^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
УДК 581.1
ULTRASTRUCTURAL CHANGES AND DYNAMIC EXPRESSIONS OF FAD7, Cu/Zn-SOD, AND MN-SOD IN Neosinocalamus affinis
UNDER COLD STRESS1
© 2014 F. Zhang*, X. Q. Zhu*, Y. L. Guo*, X. Q. Wan**, T. T. Lin**, Q. B. Chen*, M. Liu*, P. Q. Liu***
*College of Landscape Architecture, Sichuan Agricultural University, Chengdu, China **College of Forestry, Sichuan Agricultural University, Ya'an, China ***Institute of Rice Research, Sichuan Agricultural University, Chengdu, China
Received April 6, 2013
As one of the fast-growing species, bamboo plays an important role in ecological stability and wood processing industry. However, low temperature limitation is the basic problem for the cultivation and introduction of bamboo. In this study, the symptoms of cold stress influence on the native bamboo (Neosinocalamus affinis (Rendle) Keng f.) and hybrid bamboo (Bambusapervariabilis x Dendrocalamopsisgrandis) were observed under transmission electron microscope, and the dynamic responses of FAD7, Cu/Zn-SOD, and Mn-SOD genes to cold stress were identified in bamboo by real-time quantitative RT-PCR. Observation by electron microscopy indicated that bamboo is one of the most chilling-sensitive species with severe ultrastructural injury induced by chilling, but the native bamboo (N. affinis) is more cold-tolerant compared with the hybrid bamboo. Results obtained by real-time quantitative RT-PCR analysis revealed that FAD7, Cu/Zn-SOD, and Mn-SOD were all cold-inducible genes in N. affinis. In addition, dynamic response patterns of N. affinis Cu/Zn-SOD and Mn-SOD under cold stress were similar. This work is a fundamental research of hardiness physiology of bamboo and may contribute to the breeding program on obtaining transgenic bamboo species.
Keywords: Neosinocalamus affinis — cold stress — ultrastructure — qRT-PCR — fatty acid desaturase — superoxide dismutase
DOI: 10.7868/S0015330314050170
INTRODUCTION
Cold is one of the most important environmental factors affecting plant growth and crop productivity. Low temperature stress, which occurs at temperatures below 15 °C would limit the geographical distribution and growing of many plants crops and cause significant productive losses [1]. The basic classifications of cold injury are chilling injury and freezing injury. Each of them affects plants with different cold injury mode. Chilling injury occurs at the low temperature above 0°C, which results in plant physiology dysfunction, but freezing injury occurs below 0°C, which dehydrates or freezes tissues to harm plants. Cells are the basic location of plant cold injury; plasma membrane
1 This text was submitted by the authors in English.
Abbreviations'. CAT — catalase; FAD7 — gene of ro-3 fatty acid desaturase; POD — peroxidase; qRT-PCR — real-time quantitative RT-PCR; SOD — superoxide dismutase; Cu/Zn-SOD — gene of Cu, Zn-SOD; Mn-SOD - gene of manganese-SOD. Corresponding author. X. Q. Wan. College of Forestry, Sichuan Agricultural University, Ya'an, 625014 China; e-mail. zh23344547@163.com
is the first structure to be injured, and chloroplasts are the most popular organelle to be observed. When plants are suffered from cold stress, they could not properly photosynthesize and respire because of the different extent of organelle damage.
A large number ofgenes are found to be up-regulated when plants are under cold stress. These genes could be simply divided into two categories. the regulatory genes, such as those of C-repeat-binding factors (CBFs), which can switch other genes on; and the functional genes, such as those encoding fatty acid desaturases (FADs) and superoxide dismutases (SODs); they are activated by regulatory genes and could protect organisms from cold damage directly [2, 3]. Many previous studies have testified the effective influence of cold-induced genes on transgenic plants [4-7]. According to these reports, FAD was proved to be important for cold-tolerance among the numerous concerned genes and SOD isoenzyme genes are distributed in the first class of expressed sequence tags in cold-response researches [6, 7].
At low temperature, the membrane lipids in the cells of heat-loving plants would transform liquid-
crystalline phase to the gel phase, resulting in a significant reduction in membrane fluidity [8]. This transformation leads to the dysfunction of various enzyme systems in membranes and organelles. However, cold-tolerant plants could maintain the required membrane fluidity within the limited temperature range. The key factor is the fatty acid desaturase, which can catalyze the desaturation reaction of fatty acids to improve membrane fluidity. In the tobacco leaf cells under chilling stress, the relative content of polyunsaturated fatty acids increased and the proportion of saturated fatty acids decreased [9]. Numerous conserved sequences of fatty acid desaturase genes had been cloned from Arabidopsis. Kodama et al. [10] isolated plastid ®-3 fatty acid desaturase gene (FAD7) fromArabidop-sis and transferred it to tobacco to obtain enhanced plant tolerance.
Under cold stress, many ROS are generated in various metabolic pathways, especially in respiratory and photosynthetic electron transport chains; they can cause oxidative damage to many cellular components [11, 12]. The enzyme superoxide dismutase (SOD) catalyzes the fast dismutation of superoxide to hydrogen peroxide, and then peroxidase (POD) and catalase (CAT) catalyze peroxide degradation to H2O and O2. While there are excess hydrogen peroxide accumulated, POD and CAT are promoted, but SOD is inhibited. As a result, the interaction of SOD, POD, and CAT results in the ROS reducing in the cells. The dismutation of SOD needs a coenzyme factor to store electron temporary. According to the different metal ions combined to SOD as coenzyme factors, there are three SOD isozymes in plants: Fe-SOD, Cu/Zn-SOD, and Mn-SOD. As the first-line of antioxidant system, SOD is a hotspot in obtaining resistant plants.
Bamboo has more than 1250 species distributed in temperate regions and is also widely cultivated in southern China. The rapid growth, high specific gravity, and long fiber length of bamboo make it preferable for the paper and pulp production. Both native and introduced bamboo species contribute to the development and stabilization of regional economy in southern provinces of China. Unexpectedly, the unusually severe snow disaster happened in southern China caused enormous impact on bamboo species in 2008, so that many of them suffered extensive damage or death. Although many researches on cold-endurance of bamboo had been developed since the 1960s, most of them either focused on cold damage survey, selection and introduction of foreign cold-tolerant breeds or hammer at physiological assessment of cold tolerance [13, 14]. As a matter of fact, only a few papers focused on the molecular mechanisms of cold tolerance in bamboo under cold stress. The CBF1 as a regulatory gene had been cloned from bamboo in our laboratory, and the effect of cold stress on endogenous hormones and CBF1 homolog in four contrasting bamboo species had been reported [15]. But there is still only one study
published so far. In this research, FAD and SODs as two kinds of functional genes were studied in bamboo.
Neosinocalamus affinis is one of the most valuable and unique species of bamboo, which is only distributed in southern China. The hybrid bamboo (Bambusa pervariabilis x Dendrocalamopsis grandis) is famous for its fast growth, and it is widely planted for economic application. During the snow disaster in 2008, the native bamboo (N. affinis) showed much more endurance and resilience than the hybrid bamboo in Ya'an, Sichuan. In order to explore the mechanisms of bamboo responses to cold stress at subcellular and molecular levels, the ultrastructural changes and dynamic expression profiles of FAD7 (gene of ®-3 fatty acid desaturase), Cu/Zn-SOD (gene of Cu, Zn-SOD), and Mn-SOD (gene of manganese-SOD) in bamboo under cold stress were studied in this work.
MATERIALS AND METHODS
Plant materials. Two bamboo species used in this study were selected based on the economical species in Ya'an, Sichuan, China: Neosinocalamus affinis (Ren-dle) Keng f. (the native bamboo) and Bambusa pervariabilis x Dendrocalamopsis grandis (the hybrid bamboo). Both of them were collected as two-year-old dormant hardwood stems from bamboo groves in Tianquan County, Ya'an City. They were transferred into a greenhouse for growing at a daytime temperature of 25°C, 75% humidity, a 16-h photoperiod (light intensity of 12870 lx), and watering every other day.
Material treatment. The temperature in treatment was designed according to the lowest temperature of winter in Ya'an City (—4°C). For studying ultrastructural changes under cold stress, individuals of N. affinis and hybrid bamboo were transferred into a room with a low temperature of —4°C for 48 h. Meanwhile, the same number of plants of these two species were placed in another room with a temperature of 25°C for 48 h as a control. For studying gene dynamic responses to different low temperature stresses, individuals of N. affinis with uniform growth were transferred into four dark rooms with different temperatures: 25, 15, 4, and —4°C, and leaves were sampled at different time points, which were 0, 1, 3, 6, 9, 12, and 24 h after treatment. All the samples were newly expanded leaves of the same age (the third leaves counting from the top of the one-year-old branch). Each experiment was performed with three replicates.
Observation of cell ultrastructure. The youngest mature leaves of two bamboo species were sampled after 48-h treatment at —4 and 25°C. Leaves were cut to rectangles (2 x 1 mm), avoiding crosscutting the main vein. The rectangles were saved in 2.5% glutaraldehyde at 4°C for 48 h. After washing in 0.1 M phosphate buffer (pH 7.2), specimens were fixed in 1% OsO4 at 4°C for 2.5 h, washed with buffer, and then deh
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