Changes in morphological, physiological, and biochemical responses to different levels of drought stress in chinese cork oak (Quercus variabilisBl.) seedlings

ISSN 1021�4437, Russian Journal of Plant Physiology, 2013, Vol. 60, No. 5, pp. 681–692. © Pleiades Publishing, Ltd., 2013.

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1 INTRODUCTION

In arid and semi�arid regions, drought is a majorlimitation to plant survival and growth [1]. It also hasadverse effects on reforestation practice [1]. A success�ful establishing of healthy seedlings is the first step inimproving reforestation [1]. However, early dieback ofthe seedlings often leads to unsuccessful reforestationpractice in arid and semi�arid regions [2]. The mech�anisms, by which current�year seedlings adapt toincreasing drought stress in arid and semi�arid envi�ronments, should be determined to achieve successfulreforestation.

Under drought stress, plants adopt avoidance andtolerance strategies or either of the two [3]. For exam�ple, plants can avoid drought stress by maximizing

1 This text was submitted by the authors in English.

water uptake (e.g., absorbing groundwater by exten�sive, deep, or dense root systems) or minimizing waterloss (e.g., limited leaf growth, stomatal closure, and soon) [4]. Aside from these morphological changes,plants have evolved to employ various physiologicaland biochemical processes (e.g., synthesis of protec�tive solutes, defense mechanisms of enzymatic andnon�enzymatic antioxidants, and so on) as compo�nents of drought tolerance [5]. The knowledge ofdrought avoidance and tolerance strategies is helpfulin designing irrigation strategies that promote waterconservation while minimizing negative impacts ongrowth.

In general, drought causes ROS formation that candisrupt normal plant metabolism through oxidativedamages to lipids, proteins, nucleic acids, and photo�synthetic pigments [5–7]. To counteract the injuriousROS effects, plants are equipped with antioxidativesystems composed of enzymes, including superoxidedismutase (SOD), peroxidase (POD), catalase (CAT),and ascorbate peroxidase (APX), as well as non�enzy�matic antioxidants, such as ascorbic acid (ASA), glu�

Changes in Morphological, Physiological, and Biochemical Responses to Different Levels of Drought Stress in Chinese Cork

Oak (Quercus variabilis Bl.) Seedlings1 M. Wu, W. H. Zhang, C. Ma, and J. Y. Zhou

Key Laboratory of Environment and Ecology of Education Ministry in West China, College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China;

e�mail: [email protected] August 9, 2012

Abstract—Changes in growth, leaf water status, pigments, osmolytes, activities of peroxidase (POD), cata�lase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), and ascorbic acid (ASA) contentwere investigated in Chinese cork oak (Quercus variabilis Bl.) seedlings. Three�month�old seedlings were sub�jected to four drought cycles (30, 60, 90, and 120 days) and four drought intensities (80, 60, 40, and 20% fieldcapacity (FC)). The seedlings had optimal height, basal diameter, and leaf water status at 80% FC. Theseparameters significantly decreased as drought intensity increased. The total root length, diameter, and surfacearea at 60% FC significantly increased compared with those at 80% FC. However, at 40 and 20% FC theseparameters significantly decreased compared with those at 80% FC. The ratio of total root length to seedlingheight significantly increased with increasing drought intensity. The contents of chlorophyll a + b (Chla + b) andcarotenoids (Car) significantly decreased at 40 and 20% FC. However, no significant changes in Chla/Chlband Car/Chla + b ratios were observed among the four drought intensities. Comparatively, the seedlings accu�mulated more soluble sugars and proline, as well as they demonstrated the higher POD, SOD, CAT, APXactivities and ASA content at >40% FC. However, prolonged drought stress at 20% FC suppressed antioxi�dant activities and osmolyte accumulation, leading to a rapid increase in lipid peroxidation. These resultssuggest that a water supply >40% FC is required to support the growth and survival of the current�year seed�lings of Chinese cork oak

Keywords: Quercus variabilis, drought stress, antioxidants, osmolytes, leaf water relations

DOI: 10.1134/S1021443713030151

Abbreviations: APX—ascorbate peroxidase; ASA—ascorbic acid;Car—carotenoids; CAT—catalase; Chla—chlorophyll a;Chlb—chlorophyll b; Chla+b—total chlorophyll; FC—fieldcapacity; POD—peroxidase; RWC—leaf relative water content;SOD—superoxide dismutase; Ψw—leaf water potential.

RESEARCH PAPERS

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tathione, tocopherol, and carotenoids [8, 9]. As two ofthe most important compatible solutes in plants, solu�ble sugars and proline play important roles in droughttolerance strategies [7]. They stabilize membranes andmaintain protein conformation at low leaf waterpotentials [7]. Moreover, they act as effective hydroxylradical scavengers in vitro [7]. Previously, it has beenreported that some plants adapt to water stress byaltering the activities of various antioxidants or by per�forming osmotic adjustment; however, most studiesonly focused on two or three drought stress regimes [3,10, 11]. In addition, the manner, by which oak seed�lings change their morphological, physiological, andbiochemical properties under different drought stresslevels and periods, remains unclear.

Chinese cork oak (Quercus variabilis Bl.) is a decid�uous broad leaf tree in East Asia (12 to 42° N and 96°to 140° E); it plays a key role in soil remediation, ero�sion control, and in the economical and ecosystemicdevelopment in China, Korea, and Japan [12]. In thearid and semi�arid regions of Northwestern China,Chinese cork oak is often used in cork production,tannin extraction, and edible fungi cultivation [12]. Inrecent decades, overexploitation has led to thedestruction and degradation of Chinese cork oak hab�itat in the arid and semi�arid regions of NorthwesternChina [13]. These regions are characterized by lowprecipitation and high evaporation. Thus, they are notconducive for the regeneration of tree species.Although a high number of germinating seeds of Chi�nese cork oak have been observed in natural habitats(e.g., the Loess Plateau), only a few strong current�year seedlings have been found [14, 15]. Soil waterconditions have been hypothesized to play a major rolein the establishment of Chinese cork oak seedlings inarid and semi�arid regions. Therefore, studying thedrought avoidance and tolerance strategies of Chinesecork oak seedlings is crucial to ensure the expansion ofChinese cork oak cultivation and restore the naturaldistribution of Chinese cork oak population in water�limited regions. However, the mechanisms, by whichcurrent�year Chinese cork oak seedlings developdrought avoidance and tolerance strategies (includingmorphological, physiological, and biochemical modi�fications) to adapt to progressive soil drought stress,have yet to be fully elucidated [16].

In the present study, we measured a set of selectedmorphological, physiological, and biochemicalresponses of Chinese cork oak seedlings to differentdrought stress intensities. The present study aims (1)to assess the manner, in which Chinese cork oak seed�lings undergo morphological, physiological, and bio�chemical modifications to adapt to progressivedrought stress environments, and (2) to identify thepossible threshold value in soil water content. Theresults of the present study provide sufficient informa�tion on Chinese cork oak seedlings for reforestationprojects.

MATERIALS AND METHODS

Plant material and growth conditions. Seeds werecollected at the end of August and at the beginning ofOctober 2009 in Huanglong Mountain, which islocated at approximately 223 km north of Xi’an City inNorthwestern China (109°38′−110°16′ E, 35°24′−36°02′ N, altitude 962.5 m to 1 783.5 m). The studysite has a mean annual temperature of 8.6°C, a meanannual rainfall of 611.8 mm, and a mean annual evap�oration of 1585.9 mm. Deformed and damaged seedswere discarded. After air�drying, healthy seeds werestored at 0 to 4°C throughout winter. Surface soil wascollected from the study area and then mixed with thecollected sandy loam and humic soils (1 : 2 : 1, v/v/v).The soil mixture was used as the substrate for each pot.It was weighed to approximately 12.4 kg/pot and thenwatered. Water was allowed to evaporate naturallyuntil the weight of the soil was constant. The waterholding capacity was determined by obtaining the dif�ference between the soil wet weight and the soil dryweight. At 100% field capacity (FC), the mean watercontent of the soil was 34%. The chemical propertieswere as follows: pH, 7.42; organic matter, 46.89 g/kg;total N, 1.48 g/kg; total P, 0.82 g/kg; and total K,28.47 g/kg.

In 2010, the experiment was conducted in a tem�perature�controlled greenhouse at the Research Sta�tion for Vegetable Sciences, Northwest Agriculture,and Forestry University, Shaanxi Province, China(34°16′56.24″ N, 108°4′27.95″ E), where the temper�atures were maintained at 25°C (07:30 UT to19:30 UT) and 14°C (19:30 UT to 07:30 UT). Duringthe experiment, the relative humidity inside the cham�ber was 65 ± 5%. The mean photosynthetic photonflux density (PPFD) inside the greenhouse wasapproximately 66% of the mean PPFD outside. OnMarch 7, 2010, the seeds were soaked in deionizedwater for 24 h before germination on moist pledget.On March 21, 2010, germinating, healthy, and uni�form seeds were selected, and five of each were sowninto each plastic pot (28 cm × 32 cm, height × diam�eter, a total of 10 plastic pots). A total of 10 pots werewell watered (soil moisture was approximately 80%FC) to ensure healthy seedling growth. Seedlings 6 cmin height with approximately two leaves were selectedand then planted onto a uniform plant/pot (one seed�ling per pot, a total of 24 pots). The drought treatmentwas initiated on May 28, 2010.

Experimental design. The experiment was arrangedin a completely randomized design for four droughtintensities (80, 60, 40, and 20% FC, which served ascontrol (well watered), mild, moderate, and severedrought conditions, respectively). The drought stressexperiment lasted for 120 days from May 28 to Sep�tember 28, and each drought treatment was conductedon six pots. Six replicates per treatment were made tomeasure seedling aboveground growth parametersafter 30, 60, 90, and 120 days of drought stress treat�


CHANGES IN MORPHOLOGICAL, PHYSIOLOGICAL, AND BIOCHEMICAL 683

ments. Meanwhile, six replicates per treatment wereobtained from fully expanded leaves of different indi�viduals to determine leaf water relations, pigments,MDA, soluble sugars, proline, and antioxidant activi�ties after 30, 60, 90, and 120 days of drought stresstreatments. After measuring the morphological, phys�iological, and biochemical aboveground parameters ofthe seedlings, all seedlings were harvested to measureroot growth parameters. Water loss through transpira�tion was measured gravimetrically by weighing all thepots and calculating the weight loss between watering.The pots under the 80, 60, 40, and 20% FC water sup�ply regimes were subjected to compensatory irrigationafter weighing at 18:00 UT daily during the experimentto maintain the soil moisture levels at 28.2 ± 1.9%,21.1 ± 0.6%, 14.6 ± 1.2%, and 6.8 ± 0.7, respectively.To minimize soil evaporation, the soil surface was cov�ered with 4 cm of quartz gravel. In addition, 0.10 g/kgN, 0.10 g/kg P2O5, and 0.066 g/kg K2O in solutionwere prepared, with urea and KH2PO4 supplementedper pot, to ensure that all plants obtained sufficientnutrients.

Growth parameters. The seedling height was mea�sured from the base of the stem at the soil level to theterminal bud of the main stem, whereas the basaldiameter was measured at the ground line. Rootgrowth parameters included total root length, diame�ter, and surface area (sum of taproot and lateral roots).Images of the roots were recorded using a scannerModel EPSON V7000 (Epson, Japan). The rootsimages were digitized using Win�RHIZO Pro 2007asoftware (Regent Instruments, Canada). After 120days of drought treatment, the ratio of total root lengthto seedling height was determined.

Leaf water relations. Leaf water potential (Ψw) wasmeasured with a pressure chamber (Model 1000, PMSInstrument company, United States) between 08:00UT and 10:00 UT. Leaf relative water content (RWC)was calculated as follows:

RWC (%) = ((fr wt − dry wt)/(turgor wt − dry wt)) × 100.

Fresh weight was determined immediately afterharvesting, and then the leaves were floated in distilledwater for 24 h to determine turgor weight. The dryweight of the same leaves was determined after ovendrying at 70°C for 48 h.

Pigments. Chlorophyll a (Chla), chlorophyll b(Chlb), and carotenoids (Car) were extracted using80% acetone. The absorbance of the extracts was mea�sured using a Model UV�1700 spectrophotometer(Shimadzu, Japan) at 480, 649.1, and 665.1 nm. Thecontents of Chla, Chlb, total chlorophyll (Chla + b), andCar were determined according to the method ofWellburn [17].

MDA content. Approximately 0.2 g of leaves washomogenized in 2 mL of cold 10% (w/v) trichloroace�tic acid (TCA) and then centrifuged at 10000 g for15 min. A 2�mL aliquot of the supernatant was addedto a test tube with an equal volume of 10% (w/v) TCA

containing 0.65% (w/v) thiobarbituric acid. The sam�ples were boiled at 100°C for 15 min, rapidly cooled inan ice bath, and then centrifuged at 10000 g for10 min. The absorbance was read at 440, 532, and600 nm. MDA equivalents were calculated as6.425(A532 − A600) − 0.559A440 [18].

Osmolytes. Soluble sugar was determined by theanthrone method [19]. The absorbance at 640 nm wasmeasured using methanol as a blank. The concentra�tion of soluble sugar was calculated using glucose solu�tion as a standard [19].

Free proline was determined by the ninhydrinmethod [20]. Using toluene as a blank, the chro�mophore�containing toluene was read at 520 nm. Theconcentration of proline was calculated using prolineas a standard [20].

Enzyme activities and ASA content. Leaves werefrozen in liquid nitrogen immediately after harvestingand then stored at −20°C until enzyme assays.Approximately 0.2 g of the leaves was homogenized inan ice bath in 3 mL of 0.05 M Na phosphate buffer(pH 7.8) with 0.1 mM EDTA and 1% (w/v) PVP. Thehomogenate was centrifuged at 15 000 g for 15 min at4°C. The supernatant was used for enzyme activityassays at 4°C. POD activity was measured in the pres�ence of 16 mM guaiacol and 10 mM H2O2 by monitor�ing the increase in absorbance at 470 nm in phosphatebuffer (pH 7.0) [21]. CAT activity was measured in thepresence of 10 mM H2O2 by monitoring the decreasein absorbance at 240 nm in phosphate buffer (pH 7.0)[21]. One unit of SOD activity was defined as theamount of enzyme that produced a 50% inhibition ofnitroblue tetrazolium reduction at 560 nm [22]. APXactivity was measured in the presence of 0.5 mM ASAand 1.0 mM H2O2 by monitoring the decrease in absor�bance at 290 nm in phosphate buffer (pH 7.0) [23].

Approximately 0.2 g of leaves was homogenized in5 mL of cold 5% (w/v) m�phosphoric acid and thencentrifuged at 15000 g at 4°C for 15 min. The superna�tant (0.2 mL) was used to determine the content ofASA. Color was developed with 1 mL of 10% (w/v)TCA, 0.8 mL of 42% o�phosphoric acid, 0.8 mL ofα,α'�dipyridyl (1g in 75% ethanol), and 0.4 mL of 3%(w/v) FeCl3 × 6 H2O. The reaction mixtures were thenincubated at 40°C for 1 h and then quantified at525 nm [24].

Statistical analysis. One�way ANOVA followed bya least significant difference (LSD) multiple compari�son tests was used to compare the differences amongthe drought stress treatments at the 0.05 significancelevel. Relationships between osmolytes (proline andsoluble sugars) and antioxidants (POD, SOD, CAT,APX, and ASA) were determined using the Pearson’scorrelation coefficient test at the 0.05 significancelevel. Regression analysis was employed for variableswith significant correlations. Statistical analyses wereperformed using SPSS software v. 13.0 (SPSS, UnitedStates).

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RESULTS

Growth Parameters

Seedling height and basal diameter significantlydecreased along the drought stress gradient from 80 to20% FC (P < 0.001; Figs. 1a, 1b). Over time, seedlingheight and basal diameter rapidly increased at 80 and60% FC and then slightly increased at 40% FC. Bycontrast, a nearly stagnant growth was found at 20%FC after 60 days of treatment.

After 120 days of treatment, drought intensityaffected total root length, diameter, and surface area(P < 0.01; Table 1). These parameters at 60% FCincreased compared with those at 80% FC. By con�trast, the same parameters at 40 and 20% FC signifi�cantly decreased compared with those at 80% FC. The

ratio of total root length to seedling height signifi�cantly increased with increasing drought stress.

Leaf Water Relations

Leaf Ψw and RWC significantly decreased withincreasing drought stress (P < 0.001; Figs. 2a, 2b).During the entire study period, leaf Ψw and RWC keptnearly steady at 80% FC, whereas those at 60, 40, and20% FC significantly decreased.

Pigments

Seedling Chla + b and Car at 80 and 60% FC did notsignificantly differ (P > 0.05) but were greater thanthose at 40 and 20% FC ((P < 0.001; Figs. 3a, 3b).Over time, Chla + b and Car increased at 80 and 60%

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Fig. 1. Time�course of seedling height (a) and basal diameter (b) under four drought stress treatments. (1) 80% FC; (2) 60% FC; (3) 40% FC; (4) 20% FC.Data are means of six replicates, and error bars are SE. Significant differences between means of different drought regimes aremarked with different small letters according to LSD test (P < 0.05).

Table 1. Root growth parameters of Chinese cork oak seedlings under the four drought stress treatments after 120 days

Treatment, % FC Total root length, cm Total root diameter, mm Total root surface area, cm2 Total root length/height

80 26.94 ± 1.35 b 343.03 ± 15.70 a 68.48 ± 7.23 ab 0.68 ± 0.04 c

60 33.84 ± 1.35 a 360.85 ± 16.53 a 82.12 ± 6.93 a 1.16 ± 0.04 b

40 20.82 ± 2.14 b 255.83 ± 6.88 c 58.86 ± 5.26 bc 1.30 ± 0.11 ab

20 16.67 ± 0.64 c 203.01 ± 6.47 d 43.79 ± 5.57 c 1.42 ± 0.05 a

F 25.967 36.266 6.556 23.717

P *** *** ** ***

Notes: Data are means (± SE) of six replicates. Significant differences between means of different water regimes are marked with dif�ferent small letters according to LSD test (P < 0.05). The F�values are P�values derived from the one�way ANOVA. The P�valuesare denoted as **P < 0.01, ***P < 0.001.



FC but reduced at 40 and 20% FC (Figs. 3a, 3b).Chla/Chlb and Car/Chla + b ratio increased along thedrought stress gradient from 80 to 20% FC. However,no significant differences were found among the fourtreatments (Figs. 3c, 3d).

MDA Content

MDA content significantly increased with increas�ing drought stress from 80 to 20% FC (P < 0.001;Table 2). During the entire study period, the MDAcontent at 20% FC maintained a more rapid linearincrease than those under the other drought stresstreatments.

Osmolytes

Significant differences in soluble sugars and prolineaccumulation were found among the four droughtregimes (P < 0.01). The lowest accumulation of solu�ble sugars and proline were observed at 80% FC (Figs.4a, 4b). After 30 to 60 days, the accumulation of solu�ble sugars and proline significantly increased withincreasing drought stress. After 60 days, the accumula�tion at 20% FC rapidly declined. After 90 days, theaccumulations at 40% FC significantly decreased. Bycontrast, the accumulations at 60% FC linearlyincreased during the entire study period. However,after 120 days, the highest levels of soluble sugar andproline were reached at 40 and 60% FC, respectively.

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Fig. 2. Time�course of leaf water potential (Ψw) (a) and relative water content (RWC) (b) under four drought stress treatments.(1) 80% FC; (2) 60% FC; (3) 40% FC; (4) 20% FC.Data are means of six replicates, and error bars are SE. Significant differences between means of different drought regimes aremarked with different small letters according to LSD test (P < 0.05).

Table 2. MDA content in Chinese cork oak seedlings under four drought stress treatments after 30 to 120 days

Treatment, % FCMDA content, nmol/g fr wt

30 days 60 days 90 days 120 days

80 3.14 ± 0.12 a 3.43 ± 0.13 a 4.27 ± 0.28 a 4.98 ± 0.24 a

60 6.08 ± 0.21 b 8.88 ± 0.30 b 10.26 ± 0.44 b 11.80 ± 0.60 b

40 9.39 ± 0.27 c 15.52 ± 0.54 c 18.81 ± 0.15 c 26.94 ± 0.41 c

20 11.26 ± 0.13 d 19.47 ± 0.24 d 25.80 ± 0.64 d 33.76 ± 0.30 d

F 16.551 34.591 895.227 640.325

P *** *** *** ***

Notes: Data are means (± SE) of six replicates. Significant differences between means of different water regimes are marked with dif�ferent small letters according to LSD test (P < 0.05). The F�values are P�values derived from the one�way ANOVA. The P�valuesare denoted as *** P < 0.001.

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Antioxidative Defense Systems

Drought stress significantly changed POD activity(P < 0.01; Fig. 5a). After 30 to 90 days, POD activityrapidly increased under the four drought stress treat�ments, but the lowest POD activity was found at 80%FC. After 90 days of treatment, POD activity rapidlydeclined at 20 and 40% FC, whereas it slightly andrapidly increased at 60 and 80% FC. Compared withthe 80% FC treatment, POD activity at 60 and 40%FC increased by 51 and 27%, respectively, whereasthat at 20% FC reduced by 24% after 120 days of treat�ment.

SOD activity significantly changed along thedrought stress gradient (P < 0.001; Fig. 5b). During theexperimental period, SOD activity at 20% FCdecreased, whereas that under other treatmentsincreased. After 120 days of treatment, the highest andlowest SOD activities were achieved at 60 and 20% FC.

After 30 to 120 days of treatment, CAT activity at20% FC linearly decreased, whereas that at 60 and

80% FC increased (Fig. 5c). Although CAT activity at40% FC decreased after 90 days of treatment, it wasstill higher compared with that under other treatmentsduring the entire experimental period,

After 30 to 120 days of treatment, APX activity at80 and 60% FC significantly increased. After 60 daysof treatment, APX activity at 40 and 20% FCdecreased. During the entire experimental period, thegreatest and lowest APX activities were observed at 60and 20% FC, respectively (Fig. 5d).

No significant difference in ASA content wasfound among the four drought stress gradients after 30days of treatment (P > 0.05, Fig. 5e). However, after120 days of treatment, ASA at 60% FC significantlyincreased by approximately 18% compared with thatat 80% FC. By contrast, ASA at 40 and 20% FCdecreased by 16 and 33%, respectively, compared withthat at 80% FC.

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Relations between Osmolyte Accumulation and Antioxidant Activities

Pearson’s correlation coefficients revealed signifi�cant positive correlations between osmolyte accumu�lation and antioxidant activities, except between solu�ble sugar and APX (r = 0.051; P > 0.05) and betweensoluble sugar and ASA (r = 0.070; P > 0.05). Regres�sion analysis was used for the variables with significantcorrelations. Soluble sugar accumulation was linearlyand positively correlated with the activities of POD,SOD, and CAT (Figs. 6a–6c). Proline content waslinearly and positively correlated with ASA contentand POD, SOD, CAT, and APX activities (Figs. 6d–6h). SOD activity was linearly and positively corre�lated with ASA content and POD, CAT, and APXactivities (Figs. 7a–7d). POD activity was linearly andpositively correlated with CAT, APX, and ASA(Figs. 7e–7g). CAT activity was also linearly and pos�itively correlated with APX activity and ASA content(Figs. 7h–7i). In addition, APX activity was linearlyand positively correlated with ASA content (Fig. 7j).

DISCUSSION

Drought stress can induce a wide number ofresponses ranging from growth inhibition and synthe�sis of some non�toxic compounds to the increment incell osmotic potential, thereby allowing metabolicprocesses to enhance antioxidant activities [5, 9, 11].In the present study, drought adversely affected theseedling growth of Chinese cork oak. Thus, droughtstress significantly decreased the height and basaldiameter of Chinese cork oak seedlings. Compared

with 80% FC treatment, 60% FC treatment enhancedtotal root length, diameter, and surface area and main�tained strong growth in height and basal diameter dur�ing the entire study period. However, these rootparameters significantly declined at 40% FC com�pared with those at 80% FC, resulting in slow growthin height and basal diameter. The seedling height andbasal diameter stopped to grow, and the total rootlength, diameter, and surface area strongly decreasedat 20% FC as compared with 80% FC. Moreover, Chi�nese cork oak seedlings adapted to progressive droughtstress by increasing the ratio of total root length toseedling height. These results indicate that the rootorgans relative to the aboveground organs of the Chi�nese cork oak seedlings were increased in response todrought stress, because a strong root system can max�imize water uptake from the soil to guarantee the sur�vival and growth of seedlings [25]. Therefore, >40%FC (soil water content 14.6 ± 1.2%) is the thresholdsfor growth and survival for the current�year seedlingsof Chinese cork oak.

Over time, leaf Ψw and RWC showed a tendency todecrease at 60, 40, and 20% FC. Therefore, thedecrease in Ψw was not sufficient to avoid the signifi�cant water loss from the leaves in these drought�stressed seedlings. In addition, leaf RWC is an accu�rate index that indicates the plant water status [5]. LeafRWC for woody and shrubby plants reached 50 to 40%and occasionally it was as low as 30 to 20% duringsevere drought, leading to leaf senescence [1, 5].According to our data, the seedlings could still main�tain the adequate leaf water status (RWC > 60%) andslight growth at prolonged 40% FC. The sharpest

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Fig. 4. Time�course of soluble sugars (a) and proline (b) contents under four drought stress treatments.(1) 80% FC; (2) 60% FC; (3) 40% FC; (4) 20% FC.Data are means of six replicates, and error bars are SE. Significant differences between means of different drought regimes aremarked with different small letters according to LSD test (P < 0.05).

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30 60 90 120

(d)

a

AP

X a

ctiv

ity,

b

0

aaa

b

c

un

it/(

min

g f

r w

t)

b

bc c

d

b

c

b

d

6

4

2

30 60 90 120

(e)

a

AS

A c

on

ten

t, μ

g/g

fr w

t

0

a

aa

bc

b

b

c cb

daaa

a

Time, days

Fig. 5. Time�course of activities of peroxidase (POD) (a),superoxide dismutase (SOD) (b), catalase (CAT) (c), andascorbate peroxidase (APX) (d), and ascorbic acid (ASA)content (e) under four drought stress treatments. (1) 80% FC; (2) 60% FC; (3) 40% FC; (4) 20% FC.Data are means of six replicates, and error bars are SE. Sig�nificant differences between means of different droughtregimes are marked with different small letters accordingto LSD test (P < 0.05).

decline in leaf RWC (RWC < 45%) was observed atprolonged 20% FC, which resulted in severe leaf wilt�ing or shedding and stunted growth. Therefore, wespeculate that 40% FC (soil water content 14.6 ±1.2%) was the critical water content to cultivate Chi�nese cork oak seedlings. Below this value, seedlingswould be severely dehydrated.

In our study, Chla + b and Car contents at prolonged40 and 20% FC significantly reduced compared withthose at 80% FC. In addition, the contents of Chla + band Car at 40 and 20% FC treatments showed a ten�dency to decrease from 30 to 120 days. The evidentdecrease in pigment contents is a result of either slowsynthesis or fast breakdown and has been consideredto be a typical symptom of oxidative stress [26, 27].The change in Chla/Chlb ratio was not significant.However, the Chla/Chlb ratio was higher at 60, 40, and20% FC compared with that at 80% FC, implying adecrease in peripheral light�harvesting complexes.Demmig�Adams and Adams [28] considered a highChla/Chlb ratio as a decreased emphasis on light col�lection in relation to the rates of PSII photochemistry.Furthermore, Car play an important role in the photo�synthetic apparatus because they dissipate excessenergy as heat, scavenge ROS, and inhibit lipid perox�idation [26, 29]. The loss of Car content was lowerthan that of Chla + b at 60, 40, and 20% FC. Conse�quently, the Car/Chla + b ratio increased, which couldbe explained as a higher need of photoprotection byCar.

MDA content in the cell is usually used to reflectthe degree of lipid peroxidation resulting from oxida�tive stress [10]. In the present study, MDA content atprolonged 60% FC treatment kept a lower increasecompared with that at prolonged 40 and 20% FCtreatments, suggesting the better protection from lipidperoxidation damage. The better protection observedin Chinese cork oak seedlings may be attributed totheir more efficient repairing mechanisms, includingthe antioxidative system, osmotic adjustment, andphotosynthetic pigments. However, the MDA contentin the seedlings at 20% FC showed a rapid linearincrease, causing stagnant growth and leaf wilting.These findings indicate that the repairing mechanismscould not keep pace with the membrane lipid peroxi�dation. Therefore, the current�year seedlings of Chi�nese cork oak could not tolerate prolonged severedrought stress. These findings also explain why theexistence of current�year seedlings in the extremely



3000

2000

1000 0

1.5

3.0

4.5

So

lubl

e su

gar

con

ten

t,m

g/g

fr w

t

POD activity,unit/(min g fr wt)

y =

–44

.44

+ 3

47.6

5xR

2 = 0

.173

, **

*

1

23

(a)

8800

6600

4400 0

1.5

3.0

4.5

So

lubl

e su

gar

con

ten

t,m

g/g

fr w

t

SOD activity,unit/(min g fr wt)

y =

512

6.39

+ 3

70.1

6xR

2 = 0

.04,

*

(b)

8800

6600

4400 0

4060

80P

roli

ne

con

ten

t, μ

g/g

fr w

t

SOD activity,unit/(min g fr wt)

y =

348

6.62

+ 4

7.55

xR

2 = 0

.21,

***

(e)

300

200

100 0

1.5

3.0

4.5

So

lubl

e su

gar

con

ten

t,m

g/g

fr w

tCAT activity,

unit/(min g fr wt)

y =

64.

84 +

32.

58x

R2 =

0.2

2, *

**

(c)

3000

2000

1000 0

4060

80P

roli

ne

con

ten

t, μ

g/g

fr w

t


y =

629

.31

+ 2

8.63

xR

2 = 0

.31,

***

(d)

300

200

100 0

4060

80P

roli

ne

con

ten

t, μ

g/g

fr w

t

CAT activity,unit/(min g fr wt)

y =

12.

19 +

2.6

5xR

2 = 0

.39,

***

(f)

8 040

6080

Pro

lin

e co

nte

nt,

μg/

g fr

wt

APX activity,unit/(min g fr wt)

y =

1.2

2 +

0.0

4xR

2 = 0

.06,

**

(g)

6 4 2

6 040

6080

Pro

lin

e co

nte

nt,

μg/

g fr

wt

ASA content, µg/g fr wt

y =

2.8

1 +

0.1

3xR

2 = 0

.07,

**

(h)

4 2

4

2

3

4

1

Fig. 6. Relationships between soluble sugars content andperoxidase (POD) activity (a), superoxide dismutase(SOD) activity (b), and catalase (CAT) activity (c);between proline content and POD activity (d), SOD activ�ity (e), CAT activity (f), APX activity (g), and ascorbic acid(ASA) content (h). (1) 80% FC; (2) 60% FC; (3) 40% FC; (4) 20% FC.The solid lines represent the best�fit linear regressions forseedlings. The P�values are denoted as *P < 0.05; **P <0.01; ***P < 0.001.

dry natural habitats of Northwestern China (e.g., dryregions of the Loess Plateau) is unsuccessful.

The accumulation of soluble sugars and proline wasmuch higher in the Chinese cork oak seedlings at 60,40, and 20 FC than at 80% FC. This observation maybe interpreted as a mechanism to lower the osmoticpotential and contribute to osmotic adjustment [7].However, the accumulation initially declined at 40%FC after 90 days and then rapidly declined in a linearmanner at 20% FC treatment after 60 days. Thesefindings indicate that prolonged appropriate droughtstress (water supply >40% FC) could continue toimprove soluble sugar and proline accumulation tomaintain an adequate water flow into the seedling andto compensate the water loss. However, long�termexcessive drought stress (water supply ≤40% FC)finally resulted in serious oxidative damage and largelydeclined soluble sugar and proline accumulation.Meanwhile, positive linear relationships were con�firmed between soluble sugars and enzyme activities(POD, SOD, and CAT), as well as between prolinecontent and activities of enzymes (POD, SOD, CAT,APX) and ASA content. These findings demonstratethat the accumulation of osmolytes, especially pro�line, could activate antioxidant defense mechanisms.Türkan et al. [11] and Ahmed et al. [30] confirmedthat proline could preserve protein structures andenzyme activities. The water content at >40% FC (soilwater content 14.6 ± 1.2%) could maintain the growthand survival of the current�year seedlings of Chinesecork oak because of the sustained increase in osmolyteaccumulation and antioxidant activities.

The degree of damage induced by ROS could bedetermined by balancing between the ROS productionrate and their scavenging rate by enzymatic and non�enzymatic antioxidants [5, 8]. In particular, SOD isthe most effective intracellular enzyme catalyzing the

conversion of toxic to H2O2 [6]. In our study,SOD activity was enhanced at prolonged 80, 60, and40% FC treatments, which caused a more efficientdecline in oxidative damage and cellular toxicity.These findings may be ascribed to the decline in the

accumulation of H2O2, which was produced

through dismutation of catalyzed by SOD, wasscavenged by the antioxidant enzymes POD, CAT,and APX [5]. CAT converts H2O2 into water and

O2•–

O2•–

.

O2•–

690


WU et al.

3000

2000

1000 0

4000

6000

8000

SO

D,

un

it/(

min

g f

r w

t)


y =

0.2

8x –

725

.82

R2 =

0.3

0, *

**1

2

3(a)

4

300

200

100 0

4000

6000

8000

SO

D,

un

it/(

min

g f

r w

t)


y =

0.0

3x –

21.

61R

2 = 0

.52,

***

(b)

6 4 2 040

0060

0080

00S

OD

, u

nit

/(m

in g

fr

wt)


y =

9.3

7E4 x

– 2

.51

R2 =

0.3

0, *

**

(c)

6 4 2 040

0060

0080

00S

OD

, u

nit

/(m

in g

fr

wt)

ASA content,

y =

1.2

1x +

0.0

003x

R2 =

0.6

0, *

**

(d)

µg/g fr wt

300

200

100 0

1000

2000

3000

PO

D,

un

it/(

min

g f

r w

t)


y =

0.0

4x +

123

.37

R2 =

0.2

6, *

**

(e)

6 4 2 010

0020

0030

00P

OD

, u

nit

/(m

in g

fr

wt)


y =

0.0

01x

– 2

.10

R2 =

0.2

1, *

**

(f)

6 4 2 010

0020

0030

00P

OD

, u

nit

/(m

in g

fr

wt)

ASA content,µg/g fr wt

y =

3.0

2 +

0.0

01x

R2 =

0.2

9, *

**

(g)

6 4 2 010

020

030

0C

AT

, u

nit

/(m

in g

fr

wt)

y =

0.0

2x –

0.5

3R

2 = 0

.27,

***

(h)


6 4 2 010

020

030

0C

AT

, u

nit

/(m

in g

fr

wt)

y =

2.6

9 +

0.0

1xR

2 = 0

.19,

***

(i)


6 4 2 02

46

AP

X,

un

it/(

min

g f

r w

t)

y =

0.2

7x –

2.6

5R

2 = 0

.56,

***

(j)


2

3

14

Fig

. 7. R

elat

ion

ship

s be

twee

n s

uper

oxid

e di

smut

ase

(SO

D)

acti

vity

an

d pe

roxi

dase

(P

OD

) ac

tivi

ty (

a), c

atal

ase

(CA

T)

acti

vity

(b)

, asc

orba

te p

erox

idas

e (A

PX

) ac

tivi

ty (

c), a

nd

asco

rbic

aci

d (A

SA

) co

nte

nt (

d); b

etw

een

PO

D a

ctiv

ity

and

CA

T a

ctiv

ity

(e),

AP

X a

ctiv

ity

(f),

an

d A

SA

con

ten

t (g)

; bet

wee

n C

AT

act

ivit

y an

d A

PX

act

ivit

y (h

) an

d A

SA

con

ten

t(i

); b

etw

een

AP

X a

ctiv

ity

and

AS

A c

onte

nt

(j).

( 1

) 80

% F

C; (

2) 6

0% F

C;

(3)

40%

FC

; (4

) 20

% F

C.

Th

e so

lid

lin

es r

epre

sen

t th

e be

st�f

it li

nea

r re

gres

sion

s fo

r se

edli

ngs

. Th

e P

�val

ues

are

den

oted

as

***P

< 0

.001

.



molecular oxygen (O2), whereas POD decomposesH2O2 by oxidation of co�substrates, such as phenoliccompounds and/or antioxidants [11]. In addition,H2O2 is scavenged in the ascorbate–glutathione cycle,wherein APX reduces H2O2 to water using ASA as theelectron donor [8]. However, our data showed that theactivities of CAT and POD at 40% FC started todecrease after 90 days. Meanwhile, APX activity andASA content at 40% FC were significantly reducedafter 60 days, leading to disruption of H2O2 scaveng�ing. Prolonged drought at 20% FC significantlydecreased ASA content and SOD, POD, CAT, andAPX activities. These findings indicate that the scav�enging functions of the antioxidants were impaired.Moreover, SOD was linearly and positively correlatedwith POD, CAT, APX, and ASA in the current�yearseedlings of Chinese cork oak. These findings suggestthat ASA content and CAT, POD, and APX activitiesincrease with increasing SOD activity because of thehigh demand of H2O2 quenching.

CONCLUSIONS

The seedling height and basal diameter at 60% FCsignificantly declined compared with those at 80%FC. Nevertheless, the seedlings continued to grow dueto improving root growth, osmolyte accumulation,and activation of antioxidant enzymes and accumula�tion of non�enzymatic antioxidants. Under the pro�longed 40% FC drought stress, the seedlings grewslowly because of decreased root growth, as well as adecrease in the osmolyte accumulation and activitiesof POD, CAT, APX, and ASA after 60 or 90 days.When the water supply was below 40% FC, a largenumber of leaves wilted and seedlings demonstratedstagnant growth; meanwhile, the repairing mecha�nisms could not keep pace with membrane lipid per�oxidation (e.g., 20% FC). These results suggest thatthe Chinese cork oak seedlings should be planted inarid and semi�arid regions with an adequate water sup�ply >40% FC (14.6 ± 1.2%) to maintain vigorousgrowth of the current�year seedlings.

ACKNOWLEDGMENTS

We would like to thank X. Ni for his substantial helpin this study. We are also grateful to Drs. P. Deng,Y. Xue, and R. Li for their valuable advice on an earlierversion of this paper.

The present work was financially supported by theNational Natural Science Funds of China (grant no.30872018) and the Forestry Industry Research SpecialFunds for Public Welfare Projects of China (grantno. 201004011).

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Changes in morphological, physiological, and biochemical responses to different levels of drought stress in chinese cork oak (Quercus variabilisBl.) seedlings