Growth and anatomical characteristics of different water and light intensities on cork oak ( Quercus suber L . ) seedlings

This study was performed to investigate the effects of water and light intensity on the growth and anatomical aspects (that is, stomatal density and chloroplasts ultrastructure) of cork oak (Quercus suber L.) seedlings from acorns collected from Tabarka (Northern Tunisia). Seedlings, four-month after germination, were planted at 3 L pots with artificial soil and were cultivated for 5 months at the greenhouse. The experiments were divided into two treatments: Water stress (well-watered: 25 to 35% of water contents, moderate drought stress: 15 to 20%, and severe drought stress: 7 to 10%) and light intensity stress (full sunlight: 100% of full sunlight, 1,200 mol m s; moderate sunlight: 65 to 70% of full sunlight, 800 mol m s; low sunlight: 20 to 25% of full sunlight, 300 mol m s; and extremely low sunlight: 3 to 4% of full sunlight, 40 mol m s). Growth varied among water stress, but this variation was typically smaller than light intensity. Seedlings grown under well-watered treatment showed the increased above ground growth as compared with deep root growth of severe drought stress. In contrast, seedlings grown under severe drought stress increased in root growth than in the above ground part. Q. suber seedlings grown under the shade-exposed low light intensity indicated adaptation such as the increased leaf area and increased specific leaf area than the leaves grown in full sunlight. In different light intensity treatments, the highest specific leaf area indicated at the extremely low light. Seedlings grown under severe drought stress increased in root growth. The palisade parenchyma chloroplasts of well-watered treatment were well developed: their shape was regular, chloroplast envelope membranes and grana were distinct, and an abundance of starch grains and a scarce of plastoglobuli was also visible inside in the chloroplasts. In severe drought stress, the ultrastructural features of the abaxial leaf surface were folded and shrunken, and was observed in a higher stomatal frequency, the thicker palisade and spongy parenchyma (containing three layers of palisade parenchyma). The severe drought stress indicated a swelling and disruption of the thylakoid, large starch grains, increased plastoglobuli, and accumulation of the reactive oxygen species (ROS), hydrogen peroxide (H2O2). The main features in the chloroplasts ultrastructure caused by the extremely low sunlight revealed not only the higher grana stacks and the broader grana, but also the higher stacking degree of thylakoids. Furthermore, seedlings grown under the extremely low sunlight showed no starch or exhibited only little starch in the chloroplasts, occupied the intracellular space in all organelles, and presented numerous small vacuoles and lipid droplets in the cytoplasm of these cells as compared with the well-watered and the severe drought stress. These results suggested that Q. suber has a well-developed mechanism to drought compared with shade stress by structural adaptations such as a deeper root system, a smaller reduction in stomatal density, and a smaller leaf area.


INTRODUCTION
Cork oak (Quercus suber L.) is an evergreen tree of great importance in the Mediterranean area both for ecological reason and for the economic value of cork (Passarinho et al., 2006).This species plays a key role such as the maintaining biodiversity and the main socio-economic activities (Hidalgo et al., 2008).This species is a sclerophyll evergreen tree, which adapted to a 4-month hot-dry summer period with at least 450 mm mean annual rainfall or no rain, maximum temperatures approaching 35 to 40°C and midday irradiances exceeding 2,000 mol m -2 s -1 photosynthetic active radiation (PAR) and 4 -5°C mean temperature for the coldest month (Lumaret et al., 2005).
A great deal of attention has recently been focused that a remarkable decline in several cork oak woodlands have occurred by complex factors involving abiotic stresses such as extremely hot dry summer and the environmental contaminants (Faria et al., 1996(Faria et al., , 1998;;Otieno et al., 2006;David et al., 2007), the outbreaks of damaging Phytophthora species including Phytophthora cinnamomi (Brasier, 1996;Balci and Halmschlager, 2003;Moreira and Martins, 2005), and seed predation damage by livestock grazing during recent decades (Campos et al., 2008).In plants, the remarkable ability to adapt to adverse environmental stress such as water stress and light stress may be related to specific changes in the morphological, physiological, and ultrastructural level (e.g., chloroplast) of leaves (Meier and Lichtenthaler, 1981).
Among the environmental factors affecting plant activity, water stress was one of the most important limiting factors at the initial phase of plant growth and establishment (Yin et al., 2005).The chloroplasts of the plants grown under the waters stress indicate the main damages in structural changes such as the excessive swelling of the thylakoids, the distortion of the grana, and the appearance of the lipid droplets (Riatic, 1992;Lopez-Carbonell, 1994).Chloroplasts of general mesophyll cells possess few plastoglobuli, but the number and size of these lipophilic compounds increase due to degraded thylakoids in chloroplasts during leaf senescence and in etioplasts before thylakoid formation (Steinmüller, 1985;Eymery, 1999).Munne-Bosch (2001) reported that symptoms of leaf senescence were revealed in chloroplasts at drought-induced senescence, showing an accumulation of plastoglobuli in the chloroplasts, including the disruption of thylakoids and much less grana stack.
Plants exhibit many morphological and physiological adaptations to cope with these environmental stresses (Aranda et al., 2005).Mediterranean-climate sclerophyllous trees also are typically known to possess anatomical and morphological structures in their leaves or other vegetative organs as related to an important indicator of plant strategies have high resistance to desiccation during substantial water stress (e.g., leaf mass per area indicates drought acclimation, density and thickness of leaf mesophyll, scarcity of intercellular spaces, *Corresponding author.E-mail: wsy@uos.ac.kr.Tel: (82) 2 2210 5634.Fax: (82) 2 2210 2838.Kwak et al. 10965 and thick cuticles) (Lansac et al., 1994;Castro-Díez et al., 2000;Bussotti et al., 2002).Oak seedlings in the degraded Mediterranean environments possess several morphological traits for desiccation avoidance in open, high irradiance or xeric sites (e.g., deeper and well structured root system, greater leaf thickness, higher stomatal density and smaller guard cells than other hardwoods, and leaf curling developed in several southeastern evergreen oaks) (Abrams, 1990).The morphological traits focused to minimize the demand previously discussed help oak maintain a higher physiological activity under unfavorable conditions of drought interacting with shade (Sack and Grubb, 2002;Aranda et al., 2005).
Variation of morphological and anatomical features to the crucial environmental resources such as the waterdeficit and the light gradiant play a major part in the optimization of the growth and early development of seedlings (Meier and Lichtenthaler, 1981;Lichtenthaler et al., 1981;Abrams, 1990;Dickson and Tomlinson, 1996;Gregoriou et al., 2007).The shade-type chloroplasts grown under the low-light generally possess characterization such as the higher grana stacks, the higher stacking degree of thylakoids, the broader grana, and an insufficient starch as compared to those under the high-light (Meier and Lichtenthaler, 1981).
Shaded (30% of full sunlight) leaves normally display increased chlorophyll contents and are thinner thickness than those grown under natural irradiation, due to possessing two palisade cell layers instead of the three layers of the palisade parenchyma of natural irradiation (Mendes et al., 2001;Romano and Martins-Loução, 2001;Gregoriou et al., 2007).However, a detailed study of Q. suber trees in the morphological and anatomical characteristics during early plant growth and development of water and light gradiant is lacking.
Therefore, the morphological and anatomical aspects of Q. suber in response to different water and light intensity will become essential for a deeper understanding of regeneration success of forestry tree species in Mediterranean-climate ecosystems.
The main objective of this study was to investigate the growth and anatomical aspects in response to different water and light intensity on Q. suber seedlings.

Experimental description and plant material
The study was carried out on cork oak (Q.suber L.) seedlings growing in two nursery stands located in the greenhouse at the University campus (University of Seoul, Seoul).Q. suber seedlings were prepared with acorns collected from Ain Snoussi and Northern Tunisia.
Seedlings, four-months after germination, were planted in 3-L plastic pots in April 2008 and placed in greenhouse of University of Seoul.The 3-L pots is filled with an artificial soil (Perlite+Vermiculite+Peatmoss, 1:1:1 [v/v/v]) with soil pH 5, and electrical conductivity (EC) 15.4 Scm -1 .The experimental design in the greenhouse was performed with a completely randomized design of 18 replicates per treatment with three irrigations and four light regimes for five months.
Seedlings were subjected to three irrigation regimes: Wellwatered (WW, 25 to 35%), moderate drought stress (MDS, 15 to 20%), and severe drought stress (SDS, 7 to 10%) regime.Soil moisture was recorded three times a week by time domain reflectometry (TDR, Trase System I, Soil Moisture Equipment Corp. USA).Seedlings under four light regimes with 18 replicates were randomly divided according to the different layers of shade cloth screening.The light intensity (the photosynthetic photon flux density, PPFD) of full sunlight was measured on a sunny day with a quantum sensor (type SKP 215, Skye Instruments, Powys, UK).The average photosynthetic photon flux density (PPFD, Q) under each light intensity regime during a sunny day was: FL, full sunlight (100% of full sunlight, 1,200 mol m -2 s -1 ); ML, moderate sunlight (65 to 70% of full sunlight, 800 mol m -2 s -1 ); LL, low sunlight (20 to 25% of full sunlight, 300 mol m -2 s -1 ); ELL, extremely low sunlight (3 to 4% of full sunlight, 40 mol m -2 s -1 ).Seedlings were grown in a randomized block design with metal frames to produce three irrigation and four light intensity regimes.

Growth and biomass measurements
To quantify differences in growth response to water and light environment, a subset of three plants from each treatment was randomly selected for morphological and biomass measurements.
Number of leaves and height of seedligns were monitored three times with monthly intervals (June, July and August).At the end of the nursery culture, each seedling was harvested and divided into component parts, and then shoot height (cm), number of leaves, root height (cm), shoot diameter (mm), and root diameter (mm) were measured.Growth and biomass variables (that is, root, shoot, total biomass of the seedling, and shoot biomass/root biomass ratio, SRR) were also calculated for application to the analysis of growth under different water and light intensity.Leaf area was measured using an LI-3100 area meter (LI-COR Inc, Lincoln, NE, USA).In order to determine dry mass, these shoot and root biomasses were measured by weighing of final dry mass of shoot and root (after desiccation at 75°C for 48 h).

Specific leaf area (SLA)
The specific leaf area (SLA: cm 2 g -1 dry weight) was calculated as the ratio of the fresh leaf area to its dry weight of individual leaves.SLA is pivotal features in the carbon-fixation 'strategy' of a species (Davi et al., 2008).
Leaf areas per leaf dry mass (SLA, an indicator of leaf structure characteristics) are fundamental traits in the carbon economy of plants (Wright et al., 2002).Plants grown under high light generally had thick leaves with a low SLA (Björkman, 1981).

Scanning electron microscopy (SEM)
Tissue samples for the microscopic observations were randomly collected from 3 to 5 mature leaves in well-watered, severe drought stress, and extremely low sunlight regimes of Q. suber seedlings at August 2008.All anatomical parameter data were obtained using SEM images.
The sampled leaves were placed individually in polyethylene bags and immediately transported to laboratory.Upon return to the laboratory, field-collected tissue samples from leaves (sliced 1 to 2 mm thick) were immediately fixed by Karnovsky's fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2), and fixed overnight for 2 to 4 h at 4°C.Samples were then rinsed three times in 0.05 M sodium cacodylate buffer (pH 7.2) for 10 min at 4°C, post-fixed with 1% osmium tetroxide (OsO4) in 0.05 M sodium cacodylate buffer (pH 7.2) for 2 h at 4 °C, briefly rinsed again two times with distilled water at room temperature.
The tissue was then washed, and than sections were stained En bloc staining with 0.5% uranyl acetate for 30 min at 4°C.In order to dry specimen, once again they are immersed two times in 100% isoamyl acetate for 15 min using critical point dryer.The specimens were then mounted on metal stubs, coated gold-palladium alloy using a sputter-coater (JFC-1100E; JEOL, Tokyo, Japan), and viewed with the scanning electron microscope (SEM, JSM-5410LV, JEOL, Tokyo, Japan) operated at an accelerating voltage of 20 kV.Scanning microscope photographs of the abaxial epidermis were used to quantify the stomatal density (the number of stomata per unit leaf area) and the leaf stomatal size.

Light microscopy (LM) and transmission electron microscopy (TEM)
For a light microscopy (LM) and a transmission electron microscopy (TEM) observation, small pieces from the middle region of the fieldcollected lamina were fixed overnight by immersion in Karnovsky's fixative: 2% paraformaldehyde and 2% glutaraldehyde buffered with 0.05 M sodium cacodylate buffer (pH 7.2) for 2 -4 h at 4°C.
The specimens were rinsed three times in the same buffer, postfixed in 1% OsO4 (the same buffer) for 2 h at 4°C, then rinsed two times with the distilled water at room temperature, stained En bloc staining with 0.5% uranyl acetate for 30 min at 4°C and dehydrated through a graded series of ethanol (30, 50, 70, 80, 90, 100, 100, and 100%) for 10 min at room temperature, and transited two times in 100% propylene oxide for 15 min at room temperature, and embedded by a Spurr's resin.
The method of measuring infiltration was accomplished according to the following steps: The tissue pieces were placed in rubber embedding molds and then embedded in Spurr's resin for 4 h.After infiltration in resin, the sections were then polymerized overnight at 70°C.
The semi-thin resin sections of the stained preparations were observed with the light microscope.Then the total thickness, thickness of palisade parenchyma, spongy parenchyma, lower epidermis, and upper epidermis were measured.Ultrastructure morphology of the specimen was examined with a Transmission Electron Microscope (LIBRA 120;Carl Zeiss,Oberkochen,Germany).

Statistical analysis
The effects of water and light intensity stress on the different morphological and anatomical parameters of the seedlings were analysed separately for each extraction by a multifactor analysis of variance (ANOVA) using the version 9.2 of the SAS statistical software package (Systat 9.2, Systat Software Inc., Richmond, USA).The test for differences among arithmetic treatment means were performed by the least significant difference (LSD) multiple range test method.The significance levels were set at the P 0.05 level.

Growth and biomass measurements
There were many studies focusing on the morphological and physiological adaptation of Q. suber to drought and shade stress (Abrams, 1990;Dickson and Tomlinson, 1996).
The present study was undertaken to examine effects of different water and light intensity in allometric differences of the morphological and anatomical aspects of Q. suber seedlings at the end of the experiment.Growth traits of Q. suber varied among water stress (Figure 1A and Table 1).This variation, however, was typically small compared to the pronounced differences among light stress (Figure 1B and Table 2).
The growth and morphology of Q. suber was affected by the levels of water (Table 3) and light intensities (Table 4).The total biomass varied among the water treatments and light treatments at the end of the experiment (Figure 1). Figure 1 showed that shoot and root biomass accumulations were greatly reduced in wellwater treatments.However, the moderate drought stress (MDS) and the severe drought stress (SDS) showed similar values of total biomass and the lowest values were observed during the moderate drought stress (MDS) period.
The root biomass within the moderate drought stress (MDS) was found in morphological aspects with smaller allocation in the response to mild drought stress as compared with the severe drought stress (SDS).But no significant difference between MDS and SDS treatments was found (Figure 1A).
Among the environmental factors affecting plant activity, water stress was one of the most important limiting factors at the initial phase of plant growth and establishment (Yin et al., 2005).Effect of water stress exhibited changes in early growth, relative biomass accumulation and allocation in response to water stress during treatment period.
There were significant differences in shoot height, shoot diameter, and shoot/root ratio of height among water treatments.Q. suber showed significant increase in shoot height, shoot diameter, and shoot/root ratio of height under well-watered treatments compared with water-stressed treatments.The shoot height was signifycantly increased in well-watered treatment (as control, WW) while the opposite trend was observed in severe drought stress (SDS) (Table 3 and Figure 2A).The growth traits focused to minimize the demand as previously discussed help oak maintain a higher physiological activity under unfavorable conditions of drought interacting with shade (Aranda et al., 2005;Jimenez et al., 2009).In addition, the watering regimes significantly affected all these morphological properties, and the effect was also significant except for root height and root diameter.
On the other hand, there were not significant difference in root height and diameter among water treatments (WW, MDS, and SDS) at the end of the expriment.Root height and root diameter indicated lower values (13.6 ± 1.3, 8.0 ± 1.1) in SDS than in WW (14.9 ± 3.8, 9.5 ± 1.1) and in MDS (14.0 ± 0.3, 8.9 ± 2.4), respectively, although the difference was not statistically significant (Table 3).
They also showed higher shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, and shoot/root biomass ratio in well-watered (WW) treatment compare with water-stressed treatments (MDS and SDS).Consequently, the seedlings grown under well-watered (WW) showed higher aboveground and belowground biomass than those of the MDS and SDS seedlings.
However, the direction of the relationship in shoot/root ratio of biomass indicated relatively high root system in water-stressed plants compared with the well-watered control plants.The results suggest root-morphological adaptations against drought on the more arid SDS.A deep and thick root system, with deep root length density, is the important factor contributing drought avoidance mechanisms to resistance to drought stress (Abrams, 1990;Dickson and Tomlinson, 1996).
Previous studies have suggested that drought avoidance mechanisms (deep root system, leaf curling, smaller leaf area, etc.) and drought tolerance mechanisms (osmotic adjustment, stomatal control to maintain moderate photosynthetic rates, etc.) are two drought strategies of different oak species indicated in response to water stress (Dickson and Tomlinson, 1996;Loewenstein and Pallardy, 1998).The values of shoot/root biomass ratio and height showed an increase of 1.8-and 1.7-fold, respectively, in well-watered (WW) as compared to values in severe drought stress (SDS) seedlings (Tables 1 and 3).Light is one of the most important environmental factors regulating growth and development (Ma et al., 2001).Seedlings grown under shade treatments had significantly different growth than that of seedlings grown under water regimes (Tables 2 and 4).Shoot height and diameter, root height and diameter were greatly increased in moderate sunlight (ML) treatment compared with other light treatments (full sunlight, FL; low sunlight,

LL; extremely low sunlight, ELL).
The allometry of biomass such as shoot diameter, and root height was significantly decreased as gradually dense shade (Table 4 and Figure given shade treatment, seedlings of sunlight (ML) conditions showed highly increase other shade-grown seedlings (LL and ELL shoot/root ratio of height showed opposite result under shade treatment.Root height per unit shoot height (SRR, shoot/root ratio) indicated more increase in shade conditions (ML, LL, and ELL) compared with sunlight (FL) treatment, and showed highly and ELL in different shade treatments (Table In spite of the differences found in morphological shoot height and diameter, similar allocation patterns height and diameter were found in FL and ELL, respectively, which belonged to the The moderate sunlight (ML) condition showed a tendency to allocate more biomass to shoot height.Nevertheless, there were no statistically significant differences diameter, root height, and root diameter between sunlight (FL) and the moderate sunlight (ML) (Table 4).Different oak species have developed both morphological and physiological adaptations to survive and grow on their growing environmental sites.
Morphological adaptations such as a lower above ground and deeper belowground in response to water stress is commonly observed in both drought avoidance and drought tolerance (Dickson and Tomlinson, 1996) Shade-tolerant species are generally manifested as an enhanced phenotypic plasticity in leaf morphological variations in comparison with physiological variations characteristics of different water (A) and light intensities (B) on seedlings at the end of the experiment.WW, well-watered; MDS, moderate drought stress; SDS, severe drought stress; FL, full sunlight; ML, moderate sunlight; LL, low sunlight; ELL, extremely shoot height, shoot diameter, and root height was significantly decreased as Figure 2B).Within a seedlings of the moderate highly increase than ELL).However, opposite result under Root height per unit shoot height (SRR, indicated more increase in shade ) compared with the full highly in ML than LL (Table 4).In spite of the differences found in morphological shoot patterns of root and ML, LL and to the same group.condition showed a tendency to allocate more biomass to shoot height.Nevertheless, differences in shoot t, and root diameter between the full moderate sunlight (ML) treatment ).Different oak species have developed both morphological and physiological adaptations to survive growing environmental sites.Morphological adaptations such as a lower above ground and deeper belowground in response to water stress is commonly observed in both drought avoidance (Dickson and Tomlinson, 1996).rant species are generally manifested as an enhanced phenotypic plasticity in leaf morphological variations in comparison with physiological variations (Valladares et al., 2005).In the present study, cork oak seedlings grown under shade adaptation strategy such as the increased leaf area the increased specific leaf area (SLA).

Specific leaf area (SLA)
Specific leaf area (SLA) reflects alternatively the leaf thickness and the relative rates of assimilative tissues in leaves (Rhizopoulou et al., 1991) The specific leaf area (SLA) was described as the ratio of leaf area to leaf dry mass (in cm light intensity (as shade, Figure statistically significant differences, while no significantly differences in the specific leaf area (SLA) were found among all water stress (Figure (Q.s suber L.).
As shown in Figure 3, shade suber were found to a higher specific leaf area (SLA) than the leaves grown in the full sunlight shade treatments, the highest specific indicated the extremely low sunlight (ELL) treatment (Figure 3B).The differences of biomass allocation of leaf tissue, as reflected by the increased specific leaf area (SLA) at the extremely low sunlight (ELL), suggested the decreased leaf thickness (Table 6).
The reliable effect of shade has approximately the strongest negative relationships on leaf area, which is a major factor in determining the leaf (Gregoriou et al., 2007).Moreover, the most common morphological plasticity in response characteristics of different water (A) and light intensities (B) on Q. suber watered; MDS, moderate drought stress; SDS, ; LL, low sunlight; ELL, extremely .In the present study, cork oak dlings grown under shade-induced stress indicated such as the increased leaf area and the increased specific leaf area (SLA).Specific leaf area (SLA) reflects alternatively the leaf thickness and the relative rates of assimilative tissues in (Rhizopoulou et al., 1991).The specific leaf area (SLA) was described as the ratio leaf area to leaf dry mass (in cm 2 g -1 ).The different Figure 3B) treatments showed statistically significant differences, while no significantly differences in the specific leaf area (SLA) were found 3A) treatments in cork oak , shade-exposed leaves of Q. igher specific leaf area (SLA) full sunlight (FL).In different shade treatments, the highest specific leaf area (SLA) indicated the extremely low sunlight (ELL) treatment (Figure 3B).The differences of biomass allocation of leaf tissue, as reflected by the increased specific leaf area (SLA) at the extremely low sunlight (ELL), suggested the thickness (Table 6).The reliable effect of shade has approximately the strongest negative relationships on leaf area, which is a major factor in determining the leaf photosynthesis .Moreover, the most common response to shade was to possess the higher specific leaf area (SLA) (Puértolas et al., 2008).Sun and shade-exposed leaves of trees indicate complete differences in their leaf size, morphology, composition, chloroplast structure and photosynthetic capacity.As compared to shade-induced leaves, sun-exposed leaves are often characterized by thicker leaves and smaller leaf area (Lichtenthaler et al., 1981).Sack and Grubb (2002) reported that the specific leaf area (SLA) in high irradiance declined across species with with increasing drought tolerance, from Viburnum to V. lantana, to Hedera and V. tinus.In addition, each study in response to severe shade was possessing a higher SLA.

Scanning electron micrograpy (SEM)
The measurement of stomatal frequency and size was conducted after the preparation of the adaxial surface of leaf epidermis.Stomatal control in response to different water stress is the first and one of the most important steps in maintaining some degree of photosynthesis by the minimal water loss (Dickson and Tomlinson, 1996) Although the length of the guard cells was approximately equal in leaves among treatments, the number of stomata per unit leaf area was significantly among treatments (Figure 4 and Table 5).
In the severe drought stress (SDS) was observed higher stomatal frequency as compared with watered (WW) treatment, while the extremely low sunlight (ELL) treatment showed the opposite trend and H).Leaves of seedlings grown under drought stress (SDS) had approximately 19 might reduce water loss at the whole-plant level, and the higher stomatal density probably avoided water stress by possessing flexible regulation (Bacelar et al., 2004).As a result, stomatal closure significantly occurs when plants are exposed to drought, in some cases (e.g., severe drought stress) photosynthesis may be more controlled by the chloroplast's capacity for CO 2 fixation (Yordanov et al., 2000).

Light micrograpy (LM)
The leaf area was significantly smaller under severe drought stress than those under the well-watered (WW) and the extremely low sunlight (ELL) treatment, whereas the leaf thickness showed the opposite trend (Table 6).Thicker upper epidermis was slightly found under wellwatered (WW) as compared to the severe drought stress (SDS) and the extremely low sunlight (ELL) leaves of Q. suber, while no significant differences in thickness of low epidermis were found among all treatments.Epidermal thickness under well-watered (WW) treatment seedlings was approximately 10 m, and 12 m larger on the upper epidermal of leaf surfaces, respectively, compared with in severe drought stress (SDS) and in the extremely low sunlight (ELL) treatment seedlings.Total leaf thickness was significantly lower in the extremely low sunlight (ELL,250.3 ± 13.3 m) treatment than in well-watered (WW, 337.1± 13.3 m) treatment and in severe drought (SDS, 426.4 ± 7.5 m) treatment (Table 6).This difference may be attributable to the lower layer of palisade parenchyma, due to possessing only single layer of palisade parenchyma compared to the sun-leaf structure with three layers of palisade parenchyma (Figure 5E).
Under severe drought stress (SDS) treatment with drought stress leaves, the total leaf thickness was extremely thicker, due to the thicker palisade parenchyma (containing three layers of mesophyll), also due to the higher spongy and epidermal thickness (Figures 5B and  E).The parenchyma of the leaves under wellwatered(WW) and severe drought stress (SDS) treatment was more densely packed in the palisade parenchyma layer than in the spongy parenchyma, whereas a significantly increased thickness of the spongy parenchyma layer was found in leaves of the extremely low sunlight (ELL) seedlings.A leaf structure under the extremely low sunlight (ELL) was observed in crosssections, with the greater proportion of spongy parenchyma by possessing three to four layers of spongy parenchyma, but with an only single layer of the palisade parenchyma (Figures 5C and F).
Plants grown under shaded conditions (that is, a low quantum flux density) normally have a higher SLA and thinner thickness, due to only shaded leaf consists of a single layer of the palisade parenchyma than those grown under natural irradiation (Young and Smith, 1980;Figure 5. Epidermal morphology of cross-sections using light microscopy (LM) through the leaf blade region of fully expanded leaf middle region in the well-watered (WW, A), the severe drought stress (SDS, B), and the extremely low sunlight (ELL, C) leaves.Notice differences in total leaf thickness, and size, shape and arrangement of the mesophyll tissues (UP, upper epidermis; LE, lower epidermis; PP, palisade parenchyma; SP, spongy parenchyma; IS, intercellular air spaces).Note that the palisade parenchyma had only one layer in the extremely low sunlight (ELL).Magnification: A-C, × 20, bar = 100 m; 1-3, × 40, bar = 50 m.Mendes et al., 2001;Terashima et al., 2006;Gregoriou et al., 2007).
In the extremely low sunlight (ELL) treatment, intercellular air spaces were greatly observed in the extent of present in the spongy parenchyma by the elevated shade effect.The effect of shade also led to the collapse of the palisade parenchyma.Evans et al. (1977) reported that the epidermal and palisade parenchyma cells frequently collapsed adjacent to spongy parenchyma cells.DeLucia et al. (1996) also concluded that the greater proportion of spongy parenchyma was displayed in shade leaves.The greater proportion and rounded shape of the spongy parenchyma of shade leaves may increase intercellular light scattering and thus absorptance, and increase the mean pathlength of light in leaves.
As compared to shaded leaves, sun-type leaves are typically thicker with multiple and longer layers of palisade parenchyma, followed by relatively thinner spongy parenchyma (Lichtenthaler et al., 1981).Oak species have been suggested that possess various effective morphological adaptations such as thicker leaf D E F Figure 6.Ultrastructure by a t palisade parenchyma cells submitted to well chloroplasts with the distinctive thylakoids ( from well-watered leaves.chloroplast envelope; Magnification: A, × 6,000, bar = 2 m. thickness, thicker cuticle thickness, greater stomatal density and decreased stomatal size that improve drought resistance, decrease photochemical damage, and contribute to minimal degree of photosynthesis under drought stress (Abrams, 1990;Dickson and Tomlinson, 1996).As a result, Q. suber seedlings may possess microscale morphological adaptations against drought on the more arid severe drought stress, which have well developed palisade parenchyma, whereas leaf thickness generally had negative effects on the shade effect.

Transmission electron micrograpy (TEM
An environmental stress can severely disrupt the ultrastructual features of chloroplasts and parenchyma Kwak et al. 10975 Ultrastructure by a transmission electron micrography (TEM) of the Q. suber parenchyma cells submitted to well-watered (WW) leaves.Well organized chloroplasts with the distinctive thylakoids (t), grana (g), and scarce plastoglobuli ( watered leaves.st, starch grains; pg, plastoglobuli; g, grana; t, thylakoid; ; cw, cell wall; pm, plasmalemma; arrowhead = chloroplasts.Magnification: A, × 6,000, bar = 2 m; B, × 20,000, bar = 1 m; C-D, × 40,000, bar = 0.5 thickness, thicker cuticle thickness, greater stomatal density and decreased stomatal size that improve drought resistance, decrease photochemical damage, and contribute to minimal degree of photosynthesis under (Abrams, 1990; Dickson and Tomlinson, may possess the microscale morphological adaptations against drought on the more arid severe drought stress, which have well whereas leaf thickness generally had negative effects on the shade effect.

TEM)
An environmental stress can severely disrupt the ultrastructual features of chloroplasts and parenchyma cells (Ristic, 1992).The plant's ability to adapt to environmental stress such as water stress and shade stress may be related to specific changes in the morphological, physiological, and ultrastructural level (e.g., chloroplast) of leaves 1981).
At the ultrastructural level, the palisade parenchyma chloroplasts of leaves of well were well developed (Figure 6A): their shape was regular chloroplast envelope membranes and grana were distinct and abundance of starch grains and scarce plastoglobuli were also visible inside in the chloroplasts ( D).
In contrast, severe drought stress (SDS) treatment to marked transformation to the structure of chloroplasts included swelling and disruption of the aberration of the chloroplast shape Kwak et al. 10975 Q. suber watered (WW) leaves.Well organized ), and scarce plastoglobuli (pg) , thylakoid; ce, ; arrowhead = chloroplasts.D, × 40,000, bar = 0.5 cells (Ristic, 1992).The plant's ability to adapt to environmental stress such as water stress and shade stress may be related to specific changes in the morphological, physiological, and ultrastructural level (e.g., chloroplast) of leaves (Meter and Lichtenthaler, At the ultrastructural level, the palisade parenchyma chloroplasts of leaves of well-watered (WW) treatment 6A): their shape was regular; chloroplast envelope membranes and grana were distinct; and abundance of starch grains and scarce plastoglobuli were also visible inside in the chloroplasts (Figures 6B,to In contrast, severe drought stress (SDS) treatment led marked transformation to the structure of chloroplasts included swelling and disruption of the thylakoid and shape (Figure 7).In some cases, the chloroplast envelope membranes were bro and not distinctive (Figure 7A).Despite the structural change of the reduced thylakoid, their shape was even deformed morphologically, the chloroplasts were observed not only large starch grains but also an increased larger size plastoglobuli within their interior Moreover, leaves of severe drought showed the obvious symptoms of senescence accumulation of the reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), in tonoplast and plasmalemma (Figure 7B to D).
The chloroplasts of the plants grown under the stress indicate the main damages in structural changes Ultrastructure by a transmission electron micrography (TEM) of the palisade parenchyma cells submitted severe drought stress (SDS) leaves.Note the numerous plastoglobuli, the distortion of chloroplast envelope and the swelling of thylakoids.st, starch grains; pg, plastoglobuli; ce, chloroplast envelope black arrow = localization of H2O2 in tonoplast (tp) and plasmalemma ); arrowhead = chloroplasts; asterisk = swellen thylakoid.Magnification: A, × 6,000, bar = 2 m; B, × 20,000, bar = 1 m; C-D, × 40,000, bar = 0.5 m. he chloroplast envelope membranes were broken the reduced grana and even deformed the chloroplasts were observed not only large starch grains but also an increased number and a within their interior (Figure 7B).stresse (SDS) showed the obvious symptoms of senescence due to accumulation of the reactive oxygen species (ROS), ), in tonoplast and The chloroplasts of the plants grown under the water stress indicate the main damages in structural changes such as the excessive swelling of the thylakoids, the distortion of the grana, and the appearance of the lipid droplets (Riatic, 1992;Lopez-Carbonell, 1994).
In drought-stressed Solanum tuberosum higher number of plastoglobuli exhibiting a larger size was detected in chloroplasts compared with well plants (Eymery, 1999).Chloroplasts of general cells possessed a few plastoglobuli, but the number and size of these lipophilic compounds increase degraded thylakoids in chloroplasts during leaf senescence and in etioplasts before thylakoid formation (Steinmüller, 1985;Eymery, 1999 reported that symptoms of leaf senescence were revealed in chloroplasts at drought Q. palisade parenchyma cells submitted severe drought stress (SDS) leaves.Note the numerous plastoglobuli, the distortion of chloroplast envelope and the chloroplast envelope; ) and plasmalemma ); arrowhead = chloroplasts; asterisk = swellen thylakoid.Magnification: A, × welling of the thylakoids, the distortion of the grana, and the appearance of the lipid Carbonell, 1994).Solanum tuberosum plants, a higher number of plastoglobuli exhibiting a larger size chloroplasts compared with well-watered 1999).Chloroplasts of general mesophyll few plastoglobuli, but the number and size of these lipophilic compounds increased due to in chloroplasts during leaf senescence and in etioplasts before thylakoid formation 1999).Munne-Bosch (2001) ymptoms of leaf senescence were drought-induced senescence, showing an accumulation of plastoglobuli in the chloroplasts, including the disruption of thylakoids and much less grana stack.The increased number and larger size of plastoglobuli in chloroplasts may result from storage of thylakoid decreased during the severe drought stress (SDS) (Eymery, 1999; Munne-Bosch Hydrogen peroxide (H 2 O 2 ) accumulation was observed first in the xylem vessels, in the cell walls, and in the plasma membrane of mesophyll cells of severely stressed plants, and increased during the summer (Munne-Bosch, 2001).Romero (2004) also suggested that the presence cadmium-treated pea leaves indicate the accumulation in Kwak et al.
Ultrastructure by a transmission electron micrography (TEM) of the Q. suber palisade parenchyma cells submitted the extremely low sunlight (ELL) leaves.
higher stacking degree and cytoplasm with lipid droplets from the extremely low sunlight (ELL) leave.pg, plastoglobuli; g, grana; t, thylakoid; ld, lipid chloroplast envelope; pm, plasmalemma; arrowhead = chloroplasts.Magnification: A, × 6,000, bar = 2 m; B, × 20,000, bar = 1 m; C-D, × 40,000, bar = 0.5 showing an accumulation of plastoglobuli in the chloroplasts, including the disruption of thylakoids and The increased number and larger size of plastoglobuli in chloroplasts may result from storage of thylakoid decreased during the severe drought Bosch, 2001).accumulation was observed first in the xylem vessels, in the cell walls, and in the plasma membrane of mesophyll cells of severely stressed plants, and increased during the drier and hotter , 2001).Romero-Puertas et al. gested that the presence of H 2 O 2 in treated pea leaves indicate the accumulation in the plasma membrane and tonoplast of different types.
On the other hand, the extremely low sunlight (ELL followed by severe shade stress from severe damage in chloroplasts ( shape was irregular and the chloroplast envelope membranes were not apparent.The main damage to the chloroplasts by the extremely low sunlight (ELL) not only the higher grana stacks and the broader grana, but also the higher stacking degree of thylakoids ( 8C).Furthermore, another distinct difference in extremely low sunlight (ELL, starch or exhibited only little starch in the chloroplasts as compared with the well-watered Kwak et al. 10977 Q. suber palisade parenchyma cells submitted the extremely low sunlight (ELL) leaves.
and cytoplasm with lipid droplets from the , lipid arrowhead = chloroplasts.D, × 40,000, bar = 0.5 the plasma membrane and tonoplast of different types.extremely low sunlight (ELL) followed by severe shade stress (SDS) may lethally suffer from severe damage in chloroplasts (Figure 8A): Their shape was irregular and the chloroplast envelope membranes were not apparent.The main damage to the extremely low sunlight (ELL) revealed ana stacks and the broader grana, ng degree of thylakoids (Figure 8C).Furthermore, another distinct difference in the , Figure 8A) possessed no starch or exhibited only little starch in the chloroplasts as watered (WW) and the severe drought stress (SDS) (Figure 6 and 7).In addition to chloroplasts, the intracellular space occupied in nearly all organelles, otherwise numerous small vacuoles and lipid droplets were present in the cytoplasm of these cells (Figure 8D).The ultrastructure of chloroplasts grown under low light exhibited the structural differences.The shade-type chloroplasts from radish seedlings (Rapbanus sativus L.) grown under the low-light possessed characterization such as the higher grana stacks, the higher stacking degree of thylakoids, the broader grana, and an insufficient starch as compared to those under the highlight (Meier and Lichtenthaler, 1981).The restraint of starch in the chloroplasts of low light leaves may be related to the low rate of photosynthesis at the low light intensities.Chloroplasts of Euphorbia forbesii was discovered a greater thylakoid membrane development in low light than in high light (Pearcy, 1986).Pearcy (1986) also suggested that E. forbesii as a result may possess a very flexible photosynthetic apparatus to survive in the understory of shaded forests.

Conclusions
This study examined the growth and anatomical aspects to examine the response of Q. suber to water and light intensity.Specifically, it focused on processes affecting drought and shade in Q. suber.
Seedlings of Q. suber grown under the severe drought stress lead to an increased stomatal frequency due to morphological plasticity such as the deeply folded, shrunken, and the overall reduction in epidermal cell size, a decreased shoot/root ratio due to stimulation of the deeper root growth.Moreover, in severe drought stress with drought stress leaves, the total leaf thickness was extremely greater, due to the thicker palisade parenchyma (containing three layers of mesophyll), also due to the higher spongy and epidermal thickness.The seedlings rapidly develop deep root system by stress effects due to soil water deficit.Furthermore, the response of its physiological root system is ability to avoid water stress in water stress-induced seedlings.
Severe drought stress (SDS) led to a marked transformation to the structure of chloroplasts included swelling and disruption of the thylakoid and aberration of the chloroplast shape.The chloroplasts were observed not only the large starch grains but also the increased number and the larger size plastoglobuli within their interior.Moreover, leaves of severe drought stresse (SDS) showed the obvious symptoms of senescence as accumulation of the reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), in tonoplast and plasmalemma.Specially, seedlings grown under the extremely low sunlight possess one palisade cell layers instead of three layers, decreased shoot/root ratio due to stimulation of deeper root growth and increased in specific leaf area (SLA) due to the lower leaf thickness (thinner).The extremely low sunlight (ELL) may lethally suffer from the severe damage in chloroplasts: Their shape was irregular and the chloroplast envelope membranes were not apparent.The main damage to the chloroplasts by the extremely low sunlight (ELL) was revealed not only the higher grana stacks and the broader grana, but also the higher stacking degree of thylakoids.
As a result, Q. suber seedlings may possess the microscale morphological adaptations against waterdeficit on the more drought stress in Mediterraneanclimate ecosystems, which have the deeper root growth and the well developed palisade parenchyma, whereas the shade stress generally has negative effect in the growth and the physio-biochemical aspect.

Figure 1 .
Figure 1.Allometric analysis of shoot and root biomass allocation in the growth response of seedlings to different levels of water (A) and light intensities (B) on Q. suber at the end of the experiment.Means (n=5) designated with the same letter are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.WW, well-watered; MDS, moderate drought stress; SDS, severe drought stress; FL, full sunlight; ML, moderate sunlight; LL, low sunlight; ELL, extremely low sunlight.

Figure 2 .
Figure 2. Growth characteristics of different water (A) and light intensities (B) on seedlings at the end of the experiment.severe drought stress; FL, full sunlight; ML, low sunlight.

Figure 3 .
Figure 3. Effects of different water (A) and light intensities (B) for the specific leaf area (SLA, cm 2 g -1 ) on Quercus suber seedlings at the end of the experiment.Bars represent mean values (n=15); the same column letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.WW, well-watered; MDS, moderate drought stress; SDS, severe drought stress; FL, full sunlight; ML, moderate sunlight; LL, low sunlight; ELL, extremely low sunlight.

Figure 4 .
Figure 4. Stomatal frequency and shape on the abaxial surface view using microscopy (SEM) of the well the extremely low sunlight (ELL, G severely folded in severe drought stress (SDS).Magnification: A, D, and G (× 200, Scale bar 100 m); B, E, and H (× 500, bar = 50

Table 1 .
Effects of different water stresses on biomass parameters in Q. suber at the end of the experiment.Means (n=5) in a row followed by the same letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.WW, well-watered; MDS, moderate drought stress; SDS, severe drought stress. *

Table 2 .
Effects of different light intensity stresses on biomass parameters in Q. suber at the end of the experiment.
*Means (n=5) in a row followed by the same letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.FL, full sunlight; ML, medium sunlight; LL, low sunlight; ELL, extremely low sunlight.

Table 3 .
Effects of different water stresses on Q. suber seedlings growth characteristics at the end of the experiment.Means ± S.E.(n=5) in a column followed by the same letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.z WW, well-watered; MDS, moderate drought stress. y

Table 4 .
Effects of different light intensity stresses on Q. suber seedlings growth characteristics at the end of the experiment.
y Means ± S.E.(n=5) in a column followed by the same letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.z FL, full sunlight; ML, moderate sunlight; LL, low sunlight; ELL, extremely low sunlight.

Table 5 .
The stomatal frequency and the size of guard cell from abaxial epidermal surface by a scanning electron microscopy (SEM) survey on effects of water and light intensity of Q. suber seedlings.

Table 6 .
Effects of water and light intensity stress on the leaf tissue thickness present in cross-section of Q. suber leaves.
y Means ± S.E.(n=5) in a column followed by the same letters are not significantly different at P 0.05 according to the least significant difference (LSD) multiple range test.z WW, well-watered; SDS, severe drought stress; ELL, extremely low sunlight.