Protection of ultrastructure in chilling-stressed banana leaves by salicylic acid

Protection of ultrastructure in chilling-stressed banana leaves by salicylic acid 1 2 first-author 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:wangzhengxun@163.com †E-mail:wangzhengxun@163.com wangzhengxun@163.com 4 2 National Engineering Research Centre for Wheat, Henan Agricultural University, Zhengzhou 450002, China National Engineering Research Centre for Wheat, Henan Agricultural University, Zhengzhou 450002, China South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, China South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, China Department of Biology, Guangzhou University, Guangzhou 510405, China Department of Biology, Guangzhou University, Guangzhou 510405, China College of Life Science, Zhongshan University, Guangzhou 510275, China College of Life Science, Zhongshan University, Guangzhou 510275, China Objective: 2 Chilling tolerance of salicylic acid (SA) in banana seedlings ( Musa acuminata cv., Williams 8818) was investigated by changes in ultrastructure in this study. Methods: 2 Light and electron microscope observation. Results: 2 Pretreatment with 0.5 mmol/L SA under normal growth conditions (30/22 °C) by foliar spray and root irrigation resulted in many changes in ultrastructure of banana cells, such as cells separation from palisade parenchymas, the appearance of crevices in cell walls, the swelling of grana and stromal thylakoids, and a reduction in the number of starch granules. These results implied that SA treatment at 30/22 °C could be a type of stress. During 3 d of exposure to 7 °C chilling stress under low light, however, cell ultrastructure of SA-pretreated banana seedlings showed less deterioration than those of control seedlings (distilled water-pretreated). Conclusion: 2 SA could provide some protection for cell structure of chilling-stressed banana seedling. Banana ( Musa acuminata cv., Williams 8818) Chilling tolerance Salicylic acid Ultrastructure INTRODUCTION 1 Banana plants, which are very sensitive to chilling stress, are often damaged seriously by chilling episodes frequently occurring during winter or early spring every year in South China. Thus, it is important to look for different ways to improve the chilling tolerance of banana plants. To our knowledge, however, only a few such studies on these episodes have been reported (Liang et al., 1994 1994 ). Salicylic acid (SA) is a natural signaling molecule, mediating resistance in response to avirulent pathogens. In plants, SA is endogenously synthesized, and plays an essential role in the defense against pathogen attack (Cronje and Bornman, 1999 1999 ; Ding et al., 2001 2001 ; Mayer et al., 2005 2005 ; Rajjou et al., 2006 2006 ). Although past focus has been mainly on the role of SA on biotic stresses, several recent studies showed that SA can enhance plant resistance to environmental stresses, such as ultraviolet light, drought, salt, chilling and heat (Yalpani et al., 1994 1994 ; Dat et al., 1998 1998 ; Mishra and Choudhuri, 1999 1999 ; Janda et al., 1999 1999 ; Senaratna et al., 2000 2000 ; He et al., 2005 2005 ; Ogawa et al., 2005 2005 ). Our previous studies found that SA pretreatment enhanced the chilling tolerance of banana seedlings. In its physiological, chemical, and molecular bases, we found that SA inhibited leakage of electrolytes, increased the activities of cell protective enzymes, reduced the accumulation of actived oxygen species (AOS), enhanced efficiency of photosynthesis, and induced high expression of selected genes during chilling treatment (Kang et al., 2003a 2003a ; 2003b 2003b ; 2004 2004 ). Since chilling injury can also be manifested in the ultrasturctural level (Lyons, 1973 1973 ; Murata, 1990 1990 ; Kratsch and Wise, 2000 2000 ), the present study was undertaken to evaluate the alteration of cell ultrastructure by SA in relation to its chilling tolerance in banana seedling plants. And the results provided evidence of enhancing chilling tolerance induced by SA in banana seedlings via the cellular ultrastructure. MATERIALS AND METHODS 1 Plant materials 2 Tissue culture seedlings of banana ( Musa acuminata cv., Williams 8818, a chilling-sensitive cultivar) grown in plastic pots with soil and sand mixture (10:1, v/v) were used for this study. Seedlings were placed in plastic growth chamber under normal growth conditions [30/22 °C day/night, 75% relative humidity (RH), 12-h photoperiod with a PPFD of 250 μmol/(m 2 ·s)] and supplied with the same levels of water and MS nutrient. Uniform and healthy seedlings of about two month old (11 cm high) were selected for the experiments. SA treatment and chilling stress 2 The above banana seedlings were pretreated by spraying leaf-blades with SA (0.5 mmol/L in distilled water) 1 d before chilling stress with an atomizer until it ran off. At the same time, 10 ml of the SA solution was used to irrigate roots of each plant. Distilled water was applied to leaves and roots of control plants. Then, SA-pretreated and control were transferred to a climatic chamber [a PPFD of 150 μmol/(m 2 ·s), a 12-h photoperiod and 70% RH] and subjected to chilling stress at 7 °C, which was a temperature of chilling stress but no cold necrosis appeared in leaves (Kang et al., 2003a 2003a ). After 3 d of exposure to 7 °C chilling stress, the seedlings were returned to the previous growth conditions to recover for an additional 2 d. Preliminary experiments showed that treatment with 0.2 mmol/L SA or lower was not reproducibly effective, while higher concentrations (higher than 0.9 mmol/L) caused visible damage (more area of wilting or chilling necrosis) to banana seedlings (Kang et al., 2003b 2003b ). For this reason, this study was focused on SA 0.5 mmol/L. Light and electron microscope observation 2 After chilling treatment, four seedlings from each treatment were randomly selected for cell ultrastructural studies. Leaves were collected after 1 d of SA 0.5 mmol/L water solution treatment at 30/22 °C, and after 3 d of chilling stress at 7 °C. Samples from the middle portion of the last developed leaf blades were excised and fixed in cold 2% (v/v) glutaraldehyde in 0.1 mol/L potassium phosphate buffer (PBS, pH 7.2), vacuum-infiltrated until the material sank and was left overnight at 4 °C. Samples were then washed in five changes of 0.1 mol/L PBS buffer (20 min each) and postfixed in 1% OsO 4 for 16 h at 4 °C in 0.1 mol/L PBS buffer. The materials were washed with five changes of 0.1 mol/L PBS buffer, dehydrated in a graded ethanol series, and embedded in Epon812 resin. Ultra-thin sections about 80~90 nm were obtained with a diamond knife on a Leica-Ultracut S ultramicrotome, and then stained with 2% uranyl acetate for 90 min and 6% lead citrate for 10 min. Sample semimicrosections of 0.2 μm were made with Pyramitome of LKB11800, and finally examined in an OLYMPUS A 70 light microscope and a JEM-1010 (Japan) transmission electron microscope at 90 kV, respectively. At least five sections from each treatment were examined. RESULTS AND DISCUSSION 1 At light microscopic level, it was observed that the cells of palisade parenchymas in leaves of control plants arranged in a regular pattern (Figs. 1a and 1e 1a and 1e ). Treatment with 0.5 mmol/L SA at 30/22 °C for 1 d resulted in the separation of some cells from their regular sites (Figs. 1b and 1f 1b and 1f ). After 3 d of 7 °C stress, most of the cells in control seedlings separated from each other, indicating that the disassembly of palisade parenchymas occurred in this case (Figs. 1c and 1g 1c and 1g ). However, not much alteration of cell arrangement in palisade parenchymas was found in chilling stress banana seedlings with SA-pretreatment (Figs. 1d and 1h 1d and 1h ). It could perhaps be speculated that alleviated changes by SA in cell structure occurred under chilling stress. CE: Cell; F: Fibro; P: Palisade parenchymas At light electron level, it was further found that chloroplasts in mesophyll cells of control plants at 30/22 °C had many starch granules (2.5 granules in each chloroplast) (Fig. 2a 2a ) and that well-developed granal stacks were interconnected by stromal thylakoids (Figs. 2e and 2i 2e and 2i ). The chloroplast membranes were intact (Figs. 2e and 2i 2e and 2i ), as were most of mitochondrial membranes (Figs. 2m 2m ). Whereas, after the treatment with SA at 30/22 °C for 1 d, swelling and separation of 40% of chloroplant thylakoids occurred, which resulted in an increase in intrathylakoid space (Figs. 2f and 2j 2f and 2j ). At the same time, most of the starch granules disappeared in chloroplasts (0.2 granules in each chloroplast), and unexpectedly, there appeared 11~12 crevices in each cell wall (Fig. 2b 2b ), and part of about 20% mitochondrial membranes were broken and the mitochondrial matrix might mix with cytoplasm (Fig. 2n 2n ). C: Cell membrane; CH: Chloroplast; CM: Chloroplast membrane; CR: Crevice; GT: Grana thylakoid; L: Lipid droplet; M: Mitochondria; MC: Mitochondria cristae; MM: Mitochondria membrane; N: Nucleus; S: Starch; ST: Stromal thylakoid; V: Vacuole; VE: Vesicle. White lines of black panes express bars of magnification It had been reported that chloroplasts are commonly the earliest visible site of ultrastructural chilling injury in plant cells (Kratsch and Wise, 2000 2000 ). Three days of 7 °C stress caused a series of pronounced changes in the fine structure of cells both in control and SA-pretreated banana plants. In control chloroplasts, almost all of the starch granules were lost (Fig. 2c 2c ), and 90% granal and stromal thylakoids became enlarged, and presented some large vesicles in chloroplast granal and stromal thylakoids (Figs. 2g and 2k 2g and 2k ). The mitochondrial cristae remained intact, but some membranes were broken-down (Fig. 2o 2o ). In the cells of SA-pretreated leaves subjected to 7 °C, however, more starch granules were found in chloroplasts (1~2 granules in each chloroplast), and the number of crevices in each cell wall decreased to 3~4 trips (Fig. 2d 2d ). Although most of the granal and stromal thylakoids became swollen, they still remained in the ordered state (Figs. 2h and 2l 2h and 2l ). Moreover, mitochondrial membranes were kept more intact than those in the control cells in this case (Figs. 2o and 2p 2o and 2p ). In the present study, the unordered arrangement of cells, swelling of granal and stromal thylakoids (Figs. 1c and 1g 1c and 1g , and Figs. 2g and 2k 2g and 2k ) in control plants under chilling condition, suggested that leaves of banana plants were very sensitive to chilling stress. However, the less ultrastructural deterioration in SA-pretreated plants (Figs. 1d, 1h 1d, 1h , 2h and 2l 2h and 2l ) indicated that SA might provide protection for banana seedlings to chilling injury and could increase the chilling tolerance of banana seedlings. On the contrary, a series of degradative changes in cell structures after SA treatment at 30/22 °C were quite striking (Figs. 1b, 1f 1b, 1f , 2f, 2j, 2n 2f, 2j, 2n ), suggesting that SA treatment under normal growth conditions could be a type of stress. Some researchers have hypothesized that there is a common denominator, e.g. when stress treatments are applied at mild levels that do not cause appreciable damage (Janda et al., 1999 1999 ). According to this, it was proposed that plants respond with similar defense systems to a wide range of stresses such as chemical pollutants, UV radiation, low oxygen, pathogen infection, and wounding (Ding et al., 2001 2001 ). Moreover, the enhanced chilling tolerance in drought-hardened maize plants also supported this view (Scandalios, 1993 1993 ). Thus, we deduced that treatment of banana seedlings with 0.5 mmol/L SA at 30/22 °C could be a mild stress and indirectly or directly induce some of the cell defense mechanism responses providing protection against subsequent chilling stress, rather than producing a direct effect by SA itself. SA pretreatment under normal growth conditions damaged thylakoid membranes of banana chloroplasts, etc. (Figs. 2f and 2j 2f and 2j ), which were supported by the decrease in starch granules (Fig. 2b 2b ), and were in accordance with low photosynthetic activity (Kang et al., 2003b 2003b ). Moreover, more starch granules and intactness of cell ultrastructure in SA-pretreated leaves during chilling stress (Figs. 2d, 2h and 2l 2d, 2h and 2l ) might be linked to their beneficial photosynthetic function (Kang et al., 2003b 2003b ). Clearly, the appearance of numerous crevices in cell wall after SA treatment at 30/22 °C and reduction during chilling treatment were interesting. It is hard to make a relevant explanation right now. We first guessed that crevices, which appeared in SA pretreatment at normal growth conditions, might be from the toxicity of AOS (such as H 2 O 2 ) induced by SA. The reason for this supposition was that exogenous SA treatments led to an increase in H 2 O 2 levels in plant tissues (Lamb and Dixon, 1997 1997 ; Kang et al., 2003a 2003a ), and, in consequence, accumulated AOS could destroy cell structures (Prasad, 1996 1996 ). However, no crevice appeared in control plants after 3 d of 7 °C chilling stress, in which probably more AOS accumulated (Kang et al., 2003a 2003a ), and crevices in the cell walls of SA-pretreated banana became fewer at this time. So, it seemed that oxidative destruction would not be the exact reason for cell wall crevice formation. Perhaps, some effect of SA on cell walls would take place during their synthesis or expansion. Primary cell wall expansion requires a loosening of the crosslinks within the wall and an increase in cytoplasmic pressure potential. Under normal growth conditions, the destroyed cell structure might increase cytoplasmic pressure potential, and tear the cell walls, to form the crevices. Under chilling stress conditions, SA was also reported to enhance the activity and expression of alterative oxidase in plants (Gilliland et al., 2003 2003 ), which could increase heat production (Rasin et al., 1987 1987 ; van der Straeten et al., 1995 1995 ). Therefore, during chilling stress, heat production mediated through alterative respiration pathway by SA might play a role in protection against chilling and repairing the crevices of cell walls. In conclusion, SA treatment under normal growth conditions could be a mild stress, but enhance the tolerant capability of banana cells to subsequent chilling stress. We would like to express our appreciation to Prof. Zhifang Lin for help with the electron microscopy and modification of manuscript. References MJ Cronje , L Bornman . Salicylic acid influences Hsp70/Hsc70 expression in Lycopersicon esculentum : dose- and time-dependent induction or potentiation . Biochem Biophys Res Commun , 1999 . 265 ( 2 ): 422 - 427 . 10558883 DOI: 10.1006/bbrc.1999.1692 http://doi.org/10.1006/bbrc.1999.1692 . Salicylic acid influences Hsp70/Hsc70 expression in Lycopersicon esculentum: dose- and time-dependent induction or potentiation JF Dat , H Lopez-Delgado , CH Foyer , IM Scott . Parallel changes in H 2 O 2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings . Plant Physiol , 1998 . 116 ( 4 ): 1351 - 1357 . 9536052 DOI: 10.1104/pp.116.4.1351 http://doi.org/10.1104/pp.116.4.1351 . Parallel changes in H2O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings CK Ding , CY Wang , KC Gross , DL Smith . Reduction of chilling injury and transcript accumulation of heat shock proteins in tomato fruit by methyl jasmonate and methyl salicylate . Plant Sci , 2001 . 161 ( 6 ): 1153 - 1159 . DOI: 10.1016/S0168-9452(01)00521-0 http://doi.org/10.1016/S0168-9452(01)00521-0 . Reduction of chilling injury and transcript accumulation of heat shock proteins in tomato fruit by methyl jasmonate and methyl salicylate A Gilliland , DP Singh , JM Hayward , CA Moore , AM Murphy , CJ York , J Slator , JP Carr . Genetic modification of alternative respiration has differentia effects on antimycin A-induced versus salicylic acid-induced resistance to Tobacco mosaic virus . Plant Physiol , 2003 . 132 ( 3 ): 1518 - 1528 . 12857832 DOI: 10.1104/pp.102.017640 http://doi.org/10.1104/pp.102.017640 . Genetic modification of alternative respiration has differentia effects on antimycin A-induced versus salicylic acid-induced resistance to Tobacco mosaic virus YL He , YL Liu , WX Cao , MF Huai , BG Xu , BR Huang . Effects of salicylic acid on heat tolerance associated with antioxidant metabolism in Kentucky Bluegrass . Crop Sci , 2005 . 45 ( 3 ): 988 - 995 . DOI: 10.2135/cropsci2003.0678 http://doi.org/10.2135/cropsci2003.0678 . Effects of salicylic acid on heat tolerance associated with antioxidant metabolism in Kentucky Bluegrass T Janda , G Szalai , I Tari , E Paldi . Hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize ( Zea mays L.) plants . Planta , 1999 . 208 ( 2 ): 175 - 180 . DOI: 10.1007/s004250050547 http://doi.org/10.1007/s004250050547 . Hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize (Zea mays L.) plants GZ Kang , CH Wang , GC Sun , ZX Wang . Salicylic acid changes activities of H 2 O 2 -metabolizing enzymes and increases the chilling-tolerance of banana seedlings . Environ Exp Bot , 2003 . 50 9 - 15 . . Salicylic acid changes activities of H2O2-metabolizing enzymes and increases the chilling-tolerance of banana seedlings GZ Kang , ZY Ou , GC Sun , ZX Wang . Effects of salicylic acid on cell membranes and some photosynthesis of chilling-stressed banana seedlings . Acta Hort Sin , 2003 . 30 ( 2 ): 141 - 146 . (in Chinese) . Effects of salicylic acid on cell membranes and some photosynthesis of chilling-stressed banana seedlings GZ Kang , GH Zhu , XX Peng , GC Sun , ZX Wang . Isolations of salicylic acid-induced genes in chilling-stressed banana seedling leaves using mRNA differential display . J Plant Physiol Mol Biol , 2004 . 30 ( 2 ): 225 - 228 . . Isolations of salicylic acid-induced genes in chilling-stressed banana seedling leaves using mRNA differential display HA Kratsch , RR Wise . The ultrastructure of chilling stress . Plant Cell Environ , 2000 . 23 ( 4 ): 337 - 350 . DOI: 10.1046/j.1365-3040.2000.00560.x http://doi.org/10.1046/j.1365-3040.2000.00560.x . The ultrastructure of chilling stress C Lamb , RA Dixon . The oxidative burst in plant disease resistance . Annu Rev Plant Physiol Plant Mol Biol , 1997 . 48 ( 1 ): 251 - 275 . 15012264 DOI: 10.1146/annurev.arplant.48.1.251 http://doi.org/10.1146/annurev.arplant.48.1.251 . The oxidative burst in plant disease resistance LF Liang , ZH Wang , BY Zhou , HB Huang . Effects of low temperature and paclobutrazol on the activities and isozymes of peroxidase in banana leaves . J South China Agric Univ , 1994 . 15 ( 3 ): 65 - 70 . (in Chinese) . Effects of low temperature and paclobutrazol on the activities and isozymes of peroxidase in banana leaves JM Lyons . Chilling injury in plants . Annu Rev Plant Physiol , 1973 . 24 ( 1 ): 445 - 466 . DOI: 10.1146/annurev.pp.24.060173.002305 http://doi.org/10.1146/annurev.pp.24.060173.002305 . Chilling injury in plants CM Mayer , KC Lee , CA Moore , SM Wong , JP Carr . Salicylic acid-induced resistance to Cucumber mosaic vivus in Squash and Arabidopsis : contrasting mechanisms of induction and antiviral action . Mol Plant Microbe Interact , 2005 . 18 ( 5 ): 428 - 434 . 15915641 . Salicylic acid-induced resistance to Cucumber mosaic vivus in Squash and Arabidopsis: contrasting mechanisms of induction and antiviral action A Mishra , MA Choudhuri . Effects of salicylic acid on heavy metal-induced membrane deterioration mediated by lipoxygenase in rice . Biol Plant , 1999 . 42 ( 3 ): 409 - 415 . DOI: 10.1023/A:1002469303670 http://doi.org/10.1023/A:1002469303670 . Effects of salicylic acid on heavy metal-induced membrane deterioration mediated by lipoxygenase in rice T Murata . CY Wang . Relation of Chilling Stress to Membrane Permeability . Chilling Injury of Horticultural Crops , 1990 . : Boca Raton, Florida CRC Press , 201 - 209 . . Relation of Chilling Stress to Membrane Permeability D Ogawa , N Nakajima , T Sano , M Tamaoki , M Aono , A Kwbo , M Kanna , M Ioki , H Kamada , H Saji . Salicylic acid accumulation under O 3 exposure is regulated by ethylene in tobacco plants . Plant Cell Physiol , 2005 . 46 ( 7 ): 1062 - 1072 . 15870097 DOI: 10.1093/pcp/pci118 http://doi.org/10.1093/pcp/pci118 . Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants TK Prasad . Mechanisms of chilling-induced oxidative stress injury and tolerance: changes in antioxidant system, oxidation of proteins and lipids and protease activities . Plant J , 1996 . 10 ( 6 ): 1017 - 1026 . DOI: 10.1046/j.1365-313X.1996.10061017.x http://doi.org/10.1046/j.1365-313X.1996.10061017.x . Mechanisms of chilling-induced oxidative stress injury and tolerance: changes in antioxidant system, oxidation of proteins and lipids and protease activities L Rajjou , M Belghazi , R Huguet , C Robin , A Moreau , C Job , D Job . Preteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms . Plant Physiol , 2006 . 141 ( 3 ): 910 - 923 . 16679420 DOI: 10.1104/pp.106.082057 http://doi.org/10.1104/pp.106.082057 . Preteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms I Rasin , A Ehmann , WR Melander , BJD Meeuse . Salicylic acid: a natural inducer of heat production in Arum. lilies . Science , 1987 . 237 1601 - 1602 . . Salicylic acid: a natural inducer of heat production in Arum. lilies JG Scandalios . Oxygen stress and superoxide dismutases . Plant Physiol , 1993 . 101 ( 1 ): 7 - 12 . 12231660 . Oxygen stress and superoxide dismutases T Senaratna , D Touchell , T Bunn , K Dixon . Acetyl salicylic acid (aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants . Plant Growth Reg , 2000 . 30 ( 2 ): 157 - 161 . DOI: 10.1023/A:1006386800974 http://doi.org/10.1023/A:1006386800974 . Acetyl salicylic acid (aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants D van der Straeten , L Chaerle , G Sharkov , A Lamlers , M van Montagu . Salicylic acid enhances the activity of the alternative pathway of respiration in tobacco leaves and induces thermogenicity . Planta , 1995 . 196 ( 3 ): 412 - 419 . DOI: 10.1007/BF00203637 http://doi.org/10.1007/BF00203637 . Salicylic acid enhances the activity of the alternative pathway of respiration in tobacco leaves and induces thermogenicity N Yalpani , AJ Enyedi , J Leon , I Raskin . Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogen-related proteins and virus resistance in tobacco . Planta , 1994 . 193 ( 3 ): 372 - 376 . DOI: 10.1007/BF00201815 http://doi.org/10.1007/BF00201815 . Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogen-related proteins and virus resistance in tobacco 10.1631/jzus.2007.B0277 Project (No. 30471112) supported by the National Natural Science Foundation of China 2006-05-15 2006-11-27 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 08