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Genetic Diversity and Physiological Response to Drought Stress of Chamaecyparis obtuse from Six Geographical Locations
Plant Breed. Biotech. 2021;9:112-123
Published online June 1, 2021
© 2021 Korean Society of Breeding Science.

Tae-Lim Kim, Kyungmi Lee, Wonwoo Cho, Danbe Park, Il Hwan Lee, Hyemin Lim*

Division of Forest Tree Improvement, National Institute of Forest Science, Suwon 16631, Korea
Corresponding author: Hyemin Lim, supia1125@korea.kr, Tel: +82-31-290-1116, Fax: +82-31-290-1009
Received December 27, 2020; Revised April 3, 2021; Accepted April 8, 2021.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Water deficit is a critical factor obstructing the growth and survival of plant. Therefore, researchers have been trying to develop drought-resistant varieties. To find indicators of drought stress-tolerance of cypress (Chamaecyparis obtusa), we analyzed the response of cypress seedlings from six provenances of Korea (Jeju, Suwon, Seoul, Seongnam, Yong-in, and Osan) to drought stress. Additionally, the genetic diversity of C. obtusa from the six provenances were determined using microsatellite markers. We confirmed that populations from Suwon and Seongnam were relatively separated from other populations through genetic distance and cluster analysis. We examined their physiologic and metabolic responses after drought treatment for five weeks. Almost all of the cypress seedlings showed a reduced shoot growth rate under drought treatment compared to controls. In addition, temperature of drought treated cypress seedling leaves was 1.2-2°C higher than that of the controls. Almost all of the drought stress-treated cypress showed increased carbon metabolite contents and pigments. In particular, the cypress seedlings from Osan showed the highest increase in all of the measured metabolites. Therefore, it is suggested that the seedlings from Osan are susceptible to drought stress. Conversely, the seedlings from Jeju, Suwon, and Yong-in showed a lower sensitivity to drought treatment. These results indicate that the cypress trees from the six provenances have a different response to drought stress. In addition, it is confirmed that previously identified indicators of drought stress, especially those that measure total soluble sugar, carotenoid, and H2O2, can be used in the selection of drought resistance cypress. These findings may useful in studies concerned with the metabolic and physiological responses of young cypress to drought.
Keywords : Chamaecyparis obtusa, Drought, Soluble sugar, H2O2, Genetic diversity
INTRODUCTION

Plants encounter obstacles from various biotic and abiotic stresses such as plural, bacteria, salts, osmotic effect, heat, cold, drought, and floodwaters for their whole life, and they have an adaptive response to these stresses, which allow them to survive (Bray 1997). Drought is the one of the most important abiotic stress factor for plants. It severely impairs their growth, development, and repro-duction. Plants have drought tolerance, but difference species or varieties have different adaptive capabilities. Severe drought induces dehydration in plants, causing of plants to wither and die. A number of papers have shown that drought stress strongly affects the crop yield, growth inhibition, pigment synthesis, photosynthetic activity, membrane integrity, osmotic adjustment, stomatal opening, cell division, and accumulation of reactive oxygen species (ROS) in plants (Sairam and Srivastava 2001; Benjamin and Nielsen 2006; Duan et al. 2007; Shinozaki and Yamaguchi-Shinozaki 2007; Praba et al. 2009; Lum et al. 2014). There has been a progressive increase in droughts, which are becoming hotter due to climate change, and it has been predicted that there will be an increase in their frequency and severity over all the world (Naumann et al. 2018). Above all, the environmental conditions at the seedling stage are very important and strongly influence the survival and growth during the whole life of plant (Danby and Hik 2007; Sung et al. 2011). Trees have a very long growth period in one generation, so choosing a tolerant variety at the seedling stage is very important in relation to the reduction of costs and time. Therefore, researchers have been focusing on physiological and metabolic reactions caused by drought stress in seedlings and trying to find screening factors for drought-resistance.

C. obtusa (Sieb. and Zucc.) Endl. (Cupressaceae), known as Hinoki cypress is a native tree widely planted in Japan. C. obtusa is known to be planted in relatively dry places because it is tolerant to drought stress (Nagakura et al. 2004). Because of the high quality of its wood, it is used as a pillar for building, furniture, and sculpture material (Nakao et al. 2001; Choi et al. 2012). Cypress has mainly been studied in relation to material identification and content analysis. Recently, researchers have studied cypress extract and reported that cypress essential oil has anti-inflammatory effect in human health (Raha et al. 2019). Cypress has mainly been planted in the southern province of Korea since it was introduced in (Yoon 1959).

This present study used six groups of C. obtusa seedlings from six different places in Korea, to determine the change of metabolites and physiological response to drought stress in cypress. Six groups of C. obtusa were obtained from Jeju, Osan, Seoul, Seongnam, Suwon, and Yong-in, which are divided into two habitats, one in the southern part of Korea (Jeju) and the other five in the central part of Korea (Osan, Seoul, Seongnam, Suwon, and Yong-in). They have come from seed orchards that have been spatially isolated and separated for decades. We compared the 6 C. obtusa populations in terms of their genetic differences and differences in their response to drought for the selection and development of drought-resistant varieties.

MATERIALS AND METHODS

Plant materials and drought treatment

Seeds of Jeju, Suwon, Seoul, Seongnam, Yong-in, and Osan, located in South Korea, were collected from a seed orchard of C. obtusa. Then the seedlings were used in this study. In April 2019, the seeds were sown in a greenhouse. Seventeen weeks after sowing the seeds, healthy and uniform plantlets were randomly selected from each treatment group. Plants were watered to a soil moisture level of 40% one day before the drought treatment. The plants were then deprived of water for 33 days and observed during this time. Soil moisture was measured every other day using a moisture probe (ICT International Pty. Ltd., Armidale, NSW, Australia).

DNA extraction and microsatellite genotyping

Fresh leaf tissue was collected from 24 individuals from each of the 6 populations of C. obtusa and, stored at ‒80℃ until DNA extraction. The total genomic DNA was extracted using ExgeneTM Plant SV mini kits (GeneAll, Seoul, Korea). We characterized each individual at seven microsatellite loci (Table 1): Co31 (Nakao et al. 2001), Cos0319, Cos1536, Cos1951, Cos2224, Cos2610, and Cos2680 (Matsumoto et al. 2010). PCR was performed in 20-40 mL reactions containing 5 ng of template DNA, 2.5 mM of MgCl2, 0.2 mM of each dNTP, 0.2 mM of 6-FAM fluorescent dye-labeled forward primer and reverse primer (Supplementary Table S1), 1 unit of A-Star Taq DNA Polymerase (BIOFACTTM, Daejeon, Korea), and 1 × reaction buffer. The PCR cycling was conducted as follows: 2 minutes at 95℃ for predenaturation followed by 32 cycles of 30 seconds at 95℃, an annealing step of 30 seconds at 50-59℃ for each primer, and an extension step of 30 seconds at 72℃, followed by a final extension for 5 minutes at 72℃. The fluorescent PCR products were mixed with Hi-Di formamide and GeneScan 500 ROX Size Standard (Applied Biosystems). The amplified PCR products were separated electrophoretically by an ABI 3730 Genetic Analyzer and scored using the GeneMapper 5.1 software (Applied Biosystems, CA, USA).

Table 1 . Genetic characteristics of C. obtusa in Korea based on a microsatellite analysis using seven microsatellite loci.

LocusNANEHOHEFISFSTGST
Co3152.50.5620.5990.0650.144**0.121
Cos0319206.40.9240.840‒0.101*0.052**0.043
Cos1536275.70.6880.8220.162**0.070**0.058
Cos1951278.40.8680.8730.006*0.089**0.075
Cos2224114.00.7970.755‒0.0550.105**0.090
Cos2610124.10.7780.735‒0.059**0.110**0.094
Cos268072.90.6370.6410.0070.140**0.120
Overall8.34.870.7500.7520.0020.0990.084

NA: Number of alleles, HO: Observed heterozygosity, HE: Expected heterozygosity (Unbiased), FIS: Fixation index, GST: coefficient of gene differentiation (Nei 1973); *P < 0.05; **P < 0.01.



Genetic diversity and differentiation of populations

To investigate the genetic diversity in the population of C. obusa in Korea, the genetic parameters of diversity and differentiation were estimated. The estimation of the polymorphism, heterozygosity and genetic differentiation was accompanied by an exact test (Rousset 2008). GST (Nei 1973) was additionally estimated for comparison with previous studies on the species (Goudet 2003). Nei’s genetic distance (Nei 1978) between the populations was used in the neighbor-joining (NJ) tree (Saitou and Nei 1987; Felsenstein 1993). To identify the distinct genetic groups in the studied populations, the Bayesian method was applied (Pritchard et al. 2000) and lnP(K) and ΔK were calculated to select the appropriated K value (Earl 2012).

IR thermal imaging

Leaf temperatures were determined by measuring infrared (IR) digital images 33 days after the onset of drought using a Fluke TiX560 Thermal imager (Fluke Corp., Everett, WA, USA).

Measurement of the chlorophyll content

The chlorophyll content was measured as described previously (Sibley et al. 1996). For each treatment, 0.1 g of fresh sample was harvested in triplicate, mixed up thoroughly with dimethyl formamide (DMF), and centrifuged at 12,000 × g for 10 minutes at 4℃. The supernatant was used as the chlorophyll source. The chlorophyll levels were measured by reading the supernatant absorbance at 645 nm and 663 nm using a Biospectrometer (Eppendorf, Hamburg, Germany). The chlorophyll contents and their means were calculated as follows for each plant and treatment:

  • Chlorophyll a = 12.7 × A663 nm – 2.79 × A645 nm

  • Chlorophyll b = 20.7 × A645 nm ‒ 4.62 × A663 nm

  • Total chlorophyll = 17.9 × A645 nm + 8.08 × A663 nm

  • Carotenoids = (1000 × A470 nm ‒ 1.82chl a ‒ 85.02chl b)/198

  • (A, absorbance; pigment concentration in mg/g fresh weight (FW))

Extraction and measurement of soluble sugar

Glucose, fructose, and sucrose were extracted from the leaves using the method in (Lu and Sharkey 2004). The sugar concentrations were determined enzymatically using a method described (Stitt et al. 1989) and a Biospectrometer (Eppendorf, Hamburg, Germany).

The total soluble sugars were extracted from leaf tissues with 80% ethanol using a modified method of the presented (Irigoyen et al. 1992), which is as follows. After the fresh weight determination, the leaves were homogenized by grinding them in liquid nitrogen with mortar and pestle. Then 2 mL of 80% (v/v) ethanol was added and the sample was vortexed for 1 hour. After centrifugation at 6,000 × g for 10 minutes, the supernatant was collected. The super-natants were added with chloroform and completely mixed. After centrifugation at 12,000 × g for 10 minutes, 50 mL of supernatant was reacted with 4.95 mL of freshly prepared anthrone reagent (500 mg anthrone + 50 mL 72% H2SO4) at 100℃ for 15 minutes. After cooling on ice, the total soluble sugar content was determined at 620 nm using a Biospectrometer (Eppendorf, Hamburg, Germany), with glucose as the standard.

Measurement of malondialdehyde (MDA)

Samples of 0.1 g leaves were collected in triplicate and extracted with 20% TCA (w/v) and 0.5% thiobarbituric acid (TBA) (w/v), then warmed at 95℃ for 30 minutes. The reaction was stopped by placing the mixture on ice for 30 minutes then centrifuging it at 12,000 × g for 10 minutes. The absorbance of the supernatant was measured at 532 nm using a Biospectrometer (Eppendorf, Hamburg, Germany). The MDA content was derived using the method (Heath and Packer 1968).

Measurement of Proline

Proline was extracted from a sample of 0.5 g fresh leaves material samples in 3% (w/v) aqueous sulfosalicylic acid and estimated using a ninhydrin reagent according to the method (Bates et al. 1973). The absorbance of a fraction with toluene separated from the liquid phase was read at a wave length of 520 nm. The proline concentration was determined using a calibration curve and expressed as m mol proline g-1 FW.

Measurement of Hydrogen peroxide (H2O2)

The H2O2 content was determined using the following protocol, presented (Alexieva et al. 2001); 0.1 g of leaf tissue was grinded in 1 mL of 0.1% trichloroacetic acid (TCA) and homogenized, then incubated for 5 minutes at room temperature. After centrifugation at 12,000 × g for 15 minutes, the supernatant was transferred to a new 2 mL tube. The supernatant was kept in the dark for 1 hour after mixing with 0.5 mL of 100 mM K-phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide. The absorbance of the resulting solution was measured at 390 nm. The amount of hydrogen peroxide was calculated using a standard curve prepared with known concentrations of H2O2.

RESULTS

Genetic diversity and population differentiation

We collected 144 individual seedlings from 6 populations located in different regions of Korea. The number of alleles in each locus was in the range of 5 to 27 with the mean value by population of 8.3, and the average effective number of alleles was 4.87 (Table 1). The overall observed and expected heterozygosity was 0.562-0.924 and 0.599-0.873, resulting in an average value of 0.750 and 0.752, respectively. The overall FIS value was 0.002 ranging from ‒0.101 to 0.162 in each locus. The FST and GST were 0.099 and 0.084 respectively. In each population, the number of alleles and expected heterozygosity were relatively low in the populations from Seongnam, Suwon, and Yong-in compared to the other three populations (Table 2). The populations from Suwon and Seongnam were relatively apart from the other populations in terms of their Nei’s genetic distance (Fig. 1a). In the clustering analysis, a K value of 3 was selected, referring to the median of lnP(K) and ΔK. A genetic cluster of populations in Jeju, Osan, Seoul, and Yong-in was observed while the populations from Seongnam and Suwon were different in terms of their genetic admixture (Fig. 1b, c).

Table 2 . Genetic characteristics of the population of C. obtusa in Korea based on microsatellite analysis.

PopulationNANEHOHEFIS
Jeju9.35.80.744 (0.055)0.802 (0.042)0.073**
Osan9.95.50.725 (0.066)0.790 (0.039)0.081**
Seoul10.67.20.818 (0.043)0.833 (0.043)0.019*
Seongnam7.63.40.726 (0.073)0.683 (0.047)‒0.063
Suwon4.73.10.780 (0.051)0.672 (0.034)‒0.164**
Yong-in7.84.20.710 (0.098)0.733 (0.057)0.028**

NA: Number of alleles, HO: Observed heterozygosity, HE: Expected heterozygosity (Unbiased), FIS: Fixation index, GST: coefficient of gene differentiation (Nei 1973); *P < 0.05; **P < 0.01.


Figure 1. Genetic relationship and population structure of the C. obtusa from the 6 regions in Korea. (a) Unrooted Neighbor-joining tree based on the Nei’s genetic distance (Nei 1978). OS: Osan; SE: Seoul; JJ: Jeju; YI: Yong-in; SW: Suwon; SN: Seongnam. (b) The median of LnP(K) and ΔK (Evanno et al. 2005) for each K, (c) Bar plot representation for K = 3, the number on the x-axis indicates the population in Jeju, Osan, Seoul, Seongnam, Suwon, and Yong-in in the ascending order.

Growth phenotype analysis of C. obtusa seedlings under drought treatment

Six groups of cypress seedling (Jeju, Seoul, Seongnam, Suwon, Yong-in, and Osan) were stopped supplying of water for 33 days (Fig. 2a). After ten days, the water supply of the seedlings was stopped, and the soil water contents was remained below 10% for three weeks, and decreased to less than 3% after 25 days. The transpiration through stoma is related to the temperature and physiological activity of plant leaves (Brito et al. 2019). According to the infrared digital image, the control seedlings were subjected to a temperature of 32.24℃, 34.29℃, 32.71℃, 32.53℃, 32.04℃, and 31.66℃ in Jeju, Seoul, Seongnam, Suwon, Yong-in, and Osan, respectively (Fig. 2c). The Jeju, Seoul, Seongnam, Suwon, Yong-in, and Osan temperatures for the drought-treated plants were 33.44℃, 35.78℃, 34.50℃, 34.60℃, 33.46℃, and 33.32℃, res-pectively. Almost all of the seedlings showed increases of 1.2-2℃ in drought-treated plants relative to their respective controls. To evaluate the growth of C. obtusa seedlings treated with drought stress, height was measured after the drought treatment (Fig. 2b). For almost of seedlings, the growth rate showed a slight reduction in the heights of the drought-treated seedlings, compared to their respective controls. Only Suwon’s seedling showed no significant difference between the drought-treated seedling and the control sample.

Figure 2. Height growth phenotype analysis and thermal images of C. obtusa seedling under drought treatment. (a) Volumetric water content in the soil of the control- and drought-treated plant pots. The soil moisture was measured every 2 days for 33 days. The control plants were watered throughout the experiment. (b) The effect of drought on the shoot growth of the C. obtusa seedlings from Jeju, Suwon, Seoul, Seongnam, Yong-in, and Osan. The values are the means ± SD (n = 10). (c) Growth phenotypes of C. obtusa Jeju, Suwon, Seoul, Seongnam, Yong-in, and Osan seedlings were subjected to drought treatment. Control plants (left) and drought-treated plants (right) after 33 days. Infrared thermal images.

Analysis of leaf chlorophyll content

Leaf chlorophyll content is used as an indicator of photosynthetic activity, and chlorophyll levels are affected by abiotic and biotic stresses. In previous reports, chloro-phyll content was shown to be affected by the external environment (Zhao and Liu 2009). While drought stress influences the chlorophyll content in many other plants, the chlorophyll content in drought-treated seedlings signifi-cantly increased for all of the seedling groups (Table 3). Furthermore, the total chlorophyll, carotenoid, chlorophyll a/b, and chlorophyll/carotenoid in all of the drought-treated seedling groups were significantly different from those of the controls. In particularly, the Osan seedling showed the greatest increase in all chlorophyll contents compared with the other populations. These results showed that the photosynthetic pigment level in C. obtusa sensitively reacts to drought stress.

Table 3 . Effects of drought-treatment on the photosynthetic pigments in C. obtusa seedlings.

Treatmentmg/g FWz)Chl a/bz)Chl/Carz)
Chl aChl btotal Chlcarotenoids
JejuControl0.83±0.050.21±0.021.05±0.040.23±0.033.92±0.654.56±0.73
Drought1.55±0.04**0.53±0.10**2.08±0.11**0.32±0.02*3.00±0.716.37±0.77
SuwonControl0.94±0.130.21±0.101.15±0.230.23±0.0085.06±2.234.90±0.90
Drought1.69±0.06**0.60±0.10*2.29±0.15**0.35±0.02**2.85±0.496.46±0.69
SeoulControl0.93±0.080.33±0.091.27±0.170.2±0.0022.86±0.496.31±0.93
Drought2.03±0.05**0.68±0.13*2.72±0.18**0.44±0.02**3.03±0.526.18±0.80
SeongnamControl0.83±0.040.30±0.091.13±0.140.17±0.0073.00±1.006.47±1.08
Drought2.03±0.10**0.75±0.20*2.78±0.29**0.41±0.03**2.83±0.776.71±1.13
Yong-inControl0.85±0.070.30±0.071.15±0.140.18±0.0062.88±0.486.24±0.95
Drought1.41±0.12**0.44±0.04*1.86±0.16**0.32±0.02**3.16±0.135.70±0.08
OsanControl1.01±0.030.41±0.031.42±0.060.2±0.012.43±0.157.00±0.47
Drought2.57±0.09**0.90±0.07**3.47±0.17**0.51±0.007**2.85±0.146.71±0.23

z)The values are the means ± SD (n = 4); The asterisks indicate significant differences (t test; *P < 0.05, **P <0.01) between the control and drought-treated seedlings.



Determination of soluble sugars

To estimate the effect of drought stress on the carbon metabolite contents of cypress, the glucose, fructose, sucrose, and total soluble sugar contents were investigated (Fig. 3). The amounts of glucose, fructose, sucrose, and total soluble sugar in the C. obtusa seedlings were measured to determine the influence on carbon partitioning under drought stress. Almost all of the drought-treated seedlings were significantly increased in glucose contents compared with their controls. There were slight differences in the fructose content between the drought-treated seedlings relative to the controls. The sucrose contents in the drought-treated seedlings from Seoul, Yong-in, and Osan were lower than those in the control seedlings. The total soluble sugar contents in the drought-treated seedlings were higher than those in the controls. In particular, under drought treatment, the total soluble sugar content of the Osan, Seoul, and Seongnam seedlings increased significantly, compared with the controls. In many cases, the drought stress condition induced an increase in the soluble sugar concentration in plants (Rosa et al. 2009).

Figure 3. Effects of the carbohydrates in the C. obtusa seedlings subjected to drought treatment. (a) Glucose. (b) Fructose. (c) Sucrose. (d) Soluble sugars. The values are the means ± SD of three independent measurements. The asterisks indicate significant differences (t test; *P < 0.05, **P < 0.01) between the control and the drought-treated seedlings.

MDA, proline, and H2O2 levels in C. obtusa seedlings

MDA content is commonly used as an index of oxidative lipid injury and tissue damage induced by drought stress. The MDA level in the drought treated seedlings from Jeju and Yong-in was higher than that in their respective controls (Fig. 4a). The increase in the MDA contents in the drought-treated seedlings indicates that the Jeju and Yong-in seedlings were physiologically damaged by drought treatment, and oxidative injury. The accumulation of proline in plant tissues is a marker for drought stress, like the compatible osmolyte (Mafakheri et al. 2010). Almost all of the drought treated seedlings showed increases in their levels of proline, and a significant accumulation of proline was observed in the drought-treated Osan and Seoul seedlings (Fig. 4b). Drought stress is known to increase H2O2 production. All groups of seedlings that underwent drought treatment showed a higher level of H2O2 than the controls (Fig. 4c). All six seedling populations showed significant difference.

Figure 4. Measurement of the MDA, proline, and H2O2 levels. (a) MDA. (b) proline. (c) H2O2. The asterisks indicate significant differences (t test; *P < 0.05, **P < 0.01) between the control and drought-treated seedlings.
DISCUSSION

Six seedling populations of C. obtusa were subjected to drought treatment for 33 days and compared to each control plants. We analyzed the genetic differences between six regionally isolated cypresses. In addition, we compared the metabolic and physiological 6 differences between 6 cypress populations after drought treatment. We examined whether the level of the change in the photosynthetic pigment, soluble sugars, and antioxidant substances can be used to select drought-tolerant cypress plants.

The number of alleles (NA) of most of the loci in this study of Korean populations exceeded the NA previously reported in the study on the development of the markers (Matsumoto et al. 2006). In most of the loci, the observed and expected heterozygosity of these markers were lower than those reported above. The overall GST (0.084) indicated a higher level of genetic differentiation in the cypress populations in Korea than in the Japanese populations (GST =0.039) (Table 1) (Tsumura et al. 2007). This is thought to reflect the discontinued distribution of the cypress population in the mid-region of Korea. The genetic distance and cluster analysis based on the Bayesian method showed the genetic differentiation of the populations in Seongnam and Suwon from the others (Fig. 1).

Leaf temperature is related to the function of trans-piration with stoma and influences plant physiological activity (Lim et al. 2017). In the current study, most of six seedlings also showed a decrease in rate of shoot growth and an increase in leaf temperature under drought condition, except seedlings of Suwon. The control and drought-treated seedlings of Suwon did not show a significant difference in their growth rate (Fig. 2).

In many cases, the chlorophyll content of plants has been observed to decrease or remain unchanged under drought stress. However, some plants (canola, wheat, and sesame) encounter a water deficit condition, and the chlorophyll content of leaves is increased and then remains unchanged (Mensah et al. 2006; Hassanzadeh et al. 2009; Sepehri and Golparvar 2011). The above results are consistent with the results of this study confirming an increase in chlorophyll a, chlorophyll b, and the total chlorophyll content under drought treatment at seedling stage during short period (Table 3). The regulation of the carotenoid level is dependent on the species, duration and intensity of water deficits. Recent reports showed that carotenoid contents tend to decrease under mild drought stress. Conversely, when the drought stress was severe, the content tended to increase slightly (Doupis et al. 2013; Sudrajat et al. 2015). In this experiment, all seedlings under drought treatment showed an increased in their carotenoid content, and this means that the seedlings were severely affected by drought treatment.

While under drought stress, an increase in the level of soluble sugar of plants can occur, and this may be a protective reaction in maintaining their cell turgor by improving their water holding and water absorbing capacity (Ji et al. 2014; Azzeme et al. 2016; Guo et al. 2018). In the present study, the level of total soluble sugar increased under drought treatment to maintain the cell turgor (Fig. 3). In particularly, the drought-treated seedlings of Seoul, Seongnam, and Osan responded sensitively, and showed a significant increase. Glucose and sucrose were reported to play the role of substrates for osmolytes and cellular respiration to maintain cell homeostasis in plant (Gupta and Kaur 2005). By contrast, fructose is not related to osmoprotection, and is related to the synthesis of secondary metabolites (Hilal et al. 2004). Our results showed that the level of glucose increased, but the level of sucrose generally decreased under drought treatment.

Under drought stress conditions, plants contain high levels of proline as an osmolyte to balance water stress, which enables them to maintain low water potentials and take up water from the environment (Delauney and Verma 1993; Kumar et al. 2003). In this study, the level of proline increased in the drought-treated seedlings, but most of the seedlings showed a slight increase, except for the Osan seedling (Fig. 4b). The Osan seedlings showed a stronger osmotic control response under drought treatment, with an increased proline content, compare with the seedlings from the other regions.

Under drought stress conditions, the cell membrane is damaged by free radicals, resulting in the peroxidation of unsaturated fatty acids in phospholipids (Hessini et al. 2009). The level of MDA increased in all seedlings under drought treatment, but it is difficult to compare the differences between the seedlings from the different regions (Fig. 4a). Under abiotic and biotic stresses, plants incur oxidative damage to macromolecules and cell structures cause overproduction of reactive oxygen species (ROS; hydrogen peroxide, H2O2; superoxide; hydroxyl radical and singlet oxygen). The level of H2O2 increased in all seedlings under drought treatment (Fig. 4c). In particularly, the level of H2O2 in Seoul, Seongnam, and Osan seedlings were significantly higher than that of the other seedlings under drought treatment.

In this study, we analyzed the genetic diversity of C. obtusa in the plants from the 6 regions in Korea, and the genetic differences between some populations were identified. Apart from that, it was confirmed that there is a difference in the responses of the C. obtusa populations in Korea to drought stress. We confirmed that previously used indicators for drought stress in other plants can be used for the selection of drought tolerant cypress. In particularly, we observed that the measurements of carotenoid, total soluble sugar, and H2O2 showed increases in all populations under drought treatment. In addition, we observed a difference in the reaction sensitivity between populations under drought treatment. The seedlings of Seoul, Seongnam, and Osan sensitively react to drought stress, showed a significant increase in the level of contents, compared with the seedlings of Jeju, Suwon, and Yong-in. Therefore, the C. obtusa from the 6 regions in Korea can be divided into two groups, depending on the sensitivity of their reaction to drought stress. This means that it could be used as an index of susceptibility relating to the water deficit state of cypress. The differences in the physiological and metabolic response between these populations could be beneficially used in the selection and development of drought-tolerant cypress. These results may provide a better understanding of the metabolic and physiological responses of C. obtusa to drought stress and expedite the processing of selection to drought-resistant cypress.

SUPPLEMENTARY MATERIALS
pbb-9-2-112-supple-table.pdf
CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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