Introduction
The impacts of climate change on vegetation will appear as a combination of stress factors, including high temperatures, reduction of rainfall, and alterations in wildfire regimes. 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 afforestation and reforestation programs because seedlings are more prone to drought than mature trees. Drought-tolerant species 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 limited 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 ribulose diphosphate and reactive oxygen species (ROS) production from the excess excitation energy. Low growth can be due to suppression of the photosynthetic process that eventually 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 resistance to drought stress (Ge et al. 2014). Decreased chlorophyll 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 senescence (Wu et al. 2013). Relatively high RWC maintenance 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 moderate (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 protection 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-enzymatic 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 conversion 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 climate 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 capacity reduced the pressure of wood exploitation on Hyrcanian 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 done on the quantitative and qualitative characteristics of the species, mechanical properties of wood and nutrient elements (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 technique 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 midday 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 objectives of the present study were (i) to evaluate the effects of drought stress on A. subcordata and A. velutinum seedlings which are dominant species in Hyrcanian forests and have a high commercial value in wood industries, to discover their capacity to handle water deficit in the initial vegetative growth period by morphological, physiological and biochemical responses; and (ii) to determine these two species’ different adaptive responses to drought stress.
Material and methods
Plant material and drought treatments
The experiment was carried out in a greenhouse at University of Guilan, Iran (37°15´ N, 49°36´ E). The average annual 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 (temperature 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 greenhouse 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 randomized 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 capacity performed as a control, mild, moderate, and severe, respectively). Using a scale with a capacity of 40 kg, transpiration 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 severe 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 diameter and biomass (total dry mass) were recorded at the end of the experiments. Three seedlings were harvested randomly from each treatment. The leaves, stems, and roots were cut and dried in an oven at 65 °C for 48 hours to calculate root, stem, and leaf biomass (the average weight of three samples per treatment). Biomass contribution including 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 calculated 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 mortar 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 respectively by spectrophotometer (Ltd T80 + UV/VIS; PG Instruments, Leicestershire, UK) according to Lichtenthaler (1987). The chlorophyll and carotenoid concentrations expressed as mg g–1 FW were calculated as:
chl a = [(12.7 × A663 ) - ( 2.69 × A645 )] × V/1000 × W
chl b = [(22.9 × A645 ) - (4.68 × A663)] × V/1000 × W
chl a+b= [( 20.2 × A645 + 8.02 × A663) × V)]/(1000 × W)
carotenoids = ((1000 × A470 - 2.27 × chl a - 81.4 × chl b )/227) × (V/(1000 × 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 samples was extracted with 10 mL of 3% (w/v) sulfosalicylic acid; 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 calculated as:
proline (µmol g-1) = [(µg proline/mL) × (mL toluene/115)] × 5/W
Malondialdehyde (MDA) content
The extent of lipid peroxidation was evaluated as malondialdehyde (MDA) content. 100 mg leaf tissue was extracted in 2 mL 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged 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 recorded 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) = ((A532 - A600)/155)×1000 × (V /W) × D
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 homogenized with 1 mL 50 mM sodium phosphate buffer at neutral 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 14000 g for 30 min at 4 °C and the supernatant was collected 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 photochemical reduction of nitroblue tetrazolium (NBT) reduction 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 activity (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:
POD activity (µmol /g FW min) = (|A465 (t2) - A465 (t1)|) / (t2 - t1) × Vt/(E × Vs × W)
where:
A- absorbance at specific wavelength
Vt- total volume
Vs- enzyme volume
E- extinction coefficient
SOD activity (U / g FW) = (100 - [((OD control - OD sample)/OD control) × 100]) / (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 determined 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 correlation coefficients were used to calculate the bivariate relationships between some morphophysiological and biochemical traits.
Results
Growth parameters
The highest plant growth parameters (height, basal diameter, total and organ biomass and leaf area) were observed in the well-watered 100% FC treatment, while drought treatments 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, respectively in comparison with control treatment. Basal diameter 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. Biomass 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 biomass 60.9 and 64.2%, and finally total biomass 61.6 and 64.2% at 25% FC in A. velutinum and A. subcordata respectively compared to control condition (Tab. 1 andTab. 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 water contents and significantly increased by 70.9% when exposed 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, respectively. Leaf area ratio significantly decreased by 41.85 and 67.7% at 25% FC in A. velutinum and A. subcordata, respectively (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 observed 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% respectively 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% FCin A. velutinum, respectively,and 21.9, 60.9 and 53.3% in A. subcordata in the 75, 50 and 25% FC treatments, respectively, compared to control condition. Chl b content decreased 20.4 and 53% in A. velutinum and 56.8 and 52% in A. subcordata at50 and 25% FCrespectively. 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 respectively, 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 increase 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, respectively and 142.7% at 25%, whereas in A. velutinum the increase 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 activity 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 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. 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 significant 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 andTab. 6).
Discussion
Drought stress is the most adverse abiotic stress to plant growth. Permanent or temporary water shortage causes detrimental 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 severe treatments (50 and 25% FC) in comparison with control 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 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 demonstrated 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 ineffective in A. velutinum. The increase in R/S is the result of declining growth rate and biomass production and increased water uptake (Wu et al. 2008,Du et al. 2010). Many studies have shown that there is an increase in R/S ratio under 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 important adaptive trait (Fang et al. 2012).
In the present study, drought decreased LA in both species 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. Also, LAR significantly decreased under drought conditions in both species. Decreased LA usually occurs due to inhibition 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 increase in root water uptake capacity (Guo et al. 2019). SLA and LAR increased under severe stress compared to the control 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 moderate treatment (50% FC), which indicates that it probably has been able to cope with drought stress by increasing photosynthetic 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 significantly 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 decreased chlorophyll content of Juglans mandshurica, Juglans nigra and Juglans regia seedlings (Liu et al. 2019). Our results also showed that the carotenoid content was not significantly increased by drought in A. velutinum, while it was significantly decreased under moderate and severe treatment (50 and 25% FC) in A. subcordata. Reduction of carotenoids suggested that drought stress caused noticeable oxidative stress by ROS accumulation (Lei et al. 2006). The slight increase in carotenoid content in A. velutinum could suppress photosynthetic apparatus damage by oxygen consumption 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 positive correlation between carotenoids content and SOD activity 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 inthe 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 provenance Kunming (KM) had higher RWC than provenance Nanchang (NC) of Camptotheca acuminate under moderate and severe treatments (50 and 30% FC) and exhibited greater drought stress tolerance as expected given the natural habitat of this provenance. Proline content of both the species, investigated in this study, was significantly increased under drought treatments with respect to the well-watered plantsalthough the higher increase was recorded in A. velutinum comparied to A. subcordata under moderate treatment (50% FC), whereas the increment was significantly greater in A. subcordata than in A. velutinum under the severe treatment (25% FC). According to correlation analysis, 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 negatively associated with RWC in Vigna radiate.
MDA is a product of poly-unsaturated fatty acid degeneration 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 subjected 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 content 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 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 membrane 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 moderate 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) compared 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 subjected to mild and moderate water stress and a decrease under severe drought. In addition, Ge et al. (2014) demonstrated 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 membranes lipid peroxidation, more efficient repairing mechanisms, including the antioxidative system, osmotic adjustment, 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 diameter, biomass and had higher RWC and photosynthetic pigment than A. subcordata. A. velutinum also showed more efficient antioxidant systems with higher activities of POD, and a lower increase in MDA content under drought stress. Our results highlight that A. velutinum maintained stronger drought tolerance based on the measured parameters. According to these findings, it is recommended that A. velutinum plantation should have priority over A. subcordata in water deficit regions.