# Chemical Parameters of Oxidative Stress Adaptability in Beech

Authors: D. Štajner; S. Orlović; B. M. Popović; M. Kebert; S. Stojnić; B. Klašnja
Journal: Journal of Chemistry
 (2013)

---

## Abstract

The 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 influence.


---

## Body

## 1. 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, defined 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].There 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. These include drought stress [5–7] and desiccation, salt stress, chilling, heat shock, heavy metals, radiation [8], air pollutants such as ozone and SO2, 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 sylvaticaL.), 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 firewood, easily split and burning for many hours with bright but calm flames. Chips of beech wood are used in the brewing of Budweiser beer as a fining 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 fiber known as Modal. Modal has been used alone or with other fibers in household linens such as towels, bathrobes, and bed sheets, and the fabric has increased in popularity in the early 21st century. The 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. This 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]. The 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]. The 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. The 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 influence of climate and soil variables on plants, it is not simple and straightforward to assess the adaptability of beech plants in the provenance trial. The most frequently used variables for assessing adaptability in trials with young plants are survival and height [25].The impacts of specific variables on beech phenology, such as changes in the temperature, water availability, or an increase in atmospheric CO2 concentrations, have been tested on different provenances with experiments in controlled environments [26–28]. However, in case of European beech, those experiments are limited to young plants (aged up to a few years).The 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.


## 2. Material and Methods

### 2.1.  Plant Material

The experiment is the part of international provenance trial which includes 20 different beech provenances from Croatia, Bosnia, Serbia, Hungary, Germany, Austria, Romania, and Swiss (Table1).Table 1
Investigated beach provenances, country of origin, and appropriate altitude.
No.
Provenance
Country
Altitude (m)
1
Sjeverni Dilj Čaglinski
Croatia
350
2
Vrani Kamen
Croatia
600
3
Tajan, Zepce
Bosnia
700
4
Grmec, Bosanska Krupa
Bosnia
650
5
Fruška Gora
Serbia
370
6
Kopaonik
Serbia
510
7
Valkonya
Hungary
300
8
Schelklingen
Germany
650
9
Höllenbach
Germany
755
10
Hasbruch
Germany
35
11
Scharnstein, Mitterndorf
Austria
480
12
Vranica-Bistrica
Bosnia
750
13
Crni Vrh
Bosnia
500
14
Alesd
Romania
490
15
Alba-Iulia
Romania
860
16
Sihlwald
Swiss
1050
17
Avala
Serbia
475
18
Boranja
Serbia
410
19
Fruška Gora
Serbia
370
20
Cer
Serbia
745Samples were taken from six-years-old beech. 20 leaves were sampled from each of five 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). The 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.


### 2.2.  Measurements

#### 2.2.1.  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 μmol/dm3 Fe2+. FRAP value was calculated using the following formula:
(1)FRAPvalue=ΔAsampleΔAstandard.


#### 2.2.2. Determination of Lipid Peroxidation

Lipid peroxidation (LP) was determined by the thiobarbituric acid (TBA) method. Values were given as equivalent amounts of malonyldialdehyde (MDA). The calibration curve was prepared with malonyldialdehyde bis-diacetal [30, 31].


#### 2.2.3.  Determination of Proline Accumulation

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


#### 2.2.4.  Soluble Protein Content Determination

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


### 2.3.  Statistical Analysis

Results were expressed as mean ± standard error. Statistical comparisons between samples were performed with Student’st-test for independent observations. Differences were considered significant at P<0.05. Correlations between FRAP values, LP, and free proline and soluble proteins contents were established by regression analysis.


## 2.1.  Plant Material

The experiment is the part of international provenance trial which includes 20 different beech provenances from Croatia, Bosnia, Serbia, Hungary, Germany, Austria, Romania, and Swiss (Table1).Table 1
Investigated beach provenances, country of origin, and appropriate altitude.
No.
Provenance
Country
Altitude (m)
1
Sjeverni Dilj Čaglinski
Croatia
350
2
Vrani Kamen
Croatia
600
3
Tajan, Zepce
Bosnia
700
4
Grmec, Bosanska Krupa
Bosnia
650
5
Fruška Gora
Serbia
370
6
Kopaonik
Serbia
510
7
Valkonya
Hungary
300
8
Schelklingen
Germany
650
9
Höllenbach
Germany
755
10
Hasbruch
Germany
35
11
Scharnstein, Mitterndorf
Austria
480
12
Vranica-Bistrica
Bosnia
750
13
Crni Vrh
Bosnia
500
14
Alesd
Romania
490
15
Alba-Iulia
Romania
860
16
Sihlwald
Swiss
1050
17
Avala
Serbia
475
18
Boranja
Serbia
410
19
Fruška Gora
Serbia
370
20
Cer
Serbia
745Samples were taken from six-years-old beech. 20 leaves were sampled from each of five 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). The 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.


## 2.2.  Measurements

### 2.2.1.  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 μmol/dm3 Fe2+. FRAP value was calculated using the following formula:
(1)FRAPvalue=ΔAsampleΔAstandard.


### 2.2.2. Determination of Lipid Peroxidation

Lipid peroxidation (LP) was determined by the thiobarbituric acid (TBA) method. Values were given as equivalent amounts of malonyldialdehyde (MDA). The calibration curve was prepared with malonyldialdehyde bis-diacetal [30, 31].


### 2.2.3.  Determination of Proline Accumulation

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


### 2.2.4.  Soluble Protein Content Determination

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


## 2.2.1.  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 μmol/dm3 Fe2+. FRAP value was calculated using the following formula:
(1)FRAPvalue=ΔAsampleΔAstandard.


## 2.2.2. Determination of Lipid Peroxidation

Lipid peroxidation (LP) was determined by the thiobarbituric acid (TBA) method. Values were given as equivalent amounts of malonyldialdehyde (MDA). The calibration curve was prepared with malonyldialdehyde bis-diacetal [30, 31].


## 2.2.3.  Determination of Proline Accumulation

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


## 2.2.4.  Soluble Protein Content Determination

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


## 2.3.  Statistical Analysis

Results were expressed as mean ± standard error. Statistical comparisons between samples were performed with Student’st-test for independent observations. Differences were considered significant at P<0.05. Correlations between FRAP values, LP, and free proline and soluble proteins contents were established by regression analysis.


## 3. Results and Discussion

Results concerning beech provenances transplanted on locality Fruška Gora are presented in Tables2 and 3 and Figures 1 and 2. The highest level of total antioxidant capacity was observed in provenance number 16 (44.410 FRAP units) followed by provenances number 10 (43.513 FRAP units) and number 17 (40.013 FRAP units) (Table 2). The lowest LP was observed in provenance number 17 (20.701 nmol MDA/mg protein), provenances 15 (21.535 nmol MDA/mg protein) and 9 and 19 (22.232 and 22.253 nmol MDA/mg protein). Highest accumulation of free proline was in provenances 20 (6.329 μmol/mg protein) and 17 (6.061 μmol/mg protein) which is a benefit because proline may protect protein structures by maintaining their structural stability [35]. It is also known that drought stress significantly increases proline accumulation [36]. Content of soluble proteins was also highest in provenances number 20 (11.446 mg/g) and number 17 (11.360 mg/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.Table 2
Total antioxidant capacity (FRAP), lipid peroxidation (LP), and free proline and soluble proteins contents in beech provenances transplanted in forest Fruška Gora.
Locality—Fruška Gora
Provenance No.a
FRAP (FRAP units)b
LPc (mmol MDA/mg protein)
Free proline (μmol/mg protein)
Soluble proteins (mg/g)
1
32.910
±
0.202
43.423
±
0.291
3.760
±
0.131
6.515
±
0.121
2
32.090
±
0.158
37.420
±
0.891
4.5170
±
0.401
8.452
±
0.116
3
33.590
±
0.315
35.195
±
0.426
4.126
±
0.192
7.692
±
0.021
4
24.949
±
0.078
30.157
±
0.662
4.004
±
0.298
7.395
±
0.023
5
28.872
±
0.114
30.653
±
0.191
4.297
±
0.086
7.898
±
0.115
6
27.974
±
0.056
23.157
±
0.351
4.582
±
0.158
8.675
±
0.124
7
19.565
±
0.068
29.779
±
0.456
3.601
±
0.205
6.601
±
0.061
8
33.679
±
0.243
26.232
±
0.399
4.965
±
0.18
9.494
±
0.128
9
39.885
±
0.222
22.232
±
0.483
4.996
±
0.216
9.446
±
0.173
10
43.513
±
0.253
29.913
±
0.465
4.489
±
0.209
8.151
±
0.166
11
31.346
±
0.451
28.364
±
0.466
3.912
±
0.211
7.281
±
0.134
12
27.910
±
0.092
29.271
±
0.808
4.838
±
0.364
9.042
±
0.146
13
29.205
±
0.105
30.117
±
0.224
4.826
±
0.101
8.903
±
0.143
14
29.462
±
0.081
30.002
±
0.413
4.815
±
0.186
9.111
±
0.081
15
15.897
±
0.046
21.535
±
0.251
4.325
±
0.113
7.918
±
0.072
16
44.410
±
0.046
51.814
±
0.267
5.264
±
0.121
9.914
±
0.088
17
40.013
±
0.114
20.701
±
0.101
6.061
±
0.045
11.36
±
0.085
18
35.910
±
0.091
24.228
±
0.447
5.026
±
0.201
9.276
±
0.086
19
32.782
±
0.110
22.253
±
0.253
5.603
±
0.114
10.431
±
0.115
20
36.487
±
0.078
24.828
±
0.331
6.329
±
0.149
11.446
±
0.130
aNames of investigated beach provenances, country of origin, and appropriate altitude are presented in Table 1.
b1FRAP unit = 100 μmol/dm3 Fe2+.
cLP: lipid peroxidation.Table 3
Correlations between total antioxidant capacity (FRAP), lipid peroxidation (LP), and free proline and soluble proteins contents in beech provenances transplanted in forest Fruška Gora.
Variable
FRAP
LP
Free proline
Soluble proteins
FRAP
1.000000
0.377605
0.545504
0.519771
LP
0.377605
1.000000
−0.059480
−0.102208
Free proline
0.545504
−0.059480
1.000000
0.957191
Soluble proteins
0.519771
−0.102208
0.957191
1.000000
*Bolded correlations are significant atP<0.05.Significant and positive correlations between different parameters (free proline, FRAP, and soluble proteins) in beech provenances in forest Fruška Gora. *Free proline was expressed asμmol/mg protein, FRAP as FRAP units (1FRAP unit = 100 μmol/dm3 Fe2+), and soluble proteins were expressed as mg/g.Significant and positive correlations between different parameters (free proline, FRAP, and soluble proteins) in beech provenances in meadow Debeli Lug. *Free proline was expressed asμmol/mg protein, FRAP as FRAP units (1FRAP unit = 100 μmol/dm3 Fe2+), and soluble proteins were expressed as mg/g.Significant positive correlations (P<0.05) were observed between numbers of examined parameters.FRAP values were significantly positively correlated with free proline (r=0.545504) and soluble proteins content (r=0.519771). Free proline was significantly high positively correlated with soluble proteins content (r=0.957191). Results obtained by correlation analysis supported our previously presented results (Figure 1).Results concerning beech provenances transplanted on locality Debeli Lug are presented in Tables4 and 5 and Figure 2. The highest level of total antioxidant capacity was observed in provenance number 11, (58.872 FRAP units), followed by provenances number 15 (58.513 FRAP units) and number 12 (58.128 FRAP 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]. The lowest LP was observed in provenance number 20 (32.288 nmol MDA/mg protein), provenances number 1 (33.557 nmol MDA/mg protein) and number 14 (35.869 nmol 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 μmol/mg protein), number 9 (5.205 μmol/mg protein), and number 12 (4.873 μmol/mg protein). Content of soluble proteins was highest in provenances number 11 (9.842 mg/g) and number 8 (9.689 mg/g). According to our results, provenance number 11, Scharnstein, Mitterndorf, in Austria, had the best adaptability to environmental factors in meadow Debeli Lug due to high FRAP value and free proline and soluble proteins contents.Table 4
Total antioxidant capacity (FRAP), lipid peroxidation (LP), and free proline and soluble proteins contents in beech provenances transplanted in meadow Debeli Lug.
Locality—Debeli Lug
Provenance No.a
FRAP (FRAP units)b
LPc (mmol MDA/mg protein)
Free proline (μmol/mg protein)
Soluble proteins (mg/g)
1
25.769
±
0.256
33.557
±
0.327
3.754
±
0.402
7.034
±
0.065
2
27.154
±
0.146
39.733
±
0.297
3.485
±
0.365
6.377
±
0.103
3
39.372
±
0.126
35.879
±
0.45
4.090
±
0.554
7.534
±
0.105
4
42.487
±
0.248
46.982
±
0.751
3.494
±
0.924
6.566
±
0.092
5
52.090
±
0.238
47.335
±
0.188
4.490
±
0.231
8.323
±
0.151
6
42.128
±
0.173
67.590
±
0.612
3.283
±
0.753
6.143
±
0.075
7
37.501
±
0.133
44.697
±
0.202
4.045
±
0.248
7.455
±
0.101
8
43.103
±
0.189
36.781
±
0.252
4.111
±
0.312
7.514
±
0.564
9
42.769
±
0.102
47.107
±
0.119
5.205
±
0.146
9.689
±
0.075
10
31.449
±
0.101
44.156
±
0.727
3.256
±
0.894
6.062
±
0.099
11
58.872
±
0.114
48.358
±
0.159
5.277
±
0.196
9.842
±
0.218
12
58.128
±
0.167
58.179
±
0.289
4.873
±
0.355
9.135
±
0.144
13
46.269
±
0.111
38.868
±
0.151
4.132
±
0.186
7.613
±
0.092
14
34.987
±
0.071
35.869
±
0.346
3.606
±
0.426
6.653
±
0.053
15
58.513
±
0.122
61.393
±
0.369
3.915
±
0.454
7.357
±
0.084
16
45.436
±
0.078
43.943
±
0.207
4.056
±
0.255
7.573
±
0.087
17
20.010
±
0.182
38.341
±
0.774
3.446
±
0.952
6.326
±
0.073
18
29.705
±
0.11
45.139
±
0.187
3.315
±
0.23
6.161
±
0.129
19
46.385
±
0.146
45.601
±
0.441
4.113
±
0.541
7.716
±
0.179
20
32.346
±
0.135
32.288
±
0.537
4.245
±
0.661
7.713
±
0.073
aNames of investigated beach provenances, country of origin, and appropriate altitude are presented in Table 1.
b1FRAP  unit = 100 μmol/dm3Fe2+.
cLP: lipid peroxidation.Table 5
Correlations between total antioxidant capacity (FRAP), lipid peroxidation (LP), andTypeequationhere.free proline and soluble proteins contents in beech provenances transplanted in meadow Debeli Lug.
Variable
FRAP
LP
Free proline
Soluble proteins
FRAP
1.000000
0.593202
0.646462
0.672390
LP
0.593202
1.000000
0.082635
0.125785
Free proline
0.646462
0.082635
1.000000
0.997778
Soluble proteins
0.672390
0.125785
0.997778
1.000000
*Bolded correlations are significant atP<0.05.Significant positive correlations (P<0.05) were observed between examined parameters.FRAP values were significantly high positively correlated with LP (r=0.593202), free proline (r=0.646462), and soluble proteins contents (r=0.672390). Free proline was significantly high positively correlated with soluble proteins content (r=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 influence. 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]. The 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 water-stressed due to wind and ice blasting in the winter time, and colder soils reduce the water uptake of the root system, 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 significant 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]. The 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].


## 4. 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.



---

*Source: 592695-2012-09-18.xml*