Effects of drought stress on antioxidant enzyme, photosynthetic pigment and flavonoid pathway in two desert shrubs

YuBing Liu , MeiLing Liu, Bo Cao

Laboratory of Plant Stress Ecophysiology and Biotechnology, Shapotou Desert Research and Experiment Station, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, Gansu 730000, China

Effects of drought stress on antioxidant enzyme, photosynthetic pigment and flavonoid pathway in two desert shrubs

YuBing Liu*, MeiLing Liu, Bo Cao

Laboratory of Plant Stress Ecophysiology and Biotechnology, Shapotou Desert Research and Experiment Station, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, Gansu 730000, China

Antioxidant enzyme activity, photosynthetic pigment content, and free malondialdehyde (MDA), as well as flavonoid content and the key enzyme activity in the flavonoid pathway were determined in two desert shrubs,Caryopteris mongolicaBunge andReaumuria soongorica(Pall.) Maxim. under drought stress. The free MDA content was enhanced during the experimental period,which may be an indicator of oxidative stress. Superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX)activities inC. mongholicashowed a significant increase during the experiment, but catalase (CAT) activity was slightly decreased.On the other hand, POD and APX activities showed a significant increase and SOD and CAT activity data had no significant changes inR. soongorica. APX, SOD, and CAT activities were higher inR. soongoricathan inC. mongholica, but MDA content was lower, indicating that the lower values of MDA were attributed to higher activities of antioxidant enzyme inR. soongorica.Chlorophyll content decreased significantly in the two shrubs during the experiment, which indicated that there was a photoprotection mechanism through reducing light absorbance by decreasing pigments content. Caretonoids content increased inC.mongholicaand decreased inR. soongorica. The ratio of Chla/Chlb decreased significantly but caretonoids/Chl revealed a significant increase in the two shrubs, which could be explained as no decrease of peripheral light-harvesting complexes and a higher tolerance to drought. Total flavonoid content and the activities of phenylalanine ammonialyase (PAL) and chalcone isomerase(CHI) showed different changes betweenC. mongholicaandR. soongoricaafter treatment. These values decreased inR.soongoricaand increased inC. mongholicaexcept for PAL activity. However, anthocyanin content increased in the two shrubs,indicating that there was a different regulation response in the flavonoid pathway in the two shrubs under drought stress, and anthocyanin should be an important antioxidant both in the shrubs. Our results demonstrated the different responses of antioxidant defense and drought tolerance ability between the two shrubs.

antioxidant enzyme; photosynthetic pigment; flavonoid;Caryopteris mongolicaBunge;Reaumuria soongorica(Pall.) Maxim.

1. Introduction

Caryopteris mongolicaBunge, a native of Mongolia and western China, is a type of sub-shrub belonging to Verbenaceae, Caryopteris.C. mongolicais one of the valuable aromatic plants in semi-arid areas and has strong ability to survive under severe environmental conditions (Liet al., 1991).Studies on the chromosome number and karyotype ofC.mongolicaindicate that the diploid chromosome number is 26 and the basic number is 13, and the metaphase chromosomes are composed of 11 pairs of metacentric chromosomes and two pairs of submetacentric chromosomes (Guoet al., 2009).

Reaumuria soongorica(Pall.) Maxim., a perennial desiccation-tolerant semi-shrub, is exposed to multiple environmental stress conditions and is widely distributed from east to west in deserts of China. WhenR. soongoricais subjected to desiccation, its leaves wilt and it appears to die completely and the plant ceases to grow. However, upon rainfall the plant is able to recover and develop new leaves(Liuet al., 2007a, b). In recent years much attention has been paid to the physiological characteristics and molecular mechanism ofR. soongoricaadapting to the environment,such as photosynthetic plasticity, stress-resistance mechanism, and adaptation strategy (Liuet al., 2008; 2010a, b).

The objective of the present research was to illustrate the contribution of antioxidant enzyme, lipid peroxidation, photosynthetic pigment, and flavonoid pathway responsible for drought stress tolerance in these two desert shrubs.

2. Materials and methods

2.1. Plant materials and growth conditions

C. mongholicaandR. soongoricaseeds were selected from the northern foothills of Lanzhou City, Gansu, China(36o17′N, 103°48′E, 1,700-1,900 m elevation).C. mongholicawas planted in a greenhouse andR. soongoricawas planted in a field at the Botanical Garden of Lanzhou University. The selected plants were 2-year old and had been transplanted into 20-cm-diameter pots in the field and acclimated for 4 months before the beginning of the experiment in September 2009. To prevent the plants from absorbing underground water, two layers of thick plastic membrane were placed under the pots. A removable rain shed was placed over the pots to avoid precipitation. The treatment plants were dehydrated by withholding water at air temperature and ambient photoperiod, while the control materials were exposed to rain or watered routinely during the entire experimental period.

2.2. Determinations of water content in leaf and soil

Soil water content (%) was measured by three samples taken at 09:00 and dried at 105 °C until constant weight. At the same time, plant leaf water content (%) was determined in freshly cut leaves and dried at 70 °C until constant weight.The relative water content (WR) of soil and leaves was calculated by the expression:

whereWfandWdare the fresh weight and dry weight, respectively.

2.3. Determinations of photosynthetic pigment

Total chlorophylls (Chl a+b), chlorophylla(Chla),chlorophyllb(Chlb), and carotenoids (Car) were determined spectrophotometrically using 80% acetone as solvent(Lichtenthaler, 1987).

2.4. Lipid peroxidation

The level of lipid peroxidation in leaf samples was determined in terms of MDA content according to the method of Rao and Sresty (2000). Content of MDA, which is an end product of lipid peroxidation, was determined by using the thiobarbituric acid reaction. MDA concentration was calculated from the absorbance at 532 nm and measurements were corrected for nonspecific turbidity by subtracting the absorbance at 600 nm.

2.5. Enzyme extraction and activity

Leaves from plants for each treatment were immediately placed in liquid nitrogen and stored at -80 °C prior to extraction. Leaf tissue (0.5 g) was ground in a cold mortar with sand and an appropriate cold extraction buffer at 4 °C, as described below. The homogenates were centrifuged at 12,000 g for 15 min at 4 °C. Enzyme assays were performed in the supernatant. Proteins were determined according to Bradford’s method (1976), using bovine serum albumin as the standard protein. The extraction for APX and POD were performed in 50 mM potassium phosphate (pH 7) containing 1 mM AsA. For SOD assays it was 0.1 M phosphate buffer (pH 7.8) and for CAT assays it was 50 mM phosphate buffer (pH 7.0).

APX (EC 1.11.1.11) activity was measured by following the oxidation of AsA, operated by H2O2, at 290 nm and 25°C according to Nakano and Asada’s method (1981).

POD (EC 1.11.1.7) activity was determined at 25 °C following the increase in A430. 1 mL of reaction mixture contained 10 mM pyrogallol, 1.7 mM H2O2and leaf extract in 0.1 M potassium phosphate (pH 6.5). Calculations were performed using an extinction coefficient of 2.47 mM·cm.

Total SOD (EC 1.15.1.1) activity was determined by inhibition of cyt c reduction (McCord and Fridovich, 1968).The rate of cyt c reduction was measured at 550 nm for 60 s.One unit of activity was defined as the amount causing a 50% inhibition of cyt c reduction at 25 °C. SOD activity was given in units of SOD activity per milligram of total proteins(U/mg protein).

CAT (EC 1.11.1.6) activity was assayed according to the procedure described by Aebi (1984). The consumption of H2O2(10 mM) by CAT in the crude extract was monitored at 240 nm (e = 0.0394 mM·cm) for 300 s at 20 °C. CAT activity was expressed as mmol H2O2consumed (min·mg protein).

2.6. Flavonoid extraction and key enzyme activity of the flavonoid pathway

Ethanol was the extraction solvent and the extraction temperature was 75 °C, and extraction time was 2 hrs. 0.5 g fresh leaves were ground in a cold mortar with sand and 5 mL of 95% ethanol, and transferred to a tube, which was then heated to the desired temperatures. The extraction was carried out for a total of 2 hrs; the obtained filtrate was evaporated to dryness by a rotary evaporator under vacuum at 60 °C and then dissolved in 1 mL methanol.

The total flavonoid content was determined by using aluminum chloride colorimetric method (Changet al., 2002)with lutein as a standard. Lutein solutions were prepared at 12.5, 25, 50, 80, and 100 mg/mL in 80% ethanol (v/v). For analysis, 0.5 mL of the methanol samples or lutein solutions were mixed with 1.5 mL of 95% ethanol (v/v), 0.1 mL of 10% aluminum chloride (m/v), 0.1 mL of 1 mol/L of potassium acetate, and 2.8 mL of distilled water, and the mixture was incubated at room temperature for 30 min. The absorbance of the mixture was then measured by using a spectrophotometer at 510 nm. For the blank sample, 10% (m/v)aluminum chloride in the mixture solution was substituted by the same volume of distilled water.

The amount of anthocyanins was quantified as described by the method of Weiss and Halevy (1989) and modified.0.1 g fresh leaves were steeped in 1 mL of acidic methanol containing 1% HCl at 4 °C for 24 hrs. After diluting the solution to a known volume with the acidic methanol, its absorbance at 530 nm was measured. The relative anthocyanin content was expressed by OD 530 nm/g FW.

PAL (EC 4.3.1.5) activity was measured by a modified method of Tanakaet al.(1974) and Moriet al.(2000). The reaction mixture was 0.4 mL of 100 mM Tris-HCl buffer(pH 8.8), 0.2 mL of 40 mM phenylalanine, and 0.2 mL of enzyme extract. The reaction mixture was incubated for 30 min at 37 °C, and the reaction was terminated by adding 0.2 mL of 25% trichloroacetic acid. In the control PAL assay,the same amount of phenylalanine was added after termination. To remove precipitated protein, the assay mixture was centrifuged at 10,000 g for 15 min at 4 °C, and the absorbance of the supernatant was measured at 280 nm relative to the control.

CHI (EC 5.5.1.6) activity in two shrubs was measured with the method of Liet al. (2006). 2 g fresh leaves were homogenized in 6 mL of 120 mM potassium phosphate buffer (pH 8.0), containing 3 mM ethylene diaminetetraacetic acid (EDTA), 20 mM β-mercaptoethanol (β-ME), 2%hydrated polyvinyl polypyrrolidone (PVPP), and 100 μM iodoacetic acid. Protein extracts were collected after centrifugation at 12,000 g for 30 min, and their concentrations were determined by the Bradford method (1976). CHI assay was performed at 25 °C by monitoring the progression of naringenin chalcone isomerization at 390 nm on a spectrophotometer. The reaction mixture contained the following in a final volume of 1 mL: 50 mM Tris-HCl buffer (pH 7.6) containing 1% ethanol, 100 μM 4,2′,4′,6′-tetrahydroxychalcone, and an appropriate amount of protein/enzyme extract. As a control, the rate of spontaneous cyclization of the substrate was measured, which was subtracted from the rate for enzyme-added reactions.

2.7. Statistical analysis

All data were presented as means ± standard deviations of three determinations. Statistical analyses were performed using the Student’st-test and one-way analysis of variance.Multiple comparisons of means were done by the LSD (least significant difference) test. Statistical assessments of differences with the same letter between mean values were performed by Duncan’s multiple range test atP≤0.05.

3. Results

3.1. Soil water content and leaf water potential

Leaf water content decreased with the soil water content during drought treatment (Figure 1a, b). Leaf water content inC. mongholicawas lower than that inR. soongorica.After 20 days of dehydrating, soil water content dropped below 5%.

Figure 1 Variations of the leaf water content (a) in C. mongholica and R. soongorica and soil water content (b) during drought treatment

3.2. Effects on MDA and antioxidant enzymes

Figure 2a shows the changes of free MDA content.There was an increasing trend in free MDA content in both shrubs during the treatment, with the last value significantly higher than the control (corresponding to 20 d and 0 d of exposure to drought). POD and APX (Figure 2b, c) activity values in the two shrubs showed a significant increase during the experiment. Compared with POD and APX activities,SOD and CAT activity data (Figures 2d, e) revealed different trends, with an increase in SOD activity and a decrease in CAT activity inC. mongholica, and no significant changes inR. soongorica. APX, SOD, and CAT activities were higher inR. soongoricathan inC. mongholica, but MDA content was lower inR. soongorica.

Figure 2 Effects of drought stress on the content of MDA (a) and activities of POD (b), APX (c), SOD (d), and CAT (e)in leaves of C. mongholica and R. soongorica after 6, 11, and 20 days of treatment

3.3. Effect on photosynthetic pigments

Photosynthetic pigment content is shown in Figure 3.Chla, Chlb, and total chlorophyll (Figure 3a, b, and c) contents, and the ratio of Chla/Chlb (Figure 3e) displayed significant decreases in the two shrubs during the experiment.The caretonoid content exhibited an increasing trend inC.mongholicaand a declining trend inR. soongorica(Figure 3d). However, in the leaves of bothC. mongholicaandR.soongoricathe ratio of caretonoids/Chl (Figure 3f) revealed a significant increase trend.

3.4. Effect on flavonoid and key enzyme activity

Figure 4 shows the changes of the contents of total flavonoids and anthocyanin, and the activities of PAL and CHI inC. mongholicaandR. soongoricaafter treatment.InR. soongorica, total flavonoids content and PAL and CHI activity were decreased by the end of treatment. In contrast, these values increased inC. mongholica, except for PAL activity which had no significant change. Athocyanin content increased as the intensity of drought stress increased in both shrubs.

Figure 3 Effects of drought stress on the contents of chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), caretonoids (d), and the ratio of Chla/Chlb (e) and caretonoids/Chl (f) in leaves of C. mongholica and R. soongorica after 6, 11, and 20 days of treatment

4. Discussion

MDA content is usually used to measure the extent of lipid peroxidation resulting from oxidative stress (Smirnoff,1993). The increase of MDA content indicated that lipid peroxidation led to enhanced membrane breakage. After a long drought treatment,R. soongoricaexhibited lower increases of MDA content thanC. mongholica, which indicated thatR.soongoricahad higher tolerance to severe drought stress.

In a study by Limaet al. (2002), under drought conditions the activities of SOD, CAT, and APX increased to a greater extent, resulting in lower levels of lipid peroxidation and electrolyte leakage in a drought-tolerant clone than in a drought-sensitive clone ofCoffea canephora. In the present study, SOD, POD, and APX (Figures 2b, c, and d)activities inC. mongholicashowed significant increases during the experiment, but CAT activity was slightly decreased (Figure 2e). On the other hand, POD and APX activities showed significant increases while SOD and CAT activity had no significant changes inR. soongorica.APX, SOD, and CAT activity was higher inR. soongoricathan inC. mongholica, but MDA content trended lower inR. soongorica. The higher activities of antioxidant enzymes inR. soongoricaunder drought stress were attributed to lower values of MDA.

In a study by Türkanet al. (2005) the drought-resistant plantPhaseolus acutifoliusalso revealed higher activities of SOD, CAT, POD, and APX, and lower levels of lipid peroxidation than drought-susceptible plants. Khanna-Chopra and Selote (2007) attributed lower membrane injury to the higher activities of POD and APX in a drought-tolerant wheat cultivar than in a drought-sensitive cultivar under severe drought stress. In this study, it seemed that higher activities of antioxidant enzymes provided higher protection against oxidative stress inR. soongoricathan inC. mongholicaunder drought stress, as judged from lower increases of MDA.

Figure 4 Effects of drought stress on the contents of total flavonoids (a) and anthocyanin (b), and the activities of PAL (c) and CHI (d)in leaves of C. mongholica and R. soongorica, after 6, 11, and 20 days of treatment

Antioxidant action can be documented in both enzymic and nonenzymic systems, and has been reported in subcellular, cellular, and botanic studies. Dietary phytochemicals from plants have been proven to play an important role in preventing diseases in humans due to their potent antioxidant activity in nature. The functioning elements include bioactive phytochemicals like vitamins C and E, carotenoids and polyphenolics, and antioxidant activity is also attributed to chlorophylls (Usukiet al., 1984a, b; Endoet al., 1985a, b).Reduction of pigments content, as a result of either slow synthesis or fast breakdown, has been considered a typical symptom of oxidative stress (Smirnoff, 1993). Chlorophyll content decreased significantly in both shrubs during the present experiment, which indicated that there was a photoprotection mechanism through reducing light absorbance by decreasing pigments content (Munné-Bosch and Alegre, 2000;Galméset al., 2007; Elsheery and Cao, 2008). The higher ratio of Chla/Chlb was also considered to be the result of a decreased emphasis on light collection in relation to the rates of PSII photochemistry (Demmig-Adams and Adams, 1996).As drought stress intensified, the ratio of Chla/Chlb decreased significantly in both shrubs, which could be explained as no decrease of peripheral light-harvesting complexes and a higher stress tolerance in the two shrubs.

Carotenoids are known to function as collectors of light energy for photosynthesis and as quenchers of triplet chlorophyll and O2. Moreover, they dissipate excess energy via the xanthophyll cycle and can act as powerful chloroplast membrane stabilizers that partition the light-harvesting complexes (LHCs) and the lipid phase of thylakoid membranes, reducing membrane fluidity and susceptibility to lipid peroxidation (Havaux, 1998). The caretonoid content exhibited an increase trend inC. mongholicaand a contrary change inR. soongorica. However, in leaves ofC. mongholicaandR. soongoricathe ratio of caretonoids/Chl (Figure 3f) revealed a significant increasing trend. The increased ratio of Car/Chl under drought conditions indicated a higher need of photoprotection by Car (Baquedano and Castillo,2006; Elsheery and Cao, 2008).

Flavonoids are ubiquitous plant secondary products that have diverse functions in the physiology and ecology of plants.Most remarkably, flavonoids can protect plants against UV-B radiation and pathogen attack, attract pollinating insects, and act as signal molecules for initiating plant-microbe symbiotic associations (Parr and Bolwell, 2000). Flavonoids are synthesized through the phenylpropanoid pathway, which has been extensively studied (Holton and Cornish, 1995; Dixon and Steele, 1999; Koeset al., 2005). A large number of the structural genes as well as some regulatory genes in the flavonoid pathway have been isolated.

In the present work there were different changes in the content of total flavonoids and the activities of PAL and CHI betweenC. mongholicaandR. soongoricaafter treatment. InR. soongorica, these values were decreased by the end of treatment, and were increased inC. mongholicaexcept for PAL activity. But the anthocyanin content increased during the treatment in both of the shrubs, indicating that there is a different regulation response in the flavonoid pathway in the two shrubs under drought stress, and anthocyanin should be an important antioxidant in both of the two shrubs.

In conclusion, the two desert shrubs exhibited different levels of lipid peroxidation, antioxidant enzyme, photosynthetic pigment, and flavonoid pathway in response to drought stress.R. soongoricahad a higher tolerance to drought stress thanC. mongholicaeven though both of the shrubs are considered to be drought-tolerant plants.

This work was financially supported by the National Natural Science Foundation of China (No. 30800122, 31070358 and 30960065) and the West Light Foundation of the Chinese Academy of Sciences.

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10.3724/SP.J.1226.2011.00332

*Correspondence to: Dr. YuBing Liu, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences. No.320, West Donggang Road, Lanzhou, Gansu 730000, China. Tel: +86-931-4967199; Email: ybliu13@163.com

10 January 2011 Accepted: 18 March 2011