM) stimulated the accumulation of soluble sugars, whereas higher concentrations significantly reduced reducing sugars, non-reducing sugars, total soluble sugars, and starch content. These findings indicate that silver nanoparticles affect carbon allocation and storage metabolism. The observed decline in carbohydrate reserves at elevated nanoparticle concentrations suggests that carbon resources may be redirected toward alternative metabolic pathways involved in stress adaptation and cellular protection. Protein metabolism was similarly affected. Both total protein and soluble protein contents decreased substantially in nanoparticle-treated cells. Total free amino acids exhibited relatively minor changes, suggesting alterations in protein turnover and amino acid homeostasis. The reduction in protein content may result from enhanced proteolytic activity, decreased protein synthesis, or both. These responses indicate extensive metabolic reorganization under nanoparticle stress conditions. Antioxidant enzyme activities also declined significantly following silver nanoparticle treatment. Catalase and ascorbate peroxidase activities were markedly reduced, especially at higher nanoparticle concentrations. These observations suggest that tolerance to silver nanoparticles is not associated with enhanced antioxidant enzyme activity. Instead, nanoparticle exposure appears to impair antioxidant defense systems, thereby increasing susceptibility to oxidative damage. Collectively, the results demonstrate that silver nanoparticles induce extensive metabolic disturbances in D. salina. The simultaneous reduction of photosynthetic pigments, carbohydrates, proteins, amino acids, and antioxidant enzyme activities indicates that nanoparticle exposure imposes substantial physiological stress. Nevertheless, the increase in fresh biomass suggests the activation of alternative adaptive mechanisms. The findings support the hypothesis that tolerance to silver nanoparticles is achieved primarily through changes in carbon partitioning and metabolic reprogramming rather than through increased antioxidant enzyme activity or carotenoid accumulation. Conclusion In conclusion, phytosynthesized silver nanoparticles significantly affect the physiological and metabolic characteristics of Dunaliella salina. The observed responses emphasize the importance of nanoparticle properties in determining biological outcomes and highlight the complexity of nanoparticle–microalga interactions. These findings contribute to a better understanding of the ecological implications of silver nanoparticle contamination and provide valuable insights into stress-adaptation mechanisms in microalgae. Future studies should focus on identifying the specific metabolic pathways involved in carbon reallocation and evaluating long-term responses to nanoparticle exposure under environmentally relevant conditions. در پژوهش حاضر، پاسخهای بیوشیمیایی و فعالیت آنزیمهای آنتی اکسیدانی جلبک سبز تک سلولی Dunaliella salina تحت تیمار نانوذرات نقره فتوسنتز شده با عصاره آبی برگ و گل گیاه بابونه آلمانی (Matricaria comomilla) مورد بررسی قرار گرفت. سوسپانسیونهای جلبکی رشد یافته در شوری 5/1 مولار کلرید سدیم در فاز نمایی رشد در معرض غلظتهای صفر، 5، 15 و 25 پیکومولار نانوذرات نقره قرار گرفتند. تعداد سلولها، محتوای قندهای احیاکننده، غیراحیاکننده و نشاسته در پاسخ به غلظت 5 پیکومولار نانوذرات نقره افزایش یافتند و یا بدون تغییر معنیدار باقی ماندند. با وجود این، تعداد سلولها و مقدار قندهای محلول در پاسخ به غلظتهای بالاتر و میزان کلروفیل های a، b و کل، بتاکاروتن، نشاسته، پروتئینها، آمینواسید آزاد کل و فعالیت آنزیمهای کاتالاز و آسکوربات پراکسیداز در پاسخ به همه غلظتهای نانوذرات نقره به شدت کاهش یافتند، در حالی که وزن تر سلولها افزایش یافت. این نتایج نشان دادند تنش القا شده توسط تیمار نانوذرات نقره در سلولهای جلبکی به جای افزایش فعالیت آنتی اکسیدانی و یا تجمع بتاکاروتن، توسط تغییر در تسهیم کربن تحمل می شود. بنابراین به نظر میرسد که سازوکار تحمل نانوذرات نقره توسط جلبک Dunaliella با تغییرات فیزیولوژیکی و متابولیکی همراه بوده و هدایت جریان کربن به مسیرهای احتمالی دیگری مثل تجمع گلیسرول و لیپید سبب القای تحمل جلبک به نانوذرات میشود.

https://ijpb.ui.ac.ir/article_30567_78567124def471094e239332ab9ef724.pdf