- Email: [email protected]
Impact of varying elevations on growth and activities of antioxidant enzymes of some medicinal plants of Saudi Arabia
Acta Ecologica Sinica 36 (2016) 141–148
Contents lists available at ScienceDirect
Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Impact of varying elevations on growth and activities of antioxidant enzymes of some medicinal plants of Saudi Arabia M. Nasir Khan ⁎, M. Mobin, Zahid Khorshid Abbas, Khalid A. ALMutairi Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 30 December 2015 Accepted 31 December 2015 Keywords: Antioxidant enzymes Elevations Medicinal plants Secondary metabolites
a b s t r a c t Tabuk is the northwestern province of Saudi Arabia which is characterized by highly variable climatic conditions that ranges from extreme cold to extreme hot which not only supports the growth of a huge variety of medicinally important plants but also modulates the inherent medicinal potential of these plants. Keeping these points in view, the present work was designed to evaluate the effect of climatic conditions on growth physiological and biochemical attributes of five medicinal plants namely, Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. We collected each plant species from four different elevations of Tabuk region namely Jabal Al-Lawz (2580 m asl: higher elevation), Jabal Al-Harrah (1370 m asl; medium elevation), Jordan Road (760 m asl; lower elevation) and Tabuk City (760 m asl; lower elevation). The plants growing at medium elevation of 1370 m exhibited better growth and physiological performance than the others that may be ascribed to enhanced leaf water, Chl and protein content as we recorded for these plants. Maximum and minimum accumulation of secondary metabolites corresponded to the plants grown at the higher and lower altitude, respectively. The antioxidant enzyme activities were highest at 1370 m asl that might have contributed to the improved growth and physiological attributes. Consequently, the results obtained in this study strongly suggest that plants grown at medium elevation of 1370 m asl possess higher activity of antioxidant enzymes, whereas, plants at higher elevation of 2580 m are best for medicinal use because they accumulated higher level of secondary metabolites. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Tabuk region, the northwestern province of Saudi Arabia, is characterized by its diversified topography ranging from plains to the mountains of low and high elevations which create highly variable environmental conditions encompassing through extreme cold to extreme hot climate. Diversified topography coupled with varied environmental conditions supports the growth of an array of medicinal plants. Elevations drastically influence environmental factors, such as quick seasonal and daily variations in temperature, low atmospheric pressure, and depressed CO2 concentration, short period of vegetation and enhanced intensity of solar UV radiation, which set the plants under serious stress. Plants show a significant morphological, physiological and biochemical responses to increasing altitude, which include decrease in stem height, stem diameter, biomass production [1], specific leaf area, and increases in the leaf thickness [2,3]. High light intensities at higher altitude induce excessive accumulation of reactive oxygen species (ROS) leading to pigment bleaching, lipid peroxidation, protein damage, inactivation of enzyme activities and ultimately cell death [4–6]. The enzyme carbonic anhydrase (CA) is found in ⁎ Corresponding author. E-mail address: [email protected] (M.N. Khan).
http://dx.doi.org/10.1016/j.chnaes.2015.12.009 1872-2032/© 2016 Elsevier B.V. All rights reserved.
abundance in the photosynthesizing tissues of both C3 and C4 plants and regulates the availability of CO2 to ribulose bisphosphate carboxylase (rubisco) by catalyzing the reversible hydration of CO2 [7]. Higher altitude restricts uptake of CO2 that consequently decreases carbon assimilation due to non-availability of oxidized NADP+ for acceptance of electrons during photosynthesis, a significant factor accorded to the formation of ROS [8]. Therapeutic use of plants is as old as human civilization and medicinal plants are integral part of healthcare system which is witnessed by the fact that 80% of the population still relies on a traditional system of medicine, based on herbal drugs [9]. Because of no side effects, the interest in medicinal products of plant has increased considerably all over the world. Of these, Artemisia judaica L. (family Asteraceae) called as ‘wormwood’ is a perennial shrub with pubescent and fragrant leaves is a highly resistant plant against adverse environmental conditions and very effective in stabilizing the habitat. Achillea fragrantissima (family Asteraceae), has been extensively used in Arabian folk medicine due its hypoglycemic properties. Teucrium polium L. (family Lamiaceae) is one of the most common medicinal plants of Arabian Peninsula that has been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, antipyretic, antispasmodic and cholagogic properties [10]. Lavandula pubescens Decne. (family Lamiaceae), is well known for its essential and aromatic oil, used in cosmetics while
142
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
decoction of leaves is given in headache and cold [11]. Retama raetam (Forssk.) Webb & Berthel. (family Fabaceae), commonly known as ‘raetam’ or ‘broom bush’ is a shrub that grows widely in Saudi Arabian deserts. The plant possesses antibacterial, antifungal, antihypertensive, anti-oxidant, antiviral, diuretic and hypoglycemic properties [12,13]. R. raetam has long been used as abortifacient, purgative and a vermifuge in the traditional system of medicine. All these five plants viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam are commonly grown in all parts of Tabuk region and are extensively used in the traditional system of medicine of the region. Although extensive research has been carried out on the altitudinal variation in morphological, physiological and biochemical characteristics of tree plants, but meager information is available on the changes in growth, lipid peroxidation, activities of antioxidant enzymes and on the accumulation of secondary metabolites of medicinal plant species growing under stressful conditions at various altitudes. Considering the importance of above mentioned plants in view, the present study was performed to test the hypothesis that variation in altitude significantly affects the activities of antioxidant enzymes and secondary metabolite concentration which plays a role in the acclimation of plants to abiotic stress induced impairment in plants. 2. Materials and methods
the plants in blotting papers. After removing the plants from blotting papers fresh weight for each plant was recorded. Dry weight of plants was recorded after drying the plants at 80 °C for 24 h in a hot air oven. 2.3. Physiological and biochemical parameters 2.3.1. Determination of hydrogen peroxide (H2O2) content The hydrogen peroxide (H2O2) content in fresh leaf samples was determined according to Velikova et al. [14]. The content of H2O2 was calculated by comparison with a standard calibration curve, plotted by using different concentrations of H2O2. The absorbance of supernatant was recorded at 390 nm. H2O2 content was expressed as μ mol g− 1 leaf FW. 2.3.2. Estimation of leaf protein content Protein content was measured according to Bradford [15] using bovine serum albumin as standard. The absorbance was read spectrophotometrically (CE 2021, Cecil, Cambridge, England) at 595 nm. 2.3.3. Determination of carbonic anhydrase (CA) activity The activity of carbonic anhydrase (CA: E.C. 4.2.1.1) was measured using titration method of Dwivedi and Randhawa [16]. The reaction mixture was titrated against HCl using methyl red as indicator. The enzyme was expressed as μM CO2 kg−1 leaf FW s−1.
2.1. Study area The study was conducted in Tabuk region, located in the northwest of Saudi Arabia. The region is characterized by its hyper-arid climate and occasional low precipitations during December to February coupled with high evaporation rate. Plants were collected from four different locations of the region i.e. Jabal Al-Lawz (28°39′N, 35°18′E), Jabal Al-Harrah (23°5′N, 39°47′E), Jordan Road (28°56′N, 36°19′E) and Tabuk City (28°22′N, 36°36'E), which are located at an elevation of 2580 m above sea level (m asl: high), 1370 m asl (medium) and 760 m asl (low) respectively. Plant species from Tabuk City were collected within the periphery of 15 km. The purpose of plant collection from Jordan Road and Tabuk City was to compare the performance of plants growing at same elevations but at different locations. Environmental data of each collection site, shown in Table 1, were provided by the department of civil engineering, university of Tabuk, Saudi Arabia. Five plant species namely A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam were collected at flowering stage from above mentioned four sites. At each collection site four 30 m × 30 m plots, at a distance of 20 m each, were established randomly. Total 16 plots were organized during the study. Four replicates of each plant from each plot were collected randomly and four healthy plants were finally selected for assessing following parameters. 2.2. Growth parameters Growth parameters for the above mentioned plants species were assessed in terms of plant height, plant fresh weight and dry weight. For fresh weight, plants were uprooted and washed to remove surface adhered soil particles then the water was blotted by wrapping
2.3.4. Leaf chlorophyll (Chl) content Total chlorophyll content in the leaves was estimated according to Lichtenthaler and Buschmann [17]. The optical density (OD) of the pigment solution was recorded at 662 and 645 nm to determine chlorophyll a and chlorophyll b content, respectively, using a spectrophotometer (CE 2021, Cecil, Cambridge, England). Total chlorophyll content was assessed by totaling chlorophyll a and b contents. The photosynthetic pigment, thus measured, was expressed as mg g−1 leaf FW. 2.3.5. Leaf relative water content (LRWC) To evaluate water status of plants, leaf relative water content (LRWC) was measured by the method of Yamasaki and Dillenburg [18]. For each treatment 10 pieces of leaves were taken. These leaf pieces were used to measure fresh mass (FM), turgid mass(TM), and dry mass (DM). The values for FM, TM and DM were used to calculate LRWC using the equation below. LRWC ð%Þ ¼ ½ðFM−DMÞ=ðTM−DMÞ 100
2.3.6. Assay of antioxidant enzymes Leaf tissues were homogenized with three volumes (w/v) of an icecold extraction buffer (50 mM Tris–HCl, pH 7.8, 1 mM EDTA, 1 mM MgCl2 and 1.5% (w/w) polyvinylpyrrolidone). The homogenate was centrifuged at 15,000 g for 20 min at 4 °C. The supernatant was used as the crude extract for the assay of enzyme activities in unit g−1 leaf FW. Activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was determined according to Beauchamp and Fridovich [19] by following the photo-reduction of nitro blue tetrazolium (NBT). Non-illuminated and
Table 1 Climatic characteristics of four collection sites. Collection sites
Jabal Al-Lawz Jabal Al-Harrat Jordan Road Tabuk City
Characteristics Elevation (m asl)
Maximum temperature (°C)
Minimum temperature (°C)
Mean temperature (°C)
Annual rain fall (mm)
Humidity (%)
2580 (high) 1370 (medium) 760 (low) 760 (low)
34.7 42.6 43.8 43.8
−4.3 −2 2.1 2.1
16.6 19.4 20.4 20.4
38 40 46 46
31.49 35.26 33.63 33.63
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
illuminated reactions without supernatant served as calibration standards. The absorbance of the solution was measured at 560 nm. Activity of peroxidase (POX: E.C. 1.11.1.7) was assayed by the method of Upadhyaya et al. [20]. The increase in absorbance at 420 nm was recorded. Catalase (CAT: E.C. 1.11.1.6) activity was measured according to Cakmak and Marschner [21]. The decline in absorbance at 240 nm due to the decline of extinction of H2O2 was recorded. 2.3.7. Estimation of secondary metabolites Dry leaf material (1 g) was used for the estimation of artemisinin content modified to a compound Q260 and quantified using HPLC method [22]. Alkaloid extraction was performed by adopting the method of Hadi and Bremner [23]. Plant leaves were used for the estimation of flavanoid content according to aluminum chloride colorometric method of Djeridane et al. [24], essential oil content by hydrodistillation method using Clevenger apparatus. 2.4. Statistical analysis Data were analyzed statistically by using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). In applying the F-test, the error due to replicates was also determined. When F value was found to be significant at 5% level of probability, least significant difference (LSD) was calculated. There were four replicates for each treatment. 3. Results It is evident from the results that medicinal plants, collected from different locations of Tabuk region, exhibited diverse pattern of growth, physiological and biochemical attributes. 3.1. Effect of altitudinal variations on the growth parameters Plants of all the five species viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam collected from Jabal Al-Harrah (medium level: 1370 m asl) were 63.4, 49.7, 25.7, 72.6 and 207.4 cm in height, respectively, whereas the same species collected from Jabal Al-Lawz (high level: 2580 m asl) were 45.7, 32.5, 18.6, 63.4 and 180.2 cm in height, respectively. However, the plants collected from Jordan Road (low level: 760 m asl) and Tabuk City (low level: 760 m asl) did not differ significantly (Fig. 1A). Regarding fresh and dry weight, plants collected from medium elevation of Jabal Al-Harrah (1370 m asl) gave highest values for these parameters (Fig. 1B and C), whereas, these plants from higher level of Jabal Al-Lawz (2580 m asl) gave lowest values. The plants A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam from medium elevation (1370 m asl) of Jabal Al-Harrah gave 70.33, 39.76, 44.59, 65.41 and 529.50 g fresh weight and 21.56, 10.15, 12.28, 17.22 and 134.17 g dry weight respectively. However, these plant species collected from higher level (2580 m asl), gave 56.12, 31.09, 38.54, 54.29 and 478.22 g fresh weight and 13.09, 6.62, 9.96, 11.92 and 118.47 g dry weight, respectively (Fig. 1B and C). 3.2. Effect of altitudinal variations on the physiological and biochemical parameters 3.2.1. Hydrogen peroxide (H2O2) content A concomitant increase in H2O2 content was noted with increasing elevation from 760 to 2580 m asl (Fig. 1D). On the other hand, plants grown at medium elevation of Jabal Al-Harrah (1370 m asl) gave minimum values for H2O2 content. All the five plants namely A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium level gave 28.5%, 26.3%, 22.1%, 23.5% and 22.6% lower H2O2 content respectively than the same corresponding plants grown at higher level which gave highest values for H2O2 content (Fig. 1D).
143
3.2.2. Leaf protein content The data show that leaf protein content increased from lower to medium level, however, at higher level of Jabal Al-Lawz (2580 m asl), a decrease was recorded (Fig. 1E). At medium elevation of Jabal Al-Harrah (1370 m asl), an increase of 54.2%, 54.5%, 49.5%, 65.1% and 47.0% in leaf protein content was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam, respectively than the plants grown at higher elevation (Jabal Al-Lawz). However, all the plants grown at the lower elevations (Jordan Road and Tabuk City: 760 m asl) gave statistically similar performance except A. fragrantissima and L. pubescens (Fig. 1E). 3.2.3. Carbonic anhydrase (CA) activity Perusal of data (Fig. 2A) shows that all the five plants gave highest activity of CA enzyme on medium elevation of Jabal Al-Harrah (1370 m asl), whereas, least enzyme activity in all the plant species was recorded from higher elevation of Jabal Al-Lawz (2580 m asl). The plant species A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam from medium elevation of JabalAl-Harrah gave 13.7%, 11.0%, 9.5%, 9.6% and 12.4% higher values than the same plant species collected from higher level of Jabal Al-Lawz (2580 m asl), which gave lowest enzyme activity. However, plant from Jordan Road and Tabuk City showed almost equal values for CA activity (Fig. 2A). 3.2.4. Leaf chlorophyll (Chl) content We noted an increase of 71.9%, 44.2%, 55.0%, 31.8% and 44.6% in leaf Chl content in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam respectively in the plants collected from Jabal Al-Harrah (medium level: 1370 m asl) than the plants from Jabal Al-Lawz (higher level: 2580 m asl) (Fig. 2B). However, these plants from Jordan Road and Tabuk City (lower level: 760 m asl) showed a significant difference and gave 19.0% and 13.7%, 14.3% and 5.8%, 13.6% and 5.7%, 23.9% and 6.3% and 9.6% and 5.7% higher values respectively than the same plant species collected from higher elevation of Jabal Al-Lawz (Fig. 2B). 3.2.5. Leaf relative water content (LRWC) Plants growing at lower elevations (760 m asl) of Jordan Road registered maximum leaf relative water content (LRWC) followed by Tabuk City (760 m asl), whereas, plants grown at higher elevation of Jabal AlLawz (2580 m asl) exhibited minimum LRWC (Fig. 2C). A significant increase of 68.1%, 72.4%, 60.7%, 31.6% and 79.4% was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam, respectively at the lower elevation of Jordan Road than the same plants collected at higher elevation (Jabal Al-Lawz) that gave minimum values for LRWC for the investigated species (Fig. 2C). 3.2.6. Effect of altitudinal variation on the activities of antioxidant enzymes All the plants species grown at medium elevation of Jabal Al-Harrah (1370 m asl) showed highest values for SOD enzyme, while the plants grown at lower elevation of Jordan Road and Tabuk City (760 m asl), being non-significant, gave the lowest values. The five plants viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium elevation of Jabal Al-Harrah gave 19.0%, 23.0%, 21.1%, 11.3% and 10.8% more SOD activity, respectively than the plants grown at lower level of Jordan Road (Fig. 2D). As shown in Fig. 2E, the plants growing at medium level (Jabal AlHarrah) and lower level (Tabuk City) showed maximum and minimum POX activity, respectively. A significant increase of 73.2%, 56.2%, 59.2%, 76.0% and 79.3% in POX activity was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium elevation of Jabal Al-Harrah as compared with the plants grown at the lower elevation of Tabuk City. Catalase (CAT) activity also showed a similar trend as shown by SOD and POX. An enhancement in CAT activity was recorded from lower to medium elevation and again a reduction in enzyme activity was observed at higher elevation (Fig. 2F). A. judaica, A. fragrantissima,
144
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
Fig. 1. Effect of varying elevations on plant height (A), fresh weight (B) and dry weight (C), H2O2 content (D) and leaf protein content (E) of Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. (Average of four determinations is presented with T bars indicating S.E.)
T. polium, L. pubescens and R. raetam, grown at medium level (Jabal AlHarrah) gave 60.8%, 61.7%, 137.9%, 113.4% and 137.6% higher activity of CAT, respectively than the plants grown at lower level of Jordan Road (Fig. 2F). 3.2.7. Effect of altitudinal variation on secondary metabolites An increase in secondary metabolites was recorded with increase in elevation. The results showed the presence of highest level of artemisinin
content in A. judiaca grown at higher elevation of Jabal Al-Lawz (2580 m asl), which gave 13.11% more artemisinin content than the same species grown at lower elevation of Tabuk City (760 m asl) which gave lowest value (Fig. 3A). In A. fragrantissima, grown at Jabal Al-Lawz, 66.2% more alkaloid production was recorded than the plants grown at lower elevation of Jordan Road (760 m asl) exhibiting lowest alkaloid accumulation (Fig. 3B). Fig. 3C shows that highest accumulation of total flavonoids in T. polium was also recorded at higher elevation of Jabal Al-Lawz, which
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
145
Fig. 2. Effect of varying elevations on CA activity (A), leaf chlorophyll content (B), leaf relative water content(C) and activities of SOD (D), POX (E) and CAT (F) of Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. (Average of four determinations is presented with T bars indicating S.E.)
was 52.9% more than the plants collected at lower level (Jordan Road). Essential oil content was extracted from the leaves of L. pubescens. Maximum oil content was obtained from L. pubescens plants collected from Jabal Al-Lawz, whereas, minimum oil content was extracted from the plants grown at the lower level of Tabuk City. L. pubescens grown at higher elevation (Jabal Al-Lawz) gave 69.9% more oil than the plants collected from Tabuk City (Fig. 3D). Alkaloid production from R. raetam showed an increasing trend with increasing elevation. Alkaloid content in R. raetam grown at Jabal Al-Lawz was 77.8% higher than the plants collected from lower elevation of Tabuk City (Fig. 3E). Therefore, it is evident from the results that artemisinin, falvonoids, alkaloids and essential oil production showed a concomitant increase with increasing altitude (Fig. 3A−E).
4. Discussion The present study explores the response of five medicinal plants to varying environmental condition of different elevations of Tabuk region. All the five plant species studied viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam showed an improvement in growth parameters from lower to medium elevation, however, at higher elevation a significant decrease in these growth attributes was recorded. These results corroborate the findings of Prakash et al. [25] who observed a concomitant increase in morphological characteristics of plants up to certain elevation and beyond that a sudden decrease was recorded. A decrease in plant height may serve as an adaptation to environmental conditions of higher altitude that may facilitate plants to evade
146
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
Fig. 3. Effect of varying elevations on artemisinin content in Artemisia judaica (A), total alkaloids in Achillea fragrantissima (B), total flavanoids in Teucrium polium (C), essential oil content in Lavandula pubescens (D) and alkaloid content in Retama raetam (E). (Average of four determinations is presented with T bars indicating S.E.)
damaging effects of high altitude winds and to keep leaves closer to warmer soil surface [26]. Moreover, reduced plant height enabled the plants to bear less number of leaves which causes poor orientation and reduced surface area that lays down the plants to harvest lesser solar energy and ultimately reduced dry matter accumulation of studied plants. Our results also strengthen the findings of Ran et al. [27] who observed that transplantation of Abies faxoniana seedlings to a lower altitude had a significant positive effect on total biomass accumulation, while a decrease in biomass was recorded in the seedlings transplanted to a higher altitude. They also concluded that growth in height and lateral branchlet length were significantly affected by the altitude, while partitioning of biomass to roots was mainly genetically controlled. Our results corroborate the findings of Keveshk [1], who observed that Euphorbia macrostegia plants at higher altitude exhibit a decline in stem height, stem diameter, wet biomass and dry biomass than the plants at lower altitude. Therefore,
our study confirms that growth and biomass accumulation decreases with increasing altitude. Increase in elevation induced H2O2 content (Fig. 1D), one of the damaging ROS. The plant system comes under oxidative stress when scavenging system lags behind the generation of ROS, which causes damage to proteins, lipids and DNA [28]. UV radiation and low temperature are important stresses for the excessive production of H2O2 in plants [29], and chloroplasts are thought to be the major sites for H2O2 production. Reduced CO2 partial pressure at higher altitudes impedes CO2 fixation that accelerates transfer of electrons from photosystem I to molecular oxygen resulting in enhanced production of H2O2 [30].However, to cope with oxidative stress plants are equipped with antioxidant enzymes such as SOD, POX, and CAT. These enzymes prevent or alleviate the damage caused by ROS and set the plants to perform normally even under stressful conditions. The results exhibit that SOD, POX and CAT show a parallel increase in their activities with
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
increasing altitude accompanied with decreased level of H2O2 content in the plants. Protective effect of antioxidant enzymes on plants is clearly confirmed by the improved growth, dry matter and protein accumulation, CA activity and leaf Chl content. Therefore, enhanced activity of antioxidant enzymes plays a vital role in the protection of plants against high elevation-induced stress. Higher activity of SOD, CAT and ascorbate peroxidase in response to photo-oxidative stress was also reported by Vuleta et al. [31] in Iris pumila leaves. Proteins are one of the vital components of any biological system. Therefore, accumulation of proteins is of considerable importance for survival of plants under stressful conditions of high mountains. Our results corroborate the findings of Li et al. [32] who observed increased accumulation of protein at high altitude. However, Sharaf et al. [33] observed highly significant differences in protein content at different altitudes; they recorded highest protein content in T. polium at elevation from 2200 to 2400 m and minimum values at elevation from 2400 to 2600 m. It has been established that accumulated proteins fulfill additional energy requirement in response to environmental stress and serve as antioxidant enzymes, defense responsive and photosynthesisrelated proteins [32]. Regarding physiological and biochemical parameters, CA activity was highest in all the plant species collected from medium elevation, whereas a significant decrease in enzyme activity was recorded at higher elevation. The enzyme CA which catalyzes reversible hydration of CO2 and maintained its constant supply to rubisco, resulted in optimum rate of photosynthesis and thus more dry matter accumulation. As high altitudes are characterized by low partial pressure and restricted conductance of CO2, lower availability of CO2 contributes to limited CA activity which in turn limits photosynthesis [34], as reflected by reduced dry matter accumulation at higher altitude (Fig. 1C).Therefore, increased CA activity might have been one of the reasons for improved growth performance of plants at lower elevation. Chl content is another important factor that contributes to the growth and yield performance of a plant. A progressive increase in Chl content was recorded from lower to medium elevation, whereas, a sudden decline was noted at higher level (Fig. 2B). At higher altitude, plants are exposed to high light, UV-B radiation, low temperature, large diurnal temperature variations and low partial pressures of O2 and CO2 which adversely affect chlorophyll biosynthesis [35]. Moreover, chlorophyllase is a chloroplast membrane associated enzyme that has been considered to be involved in degradation of chlorophyll molecule through catalyzing dephytilation reaction. Enhanced activity of chlorophyllase has been recorded in plants exposed to several environmental stresses [36]. Our results are in agreement with the findings of Wingler et al. [37] who recorded a decrease in Chl content with increasing altitude. A significant decrease in LRWC was recorded with a concomitant increase in altitude from 760 to 2580 m asl. It has been reported that stomatal density increases with decreasing partial pressure of CO2 at higher elevations [38]. Increase in the number of stomata culminates in enhanced stomatal conductance which accelerates the rate of transpiration, as witnessed by lower value of LRWC in the plants of higher altitude (Fig. 2C). Moreover, enhanced absorption of radiation by leaves, increase in the diffusion coefficient of water vapor in air, and increased density gradient of water vapor from the leaf to the ambient air are the major factors behind the potential increase of transpiration with altitude. High wind velocity, large diurnal fluctuations in temperature and limited supply of water also contribute to low level of LRWC at higher altitudes. On the other hand, plants from higher elevation showed a decline in the activities of antioxidant enzymes resulting in increased H2O2 accumulation and thus weak protection to the plants. It is well known that SOD dismutates superoxide radicals to H2O2, whereas, CAT and POX are involved in converting H2O2 into water and oxygen. Therefore, excessive accumulation of H2O2 may be either due to enhanced activity of SOD and/or reduced activities of CAT and POX. However, results show reduced activities of all the three enzymes, therefore, CAT and
147
POX jointly contributed to excessive accumulation of H2O2 content. Moreover, a significant decrease in leaf protein content (Fig. 1E) could also contribute to a decrease in scavenging enzyme activity. The inhibition in antioxidant enzyme activities with increase in altitude was also recorded by Wang et al. [39]. High mountain plants are equipped with another defense system which gives protection against UV radiations at higher altitudes. Such a system is orchestrated by altered synthesis of secondary metabolites such as phenolics, flavanoids, and essential oils. A concomitant increase in secondary metabolites (alkaloids, flavonoids and essential oil content) was recorded from 760 to 2580 m height (Figs. 3A–E). Although, exposure of plants to UV radiations induces generation of toxic free radicals, but at the same time UV radiations has been shown to activate biosynthesis of secondary metabolites, which play an important role in the protection of plants against harmful radiations. These secondary metabolites possess the capacity to absorb UV radiation and thus, confines UV radiation away from inner cell layers [40].Luis et al. [41],observed a twofold increase in total phenolic contents of UV-B exposed rosemary plants compared with the plants grown without UV-B. A significant increase in essential oil content in L. pubescens was recorded at the higher elevation of 2580 m (Fig. 3D). The proclamation, that plants at higher altitude produce more essential oil as one of the defensive strategies against low temperature and UV radiation, is supported by the studies of Ioannidis [42] and Lianopoulou et al. [43]. It is evident from results that plants growing at higher altitude possess excessive amounts of secondary metabolites which provide an additional defense against various stresses and enable the plants to perform normally under harsh environmental conditions of higher elevations. 5. Conclusion The assessment of results shows that the plants grown at lower and medium elevation performed well in terms of growth, physiological and biochemical parameters because of the protection given by the antioxidant enzymes. However, at higher elevation plants exhibited higher values for secondary metabolites. Therefore, to put all in a nut shell it can be concluded that plants at lower and medium elevation were more protected by antioxidant enzymes than the secondary metabolites, however, reverse is true for the plants of higher elevation. Acknowledgment The support given by the Deanship of Scientific Research (DSR), University of Tabuk, Saudi Arabia (project number S-0082-1434) is gratefully acknowledged. References [1] N.M. Keveshk, Investigation of effect of altitude in some morphological characteristics and yield in Euphorbia macrostegia, Int. J. Agric. Crop Sci. 5 (16) (2013) 1779–1783. [2] A.D. Richardson, G.P. Berlyn, T.G. Gregoire, Spectral reflectance of Picea rubens (Pinaceae) and Abies balsamea (Pinaceae) needles along an elevational gradient, Mt. Moosilauke, New Hampshire, USA, Am. J. Bot. 88 (4) (2001) 667–676. [3] C. Li, G. Xu, R. Zang, et al., Sex-related differences in leaf morphological and physiological responses in Hippophae rhamnoides along an altitudinal gradient, Tree Physiol. 27 (3) (2007) 399–406. [4] K. Asada, Radical production and scavenging in the chloroplasts, in: N.R. Baker (Ed.), Advances in Photosynthesis, Vol. 5: Photosynthesis and the Environment, Kluwer Academic Publishers, Dordrecht 1996, pp. 123–150. [5] I.M. Moller, P.E. Jensen, A. Hansson, Oxidative modifications to cellular components in plants, Annu. Rev. Plant Biol. 58 (2007) 459–481. [6] H. Lantemona, A.L. Abadi, A. Rachmansyah, J. Pontoh, Impact of altitude and seasons to volume, brix content, and chemical composition of aren sap in north Sulawesi, J. Environ. Sci. Toxicol. Food Tech. 4 (2) (2013) 42–48. [7] M.R. Badger, G.D. Price, The role of carbonic anhydrase in photosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45 (1994) 369–392. [8] D. Peltzer, E. Dreyer, A. Polle, Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei, Plant Physiol. Biochem. 40 (2) (2002) 141–150.
148
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
[9] K.G. Ramawat, S. Goyal, The Indian herbal drugs scenario in global perspectives, in: K.G. Ramawat, J.M. Merillon (Eds.), Bioactive Molecules and Medicinal Plants, Springer, Berlin Heidelberg New York 2008, p. 323. [10] O. Said, K. Khalil, S. Fulder, H. Azaizeh, Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region, J. Ethnopharmacol. 83 (3) (2002) 251–265. [11] J.S. Mossa, M.A. Al-Yahya, I.A. Al-Meshal, Medicinal Plants of Saudi Arabia, King Saud University Press, Riyadh, 2000. [12] M.M. Algandaby, H.A. Alghamdi, O.M. Ashour, et al., Mechanisms of the antihyperglycemic activity of Retama raetam in streptozotocin-induced diabetic rats, Food Chem. Toxicol. 48 (8–9) (2010) 2448–2453. [13] H. Edziri, M. Mastouri, I. Cheraif, M. Aouni, Chemical composition and antibacterial, antifungal and antioxidant activities of the flower oil of Retama raetam (Forssk.) Webb from Tunisia, Nat. Prod. Res. 24 (9) (2010) 789–796. [14] V. Velikova, I. Yordanov, A. Edreva, Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines, Plant Sci. 151 (1) (2000) 59–66. [15] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Ann. Biochem. 72 (1–2) (1976) 248–254. [16] R.S. Dwivedi, N.S. Randhawa, Evaluation of rapid test for hidden hunger of zinc in plants, Plant Soil 40 (2) (1974) 445–451. [17] H.K. Lichtenthaler, C. Buschmann, Chlorophylls and carotenoids: measurement and characterization by UV–vis spectroscopy, Current Protocols in Food Analytical Chemistry, John Wiley and Sons, New York 2001, pp. F4.3.1–F4.3.8. [18] S. Yamasaki, L.C. Dillenburg, Measurements of leaf relative water content in Araucaria angustifolia, Rev. Bras. Fisiol. Veg. 11 (2) (1999) 69–75. [19] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Ann. Biochem. 44 (1) (1971) 276–287. [20] A. Upadhyaya, D. Sankhla, T.D. Davis, et al., Effect of paclobutrazol on the activities of some enzymes of activated oxygen metabolism and lipid peroxidation in senescing soybean leaves, J. Plant Physiol. 121 (5) (1985) 453–461. [21] I. Cakmak, H. Marschner, Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves, Plant Physiol. 98 (4) (1992) 1222–1227. [22] S.S. Zhao, M.Y. Zeng, Determination of qinghaosu in Artemisia annua L. by high performance liquid chromatography, J. Pharma. Anal. 6 (1986) 3–5. [23] S. Hadi, b. Bremner, Initial studies on alkaloids from Lombok medicinal plants, Molecules 6 (2) (2001) 117–129. [24] A. Djeridane, M. Yousfi, B. Nadjemi, et al., Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds, Food Chem. 97 (4) (2006) 654–660. [25] V. Prakash, H. Bisht, P. Prasad, Altitudinal variation in morpho-physiological attributes in Plantago major: selection of suitable cultivation site, Res. J. Med. Plants 5 (3) (2011) 302–311. [26] C. Körner, P. Chochrane, Influence of leaf physiognomy on leaf temperature on clear midsummer days in the snowy mountains, south-eastern Australia, Acta Oecol. 4 (2) (1983) 117–124. [27] F. Ran, X. Zhang, Y. Zhang, et al., Altitudinal variation in growth, photosynthetic capacity and water use efficiency of Abies faxoniana Rehd. et Wils. seedlings as revealed by reciprocal transplantations, Trees 27 (5) (2013) 1405–1416.
[28] K. Apel, H. Hirt, Reactive oxygen species: metabolism, oxidative stress, and signal transduction, Annu. Rev. Plant Biol. 55 (2004) 373–399. [29] E. Georgieva, D. Petrova, Z. Yordanova, Influence of cryopreservation on the antioxidative activity of in vitro cultivated Hypericum species, Biotechnol. Biotechnol. Equip. 28 (5) (2014) 863–870. [30] K. Asada, The water–water cycle in chloroplasts: scavenging of active oxygen species and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601–639. [31] A. Vuleta, S. ManitaševićJovanović, B. Tucić, How do plants cope with oxidative stress in nature? A study on the dwarf bearded iris (Iris pumila), Acta Physiol. Plant. 37 (2014) 1711. [32] X. Li, Y. Yang, L. Ma, et al., Comparative proteomics analyses of Kobresia pygmaea adaptation to environment along an elevational gradient on the central Tibetan plateau, PLoS One 9 (6) (2014), e98410. [33] A.E.A. Sharaf, Om-M.A. Khafagi, E.-B.E. Hatab, M.M. Moursy, Effect of altitudinal gradients on the content of carbohydrate, protein, proline and total phenols of some desert plants in Saint Katherine Mountain, South Sinai, Egypt, Middle-East J. Sci. Res. 14 (1) (2013) 122–129. [34] A.J. Studer, A. Gandin, A.R. Kolbe, et al., A limited role for carbonic anhydrase in C4 photosynthesis as revealed by a ca1ca2 double mutant in maize, Plant Physiol. 165 (2) (2014) 608–617. [35] Y. Li, D. Yang, S. Xiang, G. Li, Different responses in leaf pigments and leaf mass per area to altitude between evergreen and deciduous woody species, Aust. J. Bot. 61 (6) (2013) 424–435. [36] D.T. Todorov, E.N. Karanov, A.R. Smith, M.A. Hall, Chlorophyllase activity and chlorophyll content in wild and mutant plants of Arabidopsis, Biol. Plant. 46 (1) (2003) 125–127. [37] A. Wingler, M. Juvany, C. Cuthbert, S. Munné-Bosch, Adaptation to altitude affects the senescence response to chilling in the perennial plant Arabis alpina, J. Exp. Bot. 66 (1) (2015) 355–367. [38] F.I. Woodward, C.K. Kelly, The influence of CO, concentration on stomatal density, New Phytol. 131 (3) (1995) 311–327. [39] Y. Wang, W. He, H. Huang, et al., Antioxidative responses to different altitudes in leaves of alpine plant Polygonum viviparum in summer, Acta Physiol. Plant. 31 (4) (2009) 839–848. [40] C.A. Kolb, M.A. Käser, J. Kopecký, et al., Effects of natural intensities of visible and ultraviolet radiation on epidermal ultraviolet screening and photosynthesis in grape leaves, Plant Physiol. 127 (3) (2001) 863–875. [41] J.C. Luis, R.M. Perez, F.V. Gonzalez, UV-B radiation effects on foliar concentrations of rosmarinic and carnosic acids in rosemary plants, Food Chem. 101 (3) (2007) 1211–1215. [42] D. Ioannidis, L. Bonner, C.B. Johnson, UV-B is required for normal development of oil glands in Ocimum basilicum L. (sweet basil), Ann. Bot. 90 (4) (2002) 453–460. [43] V. Lianopoulou, A.M. Bosabalidis, A. Patakas, et al., Effects of chilling stress on leaf morphology, anatomy, ultra structure, gas exchange, and essential oils in the seasonally dimorphic plant Teucrium polium (Lamiaceae), Acta Physiol. Plant. 36 (8) (2014) 2271–2281.
Contents lists available at ScienceDirect
Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes
Impact of varying elevations on growth and activities of antioxidant enzymes of some medicinal plants of Saudi Arabia M. Nasir Khan ⁎, M. Mobin, Zahid Khorshid Abbas, Khalid A. ALMutairi Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 30 December 2015 Accepted 31 December 2015 Keywords: Antioxidant enzymes Elevations Medicinal plants Secondary metabolites
a b s t r a c t Tabuk is the northwestern province of Saudi Arabia which is characterized by highly variable climatic conditions that ranges from extreme cold to extreme hot which not only supports the growth of a huge variety of medicinally important plants but also modulates the inherent medicinal potential of these plants. Keeping these points in view, the present work was designed to evaluate the effect of climatic conditions on growth physiological and biochemical attributes of five medicinal plants namely, Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. We collected each plant species from four different elevations of Tabuk region namely Jabal Al-Lawz (2580 m asl: higher elevation), Jabal Al-Harrah (1370 m asl; medium elevation), Jordan Road (760 m asl; lower elevation) and Tabuk City (760 m asl; lower elevation). The plants growing at medium elevation of 1370 m exhibited better growth and physiological performance than the others that may be ascribed to enhanced leaf water, Chl and protein content as we recorded for these plants. Maximum and minimum accumulation of secondary metabolites corresponded to the plants grown at the higher and lower altitude, respectively. The antioxidant enzyme activities were highest at 1370 m asl that might have contributed to the improved growth and physiological attributes. Consequently, the results obtained in this study strongly suggest that plants grown at medium elevation of 1370 m asl possess higher activity of antioxidant enzymes, whereas, plants at higher elevation of 2580 m are best for medicinal use because they accumulated higher level of secondary metabolites. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Tabuk region, the northwestern province of Saudi Arabia, is characterized by its diversified topography ranging from plains to the mountains of low and high elevations which create highly variable environmental conditions encompassing through extreme cold to extreme hot climate. Diversified topography coupled with varied environmental conditions supports the growth of an array of medicinal plants. Elevations drastically influence environmental factors, such as quick seasonal and daily variations in temperature, low atmospheric pressure, and depressed CO2 concentration, short period of vegetation and enhanced intensity of solar UV radiation, which set the plants under serious stress. Plants show a significant morphological, physiological and biochemical responses to increasing altitude, which include decrease in stem height, stem diameter, biomass production [1], specific leaf area, and increases in the leaf thickness [2,3]. High light intensities at higher altitude induce excessive accumulation of reactive oxygen species (ROS) leading to pigment bleaching, lipid peroxidation, protein damage, inactivation of enzyme activities and ultimately cell death [4–6]. The enzyme carbonic anhydrase (CA) is found in ⁎ Corresponding author. E-mail address: [email protected] (M.N. Khan).
http://dx.doi.org/10.1016/j.chnaes.2015.12.009 1872-2032/© 2016 Elsevier B.V. All rights reserved.
abundance in the photosynthesizing tissues of both C3 and C4 plants and regulates the availability of CO2 to ribulose bisphosphate carboxylase (rubisco) by catalyzing the reversible hydration of CO2 [7]. Higher altitude restricts uptake of CO2 that consequently decreases carbon assimilation due to non-availability of oxidized NADP+ for acceptance of electrons during photosynthesis, a significant factor accorded to the formation of ROS [8]. Therapeutic use of plants is as old as human civilization and medicinal plants are integral part of healthcare system which is witnessed by the fact that 80% of the population still relies on a traditional system of medicine, based on herbal drugs [9]. Because of no side effects, the interest in medicinal products of plant has increased considerably all over the world. Of these, Artemisia judaica L. (family Asteraceae) called as ‘wormwood’ is a perennial shrub with pubescent and fragrant leaves is a highly resistant plant against adverse environmental conditions and very effective in stabilizing the habitat. Achillea fragrantissima (family Asteraceae), has been extensively used in Arabian folk medicine due its hypoglycemic properties. Teucrium polium L. (family Lamiaceae) is one of the most common medicinal plants of Arabian Peninsula that has been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, antipyretic, antispasmodic and cholagogic properties [10]. Lavandula pubescens Decne. (family Lamiaceae), is well known for its essential and aromatic oil, used in cosmetics while
142
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
decoction of leaves is given in headache and cold [11]. Retama raetam (Forssk.) Webb & Berthel. (family Fabaceae), commonly known as ‘raetam’ or ‘broom bush’ is a shrub that grows widely in Saudi Arabian deserts. The plant possesses antibacterial, antifungal, antihypertensive, anti-oxidant, antiviral, diuretic and hypoglycemic properties [12,13]. R. raetam has long been used as abortifacient, purgative and a vermifuge in the traditional system of medicine. All these five plants viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam are commonly grown in all parts of Tabuk region and are extensively used in the traditional system of medicine of the region. Although extensive research has been carried out on the altitudinal variation in morphological, physiological and biochemical characteristics of tree plants, but meager information is available on the changes in growth, lipid peroxidation, activities of antioxidant enzymes and on the accumulation of secondary metabolites of medicinal plant species growing under stressful conditions at various altitudes. Considering the importance of above mentioned plants in view, the present study was performed to test the hypothesis that variation in altitude significantly affects the activities of antioxidant enzymes and secondary metabolite concentration which plays a role in the acclimation of plants to abiotic stress induced impairment in plants. 2. Materials and methods
the plants in blotting papers. After removing the plants from blotting papers fresh weight for each plant was recorded. Dry weight of plants was recorded after drying the plants at 80 °C for 24 h in a hot air oven. 2.3. Physiological and biochemical parameters 2.3.1. Determination of hydrogen peroxide (H2O2) content The hydrogen peroxide (H2O2) content in fresh leaf samples was determined according to Velikova et al. [14]. The content of H2O2 was calculated by comparison with a standard calibration curve, plotted by using different concentrations of H2O2. The absorbance of supernatant was recorded at 390 nm. H2O2 content was expressed as μ mol g− 1 leaf FW. 2.3.2. Estimation of leaf protein content Protein content was measured according to Bradford [15] using bovine serum albumin as standard. The absorbance was read spectrophotometrically (CE 2021, Cecil, Cambridge, England) at 595 nm. 2.3.3. Determination of carbonic anhydrase (CA) activity The activity of carbonic anhydrase (CA: E.C. 4.2.1.1) was measured using titration method of Dwivedi and Randhawa [16]. The reaction mixture was titrated against HCl using methyl red as indicator. The enzyme was expressed as μM CO2 kg−1 leaf FW s−1.
2.1. Study area The study was conducted in Tabuk region, located in the northwest of Saudi Arabia. The region is characterized by its hyper-arid climate and occasional low precipitations during December to February coupled with high evaporation rate. Plants were collected from four different locations of the region i.e. Jabal Al-Lawz (28°39′N, 35°18′E), Jabal Al-Harrah (23°5′N, 39°47′E), Jordan Road (28°56′N, 36°19′E) and Tabuk City (28°22′N, 36°36'E), which are located at an elevation of 2580 m above sea level (m asl: high), 1370 m asl (medium) and 760 m asl (low) respectively. Plant species from Tabuk City were collected within the periphery of 15 km. The purpose of plant collection from Jordan Road and Tabuk City was to compare the performance of plants growing at same elevations but at different locations. Environmental data of each collection site, shown in Table 1, were provided by the department of civil engineering, university of Tabuk, Saudi Arabia. Five plant species namely A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam were collected at flowering stage from above mentioned four sites. At each collection site four 30 m × 30 m plots, at a distance of 20 m each, were established randomly. Total 16 plots were organized during the study. Four replicates of each plant from each plot were collected randomly and four healthy plants were finally selected for assessing following parameters. 2.2. Growth parameters Growth parameters for the above mentioned plants species were assessed in terms of plant height, plant fresh weight and dry weight. For fresh weight, plants were uprooted and washed to remove surface adhered soil particles then the water was blotted by wrapping
2.3.4. Leaf chlorophyll (Chl) content Total chlorophyll content in the leaves was estimated according to Lichtenthaler and Buschmann [17]. The optical density (OD) of the pigment solution was recorded at 662 and 645 nm to determine chlorophyll a and chlorophyll b content, respectively, using a spectrophotometer (CE 2021, Cecil, Cambridge, England). Total chlorophyll content was assessed by totaling chlorophyll a and b contents. The photosynthetic pigment, thus measured, was expressed as mg g−1 leaf FW. 2.3.5. Leaf relative water content (LRWC) To evaluate water status of plants, leaf relative water content (LRWC) was measured by the method of Yamasaki and Dillenburg [18]. For each treatment 10 pieces of leaves were taken. These leaf pieces were used to measure fresh mass (FM), turgid mass(TM), and dry mass (DM). The values for FM, TM and DM were used to calculate LRWC using the equation below. LRWC ð%Þ ¼ ½ðFM−DMÞ=ðTM−DMÞ 100
2.3.6. Assay of antioxidant enzymes Leaf tissues were homogenized with three volumes (w/v) of an icecold extraction buffer (50 mM Tris–HCl, pH 7.8, 1 mM EDTA, 1 mM MgCl2 and 1.5% (w/w) polyvinylpyrrolidone). The homogenate was centrifuged at 15,000 g for 20 min at 4 °C. The supernatant was used as the crude extract for the assay of enzyme activities in unit g−1 leaf FW. Activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was determined according to Beauchamp and Fridovich [19] by following the photo-reduction of nitro blue tetrazolium (NBT). Non-illuminated and
Table 1 Climatic characteristics of four collection sites. Collection sites
Jabal Al-Lawz Jabal Al-Harrat Jordan Road Tabuk City
Characteristics Elevation (m asl)
Maximum temperature (°C)
Minimum temperature (°C)
Mean temperature (°C)
Annual rain fall (mm)
Humidity (%)
2580 (high) 1370 (medium) 760 (low) 760 (low)
34.7 42.6 43.8 43.8
−4.3 −2 2.1 2.1
16.6 19.4 20.4 20.4
38 40 46 46
31.49 35.26 33.63 33.63
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
illuminated reactions without supernatant served as calibration standards. The absorbance of the solution was measured at 560 nm. Activity of peroxidase (POX: E.C. 1.11.1.7) was assayed by the method of Upadhyaya et al. [20]. The increase in absorbance at 420 nm was recorded. Catalase (CAT: E.C. 1.11.1.6) activity was measured according to Cakmak and Marschner [21]. The decline in absorbance at 240 nm due to the decline of extinction of H2O2 was recorded. 2.3.7. Estimation of secondary metabolites Dry leaf material (1 g) was used for the estimation of artemisinin content modified to a compound Q260 and quantified using HPLC method [22]. Alkaloid extraction was performed by adopting the method of Hadi and Bremner [23]. Plant leaves were used for the estimation of flavanoid content according to aluminum chloride colorometric method of Djeridane et al. [24], essential oil content by hydrodistillation method using Clevenger apparatus. 2.4. Statistical analysis Data were analyzed statistically by using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). In applying the F-test, the error due to replicates was also determined. When F value was found to be significant at 5% level of probability, least significant difference (LSD) was calculated. There were four replicates for each treatment. 3. Results It is evident from the results that medicinal plants, collected from different locations of Tabuk region, exhibited diverse pattern of growth, physiological and biochemical attributes. 3.1. Effect of altitudinal variations on the growth parameters Plants of all the five species viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam collected from Jabal Al-Harrah (medium level: 1370 m asl) were 63.4, 49.7, 25.7, 72.6 and 207.4 cm in height, respectively, whereas the same species collected from Jabal Al-Lawz (high level: 2580 m asl) were 45.7, 32.5, 18.6, 63.4 and 180.2 cm in height, respectively. However, the plants collected from Jordan Road (low level: 760 m asl) and Tabuk City (low level: 760 m asl) did not differ significantly (Fig. 1A). Regarding fresh and dry weight, plants collected from medium elevation of Jabal Al-Harrah (1370 m asl) gave highest values for these parameters (Fig. 1B and C), whereas, these plants from higher level of Jabal Al-Lawz (2580 m asl) gave lowest values. The plants A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam from medium elevation (1370 m asl) of Jabal Al-Harrah gave 70.33, 39.76, 44.59, 65.41 and 529.50 g fresh weight and 21.56, 10.15, 12.28, 17.22 and 134.17 g dry weight respectively. However, these plant species collected from higher level (2580 m asl), gave 56.12, 31.09, 38.54, 54.29 and 478.22 g fresh weight and 13.09, 6.62, 9.96, 11.92 and 118.47 g dry weight, respectively (Fig. 1B and C). 3.2. Effect of altitudinal variations on the physiological and biochemical parameters 3.2.1. Hydrogen peroxide (H2O2) content A concomitant increase in H2O2 content was noted with increasing elevation from 760 to 2580 m asl (Fig. 1D). On the other hand, plants grown at medium elevation of Jabal Al-Harrah (1370 m asl) gave minimum values for H2O2 content. All the five plants namely A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium level gave 28.5%, 26.3%, 22.1%, 23.5% and 22.6% lower H2O2 content respectively than the same corresponding plants grown at higher level which gave highest values for H2O2 content (Fig. 1D).
143
3.2.2. Leaf protein content The data show that leaf protein content increased from lower to medium level, however, at higher level of Jabal Al-Lawz (2580 m asl), a decrease was recorded (Fig. 1E). At medium elevation of Jabal Al-Harrah (1370 m asl), an increase of 54.2%, 54.5%, 49.5%, 65.1% and 47.0% in leaf protein content was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam, respectively than the plants grown at higher elevation (Jabal Al-Lawz). However, all the plants grown at the lower elevations (Jordan Road and Tabuk City: 760 m asl) gave statistically similar performance except A. fragrantissima and L. pubescens (Fig. 1E). 3.2.3. Carbonic anhydrase (CA) activity Perusal of data (Fig. 2A) shows that all the five plants gave highest activity of CA enzyme on medium elevation of Jabal Al-Harrah (1370 m asl), whereas, least enzyme activity in all the plant species was recorded from higher elevation of Jabal Al-Lawz (2580 m asl). The plant species A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam from medium elevation of JabalAl-Harrah gave 13.7%, 11.0%, 9.5%, 9.6% and 12.4% higher values than the same plant species collected from higher level of Jabal Al-Lawz (2580 m asl), which gave lowest enzyme activity. However, plant from Jordan Road and Tabuk City showed almost equal values for CA activity (Fig. 2A). 3.2.4. Leaf chlorophyll (Chl) content We noted an increase of 71.9%, 44.2%, 55.0%, 31.8% and 44.6% in leaf Chl content in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam respectively in the plants collected from Jabal Al-Harrah (medium level: 1370 m asl) than the plants from Jabal Al-Lawz (higher level: 2580 m asl) (Fig. 2B). However, these plants from Jordan Road and Tabuk City (lower level: 760 m asl) showed a significant difference and gave 19.0% and 13.7%, 14.3% and 5.8%, 13.6% and 5.7%, 23.9% and 6.3% and 9.6% and 5.7% higher values respectively than the same plant species collected from higher elevation of Jabal Al-Lawz (Fig. 2B). 3.2.5. Leaf relative water content (LRWC) Plants growing at lower elevations (760 m asl) of Jordan Road registered maximum leaf relative water content (LRWC) followed by Tabuk City (760 m asl), whereas, plants grown at higher elevation of Jabal AlLawz (2580 m asl) exhibited minimum LRWC (Fig. 2C). A significant increase of 68.1%, 72.4%, 60.7%, 31.6% and 79.4% was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam, respectively at the lower elevation of Jordan Road than the same plants collected at higher elevation (Jabal Al-Lawz) that gave minimum values for LRWC for the investigated species (Fig. 2C). 3.2.6. Effect of altitudinal variation on the activities of antioxidant enzymes All the plants species grown at medium elevation of Jabal Al-Harrah (1370 m asl) showed highest values for SOD enzyme, while the plants grown at lower elevation of Jordan Road and Tabuk City (760 m asl), being non-significant, gave the lowest values. The five plants viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium elevation of Jabal Al-Harrah gave 19.0%, 23.0%, 21.1%, 11.3% and 10.8% more SOD activity, respectively than the plants grown at lower level of Jordan Road (Fig. 2D). As shown in Fig. 2E, the plants growing at medium level (Jabal AlHarrah) and lower level (Tabuk City) showed maximum and minimum POX activity, respectively. A significant increase of 73.2%, 56.2%, 59.2%, 76.0% and 79.3% in POX activity was recorded in A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam grown at medium elevation of Jabal Al-Harrah as compared with the plants grown at the lower elevation of Tabuk City. Catalase (CAT) activity also showed a similar trend as shown by SOD and POX. An enhancement in CAT activity was recorded from lower to medium elevation and again a reduction in enzyme activity was observed at higher elevation (Fig. 2F). A. judaica, A. fragrantissima,
144
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
Fig. 1. Effect of varying elevations on plant height (A), fresh weight (B) and dry weight (C), H2O2 content (D) and leaf protein content (E) of Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. (Average of four determinations is presented with T bars indicating S.E.)
T. polium, L. pubescens and R. raetam, grown at medium level (Jabal AlHarrah) gave 60.8%, 61.7%, 137.9%, 113.4% and 137.6% higher activity of CAT, respectively than the plants grown at lower level of Jordan Road (Fig. 2F). 3.2.7. Effect of altitudinal variation on secondary metabolites An increase in secondary metabolites was recorded with increase in elevation. The results showed the presence of highest level of artemisinin
content in A. judiaca grown at higher elevation of Jabal Al-Lawz (2580 m asl), which gave 13.11% more artemisinin content than the same species grown at lower elevation of Tabuk City (760 m asl) which gave lowest value (Fig. 3A). In A. fragrantissima, grown at Jabal Al-Lawz, 66.2% more alkaloid production was recorded than the plants grown at lower elevation of Jordan Road (760 m asl) exhibiting lowest alkaloid accumulation (Fig. 3B). Fig. 3C shows that highest accumulation of total flavonoids in T. polium was also recorded at higher elevation of Jabal Al-Lawz, which
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
145
Fig. 2. Effect of varying elevations on CA activity (A), leaf chlorophyll content (B), leaf relative water content(C) and activities of SOD (D), POX (E) and CAT (F) of Artemisia judaica, Achillea fragrantissima, Teucrium polium, Lavandula pubescens and Retama raetam. (Average of four determinations is presented with T bars indicating S.E.)
was 52.9% more than the plants collected at lower level (Jordan Road). Essential oil content was extracted from the leaves of L. pubescens. Maximum oil content was obtained from L. pubescens plants collected from Jabal Al-Lawz, whereas, minimum oil content was extracted from the plants grown at the lower level of Tabuk City. L. pubescens grown at higher elevation (Jabal Al-Lawz) gave 69.9% more oil than the plants collected from Tabuk City (Fig. 3D). Alkaloid production from R. raetam showed an increasing trend with increasing elevation. Alkaloid content in R. raetam grown at Jabal Al-Lawz was 77.8% higher than the plants collected from lower elevation of Tabuk City (Fig. 3E). Therefore, it is evident from the results that artemisinin, falvonoids, alkaloids and essential oil production showed a concomitant increase with increasing altitude (Fig. 3A−E).
4. Discussion The present study explores the response of five medicinal plants to varying environmental condition of different elevations of Tabuk region. All the five plant species studied viz. A. judaica, A. fragrantissima, T. polium, L. pubescens and R. raetam showed an improvement in growth parameters from lower to medium elevation, however, at higher elevation a significant decrease in these growth attributes was recorded. These results corroborate the findings of Prakash et al. [25] who observed a concomitant increase in morphological characteristics of plants up to certain elevation and beyond that a sudden decrease was recorded. A decrease in plant height may serve as an adaptation to environmental conditions of higher altitude that may facilitate plants to evade
146
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
Fig. 3. Effect of varying elevations on artemisinin content in Artemisia judaica (A), total alkaloids in Achillea fragrantissima (B), total flavanoids in Teucrium polium (C), essential oil content in Lavandula pubescens (D) and alkaloid content in Retama raetam (E). (Average of four determinations is presented with T bars indicating S.E.)
damaging effects of high altitude winds and to keep leaves closer to warmer soil surface [26]. Moreover, reduced plant height enabled the plants to bear less number of leaves which causes poor orientation and reduced surface area that lays down the plants to harvest lesser solar energy and ultimately reduced dry matter accumulation of studied plants. Our results also strengthen the findings of Ran et al. [27] who observed that transplantation of Abies faxoniana seedlings to a lower altitude had a significant positive effect on total biomass accumulation, while a decrease in biomass was recorded in the seedlings transplanted to a higher altitude. They also concluded that growth in height and lateral branchlet length were significantly affected by the altitude, while partitioning of biomass to roots was mainly genetically controlled. Our results corroborate the findings of Keveshk [1], who observed that Euphorbia macrostegia plants at higher altitude exhibit a decline in stem height, stem diameter, wet biomass and dry biomass than the plants at lower altitude. Therefore,
our study confirms that growth and biomass accumulation decreases with increasing altitude. Increase in elevation induced H2O2 content (Fig. 1D), one of the damaging ROS. The plant system comes under oxidative stress when scavenging system lags behind the generation of ROS, which causes damage to proteins, lipids and DNA [28]. UV radiation and low temperature are important stresses for the excessive production of H2O2 in plants [29], and chloroplasts are thought to be the major sites for H2O2 production. Reduced CO2 partial pressure at higher altitudes impedes CO2 fixation that accelerates transfer of electrons from photosystem I to molecular oxygen resulting in enhanced production of H2O2 [30].However, to cope with oxidative stress plants are equipped with antioxidant enzymes such as SOD, POX, and CAT. These enzymes prevent or alleviate the damage caused by ROS and set the plants to perform normally even under stressful conditions. The results exhibit that SOD, POX and CAT show a parallel increase in their activities with
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
increasing altitude accompanied with decreased level of H2O2 content in the plants. Protective effect of antioxidant enzymes on plants is clearly confirmed by the improved growth, dry matter and protein accumulation, CA activity and leaf Chl content. Therefore, enhanced activity of antioxidant enzymes plays a vital role in the protection of plants against high elevation-induced stress. Higher activity of SOD, CAT and ascorbate peroxidase in response to photo-oxidative stress was also reported by Vuleta et al. [31] in Iris pumila leaves. Proteins are one of the vital components of any biological system. Therefore, accumulation of proteins is of considerable importance for survival of plants under stressful conditions of high mountains. Our results corroborate the findings of Li et al. [32] who observed increased accumulation of protein at high altitude. However, Sharaf et al. [33] observed highly significant differences in protein content at different altitudes; they recorded highest protein content in T. polium at elevation from 2200 to 2400 m and minimum values at elevation from 2400 to 2600 m. It has been established that accumulated proteins fulfill additional energy requirement in response to environmental stress and serve as antioxidant enzymes, defense responsive and photosynthesisrelated proteins [32]. Regarding physiological and biochemical parameters, CA activity was highest in all the plant species collected from medium elevation, whereas a significant decrease in enzyme activity was recorded at higher elevation. The enzyme CA which catalyzes reversible hydration of CO2 and maintained its constant supply to rubisco, resulted in optimum rate of photosynthesis and thus more dry matter accumulation. As high altitudes are characterized by low partial pressure and restricted conductance of CO2, lower availability of CO2 contributes to limited CA activity which in turn limits photosynthesis [34], as reflected by reduced dry matter accumulation at higher altitude (Fig. 1C).Therefore, increased CA activity might have been one of the reasons for improved growth performance of plants at lower elevation. Chl content is another important factor that contributes to the growth and yield performance of a plant. A progressive increase in Chl content was recorded from lower to medium elevation, whereas, a sudden decline was noted at higher level (Fig. 2B). At higher altitude, plants are exposed to high light, UV-B radiation, low temperature, large diurnal temperature variations and low partial pressures of O2 and CO2 which adversely affect chlorophyll biosynthesis [35]. Moreover, chlorophyllase is a chloroplast membrane associated enzyme that has been considered to be involved in degradation of chlorophyll molecule through catalyzing dephytilation reaction. Enhanced activity of chlorophyllase has been recorded in plants exposed to several environmental stresses [36]. Our results are in agreement with the findings of Wingler et al. [37] who recorded a decrease in Chl content with increasing altitude. A significant decrease in LRWC was recorded with a concomitant increase in altitude from 760 to 2580 m asl. It has been reported that stomatal density increases with decreasing partial pressure of CO2 at higher elevations [38]. Increase in the number of stomata culminates in enhanced stomatal conductance which accelerates the rate of transpiration, as witnessed by lower value of LRWC in the plants of higher altitude (Fig. 2C). Moreover, enhanced absorption of radiation by leaves, increase in the diffusion coefficient of water vapor in air, and increased density gradient of water vapor from the leaf to the ambient air are the major factors behind the potential increase of transpiration with altitude. High wind velocity, large diurnal fluctuations in temperature and limited supply of water also contribute to low level of LRWC at higher altitudes. On the other hand, plants from higher elevation showed a decline in the activities of antioxidant enzymes resulting in increased H2O2 accumulation and thus weak protection to the plants. It is well known that SOD dismutates superoxide radicals to H2O2, whereas, CAT and POX are involved in converting H2O2 into water and oxygen. Therefore, excessive accumulation of H2O2 may be either due to enhanced activity of SOD and/or reduced activities of CAT and POX. However, results show reduced activities of all the three enzymes, therefore, CAT and
147
POX jointly contributed to excessive accumulation of H2O2 content. Moreover, a significant decrease in leaf protein content (Fig. 1E) could also contribute to a decrease in scavenging enzyme activity. The inhibition in antioxidant enzyme activities with increase in altitude was also recorded by Wang et al. [39]. High mountain plants are equipped with another defense system which gives protection against UV radiations at higher altitudes. Such a system is orchestrated by altered synthesis of secondary metabolites such as phenolics, flavanoids, and essential oils. A concomitant increase in secondary metabolites (alkaloids, flavonoids and essential oil content) was recorded from 760 to 2580 m height (Figs. 3A–E). Although, exposure of plants to UV radiations induces generation of toxic free radicals, but at the same time UV radiations has been shown to activate biosynthesis of secondary metabolites, which play an important role in the protection of plants against harmful radiations. These secondary metabolites possess the capacity to absorb UV radiation and thus, confines UV radiation away from inner cell layers [40].Luis et al. [41],observed a twofold increase in total phenolic contents of UV-B exposed rosemary plants compared with the plants grown without UV-B. A significant increase in essential oil content in L. pubescens was recorded at the higher elevation of 2580 m (Fig. 3D). The proclamation, that plants at higher altitude produce more essential oil as one of the defensive strategies against low temperature and UV radiation, is supported by the studies of Ioannidis [42] and Lianopoulou et al. [43]. It is evident from results that plants growing at higher altitude possess excessive amounts of secondary metabolites which provide an additional defense against various stresses and enable the plants to perform normally under harsh environmental conditions of higher elevations. 5. Conclusion The assessment of results shows that the plants grown at lower and medium elevation performed well in terms of growth, physiological and biochemical parameters because of the protection given by the antioxidant enzymes. However, at higher elevation plants exhibited higher values for secondary metabolites. Therefore, to put all in a nut shell it can be concluded that plants at lower and medium elevation were more protected by antioxidant enzymes than the secondary metabolites, however, reverse is true for the plants of higher elevation. Acknowledgment The support given by the Deanship of Scientific Research (DSR), University of Tabuk, Saudi Arabia (project number S-0082-1434) is gratefully acknowledged. References [1] N.M. Keveshk, Investigation of effect of altitude in some morphological characteristics and yield in Euphorbia macrostegia, Int. J. Agric. Crop Sci. 5 (16) (2013) 1779–1783. [2] A.D. Richardson, G.P. Berlyn, T.G. Gregoire, Spectral reflectance of Picea rubens (Pinaceae) and Abies balsamea (Pinaceae) needles along an elevational gradient, Mt. Moosilauke, New Hampshire, USA, Am. J. Bot. 88 (4) (2001) 667–676. [3] C. Li, G. Xu, R. Zang, et al., Sex-related differences in leaf morphological and physiological responses in Hippophae rhamnoides along an altitudinal gradient, Tree Physiol. 27 (3) (2007) 399–406. [4] K. Asada, Radical production and scavenging in the chloroplasts, in: N.R. Baker (Ed.), Advances in Photosynthesis, Vol. 5: Photosynthesis and the Environment, Kluwer Academic Publishers, Dordrecht 1996, pp. 123–150. [5] I.M. Moller, P.E. Jensen, A. Hansson, Oxidative modifications to cellular components in plants, Annu. Rev. Plant Biol. 58 (2007) 459–481. [6] H. Lantemona, A.L. Abadi, A. Rachmansyah, J. Pontoh, Impact of altitude and seasons to volume, brix content, and chemical composition of aren sap in north Sulawesi, J. Environ. Sci. Toxicol. Food Tech. 4 (2) (2013) 42–48. [7] M.R. Badger, G.D. Price, The role of carbonic anhydrase in photosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45 (1994) 369–392. [8] D. Peltzer, E. Dreyer, A. Polle, Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei, Plant Physiol. Biochem. 40 (2) (2002) 141–150.
148
M.N. Khan et al. / Acta Ecologica Sinica 36 (2016) 141–148
[9] K.G. Ramawat, S. Goyal, The Indian herbal drugs scenario in global perspectives, in: K.G. Ramawat, J.M. Merillon (Eds.), Bioactive Molecules and Medicinal Plants, Springer, Berlin Heidelberg New York 2008, p. 323. [10] O. Said, K. Khalil, S. Fulder, H. Azaizeh, Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region, J. Ethnopharmacol. 83 (3) (2002) 251–265. [11] J.S. Mossa, M.A. Al-Yahya, I.A. Al-Meshal, Medicinal Plants of Saudi Arabia, King Saud University Press, Riyadh, 2000. [12] M.M. Algandaby, H.A. Alghamdi, O.M. Ashour, et al., Mechanisms of the antihyperglycemic activity of Retama raetam in streptozotocin-induced diabetic rats, Food Chem. Toxicol. 48 (8–9) (2010) 2448–2453. [13] H. Edziri, M. Mastouri, I. Cheraif, M. Aouni, Chemical composition and antibacterial, antifungal and antioxidant activities of the flower oil of Retama raetam (Forssk.) Webb from Tunisia, Nat. Prod. Res. 24 (9) (2010) 789–796. [14] V. Velikova, I. Yordanov, A. Edreva, Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines, Plant Sci. 151 (1) (2000) 59–66. [15] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Ann. Biochem. 72 (1–2) (1976) 248–254. [16] R.S. Dwivedi, N.S. Randhawa, Evaluation of rapid test for hidden hunger of zinc in plants, Plant Soil 40 (2) (1974) 445–451. [17] H.K. Lichtenthaler, C. Buschmann, Chlorophylls and carotenoids: measurement and characterization by UV–vis spectroscopy, Current Protocols in Food Analytical Chemistry, John Wiley and Sons, New York 2001, pp. F4.3.1–F4.3.8. [18] S. Yamasaki, L.C. Dillenburg, Measurements of leaf relative water content in Araucaria angustifolia, Rev. Bras. Fisiol. Veg. 11 (2) (1999) 69–75. [19] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Ann. Biochem. 44 (1) (1971) 276–287. [20] A. Upadhyaya, D. Sankhla, T.D. Davis, et al., Effect of paclobutrazol on the activities of some enzymes of activated oxygen metabolism and lipid peroxidation in senescing soybean leaves, J. Plant Physiol. 121 (5) (1985) 453–461. [21] I. Cakmak, H. Marschner, Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves, Plant Physiol. 98 (4) (1992) 1222–1227. [22] S.S. Zhao, M.Y. Zeng, Determination of qinghaosu in Artemisia annua L. by high performance liquid chromatography, J. Pharma. Anal. 6 (1986) 3–5. [23] S. Hadi, b. Bremner, Initial studies on alkaloids from Lombok medicinal plants, Molecules 6 (2) (2001) 117–129. [24] A. Djeridane, M. Yousfi, B. Nadjemi, et al., Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds, Food Chem. 97 (4) (2006) 654–660. [25] V. Prakash, H. Bisht, P. Prasad, Altitudinal variation in morpho-physiological attributes in Plantago major: selection of suitable cultivation site, Res. J. Med. Plants 5 (3) (2011) 302–311. [26] C. Körner, P. Chochrane, Influence of leaf physiognomy on leaf temperature on clear midsummer days in the snowy mountains, south-eastern Australia, Acta Oecol. 4 (2) (1983) 117–124. [27] F. Ran, X. Zhang, Y. Zhang, et al., Altitudinal variation in growth, photosynthetic capacity and water use efficiency of Abies faxoniana Rehd. et Wils. seedlings as revealed by reciprocal transplantations, Trees 27 (5) (2013) 1405–1416.
[28] K. Apel, H. Hirt, Reactive oxygen species: metabolism, oxidative stress, and signal transduction, Annu. Rev. Plant Biol. 55 (2004) 373–399. [29] E. Georgieva, D. Petrova, Z. Yordanova, Influence of cryopreservation on the antioxidative activity of in vitro cultivated Hypericum species, Biotechnol. Biotechnol. Equip. 28 (5) (2014) 863–870. [30] K. Asada, The water–water cycle in chloroplasts: scavenging of active oxygen species and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601–639. [31] A. Vuleta, S. ManitaševićJovanović, B. Tucić, How do plants cope with oxidative stress in nature? A study on the dwarf bearded iris (Iris pumila), Acta Physiol. Plant. 37 (2014) 1711. [32] X. Li, Y. Yang, L. Ma, et al., Comparative proteomics analyses of Kobresia pygmaea adaptation to environment along an elevational gradient on the central Tibetan plateau, PLoS One 9 (6) (2014), e98410. [33] A.E.A. Sharaf, Om-M.A. Khafagi, E.-B.E. Hatab, M.M. Moursy, Effect of altitudinal gradients on the content of carbohydrate, protein, proline and total phenols of some desert plants in Saint Katherine Mountain, South Sinai, Egypt, Middle-East J. Sci. Res. 14 (1) (2013) 122–129. [34] A.J. Studer, A. Gandin, A.R. Kolbe, et al., A limited role for carbonic anhydrase in C4 photosynthesis as revealed by a ca1ca2 double mutant in maize, Plant Physiol. 165 (2) (2014) 608–617. [35] Y. Li, D. Yang, S. Xiang, G. Li, Different responses in leaf pigments and leaf mass per area to altitude between evergreen and deciduous woody species, Aust. J. Bot. 61 (6) (2013) 424–435. [36] D.T. Todorov, E.N. Karanov, A.R. Smith, M.A. Hall, Chlorophyllase activity and chlorophyll content in wild and mutant plants of Arabidopsis, Biol. Plant. 46 (1) (2003) 125–127. [37] A. Wingler, M. Juvany, C. Cuthbert, S. Munné-Bosch, Adaptation to altitude affects the senescence response to chilling in the perennial plant Arabis alpina, J. Exp. Bot. 66 (1) (2015) 355–367. [38] F.I. Woodward, C.K. Kelly, The influence of CO, concentration on stomatal density, New Phytol. 131 (3) (1995) 311–327. [39] Y. Wang, W. He, H. Huang, et al., Antioxidative responses to different altitudes in leaves of alpine plant Polygonum viviparum in summer, Acta Physiol. Plant. 31 (4) (2009) 839–848. [40] C.A. Kolb, M.A. Käser, J. Kopecký, et al., Effects of natural intensities of visible and ultraviolet radiation on epidermal ultraviolet screening and photosynthesis in grape leaves, Plant Physiol. 127 (3) (2001) 863–875. [41] J.C. Luis, R.M. Perez, F.V. Gonzalez, UV-B radiation effects on foliar concentrations of rosmarinic and carnosic acids in rosemary plants, Food Chem. 101 (3) (2007) 1211–1215. [42] D. Ioannidis, L. Bonner, C.B. Johnson, UV-B is required for normal development of oil glands in Ocimum basilicum L. (sweet basil), Ann. Bot. 90 (4) (2002) 453–460. [43] V. Lianopoulou, A.M. Bosabalidis, A. Patakas, et al., Effects of chilling stress on leaf morphology, anatomy, ultra structure, gas exchange, and essential oils in the seasonally dimorphic plant Teucrium polium (Lamiaceae), Acta Physiol. Plant. 36 (8) (2014) 2271–2281.