Chemical Parameters of Oxidative Stress Adaptability in Beech

e antioxidant activity, lipid peroxidation, and contents of free proline and soluble proteins were investigated on six-year-old beech plants. Provenance Avala, in Serbia, had the best adaptability to environmental factors on locality Fruska Gora due to low lipid peroxidation, high FRAP value, and free proline and soluble proteins contents. Provenances Scharnstein and Mitterndorf, in Austria, had the best adaptability to environmental factors on locality Debeli Lug due to high FRAP value and free proline and soluble proteins contents. FRAP values in majority of provenances from locality Debeli Lug were higher. Correlations parameters were much higher between provenances in locality Debeli Lug, situated at higher altitude, which is the consequence of better adaption to environmental factors in�uence.


Introduction
Biological combustion involved in the respiration process produces harmful intermediates called reactive oxygen species (ROS).Excess of ROS can lead to cumulative damage in proteins, lipids, and DNA, resulting in so-called oxidative stress.Oxidative stress, de�ned as the imbalance between oxidants and antioxidants in favor of the oxidants [1], has been suggested to be the cause of aging [2] and various diseases in humans [3].
ere are many potential sources of ROS in plants.Some are reactions involved in normal metabolism, such as photosynthesis and respiration [4].Other sources of ROS belong to pathways enhanced during abiotic stresses.ese include drought stress [5][6][7] and desiccation, salt stress, chilling, heat shock, heavy metals, radiation [8], air pollutants such as ozone and SO 2 , mechanical stress, nutrient deprivation, pathogen attack, and high light stress [9,10].Plants suffering increased oxidative stress generally respond with increases in antioxidative systems although this response appears not always to be sufficient to prevent injury and lipid peroxidation [11,12].Beside growth and biomass production, the survival of plants requires the ability to defend themselves against adverse biotic or abiotic environmental stresses [13,14].Unfavorable conditions such as drought or air pollutants may cause increased oxidative stress.
Antioxidants from natural sources have received much attention, and efforts have been made to identify new natural resources for health-promoting antioxidant agent in human diets with economical potential for the pharmaceutical industry [15].In addition, these naturally occurring antioxidants can help to prevent oxidative damage caused by oxidative stress in plants.Hence, the balance between antioxidation and oxidation is believed to be a critical concept for maintaining a healthy biological system [16].
European beech (Fagus sylvatica L.), one of the principal tree species in Europe, has an invaluable role in many forest ecosystems.It is the most widely distributed forest tree species in Europe and is highly interesting for both economic and ecological reasons.Beech wood is excellent �rewood, easily split and burning for many hours with bright but calm �ames.Chips of beech wood are used in the brewing of Budweiser beer as a �ning agent.Beech is also used to smoke some cheeses.Some drums are made from beech.Also, beech pulp is used as the basis for manufacturing a textile �ber known as Modal.Modal has been used alone or with other �bers in household linens such as towels, bathrobes, and bed sheets, and the fabric has increased in popularity in the early 21st century.e wood is also used to make the pigment known as bistre favored by such artists as Rembrandt.
As a result of this historic bottleneck, northern populations of beech are genetically quite uniform while higher diversity is found near the southern limit of their range [17].Nevertheless, the latter are more sensitive to stressful environmental conditions since they are living under ecological conditions just barely within the limit of their requirements.is situation could become even worse if global air temperature increases, which would favor periods of drought in the Mediterranean and east European regions as well as increasing the risk of spring frost [18].e reaction of beech forests to potential climate change is one of the key issues in forestry today [19].Climate changes will negatively affect beech ecosystems, causing reduction of the beech range.Given to particular sensitivity to drought, it is projected that beech will face severe problems under global rising of temperature [20].e most endangered will be stands in the southern and south-eastern parts of present distribution range, while the conditions in the northern parts of beech distribution range may become more favorable for beech and its ecosystems.Less than 38% of Fagus sylvatica trees in Western Europe are healthy today.e health and growth of forest trees, however, are determined by a variety of natural and anthropogenic site factors and antioxidants [21].One of the main indicators of tree decline and air pollution is accelerated leaf ageing, and this process is characterized in beech leaves by antioxidants and pigment destruction.Antioxidants decreased with beech decline [18].As the beech is less tolerant to water shortage, in the southern parts of its range its competitiveness will largely be limited by increased water stress [22].Declining of the beech vitality, caused by droughts, may weaken physiological condition of populations, leading to insect and disease outbreaks [23].Furthermore, declines in the vitality of beech could result in the disappearance of beech from some habitats as a consequence of the loss of space by the competing species [24].Due to the complex in�uence of climate and soil variables on plants, it is not simple and straightforward to assess the adaptability of beech plants in the provenance trial.e most frequently used variables for assessing adaptability in trials with young plants are survival and height [25].
e impacts of speci�c variables on beech phenology, such as changes in the temperature, water availability, or an increase in atmospheric CO 2 concentrations, have been tested on different provenances with experiments in controlled environments [26][27][28].However, in case of European beech, those experiments are limited to young plants (aged up to a few years).e aim of this research was to propose efficient method for establishing the adaptability of different European beech provenances (aged up to a six years) to oxidative stress by evaluating the antioxidant properties and free proline and soluble proteins contents grown under different environmental conditions.

Material and Methods
2.1.Plant Material.e experiment is the part of international provenance trial which includes 20 different beech provenances from Croatia, Bosnia, Serbia, Hungary, Germany, Austria, Romania, and Swiss (Table 1).Samples were taken from six-years-old beech.20 leaves were sampled from each of �ve trees from the same locality in order to obtain an average sample.Plants were transplanted in a complete randomized block design in three blocks (repetitions).e trial was located within two localities: Fruška Gora (altitude 370 m) and Debeli Lug (altitude 742 m).Fresh expanded leaves from the top were used to determine all antioxidant parameters, lipid peroxidation, and free proline and soluble proteins contents.

Determination of Total Antioxidant
Capacity.Total antioxidant capacity was estimated according to the FRAP (Ferric Reducing Antioxidant Power) assay [29].Total reducing power is expressed as FRAP units.FRAP unit is equal to 100 mol/dm 3 Fe 2+ .FRAP value was calculated using the following formula: Soluble proteins = 0.35179 + 1.7967 * free proline F 1: Signi�cant and positive correlations between di�erent parameters (free proline, FRAP, and soluble proteins) in beech provenances in forest Fruška Gora.* Free proline was expressed as mol/mg protein, FRAP as FRAP units (1FRAP unit = 100 mol/dm 3 Fe 2+ ), and soluble proteins were expressed as mg/g.method.Values were given as equivalent amounts of malonyldialdehyde (MDA).e calibration curve was prepared with malonyldialdehyde bis-diacetal [30,31].

Determination of Proline Accumulation.
Proline accumulation was determined by the method as described by Paquin and Lechasseur [32].Proline was determined aer extracion with sulphosalicylic acid and reaction with ninhydrin.A standard curve of proline was used for calibration [33].

Soluble Protein Content Determination.
Soluble protein content was determined by the method of Bradford [34].
All determinations were performed in triplicate.

Statistical Analysis.
Results were expressed as mean ± standard error.Statistical comparisons between samples were performed with Student's -test for independent observations.Di�erences were considered signi�cant at   .
Correlations between FRAP values, LP, and free proline and soluble proteins contents were established by regression analysis.

Results and Discussion
Results concerning beech provenances transplanted on locality Fruška Gora are presented in Tables 2 and 3  Highest accumulation of free proline was in provenances 20 (6.329 mol/mg protein) and 17 (6.061mol/mg protein) which is a bene�t because proline may protect protein structures by maintaining their structural stability [35].It is also known that drought stress signi�cantly increases proline accumulation [36].Content of soluble proteins was also highest in provenances number 20 (11.446  Soluble proteins = −0.0581+ 1.8699 * free proline F 2: Signi�cant and positive correlations between different parameters (free proline, FRAP, and soluble proteins) in beech provenances in meadow Debeli Lug.* Free proline was expressed as mol/mg protein, FRAP as FRAP units (1FRAP unit = 100 mol/dm 3 Fe 2+ ), and soluble proteins were expressed as mg/g.17 (11.360mg/g) (Table 2).Proteins rich in proline have particular roles in the development, structure, and function of the cell walls [37].According to our results provenance number 17, Avala in Serbia, had the best adaptability to environmental factors in forest Fruška Gora due to low LP, high FRAP value, and contents of free proline and soluble proteins.
Signi�cant positive correlations (  ) were observed between numbers of examined parameters.
FRAP values were signi�cantly positively correlated with free proline (  ) and soluble proteins content (  ).Free proline was signi�cantly high positively correlated with soluble proteins content (  ).Results obtained by correlation analysis supported our previously presented results (Figure 1).
Results concerning beech provenances transplanted on locality Debeli Lug are presented in Tables 4 and 5 and Figure 2. e highest level of total antioxidant capacity was observed in provenance number 11, (58.872FRAP units), followed by provenances number 15 (58.513FRAP units) and number 12 (58.128FRAP units) (Table 4).Presented FRAP values were generally higher in provenances from meadow Debeli Lug.It has been proved that efficient antioxidative characteristics can provide better protection against oxidative stress [38], promote the growth of plants, and improve their productivity [39].It is known that antioxidant varied provenances probably due to different adaptation ability of each provenance [40].e lowest LP was observed in provenance number 20 (32.288 nmol MDA/mg protein), provenances number 1 (33.557nmol MDA/mg protein) and number 14 (35.869nmol MDA/mg protein).Increased MDA content suggested to oxidative damages in examined provenances, similarly as detected in olive trees.On the contrary low, accumulation of MDA is indicator of drought stress tolerance [35].Highest accumulation of free proline was detected in provenances No 11 (5.277 mol/mg protein), number 9 FRAP values were signi�cantly high positively correlated with LP (  0.593202), free proline (  0.646462), and soluble proteins contents (  0.672390).Free proline was signi�cantly high positively correlated with soluble proteins content (  0.997778).Results obtained by correlation analysis (Table 5) supported our previously presented results (Figure 2).It is also obvious that correlations parameters are much higher in provenances in meadow Debeli Lug from higher altitude, which is the consequence of their better adaption to environmental factors in�uence.Similar results were obtained by other plant species [41].
Antioxidant capacities of examined beech provenances not only depend on plant but also on the drought adaptation which are closely related to the environmental factors of their natural habitats which was in agreement with statements that physiological and biochemical processes of plants depend on the rapidity, severity, and duration of the drought event [42].Furthermore, no single method is sufficient because more than one type of antioxidant capacity measurement needs to be performed to take into account the various modes of action of antioxidants [43].e results suggest that beech provenances originating from the higher altitude (Debeli Lug) have a better drought tolerance due to higher FRAP values and higher correlations between oxidative stress parameters than provenances originating from the low altitude, which is in agreement with results obtained by investigation conducted on two poplar species [44].It is well known that beech originating from the higher altitude possesses a better drought tolerance and stronger drought adaptation than those beech originating from the low altitude, which can be explained by high and abundant precipitations due to much rainfall at high altitude; the trees at high altitudes may be waterstressed due to wind and ice blasting in the winter time, and colder soils reduce the water uptake of the root system, T 4: Total antioxidant capacity (FRAP), lipid peroxidation (LP), and free proline and soluble proteins contents in beech provenances transplanted in meadow Debeli Lug. and then they possessed better acclimation to drought stress than ones at low altitude [45].It was shown that although antioxidant protection was important in plants, there were signi�cant differences among the plant species.In addition to antioxidant protection, the higher soluble protein and proline contents have a very important role in the stress resistance of the woody plants [46].e results also showed that drought adaptations of beech provenances are closely related to the environmental factors.Similar conclusions were obtained by investigations of adaptability of birch (Betula pendula Roth) and aspen (Populus tremula L.) genotypes to different soil moisture conditions [47].

Conclusion
Presented results can contribute to explain differences of beech provenances in response to oxidative stress due to duration of the drought event and their individual altitude.It was proved that efficient antioxidative characteristics and proline accumulation can provide better protection against oxidative stress in leaves under drought stress.It is also established that beech originating from the higher altitude expresses a better drought tolerance than those beech originating from the low altitude.Information on the patterns of biochemical response to environmental stress provides an important tool for the improvement of environmental bioengineering strategies and reforestation programs for European beech.

T 1 :
Investigated beach provenances, country of origin, and appropriate altitude.
a Names of investigated beach provenances, country of origin, and appropriate altitude are presented in Table1.b c LP: lipid peroxidation.
a Names of investigated beach provenances, country of origin, and appropriate altitude are presented in Table1.b c LP: lipid peroxidation.* �olded correlations are signi�cant at   0.05.