60 ACTA BOT. CROAT. 82 (1), 2023 Acta Bot. Croat. 82 (1), 60–70, 2023 CODEN: ABCRA 25 DOI: 10.37427/botcro-2022-029 ISSN 0365-0588 eISSN 1847-8476 Acer velutinum Bioss. (velvet maple) seedlings are more tolerant to water deficit than Alnus subcordata C.A. Mey. (Caucasian alder) seedlings Mokarram Ravanbakhsh1, Babak Babakhani1*, Mahmood Ghasemnezhad1,2, Fariba Serpooshan1, Mohamad Hassan Biglouie1,3 1 Department of Biology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran. 2 Department of Horticultural Sciences, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. 3 Department of Water Engineering, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. Abstract – Drought stress is a major environmental factor limiting plant growth. Selection of drought-tolerant plants is of critical importance in vegetation restoration and forestation programs. Alnus subcordata and Acer velutinum are two valuable, dominant, and endemic species in the Hyrcanian forests. There are fast-growing species and significant diffuse-porous hardwood in afforestation and reforestation. One-year old seedlings of both species were exposed to four water shortage treatments (100, 75, 50 and 25% of field capacity (FC) chosen as control, mild, moderate, and se- vere) for 12 weeks. Thereafter, their morphological characteristics such as height and basal area, total and organ bio- mass (root, stem, and leaf ), leaf area (LA), specific leaf area (SLA), leaf area ratio (LAR), as well as physiological and biochemical characteristics such as relative water content (RWC), content of chlorophyll, free proline and malondial- dehyde (MDA), and superoxide dismutase (SOD) and peroxidase (POD) activity were measured. The results showed that when exposed to reduced water availability, plant height, basal diameter, total and organ biomass, LA, LAR, RWC and chlorophyll content decreased, but their proline concentration, MDA content, SOD, and POD activity increased in both species. The root to shoot ratio (R/S) and root mass ratio (RMR) increased at 50 and 25% FC treatments in A. subcordata, whereas no significant difference was found in A. velutinum under drought treatments. SLA increased significantly at 50% FC in A. velutinum and decreased in A. subcordata under drought treatments compared to control treatment. A. velutinum showed more proline content, RWC, POD, and lower increase in MDA content than A. subcordata under moderate treatment. Therefore, A. velutinum appears to possess a better mechanism to cope with drought stress. The drought tolerance of A. velutinum may enhance its potential for climatic adaptations under drier condi- tions with the ongoing climatic change. Keywords: Alnus subcordata, Acer velutinum, antioxidant enzymes, biomass, growth, water deficit Introduction The impacts of climate change on vegetation will appear as a combination of stress factors, including high tempera- tures, reduction of rainfall, and alterations in wildfire re- gimes. The principal aspect of global climate change, the frequency, and intensity of drought stress will increase in the future (Wu et al. 2017). Drought can damage afforesta- tion and reforestation programs because seedlings are more prone to drought than mature trees. Drought-tolerant spe- cies should be considered so as to contribute to sustainable forest ecosystems (Bhusal et al. 2020). Selection of drought- tolerance plants has a critical role in vegetation restoration and silvicultural strategies (Khaleghi et al. 2019). Drought affects various aspects of the plant; the roots are the first part to be affected in the face of drought. The chemical signals (abscisic acid) produced in the roots along with decreased leaf turgor and atmospheric vapor pressure can reduce stomatal conductance. The limitation associated with increased stomatal resistance (under mild to moderate water deficit), is known as a stomatal limitation. Limitation due to non-stomatal disturbance under severe drought stress (non-stomatal limitation) can be induced by the lim- ited diffusion of CO2 from the intercellular spaces to the chloroplasts or by metabolic factors such as a decrease in Rubisco activity, disturbances in the regeneration of ribu- lose diphosphate and reactive oxygen species (ROS) produc- tion from the excess excitation energy. Low growth can be * Corresponding author e-mail: babakhani_babak@yahoo.com mailto:babakhani_babak@yahoo.com RESPONSES TO WATER DEFICIT IN ACER VELUTINUM AND ALNUS SUBCORDATA ACTA BOT. CROAT. 82 (1), 2023 61 due to suppression of the photosynthetic process that even- tually reduces biomass (Du et al. 2010, Dulai et al. 2014). Chlorophyll content can directly influence photosynthetic potential and primary production. Reduction in chlorophyll content under water deficit has been regarded as a typical feature of oxidative stress (Liu et al. 2019). Photosynthetic pigment stabilization under stress conditions increases re- sistance to drought stress (Ge et al. 2014). Decreased chlo- rophyll content under water deficit was reported in such tree species as Fagus sylvatica (Gallé and Feller, 2007), Quercus variabilis (Wu et al. 2013), Alnus cremastogyne (Tariq et al. 2018), and Acer davidii (Guo et al. 2019), while no change in chlorophyll content was found in Melia azedarach (Dias et al. 2014). Relative water content (RWC) is a key indicator of degree of hydration and vital for optimal physiological functions and growth processes. RWC in woody and shrubby species reached 50 to 40% and seldom was it as low as 30 to 20% under severe water stress, which eventually causes leaf se- nescence (Wu et al. 2013). Relatively high RWC mainte- nance in water shortage is an indicator of drought tolerance (Ying et al. 2015, Toscano et al. 2016). Quercus variabilis seedlings could maintain sufficient RWC and slight growth at 40% field capacity (FC) (Wu et al. 2013). RWC of Alnus cremastogyne signifcantly decreased by 32.6% under drought (Tariq et al. 2018). Decrease of RWC in response to moder- ate (50% FC) and severe (30% FC) drought treatment in Maclura pomifera has been reported (Khaleghi et al. 2019). Resistance to biotic and abiotic stress in plants increases by the accumulation of significant amounts of free proline, soluble sugars (sucrose, glucose and fructose), and soluble proteins (maturation proteins). These compatible solutes are able to maintain the concentration of cell sap and prevent the loss of water in plasma (Mohammadkhani and Heidari 2008, Farooq et al. 2009, Guo et al. 2018). Proline functions not only as an osmolyte, but also as an antioxidant, thus helping ROS detoxification by membrane integrity protec- tion and enzyme/protein stabilization (Ghaffari et al. 2019, Khaleghi et al. 2019). The intercellular concentration of malondialdehyde (MDA), a breakdown product of lipid peroxidation, has been measured as an indicator of oxidative damage (Ge et al. 2014, Abid et al. 2018). To scavenge ROS, plants maintain an efficient antioxidant defense system including non-en- zymatic antioxidants and antioxidant enzymes (Khaleghi et al. 2019). Peroxidase (POD) and superoxide dismutase (SOD) disintegrate ROS, and therefore, protect plants from drought stress (Geng et al. 2019). SOD catalyzes the conver- sion of superoxide radical (O2•−) to molecular oxgen (O2) and hydrogen peroxide (H2O2). This H2O2 is detoxified to O2 and H2O through the activities of catalase (CAT) and POD as well as the ascorbate-glutathione (AsA-GSH) cycle (Wang et al. 2012, Abid et al. 2018). Based on climate modeling, the air temperature in Iran will rise by 2.7 °C up to 2050, which will increase the water needs of plants (Attarod et al. 2017). The Caspian forest cli- mate has become warmer and the vegetation growth trend has been upwards of about one hundred meters in the last half-century (Taleshi et al. 2018). Reforestation by Alnus subcordata C.A. Mey. (Caucasian alder), and Acer velutinum Bioss. (Persian or velvet maple) to increase production ca- pacity reduced the pressure of wood exploitation on Hyrca- nian forests (Abdolahi et al. 2017). A. subcordata and A. velutinum are the most valuable endemic species and are indigenous to the Hyrcanian province in the Euro-Siberian region. Due to their importance, numerous studies have do- ne on the quantitative and qualitative characteristics of the species, mechanical properties of wood and nutrient ele- ments (Naghdi et al. 2016, Naji et al. 2016, Tavankar et al. 2017, Ghorbani et al. 2018, Jourgholami et al. 2020). According to a few recent studies, nano priming tech- nique increased drought tolerance of A. subcordata seeds (Rahimi et al. 2016). A. subcordata as an urban tree showed limited tolerance to water deficit by determination of mid- day leaf water potential (ΨL) and stomatal conductance (gs) (Sjöman et al. 2021). However, their response to drought and the mechanism of these two species in artificial cultivation are still unclear and poorly understood. Therefore, the ob- jectives of the present study were (i) to evaluate the effects of drought stress on A. subcordata and A. velutinum seed- lings which are dominant species in Hyrcanian forests and have a high commercial value in wood industries, to dis- cover their capacity to handle water deficit in the initial veg- etative growth period by morphological, physiological and biochemical responses; and (ii) to determine these two spe- cies’ different adaptive responses to drought stress. Materials and methods Plant material and drought treatments The experiment was carried out in a greenhouse at Uni- versity of Guilan, Iran (37°15́ N, 49°36́ E). The average an- nual temperature was 15.9 °C and cumulative precipitation 1329.1 mm (Allahyari et al. 2016). One-year-old A. subcordata C.A. Mey. and A. velutinum Boiss. seedlings were obtained from a local nursery called Pilambara (37°35′ N, 49°05′ E) in Resvanshahr, Guilan Province, Iran. The seedlings were transplanted to 9 L plastic pots filled with homogenized topsoil. The plants were grown in a naturally lit greenhouse (tem perature range: 18–28 °C; relative humidity range 73–94%) in a semi-controlled environment (only sheltered from rainfall) from July 10 to October 10, 2019. The green- house was well ventilated by plastic side films being rolled around it (Guo et al. 2013). Drought treatments were performed three months after the planting of the seedlings (an acclimatization period, and when plants had produced fully expanded leaves) (Guo et al. 2013, Medeiros et al. 2013, Meng et al. 2013). A random- ized complete design with two factors (two species and four watering regimes) was employed with three replications for four water shortage treatments (100, 75, 50 and 25% of field RAVANBAKHSH M., BABAKHANI B., GHASEMNEZHAD M., SERPOOSHAN F., BIGLOUIE M. H. 62 ACTA BOT. CROAT. 82 (1), 2023 capacity performed as a control, mild, moderate, and severe, respectively). Using a scale with a capacity of 40 kg, transpi- ration water loss was measured gravimetrically by weighing all pots and re-watering with tap water every two days. The water added to each pot during the experimental period was 27, 18, 10.8 and 6.75 L for control, mild, moderate, and se- vere treatments respectively for seedlings of A. subcordata and 22.5, 15, 9, and 6 L for seedlings of A. velutinum. The evaluation was performed after three months at the end of the experiment. Growth parameters Seedling height (cm) was measured from the soil surface to the terminal bud of the main stem using a measuring tape; also, the basal diameter (mm) was measured at the ground line by electronic calipers. Plant height, basal diam- eter and biomass (total dry mass) were recorded at the end of the experiments. Three seedlings were harvested ran- domly from each treatment. The leaves, stems, and roots were cut and dried in an oven at 65 °C for 48 hours to cal- culate root, stem, and leaf biomass (the average weight of three samples per treatment). Biomass contribution includ- ing leaf mass ratio (LMR), stem mass ratio (SMR) and root mass ratio (RMR) was calculated by dividing the stem, leaf, and root biomass by the total biomass (root, stem, and leaf), respectively. Root: shoot ratio (R/S) was calculated using root biomass by total leaf and stem biomass in percentage. Leaf area (LA) was determined with a leaf scanner (model A3 Light box GCL Bubble Etch Tanks), and WinDIAS 3.2. software. Specific leaf area (SLA) was estimated by dividing the leaf area by leaf biomass, while leaf area ratio (LAR) was determined by dividing the total leaf area by every seedling total biomass (Wu et al. 2017, Zhang et al. 2019). Relative water content Ten leaf discs with a diameter of 5 mm were cut from the interveinal parts of each plant and fresh weight (FW) was determined. After that, turgor weight (TW) was calcu- lated by weighing discs dipped in water for 24 hours in the dark. Finally, leaf discs were oven dried for 24 hours at 65 °C to determine dry weight (DW). Relative water content (RWC) was measured as follows: RWC (%) = (FW-DW) / (TW-DW) × 100 (Toscano et al. 2016). Photosynthetic pigment content For the extraction of photosynthetic pigments, 200 mg liquid nitrogen frozen tissue was ground by pestle and mor- tar and pigments were extracted by adding 10 mL of 80% cold acetone. The content of chlorophyll a (chl a) and b (chl b), total chlorophyll (chl a+b) and carotenoids was measured spectrophotometrically at 663, 645 and 470 nm respective- ly by spectrophotometer (Ltd T80 + UV/VIS; PG Instru- ments, Leicestershire, UK) according to Lichtenthaler (1987). The chlorophyll and carotenoid concentrations ex- pressed as mg g–1 FW were calculated as:  = × − × × × 663 645chl (12.7 ) (2.69 ) /1000a A A V W  = × − × × × 645 663chl (22.9 ) (4.68 ) /1000b A A V W  + = × + × × × 645 663chl (20.2 8.02 ) / (1000 )a b A A V W carotenoids = × − × − × × × 4701000 2.27 chl 81.4 chl 227 1000 A a b V W where: A ‒ absorbance at specific wavelengh V ‒ final volume of chlolophyll extract in 80% acetone W ‒ fresh weight of tissue extracted Free proline concentration Free proline concentration was estimated according to Bates et al. (1973). In this method, 0.5 g of frozen leaf sam- ples was extracted with 10 mL of 3% (w/v) sulfosalicylic ac- id; 2 mL of an aliquot of the supernatant was mixed with 2 mL of acetic acid and 2 mL of ninhydrin acid incubated for 40 minutes at 100 °C. The reaction was stopped in an ice bath and the reaction mixture was obtained with 4 mL of toluene and absorbance of the top layer was measured at 520 nm. Proline concentration was calculated by a standard curve, ranging from 0 to 400 µg mL-1 that was plotted with L-proline. Free proline concentration in tissue was calcu- lated as: proline (mmol g–1) = (mmol proline/mL) ´ (mL toluene/115) ´ 5/W Malondialdehyde (MDA) content The extent of lipid peroxidation was evaluated as malo- ndialdehyde (MDA) content. 100 mg leaf tissue was extract- ed in 2 mL 0.1% (w/v) trichloroacetic acid (TCA) and cen- trifuged at 12000 g for 15 min and then 0.5 mL of the upper phase was mixed with 1.5 mL TCA 20% (w/v) containing 0.5% (w/v) thiobarbituric acid (TBA). The mixture was heated for 90 min at 90 °C and then rapidly cooled in an ice bath. Afterwards, the mixture was centrifuged at 10000 g for 5 min and the absorbance (A) of the supernatant was re- corded at 532 and 600 nm. The MDA content in tissue was calculated by an extinction coefficient of 155 mM-1 cm-1 as nmol g-1 (Chakhchar et al. 2015): MDA (nmol g–1 FW) = − × × ×532 600 1000 155 A A V D W where: V ‒ final volume of extract W ‒ fresh weight of tissue extracted D ‒ dilution factor Enzyme activities 100 mg fresh leaves was ground in liquid nitrogen using a mortar and pestle, and the ground samples were homog- enized with 1 mL 50 mM sodium phosphate buffer at neu- tral pH containing 2 mM α-dithiothreitol, 2 mM EDTA, 0.2% Triton X-100, 50 mM Tris-hydrochloric acid and 2% polyvinylpyrrolidone. The homogenate was centrifuged at RESPONSES TO WATER DEFICIT IN ACER VELUTINUM AND ALNUS SUBCORDATA ACTA BOT. CROAT. 82 (1), 2023 63 14000 g for 30 min at 4 °C and the supernatant was collect- ed and stored at −80 °C for SOD and POD activity analysis (Yang and Miao 2010, Ghaffari et al. 2019). SOD activity (EC 1.15.1.1) was evaluated by inhibition ability of the photo- chemical reduction of nitroblue tetrazolium (NBT) reduc- tion to formazan by O2•−. One unit of SOD was considered as the amount of enzyme required to cause 50% inhibition of NBT photochemical reduction which can be measured at 560 nm (Giannopolitis et al. 1977). Guaiacol peroxidase ac- tivity (POD) (EC 1.11.1.7) was assayed according to the guaiacol method (Plewa et al. 1991). POD catalyzes guaiacol to tetraguaiacol by H2O2. Absorbance was read at 465 nm for 2 min. The calculation were done through the following formulas:   =   mol POD activity g FW min m − × − × × 465 465 t s A (t2) A (t1) V t2 t1 E V W where: A – absorbance at specific wavelength Vt – total volume Vs – enzyme volume E – extinction coefficient   =   U SOD activity g FW −  − ×   × (OD control OD sample) 100 100 OD control 50 W where: OD control- absorbance in the absence of SOD OD sample- absorbance in the presence of SOD. Statistical analysis A randomized complete design was employed with three replications (n = 3). First, the variables were analyzed using one-way ANOVA (analysis of variances) with water supply regimes as factors for each species, then the main effects of drought stress and species and their interactions were deter- mined by two-way ANOVA. When significant differences occurred among treatments, means were separated by Duncan’s multiple range tests at P ≤ 0.05. Pearson’s correla- tion coefficients were used to calculate the bivariate relation- ships between some morphophysiological and biochemical traits. Results Growth parameters The highest plant growth parameters (height, basal di- ameter, total and organ biomass and leaf area) were observed in the well-watered 100% FC treatment, while drought treat- ments significantly decreased plant height, basal diameter, total and organ biomass in both species (P ≤ 0.05). Plant height decreased by 30.9, 26.6 and 16.9% when exposed to 25, 50 and 75% FC in A. subcordata respectively, and 23.3 and 17.8% in A. velutinum at 25 and 50% FC treatments, re- spectively in comparison with control treatment. Basal di- ameter decreased by 29.2, 32.7 and 13.8% at 25, 50 and 75% FC treatments in A. subcordata respectively, and 19.8% at 25% FC in A. velutinum, compared to control condition. Bio- mass traits showed a decreasing trend in both species under water treatment; namely, leaf biomass reduction was 79.1 and 80.8%, that of stem biomass was 40.5 and 75.8%, root bio- mass 60.9 and 64.2%, and finally total biomass 61.6 and 64.2% at 25% FC in A. velutinum and A. subcordata respec- tively compared to control condition (Tab. 1 and Tab. 2). Drought stress significantly decreased leaf area in both species. Leaf area decreased 71.9 and 83.6% in A. velutinum and A. subcordata, respectively, when exposed to 25% FC. Specific leaf area tended to increase with decreasing soil wa- ter contents and significantly increased by 70.9% when ex- posed to 50% FC in A. velutinum. In contrast, it decreased 19.3, 26.3 and 49.6% in A. subcordata at 75, 50 and 25% FC, Tab. 1. Effect of drought stress on height, basal diameter, leaf area (LA), special leaf area (SLA), and leaf area ratio (LAR) of A. velutinum and A. subcordata seedlings. Values are means of three replicates ± standard deviation (SD). Different capital letters indicate signifi- cant (P ≤ 0.05) differences between A. velutinum and A. subcordata subjected to the same treatment. Different lowercase letters indi- cate significant (P ≤ 0.05) differences among different treatments applied to the same species. FS: species effect, FD: drought effect, FS×FD: species × drought interaction effect. *, **, and ***: significant at P ≤ 0.05, 0.01, and 0.001, respectively. Field capacity (FC, %) Plant height (cm) Basal diameter (mm) Leaf area (cm2) Special leaf area (cm2 g–1) Leaf area ratio (cm2 g–1) Acer velutinum 100 52.75±1.96Da 14.51±0.37BCa 123.40±11.21Ba 117.23±5.22Eb 30.51±1.53Ba 75 50.75±1.24Da 14.06±0.52Ca 78.55±3.41Cb 131.84±10.34DEb 24.49±0.85BCb 50 43.33±1.44Db 13.35±0.59CDa 60.80±4.30Cb 200.35±14.53Ca 29.00±0.21Ba 25 40.44±2.23Db 11.63±0.38Db 34.62±0.81Dc 126.79±11.11DEb 17.74±1.49Cc Alnus subcordata 100 132.67±7.97Aa 18.69±0.80Aa 153.66±12.50Aa 356.28±22.99Aa 57.95±3.74Aa 75 110.12±5.54Bb 16.11±0.87Bb 78.94±7.08Cb 287.35±5.33Bab 57.46±2.35Aa 50 97.33±6.32Cab 12.58±0.62CDc 37.36±3.90Dc 262.53±30.17Bb 31.78±7.17Bb 25 90.89±5.37Cc 13.23±0.70CDc 25.13±2.39Dc 179.33±0.05CDc 18.70±0.90Cb FS 328.49*** 15.95*** 0.01ns 98.39*** 53.38*** FD 13.68*** 65.12*** 93.90*** 8.89** 28.64*** FS × FD 3.98* 18.00** 5.40** 11.69*** 14.17*** RAVANBAKHSH M., BABAKHANI B., GHASEMNEZHAD M., SERPOOSHAN F., BIGLOUIE M. H. 64 ACTA BOT. CROAT. 82 (1), 2023 respectively. Leaf area ratio significantly decreased by 41.85 and 67.7% at 25% FC in A. velutinum and A. subcordata, re- spectively (Tab. 1). The biomass contribution was significantly affected by changes in water availability. R/S increased by 45 and 53.3% in A. subcordata under moderate and severe treatments, while no significant difference among drought treatments was found in A. velutinum. RMR increased with reduced water availability in A. subcordata. The enhancement was 24.4% at 50% FC and 28.2% at 25% FC in comparison with control treatment, whereas no significant diffrence was ob- served in A. velutinum. Drought stress markedly decreased LMR by 45.9 and 44.1% when exposed to 25 and 50% FC in A. velutinum respectively, and 32.1 and 27.3% in A. subcordata in the 25 and 50% FC treatments, respectively in comparison with control treatment. SMR in A. velutinum significantly increased in all treatments in comparison with control treatment, while it showed a reduction tendency in A. subcordata (Tab. 3). Relative water content and photosynthetic pigment content RWC showed significant decreases of 24.9 and 33.5% re- spectively at 50 and 25% FC in A. subcordata, whereas in A. velutinum the only significant decrease was of 27.3% at 25% FC compared with the well-watered seedlings (Tab. 4). Chl a content was reduced by 24 and 28% at 50 and 25% FC in A. velutinum, respectively, and 21.9, 60.9 and 53.3% in A. subcordata in the 75, 50 and 25% FC treatments, re- spectively, compared to control condition. Chl b content de- Tab. 2. Effect of drought stress on biomass in A. velutinum and A. subcordata seedlings. Values are means of three replicates ± stand- ard deviation (SD). Different capital letters indicate significant (P ≤ 0.05) differences between A. velutinum and A. subcordata applied to the same treatment. Different lowercase letters indicate significant (P ≤ 0.05) differences among different treatments applied to the same species. FS: species effect, FD: drought effect, FS×FD: species × drought interaction effect. *, **, and ***: significant at P ≤ 0.05, 0.01, and 0.001, respectively. Field capacity (FC, %) Root biomass (g) Leaf biomass (g) Stem biomass (g) Total biomass (g) Acer velutinum 100 33.33±3.38Ba 16.00±0.58Ba 12.33±1.20DEa 61.67±3.76Ca 75 22.67±1.45CDb 8.00±0.58Cb 12.00±0.58DEa 42.67±1.45DEb 50 13.00±0.58Ec 3.67±0.33Dc 8.33±0.33DEb 25.00±0.58FGc 25 13.00±0.58Ec 3.33±0.88Dc 7.33±0.33Eb 23.67±1.45Gc Alnus subcordata 100 46.66±2.90Aa 20.33±0.66Aa 58.00±4.16Aa 125.00±4.00Aa 75 27.33±1.66Bb 14.33±1.20Bb 30.00±3.05Bb 71.66±5.48Bb 50 21.33±2.02CDbc 5.33±0.33Dc 19.00±1.15Cc 45.66±2.40Dc 25 16.66±2.18DEc 3.90±0.92Dc 14.00±0.57CDc 34.56±3.47EFc FS 26.21*** 44.43*** 211.64*** 185.72 *** FD 60.19*** 195.72*** 61.62*** 162.89*** FS × FD 2.23ns 7.25 ** 39.88 *** 25.16*** Tab. 3. Effect of drought stress on biomass partitioning rate of A. velutinum and A. subcordata seedlings. Values are means of three replicates ± standard deviation (SD). Different capital letters indicate significant (P ≤ 0.05) differences between A. velutinum and A. subcordata subjected to the same treatment. Different lowercase letters indicate significant (P ≤ 0.05) differences among different treatments applied to the same species. FS: species effect, FD: drought effect, FS×FD: species × drought interaction effect. *, **, and ***: significant at P ≤ 0.05, 0.01, and 0.001, respectively. Field capacity (%) Root to shoot ratio (R/S) Leaf mass ratio (LMR) Stem mass ratio (SMR) Root mass ratio (RMR) Acer velutinum 100 1.18±0.12Aa 26.19±2.12Aa 19.96±1.33Ca 53.85±2.60ABa 75 1.13±0.07ABa 18.73±1.09BCb 28.25±2.14Bb 53.01±1.62ABa 50 1.09±0.08ABCa 14.62±1.07CDEb 33.36±1.50Bb 52.01±2.00ABCa 25 1.22±0.05Aa 14.16±1.48DEb 30.78±1.92Bb 55.05±1.04Aa Alnus subcordata 100 0.60±0.06Db 16.26±0.01BCDa 46.33±2.42Aa 37.39±2.43Db 75 0.62±0.03Db 19.97±0.44Ba 41.75±1.60Aa 38.26±1.31Db 50 0.87±0.08Ca 11.82±1.40Eb 41.64±2.25Aa 46.53±2.25Ca 25 0.92±0.05BCa 11.03±1.80Eb 41.03±2.91Aa 47.94±1.37BCa Fs 56.18 *** 14.74 *** 105.59 *** 65.76 *** FD 2.90 ns 20.74 *** 1.63 ns 4.58* FS × FD 2.58 ns 5.90** 8.20 ** 4.09 * RESPONSES TO WATER DEFICIT IN ACER VELUTINUM AND ALNUS SUBCORDATA ACTA BOT. CROAT. 82 (1), 2023 65 creased 20.4 and 53% in A. velutinum and 56.8 and 52% in A. subcordata at 50 and 25% FC respectively. Chl a+b decreased by 20, 60 and 53.3% when exposed to 75, 50 and 25% FC in A. subcordata, respectively, and 25.5 and 36.2% in A. velutinum in the 25 and 50% FC treatments respec- tively, in comparison with control treatment. The content of carotenoids significantly decreased under drought in A. subcordata, where the reduction was 50 and 38.5% at 50 and 25% FC, whereas A. velutinum showed a tendency to in- crease in carotenoids under drought stress (Tab. 4). Biochemical responses In the leaves of both species, increase in proline content was recorded upon stress treatments. Proline content in A. velutinum leaves increased 22.1 and 132.6% at 75 and 50% FC, respectively and 136.8% at 25% FC. In A. subcordata the increase was 34.9 and 62.2% at 75 and 50% FC, respectiely and 169.8% at 25% FC in comparison with control treatment (Fig. 1A). The MDA content increased substantially as drought stress progressed in both species. In A. subcordata the increase was 93.7 and 133.8% at 75 and 50% FC, respec- Tab. 4. Effect of drought stress on photosynthetic pigments content, and RWC of A. velutinum and A. subcordata seedlings. Values are means of three replicates ± standard deviation (SD). Different capital letters indicate significant (P ≤ 0.05) differences between A. velutinum and A. subcordata subjected to the same treatment. Different lowercase letters indicate significant (P ≤ 0.05) differences among different treatments applied to the same species. FW: fresh weight, RWC: relative water content; Fs: species effect, FD: drought effect, FS×FD: species × drought interaction effect. *, **, and ***: significant at P ≤ 0.05, 0.01, and 0.001, respectively. Field capacity (%) Chlorophyll a (mg g–1 FW) Chlorophyll b (mg g–1 FW) Total chlorophyll (mg g–1 FW) Total carotenoids (mg g–1 FW) RWC (%) Acer velutinum 100 1.00±0.05ABab 0.49±0.13Aab 1.49±0.07ABab 0.18±0.02BCDa 72.58±3.82Aa 75 1.30±0.20Aa 0.53±0.03Aa 1.84±0.23Aa 0.22±0.03ABCa 75.08±2.66Aa 50 0.76±0.07BCb 0.39±0.09ABCab 1.11±0.01Cbc 0.25±0.02ABa 69.20±3.82Aa 25 0.72±0.10BCDb 0.23±0.03BCb 0.95±0.12CDc 0.25±0.02ABa 52.77±1.36Bb Alnus subcordata 100 1.05±0.08ABa 0.44±0.03Aa 1.50±0.06Aa 0.26±0.02Aa 70.38±3.00Aa 75 0.82±0.12BCb 0.40±0.06Aba 1.23±0.11BCb 0.21±0.00ABCb 67.61±2.73Aa 50 0.41±0.00Dc 0.19±0.01Cb 0.60±0.02Dc 0.13±0.0 Dc 52.83±3.08Bb 25 0.49±0.04CDc 0.21±0.02BCb 0.70±0.07Dc 0.16±0.00CDc 46.77±3.08Bb Fs 11.55** 5.21 * 18.36 ** 5.54 * 13.99** FD 12.69 *** 8.13 ** 23.72 *** 0.82 ns 23.10 *** FS × FD 2.42 ns 0.81 ns 3.03ns 7.69 ** 1.94 ns Fig. 1. Changes in proline (A), malondialdehyde (MDA) (B), superoxide dismutase (SOD) (C) and guaiacol peroxidase (POD) measured in leaves from A. velutinum and A. subcordata seedlings subjected to four drought treatments (100, 75, 50 and 25% of field capacity – FC). Values are means of three replicates ± standard deviation (SD). Different capital letters indicate significant (P ≤ 0.05) differences between A. velutinum and A. subcordata subjected to the same treatment. Different lowercase letters indicate significant (P ≤ 0.05) differences among the different treatments to which the same species were subjected. RAVANBAKHSH M., BABAKHANI B., GHASEMNEZHAD M., SERPOOSHAN F., BIGLOUIE M. H. 66 ACTA BOT. CROAT. 82 (1), 2023 tively and 142.7% at 25%, whereas in A. velutinum the in- crease was 60.5 and 65% at 50 and 25% FC (Fig. 1B). In A. velutinum, SOD activity increased 12 and 8.9% at 50 and 25% FC, respectively. In A. subcordata, SOD activ- ity was significantly increased by 36, 25 and 20.9% at 75, 50 and 25% FC, respectively (Fig. 1C). POD activity in A. velutinum increased by 113 and 327% at 75 and 50% FC , respectively and 40% at 25% FC, whereas the values in A. subcordata were increased by 148 and 140% at 75 and 50% FC, respectively (Fig. 1D). Correlation analysis Correlation analysis indicated that there was a signifi- cant and positive correlation between SLA and chl a, chl b and chl a+b in A. subcordata, but there was no significant correlation between SLA and chl concentration in A. velutinum. Correlation analysis revealed that there was a significant and positive correlation between SOD and POD activities also, between proline and chl a, chl a+b in both species. According to correlation analysis there was no sig- nificant correlation between RWC and proline in A. velutinum but also, there was a negative correlation between RWC and proline in A. subcordata. Correlation analysis also revealed that there was a significant and positive correlation between carotenoid content and SOD activity in A. velutinum (Tab. 5 and Tab. 6). Discussion Drought stress is the most adverse abiotic stress to plant growth. Permanent or temporary water shortage causes det- rimental effects on plant growth and development (Tariq et al. 2018; Du et al. 2019). Height, total and organ biomass of both species signifcantly declined under moderate and se- vere treatments (50 and 25% FC) in comparison with con- trol treatment. Basal diameter signifcantly decreased under moderate and severe treatments (50 and 25% FC) in A. subcordata and just reduced under severe treatments (25% FC) in A. velutinum. These results are in accordance with Tab. 5. Correlation analysis among some morphophysiological and biochemical traits in Acer velutinum under drought stress condi- tions. Each square indicates the Pearson correlation coefficient of a pair of parameters. Leaf area: LA, specific leaf area: SLA, relative water content: RWC, chlorophyll a: chl a, chlorophyll b: chl b, total chlorophyll: chl a+b, and carotenoids: car, free proline: pro, malondialdehyde: MDA, peroxidase: POD, superoxide dismutase: SOD. ** and * indicate a significant correlation between control and drought treatments at P ≤ 0.01 and P ≤ 0.05, respectively. LA SLA car SOD POD MDA pro RWC chl a chl b chl a+b LA 1.000 –0.293 –0.476 –0.695* –0.306 –0.719** –0.803** 0.607* 0.466 0.426 0.524 SLA 1.000 0.300 0.632* 0.779** 0.323 0.427 0.047 –0.256 –0.196 –0.294 car 1.000 0.605* 0.365 0.343 0.415 –0.213 –0.096 –0.229 –0.181 SOD 1.000 0.624* 0.638* 0.771** –0.150 –0.224 –0.298 –0.314 POD 1.000 0.366 0.487 0.151 –0.332 –0.042 –0.293 MDA 1.000 0.910** –0.524 –0.585* –0.523 –0.651* pro 1.000 –0.540 –0.651* –0.420 –0.667* RWC 1.000 0.596* 0.768** 0.735** chl a 1.000 0.473 0.939** chl b 1.000 0.745** chl a+b 1.000 Tab. 6. Correlation analysis among some morphophysiological and biochemical traits in Alnus subcordata under drought. Each square indicates the Pearson correlation coefficient of a pair of parameters. Leaf area: LA, specific leaf area: SLA, relative water con- tent: RWC, chlorophyll a: chl a, chlorophyll b: chl b, total chlorophyll: chl a+b, and carotenoids: car, free proline: pro, malondialde- hyde: MDA, peroxidase: POD, superoxide dismutase: SOD. ** and * indicate a significant correlation between control and drought treatments at P ≤ 0.01 and P ≤ 0.05, respectively. LA SLA car SOD POD MDA pro RWC chl a chl b chl a+b LA 1.000 0.836** 0.873** –0.566 –0.314 –0.826** –0.757** 0.775** 0.869** 0.758** 0.889** SLA 1.000 0.648* –0.300 0.094 –0.797** –0.746** 0.746** 0.632* 0.607* 0.665* car –0.559 –0.330 –0.716** –0.626* 0.799** 0.944** 0.698* 0.925** SOD 1.000 0.705* 0.444 0.295 –0.250 –0.538 –0.210 –0.466 POD 1.000 0.281 –0.191 0.113 –0.309 –0.182 –0.288 MDA 1.000 0.543 –0.702* –0.630* –0.697* –0.692* pro 1.000 –0.812** –0.688* –0.655* –0.721** RWC 1.000 0.749** 0.684* 0.776** chl a 1.000 0.725** 0.974** chl b 1.000 0.861** chl a+b 1.000 RESPONSES TO WATER DEFICIT IN ACER VELUTINUM AND ALNUS SUBCORDATA ACTA BOT. CROAT. 82 (1), 2023 67 previous studies on Salix paraqplesia and Hippophae rhamnoides (Fang et al. 2012) as well as Prunus sargentii and Larix kaempferi seedlings (Bhusal et al. 2020) which dem- onstrated that drought significantly reduced seedling growth and biomass. We found that drought treatment significantly increased the R/S and RMR in A. subcordata. It was statistically inef- fective in A. velutinum. The increase in R/S is the result of declining growth rate and biomass production and in- creased water uptake (Wu et al. 2008, Du et al. 2010). Many studies have shown that there is an increase in R/S ratio un- der water stress (Fang et al. 2012; Guo et al. 2019, Zhang et al. 2019). More biomass allocation to belowground organs and maintainance of higher R/S can be indicated as an im- portant adaptive trait (Fang et al. 2012). In the present study, drought decreased LA in both spe- cies under drought stress. SLA showed an increasing trend in A. velutinum under drought stress treatments. However, it decreased in all drought treatments in A. subcordata. Al- so, LAR significantly decreased under drought conditions in both species. Decreased LA usually occurs due to inhibi- tion of leaf development, loss of access to photosynthetic products to make new cells (Tariq et al. 2018). Some plant species adjust LA to prevent transpiration or a relative in- crease in root water uptake capacity (Guo et al. 2019). SLA and LAR increased under severe stress compared to the con- trol in Jatropha curcas seedlings, which is considered a drought-tolerant plant (Díaz-lópez et al. 2012). In our study, A. velutinum significantly increased the SLA under moder- ate treatment (50% FC), which indicates that it probably has been able to cope with drought stress by increasing photo- synthetic capacity and carbon assimilation (Wu et al. 2017, Barros et al. 2020). Correlation analysis indicated that there was a significant and positive correlation between SLA and chl a, chl b and chl a+b in A. subcordata, but there was no significant correlation between SLA and chl concentration in A.velutinum. We found that chl a, chl b, and chl a+b content signifi- cantly decreased under drought stress in both species. A. velutinum had a higher chlorophyll content (chl a, chl b, and chl a+b) than A. subcordata under moderate and severe treatment (50 nd 25% FC). According Lei et al. (2006), the dry climate population of Populus przewalskii had higher chlorophyll content than the wet climate population under the drought treatment. Drought stress also significantly de- creased chlorophyll content of Juglans mandshurica, Juglans nigra and Juglans regia seedlings (Liu et al. 2019). Our re- sults also showed that the carotenoid content was not sig- nificantly increased by drought in A. velutinum, while it was significantly decreased under moderate and severe treat- ment (50 and 25% FC) in A. subcordata. Reduction of carot- enoids suggested that drought stress caused noticeable oxi- dative stress by ROS accumulation (Lei et al. 2006). The slight increase in carotenoid content in A. velutinum could suppress photosynthetic apparatus damage by oxygen con- sumption in xanthophyll cycle or detoxification of ROS (Ashraf and Harris, 2013, Medeiros et al. 2013). Correlation analysis also revealed that there was a significant and posi- tive correlation between carotenoids content and SOD ac- tivity in A. velutinum. In our study, A. velutinum seedlings showed a decline in RWC only under the severe treatment (25% FC), whereas A. subcordata showed a significant decrease in the moderate and severe treatments (50 and 25% FC, respectively). Díaz- López et al. (2012) indicated that Jatropha curcas can be considered a drought-resistant species as it has been able to sustain its RWC level under mild to severe stress drought treatments. Moreover, Ying et al. (2015) suggested that prov- enance Kunming (KM) had higher RWC than provenance Nanchang (NC) of Camptotheca acuminate under moderate and severe treatments (50 and 30% FC) and exhibited great- er drought stress tolerance as expected given the natural habitat of this provenance. Proline content of both the spe- cies, investigated in this study, was significantly increased under drought treatments with respect to the well-watered plants although the higher increase was recorded in A. velutinum comparied to A. subcordata under moderate treatment (50% FC), whereas the increment was significant- ly greater in A. subcordata than in A. velutinum under the severe treatment (25% FC). According to correlation analy- sis, there was no significant correlation between RWC and proline content in A. velutinum, while negative correlation between RWC and proline was recorded in A. subcordata. Ashrafi et al. (2018) reported a negative correlation between RWC and osmoprotectants in Thymus vulgaris and T. kotschyanus, and found that osmoprotectants accumulate by reduction of RWC to maintain plant water. Similarly, Bangar et al. (2019) found that proline content was nega- tively associated with RWC in Vigna radiate. MDA is a product of poly-unsaturated fatty acid degen- eration in phospholipids of cellular membrane, and is used as an index of oxidative stress magnitude under drought (Wang et al. 2012, Guo et al. 2018). MDA content increased along with the drought stress in both species in this study. The significant increase of MDA content with progressive drought stress, suggests that drought stress caused oxidative damage. Our results, according to Wu et al. (2013) in Quercus variabilis and Tariq et al. (2018) in Alnus cremastogyne sub- jected to drought stress, showed an increase of MDA content. In A. velutinum, the values increased under moderate and severe treatment (50 and 25% FC), while in A. subcordata MDA content was elevated upon all drought treatments. The increases in MDA content in A. velutinum were lower than those in A. subcordata. This indicated that drought led to more damage in the cellular membranes under stress treatments in A. subcordata. Similarly, Ying et al. (2015) found that drought stress significantly increased MDA con- tent in Camptotheca acuminata provenance KM and NC and the increases in MDA content in provenance KM were lower than those in provenance NC. They suggested that the less production of ROS in provenance KM under water deficit led to better membrane integrity. The ability of antioxidant enzymes to eliminate ROS and reduce its harmful effects may be related to plant RAVANBAKHSH M., BABAKHANI B., GHASEMNEZHAD M., SERPOOSHAN F., BIGLOUIE M. H. 68 ACTA BOT. CROAT. 82 (1), 2023 drought resistance (Anjum et al. 2011). High accumulation of ROS initiated and accelerated lipid peroxidation. POD plays an essential role in reducing the accumulation of H2O2, reducing MDA content and maintaining cell mem- brane integrity. Increased SOD and POD activity in stress treatments reflects an increase in ROS removal capacity and thus a reduction in membrane lipid damage (Ge et al. 2014, Guo et al. 2018). Toscano et al. (2016) suggested that Eugenia uniflora and Photinia × fraseri subjected to mild and mod- erate water stress showed increasing activities of antioxidant enzymes. We found that drought stress induced POD and SOD activity in both species under drought treatments in our study, although the highest activities were measured under mild and moderate treatments (75 and 50% FC) com- pared to the control. Our results are in good accordance with those published by Ge et al. (2014), who reported an increase of POD and SOD activities in Phoebe bournei sub- jected to mild and moderate water stress and a decrease un- der severe drought. In addition, Ge et al. (2014) demonstrat- ed that the increase in MDA content acts as a feedback mechanism to control the activities of antioxidant enzymes. In our study, A. veltinum showed higher POD activity and lower increment of MDA than A. subcordata under the moderate and severe treatments. Similarly, Wang et al. (2012) found that a stronger protective mechanism by a drought-tolerant apple rootstock (Malus prunifolia) than in a sensitive-tolerant apple rootstock (Malus hupehensis) can be ascribed to lower MDA content, higher values for leaf RWC, and greater antioxidative defense system. Wu et al. (2013) has also shown that the MDA content at 60% FC treatment kept a lower increase compared with 40 and 20% FC treatments, indicating better protection against mem- branes lipid peroxidation, more efficient repairing mecha- nisms, including the antioxidative system, osmotic adjust- ment, and photosynthetic pigments in Quercus variabilis seedlings. Conclusion The present study concluded that although there were common responses in investigated parameters between two Hyrcanian endemic species i.e., A. velutinum and A. subcordata, certain different responses were also recorded under drought stress. Our results demonstrated that drought stress significantly reduced growth, biomass and photosynthetic pigment content, but increased free proline content, POD and SOD activities in both species. A. velutinum showed a slight reduction in seedlings height, basal diam- eter, biomass and had higher RWC and photosynthetic pig- ment than A. subcordata. 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