鈣與植物乙烯反應的關(guān)系研究

植物學(xué)報Acta Botanica Sinica 2002 ,44( 4):422 - 426Relationship Between Ca2 + and Plant Ethylene ResponseZHANG Yan-Ping , ZHU Ben-Zhong , LUO Yun-Bo'( Cllge of Food Sciences ,China Agriculural Uniersty , Bejing 1004 , China )Abstract : The relationship between Ca2 + and ethylene response was investigated through analyzing the effectofCa+ on the response to ethylene in etiolated tomato ( Lycopersicon esculentum Mill cv. Lichun ) seedlinggrown in darkness .When the etiolated tomato seedlings were treated with different concentrations of Ca2+ , thetriple response" phenotype , ethylene production , the expression of ethylene receptor gene NEVER- RIPE( NR ) and the content of cytosolic CaM were determined. With the concentration of Ca2+ in the culture medi-um increasing from 0 mmol/L to 3.8 mmol/L , the triple response" phenotype of etiolated tomato seedling wascorrespondingly strengthened ; meanwhile the ethylene production , the amount of NR gene expression and theconcentration of CaM increased respectively. However , when the concentration of Ca- + was increased from 3.8 mmol/L to 10 mmol/L , the phenotype of° triple response" , ethylene production , NR gene expression , andthe CaM content didn' t increase further , but decreased consequently. The results indicated that the effect ofCa2 + on the ethylene triple response in etiolated tomato seedling was relevant to ethylene biosynthesis and ethy-lene receptor gene expression which were influenced by applied Ca2+ , and these effects might be mediatedthrough the change of CaM concentration in plant cell.Key words : Ca2+ ; triple response ; ethylene signal transduction ; CaMCytosolic Ca2 + is a well-known and important second .andtreatedwithwaterat55C-60Cfor15min，andmessenger in plant cell signal transductiort11 , and it isallowed to imbibe in water at room temperature for 10 h ,involved in a wide range of physiological and biochemicalthen the seeds were surface sterilized with 70% ethanolprocesses in plant growth and development 2] , includingfor5 min and with 1% AgNO3 for 25 min，rinsed threeits role on the physiological action of ethylene. For exam-times with sterilized water. Sterilized tomato seeds wereple, Ca2 + regulates the plant pathogen-related gene ex-spread onto MS agar medium( MS salt except Ca2+ ,2%pression through influencing ethylene signal transductionsucrose ,0. 8% agar at pH5.8 ) with different concentra-pathwayCa2+ promotes the ethylene production intion of CaCl2 including 0 mmol/L, 0. 1 mmol/L, 1tomato fruit 41 , pea hypocotyt] and apple tissue 61 , andmmol/L ,3. 8 mmol/L and 10 mmol/L. The MS mediumplays a role in ACO gene expression regulated by ethylenewith 0 mmol/L Ca2+ was used as the control and the 3.8in pea seedling 77. However , the relationship betweenmmol/L Ca2+ was the normal concentration in MS medi-Ca2 + and ethylene response remains unclear and there isum. The tested plants were divided into two groups , instill no new research data to elucidate the pathway thatone group the MS medium was supplemented with 1. 5μL/L of ethephon , a chemical ethylene releaser , and noCa4+ influences the plant ethylene response." Triple re-ethephon in the other group. The seeds were germinatedsponse”, a typical method for investigating ethylene reandgrewinadarkincubatorat25Cfor10d,andthesponse of dicotyleon plants , refers to the inhibition ofseedlings were used for the determination of ethylene re-hypocotyl elongation , swelling of seedling hypocotyl andsponse .exaggeratied curvature of apical hook of the seedling1.2 Measurement of" triple response" indexgrown in darkness and in the presence of ethylene8. InThe length of the hypocotyl of etiolated tomatothis study , we investigated the relationship of Ca2 + andseedling was measured with a vernier caliper，the width ofethylene response by analyzing the change of phenotype ofhypocotyl was measured with a vermier caliper for diameterethylene' triple response' ' , ethylene release , ethylene re-of hypocotyl and the curvation of apical hook was mea-ceptor gene expression and the concentration of cytosolicsured with a protractor .CaM in dark-grown tomato seedling treated with different1.3Determination of ethyleneconcentration of exogenous Ca2+ .A 100 μL gas sample was withdrawn from the sealedcontainer with a hypodermic syringe and ethylene was as-1 Materials and Methodssayed中國煤化工graph equipped with a3-1.1 Plant materialsmeterJYHCNMHGionization detector. ThePlump tomato ( Lycopersicon esculentum Mill cv.carrier gas ao T2 al u. u ng/ .m^the rate of flow 0Lichun) seeds in uniform size and shape were selectedburning gas H2 was 50 mL/ min , column temperature 75Received : 2001-03-26 Accepted : 2001-12-19Supported by the丹物理Basic Research and Development Plan of China( G199901 1701 ) and National OUutstanding Youth Fund of China( 39825118 ).* Author for orepondance. E-mail: < yunbol @ public3. bta. net. cn>ZHANG Yan-Ping et al : Relationship Between Ca2 + and Plant Ethylene Response423C , detection temperature 130 C. The determination ofethylene from each container was repeated three times .1.4 RNA isolation and Northern blot hybridizationTotal RNA from 10-day-old dark-grown etiolatedtomato seedling was isolated and analyzed with Northernblot according to Zhang et at 9]. The probe for radiolabeland hybridization was 690 bp NR gene fragment amplifiedwith a pair of primers ( 5' CCGAATTCTCTTTGGGAC-GAAACGAGATA3'，5'CC GGTACCATTTGTATTGCTT-236CAGGGCTA3' ), which was radiolabeled using RandomPrimer DNA Labeling Kit Ver. 2 from Japanese TaKaRaFig.1.company . The relative expression level was indicated withsponse" of tomato seedlings in MS media without extended ethyleneapplication in the dark .the ratio of the amount of NR gene expression in treated1- 6 represented that the concentration of Ca2+ was0,0.1.1,seedling to that in seedling without Ca2+ treatment in the3.8 , 10 , 100 mmol/L , respectively.absence of ethephon.1.5 The extract and assay of CaMWhen 1.5 μL/L ethephon was applied in MS cultureTwo grams of etiolated tomato seedling were homoge-triple responseof tomato etiolatednized in a twofold volumes ( W/V ) of extracted bufferseedling also presented markedly strengthening tendencycontaining 10 mmol/L imidazole , 1 mmol/L ethylenegly-with Ca2 + concentration increasing in MS medium becol bis ( 2-aminoethyl-ether ) tetraacetic acid( EGTA ),1 .tween 0- 3.8 mmol/L similar to that of seedling grown inmmol/L β-mercaptoethanol ,10 mmol/L MgCl2 and 0. 15the absence of applied ethephon( Fig.2 ). Similarly themmol/L NaCl ( pH 7. 5 ), and placed into centrifugetriple response” was not further strengthened when thetube , incubated at 95 C - 100 C for 3 min and cooledconcentration of Ca2 + was increased up to 10 mmol/L ,in cold water quickly ，followed by centrifugation atwhich was also identical with that of seedlings grown in10 000g for 30 min. The supermatent was collected andthe absence of ethephon .determined for CaM concentration by competitive ELISA2 Results2.1Effect of Ca2 + on ethylene" triple response"In the absence of ethylene , the growth of tomatoseedling in darkness on MS medium without Ca2+ was ex-5tremely inhibited( Fig. 1 and Table 1 ), indicating thatCa2+ was necessary for tomato seedling grown in dark-Fig.2. Efct of different concentration of Ca2+ on the”triple re-sponse" of tomato seedlings in MS media in the presence of ethep-nessThe phenotype of" triple response" of tomato etiolat-hone 1.5 pμL/L ethephon in the dark.ed seedling was markedly strengthened with Ca2 + concen-1- 6 represented that the concentration of Ca2+ was 0 ,0.1，1，tration increasing in the culture medium from 0.1 mmol/L3.8 , 10，100 mmol/L，respectively.to 3.8 mmol/L，including inhibition of hypocotyl elonga-2.2 Efect of Ca2+ on ethylene production of etiolat-tion , swelling of hypocotyl and increasing of curvature ofapical hook( Fig.1 and Table 1 ). However , the pheno-ed seedling of tomatoThe amount of ethylene released by etiolated seedlingtype of" triple response" of etiolated tomato seedling wasof tomato increased markedly when the concentration ofnot further strengthened with the increasing concentrationof Ca2+ in the medium up to 10 mmol/L , the phenotypeCa2+ increased between 0 - 3.8 mmol/L whether ethep-of" triple response” was weaker than that of seedlinghon was added or not , and decreased with the increase ofCa2+ concentration in the medium( Fig.3 ). These results .grown in the medium with 3.8 mmol/L Ca2 +Table 1 Efect of Ca2 + on ethylene induced triple response" in tomato seedlings- Ethylene+ Ethylene .Content of Ca2+Length .WidthHook angle(mmoLI)( cm)(0)中國煤化工(°)00.728+0.008 0.065+4.43x10-3 10.000+ 0.000MYH0003116. .090+ 24. 5850.110.325+2.100 0.077+8.14x 10- s10. 303土3.780CNM H Go003124.333土21.1661.03.634士0.7660. 107+0.0002 74.545 土20.4001.716士0.0800.211 +0.000 1136. 515土15. I953.82. 131 +0.2530. 190+0.0001152 .638土18.600426土0.0500.225+0.000 1162. .421 士13.51410.02.287 +0.1850. 179 +0.000 03146.757+ 15. 1001.443士+0.0410.213+0.0014 158 .969土11.1550+00+0.Thirty-two seedlings in each treatment were used for quantification. Value indicated the mean士standard error of 3 replicates. 0士0 indicated the seeds did notgeminate. Seli產(chǎn)數據10-d-old at the time of measurement.424植物學(xué)報Acta Botanica Sinica Vol.44 No.4 2002production and ethylene receptor gene expression with dif-6口 Aplied m ethylemeferent concentration of Ca- +5 |■Applied ethylene18s rRNA名2。4.000.11.03.810.0 100. 0Ca” (mol/L)王2.52. 0E 1.5Fig.3. Ethylene production of tomato seedlings treated with differ-L.0ent Ca4+ concentration in the presence or absence of non-exogenousethylene.indicated that Ca2+ in the culture medium at concentra-tions between 0- 3.8 mmol/L promoted the production ofFig.4. Effect of diferent Ca2 + concentration on the expression ofethylene by tomato seedling grown in the dark.NR and the relative RNA level in the tomato seedlings in the ab-2.3 Effect of Ca2+ on the expression of ethylene re-sence of exogenous ethylene .Total RNA was isolated from 10- day-old dark- grown seedlings andceptor gene10 1g of total RNA were used for Northerm blot. The Ca2 + concen-The expression of ethylene receptor gene NR in etio-lated seedling of tomato was present in the condition of 0 ,trationof 1 ,2 ,3 ,4 ,5 were 0 ,0.1 ,1 ,3.8 , 10 mmol/L , re-spectively.0.1 ,1 ,3.8 and 10 mmol/L Ca2+ , respectively ( Fig.4 ). With concentration of Ca2 + increased from 0 mmol/L12( control )to 3.8 mmol/L , the relative amount of expres-10口A(yíng)pplled no ethy lene■Applled ethylenesion of NR gene correspondingly increased from 1 to3.5458 , and the amount of NR gene expression with 1mmol/L Ca2 + added to the medium was twice as much asthat with 0.1 mmol/L Ca2+ . But when the concentrationd dddof Ca2+ was increased to 10 mmol/L, NR gene expres-3.810.0sion decreased to 2.803 0 ,still 2.8 fold increase of theca”(mmo1/L)control. These data indicated that expression of NR genein etiolated seedling of tomato was obviously affected byFig.5. Concentration of CaM of tomato seedlings treated with dif-diferent concentration of added Ca2+ , and the tendencyferent concentration of Ca2+ in the presence and absence of exoge-of change was consistent with the effect of" triple re-nous ethylene.sponse'2.4 Change of CaM concentration in the presence of3 DiscussionCa2+and ethyleneThe change of CaM content with different concentra-It is well-known that Ca2+ plays a vital role in thetion of Ca2+ and ethylene was shown in Fig. 5. Withoutphysiological processes in plant growth and develop-the ethephon and cytosolic CaM content in seedlings in-mentEthylene , one of the five important plant hor-creased continuously when Ca4+ in MS medium increasedmones，also is involved in extensive physiological re-from 0 mmol/L to 3. 8 mmol/L. However the concentra-sponses in plant development 11]. However , there are stilltion of CaM began to decrease when Ca2+ concentrationfew reports about the direct regulation of Ca2 + on ethyleneincreased from 3. 8 mmol/L to 10 mmol/L. This changephysiology and response. Previous studies showed thatindicated that the change of CaM content was not correlat-Ca2+ played an important role in the accumulation ofed with that of cytosolic Ca2 + ,but the tendency of changeethylene- inducible pathogensis-related gene expressiorfwas similar to ethylene production( Fig. 3 ) and NR geneand in Su-Hwan s experiment 71，expression of ACO geneexpression( Fig. 4 ). When etiolated seedling of tomatoinducible by ethylene was influenced with different con-was grown in MS medium with 1.5 μL/L ethephon , thecentra中國煤化工helator , suggesting thatCaM content was higher than that without ethephon at dif-|YHCNM H Gthylene. In this study ,ferent Ca2 + concentrations in the medium. But the patternwe treated ne enolatea tomato seedling with a series ofof change was the same , i. e. the content of CaM in-concentration of Ca2+ from 0 to 10 mmol/L in the MScreased when Ca2 + concentration increased from0 to 3.8medium ,and all led to the strengthening of the phenotypemmol/L and began to decrease when Ca + concentrationof ethylene triple response , including inhibition of elonga-increased to 10 mmol/L. Also , this pattern of change wastion of hypocotyl , swelling of the hypocotyl and exaggerat-present inr' thes PHenotype of triple response , ethyleneed curvature of the apical hook. All these data indicatedZHANG Yan-Ping et al : Relationship Between Ca2 + and Plant Ethylene Response425that Ca2+ affected the function of ethylene and the re-CaM content. However ，there was still no informationsponse of plant to ethylene .about the effect of ethylene on the change of cytosolicResponse of the plant to ethylene is brought aboutCaM content. In our experiment ，the change pattern ofthrough the combination of biosynthesis and perception ofethylene”triple response" , ethylene production , ethyleneethylene and the pathway of signal transduction. It is stillreceptor gene NR expression after Ca4+ treatment wasunclear whether Ca2 + influences the ethylene responseidentical to that of cytosolic CaM content , implying thatthrough regulating the ethylene synthesis pathway or ethy-the effect of Ca2+ on the” triple response" of etiolatedlene signal transduction pathway ，or both. Burns andtomato seedling might be related to cytosolic CaM. Mean-Evensen' s 6J study indicated that ethylene production waswhile , increase of cytosolic CaM content and strengthen-promoted in mungbean hypocotyl treated with 0.1 mmol/Ling of etiolated tomato seedling' triple response" engenderCa2+ , and Fergusort 12 J found that ethylene production inwith exogenous ethylene treatment ( Fig.5 and Table 1 )cucumber cytoledons decreased when treated with 1indicated that ethylene alike to other plant hormones un-mmol/L Ca+. In our study，we found that ethylene pro-dertook physiological role as well as improved the cytosolicduction in etiolated tomato seedling all increased in differ-CaM content.ent extent treated with different concentration of appliedIn conclusion , exogenous Ca2+ brought about theCa2+ , suggesting that Ca2 * treatment could promote ethy-change of" triple response" , ethylene release , ethyleneene production in etiolated tomato seedling. About thereceptor gene expression and cytosolic CaM content in eti-relationship of Ca2 + and ethylene signal transduction , Razolated tomato seedling. However , the mechanism of Ca2 +and Fluht 3] discovered that Ca2+ was involved in ethy-on ethylene biosynthesis and somewhere in the pathway oflene action and signal transduction which regulated theethylene signal transduction still need further investiga-tion .expression of pathogen-related gene expression , and Su-Hwart 77 found that Ca2 * might regulate the ethylene sig-Acknowledgements: We thank Prof. SUN Da-Ye andnal transduction pathway in pea. Our results showedProf. BAI Juan in Hebei Normal University for technicalchange of expression of ethylene receptor NR gene wasassistance and determination of CaM.brought about by Ca2treatment , indicating that Ca2 +might influence ethylene perception and signal transduc-References :tion pathway in etiolated tomato seedling.In the condition of 10 mmol/L Ca2+ , the ethylene[ 1 ] Bush D S. Calcium regulation in plant cells and its role insigalling. Annu Rev Plant Plysiol Plant Mol Biol, 1995 ,production decreased in detached cucumber leaf in Fergu-46:95- 122.:on' s experiment 121，and ethylene production in pea[2] LeeJS, Mulkey TJ, Evans M L. Reversible loss of grav-hypocotyl increased along with the increase of Ca2 + con-itropic sensitivity in maize roots after tip application of calci-centratiort51. In this study , all concentrations of Ca2 +um chelators. Science, 1983, 220: 1375 - 1376.tested stimulated the biosynthesis of ethylene , and the[3 ] Raz J, Fluhr J. Calcium requirement for ethylene- dependentresponse. Plant Cell, 1992, 5:1123-1130.amount of increase was different with different concentra-[4] Suwwan M A, Poovaiah B W. Association between elemen-tion of Ca2+ . Ethylene released by etiolated tomatotal content and fruit ripening in rin and nomal tomatoes .seedling continuously increased with Ca-+ concentrationPlant Plhysiol, 1978, 61:883 - 885.from 0 to 3.8 mmol/L. However with 10 mmol/L Ca2+ ,.[5]Leshem Y Y, Sridnara S. Involvement of caleium andcalmodulin in membrane deterioration during senescence ofalthough ethylene production was higher than that of con-pea foliage. Plant Plysiol, 1984, 75:239 - 335.trol , the increase was less than that of 3.8 mmol/L Ca2 +[6] BurnsJ K, Evensen K B. Ca2+ efeetse on ethylene, car-( Fig.3 ). Futhemore , the effect of Ca2+ on the expres-bone dioxide and 1-aminocyclopropane- 1-carboxylic acidsion of ethylene receptor NR gene was changed similarlysynthase activity. Physiol Plant, 1986, 66:609 - 625.( Fig.3 ). Burm and Evenserf 6] treated tomato pericarp[ 7 ] Su-Hwan K, Sun H L. The requirements for Ca2+，proteinphosphorylation, and dephosphorylation for ethylene signaltissue discs with 5 mmol/L CaCl. Before , in the begin-transduction in Pisum satium L. Plant Cell Physiol, 1997 ,ing and at peak , and 1 d,2 d after climateric , it was38:1142- 1149.found that ethylene production increased 2% , 20% ,[8 ] Neljubov D. Ueber die Horizontal Nutation der Stengel von33% ,39% and 50% respectively , but no increase withPisum satium und Einiger Anderer. Pflanzen, 1901, 10:10 mmol/L and 50 mmol/L CaCl2. Therefore , the effect128- 139.of Ca2 + on ethylene biosynthesis and signal transduction[9] ZhangJ-S, GuJ, Chen S_Y. A gene encoding a truncatednf Ruhionn io tnnscribed and salt- inducible inmight be related to concentration.中國煤化工91:361 - 366.Many physiological roles of Ca2+ were carried out in[10]umoassay of calmodulin. Ac-lant cell through the combination of Ca2+ and its recep-YHCN M H GHass1988, 8:54- 58. (intor CaMF 1314]. Wu and Lit 15] pointed out that increase[11]Chinese with English abstract)Abeles F B, MorganR W, Saltveit M E. Ethylene in Plantof the cytosolic CaM content was relevant to the ripeningBiology. San Diego: Academic Press, 1992. 120 - 220.and senescence process of tomato fruit ，and Fell and[12] Ferguson I B. Calcium in plant senescence and fruit ripen-SmitF 16 17].。found. that IAA and GA developed itsing. Plant Cell Enwiron, 1983, 7:477 - 489.physiological' 7t accompanying the increase of cytosolic[13] Roberts D M, Hamon A C. Calcium- modulated proteins:426植物學(xué)報Acta Botanica Sinica Vol.44 No.4 2002targets of intracellular calcium signals in higher plants. An-stract”ruu Rev Plant Physiol Plant Mol Biol, 1992, 43:375 - 414.[16]Fell H. Auxin causes osilltions of cytosolic free calcium[14]Dieter P. Calmodulin and calmodulin-mediated processes inand pH in Zea mays coleoptiles. Planta， 1988, 174:495plants. Plant Cell Eniron, 1984, 7:371 - 380.- 499.[15] Wu Y-M(吳有梅), Liu Yu(劉愚). The relationship be-[17] SmithSJ, Walker R P, Beale M H, Hooley R. Biologicaltween the concentration of CaM and ethylene production inactivity of some gibrellins and gibberellin derivatives inthe ripening of tonato fruit. Acta Plant Plysiol (植物生理aleurome cells and protoplasts of Arena fatua. Phytochem-學(xué)報), 1990, 16:245 - 250. (in Chinese with English ab-istry, 1993, 33:17- 20.鈣與植物乙烯反應的關(guān)系研究張艷萍朱本忠羅云波(中國農業(yè)大學(xué)食品學(xué)院, 北京10094 )摘要:研究了Ca2+ 對番茄( Lyopersicon esculentum Mill ev. Lichun 黃化幼苗乙烯反應的影響。通過(guò)測定不同Ca2+濃度條件下番茄黃化幼苗的三重反應"、內源乙烯釋放量、乙烯受體基因NEVER-RIPE(NR表達量及胞內CaM含量的變化結果發(fā)現隨著(zhù)培養基中Ca2+濃度從0 mmol/L增加到3. 8 mmo/L番茄黃化幼苗的三重反應”表型明顯增強,內源乙烯釋放量、NR基因的表達量及胞內CaM的含量都有不同程度的增加當Ca2+濃度由3.8mmol/L進(jìn)--步增加到10mmol/L時(shí)番茄黃化幼苗三重反應”表型受到抑制,內源乙烯釋放量、NR基因的表達量及胞內CaM的含量都有所下降。因此,Ca2+ 對番茄黃化幼苗三重反應"的影響與Ca2+調節內源乙烯合成和乙烯受體基因的表達有關(guān)而且Ca2+可能是通過(guò)CaM含量的變化來(lái)調節乙烯作用的。關(guān)鍵詞: Ca2+ ;三重反應;乙烯信號轉導;CaM中圖分類(lèi)號:Q945 .文獻標識碼:A文章編號:0577-7496( 2002 )4-0422-05收稿日期2001-03-26接收日 期2001-12-19基金項目國家重點(diǎn)基礎研究規劃發(fā)展項目( G1999011701 )和國家杰出青年基金( 39825118 )*通訊作者。FE-mail : < yunbol @ public3. bta. net. cn> .(責任編輯:賀萍)中國煤化工MHCNM HG