70 ACTA BOT. CROAT. 84 (1), 2025 Acta Bot. Croat. 84 (1), 70–80, 2025 CODEN: ABCRA 25 DOI: 10.37427/botcro-2025-015 ISSN 0365-0588 eISSN 1847-8476 Combined application of rutin and silicon sustains maize seedlings osmotic stress tolerance by improving photosynthetic capacity and chlorophyll metabolism Asiye Sezgin Muslu1*, Cansu Altuntaş2, Namuun Altansambar1,3, Mehmet Demiralay4, Asim Kadıoğlu1 1 Karadeniz Technical University, Faculty of Science, Department of Biology, 61080 Trabzon, Türkiye 2 Medical-Aromatic Plants Application and Research Center, Artvin Coruh University, 08100 Artvin, Türkiye 3 National University of Mongolia, School of Art and Sciences, Department of Biology, 14201 Ulaanbaatar, Mongolia 4 Artvin Coruh University, Faculty of Forestry, Department of Forestry Engineering, 08000 Artvin, Türkiye Abstract – In the current study, the role of external applications of rutin (Rut) and silicon (Si) in stress tolerance was investigated. Although it is known that Si has a role in improving plant defense against a variety of stresses, the role of Rut application in stress response remains unclear. Therefore, the current study was designed to evaluate the function of the synergistic effect of combined Rut and Si applications on the photosynthetic capacity of maize seedlings under osmotic stress. Twenty-one-day-old seedlings were treated with Rut (60 mg L-1) and Si (1 mM), and exposed to osmotic stress (induced by 10% and 15% (w/v) polyethylene glycol) for 48 h. The individual application of Rut and Si and especially the simultaneous treatment of Rut+Si improved the gas exchange parameters, chlorophyll content, photosystem II (PSII) activity, Rubisco enzyme activity, and the expression levels of magnesium chelatase and Rubisco genes, but decreased the expression of chlorophyllase gene under osmotic stress in comparison to osmotic stress alone. These findings suggest that exogenous Rut and Si can improve photosynthetic capacity in maize seedlings exposed to osmotic stress by increasing PSII activity and the expression of genes involved in photosynthesis and chlorophyll metabolism, as well as reducing chlorophyll degradation. The simultaneous treatment of Rut+Si may be useful in developing osmotic stress tolerance of plants. Keywords: chlorophyllase, chlorophyll fluorescence parameters, gas exchange parameters, gene expression, magnesium chelatase, Rubisco Introduction Water deficit, without a doubt one of the most critical stress situations, having a significant impact on crop growth and development and thus affecting crop productivity, and as a result, food security, is becoming a major concern around the world. It causes changes in fundamental plant morphophysiology and biochemistry and water loss, which reduces stomatal opening, chlorophyll content, and photo- synthesis rate, potentially reducing plant growth and pro- ductivity (Xiang et al. 2013, Iqbal et al. 2020). Photosynthesis is a multi-step process that turns light energy into chemical energy, including photosynthetic electron transport and the carbon reduction cycle (Berry et al. 2013). In many plants, water-deficit stress causes stomatal closure, a decrease in transpiration rate and carbon dioxide assimilation capacity, and reduces the activities of photosynthetic carbon reduction cycle enzymes, including 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) (Chaitanya et al. 2003), as well as the efficiency of photosynthetic electron transport and photosystem II (Xiang et al. 2013). In plants, Rubisco is composed of eight large subunits (LSUs) encoded by chloroplast rbcL gene and eight small subunits (SSUs) encoded by a family of nuclear rbcS genes (Lin et al. 2020). Water-deficit stress considerably reduces the amount of chlorophyll. Chlorophyll metabolism may significantly affect the assembly of photosynthetic machineries as well as * Corresponding author e-mail: asiyeszgn@outlook.com mailto:asiyeszgn@outlook.com RUTIN AND SILICON IMPROVE OSMOTIC STRESS TOLERANCE IN MAIZE ACTA BOT. CROAT. 84 (1), 2025 71 communication between chloroplasts and nuclei (Flexas et al. 2006, Tanaka and Tanaka 2006). The incorporation of Mg2+ into protoporphyrin IX, catalyzed by magnesium protoporphyrin IX-chelatase (Mg-Ch), a three-subunit (ChlI, ChlD, and ChlH) enzyme, is the first unique step in chlorophyll biosynthesis. Chlorophyllase (Chlase), which catalyzes ester bond hydrolysis to produce protochlorophyl- lide and phytol, is the first enzyme thought to be involved in chlorophyll degradation (Santos 2004). Water- deficit stress-induced increase in Chlase activity and gene expres- sion may result in a loss of chlorophyll accumulation (Banaś et al. 2011). Low Mg-Ch activity was also linked to a lack of chlorophyll. Under magnesium deficiency, reduced expres- sion levels of Mg-Ch and chlorophyll synthase, inhibited chlorophyll synthesis (Zhou et al. 2011). Silicon (Si) has attracted a lot of attention because it has been shown to improve plant tolerance to a wide range of abiotic stress factors (Coskun et al. 2019). Several studies have shown that Si can increase a plant's ability to withstand drought by either speeding up photosynthetic activity or slowing down transpiration. Si enhances crop growth, pro- duction, and quality by affecting photosynthetic activity, nitrogen uptake, and resilience to stress factors (Cooke and Leishman 2011, Liang et al. 2015, Ahanger et al. 2020). It was reported that Si mitigates low phosphorus stress by improv- ing photosynthetic capacity, antioxidant potential, and nutrient homeostasis (Zhang et al. 2019). Due to improved water retention, Si reduces drought stress in a variety of plants (Gong and Chen 2012, Liu et al. 2014, Khan et al. 2020, Desoky et al. 2020, Verma et al. 2020). Rutin (Rut), a flavonoid phenolic compound found in plants such as asparagus (Wang et al. 2003), has antioxidant properties and has been shown to reduce lipid peroxidation (Yang et al. 2008). In comparison to other antioxidant com- pounds, little research has been conducted to determine the effects of rutin on stressed plants (Ferdinando et al. 2012, Ismail et al. 2015, Singh et al. 2017). In recent studies, it was revealed that rutin improved salt stress tolerance in maize seedlings by modulating osmolyte accumulation and anti- oxidant capacity (Sezgin Muslu 2024). The roles of exogenous rutin in protecting plants from abiotic stress factors remain to be fully determined. More- over, reports showing the effects of rutin and silicon appli- cation alone and/or in combination on osmotic stress are limited. In a previous study, the combined application of rutin and silicon alleviated osmotic stress in maize seed- lings by triggering the accumulation of osmolytes and antioxidants’ defense mechanisms (Altansambar et al. 2024). However, no attempt has been undertaken to deter- mine how combined application of silicon and rutin affects photosynthesis in plants subjected to osmotic stress. There is also insufficient evidence to explain the effects of rutin and silicon applications on the activities and gene expres- sions of some key enzymes involved in chlorophyll metabo- lism and photosynthetic processes. Therefore, in the current study, it was hypothesized that (1) Rut and Si might sustain maize seedlings’ osmotic stress tolerance by improving photosynthetic capacity and chlorophyll metabolism, and (2) Rut might play an important role in the prominent effects of combined applications of Rut and Si in relieving osmotic stress. Our study will provide new information on the changes in photosynthetic capacity and chlorophyll metabo- lism of Rut and Si at the biochemical and molecular level. Material and methods Plant material, experimental conditions and treatments Zea mays L. seedlings were grown hydroponically in Hoa- gland’s solution (Hoagland and Arnon 1950) in a growth chamber (day/night temperatures of 25/22 °C, 60 ± 2% rela- tive humidity, and photosynthetic photon flux density of 400 µmol m-2 s-1 with a 16-h photoperiod). After 21 days of growth, rutin (Rut, 60 mg L-1) and silicon (Si, 1 mM) were applied to the seedlings. In our previous study, with concentrations of rutin (30, 60, and 90 mg L-1) and silicon (0,5, 1, and 2 mM), plants under osmotic stress (10% and 15% (w/v) polyethylene glycol) pretreated with 60 mg L-1 Rut and 1 mM Si were found to better maintain water status and had lower membrane damage (Altansambar et al. 2024). Si treatment was added using calcium silicate (CaSiO3) (Sigma Aldrich, USA). For Rut treatment, rutin hydrate was obtained from Sigma Aldrich, USA. After 24 h, seedlings were exposed to osmotic stress in- duced by adding 10 and 15% (w/v) polyethylene glycol (PEG6000) to Hoagland’s solution for 48 h. Seedlings treated with Hoagland’s solution without PEG were used as the con- trol group. Therefore, we designed nine different treatments: (1) mock; (2) moderate stress: 10% PEG; (3) severe stress: 15% PEG; (4) Rut application before moderate osmotic stress (Rut + 10% PEG); (5) Si application before moderate osmotic stress (Si + 10% PEG), (6) Rut and Si application before moderate osmotic stress (Rut + Si + 10% PEG); (7) Rut application be- fore severe osmotic stress (Rut + 15% PEG); (8) Si application before severe osmotic stress (Si + 15% PEG); (9) Rut and Si application before severe osmotic stress (Rut + Si + 15% PEG). The experimental plan was arranged in a completely ran- domized design with five replicates, providing a total of five containers with a total of five plants per treatment. After treatments, 24-day-old seedlings were harvested and subject- ed to biochemical and molecular analysis.  Determination of total chlorophyll content The total chlorophyll content was determined using Ar- non's method (1949). At 0-4 °C, fresh leaf samples (0.1 g) were extracted with 80% acetone. The extracts were centri- fuged for 10 min at 15000 g. A spectrophotometer was used to measure absorbance of the supernatant at 645 and 663 nm. The amount of total chlorophyll (mg chlorophyll per fresh tissue) was calculated using the following equation: mg chlorophyll g–1 FW = (20.2 × (A645) + 8.02× (A663) × V 1000 W× where, A is the absorbance at specific wavelengths; V is the final volume of chlorophyll extract in 80% acetone and W is the fresh weight. SEZGIN MUSLU A., ALTUNTAŞ C., ALTANSAMBAR N., DEMIRALAY M., KADIOĞLU A. 72 ACTA BOT. CROAT. 84 (1), 2025 Determination of gas exchange parameters The LI-6800 Portable Photosynthesis System (LI-COR Biosciences, Inc., Lincoln, NE, USA) was used to quantify the net photosynthetic rate (Pn), transpiration (E), stomatal conductance (gsw), and intercellular CO2 concentration (Ci) of Zea mays. Five plants were included in each group, and ten measurements were taken manually from the upper most fully developed third leaf of each plant at 5-second in- tervals. The following circumstances were used for the mea- surements: light intensity of 250 µmol m−2 s −1 (Demiralay 2022), block and leaf temperatures of 25 °C, and relative hu- midity of 60%. The integrated CO2 mixer in the portable photosynthetic system allowed for independent adjustment of the CO2 concentration. After the leaf was clamped, it was held for at least 30 min until the values of reference CO2 and sample CO2 reached equilibrium at 400 µmol mol–1 CO2. Determination of chlorophyll fluorescence parameters A Multi-Mode Chlorophyll Fluorometer (OS5p, Opti- Sciences, Inc., Hudson, NJ, USA) was used to measure chlorophyll fluorescence (CF). Three seedlings were chosen at random for each group and used to measure CF param- eters. The leaves were maintained in the dark for 20 min before the Chl fluorescence was measured. A modest red light (0.1 µmol m−2 s −1) was used to determine the minimal fluorescence (F0) after 20 min of acclimation to darkness using the dark leaf clip. The maximum fluorescence (Fm) was then measured using an 8 sec saturating pulse (8000 µmol m−2 s−1) (Nar et al. 2009). Then, in plants exposed to actinic light (500 μmol m–2 s–1) (Chen et al. 2019) Fm', the maximum fluorescence observed in a light-adapted state when all PSII reaction centers are closed and Fs, the fluores- cence level measured under steady-state photosynthesis conditions (Krause and Weis 1991), were also determined. The fluorescence parameters were calculated using the following formulas (van Kooten and Snel 1990): the maxi- mum quantum yield of PSII photochemistry, Fv/Fm = (Fm – F0)/Fm; photochemical quenching of variable chlorophyll fluorescence, qP = (F'm –Fs)/(F'm–F'0); and nonphoto chemical chlorophyll fluorescence quenching, NPQ = (Fm-F'm)/Fm. The effective quantum yield of ФPSII = (Fm'-Fs)/Fm') of Genty et al. (1989), and electron transfer rate (ETR) of Nar et al. (2009) were also determined. Determination of Rubisco activity Extraction buffer (50 mM Tris-HCl, 0.1% (w/v) mercap- toethanol, 12% (v/v) glycerol, 10 mM magnesium chloride (MgCl2), 1 mM EDTA, and 1% (w/v) polyvinylpolypyrrol- idone (PVPP-40) was used to prepare extracts from the samples (Parry et al. 1997). The protein contents of the extracts were measured as described by Bradford (1976), with bovine serum albumin as a standard. Rubisco activity was measured according to Sawada et al. (2003) method. The activity was measured at 25°C for 5 min by adding 100 µl of supernatant into 900 µl of assay buffer containing 50 mM of HEPES-KOH (pH 8.0), 1 mM of EDTA, 20 mM of MgCl2, 25 mM of dithioerythritol, 10 mM of NaHCO3, 5 mM of ATP, 0.15 mM of nicotinamide adenine dinucleo- tide (NADH), 5 mM of creatine phosphate, 0.6 mM of ribulose-1,5-bisphosphate (RuBP), 10 units of phospho- creatine kinase, 10 units of glyceraldehyde-3-phosphate dehydrogenase and 10 units of phosphoglycerate kinase. The enzymatic activity was determined via the decrease in absorbance at 340 nm using extinction coefficient of 6.22 mM−1 cm−1. Total Rubisco activity was expressed in units per milligram of proteins. Determination of chlorophyllase activity To determine the activity of chlorophyllase (Chlase), extraction was performed by a slight modification of the method described by Yang et al. (2004). The leaf samples (0.1 g) were homogenized with extraction buffer (5 mM potassium phosphate buffer (pH 7), 50 mM KCl, and 0.24% Triton X-100). After extraction, to remove chlorophyll, the samples were treated with cold acetone, followed by incuba- tion at 30 °C for 30 min in the dark. The samples were then centrifuged at 15000 g for 15 min. Chlase activity was determined by adapting the method of McFeeters et al. (1971). A standard reaction mixture was prepared contain- ing the reaction buffer (100 mM sodium phosphate buffer (pH 7), 0.24% Triton X-100) (2 mL), 1 μmol mL-1 chlorophyll a (0.2 mL) as a substrate and supernatant (0.3 mL), and the reaction was stopped using 0.5 mL of 10 mM KOH with in- cubation at 30 °C for 30 min. After that, 5 mL of hexane/ acetone (3:2, v/v) was added to 1 mL of reaction medium. The content of chlorophyllide a in the acetone phase was determined spectrophotometrically at 667 nm using extinc- tion coefficient of 74.9 mM−1 cm−1. Chlase activity was expressed as production of chlorophyllide a. Proteins were determined by the method of Bradford (1976). The activity was expressed in units per milligram of proteins. Determination of the expression levels of the rbcL, rbcS, Mg-ChlI, and Chlase genes Fresh samples (0.1 g) were used for total RNA isolation. After the samples were broken down in a tissue homogenizer, total RNA isolation was performed using a total RNA isola- tion kit (Favorgen FavorPrep Plant Total RNA Mini Kit) following the kit's protocol. A NanoDrop spectrophotom- eter was used to determine the quantity and purity of the RNA samples. The RNA samples were assessed for purity before being stored at -80 °C for cDNA extraction. From the isolated total RNA samples, 2000 ng of cDNA was obtained per group using the High Capacity cDNA Reverse Transcrip- tion Kit from Applied Biosystems USA. The synthesized cDNAs were stored at -20 °C until real time PCR analyses were performed. The resulting cDNAs were used to identify gene expres- sion using real-time PCR. 5 HOT FIREPol EvaGreen qPCR Supermix and the CFX Connect Real-Time PCR System were used for analysis. The real-time PCR protocol was modified from the Solis BioDyne instructions: 12 min at RUTIN AND SILICON IMPROVE OSMOTIC STRESS TOLERANCE IN MAIZE ACTA BOT. CROAT. 84 (1), 2025 73 95 °C, 45 cycles of 15 sec at 95 °C, 30 sec at 60 °C, 30 sec at 72 °C, and 0.5 °C increments from 65 °C to 95 °C for the melt curve. Each biological repeat was examined as three technical replications, with the average technical error ac- cepted as 0.5 (± 1) Cq values. Furthermore, gene-specific primers were used to investigate the levels of expression of the genes (Tab. 1). The data obtained as a result of the anal- ysis were normalized to the β-actin reference gene and pre- sented as relative gene expression using the 2-ΔΔCT method, following the protocol outlined by Bookout and Mangels- dorf (2003). Statistical analysis All experiments were performed five times with five bi- ological replicates in total. The Shapiro-Wilk normality test was used to test the normal distribution of variables. A two- way ANOVA and Tukey's multiple range test was performed at the 0.05 (5%) level using IBM SPSS 23.0 Statistics Package (SPPS Corp., Chicago, IL, USA). Results Total chlorophyll content According to the results of two-way ANOVA (Tab. 2), the total chlorophyll content was significantly (P < 0.001) affected by osmotic stress conditions (OS) and treatments with Rut, Si or both (T). Moreover, the interaction between OS and T factors was also highly significant for the total chlorophyll content, suggesting that the effect of one factor depends on the level of the other factor. As shown in Fig. 1, the differences in the concentration of the osmotic stress (10 or 15% PEG) had a significant (P < 0.05) effect on the total chlorophyll content of maize leaves. The stressed seedlings showed 47.25 and 162.02% lower total chlorophyll levels, respectively, in comparison with the Tab. 1. The sequences of specific primers of genes, used for qRT- PCR analysis. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), ribulose-1,5-bisphosphate carboxylase/oxy- genase small subunit (rbcS), magnesium chelatase subunit I (Mg- ChlI), chlorophyllase (Chlase). Primers Sequences Actin (ACT), forward Actin (ACT), reverse 5'-GAAGATCACCCTGTGCTGCT-3' 5'-ACCAGTTGTTCGCCCACTAG-3' rbcL, forward rbcL, reverse 5'-AAAGCCTTACGCGCTCTACGT-3' 5'-CGGACCTTGGAAAGTTTTTGAA-3' rbcS, forward rbcS, reverse 5'-ATGTGGAAGCTGCCCATGTT-3' 5'-GCCTCCTGCAGCTCCTTGTA-3' Mg-ChlI, forward Mg-ChlI, reverse 5'-TGTATGCTGCTCGAGTTGCA-3' 5'-CTTGCTGCTGATCCTGTGGA-3' Chlase, forward Chlase, reverse 5'-ACACCACCGAGGAGATCAAC -3' 5'-GTCCAGCTCGTCGTAGAAGG -3' Tab. 2. Results of two-way ANOVA (P-Values, F ratios) for the independent osmotic stress conditions (OS), treatment (T) and osmotic stress × treatment interactions (OS x T). Pn – net photosynthesis, E – transpiration, gsw – stomatal conductance, Ci – intercellular CO2 concentration, Fv/Fm – maximum quantum yield of PSII photochemistry, ФPSII – the effective quantum yield of PSII, qP – photo chemical quenching, NPQ – non-photochemical quenching, rbcL – Rubisco large subunit, rbcS – Rubisco small subunit, Mg-ChlI – magnesium chelatase subunit I, Chlase – chlorophyllase. * indicate significant difference at P < 0.05; ** indicate significant difference at P < 0.01; *** indicate significant difference at P < 0.001. Varience sources of two – way ANOVA Experimental parameters OS (3 levels) T (3 levels) OS × T F P-Value F P-Value F P-Value Total chlorophyll content 2218.166*** <0.001 356.934*** <0.001 41.695*** <0.001 Pn 716.197*** <0.001 81.093*** <0.001 8.901*** <0.001 E 620.588*** <0.001 32.367*** <0.001 8.276** 0.001 gsw 98.33*** <0.001 114.129*** <0.001 91.277*** <0.001 Ci 127.886*** <0.001 78.912*** <0.001 10.515*** <0.001 Fv/Fm 205.39*** <0.001 101.56*** <0.001 61.83*** <0.001 ФPSII 9.86** 0.006 58.69*** <0.001 8.77*** <0.001 ETR 26.45** 0.006 32.99*** <0.001 19.88*** <0.001 qP 45.144*** <0.001 63.64*** <0.001 5.47** 0.007 NPQ 36.78** 0.007 47.66*** <0.001 27.184*** <0.001 Rubisco activity 1132.542*** <0.001 226.229*** <0.001 33.179*** <0.001 Chlase activity 3201.211*** <0.001 97.006*** <0.001 26.456*** <0.001 rbcL gene expression 12358.673*** <0.001 1712.888*** <0.001 113.346*** <0.001 rbcS gene expression 10641.439*** <0.001 613.537*** <0.001 65.199*** <0.001 Mg-ChlI gene expression 269.346*** <0.001 7.005** 0.003 0.399 0.755 Chlase gene expression 1652.202*** <0.001 35.847*** <0.001 13.655*** <0.001 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11018582/#CR6 SEZGIN MUSLU A., ALTUNTAŞ C., ALTANSAMBAR N., DEMIRALAY M., KADIOĞLU A. 74 ACTA BOT. CROAT. 84 (1), 2025 control group. However, the total chlorophyll content was significantly enhanced (P < 0.05) by the application of Rut or Si alone and in combination under both osmotic stress treatments, especially moderate stress. Additionally, compared to Rut or Si alone, the combination of Rut and Si was found to further increase the total chlorophyll content (Fig. 1). Gas exchange parameters According to results of two-way ANOVA (Tab. 2), both OS and T had highly significant effects (P < 0.001) on net photosynthesis (Pn) and the transpiration rate (E). Similar- ly, OS and T both significantly affected (P < 0.001) stomatal conductance (gsw) and intercellular CO2 concentration (Ci). The interaction between OS × T was also significant for Pn, E, gsw and Ci, though the F-value for the interaction was small compared to the OS and T factors, indicating a less strong but still significant interaction (P ≤ 0.001). The Pn, E, gsw and Ci values were significantly decreased (P < 0.05) with 10% and 15% PEG treatments compared to the non-stress group. The decrease in these values was high- est in the 15% PEG treated group. However, exogenous Rut, Si and Rut+Si treatments significantly increased (P < 0.05) Pn, E, gsw, and Ci under both osmotic stress conditions. However, the highest values of Pn and E were observed in Rut, Si and Rut+Si treatments under 10% PEG. Furthermore, the highest value of gsw was observed in Si and Rut+Si treatments and the highest value of Ci were detected in Rut+Si treatment under 10% PEG, which were significantly (P < 0.05) different from 15% PEG conditions (Tab. 3). Chlorophyll fluorescence parameters Two-way ANOVA analysis (Tab. 2) showed that the con- tent of all chlorophyll f luorescence parameters (Fv/Fm, ФPSII, ETR, qP, and NPQ) was significantly different in re- spect of osmotic stress conditions (OS), treatments (T) and their interactions (OS × T). As shown in Tab. 4, the values of Fv/Fm, ФPSII, ETR, qP were significantly (P < 0.05) reduced under both 10% and 15% PEG treatments compared to the non-stressed group. In contrast, values of NPQ were significantly (P < 0.05) increased under both osmotic stresses compared to the non- stress group. Application of Rut or Si, and especially their combination significantly increased values of Fv/Fm, ФPSII and qP under both osmotic stresses, although values were still not as high as in control. In contrast, NPQ values were lower after application of Rut or Si, and especially of a com- bination of the two, than under osmotic stress alone. Rubisco activity According to results of two-way ANOVA (Tab. 2) the Rubisco activity was significantly affected (P < 0.001) by OS and T as well as their interaction (OS × T) indicating strong effects. Compared to non-stressed plants, osmotic stress signif- icantly reduced the activity of Rubisco, with severe stress Fig. 1. Effect of the treatments (T) with rutin (Rut), silicon (Si), and their combination (Rut+Si) on total chlorophyll content of maize seedlings under osmotic stress (OS) conditions (10% or 15% poly- ethylene glycol - PEG). Vertical bars represent standard deviations of the means, N = 5. Data were subjected to two-way ANOVA while Tukey's multiple range test was used for determinating the differ- ences among means for interaction OS × T. Values marked with the different letters denote significant difference (P < 0.05). Tab. 3. Effect of the treatments (T) with rutin (Rut), silicon (Si), and their combination (Rut+Si) on net photosynthesis (Pn), transpi- ration (E), stomatal conductance (gs) and intercellular CO2 concentration (Ci) of maize seedlings under osmotic stress (OS) condi- tions (10% or 15% polyethylene glycol - PEG). All values are presented as means ± standard deviation, N = 5. Data were subjected to a two-way ANOVA and Tukey's multiple range test was used to determine the differences among means for interaction OS × T. Val- ues marked with the different letters denote significant difference (P < 0.05). Applications Net photosynthesis (µmol CO2 m−2 s−1) Transpiration (mmol H2O m−2 s−1) Stomatal conductance (mmol H2O m−2 s−1) Intercellular CO2 concentration (µmol mol-1) Control 19.53 ± 0.18a 0.0013 ± 0.0008a 0.092 ± 0.001a 47.52 ± 1.23a 10 % PEG 11.00 ± 0.36c 0.0008 ± 0.00005c 0.041 ± 0.002f 38.06 ± 1.21e Rut + 10 % PEG 12.58 ± 0.12b 0.0009 ± 0.00001b 0.046 ± 0.0001d 43.45 ± 0.15c Si + 10% PEG 12.56 ± 0.36b 0.0008 ± 0.00002bc 0.06 ± 0.001b 43.07 ± 0.2c Rut + Si + 10% PEG 12.72 ± 0.23b 0.0009 ± 0.00002b 0.059 ± 0.001b 45.24 ± 1.23b 15% PEG 8.46 ± 0.08f 0.0003 ± 0.00004h 0.036 ± 0.0021g 33.46 ± 1.22g Rut + 15% PEG 9.2 ± 0.23e 0.0006 ± 0.00002e 0.057 ± 0.001c 35.73 ± 0.21f Si + 15% PEG 10.36 ± 0.14cd 0.0004 ± 0.00001g 0.045 ± 0.0002e 41.23 ± 1.12d Rut + Si + 15% PEG 10.58 ± 0.23c 0.0005 ± 0.00001h 0.044 ± 0.001e 41.93 ± 0.82d RUTIN AND SILICON IMPROVE OSMOTIC STRESS TOLERANCE IN MAIZE ACTA BOT. CROAT. 84 (1), 2025 75 (15% PEG) having a more prominent effect. Exogenous ap- plications of Rut, Si, and Rut+Si significantly (P < 0.05) in- creased Rubisco activity under both osmotic stress condi- tions, especially moderate stress (10% PEG), compared to the same level of stress alone. Moreover, compared to Rut or Si alone, the combination of Rut and Si was found to fur- ther increase Rubisco activity (Fig. 2). Chlorophyllase activity Two-way ANOVA analysis showed that the chlorophyl- lase activity was significantly affected by OS and T factors as well as OS × T (P < 0.001) indicating strong effects (Tab. 2). Both levels of osmotic stress, but especially severe stress (15% PEG) significantly (P < 0.05) increased chlorophyllase activity, compared to the control. Exogenous treatments with Rut and Si decreased the chlorophyllase activity under osmotic stress, compared to the seedlings exposed to the same level of osmotic stress alone but more efficiently at moderate stress. Also, compared to Si alone, seedlings treat- ed with Rut+Si exhibited an even more significant (P < 0.05) decrease in chlorophyllase activity under stress conditions (Fig. 2). The expression levels of rbcL, rbcS, Mg-ChlI and Chlase genes According to results of two-way ANOVA, the expression levels of the rbcL and rbcS, endcoding large and small Rubis- co subunits, respectively, were significantly different (P < 0.001) in respect of osmotic stress conditions (OS), treat- ments (T) and their interactions (OS × T) (Tab. 2). As com- pared to the control, the expression levels of rbcL and rbcS were significantly down-regulated by 10 and 15% PEG. Ex- ogenous applications of Rut, Si, and especially Rut+Si sig- nificantly up-regulated the expression levels of rbcL and rbcS under moderate and severe osmotic stress as compared to the same level of stress alone (Fig. 3). Two-way ANOVA analysis showed that the effect of OS factor on the expression levels of the Mg-ChlI was signifi- cant with P < 0.001 as well as the effects of T with P = 0.003 (Tab. 2). The expression levels of the Mg-ChlI in maize seed- lings treated with 10 and 15% PEG were significantly lower than in the control. In addition, under moderate and severe osmotic stress conditions, the expression levels of Mg-ChlI in seedlings pretreated with Rut, Si, and Rut+Si were higher than under moderate and severe osmotic stress (Fig. 3). According to results of two-way ANOVA, the expression level Chlase was significantly different (P < 0.001) in respect of osmotic stress conditions (OS), treatments (T) and their interactions (OS × T) (Tab. 2). Both levels of osmotic stress caused a significant increase in the expression level of Tab. 4. Effect of the treatments (T) with rutin (Rut), silicon (Si), and their combination (Rut+Si) on maximum efficiency of PSII (Fv/Fm), the effective quantum yield of PSII (ФPSII), photoche mical quenching (qP), non-photochemical quenching (NPQ) of maize seedlings under osmotic stress conditions (OS) (10% or 15% polyethylene glycol – PEG). All values are presented as means ± standard deviation, N = 5. Data were subjected to a two-way ANOVA and Tukey's multiple range test was used to determine the differences among means for interaction OS × T. Values marked with the different letters denote significant difference (P < 0.05). Applications Fv/Fm ФPSII ETR qP NPQ Control 0.754 ± 0.004a 0.598 ± 0.037a 24.03 ± 1.01a 0.852 ± 0.01a 0.145 ± 0.015e 10 % PEG 0.455 ± 0.01e 0.32 ± 0.03e 13.55 ± 1.6g 0.710 ± 0.02f 0.345 ± 0.008a Rut + 10 % PEG 0.536 ± 0.002d 0.477 ± 0.006b 19.3 ± 0.9bc 0.822 ± 0.01c 0.285 ± 0.007bc Si + 10% PEG 0.672 ± 0.02c 0.425 ± 0.002c 17.8 ± 0.1de 0.821 ± 0.01c 0.225 ± 0.006d Rut + Si + 10% PEG 0.71 ± 0.002b 0.476 ± 0.004b 20.4 ± 0.1b 0.842 ± 0.01b 0.285 ± 0.01bc 15% PEG 0.452 ± 0.01e 0.404 ± 0.002d 16.7 ± 0.4f 0.713 ± 0.007f 0.33 ± 0.009a Rut + 15% PEG 0.529 ± 0.03d 0.469 ± 0.006b 18.1 ± 00.4cd 0.759 ± 0.007e 0.243 ± 0.01d Si + 15% PEG 0.471 ± 0.02e 0.431 ± 0.005c 17.9 ± 0.1de 0.802 ± 0.04d 0.303 ± 0.01b Rut + Si + 15% PEG 0.526 ± 0.01d 0.482 ± 0.01b 17.05 ± 0.35de 0.817 ± 0.004c 0.265 ± 0.02c Fig. 2. Effect of the treatments (T) with rutin (Rut), silicon (Si), and their combination (Rut+Si) on Rubisco activity (A) and chlo- rophyllase (Chlase) activity (B) of maize seedlings under osmotic stress (OS) conditions (10% or 15% polyethylene glycol – PEG). Vertical bars represent standard deviations of the means, N = 5. Data were subjected to a two-way ANOVA and Tukey's multiple range test was used to determine the differences among means for interaction OS × T. Values marked with the different letters de- note significant differences (P < 0.05). SEZGIN MUSLU A., ALTUNTAŞ C., ALTANSAMBAR N., DEMIRALAY M., KADIOĞLU A. 76 ACTA BOT. CROAT. 84 (1), 2025 Chlase, compared to the control. Under moderate and se- vere osmotic stress, exogenous application of Rut, Si, and especially Rut+Si significantly down-regulated the expres- sion level of Chlase (Fig. 3). Discussion Chlorophyll is required to convert light energy to chem- ical energy, and its depletion limits the photosynthetic pro- cess in plants (Kalaji et al. 2017). In current study, two-way ANOVA analysis of variance revealed that both indepen- dent variables (OS and T) had significant effects on the total chlorophyll content. Total chlorophyll content decreased in seedlings exposed to osmotic stress, while Rut, Si, and es- pecially Rut+Si applications reduced the negative effects of osmotic stress on chlorophyll content. Consistently with our findings, Si- treated wheat and Eruca sativa L. plants showed a significant increase in chlorophyll content (Maghsoudi et al. 2015, Bukhari et al. 2021). Moreover, Si application improved the chlorophyll content in Fagopyrum esculentum M. plants under aluminium stress (Dar et al. 2022). In another study, total content of chlorophyll in Oryza sativa L., leaves increased during treatment with rutin (Singh et al. 2017). The chlorophyll content could decline due to in- creased Chlase activity under abiotic stressors (Dawood et al. 2014). Increased Chlase activity has been correlated with chlorophyll degradation in plants under osmotic stress con- ditions (Altuntaş et al. 2020), which is agreement with our results in maize seedlings where osmotic stress conditions induced significant enhancement of Chlase activity. How- ever, Chlase activity significantly decreased in seedlings pretreated with Rut, Si, and especially Rut+Si under osmot- ic stress thus contributing to the higher content of chloro- phyll content observed. In mustard seedlings exposed to salinity and drought stress, it was found that Chlase activ- ity and chlorophyll degradation were decreased by Si (Alam- ri et al. 2020). Moreover, we also observed a correlation of Chlase activity with gene expression of Chlase i.e. Chlase activity decreased and the Chlase gene expression was con- siderably down-regulated in seeedlings under osmotic stress pretreated with Rut, Si, and Rut+Si. Therefore, we can hy- pothesize that Rut, Si and Rut+Si may have adjusted the transcript levels of genes encoding the Chlase that degrades chlorophyll, which can prevent the bleaching of chlorophyll and preserve photosynthetic activity. In the production of chlorophyll, a crucial regulation and enzymatic step is cat- alyzed by the heterotrimeric enzyme complex known as Mg-Chl (Rissler et al. 2002). Overexpression of the Mg-che- lator H subunit in guard cells has been shown to increase drought tolerance in Arabidopsis thaliana (Tsuzuki et al. 2013). Another study also showed that Si application upreg- Fig. 3. Effect of the treatments (T) with rutin (Rut), silicon (Si), and their combination (Rut+Si) on the expression levels of of the rbcL (A) and rbcS (B), Mg-ChlI (C) and Chlase (D) genes of maize seedlings under osmotic stress (OS) conditions (10% or 15% polyethylene glycol - PEG). Vertical bars represent standard deviations of the means, N = 5. Data were subjected to two-way ANOVA and Tukey's multiple range test was used for determinating the differences among means for interaction OS × T. Values marked with the different letters denote significant differences (P < 0.05). RUTIN AND SILICON IMPROVE OSMOTIC STRESS TOLERANCE IN MAIZE ACTA BOT. CROAT. 84 (1), 2025 77 ulated the expression of genes encoding enzymes in chloro- phyll synthesis, Mg-Chl and chlorophyll oxygenase in cu- cumber seedlings under excess nitrate stress. In our study, the expression of the Mg-ChlI gene was significantly upreg- ulated in Rut, Si, and Rut+Si applied maize seedlings under osmotic stress. As a result, we can conclude that increased Mg-ChlI gene expression and decreased Chlase expression can confer stress tolerance to maize seedlings. Damage to photosynthesis can reduce chlorophyll con- tent, causing the chloroplast bilayer membrane to rupture and disrupting coordination between the two photosys- tems, ultimately decreasing the photosynthesis rate (Lawlor and Cornic 2002, Chaves and Oliveira 2004). Lower Pn could be due to the fact that osmotic stress can lead to a de- crease in gsw, Ci, and E as a result of stomatal closure (Chaves et al. 2003). Similarly, in our study, osmotic stress conditions (10% and 15% PEG) significantly decreased val- ues of gas exchange parameters. The decreases were found to be higher at 15% PEG compared to 10% PEG application because severe drought stress can lead to structural and bio- chemical impairments in light-dependent reactions and carboxylation processes (Ghotbi-Ravandi et al. 2014). Many researchers have reported that exogenous applications of Si can enhance photosynthesis performance under osmotic stress (Maghsoudi et al. 2016, Li et al. 2018, Li et al. 2022, Mavondo-She et al. 2024). Like those studies, our results showed that Rut, Si and Rut+Si significantly increased all of the gas exchange parameters in maize seedlings. The results suggest that exogenous treatments of Rut, Si and Rut+Si can mitigate the adverse effects of osmotic stress on gas ex- change due to more efficient light use and improved chlo- rophyll metabolism through the regulation of the Mg-ChlI and Chlase gene. Hence, those applications can maintain higher chlorophyll content, preserve the photosynthetic ma- chinery and enhance overall photosynthetic performance. Osmotic stress can have a significant impact on plant photosynthesis resulting in changed chlorophyll fluores- cence (Chen et al. 2021). However, exogenous applications of various compounds have been shown to mitigate the neg- ative effects of osmotic stress and enhance chlorophyll fluo- rescence parameters due to improved photosynthetic per- formance and overall plant resilience (Hayat and Ahmad 2007, Ashraf and Foolad 2007, Ahmed et al. 2019, Hussain et al. 2023). Many researchers have reported that chloro- phyll fluorescence parameters are valuable for assessing PSII activity and functioning of the photosynthetic apparatus in plants under osmotic stress. The maximum quantum effi- ciency of PSII is linked to photosynthetic efficiency in leaves (Baker and Rosenqvist 2004, Baker 2008) and the Fv/Fm ra- tio serves as an indicator of photoinhibition or stress dam- age. In the present study, 10% and 15% PEG reduced Fv/Fm, ΦPSII, and qP values, indicating a potential decline in pho- tosynthetic activity. The reduction in Fv/Fm is likely linked to decreased activity of PSII reaction centers and/or de- creased energy transfer efficiency within reaction centers as well as reduced gsw and CO2 availability, suggesting photo- inhibition. An increase in NPQ indicating enhanced energy dissipation, as a mechanism to avoid photodamage, ob- served in stressed plants confirms these results. Addition- ally, the reductions in ΦPSII and qP may be associated with the changes in Fv/Fm (Maxwell and Johnson 2000). These declines are probably due to altered chlorophyll content, leading to the conclusion that osmotic stress inhibits PSII activity as previously established for maize (Altuntaş et al. 2020). However, applications of Rut, Si, and the combina- tion of Rut+Si mitigated the impairment of photosynthetic parameters, as evidenced by enhanced electron transport under osmotic stress, and improved PSII efficiency. Abiotic stressors can also reduce photosynthesis by re- ducing the activity of Rubisco which is the enzyme that fixes CO2 and catalyzes photo-respiratory carbon oxidation (Ab- dulbaki et al. 2022). We determined that osmotic stress in maize seedlings negatively affected the activity of Rubisco while application of Rut, Si, and Rut+Si alleviated this neg- ative effect. Especially effective was the combination of Rut and Si under moderate osmotic stress. Similarly, Si applica- tion increased Rubisco activity in cucumber seedlings un- der cinnamic-acid-induced stress (Lyu et al. 2022). Rubisco biosynthesis requires a high number of chaperones and in- volves eight large (Rubisco LSU) and eight small subunits (Rubisco SSU) (Lin et al. 2020). The abundances of tran- scripts of rbcL encoding LSU and rbcS encoding SSU could enhance photosynthetic properties, photosynthetic efficien- cy or capacity (Chen et al. 2015). In the current study, it was found that the expression levels of rbcL and rbcS were down- regulated in the maize seedlings under osmotic stress con- ditions which correlates with decreased activity of Rubisco. Rut, Si, and Rut+Si applications significantly upregulated the expression of rbcL and rbcS genes in seedlings exposed to osmotic stress, consequently also increasing Rubisco ac- tivity. Consistently with our study, Si application signifi- cantly upregulated the expression of genes encoding Rubis- co subunits in tomato seedlings under low-calcium stress (Li et al. 2022). No study has been found in the literature on the way in which Rut application affects the activities of photosynthetic enzymes and the expression of genes encod- ing these enzymes. In our study, it was revealed that the al- leviating effect of Si was further stimulated by Rut. We found that increased expression of genes involved in the regulation of the activity of major photosynthetic enzymes and genes encoding Rubisco following combined applica- tion of Rut+Si could be one of the primary causes of en- hanced photosynthesis under osmotic stress, suggesting that the synergistic effect of Rut and Si may positively mod- ulate CO2 assimilation. In conclusion, the results of this study show that the ap- plication of Rut, Si, or Rut+Si to maize plants under osmot- ic stress can suppress chlorophyll degradation and stimulate chlorophyll synthesis and Rubisco activity, thereby preserv- ing photosynthetic activity. Rut, Si, and especially Rut+Si applications have the potential to maintain photosynthesis or alleviate photosynthetic damage in plants under osmot- ic stress. Our research was aimed at a better understanding of the key mechanisms underlying Rut+Si-mediated osmot- SEZGIN MUSLU A., ALTUNTAŞ C., ALTANSAMBAR N., DEMIRALAY M., KADIOĞLU A. 78 ACTA BOT. 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