Characteristics of transgenic tomatoes antisensed for the ethylene receptor genes LeETR1 and LeETR2

Characteristics of transgenic tomatoes antisensed for the ethylene receptor genes LeETR1 and LeETR2 1 2 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:tjying@hzcnc.com †E-mail:tjying@hzcnc.com tjying@hzcnc.com 2 1 1 School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China Department of Biological and Chemical Engineering, Guangxi University of Technology, Liuzhou 545006, China Department of Biological and Chemical Engineering, Guangxi University of Technology, Liuzhou 545006, China Two stable transformed lines containing antisense LeETR1 or LeETR2 sequences and their hybridized line were investigated to determine the effect of LeETR1 and LeETR2 specificity in the ethylene receptor family in tomato ( Lycopersicon esculentum Mill.) on ethylene signaling. The transgenic line ale1 containing antisense LeETR1 displayed shorter length of seedling grown in the dark and adult plant in the light, severe epinastic petiole, and accelerated abscission of petiole explant and senescence of flower explant, compared with its wild type B1. The transgenic line ale2 containing antisense LeETR2 also exhibited shorter hypocotyls and slightly accelerated abscission. The phenotypes of cross line dale of LeETR1 and LeETR2 were close to ale1 in many aspects. These results suggested that LeETR1 probably plays a relatively important role in ethylene signaling of tomato growth and development. Antisense transformation Ethylene receptor Ethylene response Tomato INTRODUCTION 1 Ethylene is a gaseous plant hormone affecting many developmental processes and fitness responses, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to stress and pathogen attack (Bleecker and Kende, 2000 2000 ; Johnson and Ecker, 1998 1998 ). According to the negatively regulating model, ethylene receptors combining with CTR1 negatively regulate ethylene response. Triple or quadruple loss-of-function mutants display a constitutive ethylene response phenotype (Bleecker and Kende, 2000 2000 ; Chang and Stadler, 2001 2001 ). It was reported that single or double loss-of-function receptor mutants displayed ethylene response phenotypes (Hua and Meyerowitz, 1998 1998 ). Carefully analysis of receptor null mutants revealed that loss of even one ethylene receptor, specifically ETR1 , results in a significant increase in ethylene responsiveness in Arabidopsis (Cancel and Larsen, 2002 2002 ). In tomato, the ethylene receptor gene family presently consists of six members LeETR1 ~ LeETR6 (Wilkinson et al., 1995 1995 ; Zhou et al., 1996a 1996a ; 1996b 1996b ; Lashbrook et al., 1998 1998 ; Tieman and Klee, 1999 1999 ; Klee and Tieman, 2002 2002 ). The proteins encoded by these genes are structurally diverse and at worst, are less than 50% identical. Each receptor gene has a distinct pattern of expression throughout development and in response to external stimuli. LeETR1 and LeETR2 are constitutively expressed in all tissues throughout development with LeETR1 expression about 5-fold that of LeETR2 . In contrast, expression patterns of the other four genes are highly regulated. NR ( never-ripe ) is constitutively expressed, but highly expressed in pedicel abscission zone and during fruit development (Wilkinson et al., 1995 1995 ; Lashbrook et al., 1998 1998 ). As fruits approach maturity, expression of several receptors increases. NR , LeETR4 and LeETR5 are induced by fruit ripening, organ senescence and abscission and pathogen infection, and are at a lower level in vegetative tissue (Tieman et al., 2000 2000 ; Ciardi et al., 2000 2000 ; Tieman and Klee, 1999 1999 ). The pathogen inducibility of LeETR4 is associated with increased ethylene synthesis following infection (Ciardi et al., 2001 2001 ). Loss of LeETR4 results in dramatic morphological changes associated with increased ethylene responsiveness. Strongly epinastic petiole, accelerated senescence of flower and delayed ripening of fruit were observed in the transgenic tomato mutant resulting from transcript reduction of LeETR4 (Tieman et al., 2000 2000 ). Some expected and unexpected phenotypes related to ethylene response, such as shorter internodes and delayed abscission, resulted from the ethylene receptor gene LeETR1 transgenic tomato (Whitelaw et al., 2002 2002 ) and reduced expression of LeETR1 in tomato. The increased ethylene responses of ETR1 loss-of-function mutant in Arabidopsis indicated that ETR1 may play more prominent role than the other receptors in ethylene signaling. However, previous result of LeETR1 antisense expression is in part against the negative regulation model of ethylene receptors in ethylene signaling, such as delayed abscission (Whitelaw et al., 2002 2002 ), which needs to be explained by more research information. The amino acid sequences of LeETR1 and LeETR2 protein are most closely related to ETR1 of Arabidopsis and are highly similar to each other (Zhou et al., 1996a 1996a ; 1996b 1996b ; Lashbrook et al., 1998 1998 ). They constitutively express in all tomato tissues throughout development (Lashbrook et al., 1998 1998 ). But their functional significance in ethylene receptor family is still unclear. Two transgenic lines of tomato, ale1 and ale2 , were prepared by introducing the antisense copy of LeETR1 or LeETR2 separately. A crossed line dale was obtained by crossing the ale1 and ale2 . MATERIALS AND METHEODS 1 The regions from 2351~2664 bp of LeETR1 (GeneBank, U41103) and 2125~2390 bp of LeETR2 (GeneBank, AF043085) genes were separately cloned into vector PCR®2.1 with Bam HI and Xba I. The cloned sequence was then reversely subcloned into the intermedium expressional vector pPZP111 between the sites of Hin dIII and Eco RI. The antisense sequence containing cauliflower mosaic virus (CaMV) 35S promoter, NPT II and GUS gene at the pPZP111 (Hajdukiewicz et al., 1994 1994 ) was transferred into tomato ( Lycopersicon esculentum Mill.) line B1 through Agrobacterium tumefaciens LAB4404 (Zheng et al., 2001 2001 ). Two transformed lines, ale1 and ale2 , respectively containing antisense copy of 3′-untranslated portion of LeETR1 or LeETR2 were obtained through three generations screen by kanamycin resistance, PCR (polymerase chain reaction) and GUS (β-glucuronidase gene) assay. The line dale was from the hybridization of ale1 with ale2 . The T4 of ale1 and ale2 , T0 of dale and wild line were grown in a plastic canopy, every plant in each line was assayed for the marked gene NPT II by PCR (Klimyuk et al., 1993 1993 ) with 5′-CCA CCA TGA TAT TCG GCA AG-3′ (AF234316: bases 9168~9187) as the left primer and 5′-GTG GAG AGG CTA TTC GGC TA-3′ (AF234316: bases 9717~9698) as the right primer (Wang et al., 1993 1993 ). For the etiolated seedlings experiment, seeds were surface-sterilized with 10% bleach then stored at 4 °C for 4 d in the dark to synchronize germination. Seeds were then sown in plant nutrient agar medium in vials with 0.5% (w/v) Suc, 0.5% (w/v) agar and other nutrient elements (Lincoln et al., 1992 1992 ). Seedlings were germinated for 5 d in the dark in air with or without 10 µl/L ethylene at 24 °C. Lengths and diameters of hypocotyls were measured. Thirty seeds were in each vial with three vials used for each line. Ten plants in each vial and a total of thirty in each line were measured. Leaf abscission zone explants used for the experiment consisted of a stem segment with one petiole still attached and leaflets removed. Flower explants for the senescence experiment contained a single freshly opened flower and its pedicel. Stem sect or flower pedicel base were inserted into 0.8% sterilized agar contained in a sealed 250 ml-bottle with a cap embedding a rubber plug in the center. A pre-determined quantity of ethylene, necessary to achieve a concentration of 10 µl/L was injected into the sealed bottle through the rubber plug. Fifteen samples were in each bottle and three bottles were used for each line. The numbers of abscission or senescence samples were recorded at certain time in each bottle. About 10~15 adult plants in each line were randomly selected for the determination of stem length and diameter. RESULTS 1 Transgenic lines were ascertained by PCR and Southern-blot analysis 2 Each plant of ale1 , ale2 , and dale were further identified for the presence of NPT II gene by PCR. Positive results were obtained from all assayed plants, which confirmed the homozygosity of the antisense transgenic lines (Fig. 1 1 ) 1~3: ale1 ; 5~6: ale2 ; 4, 8~9: B1; 10: Marker Length of etiolated seedlings and adult plant 2 All hypocotyls of etiolated seedlings of ale1 , ale2 and dale , exposed to air either or not containing 10 µl/L of ethylene, were shorter and thicker than those of their wild type B1 (Fig. 2 2 ). All of the three positive transgenic lines were shortened by at least 43% compared with wild type. Adult plants of ale1 , ale2 and dale were also shortened and thickened to different extents (Fig. 3 3 ). ale1 displayed the shortest length and was shortened by 36.4% in relation to that of B1; ale2 and dale were also shortened to some degree. The diameter of ale1 and dale stems increased by 19.2% and 20.8% respectively compared with B1. Abscission of petiole explants 2 The abscission of petiole explants of ale1 , ale2 and dale were significantly accelerated, not only in 10 µl/L ethylene but also in air (Fig. 4a 4a ). The abscission rates of all transgenic lines rapidly increased within the first 24 h and were much higher than those of contrast line B1 throughout the investigating period. ale1 and dale showed significantly higher abscission level than ale2 did during the period of investigation. Senescence of flowers 2 Accelerated senescences of flower explants of ale1 and dale were observed when they were exposed to air or ethylene (Fig. 4b 4b ). All ale1 flower explants senesced within 48 h when exposed to air without ethylene and with 24 h when exposed to ethylene. The samples of ale1 and dale displayed much higher senescence rate than other lines did. The result suggested that both sensitivity and response to ethylene of ale1 and dale flower senescence might be increased. DISCUSSION 1 According to the negative regulating model, single or double loss-of-function ethylene receptor mutants showed no significant ethylene response phenotype. Triple or quadruple loss-of-function mutants displayed a constitutive ethylene response phenotype. However the loss-of-function mutants in the ethylene receptor ETR1 caused enhanced sensitivity and exaggerated response to ethylene in Arabidopsis . An apparent growth defect resulted from enhanced response to ethylene, and increased sensitivity was also observed (Cancel and Larsen, 2002 2002 ). Some expected and unexpected phenotypes in relation to ethylene response, such as shorter internodes and delayed abscission, resulted from the ethylene receptor LeETR1 transgenic tomato (Whitelaw et al., 2002 2002 ). Strongly epinastic petioles, accelerated senescence of flowers and delayed ripening of fruit were observed in the transgenic tomato mutant of transcript reduction of LeETR4 (Tieman et al., 2000 2000 ). In this study, more typical phenotypes in relation to ethylene response, such as the shortened and thickened hypocotyls of etiolated seedlings and adult plants, and accelerated abscission of petioles and senescence of flowers, were observed in the transgenic lines ale1 , ale2 and their crossed lines dale . These phenotypes suggest that either sensitivity or response to ethylene is enhanced in these transgenic lines. LeETR1 and LeETR2 in tomato are highly homologous. The ale1 and ale2 displayed some similar ethylene response phenotypes, but ale1 showed more intensive ethylene response in many aspects than the ale2 did. The characteristics of dale were more close to those of ale1 . From the significant effects, especially accelerated abscission resulting from LeETR1 transgenic expression, it is supposed that LeETR1 possibly plays more important role on the ethylene signaling pathway LeETR family. It may also be inferred that each member in LeETR family has some specific functions and is not totally redundant from this study and previous reports (Tieman et al., 2000 2000 ; Whitelaw et al., 2002 2002 ). There may be special or temporal specific function for different receptor. References AB Bleecker , H Kende . Ethylene: a gaseous signal molecule in plants . Annu Rev Cell Dev Biol , 2000 . 16 ( 1 ): 1 - 18 . 11031228 DOI: 10.1146/annurev.cellbio.16.1.1 http://doi.org/10.1146/annurev.cellbio.16.1.1 . Ethylene: a gaseous signal molecule in plants JD Cancel , PB Larsen . Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis . Plant Physiol , 2002 . 129 ( 4 ): 1557 - 1567 . 12177468 DOI: 10.1104/pp.003780 http://doi.org/10.1104/pp.003780 . Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis C Chang , R Stadler . Ethylene hormone receptor action in Arabidopsis . Bioessays , 2001 . 23 ( 7 ): 619 - 627 . 11462215 DOI: 10.1002/bies.1087 http://doi.org/10.1002/bies.1087 . Ethylene hormone receptor action in Arabidopsis JA Ciardi , DM Tieman , ST Lund , JB Jones , RE Stall , HJ Klee . Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression . Plant Physiol , 2000 . 123 ( 1 ): 81 - 92 . 10806227 DOI: 10.1104/pp.123.1.81 http://doi.org/10.1104/pp.123.1.81 . Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression J Ciardi , D Tieman , J Jones , HJ Klee . Reduced expression of the tomato ethylene receptor LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria . Mol . Plant Microbe Interactions , 2001 . 14 ( 4 ): 487 - 495 . . Reduced expression of the tomato ethylene receptor LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria. Mol P Hajdukiewicz , Z Svab , P Maliga . The small, versatile pPZP family of Agrobacterium binary vectors . Plant Mol Biol , 1994 . 25 ( 6 ): 989 - 994 . 7919218 DOI: 10.1007/BF00014672 http://doi.org/10.1007/BF00014672 . The small, versatile pPZP family of Agrobacterium binary vectors J Hua , EM Meyerowitz . Ethylene responses are negatively regulated by a receptor gene family in Arabisopsis thaliana . Cell , 1998 . 94 ( 2 ): 261 - 271 . 9695954 DOI: 10.1016/S0092-8674(00)81425-7 http://doi.org/10.1016/S0092-8674(00)81425-7 . Ethylene responses are negatively regulated by a receptor gene family in Arabisopsis thaliana PR Johnson , JR Ecker . The ethylene gas signal transduction pathway: a molecular perspective . Annu Rev Genet , 1998 . 32 ( 1 ): 227 - 254 . 9928480 DOI: 10.1146/annurev.genet.32.1.227 http://doi.org/10.1146/annurev.genet.32.1.227 . The ethylene gas signal transduction pathway: a molecular perspective H Klee , D Tieman . The tomato ethylene receptor gene family: form and function . Physiol Plant , 2002 . 115 ( 3 ): 336 - 341 . 12081525 DOI: 10.1034/j.1399-3054.2002.1150302.x http://doi.org/10.1034/j.1399-3054.2002.1150302.x . The tomato ethylene receptor gene family: form and function VL Klimyuk , BJ Carroll , CM Thomas , JDG Jones . Alkali treatment for rapid preparation of plant material for reliable PCR analysis . Plant J , 1993 . 3 ( 3 ): 493 - 494 . 8220456 DOI: 10.1046/j.1365-313X.1993.t01-26-00999.x http://doi.org/10.1046/j.1365-313X.1993.t01-26-00999.x . Alkali treatment for rapid preparation of plant material for reliable PCR analysis CC Lashbrook , DM Tieman , HJ Klee . Differential regulation of the tomato ETR gene family throughout lant development . Plant J , 1998 . 15 ( 2 ): 243 - 252 . 9721682 DOI: 10.1046/j.1365-313X.1998.00202.x http://doi.org/10.1046/j.1365-313X.1998.00202.x . Differential regulation of the tomato ETR gene family throughout lant development C Lincoln , J Turner , M Estelle . Hormone-resistant mutants of Arabidopsis have an attenuated response to Agrobacterium strains . Plant Physiol , 1992 . 98 ( 3 ): 979 - 983 . 16668774 . Hormone-resistant mutants of Arabidopsis have an attenuated response to Agrobacterium strains DM Tieman , HJ Klee . Differential expression of two novel members of the tomato ethylene-receptor family . Plant Physiol , 1999 . 120 ( 1 ): 165 - 172 . 10318694 DOI: 10.1104/pp.120.1.165 http://doi.org/10.1104/pp.120.1.165 . Differential expression of two novel members of the tomato ethylene-receptor family DM Tieman , MG Taylor , JA Ciardi , HJ Klee . The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family . Proc Nat Acad Sci USA , 2000 . 97 ( 10 ): 5663 - 5668 . 10792050 DOI: 10.1073/pnas.090550597 http://doi.org/10.1073/pnas.090550597 . The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family H Wang , MQ Qi , AC Cutler . A simple method of preparing plant samples for PCR . Nucleic Acid Research , 1993 . 21 ( 17 ): 4153 - 4154 . . A simple method of preparing plant samples for PCR CA Whitelaw , NN Lyssenko , L Chen , D Zhou , AK Mattoo , ML Tucker . Delayed abscission and shorter internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato . Plant Physiol , 2002 . 128 ( 3 ): 978 - 987 . 11891253 DOI: 10.1104/pp.010782 http://doi.org/10.1104/pp.010782 . Delayed abscission and shorter internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato JQ Wilkinson , MB Lanahan , HC Yen , JJ Giavannoni , HJ Klee . An ethylene-inducible component of signal transduction encoded by never-ripe . Science , 1995 . 270 ( 5243 ): 1807 - 1809 . 8525371 . An ethylene-inducible component of signal transduction encoded by never-ripe TS Zheng , TJ Ying , GW Zeng , GQ He . Expression of ethylene receptor gene LeETR2 in tomato mutant Epi and construction of its antisense expression vector . J Zhejiang Univ (Agr and Life Sci) , 2001 . 27 ( 2 ): 156 - 160 . (in Chinese) . Expression of ethylene receptor gene LeETR2 in tomato mutant Epi and construction of its antisense expression vector D Zhou , P Kalaitzis , AK Mattoo , ML Tucker . The mRNA for an ETR1 homologue is constitutively expressed vegetative and reproductive tissue . Plant Mol Biol , 1996 . 30 ( 6 ): 1331 - 1338 . 8704141 DOI: 10.1007/BF00019564 http://doi.org/10.1007/BF00019564 . The mRNA for an ETR1 homologue is constitutively expressed vegetative and reproductive tissue D Zhou , AK Mattoo , ML Tucker . Molecular cloning of a tomato cDNA (accession No. U47279) encoding an ethylene receptor . Plant Physiol , 1996 . 110 ( 4 ): 1435 . Molecular cloning of a tomato cDNA (accession No. U47279) encoding an ethylene receptor 10.1631/jzus.2006.B0591 Project (No. 30371001) supported by the National Natural Science Foundation of China 2005-08-23 2006-02-09 2006-06-14 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 07