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Jasmonates are essential factors inducing gummosis in tulips: mode of action of jasmonates focusing on sugar metabolism
ARTICLE IN PRESS Journal of Plant Physiology 162 (2005) 495—505
www.elsevier.de/jplph
Jasmonates are essential factors inducing gummosis in tulips: mode of action of jasmonates focusing on sugar metabolism Edyta Skrzypeka,b, Kensuke Miyamotoa, Marian Saniewskic, Junichi Uedaa, a
College of Integrated Arts and Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan b Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland c Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland Received 18 June 2004; accepted 8 September 2004
KEYWORDS Ethylene; Glucuronoarabinoxylan; Gummosis (gum formation); Jasmonate; Jasmonic acid; Methyl jasmonate; Sugar metabolism; Tulipa gesneriana
Summary The purpose of this study was to know the mechanism of jasmonates to induce gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) shoots, especially on the focus of sugar metabolism. Gummosis in the first internode of tulip plants was induced by the application of methyl jasmonate (JA-Me, 1% w/w in lanolin) and jasmonic acid (JA, 1% w/w in lanolin) 5 days after application and strongly stimulated by the simultaneous application of ethylene-releasing compound, ethephon (2-chloroethylphosphonic acid, 1% w/w in lanolin), although ethephon alone had little effect. JA-Me stimulated ethylene production of the first internodes of tulips, ethylene production increasing up to more than 5 times at day 1 and day 3 after the application. On the other hand, application of ethephon did not increase endogenous levels of jasmonates in tulip stems. Analysis of composition of tulip gums revealed that they were consisted of glucuronoarabinoxylan with an average molecular weight of ca. 700 kDa. JA-Me strongly decreased the total amount of soluble sugars in tulip stems even in 1 day after application, being ca. 50% of initial values 5 days after application, but ethephon did not. However, both JA-Me and ethephon had almost no effect on the neutral sugar compositions of soluble sugars mainly consisting of glucose, mannose and xylose in ratio of 20:2:1 and traces of arabinose. Both
Abbreviations: Ethephon, 2-chloroethylphosphonic acid; JA, jasmonic acid; JA-Me, methyl jasmonate Corresponding author. Tel.: +81 72 254 9733; fax: +81 72 254 9932. E-mail address: [email protected] (J. Ueda). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.09.007
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E. Skrzypek et al. JA-Me and ethephon applied exogenously stimulated senescence of tulip shoots shown by the loss of chlorophyll. These results strongly suggest that the essential factor of gummosis in tulips is jasmonates affecting the sugar metabolism in tulip shoots. The mode of action of jasmonates to induce gummosis of tulip shoots is discussed in relation to ethylene production, sugar metabolism and senescence. & 2005 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Gummosis is the process of the accumulation and exudation of gums mainly consisted of polysaccharides including uronic acids in some plants species. The gummosis has been known as a common response to various environmental stresses such as wounding, pathogen infection or insect attack. Gummosis in stone fruit trees has considered to be regulated by ethylene (Nair et al., 1980; Olien and Bukovac, 1982; Boothby, 1983; Morrison et al., 1987). Not only in stone fruit trees but also in tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs, infection of Fusarium oxysporum f. sp. tulipae and application of ethylene or ethylene-releasing compound, ethephon, have been found to induce gums (Kamerbeek and De Munk, 1976; De Hertogh et al., 1980; De Munk and Saniewski, 1989; De Wild et al., 2002). Recently, jasmonic acid (JA), methyl jasmonate (JA-Me) and their related compounds designated as jasmonates have been demonstrated to play an important role in signal transduction pathway in response to stresses, resulting in significant physiological phenomena (Koiwa et al., 1997). Jasmonates have also been found to induce gums in various species of stone fruit trees such as plum, peach, cherry and apricot (Saniewski et al., 1998a, 2002, 2003; Ueda et al., 2003) and in tulips (Saniewski and Puchalski, 1988; Saniewski et al., 1998b, 2000). It is known that ethylene acts together with JA-Me and intensify effect of JA-Me action in gummosis and that both of them are implicated in promotion of senescence (Saniewski et al., 1998b). To understand the mechanism of JA-Me to induce gummosis and their interaction with ethylene in tulips, we investigated the effects of JA-Me and/or ethephon on qualitative and quantitative changes in soluble sugars, endogenous levels of these hormones and senescence in stems of tulip plants as well as sugar composition of gums and cell walls of tulips, and molecular weight of gums. Possible mode of action of jasmonates in inducing gum formation is also discussed.
Plant materials The experiments were performed with the bulbs of tulip (T. gesneriana L. cv. Apeldoorn) from commercial stock. After lifting, the bulbs with a circumference of 10–12 cm stored at 18–20 1C were cooled at 5 1C for a minimum 12 weeks. After removal to the tunics, tulip bulbs were planted in pots individually and cultivated at the temperature 17–20 1C under natural daylight conditions in a greenhouse. Tulip plants at the age of ca. 3–5 days before flowering were used. JA-Me (1% w/w) and/ or ethephon (1% w/w) were applied as a ring (2–3 mm in width) of lanolin paste to the first internode of stem of intact tulips. After appropriate incubation gum formation was observed optically, then the 1st internode segments prepared from the plants were subjected for various analyses. A lot of 3–8 plants were used in each treatment.
Analysis of soluble sugars Soluble sugars in the first internode segments of tulips were extracted with 80% aqueous EtOH. The amounts of total and reducing sugars were determined by phenol–sulfuric (Dubois et al., 1956) and Somogyi–Nelson methods (Somogyi, 1952), respectively. The amounts of sucrose were estimated by the changes in reducing sugars before and after invertase treatments. The amounts of uronic acids were also determined by carbazole–sulfuric acid method (Galambos, 1967). Soluble neutral sugars together with inositol as an internal standard were reduced with sodium borohydride and acetylated with acetic anhydride in the presence of N-methylimidazole. Qualitative and quantitative analysis of acetylated sugars were determined with a gas–liquid chromatography according to the method reported previously (Ueda et al., 1995, 2002).
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Gel permeation chromatography
Determination of jasmonates
Molecular mass of tulip gums was estimated by a gel permeation chromatography according to the method by Wakabayashi et al. (1997). Tulip gums were dissolved in potassium phosphate buffer (50 mM, pH 7.2). A portion of the solution (ca. 300 mL, approximately 0.3 mg Glc equivalent) was subjected to a high performance liquid chromatograph (LC-6A, Shimadzu Co. Ltd., Kyoto, Japan) equipped with a gel-permeation column (TSK-gel G5000PW, Tosoh Co. Ltd., Tokyo, Japan) and a refractive index detector (RID-6A, Shimadzu Co. Ltd., Kyoto, Japan). The sample was eluted with the same buffer at a flow rate of 1 mL/min. Fractions of 0.5 mL were collected with a fraction collector (Model 203, Gilson, Middleton, WI, USA). The elution pattern was monitored by a refractive index detector. The contents of total sugars and uronic acids in each fraction were determined by phenol-sulfuric method and carbazole-sulfuric acid method, respectively. The weight-average molecular mass of gums was calculated from the equation reported by Nishitani and Masuda (1981). Dextrans (Sigma) of 10, 40, 70, 120 and 500 kDa were used as molecular mass markers. After hydrolysis of polysaccharides in each fraction with 2 N trifluoroacetic acid and acetylation (Albersheim et al., 1967), neutral sugar composition of gums was determined by a gas-chromatography as mentioned above.
First internode segments prepared from tulips treated with or without ethephon were extracted twice with 80% aqueous EtOH at 4 1C. Alcoholic extracts were concentrated in vacuo to give an aqueous residue. Deuterium-labeled JA (d2-JA) and JA-Me (d2-JA-Me) were added to the aqueous residue as internal standards, then followed by partitioning with diethyl ether. After partitioning with diethyl ether at pH 7, the aqueous phase was adjusted to pH 3 with HCl and then partitioned with diethyl ether in the usual way, giving the diethyl ether-soluble neutral and acidic materials, respectively. These materials were further purified with silica gel thin-layer chromatography according to the method reported previously (Ueda et al., 1994). Both fractions eluted from zones corresponding to authentic JA and JA-Me in silica gel thin-layer chromatograms were methylated with ethereal diazomethane. Qualitative and quantitative analyses of JA and JA-Me were carried out using a Finnigan GCQ gas–liquid chromatography–mass spectrometry according to the same procedure reported previously (Ueda et al., 1991). Experiments were carried out twice and results represented as the total amounts of JA plus JA-Me were expressed an average of two independent experiments. Quite similar results were obtained in these experiments.
Cell wall polysaccharides
Ethylene production
Analyses of cell wall polysaccharides were carried out according to the method by Nishitani and Masuda (1981). Methanol-killed segments of stems and leaves of tulips were homogenized. The homogenates were sufficiently washed with cold water, acetone and then methanol-chloroform, successively. The homogenates were treated with a-amylase for 3 h at 37 1C to remove starch and then centrifuged. The pellet was extracted with ammonium oxalate, then centrifuged. The supernatant contained pectic polysaccharides. The pellet residue was extracted with 4% KOH and then 24% KOH, each supernatant giving hemicellulose I (HC-I) and hemicellulose II (HC-II) fractions, respectively. The residue was recognized as an acellulosic fraction. The amount of polysaccharides in each fraction was determined by phenol–sulfuric acid method and carbazole-sulfuric acid method. Neutral sugar compositions of pectic, HC-I and HC-II polysaccharides were determined by a gas–liquid chromatography after hydrolysis and acetylation as mentioned above.
First internode segment (2 cm in length) prepared from tulips treated with or without JA-Me was incubated in a 20-mL beaker. Each beaker was sealed with Parafilm, and then incubated at 25 1C in the dark. After 2-h incubation, 1 mL of the gas from the headspace of the beaker was sampled. Determination of ethylene production was carried out according to the method previously reported (Ueda et al., 1996) using a Hitachi 163 gas–liquid chromatograph equipped with a hydrogen flame ionizing detector. A glass column (5 mm 2 m) packed with Porapak N was used.
Chlorophyll content The total amounts of chlorophyll of first internode segment (2 cm in length) prepared from tulips treated with or without JA-Me in the presence or absence of ethephon was extracted with 10 mL of 80% aqueous EtOH overnight. Absorbance of the extracts was measured at 665 nm using a Shimadzu
ARTICLE IN PRESS 498 UV-1200 spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan).
Results Gum formation The application of lanolin alone to the first internode of intact tulip plants had no effect on gum formation during 7-day incubation. On the other hand, the application of JA-Me at 1% (w/w in lanolin) to the first internode of intact tulip plants induced gum formation 5 days after application, gums being exuded in the place of the application on the first internode and infiltrated into the basal part of the first leaf (Fig. 1) as reported previously (Saniewski et al., 1998b, 2000). Similar to JA-Me, the application of JA also induced gum formation in tulip shoots, activities of JA-Me and JA to induce gum formation in tulip shoots being almost same. Application of ethephon together with JA-Me strikingly stimulated gum formation, while ethephon alone had little effect, suggesting that jasmonates but not ethylene play an essential role for gum formation in tulip shoots (all data not shown).
Effect of JA-Me on ethylene, and vise versa To better understand the mechanism of interaction of jasmontes and ethylene on gum formation, effect of JA-Me on ethylene production in tulip stems, and that of ethephon on endogenous levels of jasmonates were examined. Lanolin alone had no effect on ethylene production in tulip stems as
E. Skrzypek et al. shown in Table 1. On the other hand, the application of JA-Me significantly increased ethylene production. The magnitude of ethylene production was observed during first 3 days after the application of JA-Me, the level being almost 5 times higher than that of control. During the following days, the production of ethylene decreased gradually but it was still higher in comparison with control level. In this study, we have confirmed the presence of JA and JA-Me in tulip stems using GC-MS (data not shown) and estimated the endogenous level of jasmonates by deuterium-labeled JA and JA-Me as internal standards. The content of jasmonates designated as JA plus JA-Me in tulip stems increased after treatment of lanolin only and ethephon until day 5, then decreased at day 7. The application of ethephon (1% w/w in lanolin) did not increase the endogenous level of jasmonates in the internode of tulips in comparison with control one (Table 2).
Molecular mass of tulip gums and their sugar composition Fig. 2 shows molecular mass distribution of tulip gums analyzed by a gel permeation chromatography. Tulip gums were consisted of almost homogeneous polysaccharides with an average molecular mass of ca. 700 kDa. The ratio of neutral sugars and uronic acids was ca. 2.5:1. Analysis of neutral sugar composition of tulip gums revealed that arabinose and xylose existed at the ratio of ca. 2: 3 (Table 3). These results suggest that tulip gums are consisted of glucuronoarabinoxylan with an average molecular mass of ca. 700 kDa.
Figure 1. Gum exuded on the first internode (left) and infiltrated in the basal part of leaf (right) in tulips in response to simultaneous application of JA-Me and ethephon. Photographs were taken 5 days after the application of JA-Me (1% w/ w in lanolin) and ethephon (1% w/w in lanolin).
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips Table 1.
499
Effect of JA-Me on ethylene production in the first internodes of tulips
Treatment
Ethylene production, nl/g/h Days after treatment
Control (lanolin alone) JA-Me
1
3
5
7
1.1770.03 5.4370.59
0.7270.03 5.2170.32
1.2670.09 3.0570.35
0.7570.02 1.1470.11
After appropriate incubation of intact tulips treated with or without JA-Me (1% w/w in lanolin), the first internode segment (2 cm in length) was prepared and subjected to determination of ethylene production. Results were indicated as the average with standard errors (n ¼ 8; control; n ¼ 3; JA-Me).
Table 2.
Effect of ethephon on endogenous level of jasmonates in tulip stems
Treatment
Jasmonate content, ng/g FW Days after treatment
Control (lanolin alone) Ethephon
1
3
5
7
53.8 31.1
86.0 72.6
75.3 82.8
62.9 64.6
After appropriate incubation of intact tulips treated with or without ethephon (1% w/w in lanolin), first internode segments (2 cm in length) were prepared and subjected to determination of endogenous levels of JA and JA-Me. Results are expressed as jasmonates, the sum of JA and JA-Me, and mean of two individual experiments.
Distribution of polysaccharides or uronic acids, %
V
0
500
70
Effects of JA-Me and ethephon on soluble sugars
10
16 12 8 4 0
Relative amounts
60 Total sugars Uronic acids 40
20
0 10
15 20 Elution volume, ml
25
Figure 2. Molecular mass distribution of tulip gums. The amounts of total sugars and uronic acids in each fraction (0.5 mL) obtained by the gel permeation chromatography (see Materials and methods) were determined by the phenol–sulfuric acid method and the carbazole-sulfuric acid method, respectively. The elution position of molecular mass standards (dextrans of 500, 70 and 10 kDa) and the void volume (V0) are shown at the top.
To understand the regulatory role of JA-Me on gum formation, effects of JA-Me on the soluble sugars as substrates for polysaccharide synthesis was examined in comparison with that of ethephon, since tulip gums demonstrated to be mainly consisted of polysaccharide, glucuronoarabinoxylans (Fig. 2 and Table 3). The total amounts of soluble sugars estimated by the phenol–sulfuric acid method were almost equivalent to those of reducing sugars estimated by the Somogyi–Nelson method in soluble sugar fractions after the treatment of invertase, suggesting that polysaccharides exist little in soluble sugar fraction. Changes in the amounts of reducing sugars before and after invertase treatments determined by the Somogyi–Nelson method showed that almost all soluble sugars were reducing sugars while only small amount of sucrose existed (Fig. 3). As shown in Fig. 3, the level of sucrose and soluble reducing sugars in tulip stem segments prepared from the tulip plants treated with lanolin alone were almost constant during the incubation for 7 days. On the other hand, JA-Me strongly reduced the amounts of reducing sugars and sucrose in the first internodes of tulips even in 1
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E. Skrzypek et al.
Table 3.
Neutral sugar composition of tulip gums
Fractions, elution volume (mL)
Neutral sugar composition, %
15.0–16.5
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
nd
nd
39.574.0
60.574.0
nd
nd
nd
Reducing sugars, mg/g FW
Sucrose, mg/g FW
Neutral sugars in the fractions (elution volume 15.0–15.5, 15.5–16.0, 16.0–16.5) corresponding to the peak of chromatogram were analyzed by a gas–liquid chromatography after hydrolysis and acetylation. Values are expressed as the average with standard errors (n ¼ 3). nd indicates no sugar detected. Elution volume is same as in Fig. 2.
day after the application. The total amounts of reducing sugars in tulip stem treated with JA-Me were gradually reduced, it being ca. 35% at the end of the experiment of day 7 in comparison with control (untreated plants) value. On the other hand, ethephon had almost no effect on the amount of soluble reducing sugars, but interestingly it reduced the amount of sucrose. These results suggest that JA-Me affects soluble sugar composition qualitatively and quantitatively. Figure 4 shows the neutral sugar composition of soluble reducing sugars of the internodes of tulips. Neutral sugars in soluble reducing sugar fraction of the first internode of tulips treated with lanolin alone consisted of mainly glucose, mannose and xylose in the ratio 20:2:1, and traces of arabinose. Both JA-Me and ethephon little affected the neutral sugar composition.
20 Controls
JA-Me
Ethephon
10
0 60
40
20
0
1
3 5 Days after treatment
7
Figure 3. Effect of JA-Me (1%) and ethephon (1%) on sucrose level and reducing sugar content in tulip stems. Bars indicate means with standard errors (n ¼ 8, control; n ¼ 5, ethephon; n ¼ 3; JA-Me). The amount of sucrose was estimated by the changes in reducing sugars before and after invertase treatments.
Cell wall polysaccharide of tulips Glucuronoarabinoxylan has been demonstrated to be one of constituents of cell wall polysaccharides. In order to compare the sugar compositions between cell wall polysaccharides and gums, cell
100 Control JA-Me Neutral sugar composition, %
80
Ethephon
60
40
20 ND
ND
Rha
Fuc
ND
0 Ara
Xyl
Man
Gal
Glc
Figure 4. Effect of JA-Me (1%) and ethephon (1%) on neutral sugar composition of soluble sugar fraction in tulip stems. Bars indicate means with standard errors (n ¼ 8; control; n ¼ 5; ethephon; n ¼ 3; JA-Me).
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips wall polysaccharides were fractionated into pectic, HC-I and HC-II, and cellulosic polysaccharides. Cell wall polysaccharides in stems were mainly consisted of cellulose, and small amount of pectic, HCI and HC-II polysaccharides, while the ratio of cellulose in leaves was smaller than that of stems (Fig. 5). As shown in neutral sugar composition of polysaccharides in pectic, HC-I and HC-II fractions, quite heterogeneous polysaccharides existed in
100 Leaves Stems
Polysaccharide composition, %
80
60
40
Cellulose
HC−II:−NS
HC−I: UA
HC−I: NS
Pectin: UA
Pectin: NS
0
HC−II: UA
20
Figure 5. Polysaccharide composition of tulip cell walls. After fractionation into pectic, HC-I, HC-II and cellulosic polysaccharides, the amounts of neutral sugars and uronic acids were determine.
60
each fraction (Fig. 6). This suggests that matrix cell wall polysaccharides differently from gums are consisted of various kinds of polysaccharides.
Discussion Interaction of JA-Me and ethylene on gum formation in tulips The application of JA-Me to the first internode of tulip stems strongly induced gum formation (Fig. 1). The application of JA also induced gum formation in tulip shoots. Activity of JA to induce gummosis in tulip shoots was almost equivalent to that of JA-Me. On the other hand, the application of ethephon, ethylene-generating substance, induced no gums in tulip stems although JA-Me strongly promoted ethylene production which might be induced by wounding just after excision of the first internode of tulip stems. These results indicate that jasmonates, but not ethylene, play an essential role for gum formation in tulip shoots. Since simultaneous application of JA-Me and ethephon stimulated gum formation in tulip shoots compared to the case of JA-Me alone, it is possible to say that ethylene is really involved in gum formation in tulip. JA-Me has been reported to stimulate ACC synthase and ACC oxidase activities and/or ethylene production in various plant species (Saniewski, 1997). In tulip stems, biosynthesis of ethylene was also substantially affected by the application of JA-Me (Table 1) probably by stimulating of ACC synthase and ACC oxidase activities as
HC − I
Pectin
501
HC − II
Leaves
Neutral sugar composition, %
Stems
40
20
0
Ara
Rha Fuc
Man Xyl
Glc Gal
Rha
Ara Fuc
Man Glc Xyl Gal
Rha
Ara Fuc
Man Xyl
Glc Gal
Figure 6. Neutral sugar composition of pectic, HC-I and HC-II polysaccharides of tulip cell walls. After hydrolysis and acetylation, neutral sugar composition was determined by a gas–liquid chromatography.
ARTICLE IN PRESS 502 suggested above. This result also suggests that JAMe applied exogenously regulates gum formation by interacting with endogenous ethylene induced by JA-Me itself. On the contrary to the application of JA-Me, the application of ethephon (1% w/w in lanolin) did not affect the endogenous level of jasmonates designated as the total amounts of JA and JA-Me in the internode of tulips (Table 2), suggesting that the synergistic effect of ethylene on gum formation is not mediated by the increase in endogenous level of jasmonates. The mode of action of ethylene to stimulate gum formation in tulip shoots in the presence of JA-Me has not been clear yet. It is probable that there are an interaction and/or a cross-talk in signal transduction pathways between jasmonates and ethylene. Hung and Kao (1996) reported that ethylene increases susceptibility to JA-Me on the process of jasmonates-promoting senescence in detached maize leaves. Similar explanation will be possible on the process of gummosis in tulip shoots. Emery and Reid (1996) have also reported that simultaneous application of JA-Me and ACC caused dramatic degradation of cell membrane as indicated by an increase in conductivity in sunflower seedlings and neither the application of ACC alone nor that of JA-Me with ethylene inhibitors evoked such an increase in conductivity. This fact also raises the possibility that ethylene together with or without JA-Me stimulates exudation process of gums by inducing loosening of cell membranes.
Chemical composition of tulip gums We have previously suggested that tulip gums consisted of glucuronoarabinoxylans by analyses of uronic acid and neutral sugar contents and the neutral sugar composition after direct hydrolysis with trifluoroacetic acid (Saniewski et al., 2000). As shown in the molecular mass distribution (Fig. 2) and neutral sugar composition (Table 3) tulip gums are suggested to be homogenous polysaccharides consisted of glucuronoarbinoxylan with an average molecular weight of ca. 700 kDa, while uronic acids have not identified yet. Glucuronoarabinoxylan is well known as one of the components of cell wall polysaccharides (Darvell et al., 1980), and in barley coleoptiles, gel permeation chromatography showed that polysaccharides composed of Ara and Xyl were at least two species with different Ara/Xyl ratios and different molecular weight (Sakurai and Kuraishi, 1984). Glucuronoarabinoxylans of cell wall polysaccharides isolated from oat coleoptiles did not bind to
E. Skrzypek et al. cellulose in vitro (Wada and Ray, 1978), being possible that steric hindrance by a high percentage of arabinosyl side chains may be responsible for the inability of this polysaccharide to bind cellulose. Since gums exude from tissues, similar inability to bind cell wall polysaccharides due to side chain structure is possible. On the other hand, it has been reported that during the ripening of avocado fruit, water-soluble polyuronide of pectic polysaccharides increased, concomitant with marked downshifts in molecular mass (Huber and O’Donoghue, 1993, Wakabayashi et al., 2000). This result suggests that molecular mass of glucuronoarabinoxylans is related to solubilization of polysaccharides. Since cell wall polysaccharides containing uronic acids were extracted into pectic, HC-I and HC-II fractions (Fig. 5) and analysis of neutral sugar composition of these fractions indicates that heterogenous molecules existed (Fig. 6), while chemical composition of glucuronoarabinoxylans of tulip cell walls has been unclear. In oat coleoptile cell wall, glucoronoarabinoxylan contained about 5–10% glucuronic acid, arabinose in amount almost equal to xylose, and small amount of galactose (Wada and Ray, 1978). The ratio of uronic acid, arabinose and xylose of tulip gums were quite different from that of glucuronoarabinoxylan of oat coleoptiles. Composition of gum polysaccharides were quite different from that of matrix polysaccharides in tulip cell walls (Figs. 2 and 6, Table 3), suggesting that gum is not merely derived from degraded product of cell wall polysaccharides, but rather newly synthesized polysaccharides. Further structure studies of gum polysaccharides in comparison with cell wall glucuronoarabinoxylans will be required from the aspect of sugar composition as well as molecular mass.
Effects of JA-Me on soluble sugars in relation to gum formation JA-Me substantially reduced the amount of glucose, the main component of soluble neutral sugar fraction, and sucrose (Fig. 3). On the contrary, ethephon did not affect the total amount of sugars in soluble sugar fraction and had no influence on soluble neutral sugar composition (Figs. 3 and 4). Nair et al. (1980) reported that degradation products of starch grains and cells seem to contribute to the gum production in Azadirachta indica. These results suggest that changes in sugar metabolism in response to JA-Me seem to contribute to gum formation in tulips,
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Control 80
JA−Me
Chlorophyll retention, %
Ethephon
60
40
20
0
1
3 5 Days after treatment
7
Figure 7. Effect of JA-Me (1%) and ethephon (1%) on chlorophyll degradation in tulip stems. Bars indicate means with standard errors (n ¼ 8; control; n ¼ 5; ethephon; n ¼ 3; JA-Me).
Ethylene
JA−Me
Senescence (loss of chlorophyll)
Sugar metabolism
Gum formation
Figure 8. A possible mode of action of jasmonates and ethylene to induce gummosis in tulip shoots in relation to sugar metabolism.
since the major resource of polysaccharides of gums in tulips might be glucose as well as that of cell walls and starch. Senescence is one of the physiological processes programmed into cell death. Ethylene has been found to promote senescence. Since JA-Me was also first isolated as a senescence-promoting substance (Ueda and Kato, 1981, Ueda et al., 1981), both of them are considered to play an important role in senescence. As shown in Fig. 7, the applications of JA-Me and ethephon substantially enhanced senescence of tulip shoots represented as the loss of
chlorophyll. Since both JA-Me and ethephon substantially reduced the amount of sucrose (Fig. 3), senescence seems to be related to sucrose metabolism. However, JA-Me induced gum formation and decreased the total amount of soluble neutral sugars in the first internode of tulip shoots, but ethephon did not affect both gummosis and soluble neutral sugar levels. These facts together with the results of the total amounts of soluble sugar described above strongly suggest that regulatory mechanisms of JA-Me for gummosis are not associated with senescence. In conclusion jasmonates are essential factors to induce gums in tulip shoots and ethylene also shows synergistic effects on gum formation in the presence of jasmonates. Based on the results in this study, a possible mode of action of jasmonates and ethylene to induce gums in tulip shoots due to changing sugar metabolism is proposed in Fig. 8.
Acknowledgments The authors thank Dr. Hideharu Seto (RIKEN) for kindly providing d2-JA and d2-JA-Me. The authors also thank Prof. Takayuki Hoson, Dr. Kazuhiko Wakabayashi and Dr. Kouichi Soga (Osaka City University) for the use of gel permeation chromatograph for the analysis of molecular mass distribution of tulip gums and invaluable suggestions. This work was supported by a Grant-in-Aid for Scientific
ARTICLE IN PRESS 504 Research to KM (No. 14560030) and JU (No. 03735) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The stay of ES in Japan was supported by the Japan Society for Promotion of Science.
References Albersheim P, Nevins DJ, English PD, Karr A. A method for the analysis of sugars in plant cell wall polysaccharides by gas–liquid chromatography. Carbohydr Res 1967;5:340–5. Boothby D. Gummosis of stone-fruit trees and their fruits. J Sci Food Agric 1983;34:1–7. Darvell A, McNeil M, Albersheim P, Delmer DP. The primary cell walls of flowering plants. Tolbert NE, editor. The biochemistry of plants, vol. 1. New York: Academic Press; 1980. p. 91–162. De Hertogh AA, Dilley DR, Blakely N. Response variation of tulip cultivars to exogenous ethylene. Acta Hort 1980;109:205–10. De Munk WJ, Saniewski M. Gummosis in tulips under the influence of ethephon. Sci Hort 1989;40:153–62. De Wild HPJ, Gude H, Peppelenbos W. Carbon dioxide and ethylene interactions in tulip bulbs. Physiol Plant 2002;114:320–6. Dubois M, Gilles KA, Hamilton JK, Roberts PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350–6. Emery RJN, Reid DM. Methyl jasmonate effects on ethylene synthesis and organ-specific senescence in Helianthus annuus seedlings. Plant Growth Regul 1996;18:213–22. Galambos JT. The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole color of sugars. Anal Biochem 1967;19: 119–32. Huber DJ, O’Donoghue EM. Polyuronides in avocado (Persea Americana) and tomato (Lycopersicon esculentum) fruits exhibit markedly different patterns of molecular weight downshifts during ripening. Plant Physiol 1993;102:473–80. Hung KT, Kao CH. Promotive effect of jasmonates on the senescence of detached maize leaves. Plant Growth Regul 1996;19:77–83. Kamerbeek GA, De Munk WJ. A review of ethylene effects in bulbous plants. Sci Hort 1976;4:101–15. Koiwa H, Bressan RA, Hasegawa PA. Regulation of proteinase inhibitors and plant defense. Trends Plant Sci 1997;2:379–84. Morrison JC, Labavitch JM, Greve CL. The role of ethylene in initiating gum duct formation in almond fruit. J Am Soc Hort Sci 1987;112(2):364–7. Nair MNB, Patel KR, Shah JJ, Pandalai RC. Effect of ethephon (2-chloroethylphosphonic acid) on gummosis in the bark of Azadirachta indica. Indian J Exp Biol 1980;18:500–3. Nishitani K, Masuda Y. Auxin-induced changes in the cell wall structure: changes in the sugar compositions,
E. Skrzypek et al. intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis. Physiol Plant 1981;52:482–94. Olien W, Bukovac MJ. Ethephon-induced gummosis in sour cherry (Prunus cerasus L.) I. Effect on xylem function and shoot water status. Plant Physiol 1982; 70:547–55. Sakurai N, Kuraishi S. Sugar compositions, intrinsic viscosities and molecular weights of hemicellulosic polysaccharides of the coleoptile cell walls in a semibrachytic and a normal type barley. Plant Cell Physiol 1984;25:955–63. Saniewski M. The role of jasmonates in ethylene biosynthesis. In: Kanellis AK, et al. editors. Biology and biotechnology of the plant hormone ethylene. Dordrecht: Kluwer; 1997. p. 39–45. Saniewski M, Miyamoto K, Ueda J. Methyl jasmonate induces gums and stimulates anthocyanin accumulation in peach shoots. J Plant Growth Regul 1998a; 17:121–4. Saniewski M, Miyamoto K, Ueda J. Gum formation by methyl jasmonate in tulip shoots is stimulated by ethylene. J Plant Growth Regul 1998b;17:179–83. Saniewski M, Puchalski J. The induction of gum formation in the leaf, stem and bulb by methyl jasmonate in tulips. Bull Pol Acad Sci Biol Sci 1988;36:35–8. Saniewski M, Ueda J, Horbowicz M, Miyamoto K, Puchalski J. Gum in apricot (Prunus armeniaca L.) shoots induced by methyl jasmonate. Acta Agrobot 2002;54:27–34. Saniewski M, Ueda J, Miyamoto K. Gum induction by methyl jasmonate in tulip stem: relevance to its chemical composition. Acta Hort 2000;515:39–48. Saniewski M, Ueda J, Miyamoto K, Urbanek H. Interactions between ethylene and other plant hormones in regulation of plant growth and development in natural conditions and under abiotic and biotic stresses. In: Vendrell M, et al. editors. Biology and biotechnology of the plant hormone ethylene III. Ohmsha: IOS Press; 2003. p. 263–70. Somogyi M. Notes on sugar determination. J Biol Chem 1952;195:19–23. Ueda J, Kato J. Promotive effect of methyl jasmonate on oat leaf senescence in the light. Z Pflanzenphysiol 1981;103:357–9. Ueda J, Kato J, Yamane H, Takahashi N. Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence. Physiol Plant 1981;52:305–9. Ueda J, Miyamoto K, Hashimoto M. Jasmonates promote abscission in bean petiole explants: its relationship to the metabolism of cell wall polysaccharides and cellulase activity. J Plant Growth Regul 1996;15: 189–95. Ueda J, Miyamoto K, Kamisaka S. Separation of a new type of plant growth regulator, jasmonates, by chromatographic procedures. J Chromatogr 1994;658: 129–42. Ueda J, Miyamoto K, Kamisaka S. Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips segments by jasmonic acid: relevance to its growth inhibition. J Plant Growth Regul 1995;14: 69–76. Ueda J, Miyamoto K, Saniewski M. Gum formation and leaf abscission in ornamental Japanese cherry (Prunus yedoensis): a possible role of ethylene and jasmonates in these processes. In: Vendrell M, et al. editors. Biology and biotechnology of the plant hormone ethylene III. Ohmsha: IOS Press; 2003. p. 263–70. Ueda J, Miyamoto K, Saniewski M, Hoshino T, Utsunomiya M, Okubo H. Chemical compositions of exudates from succulent plants induced by methyl jasmonate. Acta Physiol Plant 2002;24:187–94.
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www.elsevier.de/jplph
Jasmonates are essential factors inducing gummosis in tulips: mode of action of jasmonates focusing on sugar metabolism Edyta Skrzypeka,b, Kensuke Miyamotoa, Marian Saniewskic, Junichi Uedaa, a
College of Integrated Arts and Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan b Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland c Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland Received 18 June 2004; accepted 8 September 2004
KEYWORDS Ethylene; Glucuronoarabinoxylan; Gummosis (gum formation); Jasmonate; Jasmonic acid; Methyl jasmonate; Sugar metabolism; Tulipa gesneriana
Summary The purpose of this study was to know the mechanism of jasmonates to induce gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) shoots, especially on the focus of sugar metabolism. Gummosis in the first internode of tulip plants was induced by the application of methyl jasmonate (JA-Me, 1% w/w in lanolin) and jasmonic acid (JA, 1% w/w in lanolin) 5 days after application and strongly stimulated by the simultaneous application of ethylene-releasing compound, ethephon (2-chloroethylphosphonic acid, 1% w/w in lanolin), although ethephon alone had little effect. JA-Me stimulated ethylene production of the first internodes of tulips, ethylene production increasing up to more than 5 times at day 1 and day 3 after the application. On the other hand, application of ethephon did not increase endogenous levels of jasmonates in tulip stems. Analysis of composition of tulip gums revealed that they were consisted of glucuronoarabinoxylan with an average molecular weight of ca. 700 kDa. JA-Me strongly decreased the total amount of soluble sugars in tulip stems even in 1 day after application, being ca. 50% of initial values 5 days after application, but ethephon did not. However, both JA-Me and ethephon had almost no effect on the neutral sugar compositions of soluble sugars mainly consisting of glucose, mannose and xylose in ratio of 20:2:1 and traces of arabinose. Both
Abbreviations: Ethephon, 2-chloroethylphosphonic acid; JA, jasmonic acid; JA-Me, methyl jasmonate Corresponding author. Tel.: +81 72 254 9733; fax: +81 72 254 9932. E-mail address: [email protected] (J. Ueda). 0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.09.007
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E. Skrzypek et al. JA-Me and ethephon applied exogenously stimulated senescence of tulip shoots shown by the loss of chlorophyll. These results strongly suggest that the essential factor of gummosis in tulips is jasmonates affecting the sugar metabolism in tulip shoots. The mode of action of jasmonates to induce gummosis of tulip shoots is discussed in relation to ethylene production, sugar metabolism and senescence. & 2005 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Gummosis is the process of the accumulation and exudation of gums mainly consisted of polysaccharides including uronic acids in some plants species. The gummosis has been known as a common response to various environmental stresses such as wounding, pathogen infection or insect attack. Gummosis in stone fruit trees has considered to be regulated by ethylene (Nair et al., 1980; Olien and Bukovac, 1982; Boothby, 1983; Morrison et al., 1987). Not only in stone fruit trees but also in tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs, infection of Fusarium oxysporum f. sp. tulipae and application of ethylene or ethylene-releasing compound, ethephon, have been found to induce gums (Kamerbeek and De Munk, 1976; De Hertogh et al., 1980; De Munk and Saniewski, 1989; De Wild et al., 2002). Recently, jasmonic acid (JA), methyl jasmonate (JA-Me) and their related compounds designated as jasmonates have been demonstrated to play an important role in signal transduction pathway in response to stresses, resulting in significant physiological phenomena (Koiwa et al., 1997). Jasmonates have also been found to induce gums in various species of stone fruit trees such as plum, peach, cherry and apricot (Saniewski et al., 1998a, 2002, 2003; Ueda et al., 2003) and in tulips (Saniewski and Puchalski, 1988; Saniewski et al., 1998b, 2000). It is known that ethylene acts together with JA-Me and intensify effect of JA-Me action in gummosis and that both of them are implicated in promotion of senescence (Saniewski et al., 1998b). To understand the mechanism of JA-Me to induce gummosis and their interaction with ethylene in tulips, we investigated the effects of JA-Me and/or ethephon on qualitative and quantitative changes in soluble sugars, endogenous levels of these hormones and senescence in stems of tulip plants as well as sugar composition of gums and cell walls of tulips, and molecular weight of gums. Possible mode of action of jasmonates in inducing gum formation is also discussed.
Plant materials The experiments were performed with the bulbs of tulip (T. gesneriana L. cv. Apeldoorn) from commercial stock. After lifting, the bulbs with a circumference of 10–12 cm stored at 18–20 1C were cooled at 5 1C for a minimum 12 weeks. After removal to the tunics, tulip bulbs were planted in pots individually and cultivated at the temperature 17–20 1C under natural daylight conditions in a greenhouse. Tulip plants at the age of ca. 3–5 days before flowering were used. JA-Me (1% w/w) and/ or ethephon (1% w/w) were applied as a ring (2–3 mm in width) of lanolin paste to the first internode of stem of intact tulips. After appropriate incubation gum formation was observed optically, then the 1st internode segments prepared from the plants were subjected for various analyses. A lot of 3–8 plants were used in each treatment.
Analysis of soluble sugars Soluble sugars in the first internode segments of tulips were extracted with 80% aqueous EtOH. The amounts of total and reducing sugars were determined by phenol–sulfuric (Dubois et al., 1956) and Somogyi–Nelson methods (Somogyi, 1952), respectively. The amounts of sucrose were estimated by the changes in reducing sugars before and after invertase treatments. The amounts of uronic acids were also determined by carbazole–sulfuric acid method (Galambos, 1967). Soluble neutral sugars together with inositol as an internal standard were reduced with sodium borohydride and acetylated with acetic anhydride in the presence of N-methylimidazole. Qualitative and quantitative analysis of acetylated sugars were determined with a gas–liquid chromatography according to the method reported previously (Ueda et al., 1995, 2002).
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Gel permeation chromatography
Determination of jasmonates
Molecular mass of tulip gums was estimated by a gel permeation chromatography according to the method by Wakabayashi et al. (1997). Tulip gums were dissolved in potassium phosphate buffer (50 mM, pH 7.2). A portion of the solution (ca. 300 mL, approximately 0.3 mg Glc equivalent) was subjected to a high performance liquid chromatograph (LC-6A, Shimadzu Co. Ltd., Kyoto, Japan) equipped with a gel-permeation column (TSK-gel G5000PW, Tosoh Co. Ltd., Tokyo, Japan) and a refractive index detector (RID-6A, Shimadzu Co. Ltd., Kyoto, Japan). The sample was eluted with the same buffer at a flow rate of 1 mL/min. Fractions of 0.5 mL were collected with a fraction collector (Model 203, Gilson, Middleton, WI, USA). The elution pattern was monitored by a refractive index detector. The contents of total sugars and uronic acids in each fraction were determined by phenol-sulfuric method and carbazole-sulfuric acid method, respectively. The weight-average molecular mass of gums was calculated from the equation reported by Nishitani and Masuda (1981). Dextrans (Sigma) of 10, 40, 70, 120 and 500 kDa were used as molecular mass markers. After hydrolysis of polysaccharides in each fraction with 2 N trifluoroacetic acid and acetylation (Albersheim et al., 1967), neutral sugar composition of gums was determined by a gas-chromatography as mentioned above.
First internode segments prepared from tulips treated with or without ethephon were extracted twice with 80% aqueous EtOH at 4 1C. Alcoholic extracts were concentrated in vacuo to give an aqueous residue. Deuterium-labeled JA (d2-JA) and JA-Me (d2-JA-Me) were added to the aqueous residue as internal standards, then followed by partitioning with diethyl ether. After partitioning with diethyl ether at pH 7, the aqueous phase was adjusted to pH 3 with HCl and then partitioned with diethyl ether in the usual way, giving the diethyl ether-soluble neutral and acidic materials, respectively. These materials were further purified with silica gel thin-layer chromatography according to the method reported previously (Ueda et al., 1994). Both fractions eluted from zones corresponding to authentic JA and JA-Me in silica gel thin-layer chromatograms were methylated with ethereal diazomethane. Qualitative and quantitative analyses of JA and JA-Me were carried out using a Finnigan GCQ gas–liquid chromatography–mass spectrometry according to the same procedure reported previously (Ueda et al., 1991). Experiments were carried out twice and results represented as the total amounts of JA plus JA-Me were expressed an average of two independent experiments. Quite similar results were obtained in these experiments.
Cell wall polysaccharides
Ethylene production
Analyses of cell wall polysaccharides were carried out according to the method by Nishitani and Masuda (1981). Methanol-killed segments of stems and leaves of tulips were homogenized. The homogenates were sufficiently washed with cold water, acetone and then methanol-chloroform, successively. The homogenates were treated with a-amylase for 3 h at 37 1C to remove starch and then centrifuged. The pellet was extracted with ammonium oxalate, then centrifuged. The supernatant contained pectic polysaccharides. The pellet residue was extracted with 4% KOH and then 24% KOH, each supernatant giving hemicellulose I (HC-I) and hemicellulose II (HC-II) fractions, respectively. The residue was recognized as an acellulosic fraction. The amount of polysaccharides in each fraction was determined by phenol–sulfuric acid method and carbazole-sulfuric acid method. Neutral sugar compositions of pectic, HC-I and HC-II polysaccharides were determined by a gas–liquid chromatography after hydrolysis and acetylation as mentioned above.
First internode segment (2 cm in length) prepared from tulips treated with or without JA-Me was incubated in a 20-mL beaker. Each beaker was sealed with Parafilm, and then incubated at 25 1C in the dark. After 2-h incubation, 1 mL of the gas from the headspace of the beaker was sampled. Determination of ethylene production was carried out according to the method previously reported (Ueda et al., 1996) using a Hitachi 163 gas–liquid chromatograph equipped with a hydrogen flame ionizing detector. A glass column (5 mm 2 m) packed with Porapak N was used.
Chlorophyll content The total amounts of chlorophyll of first internode segment (2 cm in length) prepared from tulips treated with or without JA-Me in the presence or absence of ethephon was extracted with 10 mL of 80% aqueous EtOH overnight. Absorbance of the extracts was measured at 665 nm using a Shimadzu
ARTICLE IN PRESS 498 UV-1200 spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan).
Results Gum formation The application of lanolin alone to the first internode of intact tulip plants had no effect on gum formation during 7-day incubation. On the other hand, the application of JA-Me at 1% (w/w in lanolin) to the first internode of intact tulip plants induced gum formation 5 days after application, gums being exuded in the place of the application on the first internode and infiltrated into the basal part of the first leaf (Fig. 1) as reported previously (Saniewski et al., 1998b, 2000). Similar to JA-Me, the application of JA also induced gum formation in tulip shoots, activities of JA-Me and JA to induce gum formation in tulip shoots being almost same. Application of ethephon together with JA-Me strikingly stimulated gum formation, while ethephon alone had little effect, suggesting that jasmonates but not ethylene play an essential role for gum formation in tulip shoots (all data not shown).
Effect of JA-Me on ethylene, and vise versa To better understand the mechanism of interaction of jasmontes and ethylene on gum formation, effect of JA-Me on ethylene production in tulip stems, and that of ethephon on endogenous levels of jasmonates were examined. Lanolin alone had no effect on ethylene production in tulip stems as
E. Skrzypek et al. shown in Table 1. On the other hand, the application of JA-Me significantly increased ethylene production. The magnitude of ethylene production was observed during first 3 days after the application of JA-Me, the level being almost 5 times higher than that of control. During the following days, the production of ethylene decreased gradually but it was still higher in comparison with control level. In this study, we have confirmed the presence of JA and JA-Me in tulip stems using GC-MS (data not shown) and estimated the endogenous level of jasmonates by deuterium-labeled JA and JA-Me as internal standards. The content of jasmonates designated as JA plus JA-Me in tulip stems increased after treatment of lanolin only and ethephon until day 5, then decreased at day 7. The application of ethephon (1% w/w in lanolin) did not increase the endogenous level of jasmonates in the internode of tulips in comparison with control one (Table 2).
Molecular mass of tulip gums and their sugar composition Fig. 2 shows molecular mass distribution of tulip gums analyzed by a gel permeation chromatography. Tulip gums were consisted of almost homogeneous polysaccharides with an average molecular mass of ca. 700 kDa. The ratio of neutral sugars and uronic acids was ca. 2.5:1. Analysis of neutral sugar composition of tulip gums revealed that arabinose and xylose existed at the ratio of ca. 2: 3 (Table 3). These results suggest that tulip gums are consisted of glucuronoarabinoxylan with an average molecular mass of ca. 700 kDa.
Figure 1. Gum exuded on the first internode (left) and infiltrated in the basal part of leaf (right) in tulips in response to simultaneous application of JA-Me and ethephon. Photographs were taken 5 days after the application of JA-Me (1% w/ w in lanolin) and ethephon (1% w/w in lanolin).
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips Table 1.
499
Effect of JA-Me on ethylene production in the first internodes of tulips
Treatment
Ethylene production, nl/g/h Days after treatment
Control (lanolin alone) JA-Me
1
3
5
7
1.1770.03 5.4370.59
0.7270.03 5.2170.32
1.2670.09 3.0570.35
0.7570.02 1.1470.11
After appropriate incubation of intact tulips treated with or without JA-Me (1% w/w in lanolin), the first internode segment (2 cm in length) was prepared and subjected to determination of ethylene production. Results were indicated as the average with standard errors (n ¼ 8; control; n ¼ 3; JA-Me).
Table 2.
Effect of ethephon on endogenous level of jasmonates in tulip stems
Treatment
Jasmonate content, ng/g FW Days after treatment
Control (lanolin alone) Ethephon
1
3
5
7
53.8 31.1
86.0 72.6
75.3 82.8
62.9 64.6
After appropriate incubation of intact tulips treated with or without ethephon (1% w/w in lanolin), first internode segments (2 cm in length) were prepared and subjected to determination of endogenous levels of JA and JA-Me. Results are expressed as jasmonates, the sum of JA and JA-Me, and mean of two individual experiments.
Distribution of polysaccharides or uronic acids, %
V
0
500
70
Effects of JA-Me and ethephon on soluble sugars
10
16 12 8 4 0
Relative amounts
60 Total sugars Uronic acids 40
20
0 10
15 20 Elution volume, ml
25
Figure 2. Molecular mass distribution of tulip gums. The amounts of total sugars and uronic acids in each fraction (0.5 mL) obtained by the gel permeation chromatography (see Materials and methods) were determined by the phenol–sulfuric acid method and the carbazole-sulfuric acid method, respectively. The elution position of molecular mass standards (dextrans of 500, 70 and 10 kDa) and the void volume (V0) are shown at the top.
To understand the regulatory role of JA-Me on gum formation, effects of JA-Me on the soluble sugars as substrates for polysaccharide synthesis was examined in comparison with that of ethephon, since tulip gums demonstrated to be mainly consisted of polysaccharide, glucuronoarabinoxylans (Fig. 2 and Table 3). The total amounts of soluble sugars estimated by the phenol–sulfuric acid method were almost equivalent to those of reducing sugars estimated by the Somogyi–Nelson method in soluble sugar fractions after the treatment of invertase, suggesting that polysaccharides exist little in soluble sugar fraction. Changes in the amounts of reducing sugars before and after invertase treatments determined by the Somogyi–Nelson method showed that almost all soluble sugars were reducing sugars while only small amount of sucrose existed (Fig. 3). As shown in Fig. 3, the level of sucrose and soluble reducing sugars in tulip stem segments prepared from the tulip plants treated with lanolin alone were almost constant during the incubation for 7 days. On the other hand, JA-Me strongly reduced the amounts of reducing sugars and sucrose in the first internodes of tulips even in 1
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E. Skrzypek et al.
Table 3.
Neutral sugar composition of tulip gums
Fractions, elution volume (mL)
Neutral sugar composition, %
15.0–16.5
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
nd
nd
39.574.0
60.574.0
nd
nd
nd
Reducing sugars, mg/g FW
Sucrose, mg/g FW
Neutral sugars in the fractions (elution volume 15.0–15.5, 15.5–16.0, 16.0–16.5) corresponding to the peak of chromatogram were analyzed by a gas–liquid chromatography after hydrolysis and acetylation. Values are expressed as the average with standard errors (n ¼ 3). nd indicates no sugar detected. Elution volume is same as in Fig. 2.
day after the application. The total amounts of reducing sugars in tulip stem treated with JA-Me were gradually reduced, it being ca. 35% at the end of the experiment of day 7 in comparison with control (untreated plants) value. On the other hand, ethephon had almost no effect on the amount of soluble reducing sugars, but interestingly it reduced the amount of sucrose. These results suggest that JA-Me affects soluble sugar composition qualitatively and quantitatively. Figure 4 shows the neutral sugar composition of soluble reducing sugars of the internodes of tulips. Neutral sugars in soluble reducing sugar fraction of the first internode of tulips treated with lanolin alone consisted of mainly glucose, mannose and xylose in the ratio 20:2:1, and traces of arabinose. Both JA-Me and ethephon little affected the neutral sugar composition.
20 Controls
JA-Me
Ethephon
10
0 60
40
20
0
1
3 5 Days after treatment
7
Figure 3. Effect of JA-Me (1%) and ethephon (1%) on sucrose level and reducing sugar content in tulip stems. Bars indicate means with standard errors (n ¼ 8, control; n ¼ 5, ethephon; n ¼ 3; JA-Me). The amount of sucrose was estimated by the changes in reducing sugars before and after invertase treatments.
Cell wall polysaccharide of tulips Glucuronoarabinoxylan has been demonstrated to be one of constituents of cell wall polysaccharides. In order to compare the sugar compositions between cell wall polysaccharides and gums, cell
100 Control JA-Me Neutral sugar composition, %
80
Ethephon
60
40
20 ND
ND
Rha
Fuc
ND
0 Ara
Xyl
Man
Gal
Glc
Figure 4. Effect of JA-Me (1%) and ethephon (1%) on neutral sugar composition of soluble sugar fraction in tulip stems. Bars indicate means with standard errors (n ¼ 8; control; n ¼ 5; ethephon; n ¼ 3; JA-Me).
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips wall polysaccharides were fractionated into pectic, HC-I and HC-II, and cellulosic polysaccharides. Cell wall polysaccharides in stems were mainly consisted of cellulose, and small amount of pectic, HCI and HC-II polysaccharides, while the ratio of cellulose in leaves was smaller than that of stems (Fig. 5). As shown in neutral sugar composition of polysaccharides in pectic, HC-I and HC-II fractions, quite heterogeneous polysaccharides existed in
100 Leaves Stems
Polysaccharide composition, %
80
60
40
Cellulose
HC−II:−NS
HC−I: UA
HC−I: NS
Pectin: UA
Pectin: NS
0
HC−II: UA
20
Figure 5. Polysaccharide composition of tulip cell walls. After fractionation into pectic, HC-I, HC-II and cellulosic polysaccharides, the amounts of neutral sugars and uronic acids were determine.
60
each fraction (Fig. 6). This suggests that matrix cell wall polysaccharides differently from gums are consisted of various kinds of polysaccharides.
Discussion Interaction of JA-Me and ethylene on gum formation in tulips The application of JA-Me to the first internode of tulip stems strongly induced gum formation (Fig. 1). The application of JA also induced gum formation in tulip shoots. Activity of JA to induce gummosis in tulip shoots was almost equivalent to that of JA-Me. On the other hand, the application of ethephon, ethylene-generating substance, induced no gums in tulip stems although JA-Me strongly promoted ethylene production which might be induced by wounding just after excision of the first internode of tulip stems. These results indicate that jasmonates, but not ethylene, play an essential role for gum formation in tulip shoots. Since simultaneous application of JA-Me and ethephon stimulated gum formation in tulip shoots compared to the case of JA-Me alone, it is possible to say that ethylene is really involved in gum formation in tulip. JA-Me has been reported to stimulate ACC synthase and ACC oxidase activities and/or ethylene production in various plant species (Saniewski, 1997). In tulip stems, biosynthesis of ethylene was also substantially affected by the application of JA-Me (Table 1) probably by stimulating of ACC synthase and ACC oxidase activities as
HC − I
Pectin
501
HC − II
Leaves
Neutral sugar composition, %
Stems
40
20
0
Ara
Rha Fuc
Man Xyl
Glc Gal
Rha
Ara Fuc
Man Glc Xyl Gal
Rha
Ara Fuc
Man Xyl
Glc Gal
Figure 6. Neutral sugar composition of pectic, HC-I and HC-II polysaccharides of tulip cell walls. After hydrolysis and acetylation, neutral sugar composition was determined by a gas–liquid chromatography.
ARTICLE IN PRESS 502 suggested above. This result also suggests that JAMe applied exogenously regulates gum formation by interacting with endogenous ethylene induced by JA-Me itself. On the contrary to the application of JA-Me, the application of ethephon (1% w/w in lanolin) did not affect the endogenous level of jasmonates designated as the total amounts of JA and JA-Me in the internode of tulips (Table 2), suggesting that the synergistic effect of ethylene on gum formation is not mediated by the increase in endogenous level of jasmonates. The mode of action of ethylene to stimulate gum formation in tulip shoots in the presence of JA-Me has not been clear yet. It is probable that there are an interaction and/or a cross-talk in signal transduction pathways between jasmonates and ethylene. Hung and Kao (1996) reported that ethylene increases susceptibility to JA-Me on the process of jasmonates-promoting senescence in detached maize leaves. Similar explanation will be possible on the process of gummosis in tulip shoots. Emery and Reid (1996) have also reported that simultaneous application of JA-Me and ACC caused dramatic degradation of cell membrane as indicated by an increase in conductivity in sunflower seedlings and neither the application of ACC alone nor that of JA-Me with ethylene inhibitors evoked such an increase in conductivity. This fact also raises the possibility that ethylene together with or without JA-Me stimulates exudation process of gums by inducing loosening of cell membranes.
Chemical composition of tulip gums We have previously suggested that tulip gums consisted of glucuronoarabinoxylans by analyses of uronic acid and neutral sugar contents and the neutral sugar composition after direct hydrolysis with trifluoroacetic acid (Saniewski et al., 2000). As shown in the molecular mass distribution (Fig. 2) and neutral sugar composition (Table 3) tulip gums are suggested to be homogenous polysaccharides consisted of glucuronoarbinoxylan with an average molecular weight of ca. 700 kDa, while uronic acids have not identified yet. Glucuronoarabinoxylan is well known as one of the components of cell wall polysaccharides (Darvell et al., 1980), and in barley coleoptiles, gel permeation chromatography showed that polysaccharides composed of Ara and Xyl were at least two species with different Ara/Xyl ratios and different molecular weight (Sakurai and Kuraishi, 1984). Glucuronoarabinoxylans of cell wall polysaccharides isolated from oat coleoptiles did not bind to
E. Skrzypek et al. cellulose in vitro (Wada and Ray, 1978), being possible that steric hindrance by a high percentage of arabinosyl side chains may be responsible for the inability of this polysaccharide to bind cellulose. Since gums exude from tissues, similar inability to bind cell wall polysaccharides due to side chain structure is possible. On the other hand, it has been reported that during the ripening of avocado fruit, water-soluble polyuronide of pectic polysaccharides increased, concomitant with marked downshifts in molecular mass (Huber and O’Donoghue, 1993, Wakabayashi et al., 2000). This result suggests that molecular mass of glucuronoarabinoxylans is related to solubilization of polysaccharides. Since cell wall polysaccharides containing uronic acids were extracted into pectic, HC-I and HC-II fractions (Fig. 5) and analysis of neutral sugar composition of these fractions indicates that heterogenous molecules existed (Fig. 6), while chemical composition of glucuronoarabinoxylans of tulip cell walls has been unclear. In oat coleoptile cell wall, glucoronoarabinoxylan contained about 5–10% glucuronic acid, arabinose in amount almost equal to xylose, and small amount of galactose (Wada and Ray, 1978). The ratio of uronic acid, arabinose and xylose of tulip gums were quite different from that of glucuronoarabinoxylan of oat coleoptiles. Composition of gum polysaccharides were quite different from that of matrix polysaccharides in tulip cell walls (Figs. 2 and 6, Table 3), suggesting that gum is not merely derived from degraded product of cell wall polysaccharides, but rather newly synthesized polysaccharides. Further structure studies of gum polysaccharides in comparison with cell wall glucuronoarabinoxylans will be required from the aspect of sugar composition as well as molecular mass.
Effects of JA-Me on soluble sugars in relation to gum formation JA-Me substantially reduced the amount of glucose, the main component of soluble neutral sugar fraction, and sucrose (Fig. 3). On the contrary, ethephon did not affect the total amount of sugars in soluble sugar fraction and had no influence on soluble neutral sugar composition (Figs. 3 and 4). Nair et al. (1980) reported that degradation products of starch grains and cells seem to contribute to the gum production in Azadirachta indica. These results suggest that changes in sugar metabolism in response to JA-Me seem to contribute to gum formation in tulips,
ARTICLE IN PRESS Jasmonates are essential factors inducing gummosis in tulips
503
Control 80
JA−Me
Chlorophyll retention, %
Ethephon
60
40
20
0
1
3 5 Days after treatment
7
Figure 7. Effect of JA-Me (1%) and ethephon (1%) on chlorophyll degradation in tulip stems. Bars indicate means with standard errors (n ¼ 8; control; n ¼ 5; ethephon; n ¼ 3; JA-Me).
Ethylene
JA−Me
Senescence (loss of chlorophyll)
Sugar metabolism
Gum formation
Figure 8. A possible mode of action of jasmonates and ethylene to induce gummosis in tulip shoots in relation to sugar metabolism.
since the major resource of polysaccharides of gums in tulips might be glucose as well as that of cell walls and starch. Senescence is one of the physiological processes programmed into cell death. Ethylene has been found to promote senescence. Since JA-Me was also first isolated as a senescence-promoting substance (Ueda and Kato, 1981, Ueda et al., 1981), both of them are considered to play an important role in senescence. As shown in Fig. 7, the applications of JA-Me and ethephon substantially enhanced senescence of tulip shoots represented as the loss of
chlorophyll. Since both JA-Me and ethephon substantially reduced the amount of sucrose (Fig. 3), senescence seems to be related to sucrose metabolism. However, JA-Me induced gum formation and decreased the total amount of soluble neutral sugars in the first internode of tulip shoots, but ethephon did not affect both gummosis and soluble neutral sugar levels. These facts together with the results of the total amounts of soluble sugar described above strongly suggest that regulatory mechanisms of JA-Me for gummosis are not associated with senescence. In conclusion jasmonates are essential factors to induce gums in tulip shoots and ethylene also shows synergistic effects on gum formation in the presence of jasmonates. Based on the results in this study, a possible mode of action of jasmonates and ethylene to induce gums in tulip shoots due to changing sugar metabolism is proposed in Fig. 8.
Acknowledgments The authors thank Dr. Hideharu Seto (RIKEN) for kindly providing d2-JA and d2-JA-Me. The authors also thank Prof. Takayuki Hoson, Dr. Kazuhiko Wakabayashi and Dr. Kouichi Soga (Osaka City University) for the use of gel permeation chromatograph for the analysis of molecular mass distribution of tulip gums and invaluable suggestions. This work was supported by a Grant-in-Aid for Scientific
ARTICLE IN PRESS 504 Research to KM (No. 14560030) and JU (No. 03735) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The stay of ES in Japan was supported by the Japan Society for Promotion of Science.
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