Gummosis in grape hyacinth bulbs,hormonal rugulation and chemical composition of gums

REGULAR PAPER

Gummosis in grape hyacinth (Muscari armeniacum )bulbs:hormonal regulation and chemical composition of gums

Kensuke Miyamoto ?Toshihisa Kotake ?Makiko Sasamoto ?Marian Saniewski ?Junichi Ueda

Received:5December 2008/Accepted:14October 2009/Published online:26November 2009óThe Botanical Society of Japan and Springer 2009

Abstract The purpose of this study was to investigate the hormonal regulation of gummosis in grape hyacinth (Muscari armeniacum )bulbs,focusing especially on the chemical composition of the gums.The application of ethephon (2-chloroethylphosphonic acid),an ethylene-releasing compound,at 1%and 2%(w/w)in lanolin as well as ethylene induced gummosis in the bulbs within several days.Methyl jasmonate (JA-Me,0.1–2%in lanolin)alone had no effect on gummosis.However,simultaneous

application of JA-Me and ethephon led to extreme stimu-lation of ethephon-induced gummosis.Ethephon-induced gummosis in the bulbs depended on the maturation stage of the bulbs,increasing from April to July,but decreasing from August to September.Regardless of the presence of JA-Me,the application of ethephon to the in?orescence axis of grape hyacinths did not induce gummosis.Gel permeation chromatography analysis revealed that gums were homogenous polysaccharides with an average molecular mass of ca.8.3kDa.Analysis of the sugar composition of the gums after hydrolysis revealed that the molar ratio of Rha:Ara:Gal:GalA:GlcA was 25:10:40:7:15.These results suggest that principal factors of gummosis as well as the chemical composition of gums differ between species of bulbous plants.

Keywords Ethephon (2-chloroethylphosphonic acid)áEthylene áGrape hyacinth (Muscari armeniacum )áGummosis áMethyl jasmonate áSugar composition Abbreviations

HPAEC-PAD High performance anion-exchange

chromatography with pulsed amperometric detection

JA Jasmonic acid JA-Me Methyl jasmonate JAs Jasmonates

Introduction

Gummosis,the process of the accumulation and exudation of gums (which are complexes of different substances but

This manuscript is dedicated to the memory of the late Dr.Alicja Saniewska.

K.Miyamoto (&)

Faculty of Liberal Arts and Sciences,

Osaka Prefecture University,1-1Gakuen-cho,Naka-ku,Sakai,Osaka 599-8531,Japan e-mail:miyamoto@las.osakafu-u.ac.jp

K.Miyamoto áJ.Ueda

Graduate School of Science,Osaka Prefecture University,1-1Gakuen-cho,Naka-ku,Sakai,Osaka 599-8531,Japan T.Kotake

Graduate School of Science and Engineering,Saitama University,255Shimo-okubo,Sakura-ku,Saitama 338-8570,Japan

M.Sasamoto

Faculty of Science,Osaka Women’s University,

1-1Gakuen-cho,Naka-ku,Sakai,Osaka 599-8531,Japan M.Saniewski

Research Institute of Pomology and Floriculture,Pomologiczna 18,96-100Skierniewice,Poland

M.Saniewski

Botanical Garden-Centre for Biological Diversity Conservation,Polish Academy of Sciences,Prawdziwka 2,02-973Warszawa,Poland

J Plant Res (2010)123:363–370DOI 10.1007/s10265-009-0273-1

with most constituents being polysaccharides),is known to be a common response to various environmental stresses such as wounding,pathogen infection or insect attack in some plant species(Boothby1983).Although the physiological role of gums in plants is not clearly understood,it is believed that gums function to limit the spread of fungal and bacterial pathogens and insects by isolating infected and infested tissues from healthy tissue. Gums may also have a protective role in particular in preventing entry of pathogens and insects to injured tis-sues and preventing moisture loss from damaged tissues (Olien and Bukovac1982;Boothby1983).Thus,forma-tion of gums,as a defense reaction following pathogen infection or insect infestation is a positive process for plants.On the other hand,various gums are used practically by man in different areas(Verbeken et al. 2003).Some gums,such as gum arabic(wound exudate from Acacia senegal),gum tragacanth(Asiatic Astragalus spp.)and gum karaya(Sterculia spp.),are permitted for pharmaceutical and food use(Anderson and Weiping 1990).

Hormonal regulation of gummosis has been studied intensively in stone fruit trees and fruits of the family Rosaceae(Olien and Bukovac1982;Boothby1983; Morrison et al.1987),mesquite(Greenwood and Morey 1979;Orozco-Villafuerte et al.2003),Citrus(Gedalovich and Fahn1985),Azadirachta indica(Nair et al.1980) and others,suggesting that ethylene is an essential factor to induce gummosis.As well as ethylene,jasmonic acid (JA)and methyl jasmonate(JA-Me)designated as jasmonates(JAs)have been demonstrated to play an important role in signal transduction pathways in response to stresses,resulting in signi?cant physiological phenomena(Koiwa et al.1997).JAs also induce gum-mosis and interact with ethylene in gummosis in various species of stone fruit trees such as plum,peach,cherry and apricot(Saniewski et al.1998a,2002,2003;Ueda et al.2003).

In bulbous plants such as tulip(Tulipa gesneriana), grape hyacinth(Muscari armeniacum)and Narcissus, gummosis also takes place(Moore1949;Rees1972, Miyamoto et al.2008).In tulip bulbs,infection with Fusarium oxysporum f.sp.tulipae and the application of ethylene(Kamerbeek and De Munk1976;De Hertogh et al.1980;De Munk and Saniewski1989;De Wild et al.2002)or JAs(Saniewski and Puchalski1988; Saniewski et al.1998b,2000;Skrzypek et al.2005a,b,c) led to the production of large quantities of gums,sug-gesting that pathogen-induced ethylene or JAs in plant tissues,or production of these compounds by pathogens is involved in gummosis(for a review,see Saniewski et al.2007).On the other hand,in shoots of tulips,it has been reported that JAs induced gummosis but ethylene did not,whereas ethylene synergistically stimulated gummosis induced by JAs,suggesting that JAs are essential or principal factors inducing gummosis in tulip shoots(Saniewski and Puchalski1988;Skrzypek et al. 2005a,b,c).

The hormonal regulation of gummosis in other bulbous plants is much less studied.To investigate the hormonal regulation of gummosis in other bulbous plants,the effects of exogenous application of these plant hormones on gummosis in grape hyacinth(Muscari armeniacum)were studied here,focusing on the interactions of these hor-mones.Furthermore,to gain insight into the metabolism relating to gummosis,the chemical composition of gums was also studied.

Materials and methods

Plant materials

Bulbs and in?orescence axes of grape hyacinth(Muscari armeniacum)plants were subjected to treatment with plant hormones.Between April and September,grape hyacinth bulbs with a circumference of4–5cm were dug out from ?eld,and treated with ethephon(2-chloroethylphosphonic acid,1%and2%,w/w in lanolin)and/or JA-Me(0.1%, 0.5%,1%and2%,w/w in lanolin)as a lanolin paste(ca. 350mg)around the scale near the basal plate of the bulb.The treated bulbs were kept in room conditions at 17–22°C.

In addition,ethephon and/or JA-Me were applied as a ring(2–3mm in width)of lanolin paste(ca30mg)to the upper part of the in?orescence axis of intact grape hyacinth plants grown in an experimental?eld.These experiments were performed in2005,2006and2007.The treatment to the in?orescence axis was also carried out after lifting the bulbs from the experimental?eld to the laboratory condi-tions.These treated plants were incubated in100-ml beakers containing an appropriate volume of distilled water under continuous white?orescent light conditions(ca. 5W/m2)at23°C.

At7–10days after treatment,gummosis on the treated bulbs and plants was assessed visually and photographed. In some cases gums exuded onto the surface of the bulbs by the plant hormone treatment were collected and dried, before being weighed.Batches of25bulbs or5plants were used for each treatment.

Sugar and protein analyses of gums in grape hyacinth bulbs

Gums were dissolved in hot water,and the solution was then centrifuged at3,000g for10min.Almost all of the

gums were recovered in the supernatant.The contents of total sugars and uronic acids in grape hyacinth gums were determined by the phenol–sulfuric acid method(Dubois et al.1956)using glucose(Glc)as a standard,and the carbazole–sulfuric acid method using glucuronic acid (GlcA)as a standard(Galambos1967),respectively.The concentration of proteins in grape hyacinth gums was determined using the Bio-Rad Protein Assay(Bio-Rad, Hercules,CA)using bovine serum albumin(BSA)as a standard.

The molecular mass of gums in bulbs was estimated by gel permeation chromatography according to the method of Wakabayashi et al.(1997).Gums produced by the application of ethephon were dissolved in distilled hot water.A portion of the solution(approximately Glc equivalent to0.5mg)was subjected to a Shimadzu LC-6A high performance liquid chromatography(Shimadzu, Kyoto,Japan)equipped with a refractive index detector (RID-6A,Shimadzu).A Tosoh TSK-gel G5000PW gel-permeation column(Tosoh,Tokyo,Japan)was used.The sample was eluted with potassium phosphate buffer (50mM,pH7.2)at a?ow rate of1ml/min.Fractions (0.5ml volume)of the sample were collected using a fraction collector(Model203,Gilson,Middleton,WI). The contents of total sugars and uronic acids in each fraction were determined by the phenol–sulfuric acid method(Dubois et al.1956)and the carbazole–sulfuric acid method(Galambos1967),respectively.The weight-average molecular mass of gums was calculated from the equation reported by Nishitani and Masuda(1981).Dex-trans(Sigma)of4,10,40,70,120and500kDa were used as molecular mass markers.The polysaccharide fractions corresponding to the peak were collected.After hydrolysis of polysaccharides in the fractions with2N tri?uoroacetic acid(TFA),soluble neutral sugars together with inositol as an internal standard were reduced with sodium borohy-dride and acetylated with acetic anhydride(Albersheim et al.1967).Qualitative and quantitative analyses of alditol acetates were carried out using a Shimadzu GC-14 gas–liquid chromatograph?tted with a hydrogen?ame ion detector(Shimadzu)according to the method reported previously(Skrzypek et al.2005c).

Sugar composition of hydrolyzed grape hyacinth gums was analyzed by high performance anion-exchange chro-matography with pulsed amperometric detection(HPAEC-PAD)using a Dionex DX-500liquid chromatograph?tted with a CarboPac PA-1column(49250mm,Dionex Japan,Osaka,Japan)as described by Ishikawa et al.(2000) and Konishi et al.(2008).A portion of the gum solution dissolved in distilled hot water was air-dried,then sub-jected to acid hydrolysis with2N TFA at121°C for1h. The samples were air-dried,then subjected to HPAEC-PAD analysis.Results and discussion

Hormonal regulation of gummosis in grape hyacinth bulbs

As well as in gummosis in tulips,gummosis in grape hyacinth—one of the important?oricultural bulbous plants—has also been reported(Moore1949;Rees1972, Miyamoto et al.2008).However,hormonal regulation of gummosis in grape hyacinth remains unclear.As shown in Fig.1a and Table1,the application of ethephon,an eth-ylene-releasing compound,at1%and2%(w/w)in lanolin to the basal plate of bulbs induced gummosis within several days after application,gums being exuded around the basal plate,the place of the application,whereas application of lanolin alone to the bulbs had no effect on gummosis.The application of gaseous ethylene at0.001and0.01%(v/v) was also found to induce gummosis in grape hyacinth bulbs (data not

shown).

Control Ethephon 1%JA Me 1%

+

a

Ethephon 1%Ethephon 2

%

JA Me 1%JA Me 2

% Control

b

Fig.1Effects of ethephon and/or methyl-jasmonate(JA-Me)on gummosis in grape hyacinth bulbs.a Grape hyacinth bulbs were treated around the scale near the basal plate with lanolin paste supplemented with ethephon or JA-Me at concentrations of1%and 2%(w/w)in lanolin.b Grape hyacinth bulbs were also treated with ethephon(1%,w/w)together with JA-Me(1%,w/w).The treated bulbs were kept in room conditions at17–22°C.The bulbs were photographed after incubation for7–10days.These experiments were carried out in July

On the other hand,JA-Me applied alone at concentra-tions of0.1%to2%(w/w)in lanolin had no effect on gummosis in grape hyacinth bulbs(Fig.1a,Table1). Simultaneous application of JA-Me and ethephon led to extreme stimulation of gummosis compared to ethephon treatment alone(Fig.1b,Table1).This simultaneous application also induced browning of the bulbs,whereas ethephon or JA alone induced little browning of the bulbs.

In tulip bulbs,application of ethylene and/or JAs indu-ces gummosis,indicating that gummosis is under the reg-ulation of these plant hormones as suggested previously (Saniewski et al.2000,2007;Skrzypek et al.2005a,b,c). Furthermore,in tulip shoots,JAs induced gummosis but ethephon did not,suggesting that JAs are essential factors in gummosis induction(Skrzypek et al.2005a,b,c).In contrast to tulip shoots,JA-Me applied alone had no effect on gummosis(Fig.1a;Tables1,2);however,simultaneous application of ethylene and JA-Me resulted in substantial induction of gummosis in grape hyacinth bulbs.Based on that fact,it is possible to say that JAs and ethylene are indeed involved in gummosis in grape hyacinth bulbs, although the principal factor inducing gummosis is thought to differ from that of tulips.

The mode of action of JAs in stimulating gum formation in grape hyacinth bulbs in the presence of ethylene remains as yet unclear.JA-Me has been reported to stimulate 1-aminocyclopropane-1-carboxylic acid(ACC)synthase and ACC oxidase activities and/or ethylene production in vari-ous plant species(Emery and Reid1996;Saniewski1997). Also in tulip plants,JA-Me stimulates the evolution of ethylene and the activity of ACC oxidase during gum ind-uction(Saniewski and We?grzynowicz-Lesiak1994,1995;

Table1Effects of ethephon and/or methyl-jasmonate(JA-Me)on gummosis in grape hyacinth plants

Gummosis Control Ethephon JA-Me Ethephon1%?JA-Me

1%2%0.1%0.5%1%2%0.1%0.5%1%2%

Bulbs-a???----??????????????? In?orescence axis----------–

Formation of gum was observed visually7–10days after treatment

a-No gummosis,?to????increasing degrees of gum production

Table2Effects of ethephon on gum production in different stages of grape hyacinth,starting from13April(?owering time in?eld)until late September

Treatment time Gum production

(Exp.1)

Gum production,dry weight mg/bulb(Exp.2)a

Ethephon1%Ethephon

2%

Ethephon2%?JA-Me

0.1%

Ethephon2%?JA-Me

0.5%

Ethephon

2%?JA-Me1%

13April No gums0===

18May=b0.162===

28May=0.161===

30May=0.169===

4June=0.125a0.216b0.178ab0.137a

15June=0.179===

3July=0.049a0.199b0.152b0.092a

20July Gums====

29July Gums====

6August Gums0.086a0.165b0.173b0.172b

31August=====

2September Traces====

5September=0.039a0.093b0.091b0.148c

22September Traces====

After appropriate incubation,gums formed were optically observed(Exp.1),or collected and weighed(Exp.2).Since no gum production after treatment with or without JA-Me in these terms,these results were not shown in the table

a In Exp.2,to clarify the effect of simultaneous application of these plant hormones,data were subjected to an analysis of variance and evaluated using Duncan’s multiple range test.Ten bulbs per treatment were used.Lower case letters(a,

b and c)indicate mean separation in each treatment by Duncan’s multiple range test at5%signi?cance

b Experiment was not carried out at this treatment time

Skrzypek et al.2005c).It is possible that the level of endogenous ethylene induced by JAs is too low to induce gummosis,as suggested in ornamental Japanese cherry shoots(Ueda et al.2003),although the effect of JAs on ethylene production in grape hyacinth bulbs has not yet been determined.

Since simultaneous application of JA-Me led to extreme stimulation of ethephon-induced gummosis(Fig.1b, Table1),it is probable that there is an interaction and/or cross-talk in signal transduction pathways between ethyl-ene and JAs in gummosis.As suggested above,JAs show a synergistic effect in ways other than through stimulation of ethylene production.Hung and Kao(1996)reported that ethylene increases susceptibility to JA-Me on the process of JAs-promoted senescence in detached maize leaves.The converse explanation,i.e.,that JAs increase susceptibility and/or responsiveness to ethylene,could possibly explain the process of ethylene-inducing gummosis in grape hya-cinth bulbs.

In tulip shoots,JA-Me affects sugar metabolism sub-stantially,resulting in the reduction of soluble sugars such as sucrose and reducing sugars(Skrzypek et al.2005a,c). Emery and Reid(1996)reported that simultaneous appli-cation of ACC and JA-Me caused a dramatic degradation of cell membranes,as indicated by an increase in conductivity in sun?ower seedlings.JAs also potentially play an impor-tant role in gummosis triggered by ethylene through modi-fying sugar metabolism or stimulate the exudation process of gums by inducing loosening of the cell membrane.Fur-ther studies on these processes will be required. Susceptibility and/or responsiveness of organs

to ethylene and/or JA-Me in induction of gummosis

in grape hyacinth plants

In tulips,gummosis has also been observed in the stem after treatment with JA-Me as described above(Saniewski and Puchalski1988;Skrzypek et al.2005a,b,c).Since only JA-Me was effective in inducing gummosis in tulip shoots,JAs but not ethylene were proposed as the essential factor to induce gummosis(Skrzypek et al.2005a,b,c).In contrast to the bulbs,in the in?orescence axis of grape hyacinths neither the application of ethephon nor the simultaneous application of ethephon and JA-Me induced gummosis(Table1).This result suggests that the ability of organs to induce gummosis differs in grape hyacinth plants and tulips.

Seasonal variations in ethephon-induced gummosis

in grape hyacinth bulbs

In tulip bulbs,seasonal variation in the ability of JA-Me and/or ethephon to induce gums has been demonstrated (Saniewski et al.2004,2007).Seasonal variation in ethe-phon-induced gummosis in grape hyacinth bulbs was studied.As shown in Table2,ethephon-induced gummosis in grape hyacinth bulbs was not observed in April.How-ever,from mid-May to June,gummosis in response to the application of ethephon was found to increase with the advance of the season.Thereafter,ethephon-induced gummosis was reduced.Similar seasonal variation in induced gummosis was also observed in response to simultaneous application of JA-Me and ethephon.Devel-opmental stages are connected with?ower bud forma-tion—a process that ends at the end of August—then maturation of grape hyacinth bulbs starts.These results suggest that the susceptibility and/or responsiveness of plant tissues,which are closely related to developmental stages or maturation of bulbs,to ethephon and/or JA are important for gummosis in grape hyacinth bulbs.

As described above,susceptibility and/or responsiveness to ethephon,in the presence or absence of JA-Me,to induce gummosis differed at different growth stages of grape hyacinth bulbs.In tulip bulbs,the ability of JA-Me and/or ethephon to induce gums was also found to exhibit seasonal variation(Saniewski et al.2004,2007),and JA-Me-induced gummosis was suggested to be related to sugar metabolism,since JA-Me affects the level of soluble sugars (Skrzypek et al.2005a,c).These results suggest that the susceptibility and/or responsiveness of plant tissues to ethephon and/or JAs that are important for gummosis in grape hyacinth bulbs are closely related to developmental stages or maturation of bulbs.Changes in the quality and/or quantity of storage materials such as carbohydrates,as well as changes in endogenous hormones in the bulbs during growth and development,are possibly involved in sus-ceptibility to ethylene.Further studies from the viewpoint of endogenous hormonal status or storage sugars will be required to clarify the seasonal variation of gummosis. Molecular mass of grape hyacinth gums and their sugar composition

The molecular mass distribution of grape hyacinth gums was analyzed by gel permeation chromatography.Grape hyacinth gums consisted of almost homogenous polysac-charides with an average molecular mass of8.3kDa, since the peak of distributions of total sugars accorded with that of uronic acids(Fig.2).After hydrolysis of polysaccharides in fractions corresponding to the peak, sugars were reduced,acetylated and subjected to analysis of neutral sugar composition using gas–liquid chroma-tography.Analysis of the neutral sugar composition of grape hyacinth gums revealed that grape hyacinth gums were rich in Rha,Gal and Ara as neutral sugars(data not shown).

In order to identify uronic acids as well as neutral sug-ars,grape hyacinth gums hydrolyzed with2N TFA at 121°C for1h were subjected to analysis by HPAEC-PAD (Fig.3).As shown in Table3,the molar ratio of Rha: Ara:Gal:GalA:GlcA was25:10:40:7:15.The neutral sugar composition as determined by HPAEC-PAD was almost the same as that in the fractions corresponding to the peak of gel permeation chromatography(data not shown).Grape hyacinth gums also contained proteins,the ratio of sugars to proteins being ca.94:6.

The chemical composition of the exuded gums demon-strated that the gums of stone-fruit trees are complex branched hetero-polysaccharides comprising residues of Gal,Ara and GlcA,with other sugars also present in small or trace quantities(Boothby1983).Those of mesquite were demonstrated to consist mainly of?ve principal compo-nents varying in molecular mass from35to940kDa,with GlcA,Gal and Ara being the major constituents(Orozco-Villafuerte et al.2003).On the other hand,analysis by gel permeation chromatography revealed that gums of grape hyacinths(Fig.2)and tulips(Skrzypek et al.2005c)were almost homogenous polysaccharides from the point of view of molecular mass,although their average molecular mass were quite different.

While grape hyacinth gums were polysaccharides with a weight-average molecular mass of ca.8.3kDa(Fig.2), HPAEC-PAD analysis of the sugar composition of gums revealed the molar ratio of Rha:Ara:Gal:GalA:GlcA to be 25:10:40:7:15.Our previous study demonstrated gums of tulips to consist of Ara,Xyl and uronic acids with an average molecular mass of ca.700kDa,suggesting that tulip gums are glucuronoarabinoxylans(Skrzypek et al. 2005c).Based on the different sugar compositions and molecular mass distribution of the different gums,sugar metabolism relating to gummosis is quite different in dif-ferent species of bulbous plants.

As a pectic polysaccharide,rhamnogalacturonan has been demonstrated to contain Rha and GalA as a backbone with additional side chains of Ara and Gal(Darvill et al. 1980).Vinod et al.(2008)reported that gums of Cochlo-spermum gossypium consist mainly of Rha,Gal and uronic acids,suggesting a rhamnogalacturonan-type polysaccha-ride.On the other hand,gums from Leucaena glabrata and Spondia purpurea have been demonstrated to consist of Gal,Ara,and GalA at a ratio of25:60:12and61:17:22, respectively,and to have good solubility in water(Perez et al.1995).Gums from Combretum paniculatum were also demonstrated to contain Gal,Ara,Rha,Man,GlcA and Me-GlcA at a ratio of31:29:17:4:14(Anderson and Wei-ping1990).Arabinogalactan-proteins(AGPs)are charac-terized by large carbohydrate components rich in Gal and Ara,and protein components whose content is generally less than10%of total weight(Fincher et al.1983).The carbohydrate moieties of AGPs have been demonstrated to consist mainly of Gal and Ara,although other monosac-charides that can be present include Rha(up to11%),Man (up to16%),Xyl(up to7%),Glc(up to4%),GlcA and its methyl derivatives(up to28%),and GalA and its methyl derivatives(up to26%;Fincher et al.1983).The glycosyl composition of AGPs in gum arabic—the wound exudate from Acacia senegal—has been demonstrated to comprise Rha,Ara,Gal and GlcA at a ratio of ca.10:26:40:9(Qi et al.1991)or ca.10:36:36:9(Goodrum et al.2000

).

Osman et al.(1995)reported that gum arabic can been eluted in several fractions by anion-exchange chromatog-raphy,and that the carbohydrate composition of all frac-tions remained relatively constant with each containing similar proportions of Rha(12–16%),Ara(22–27%),Gal (32–42%),and GlcA(9–20%).Grape hyacinth gums are rich in Rha,Ara,Gal and GlcA,and contain protein components at ca.6%;however,they are also rich in GalA as described above(Table3).The relatively high content of Rha,Ara,Gal and GlcA suggests that grape hyacinth gums consist of AGPs whose uronic acids are generally GlcA and its derivatives(Fincher et al.1983),but in this experiment we cannot exclude the possibility that the gum also contains rhamnogalacturonan,which is generally rich in Rha,Gal and GalA.To identify the gum molecules, further study including sugar linkage analysis,Yariv-reactivity,and enzymatic digestion would be required.

In conclusion,gums of grape hyacinth consist of com-plex polysaccharides rich in uronic acids with an average molecular mass of8.3kDa including Rha,Ara,Gal,GalA and GlcA(at a molar ratio of ca.25:10:40:7:15).Gum-mosis in bulbous plants is regulated by the interaction of ethylene and JAs,but the principal factor of gummosis in bulbous plants differs in different plant species.The prin-cipal factor of gummosis in grape hyacinths is ethylene rather than JAs.Gums formed in grape hyacinth bulbs also differ from gums in tulip shoots in their chemical charac-teristics such as molecular mass and sugar composition. Acknowledgments The authors thank Prof.Takayuki Hoson and Dr.Kouichi Soga(Osaka City University)for use of the gel perme-ation chromatograph for the analysis of molecular mass distribution of grape hyacinth gums,and for their invaluable suggestions.This work was supported partially by a Grant-in-Aid for Scienti?c Research from the Ministry of Education,Culture,Sports,Science and Technology,Japan,and a Special Research Grant from Osaka Prefecture University to K.M.

References

Albersheim O,Nevins DJ,English PD,Karr A(1967)A method for the analysis of sugars in plant cell wall polysaccharides by gas–liquid chromatography.Carbohydr Res5:340–345

Anderson DMW,Weiping W(1990)Composition of the gum from Combretum paniculatum and other gums which are not permitted food additives.Phytochemistry29:1193–1195Boothby D(1983)Gummosis of stone-fruit trees and their fruits.J Sci Food Agric34:1–7

Darvill A,McNeil M,Albershim P,Delmer DP(1980)The primary cell walls of?owering plants.In:Tolbert NE(ed)The biochemistry of plants,vol1.Academic,New York,pp91–162 De Hertogh AA,Dilley DR,Blakely N(1980)Response variation of tulip cultivars to exogenous ethylene.Acta Hortic109:205–210 De Munk W,Saniewski M(1989)Gummosis in tulips under the in?uence of ethephon.Sci Hortic40:153–162

De Wild HPJ,Gude H,Peppelenbos W(2002)Carbon dioxide and ethylene interactions in tulip bulbs.Physiol Plant114:320–326 Dubois M,Gilles KA,Hamilton JK,Roberts PA,Smith F(1956) Colorimetric method for determination of sugars and related substances.Anal Chem28:350–356

Emery RJN,Reid DM(1996)Methyl jasmonate effects on ethylene synthesis and organ-speci?c senescence in Helianthus annuus seedlings.Plant Growth Regul18:213–222

Fincher GB,Stone BA,Clarke AE(1983)Arabinogalactan-proteins: structure,biosynthesis,and function.Annu Rev Plant Physiol 34:47–70

Galambos JT(1967)The reaction of carbazole with carbohydrates.I.

Effect of borate and sulfamate on the carbazole color of sugars.

Anal Biochem19:119–132

Gedalovich E,Fahn A(1985)Ethylene and gum duct formation in citrus.Ann Bot56:571–577

Goodrum LJ,Patel A,Leykam JF,Kieliszewski MJ(2000)Gum arabic glycoprotein contains glycomodules of both extension and arabinogalactan-glycoproteins.Phytochemistry54:99–106 Greenwood C,Morey P(1979)Gummosis in honey mesquite.Bot Gaz140:32–38

Hung KT,Kao CH(1996)Promotive effect of jasmonates on the senescence of detached maize leaves.Plant Growth Regul 19:77–83

Ishikawa M,Kuroyama H,Takeuchi Y,Tsumuraya Y(2000) Characterization of pectin methyltransferase from soybean hypocotyls.Planta210:782–791

Kamerbeek GA,De Munk WJ(1976)A review of ethylene effects in bulbous plants.Sci Hortic4:101–115

Koiwa H,Bressan RA,Hasegawa PA(1997)Regulation of proteinase inhibitors and plant defense.Trends Plant Sci2:379–384 Konishi T,Kotake T,Soraya D,Matsuoka K,Koyama T,Kaneko S, Igarashi K,Samejima M,Tsumuraya Y(2008)Properties of family79b-glucuronidases that hydrolyze b-glucuronosyl and 4-O-methyl-b-glucuronosyl residues of arabinogalactan-protein.

Carbohydr Res343:1191–1201

Miyamoto K,Sasamoto M,Saniewski M,Ueda J(2008)Interaction of ethylene and methyl jasmonates on gummosis in grape hyacinth bulbs(Muscari armeniacum):relevance to chemical composition of gums.In:Abstracts of the10th International symposium on?ower bulbs and herbaceous perennials.p106 Moore WC(1949)Diseases of bulbs.Ministry of Agriculture and Fisheries Bulletin117,His Majesty’s Stationery Of?ce,London Morrison JC,Labavitch JM,Greve CL(1987)The role of ethylene in initiating gum duct formation in almond fruit.J Am Soc Hortic Sci112:364–367

Table3Sugar composition of grape hyacinth gums analyzed by high performance anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD)after hydrolysis

Sugar composition(mol%)

Fuc Rha Ara Gal Glc Man Xyl GalA GlcA

1.9±0.225.7±0.510.1±1.139.8±1.0ND a ND0.4±0.37.1±0.414.9±0.4

Data are means±standard error(n=3)

a Not detected

Nair MNB,Patel KR,Shah JJ,Pandalai RC(1980)Effect of ethephon (2-chloroethylphosphonic acid)on gummosis in the bark of Azadirachta indica.Indian J Exp Biol18:500–503

Nishitani K,Masuda Y(1981)Auxin-induced changes in the cell wall structure:changes in the sugar composition,intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyls cell wall of Vigna angularis.Physiol Plant52:482–494

Olien W,Bukovac MJ(1982)Ethephon-induced gummosis in sour cherry(Prunus cerasus L.)I.Effect on xylem function and shoot water status.Plant Physiol70:547–555

Orozco-Villafuerte J,Cruz-Sosa F,Ponce-Alquicira E,Vernon-Carter EJ(2003)Mesquite gum:fraction and characterization of the gum exuded from Prosopis laevigata obtained from plant tissue culture and from wild trees.Carbohydr Polym54(3):327–333 Osman ME,Menziez AR,Martin BA,Williams PA,Phillips GO, Baldwin TC(1995)Characterization of gum Arabic fractions obtained by anion-exchange chromatography.Phytochemistry 38:409–417

Perez GRM,Sanchez AJ,Perez GS,Vargas SR(1995)An analytical study of gums from Leucaena globrata and Spondia purpurea.

J Sci Food Agric68:39–41

Qi W,Fong C,Lamport DTA(1991)Gum arabic glycoprotein is a twisted hairly rope.A new model based on O-galactosylhydrox-yproline as the polysaccharide attachment site.Plant Physiol 96:848–855

Rees AR(1972)Physiological disorders.I Gummosis.In:Rees AR (ed)The growth of bulbs.Applied aspects of the physiology of ornamental bulbous crop plants.Academic,London,pp245–246 Saniewski M(1997)The role of jasmonates in ethylene biosynthesis.

In:Kanellis AK et al(eds)Biology and biotechnology of the plant hormone ethylene.Kluwer,Dordrecht,pp39–45 Saniewski M,Puchalski J(1988)The induction of gum formation in the leaf,stem and bulb by methyl jasmonate in tulips.Bull Pol Acad Sci Biol Sci36:35–38

Saniewski M,We?grzynowicz-Lesiak E(1994)Is ethylene responsible for gum formation induced by methyl jasmonate in tulip stem?

J Fruit Ornam Plant Res2:79–80

Saniewski M,We?grzynowicz-Lesiak E(1995)The role of ethylene in methyl jasmonate-induced gum formation in stem of tulips.Acta Hortic394:305–313

Saniewski M,Miyamoto K,Ueda J(1998a)Methyl jasmonate induces gums and stimulates anthocyanin accumulation in peach shoots.J Plant Growth Regul17:121–124

Saniewski M,Miyamoto K,Ueda J(1998b)Gum formation by methyl jasmonate in tulip shoots is stimulated by ethylene.

J Plant Growth Regul17:179–183Saniewski M,Ueda J,Miyamoto K(2000)Gum induction by methyl jasmonate in tulip stem:relevance to its chemical composition.

Acta Hortic515:39–48

Saniewski M,Ueda J,Horbowicz M,Miyamoto K,Puchalski J(2002) Gum in apricot(Prunus armeniaca L.)shoots induced by methyl jasmonate.Acta Agrobot54:27–34

Saniewski M,Ueda J,Miyamoto K,Urbanek H(2003)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(eds)Biology and biotechnology of the plant hormone ethylene III.IOS,Amster-dam,pp263–270.ISBN1586033468

Saniewski M,Ueda J,Miyamoto K,Okubo H,Puchalski J(2004) Interaction of methyl jasmonate and ethephon in gum formation in tulip bulbs.J Fac Agric Kyushu Univ49:207–215 Saniewski M,Miyamoto K,Okubo H,Saniewska A,Puchalski J, Ueda J(2007)Gummosis in tulip(Tulipa gesneriana L.):focus on hormonal regulation and carbohydrate metabolism.Floric Ornam Biotechnol1:34–40

Skrzypek E,Miyamoto K,Saniewski M,Higuchi N,Ueda J(2005a) Gum formation in tulips affected by methyl jasmonate requires changes in sugar metabolism.Acta Hortic673:215–222 Skrzypek E,Miyamoto K,Saniewski M,Ueda J(2005b)Identi?ca-tion of jasmonic acid and its methyl ester as gum-inducing factors in tulips.J Plant Res118:27–30

Skrzypek E,Miyamoto K,Saniewski M,Ueda J(2005c)Jasmonates are essential factors inducing gummosis in tulips:mode of action of jasmonates focusing on sugar metabolism.J Plant Physiol 162:495–505

Ueda J,Miyamoto K,Saniewski M(2003)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(eds)Biology and biotechnology of the plant hormone ethylene III.IOS,Amsterdam,pp263–270.ISBN1 586033468

Verbeken D,Dierckx S,Devettinck K(2003)Exudate gums: occurrence,production,and applications.Appl Microbiol Bio-technol63:10–21

Vinod VT,Sashidhar RB,Sarma VU,Vijaya Saradhi UV(2008) Compositional analysis and rheological properties of gum kondagogu(Cochlospermum gossypium):a tree gum from India.

J Agric Food Chem56:2199–2207

Wakabayashi K,Hoson T,Kamisaka S(1997)Suppression of cell wall stiffening along coleoptiles of wheat(Triticum aesativum L.)seedlings grown under osmotic stress conditions.J Plant Res 110:311–316