Scientia Silvae Sinicae  2009, Vol. 45 Issue (8): 50-55   PDF    


Zhang Min, Li Rongjin, Huang Libin, Ji Yonghua, Dou Quanqin, Qian Meng, Fang Yanming
张敏, 李荣锦, 黄利斌, 季永华, 窦全琴, 钱猛, 方炎明
Physiological and Biochemical Response of Tonoplast Vesicles Isolated from Broussonetia papyrifera to NaCl Stress
Scientia Silvae Sinicae, 2009, 45(8): 50-55.
林业科学, 2009, 45(8): 50-55.




Min Zhang
Rongjin Li
Libin Huang
Yonghua Ji
Quanqin Dou
Meng Qian
Yanming Fang

张敏1,2, 李荣锦2, 黄利斌2, 季永华2, 窦全琴2, 钱猛3, 方炎明1     
1. 南京林业大学森林资源与环境学院 南京 210037;
2. 江苏省林业科学研究院 南京 211153;
3. 南京农业大学生命科学学院生命科学实验中心 南京 210095
摘要:以构树幼苗根组织和叶组织为试材, 测定不同浓度NaCl胁迫下液泡膜H+-ATPase的活性、液泡膜脂肪酸组成和膜的流动性. 结果表明: 在低浓度NaCl胁迫下, 根组织中液泡膜H+-ATPase的活性有所增加, 当胁迫浓度为150 mmol·L-1时, 活性又有所下降, 但接近对照水平. 而叶组织液泡膜H+-ATPase的活性在100 mmol·L-1时降低, 高浓度NaCl(150 mmol·L-1)处理后又有所升高. 液泡膜脂肪酸基本组成为C16: 0, C16: 1, C18: 0, C18: 1, C18: 2和C20: 0. 盐胁迫后构树幼苗液泡膜脂肪酸组分相对含量发生变化. 根组织液泡膜脂肪酸不饱和度下降, 150 mmol·L-1 NaCl处理后有所升高; 而在叶组织中, 液泡膜脂肪酸不饱和度在50 mmol·L-1 NaCl处理后下降, 随着盐胁迫浓度的增加, 不饱和度又高于对照. NaCl胁迫下, 构树液泡膜流动性和脂肪酸不饱和度的变化趋势相一致. 脂肪酸不饱和度下降时, 膜流动性下降, 反之则膜流动性上升. 研究结果为深入了解木本植物液泡膜H+-ATPase活性、脂肪酸组成和膜的流动性在盐胁迫下的适应及其3者的相互关系提供了参考.
关键词构树    盐胁迫    液泡膜    脂肪酸    膜流动性    H+-ATPase    
Physiological and Biochemical Response of Tonoplast Vesicles Isolated from Broussonetia papyrifera to NaCl Stress
Zhang Min1,2, Li Rongjin2, Huang Libin2, Ji Yonghua2, Dou Quanqin2, Qian Meng3, Fang Yanming1    
1. College of Forestry Resources and Environment, Nanjing Forestry University Nanjing 210037;
2. Jiangsu Academy of Forestry Nanjing 211153;
3. Life Science Laboratory Center, Nanjing Agricultural University Nanjing 210095
Abstract: The activity of H+-ATPase, fatty acid composition and membrane fluidity of tonoplast vesicles isolated from roots and leaves of Broussonetia papyrifera plantlets under salt stress with different concentrations of NaCl were investigated. The results showed that V-H+-ATPase activity in roots increased at low level of NaCl concentration (50 mmol·L-1). When NaCl concentration exceeds 100 mmol·L-1, the activity was similar with control. In leaves, V-H+-ATPase activity decreased at 100 mmol·L-1 NaCl, but increased at higher levels of NaCl (150 mmol·L-1). It was found that the fatty acid composition of tonoplast was consisted of C16: 0, C16: 1, C18: 0, C18: 1, C18: 2 and C20: 0. Alterations in relative content of tonoplast fatty acid composition under NaCl stress were observed. In roots the extent of unsaturation of tonoplast fatty acids decreased, and then it increased under 150 mmol·L-1 NaCl. In leaves, the extent of unsaturation of tonoplast fatty acids decreased under 50 mmol·L-1 NaCl treatment, however the extent increaseal with the increase of salinity, leaves and was higher than that in control. The changes in tonoplast fluidity coincided with those in unsaturation of fatty acids under NaCl stress. Tonoplast fluidity decreased with the decline of unsaturation of fatty acids. The objectives of this study were to gain better understand of the adaptive mechanism of H+-ATPase, fatty acid composition and membrane fluidity of tonoplast of woody plants and their relationship under salt stress.
Key words: Broussonetia papyrifera     salt stress     tonoplast     fatty acid     membrane fluidity     H+-ATPase    

Salinity is one of the major abiotic stresses, which has a major impact on plant production and productivity. Excess amount of salt in the soil adversely affects plant growth and development (Sairam et al., 2004). It is now generally accepted that the cell membrane is the primary site of injury when plants are subjected to salt stress (Surjus et al., 1996). Membranes play an important role in many cellular activities including ion transport, proton-pumping ATPase, signal transduction, etc (Huang, 1996). It is not until recently that the significance of membrane lipids in plant salinity tolerance has been appreciated. In the previous studies some researchers have investigated the changes in fatty acids, phospholipids and sterols (Surjus et al., 1996; Wu et al., 2005; Liang et al., 2005) in order to establish a relationship between lipid alteration and salt stress in plants. However, the results are often controversial (Surjus et al., 1996).

The ability of plants to grow in high NaCl concentrations is associated with the ability of the plants to transport, compartmentalize or extrude Na+ (Apse et al., 2007). The pumping of Na+ into the vacuole is catalyzed by vascular Na+/H+ antiporter (Tester et al., 2003). H+-electrochemical potential gradient, which provides the driving force, was initially established by the H+-ATPase and H+-pyrophosphatase on the tonoplast (Tester et al., 2003). Vacuolar H+-ATPases (V-H+-ATPase) are multi-subunit enzymes that pump protons into intracellular compartments (Coker et al., 2003). V-H+-ATPase plays important roles in pH homeostasis and various stress responses (Coker et al., 2003; Sze et al., 2002). The V-H+-ATPase has been reported to be involved in salt tolerance in the previous studies (Liang et al., 2005; Zhang et al., 2002; Yu et al., 2005).

Membrane structural and functional stability is crucial in plant adaptation to abiotic stresses (Liang et al., 2005). The maintains of membrane fluidity is a prerequisite for the function, viability, growth and reproduction of cells (Lu et al., 2006). Changes in membrane fluidity may affect the membrane micro-environment surrounding proteins, which in turn influences membrane functions such as carrier-mediated transport and the activity of membrane bound enzymes including ATPase activity (Surjus et al., 1996).

Until now, there is few studies have been carried out to analyze the changes of lipid composition and membrane fluidity of woody plants under salt stress. In this study, the tonoplast vesicles from the leaves and roots of Broussonetia papyrifera grew under NaCl stress were isolated and the effect of NaCl on tonoplast H+-ATPase activity and membrane fatty acid composition were investigated. The membrane fluidity of tonoplast vesicles was also studied. The aim of our study is to gain better understanding of the mechanisms of salt tolerance in woody plant.

1 Materials and methods 1.1 Plant material

B. papyrifera used in this study was introduced from Japanes and was cultivated in the nursey of Jiangsu Academy of Forestry. Uniform-sized B. papyrifera in vitro regenerated rooting plantlets were planted into plastic pots filled with 500 mL of half-strength MS solutions in a growth chamber. The nutrient solution was renewed every other day. The experimental design consisted of a control (no NaCl) and three treatments (50, 100 and 150 mmol·L-1 NaCl) and was established in a randomized design with three replicates. After five days treatment the roots and leaves were harvested for isolation of tonoplast vesicles.

1.2 Isolation of tonoplast-enriched vesicles

Tonoplast vesicles were isolated according to the previously reported method (Liang et al., 2005) with minor modifications. Roots and leaves were homogenized with an ice-cold mortar and pestle in the homogenization buffer (50 mmol·L-1 HEPES-Tris, pH 7.6, 250 mmol·L-1 sorbitol, 125 mmol·L-1 KCl, 5 mmol·L-1 EGTA, 2.5 mmol·L-1 K2S2O5, 2 mmol·L-1 PMSF, 1.5% PVP, 0.1% BSA, 2 mmol·L-1 DTT). The homogenates were filtered through four layers of cheesecloth and subjected to differential centrifugation at 10 000×g for 15 min and subsequently at 50 000×g for 30 min at 4 ℃. The 10 000×g to 50 000×g pellets were suspended in 6 mL of buffer A (300 mmol·L-1 sucrose, 10 mmol·L-1 KCl, 1 mmol·L-1 EGTA, and 2 mmol·L -1 DTT, pH 7.8). Then 4 mL of buffer B (250 mmol·L-1 sorbitol, 1 mmol·L-1 EGTA, 5 mmol·L-1 HEPES, pH 7.3) were layered over the buffer A gradient. The gradients were then subjected to ultracentrifugation (100 000×g, 2 h, 4 ℃) with a Ti90 rotor in an Optima L-80XP ultracentrifuge (Beckman Coulter Inc., Fullerton, CA, USA). Vesicles banding at the buffer A/B interface were collected and stored at -80 ℃ until use.

1.3 Determination of H+-ATPase activity in tonoplast

Activity of V-H+-ATPase was measured according to Shi et al. (2007) with minor modification. The reaction was carried out in a 0.5 mL mixture (30 mmol·L-1 HEPES-Tris, pH7.5, 3 mmol·L-1 MgSO4, 100 mmol·L-1 KCl, 0.5 mmol·L-1 NaN3; 0.1 mmol·L-1 Na3VO4, 0.1 mmol·L-1 (NH4)4MoO4, 0.02% Triton X-100, 3 mmol·L-1 ATPNa2). Reaction was started by adding 50 μL tonoplast vesicles. The reaction was terminated by the addition of 0.1 mL of 10% SDS after incubation at 37 ℃ for 30 min. The release of Pi from ATP was determined by the Fiske-Subbarow reagent (Fiske et al., 1925), using NaH2PO4 as the standard.

1.4 Tonoplast fatty acid analysis

Fatty acids of tonoplast vesicles were analyzed using the method described by Liang et al. (2005) with some modifications. Fatty acids were methylated with boron trifluoride-ethyl ether/methanol (1:3, v/v) including 10 μg of heptadecanoic acid served as internal standard. The fatty acid methyl esters were then analyzed by Thermo Trace GC Ultra gas chromatograph equiped with a flame ionization detector (FID) and an Sp-2560 quartz capillary column (100 m×0.25 mm×0.2 μm). Highly pure N2 was used as the carrier gas. The injection and detection temperature were both set at 220 ℃. The following temperature program was used: The initiation temperature was set at 140 ℃ and maintained for 5 min, followed by 4 ℃·min-1 to 220 ℃ for 25 min. Quantitation was achieved by normalization with an internal standard of heptadecanoic methyl ester. The fatty acid composition was expressed as mol %.

1.5 Determination of tonoplast fluidity

DPH (1, 6-diphenyl-1, 3, 5-hexatriene) was used as a fluorescent probe to determine the fluidity of tonoplast vesicles in this study. A stock solution of DPH (2 mmol·L-1) was obtained by dissolving DPH in tetrahydrofuran. For lebeling of tonoplast vesicles, DPH was used at a concentration of 2 μmol·L-1. The mixture was incubated at 37 ℃ for 1 h in the dark. The fluorescence polarization of DPH was determined on a Hitachi 850 fluorescence spectrometer fitted with a polarization attachment as described by Pang et al. (2005). The samples were excited at 360 nm, and the emissions at 429 nm were recorded. Both excitation and emission slits were set at 5 nm. The degree of fluorescence polarization (P) was calculated according to the following formula: P=(I//-I)/(I//+GI), in which I// and I are the fluorescence intensities measured with parallel and perpendicular oriented polarizers, respectively, and G is the calibration factor. Here, G = I/ I//.

1.6 Statistical analysis

Statistical analysis was carried out by one-way ANOVA using SPSS 10.0 software to determine the different significance. When the ANOVA was significant at P < 0.05, the Duncan multiple range test was used for mean comparison. Data presented were mean ±SD of three experiments.

2 Results 2.1 Effect of NaCl stress on V-H+-ATPase activity from roots and leaves of B.papyrifera

To assess the influence of NaCl on the activity V-H+-ATPase, B. papyrifera were treated with different concentrations of NaCl and tonoplast vesicles were isolated from the roots and leaves. The results showed that the activity of V-H+-ATPase in the roots increased with the increase of NaCl concentration. And then V-H+-ATPase activity decreased when NaCl concentration reached 150 mmol·L-1, but similar with the control (Fig. 1). However, the changes of V-H+-ATPase activity in the leaves were not consistent with the roots. In leaves, V-H+-ATPase had no significant difference to control when treated with low NaCl concentration. At salinity of 100 mmol·L-1 the activity decreased significantly and reached the lowest level. After then, the activity recovered at highest concentration of NaCl (150 mmol·L-1) and it almost close to the level as control.

Fig.1 Changes in V-H+-ATPase activity from roots and leaves of Broussonetia papyrifera Each measurement was conducted with three replicates, and data were mean ±SD. Different letters indicate significant difference by Duncan's multiple range test at 0.05 level.
2.2 Changes in fatty acid composition of tonoplast

The analysis of fatty acid composition of the tonoplast vesicles lipid extract revealed that the fatty acids in tonoplast of B. papyrifera roots and leaves mainly consisted of C16:0, C16:1, C18:0, C18:1, C18:2 and C20:0, whereas C18:3 was found in the roots rather than in the leaves (Tab. 1 and 2). The ratio of unsaturated fatty acids to saturated fatty acids (U/S) decreased in the tonoplasts of roots and leaves at lower levels of NaCl. While the U/S of roots increased and was slightly higher than control under 150 mmol·L-1 NaCl. In leaves, the U/S increased gradually at 100 and 150 mmol·L-1 NaCl treatment (Tab. 1). Moreover, the index of unsaturated fatty acids (IUFA) decreased in the roots after NaCl treatmen. In contrast, the IUFA decreased in the leaves under 50 mmol·L-1 NaCl, and then increased under 100 and 150 mmol·L-1 NaCl (Tab. 2).

Tab.1 Effect of NaCl on fatty acid composition of tonoplast isolated from roots of B. papyrifera
Tab.2 Effect of NaCl on fatty acid composition of tonoplast isolated from leaves of B. papyrifera
2.3 NaCl treatment changed the fluidity of tonoplast vesicles

The tonoplast vesicles were labeled with the fluorescent reagent DPH to evaluate the changes in the fluidity of tonoplast vesicles under NaCl stress. The fluorescence labeled tonoplast vesicles were scanned with a fluorescence spectrometer, and the peak value of excitation and emission was found at 360 and 429 nm, respectively (Fig. 2 A and B). The membrane fluidity was detected by the steady-state fluorescence polarization measurements. The results in Tab. 3 demonstrated that in the roots the P value increased under 50 and 100 mmol·L-1 NaCl, which indicated the decrease in tonoplast fluidity. And tonoplast fluidity increased when NaCl concentration reached 150 mmol·L-1. Whereas, in the leaves tonoplast fluidity decreased under 50 mmol·L-1 NaCl, and then it increased with the increase of NaCl concentration.

Fig.2 Excitation (A) and emission (B) spectrum of fluorescence labeled tonoplast vesicle
Tab.3 Changes in tonoplast fluidity of roots and leaves from B.papyrifera
3 Discussion

The fundamental basis of the adaptation of plants to salinity stress is the control of transport of salt across membranes. Two major membranes of plant cells, the plasmalemma and the tonoplast, are particularly involved in the process of compartmentation of the NaCl taken up (Lüttge, 1993). The tonoplasts of plant cells contain proteins catalyzing primary-and secondary-active processes of ion transport, which are essential in the salt compartmentation involved in adaptation to salinity (Lüttge, 1993). V-H+-ATPase plays a fundamental role in energizing Na+/H+ antiport activity in cells accumulating significant quantities of NaCl (Zhang et al., 2002). It has been shown that the activity of the V-H+-ATPase increases in the tonoplast vesicles isolated from barly roots under treatment with NaCl (Zhang et al., 2002). In this study, we found that at low concentrations of NaCl treatment the activity of V-H+-ATPase isolated from the roots of B. papyrifera was stimulated. And the activity decreased slightly under 150 mmol·L-1 NaCl, but not significant compared with the control. However, the changes in the activity of V-H+-ATPase isolated from the leaves were different from the roots. The V-H+-ATPase activity in the leaves decreased in response to NaCl stress. These differences observed may be because that differentiated cells in various plant tissues and organs may function differently in coordination with neighboring cells to achieve salinity tolerance (Wu et al., 2005).

Membranes have been shown to play an important role in the ability of plants to cope with salinity (Mansour et al., 2005). Various kinds of environmental stress, such as temperature stress and osmotic stress, cause alterations in the physical properties of the membrane lipids in living cells (Los et al., 2004). The physical state of membrane lipids also acts directly to regulate the activity of membrane-bound proteins, such as the ion channels (Sukharev, 1999), sensor proteins (Sugiura et al., 1994), and proton-pumping ATPase (Surjus et al., 1996). Determining how salinity affects the membrane composition of the woody plant B. papyrifera will increase our understanding of plant salt tolerance. We observed that in the roots, NaCl concentration at which H+-ATPase activity increased was identical with that at which tonoplast fluidity decreased. This phenomenon was also reported in the tonoplast isolated from barley roots under salinity (Zhang et al., 2002). The previous study has suggested that proper fluidity of membrane was important for optimal structure and high activity of H+-ATPase (Zhang et al., 2002). Moreover, we found that changes in the activity of V-H+-ATPase in the leaves was not consistent with the roots. V-H+-ATPase activity declined with the increase in NaCl concentration.

Traditionally, alterations in fatty acid unsaturation degree are related to changes in membrane fluidity, and it is known that a decrease in fatty acid unsaturation results in a decrease in membrane fluidity (Navari-Izzo et al., 2000; Quartacci et al., 2002). In our study, the observed decline in tonoplast fluidity might due to the decrease in the ratio of unsaturated fatty acids to saturated fatty acids (U/S).

In a previous review, Munns (1993) has concluded that "Advances in salt tolerance at the molecular level will be in manipulating the expression and structure of proteins that control the transport of salt across membranes". Therefore, it is of great important to investigate the changes of the V-H+-ATPase under salinity, especially in woody plants.

In conclusion, the detailed analysis of the isolated tonoplast vesicle component may provide valuable information with respect to the physiological responses of plants to salt stress.

Apse M P, Blumward E. 2007. Na+ transport in plants. FEBS Letter, 581: 2247-2254. DOI:10.1016/j.febslet.2007.04.014
Coker J S, Jones D, Davies E. 2003. Identification, conservation, and relative expression of V-ATPase cDNAs in tomato plants. Plant Molecular Biology Reporter, 21: 145-158. DOI:10.1007/BF02774241
Fiske C H, Subbarow Y. 1925. The colorimetric determination of phosphorus. Journal of Biological Chemistry, 66: 375-400.
Huang C Y. 1996. Salt-stress induces lipid degradation and lipid phase transition in plasma membrane of soybean plants. Taiwania, 41: 96-104.
Liang Y C, Zhang W H, Chen Q, et al. 2005. Effects of silicon on H+-ATPase and H+-APase activity, fatty acid composition and fluidity of tonoplast vesicles from roots of salt-stressed barley (Hordeum vulgare L.). Environmental and Experimental Botany, 53: 29-37. DOI:10.1016/j.envexpbot.2004.02.010
Los D A, Murata N. 2004. Membrane fluidity and its roles in the perception of environmental signals. Biochimica et Biophysica Acta, 1666: 142-157. DOI:10.1016/j.bbamem.2004.08.002
Lu Y N, Qiu Q Y, Wang Y, et al. 2006. Effects of phellodendron and its main components on the cell membrane fluidity. Chinese Journal of pathophisiology, 22: 156-159.
Lüttge U. 1993. Plant cell membranes and salinity: structural, biochemical and biophysical changes. R Bras Fisiol Veg, 5: 217-224.
Mansour M M F, Salama K H A, Al-Mutawa M M, et al. 2002. Effect of NaCl and polyamines on plasma membrane lipids of wheat roots. Biologia Plantarum, 45: 235-239. DOI:10.1023/A:1015144607333
Munns R. 1993. Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell & Environment, 16: 15-24.
Navari-Izzo F, Quartacci M F, Pinzino C, et al. 2000. Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration. Plant Physiology, 124: 1427-1436. DOI:10.1104/pp.124.3.1427
Pang Y H, Zhu H, Wu P, et al. 2005. The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains. FEBS Letters, 579: 2397-2403. DOI:10.1016/j.febslet.2005.03.038
Quartacci M F, Glišić O, Stevanović B, et al. 2002. Plasma membrane lipids in the resurrection plant Ramonda serbica following de hydration and rehydration. Journal of Experimental Botany, 53: 2159-2166. DOI:10.1093/jxb/erf076
Sairam R K, Tyagi A. 2004. Physiology and molecular biology of salinity stress tolerance in plants. Current Science, 86: 407-421.
Shi Q H, Ding F, Wang X F, et al. 2007. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiology and Biochemistry, 45: 542-550. DOI:10.1016/j.plaphy.2007.05.005
Sugiura A, Hirokawa K, Nakashima K, et al. 1994. Signal-sensing mechanisms of the putative osmosensor KdpD in Escherichia coli. Molecular Microbiology, 14: 929-938. DOI:10.1111/mmi.1994.14.issue-5
Sukharev S. 1999. Mechanosensitive channels in bacteria as membrane tension reporters. FASEB Journal, 13(Suppl.): S55-S61.
Surjus A, Durand M. 1996. Lipid changes in soybean root membranes in response to salt treatment. Journal of Experimental Botany, 47: 17-23. DOI:10.1093/jxb/47.1.17
Sze H, Schumacher K, Müller M L, et al. 2002. A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H+-ATPase. Trends in Plant Science, 7: 157-161. DOI:10.1016/S1360-1385(02)02240-9
Tester M, Davenport R. 2003. Na+ tolerance and Na+ transport in higher plants. Annals of Botany, 91: 503-527. DOI:10.1093/aob/mcg058
Wu J L, Seliskar D M, Gallagher J L. 2005. The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress. American Journal of Botany, 92: 852-858. DOI:10.3732/ajb.92.5.852
Yu B J, Lam H M, Shao G H, et al. 2005. Effects of salinity on activities of H+-ATPase, H+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean (Glycine max L.) seedlings. Journal of Environmental Sciences, 17: 259-262.
Zhang W H, Liu Y L. 2002. Relationship between tonoplast H+-ATPase activity, ion uptake and Calcium in barley roots under NaCl stress. Acta Botanica Sinica, 44: 667-672.