Physiological and biochemical responses of Quercus brantii seedlings to water deficit stress

[Research]

Physiological and biochemical responses of Quercus brantii seedlings to water deficit stress

Zolfaghari R.¹*, Akbarinia M.²

1. Department of Forestry, Faculty of Agriculture and Forestry, Yasouj University, Yasouj, Iran

2. Department of Forestry, Faculty of Natural Resources, Tarbiat Modarres University, Noor, Iran

ABSTRACT

Water shortage is one of the most important environmental stresses in Mediterranean regions. Poor seedling quality may account for the failure of oak regeneration. To determine the best seed origin of Quercus brantii, we investigated on seedlings collected from 20 mother trees in the Zagros Mountain forests 700 to 2200 m altitudes above sea level. Seedlings from different altitudes were irrigated at 25%, 50%, 75% and 100% of field capacity (FC), from June through the end of August 2005, and then their growth, physiological and biochemical parameters were examined. The results showed that the activity of peroxidase (PO), superoxide dismutase (SOD), and amylase, as well as the rate of membrane lipid peroxidation and the content of lignin were not affected by water deficit stress. However, the survival and growth rates were reduced below 50% FC. Seedlings originated from lower altitudes had higher growth and survival rate than those from higher altitudes below 50% of FC. The seedlings grown under 50% FC had also high phosphorus and water soluble carbohydrate contents. In conclusion, the present study showed that the seedlings from lower altitudes, which their mother trees grown under warmer climate condition in growth season, were more resistant to water deficit due to higher root to shoot ratio, phosphorus and water soluble carbohydrate contents under water deficits.

INTRODUCTION

Seedling stage is an important and usually a critical phase in the regeneration of woody plants under natural conditions. So, the risk of environmental stresses is very high at this stage. Mediterranean environment character- rized by important seasonal and episodic droughts (Tatarinov et al. 2016). Water availability is a limiting factor for the regeneration of oak and other woody plants (Aschmann 1984). In general, drought stress reduces plant growth (Clark et al. 2000).

Drought is a complex physico–chemical process in which many biological macrom- olecules and small molecules e.g. nucleic acids, proteins, carbohydrates, lipids, hormones, ions, free radicals and mineral elements are involved (Hong Bo shao et al. 2005). Some plants can tolerate increasing drought stress by decreasing osmotic potential or via accumulating solutes in the tissues (Morgan 1984).

Tyree & Jarvis (1982) suggested that low osmotic potential enhances the ability of plants to take water up from dry soil and its role is as important as the growth of root. Low soil moisture can also affect the rate of nutrient flow from the soil to the roots as well as alter the relative availability of nutrients in soil solution (Misra & Tyler, 1999). Furthermore, on dry sites a high K⁺ level in the plants could increase seedling drought resistance. In Douglas-fir, Pseudotsuga menziesii Mirb. Seedlings, increased K⁺ concentration resulted in greater water-use efficiency (Haase & Rose, 1994). On the other hand, one of the earliest responses of plants to pathogens, wounding, drought, extremes of temperature or physical and chemical stress is the accumulation of reactive oxygen species (ROS) such as superoxide, hydroxyl radicals, hydrogen peroxide and singlet oxygen (Jiang & Zhang 2004). Antioxidant defense enzymes i.e., superoxide distumase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), glutathione reductase (GR) and monod- ehydroascorbate reductase (MDAR) are of the system designed to minimize the concentrations of ROS. SOD catalyzes the superoxide into oxygen and hydrogen peroxide (Perry et al. 2011). Catalase and peroxidases can ellipse H₂O₂ (Peltzer et al. 2002).

So, POD decomposes H₂O₂ by oxidation of co-substrates such as phenolic compounds and/or antioxidants (Blokhina et al. 2003). Hence with identification and quantification of morpho- logical and physiological characteristics of the seedlings in any environment, we can warrant plantation success (Blokhina et al. 2003).

Persian oak, Quercus branti Lindl., is one of the most important species in the Zagros mountains forests steppe, west of Iran. This forest is characterized by Mediterranean climate and extreme summer aridity (Fig. 1). Over 1.7 million ha of the Zagros forests has been destroyed since 1962 (Ghazanfari et al. 2004). Q. branti distributes on different altitudes (Heydari et al. 2013).

Investigation of the tree populations’ responses to environmental variable will give us a better understanding of their adaptive processes in different environments. With this knowledge, we are better equipped to design breeding programs, hence decreasing the effects of summer drought (Modrzynski & Eriksson 2002). In this study, we selected oak acorns from 20 mother trees at different altitudes (700- 2200 m) and exposed them to different levels of water deficit. Hypothetically, the populations of Q. branti in lower altitudes origin are more resistant to water deficit.

Also, because of the large number of responses that can occur under water deficit condition, the aims of the present study were: 1) to assess the effect of water deficit stress on growth, nutrition, soluble carbohydrate accumulation and antioxidant system of Q. branti seedlings. 2) to compare the Q. branti seedlings from different altitudes with each other in relation to water deficit tolerance.

MATERIALS AND METHODS

Plant materials

Acorns of Q. brantii of 20 mother trees were collected from Zagros forests in Iran. The mother trees were located in three altitude classes from 700 through 2200m (700-1200m, 1200-1700m and 1700-2200m).

Acorns were sown in March 2005 in 200 cm³ pots containing the Zagros forest soil modified by 20% sand in volume (45% sand, 30.7% silt, and 25.3% clay). Then seedlings were daily irrigated until June 2005.

The water deficit experiment was started in June 2005. Uniformed in height and viable seedlings were selected for this experiment and then those randomly divided into four irrigation level treatments.

Irrigation treatments

The soil field capacity (FC) was determined for 200 cm³ pots containing local forest soil by weighting. Uniformed seedlings were subjected to four irrigation level treatments including 100% FC (control), 75%, 50% and 25% of FC in outdoor condition (n = 10 of each mother tree).

Seedlings were irrigated to field capacity every 2 days by weighting the pot plus plant at field capacity during the experiment (from June through end of August 2005).

All seedlings were harvested at the end of the experiment and divided into leaves, stem and roots. Leaves of the seedlings were frozen with liquid N₂ and kept at -80 °C for further experiments.

Each experiment was conducted using three seedlings from each mother tree.

Growth and survival rate measurements

Fresh weight of leaf, shoot and root were measured. Dry weight of the samples was also determined after oven-drying at 80 °C for 24 hours. The ratio of root/shoot and biomass of the plants were then calculated.

Survival rate (%) of the seedlings from different mother trees were measured by calculating difference numbers of alive seedlings at the beginning and end of the water deficit treatments.

Lipid peroxidation

The level of the peroxidation of membrane lipids was determined by measuring malondialdehyde (MDA) as final product of lipid peroxidation. Samples were homogenized in an aquatic solution of Trichloroacetic acid (TCA) (10% w/v). The homogenate was centrifuged at 15000 × g for 10 min and 1 ml of the supernatant was added to 1 ml of 2-Thiobarbituric acid (TBA) 0.5%. The mixture was incubated at 100 °C in a water bath for 30 min. and the reaction was ceased by ice-water bath. The absorbance was read at 532 nm followed by correction for the non-specific absorbance at 600 nm. The amount of MDA-TBA complex was calculated from the extinction coefficient of 155mM^-1cm^-1 (Vos et al. 1991).

Extraction and determination of antioxidant enzymes

Frozen samples (200 mg fresh weight) were homogenized in 3 ml Hydroxyethylpiperazine ethane sulfonic acid-KOH buffers (HEPES-KOH) (pH 7.8) containing 0.1 mM Ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 15000 × g for 15 min. All operations were performed at 4 °C. The supernatant was used for SOD activity (Giannopolitis & Ries 1977). Reaction mixture (3 ml) consisted of 50 mM HEPES-KOH buffer (pH 7.8), 0.1 mM EDTA, 50 mM Na₂CO₃ (pH 10.2), 12 mM L-methionine, 75 µM NBT, aliquots of enzyme extract and 1 µM riboflavin. One unit SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of NBT reduction measured at 560 nm. The protein content was determined by the method of Bradford (1976)using bovine serum albumin as a standard.

Peroxidase (PO) was extracted and determine in three fractions: the soluble (SPO), ionically-(IPO) and covalently-(CPO) bound fractions. SPO is involved in the stress response. IPO and CPO are supposed to be more related to the lignification and suberization of the cells (Abdolmaleki et al. 2007).  Frozen samples were homogenized in 50 mM Tris-maleate buffer (pH 6.0) and centrifuged at 1000 × g for 10 min at 2 °C. The supernatant was re-centrifuged at 18000 × g for 20 min at 2 °C. This second supernatant was used to assay SPO. Pellets of the first and the second centrifugations were pooled, incubated with 0.2 M CaCl₂ for 2 h at room temperature and then centrifuged at 18000 × g for 20 min at 2 °C. The supernatant was used to measure the activity of IPO.  The pellet was used directly for CPO assay.  SPO activity was assayed in 60 mM K-phosphate buffer (pH 6.1) containing 28 mM guaiacol and 5 mM H₂O₂. The increase in the absorbance was recorded at 470 nm. For IPO and CPO assay the final reaction mixture (3ml) contained 41.6 µM syringaldazine, forty mM Tris-maleate buffer (pH 6.0) and 16 mM H₂O₂.  Activity of IPO was expressed as the increase in absorbance at 530 nm per min per mg protein and of CPO was expressed as the increase in absorbance at 530 nm against cell wall dry weight (Ghanati et al. 2005).

Determination of amylase

Iodometric method was used to determine the endo-amylase activities (Douglas & Arshad 1996). The starch solution (1.5 g L^-1) was made by dissolving starch in 2 M NaOH solution at room temperature, followed by the addition of an equal volume of 2 M HCl. The resulting solution was then heated to a clear solution before its pH was adjusted to desired value by addition of 2 M NaOH. Final solutions were made by dilution with buffer at the desired pH. A solution (126.9 mg L^-1), containing the same volume of 0.1 M iodine and 5 g 100 mL^-1 potassium iodide, was diluted with cold distilled water to obtain a cold iodine solution (around 2 °C). Activity of Amylase were determined at 25 °C by allowing the enzyme solution in buffer (0.5 mL) to react with the starch solution (5 mL) for 2 min. The reaction was then stopped by adding 2.5 mL of cold iodine solution, which converts the non-hydrolyzed starch to starch-iodine blue complex. The resulting solution was afterwards placed in a thermostatic bath at 25 °C for 10 min and its absorbance was measured at 615 nm. The amount of the hydrolyzed starch (HS%) was determined as the absorbance value of the hydrolyzed starch, relative to a blank value of non-hydrolyzed starch solution, prepared without the addition of the enzyme (Douglas & Arshad 1996).

Determination of lignin

Cell walls of Q. brantii seedlings were isolated by sequential washing of filtered cells with 10 volumes of Na-Phosphate buffer (0.1 mM, pH 6.8), EtOH, CHCl₃-MeOH (2:1 v/v) and acetone followed by air-drying. Lignin content was measured via a modified acetyl bromide procedure (Iiyama & Wallis 1990). In brief, 6 mg of fine-powdered wall preparation was treated with a mixture (total of 2.5 ml) of 25% (w/w) AcBr in HOAc and 0.1 ml of 70% HClO₄ at 70 °C for 30 min with shaking at 10 min intervals. After cooling with ice, the digestion mixture was transferred to a 25 ml volumetric flask containing 5 ml of 2 M NaOH and 6 ml HOAc and made up to 25 ml. The lignin content was determined by measuring the absorbance at 280 nm using a specific absorption of 20.0 g^-1 L cm^-1 (Iiyama & Wallis 1990).

Nutrient measurements

Leaves of the seedlings were dried at 80 °C in oven for 24 h and then ground in a Wiley mill (20 mesh screens). Ca, an Mg and K concentration of the leaves were measured by digesting with perchloric acid and then analyzing with atomic absorption spectrophotometer (Shimadzu AA-670, Japan) and was expressed against leaf dry weight.

One pool of three leaves was analyzed for phosphorous concentration of whole plant colorimetrically (absorption 400 nm) using vanadat-molybdate (Kennedy 1984).

Determination of the soluble carbohydrate accumulation content (glucose, rhamnose and mannose)

Frozen samples (100 mg fresh weight of leaf) were homogenized in 3 ml distilled water. The homogenates were filtered by filter paper. Sugar content of filtrates was determined colorimetrically according to phenol-sulfuric method (Debois et al. 1956). Glucose, rhamnose and mannose contents were determined at 490, 480 and 490 nm respectively. A standard curve was obtained at various concentrations (ranging from 0 to 30 µg mL^-1).

Statistical analysis

The data were analyzed as a factorial design with two factors of irrigation treatment (100, 75 and 50% of field capacity) and altitude classes (low, middle and high). The putative outliers were observed by box plot and homogeneity and normality of the data distribution were examined using Kolmogorov-Smirnov test. Data were subjected to the analysis of variance (GLM) for a randomized complete block design (Table1). The differences between means were measured by Tukey test at the 5% significant level. SPSS software was used for all data analyses.

RESULTS and DISCUSSION

The effects of water deficit were investigated on Q. brantii seedlings of different mother trees from different altitudes at four treatments during three growing months. The gradual exposing of oak seedlings to water shortage up to 25% FC within three months was not endurable for Q. brantii. The results also showed no significant differences concerning to the  growth parameters and survival rates (%) between the seedlings of control (100% FC) and 75% FC, whereas the leaf and stem fresh weight were significantly lower at 75% FC compared to the control (Table 2), because leaf and stem expansion is more susceptible to drought (Ings et al., 2013). By increasing water deficit up to 50% FC, however, all growth parameters and survival rates were decreased considerably in comparison with other treatments. Water deficit stress has been known to limit carbon fixation, growth and net primary production (Dawson 1993). The rate of root weight also decreased by drought at 50% FC (Table 2). In accordance with our results, Fort et al. (1997) reported that the growth of the root in Q. robur L.seedlings decreased under water deficit condition.

Table 1. The significance of different sources of variation for water regimes, interaction of water regimes × altitudes.

** P ≤ 0.01, * P ≤ 0.05, ns: not significant.

Table 2.  Mean measured parameters of Q. brantii seedlings under different water regime treatments.

Different letters in each row indicate significant mean differences at p ≤ 0.05 based on Tukey test.

Growth parameters of water shortage-treated seedlings were also impressed by different altitudes of the locations where seeds had been collected (Ma et al. 2014). The seedlings originated from the lower altitudes showed more biomass at 50% FC and root/shoot ratio at 75% FC than the seedlings originated from higher ones (Table 3).

Volaire & Thomas (1995) found that drought-resistant population had significantly more root and aerial biomass than drought–susceptible population. Also, study on Picea abies populations from different altitudes showed that there was a correlation between root size and resistant to drought (Modrzynski & Eriksson 2002). This may be related to the fact that mother trees of these seeds belonged to the higher temperature (lower altitudes) in natural environment, so they responded to water deficit earlier and fasterparticularly compared to higher altitudes.  

Water deficit did not affect the activities of SOD, SPO, IPO, CPO and amylase of Q. branti seedlings at different treatments after approximately 3 months experiment (Table 2). There was also no significant difference in MDA content of Q. brantii at different treatments, so the oxidation of membrane lipids was not different among the treatments. In accordance with our results, Pinheiro et al. (2004) did not find a general link between MDA and drought tolerance when four clones of Coffea canephora were subjected to long period drought. In contrast, by studding of two clones of Coffea canephora in rapid drought stress, Lima et al. (2002) proposed that drought tolerance might at least in part be associated with enhanced activity of antioxidant enzymes.

Smironff & Colombe (1998) also showed that MDA content increased in the early period of drought but then decreased. Therefore, the Q. brantii seedlings might gradually be adjusted to changing conditions, whereas in the other cases like coffee, the change is relatively abrupt and might exceed the metabolic capacity of plants to acclimate (schwanz et al. 1996). We also observed a gradual increase in glucose, rhamnose and mannose content at different water deficit treatments and the highest amount was at 50% FC (Tables 2).

Therefore, at this treatment level, the osmotic adjustment was possibly due to the accumulation of glucose, mannose and rhamnose (Slama et al. 2007). Hartmann et al. (2013) also found an elevated concentration of sugar in organs of Picea abies tree during drought period.

Also, interaction between different altitudes and water deficit was significant for soluble carbohydrate content (Table 1). So that, it was significantly higher in high altitude seedlings than in lower ones at 50% FC (Table 4).

A shift in carbohydrate formation from starch and sucrose to soluble glucose and fructose is a process which induces a biochemical adaptation to water deficit for osmotic adjustment (Epron & Dreyer 1996). In the present study, it occurred in the higher altitude seedlings earlier, because they are more susceptible to drought stress than the lower altitude ones.

According to the results, the foliage nutrient concentrations of Ca, Mg and K were not changed under drought treatments (Table 2),  suggesting that either high availability of nutrients in pot experiment or equivalent reduction rate between growth and nutrient uptake can postpone the cation depletion of plants under water shortage (Chołuj et al. 2008). Haase & rose (1993) also obtained a similar result with Douglas-fir seedlings. So, they concluded that Ca and Mg are relatively immobile and can be uptaken only by increased soil water content. However, there was also a tendency toward the high P concentration in the seedling leaves grown under water stress treatment, such that the highest amount observed at 50% FC treatments. Baruch (1994) observed an increase in P concentration under drought treatment, because the uptake of phosphorus by root is less susceptible to drought conditions (Volaire & Thomas 1995). So, due to reducing in growth rateunder water stress, phosphorus can accumulate in leaves. Nambiar & Fife (1987) also found that rapid replenishment of N and P into the needles coincided with the time of slow shoot growth under water deficit.

Table 3. Mean growth parameters and survival rate (%) of the Q. brantii seedlings from different altitudes.

Different letters in each row indicate significant mean differences at p ≤ 0.05 based on Tukey test. 

Table 4. Mean glucose, rhamnose and mannoseof the Q. brantii seedlings under different water regimes and altitudes.

Different letters in each column indicate significant mean differences at p ≤ 0.05 based on Tukey test. 

Fig. 1. Precipitation and temperature of different altitudes of Zagros Mountains forests in different months.