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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.51 No.3 pp.37-48
DOI : https://doi.org/10.14397/jals.2017.51.3.37

Physiological Response of OsHSP26 Overexpressed Transgenic Poplar(Populus alba L.) on Temperature Stress

Dong Jin Park1,3 , Chang-Mi Heo2 , Woo Hyung Yang3, Seong Hyeon Yong3, Byung-hyun Lee4, Myung-Suk Choi1,3*
1Institute of Agriculture & Life Science, Gyeongsang National University, Jinju, 52828, Korea
2Gyeongsangnamdo Forest Environment Research Institution, Jinju, 52615, Korea
3Divison of environmental forest science, Gyeongsang National University, Jinju, 52828, Korea
4Division of Applied Life Science(BK 21), PMBBRC, Gyeongsang National University, Jinju, 52828, Korea

† These authors contributed equally to this work.

Corresponding author: Myung-Suk Choi +82-55-772-1856+82-55-772-1859mschoi@gnu.ac.kr
February 15, 2017 April 3, 2017 April 5, 2017

Abstract

The ability of plants to endure environmental stress factors, which are going to be more severe due to global warming, is important especially for forest plants. Because obtain trait of resistance to temperature using conventional breeding for woody plants is a time consuming way. In this study, chloroplast-localized OsHSP26 gene was overexpressed in Populus alba L. to breed tolerant transgenic poplar to temperature stress. The plantlets of OsHSP26-overexpressed transgenic poplar showed more heathy phenotypic response than wild-type plants under both prolonged low- and high-temperature stress. While the SPAD value, which refers chlorophyll content, in wild-type plants decreased depending on the exposure time to the temperature stress, higher SPAD value were shown in the transgenic plants. The contents of total phenolic compounds in the transgenic plants were lower than those of the wild-type plants, and not significantly changed except in the treatment of prolonged low-temperature. However, the total flavonoids contents of the transgenic plants were dramatically increased under prolonged temperature stress. The DPPH scavenging activities of the transgenic plants were higher than those of the wild-type plants under temperature stress. Consequently, it was revealed that overexpressing OsHSP26 allow for P. alba to be tolerant to temperature stress.


초록


    Introduction

    Since temperature stress factor are getting more severe by global warming, inhibitions in growths and productivity have occurred not only for commercial crops but also for forest trees. The inhibition in growths of forest trees was a major reason for forest decline. Allen et al.(2010) reported that the forest declines by climate change have happen globally. On global warming, the roles of forest are emphasized because forest trees have also played a role of an important carbon sink, not only for timber industry. However, the outputs of breeding temperature stress tolerant trees have been poorly reported even though there are many studies for understanding physiological, metabolic and molecular responses in forest trees on temperature stress such as poplar family and pine tree family(Percival & Noviss, 2010; Popko et al., 2010; Grene et al., 2012; Song et al. 2014; Jia et al., 2015). In conventional breeding methods, the long life cycle of forest trees has been noted as a major reason for a limit of tree breeding.

    Molecular breeding has been suggested for a counterplan for conventional breeding. genetically modified crops for enhancing productivity have been developed in many commercial species such as rice, barely, wheat, maize etc.(Baker et al., 2006; Jiao et al., 2010; Kogel et al., 2010; Rivera et al., 2013). Especially, the molecular breeding is anticipated as the way to breed temperature stress tolerant trees efficiently. PeBREB2L-overexpressed poplar transgenic plants were tolerant to dehydration, salinity and freezing stress(Chen et al., 2011). Overexpressing exogenous Cry1Ac, Cry3A, BADH genes in transgenic lines of Populus euramericana showed insect-resistance and salt-tolerant(Yang et al., 2016).

    Heat shock proteins(HSPs) are known as a molecular chaperone and wildly conserved in various plant species. In an earlier study, HSPs were related to responding to heat shock in plants(Ritossa F, 1962). However, it has been revealed that HSPs are involved in protecting plants from various abiotic stress such as cold, UV light, wound healing, tissue remodeling, or biotic stresses(Lindquist & Craig, 1988; Vierling, 1991; Boston et al., 1996). Therefore, over-expressing HSPs in commercial crops has attempted for enhancing their tolerance to abiotic stress factors(Sato & Yokoya, 2008; Chauhan et al., 2012; Wang et al., 2015).

    Low molecular HSPs, as classified as small heat shock proteins(sHSPs), also function as molecular chaperones similar to the other HSPs. CsHSP17.5 which is isolated from Castanea sativa is upregulated by freezing stress and function for acquiring freezing stress(Lopez-Matas et al., 2004). Polenta et al.(2007) reported that they isolate 4 major thermal- inducible proteins(HSPC1, HSPC2, HSPC3 and HSPC4), which molecular weights are 21 kDa, from tomato pericarp. Especially, it has been published that OsHSP26 is up-regulated by high temperature and oxidative stress(Lee et al., 2000), and the OsHSP26 overexpressed tall fescue transgenic plants are tolerant to heat and oxidative stress(Kim et al., 2012). Therefore, we hypothesized that overexpressing OsHSP26 allow tolerance for forest tree to temperature stress. This study was carried out to establish OsHSP26 overexpressed transgenic poplar. Moreover, we figured out the appearance of the transgenic poplar on temperature stress.

    Material and methods

    1Plant material and in vitro culture conditions

    P. alba leaf segments(approximately 1×1cm) were surface-sterilized in solution 1%(v/v) sodium hypochlorite for 10 min and then rinsed three times with sterile distilled water. After surface sterilization, the leaf segments were placed into vessels containing 50 ml MS solid medium containing 0.5 mg/L BAP for inducing regenerated plantlets. To obtain the in vitro plantlets the vessels containing the leaf segments were incubated at 25℃ under light from cool-white fluorescent lamps(2500 Lux) with 16/8 hr(light/dark) period for 5-6 weeks.

    The basal medium used throughout the experiments consisted of MS inorganic salts, 3% sucrose, and 4 g/L gelrite. The pH of all media was adjusted to 5.8 before autoclaving. Twenty-five ml of medium was dispensed into each plastic Petri dish(87x15 mm). Unless mentioned otherwise, all cultures were maintained in the light(approximately 2500 Lux from cool-white fluorescent lamps with 16-h photoperiods) at 25°C.

    2Expression vector and Agrobacterium-mediated transformation

    The OsHSP26 cDNA(GenBank accession, AB020973) was isolated and cloned by screening a cDNA library of heat-treated rice leaves(Lee et al., 2000). The OsHSP26 cDNA fragment was ligated into the pIG121Hm vector under the control of the CaMV 35S promoter(Fig. 1). Agrobacterium tumefaciens strain EHA105 was used for the genetic transformation that was performed according to previous report(Lee et al., 2004).

    Chloroplast-localized OsHSP26 gene was transformed to P. alba. The leaf and stem were cut into 3-5 cm segments, inoculated with the diluted bacterial suspension for 5 min, and transferred to a solid co-cultivation medium consisting of MS medium supplemented with combinations of 0.5 mg/L BA, 500 mg/L carbenicillin. The in vitro plantlets which were screened by carbenicillin resistance were collected, and used for further experiments.

    3Temperatures stress treatments

    Wild-type and transgenic plants were exposed to high- and low-temperature by being placed in plant growth chamber(SH-301, Seyoung Science Co., Korea) for 24 and 72 hrs in each temperature stress. For control, the wild type and transgenic plants were put in 25℃.

    4Assessment of chlorophyll contents

    Profiles of leaf chlorophyll content were measured with a SPAD-502 portable chlorophyll meter(Minolta Co., Ltd., Japan). The SPAD-502 portable chlorophyll meter provided a rapid and non-destructive estimate of leaf chlorophyll content. The SPAD values were repeatedly taken at the center of the leaves throughout the experiments.

    5Determination of moisture content

    The calculation of the moisture content was based on a 1 g sample. The experiment was conducted in a completely randomized delineation with three repetitions and the treatments were arranged in a 2 factorial scheme constituted by the treatment temperature(4 and 27°C). The quantity of moisture was deducted from the loss in weight obtained by the oven method to calculate the moisture content. Moisture content, in percent, wet basis, is defined as follows:

    [(fresh weight - dry weight)/ fresh weight] × 100

    Where the total is a mass of the grain sample and is the mass of the dry material. The wet and dry weights of grain samples are determined by weighing with an analytical balance and standard oven drying procedures(ASAE Standards, 1996).

    6Determination of total polyphenol content

    Fresh leaves(FW, 500 mg) were homogenized and extracted with 25 ml of 70% methanol at room temperature for 24 hour. The extracts were filtered through a filter paper and condense(and methanol remove) with a rotary evaporator under reduced pressure. Samples were spectrophotometrically analyzed for measuring contents of total phenolic compounds by a modified Folin and Denis method(Folin & Denis, 1915). The volume of 0.2 ml sample extract and 4ml distil water were added to a test tube, followed by addition of 0.2 ml of Folin-Ciocalte'u reagent. They were mixed well and then allowed to stand 3 min before 0.4 ml of 20% sodium carbonate solution was added. The mixture was incubated for 60 min at room temperature in the dark, and then the absorbance was measured at 765 nm using a spectrophotometer. The measurement was compared to a standard curve of prepared garlic acid solution and expressed as means μg of garlic acid equivalents per g for the triplicate extracts.

    7Determination of total flavonoids content

    The total flavonoid content was determined according to the aluminum chloride colorimetric method described by Chang et al.(2002). Briefly, aliquots of 0.1 g of vegetable and fruit samples were, respectively, dissolved in 1 ml deionized water. This solution(0.5 ml) was mixed with 1.5 ml of 95% alcohol, 0.1 ml of 10% aluminum chloride hexahydrate (AlCl3), 0.1 ml of 1 M potassium acetate (CH3COOK), and 2.8 ml of deionized water. After incubation at room temperature for 40 min, the reaction mixture absorbance was measured at 415 nm against a deionized water blank on a spectrophotometer (Hitachi, Model 100-20). Quercetin was chosen as a standard. Using a seven-point standard curve(0-50 mg/l), the levels of total flavonoid contents in fruits and vegetables were determined in triplicate, respectively. The data were then converted into μg quercetin equivalents(QE)/ g fresh matter from fruit or vegetables based on the moisture content of lyophilized powder and fresh fruit and vegetable materials.

    8Determination of antioxidant activity by the DPPH radical scavenging method

    The antioxidant activity of samples was measured by the method of Blois(1958) with some modifications. A test sample solution(final extract concentration 1mg/ml) 50 μl was added to 2.0x10-4M DPPH ethanol solution 500 μl. The solution was rapidly mixed and after a stand at room temperature in the dark for 30min. The decrease in absorbance at 517 nm was measured using an Eliser-reader(anthos-2020 anthos labtec. instruments). BHT(butylated hydroxyanisole) was used as a positive control and methanol as a blank, distilled water was used as a control instead of extract. Antioxidant activity was expressed as percentage inhibition of the DPPH radical and was determined by the following equation(Yen & Duh, 1994):

    AA ( % ) = ( Abs control Abs sample / Abs control ) × 100

    Results and discussion

    1Phenotypic responses on temperature stresses

    To assess the functions of OsHSP26 in P. alba, the transgenic P. alba were tested on temperature stress(Fig. 2). On normal condition, there were no differences in the phenotype of both the wild-type and transgenic plants(Fig. 2. A and G). However, in the conditions of both chilling and heat stress, sensitive phenotypic responses in the wild-type plants were shown. In the 4°C chamber for treating chilling stress, wilted leaves were founded within 24 hours(Fig. 2. B). In the chilling condition for 72 hours, necrosis in leaves of the wild-type plants was observed(Fig. 2. C). On the other hands, no damage was observed in the transgenic plant under the chilling condition for 24 and 72 hours(Fig. 2. H and I). In the condition of high temperature, transgenic plants were more tolerant than wild-type plant. The leaves of wild-type plant were wilted within 24 hours, and severe damages were shown in 72 hours(Fig. 2. E and F). However, no damage was found in the transgenic plants in heat stress for 24 hours(Fig. 2. H). Although wilted leaves of the transgenic plants were shown in 72 hours, the inhibition was less than the wild-type in the same condition(Fig 2. F and J). These results corresponded to the results which OsHSP26 overexpressed tall fescue shows more tolerant phenotypic response than wild type under heat stress(Lee et al., 2004). Moreover, it has been reported that molecular chaperons such as HSPs are mediated to cold stress response in plants(Guy 1999; Ujaji et al., 1999). Therefore, it is suggested that the tolerance to temperature stress were given by the over-expressed OsHSP26 in the transgenic poplar. 3

    2Physiological parameters on temperature stresses

    The SPAD value of the wild-type in normal condition was 36.07. The SPAD values were decreased in both temperature stresses in the proportion of the exposure time on temperature stress. However, a different pattern was shown in the transgenic plants. The SPAD value of transgenic plants in the normal condition was 38.33, which is higher than one of the wild-type. The SPAD values of the transgenic plant on chilling stress were 29.10(24 hrs) and 33.77(72 hrs), respectively. On high temperature, the SPAD values were 29.97(24 hrs) and 34.83(72 hrs), respectively. The SPAD values in the transgenic plants on the temperature stress conditions were decreased highly in 24 hours. However, the values of the transgenic on both temperature conditions were recovered in 72 hours. This result suggests that the transgenic plants can more rapidly adapt to temperature stress conditions.

    Generally, temperature stress influences plant photosynthesis and light utilization(Glaszmann et al., 1990; Wu et al., 1997; Salvucci & Crafts-Brandner 2004; Feng et al., 2014). The SPAD values, which refer to chlorophyll contents, were measured to assess how the transgenic plants maintain their health on both low and high temperature and compared with the wild-type plants. In this study, the SPAD values of the transgenic plant under the prolonged temperature stress were higher than those of wild-type. Gosavi et al.(2014) reported that less reduction in chlorophyll contents and higher number of HSPs were induced in the drought tolerant genotype of Sorghum under heat stress than the drought sensitive genotypes. Thus, it seemed that the over-expressed OsHSP26 prevents the inhibition in chlorophyll contents by temperature.

    We monitored the water contents of the both wild-type and transgenic plants because plant water status is most important variable under changing ambient temperature(Mazorra et al., 2002). Under field condition, high-temperature stress is frequently associated with reduced water availability(Simoes- Araujo et al., 2003). Significant changes in the water contents were not observed even in the wild-type plants. About 90% of the water content in normal condition was dropped to 86.9% for 24 hours, and 86.1% for 72 hours, respectively, under chilling condition. Responding to heat stress, similarly, the water contents were decreased to 87.5 for 24 hours, and 86.9% for 72 hours, respectively. The water content of the transgenic in the normal condition was 89.7, and it was decreased to 89.2%(24 hrs) and 86.4%(72%) under chilling stress. In high-temperature condition, the water content of the transgenic plants was 87.8% for 24 hours and 87.0% for 72 hours, respectively. The measurements of the water contents were conducted using in vitro cultured plantlets. In general, the inside of vessels for in vitro culture is highly humid. Therefore, it seemed that the features of the in vitro culture caused no significant differences in the water contents between wild-type and transgenic plants under temperature stress.

    3Metabolic responses on temperature stresses

    It is well known that secondary metabolites such as phenolics including flavonoids, anthocyanins, and plant steroids are also significantly involved in plant responses to heat stress and generally play roles in abiotic stress responses generally associated with tolerance to heat(Wahid, 2007). Also, it is known that the phenolic compounds are accumulated in plants when they are exposed to temperature stress(Levine et al., 1993; Rivero et al., 2001). Thus, the phenolic compound accumulations in the transgenic plants under temperature stress were determined and compared to those in the wild-type plants. On total phenolic compounds analysis, the content of the wild-type in the normal condition was 4113.3(μg/g FW), and it was up-regulated to 4451.1 (μg/g FW) for 24 hours, and 4554.7(μg/g FW) for 72 hours in chilling condition. Similarly, the total phenolic compounds content under high temperature was changed to 4222.5(μg/g FW) for 24 hours, and 4376.8(μg/g FW) for 72 hours, respectively. In transgenic line, the total phenolic compounds content in normal condition was 3416.6(μg/g FW), and it was slightly accumulated in 24 hours in the chilling condition(3611.8 μg/g FW). However, the content was significantly increased in the chilling condition within 72 hours(4556.9 μg/g FW). In the high-temperature condition, the content was not almost changed within 24 hours(3408.3 μg/g FW), and slight accumulation in the content of total phenolic compounds was shown in 72 hours(3552.5 μg/g FW).

    On the other hands, enhancement of total flavonoids in the transgenic plants under prolonged temperature stress. The total flavonoids content of the wild-type in the normal condition was 801.63 μg/g FW. And it was significantly increased up to 2882.2 μg/g FW for 72 hours in the chilling condition while a slight reduction was shown in 24 hours(779.1 μg/g FW). In the high temperature, the total flavonoid content was accumulated up to 1120.2 μg/g FW for 24 hours and 2666.9 μg/g FW for 72 hours, respectively. For the transgenic plant, the total flavonoids content was 1122.4 μg/g FW in the normal condition, and it was higher than one of wild-type in the same condition. The contents of transgenic in the both stress condition for 24 hours were 785.1 μg/g FW in the chilling condition, and 978.8 μg/g FW in high temperature, respectively. However, in the prolonged temperature stress conditions, the total flavonoids were highly accumulated as much as 3-folds(3358.3 μg/g FW) in low temperature, and 2.5-folds(2624.3 μg/g FW) in the high temperature.

    Amarowicz et al.(2010) reported that low-temperature stress decreased the content of phenolic compounds and condensed tannins in the grapevine leaf extract, but the composition ratio of garlic, caffeic, and ferulic acids in the extract was higher than the control extract. Likewise, the results showed that the total flavonoids contents were highly accumulated under prolonged temperature stress whereas there was no significant difference in total phenolic compounds. Thus, it is considered that change in the composition of phenolic compounds was caused by temperature stress, and it allowed the transgenic plants to be tolerant to the temperature stress.

    4Antioxidant activities on temperature stress

    DPPH scavenging activity assay has been used to assess antioxidant activities of plants(Sanna et al., 2012; Aksoy et al., 2013; Sowndhararajan & Kang, 2013). Thus, we compared the antioxidant activities of the wild-type and transgenic plants using the DPPH scavenging activity assay under temperature stress(Fig. 4.). The DPPH scavenging activities of methanol extracts of wild-type plants in the normal condition were 20.1%(1000 ppm), 32.3%(2000 ppm), and 38.9%(4000 ppm), respectively. The activities of wild-type plants under the temperature stress within 24 hours generally lower than those activities in the normal condition: 14.8%(1000 ppm), 24.3%(2000 ppm), and 38.2%(4000 ppm) in 4°C; 15.3%(1000 ppm), 29.9%(2000 ppm), and 38.7%(4000 ppm) in 40°C. Under chilling stress for 72 hrs, the scavenging activities of the wild plant at every concentration were lower than those in the normal condition except 4000 ppm extract: 18.2%(1000 ppm), 30.7%(2000 ppm), and 31.7%(4000 ppm). In the treatment of high temperature for 72 hours, the DPPH scavenging activities were lower than the activities of the methanol extracts from the normally grown wild-type: 19.5%(1000 ppm), 29.3%(2000 ppm), and 30.5%(4000 ppm).

    The DPPH scavenging activities of transgenic in normal condition were lower than wild-type: 11.9% (1000 ppm), 17.4%(2000 ppm), and 39.9%(4000 ppm). However, difference in the activities was shown under temperature stress conditions. For 24 hours of temperature stress, the activities were higher than the wild-type plants except 1000 ppm extracts. Higher activities of DPPH scavenging in every concentration of extracts were shown in both low and high temperature conditions: 21.7%(1000 ppm; 4°C), 31.0% (2000 ppm; 4°C), 38.7%(4000 ppm; 4°C), 19.9%(1000 ppm; 40°C), 20.9%(2000 ppm; 40°C), and 53.2%(4000 ppm; 40°C).

    Under abiotic stress, radical oxygen species are accumulated in the plant cells, and cause damages in a cellular structure including membranes, nucleotides, and proteins. Therefore, antioxidant activity is important for plants to overcome oxidative stress caused by temperature stress factors. In this study, the DPPH scavenging activities of the transgenic were higher than those of wild-type plants. Lee et al.(2013) revealed that the expressions of HSPs are related to the expression of antioxidant genes in rice under heat stress. Moreover, it was reported that the DPPH scavenging activities are enhanced to adapt to heat and chilling stress in rice(Kang & Saltveit, 2002). Therefore, it is considered that the higher DPPH scavenging activities are originated in the overexpression of OsHSP26 in the transgenic plants. Fig. 5

    Acknowledgement

    This work was supported by the Forest Science Technology and Development Grant funded by the Korea Forest Service(S211216L020120).

    Figure

    JALS-51-37_F1.gif

    Diagrammatic representation of the binary vector pIG121Hm/OsHSP26. T-DNA region of the pIG121Hm plasmid used for the P. alba transformation. RB, right border; LB, left border; NPTⅡ, NOS, nopaline synthase(nos) gene promoter bar, neomycin phosphotransferase; phosphinothricin acetyltransferase; 35S, CaMV 35S promoter; OsHSP26, rice chloroplast localized small heat shock protein 26.

    JALS-51-37_F2.gif

    Phenotypic response of the OsHSP26-overexpressed transgenic poplar under temperature stress. (A) Wild-type in vitro plantlets under normal condition. (B) under chilling condition for 24 hrs. (C) for 74 hrs. (D) Wild-type plantlets under normal condition. (E) under high-temperature condition for 24 hrs. (F) for 74 hrs. (G) OsHSP26 transgenic plantlet under normal condition. (H) under chilling condition for 24 hrs. (I) for 72 hrs. (J) OsHSP26 transgenic plantlet under normal condition. (K) under high-temerature condition for 24 hrs. (L) for 72 hrs.

    JALS-51-37_F3.gif

    Physiological responses of the OsHSP26-overexpressed transgenic poplar under temperature stress. (A) Changes in chlorophyll contents of the OsHSP26-overexpressed transgenic plant by temperature stress. (B) Changes in chlorophyll contents of the OsHSP26-overexpressed transgenic plant by temperature stress.

    JALS-51-37_F4.gif

    Metabolic responses of the OsHSP26-overexpressed transgenic poplar under temperature stress. (A) Changes in phenolic compound contents of the OsHSP26-overexpressed transgenic plant by temperature stress. (B) Changes in total flavonoid contents of the OsHSP26-overexpressed transgenic plant by temperature stress.

    JALS-51-37_F5.gif

    Antioxidant activity of the OsHSP26-overexpressed transgenic poplar under temperature stress. (A) DPPH scavenging activities of the 1000 ppm extracts. (B) of the 2000 ppm extracts. (C) of the 4000 ppm extracts.

    Table

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