- Email: [email protected]
Tomato alcohol dehydrogenase
Volume 293, number 1,2,3, 39-42 FEBS 10481 Q 199 I Federation of European Eiochemical Societies 0014575:3.‘3l/S3.50
Tomato
aicohoi
December
1991
dehyckogenase
Expression during fruit ripening and under hypoxic conditions Dominique
Van Der Straeten, Lrrborn/oriu~~
Renato
A. Rodrigues
voor Gorericu.
Pousada,
Jan Gielen and Marc Van Montagu
GCIII, B-9000
ti~~irrrsi~eit
Grttr,
Bclgirm
Received I :! July 1991: revised version received 35 October I991 WC have isolated and scqucnccd a partial tomato alcohol dehydrogcnasc (/It/h) cDNA clone. Expression of tomato clc/h was studied at the messenger RNA lcvcl in seedlings. roots. and fruit. High induction was obscrvcd under hypoxic conditions. both in tomato seedlings and in roots. In addition. the Al//t mRNA was present a~ the mature green and pink stage ofthc tomato fruit. and was highly induced in late ripening. Morcowzr. an artiftcial ripening trcatmcnt rcsultcd in at IC;ISI50.fold induction compared to the mature green mRNA Icvcl. Gcnomic DNA gel bloomingsugestcd the preswcc of a multigcnc family for ,Il/lf in tomato. Alcoho! dchydrogcnasc;
Anacrobiosis:
Glycolysis:
1. INTRODUCTION
Alcohol dehydrogenase (ADH: alcohol:NAD+ oxidoreductase: EC 1. I, I. 1) is a terminal step in glycolysis. leading to ethanolic fermentation under anaerobic conditions, The enzyme from horse liver has been structurally characterized at the three-dimensional level [I]. Plant ADH has been studied in quite some detail at the molecular level. Genes encoding alcohol dchydrogenasc have been cloned and sequenced both from monocotyledonous (maize [I?]; barley [3]; rice [4.5]) and dicotyledonous species (A~~~bi~o/~sisthliutut [G]; pea [7]; potato [8]). Under hypoxic conditions, plants shift their metabolism from aerobic to fermentative pathways. Alcohol dehydrogcnase has been identified as one of the major anaerobic polypeptides (ANPs; [9]), and the cisacting controlling elements responsible for high anaerobic Ad/t mRNA induction have been extensively characterized [IO.1 I]. Recently it has become clear that Ad/r is not only responsive to anaerobic stress, but to a wide range of stresses, including elicitors, salicylic acid, UV light [8]. and loin temperature treatment [I 21. An interesting and
so far almost unexplored research area concerns the molecular aspects of glycolysis in general and ADH in particular, during the ripening process of fruits and vegetables. This could be important in storage under controlled al:d modified atmospheres, where hypoxia could lead to dramatic increases of mRNA and/or protein levels. thus having an impact on the flavour Corrc.~llo,nlr~,irr otlrlrcss: M. Van hlontagu. Laborutorium voor Gcnctica. Uniwrsiteit Gent, K.L. Lcdcgnnckstraat 35, U-9000 Gent. Eel-
giunl. Fax: (32) (91) 64 5349.
Ripening:
L~wpersicorl
rscdtwfrurr
of the fruit. In this report. we present cloning of a partial tomato Ah cDNA and characterize its expression under anaerobiosis in tomato seedlings and roots as well as in ripening tomato fruit.
quality
2. MATERIALS 2.I. Phi rnarwirrl.
AND METHODS
growlr codi~iot~s. mul urnrwohic itrhcriotrs (L~wpmico~r m-u/mr~rrrr cv. Supersonic)
Tomato plants were grown a~ 24’C and 80% humidity. Hypoxic induction of I3-day-old. dark-grown tomal secdlinps was by complete submergcncc for 20 h in IO mM Tris-HCI. pH 7.0, containing 75 pgfml cltloramphcnicol. as described by Springer CI al. 1131. When applied IO l.5-month-old tomato plants. waterlogging conditions were simulated by placing them in il IOO-liter tank filled with water to 3 cm above the soil surface for 40 h at ll°C. Tomato bruits (green. pink. or red; cv. Orlando) were obtained from a local market. Artilicial ripening was by a combination of I6 h incubation in 50 mM LiCI. 0.5 mM indolcacctic acid. 0. I mM bcnzyladeninc, and 0.6 mM aminooxyaccticacid. followed by 2 h of wounding (as modilicd from [14]). 1.1. Currsrrrtcriurr of N IOIIIOIO cD!VA lirinrr.v The construction of a tomato cDNA library lioni RNA induced .^., . I'.!__._ __ ..,_,I CI.", l...hr:A(;~~,;m. unucr arumal rlprlwll; cljhwubm. 03 HS~, ;; llllcl ,n,Y.aUI.-U..U.. conditions have been described previously [IS].
The tomato ADH cDNA insert was subcloned as en EcoRI fragmcnt in pUCI8. and named TADH I. Scqucnces were obtained by the didcoxy chain termination method [ IG]. 2.4. Mrcleic ~cltl gd blorliug urirl I~ylwidiWio~r Gcnomic DNA was essentially prepared as described [ 171. followed by a CsCl gradient. DNA gels wrc blotted onto Hybond N’ (Amcrsham) and the probe labelled by using a Mcgaprimc kit according IO lhc tnanubcturcr’s specificulions (Amcrshnm). Hybridization was carried out at 60°C mainly as described [ Ia]. Tolal RNA was purified as reported [l9]. and the poly(A)’ fraclion prepared on oligo-dT ccllulosc [SO]. Six pcrccnt rormaldehydc gels
39
Volume
295,
number 1.2.3
FEBSLETTERS
December 1991
1234567 --
were blottedon Hybond N (Amtrsham).RNX gel blothybridization was at PC in jx SSPEiO.S% SDS!50%formnmidcl% Denhardt's solutioniO.l mg'ml ofdcnaturcd herring sperm DNA [IS].For RNA slotblotnnnlysis. samples wcrc prcparcd and fixed01110 I-lybotid N' as recommended by Amersham.
--m-_. ._
__,“%--.,*
-.,_.
.l,.. ....
kb
L
3. RESULTS
IO
3.1. Screenittg of a ?.gtli tottwto cDNA IiDrary ami seqtrettcittg of rhc Adh clone Upon screening a tomato cDNA library for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase [15]. one positive clone was found which contained an extra fragment. identified as AC/IIafter sequence analysis and comparison with sequences in the database. The fragment was subcloned in pUCI8 and named TADH 1. The sequence is presented in Fig. I. The cDNA of 594 bp includes part of the coding region (i-390 bp. containing the last 130 amino acids) and possesses an untranslated sequence of 204 bp. Both the catalytic domain and coenzyme binding domain of the ADH protein reside, at least in part. in the peptide encoded by TADHI. This explains the high degree of similarity with other AP!I species. On the amino acid level, TADH! is 96% homologous to potato ADH, and 79-
8.0
1lC
-1.0
-a ,’
. ..
.
.;._
45 54 21 36 9 18 CCT AM CCA ClT.CM GAG GTA ATTCCT GAG ATCAC7 GAT GGC GGA GTC GAT AGG
--0.5
:,
RKPVQEVIAEHTDGGVDR 99 108 12 81 90 63 ACTGTGGM TGtACT ‘XT CAC All CAT GCl'A;GAll'TCA GCA TTT GM TGT GTC s v E c P GE 1 D A HI S A F E C V
Fig. 7.Gcnoniic DNA gelblol analysisof tomato. Ten pg oTlo:al DNA twSdigjtcdwith &rrrrHl (lane I).EcoRI(lanc2). I-li,rdIll (Ian:
153 162 135 144 117 126 CAT GAT KC TGG GGA GI'G Ccc GTT CTT GTT CU GTA CCC CAT AAA GM GCTGTG
3).;md ~/~~~l(Iitne 1).One, Iwo. and livecopy cquivalcnts oTTADHl arc in laws 5, 6 and 7, rcspcctively. The EcoRI-;Vcol fragment of TADHl(380bp)was nicgaprimclabclicdand uscdasa probe. Exposure was for 36 h on flash-sensitized film.
liDGWGVAVLVGVPBKEAV 207 216 171 189 198 180 Tl'C MC ACACATCCl'CIG AAClWl'lB AATGEACGG ACTCl'C AAAGGAACClX PKTUPLNFLNERTLKGTF 243 261 270 225 234 252 lTTCGAMC TACAAACCTCGTT'XGATATTCCTTGTGTPGTTGAGAAATACATG FGNYRPRSDIPCVVEKYH 297 279 288 306 315 324 MC AAAGM CITGM TIGGAGAAA'IWATCACTCATACAClTCCATITGCTGM !! K s L e L E R F I T A T L P F A E 369 3'18 342 351 333 360 ATCMT MC CCTRC CAT TCA ATGCl'GMC CGAGM GGCClTCGTTGCATCATC INKAPDLHLKGEGLRCII 433 443 403 413 423 381 ACC ATG Ccc GACTM ACETCKl%ThGAAMCGACACl'G~AGAAAAAAGACCMT T H A D.
84% homologous to maize ADH2 and ADH I, respcctively. On the DNA level these homologies are 96% between tomato and potato and 71 c/c between tomato and maize. 3.2. Detectiott of Adh sc~tret~cs it? totttato gctlotuic D&i
Genomic DNA gel blot analysis with the coding part of TADHI as a probe revealed a fairly complex hybridization pattern (Fig. 2). Restriction nf tomato gcnomic DNA with BatttI-II, EcoRI, Nittdlll, and Xhnl showed 3-G hybridizing bands, all of which had intensities of one or two copy equivalents. This complexity indicated the presence of a multigene family in tomato, consistent with the three M/I genes found in potato, another genus of the Solatraceue family [S].
493 503 513 473 483 453 463 AAAlTGTCACi%TCTIAlTlWClTKGTUTlW'l7G~~TTGTAACATTCCATCCATGTC~~TCTTT 563 573 583 543 553 523 533 3.2. Atralysis of ~GCTCITITCCPTAGAn;m;TCA~AT~~~A~~~T~~~TT~~CM tttRNA he/ 593 AAAMAMW The anaerobic Fig. I. Nuclcolidctenddcduccd aminoncid scqucnccs of the tomato ADH cDNA TADHI (EMBL accessionnumber XGOGOO).
40
the espessiot~
of tomato A DH at the
induction of A& messenger was tested in tomato seedlings and roots. As shown in Fig. 3A, a l.4-kb mRNA was detected at low levels in uninduced
Solulne
295. number
FEBS
C
LETTERS
December
I991
resuits of the DNA gel blot. it is very likely that the probe revealed all classes of Arlll mRNA.
kb 4. DISCUSSION 1
Fig. 3. Expression of tomato ADH
under anaerobic conditions (A).
during ripcuing (B and C). and after wounding (C). A J’P-labcllcd EwRI-Ncol fraatnent ol’TADH I was used as a probe. Hvbridization was at 42OC in ibuffer comaining 50% formamibc. (A) KNA gel blot of submcrgcd etiolatcd tomato seedling and waterlogged tomato rools. Each lanr contains 100 fig of total RNA. (Lane I) Etiolaled seedlings: (lime 2) after 20 h submcrgcncc: (Ianc 3) root tissue; (lane 4) root tissc:: alicr 40 h walerlo_rzin_r. Esposurc was 24 h on llashscnsilizcd film. (B) RNA blol analysis or IOIIMIO fruit ;II diffcrcnl siagcs. Each lane conrains IO pugor poly(A)’ RNA. (Lane I) Green tissue: (IiInc 2) pink tissue; (law 3) artiliciully ripcncd pink [issue. Esposuru WiIs2-t h on flash-scnsilizcd film. (C) RXA SIOI blot analysis of naturally and arLificially ripcncd tomato fruit. or afrcr wounding. Each Ianc contains 25 pugor total RNA. (I) Green lissuc; (2) pink tissue: (3) red ripe tissue; (4) arlifici;tlly ripened pink tissue: (5j pink tissue aficr 8 h wounding wcatmcm. Esposurc was 60 II on flashscnsilizcd film.
tissues. After 20 h of submergence of tomato seedlings. the Ad/? mRNA level was at least IO-fold increased, whereas ttt least 30-fold induction was observed in waterlogged tomato roots after 40 h. Fig. 38 presents an RNA gel blot of tomato fruit at different ripening stages. Very little if any difference was seen between mRNA levels in mature green and pink tomatoes, By contrast, there was a dramatic increase of Ad messenger when fruits were artificially ripened. At least 50-fold induction compared to the level in green tissue was observed. This induction range is comparable to that demonstrated for ACC synthasr under the same conditions [IS]. From Fig. 3C it was concluded that approximately the same induction of M/r messenger was reached upon natural ripening. The highest induction was observed after 8 h of wounding of pink bruit tissue. In all cases, the RNA blots were probed with a coding region fragment of TADH I (IZcoRM’coI fragment, 380 bp). Because _#ir/lrgents were found to be highly conserved ttmong different species [4,8], and given the
In this paper we report cDNA cloning and expression analysis of tomato Ad/t. The partial cDNA covers the last third of the ADH protein and is highly conserved with both monocotyledonous and dicotyledonous Adz genes (79-96%). RNA gel blot analysis indicated the existence of a l.4-kb messenger, the same size as was found for the Arubidopsis rhlimcr AC//I mRNA [6]. High induction of tomato Ad mRNA was observed under anaerobiosis, as expected for this terminal step of glycolysis [lo]. Comparable induction levels have been reported for Adit from several plants [2,4,6-81. Hypoxia is not only created under conditions of waterlogging, but also occurs when fruits and vegetables are stored under controlled or modified atmosphere. Research concerning glycolytic activities during fruit ripening and storage has so Tar been limited to the analysis of enzymatic activities [21-251. Glycolytic carbon flux was reported to increase 2-3-fold during ripening [21]. Enhancement of gycolytic activity was detected both in ethylene-treated [32] and in naturally ripened fruit [23,2-1]. A recent paper [25] described the enhancement of ADH activity in avocado fruit ripened in air and under hypoxia. Our results on accumulation of Ad/r mRNA during tomato fruit ripening are consistent with those data. There is a low constitutivc expression of Ad/t mRNA in mature green tomato fruit, and in the pink stage. The fact that the hybridization occurred to a broad rather than a discrete band may indicate the presence of mRNAs from several genes. At the protein level. new ADH isozymes have also been found in avocado late in ripening [35]. Under both natural and artificial ripening conditions, tomato Ad mRNA accumulated to a level at least 50-fold over green fruit. High induction under artificial ripening conditions is probably the result of the combined effects of anoxia, with auxin [4] and wounding. An 8-h wounding treatment of pink tomato fruit was also shown to result in A& messenger ieveis compiirabie io i!iose iii rii;cnifig fruit. Recent!y, potato Ad/? was shown to be responsive to different stresses [8]. Further research will be needed to investigate possible effects of difl’erent inducing factors of AtIll during the ripening process. This is of particular importance with respect to the maintenance of Ravour of fruits during post-harvest periods. ,lrk,~on,/~~/~~~rr~crrrs: WC thank hlartinc De Cock for linal lay-am or ihc manuscripl and our arl team ror the prcparalion of rhc ligurcs. D.V.D.S. is a Senior Rcscarch Assistant of the Nalional Fund l’or Fcicnlilic Rcscarch (Belgium). R.A.R.P. is indebted IO 111~Programa Cicncin of Juma Nacionul dc lnvcsligacao Cicmifica c Tccnok&ca (Portugal) for a rcllowship.
41
Volume
295. number
12.3
FEBS LETTERS
REFERENCES ii] Ekiund.
H.. Nordsirbm. B.. Zeppenzittirl.. E.. Siidcrlund. G.. Ohlsson. I.. Boise. T.. Soderbcrg. B.O.. Tapia. 0. and Briindcn. Cl. (1976) J. Mol. Biol. 102. 27-59. [2] Dennis. ES.. Sachs. M.M.. Gerlach. W.L.. Finnegan. E.J. and Peacock. W.J. (1985) Nucleic Acids Res. 13. 727-743. [3] Trick. M.. Dennis. E.S.. Edwards. K.J.R. and Peacock. W.J. (1988) Plant Mol. Biol. II. 147-160. [41 Xie. Y. and ‘%‘I!. R. (1939) Plant Mol. Biol. 13. 53-68. Xie. Y. and WU. R. (1990) Gene 87. 185-191. ‘r:; Chang. C. and Moycrowitz. E.%l. (1986) Proc. Natl. Acad. Sci. USA 83. 1408-1112. [71 Llewellyn. D.J.. Finnegan. E.J.. Ellis. J.G.. Dennis. E.S. and Peacock. W.J. (1957) J. 3101. Biol. 195. 113-123. [Sl Matton. D.P.. Constabel. P. and Brisson. N. (i99C) Plant Mol. Biol. 14. 775-783. t91 Sachs, M.M.. Freeling. M. and Okimoto. R. (1980) Cell 20.761767. [lOI Dennis. E.S.. Walker. J.C.. Llewellyn. D.J.. Ellis. J.G.. SingIt. K.. Tokuhisa. J.G.. Wolstenholmc. D.R. and Peacock. W.J. (1987) in: Plant Molecular Biology (van Wettstcin, D. and Chua. N.-H. cds.) pp. 407-417. Plenum Press. New York. [l II Olive. M.R.. Walker. J.C., Singh. K.. Dennis. E.S. and Peacock. W.J. (1990) Plant Mol. Biol. IS. 593-604. 1121Christie. P.J.. Hahn. M. and Walbot. V. (1991) Plant Physic!. 95. 699-706. [I31 Springer. B.. Werr. W.. StarlinBer. P.. Bennett. DC.. Zokolica. M. and Frerlinp. M. (198G) Mol. Gen. Gcnct. 305. 461-465.
42
December
1991
[id] S;IIO. T. and Thcologis. A. (1989) Proc. Karl. Acad. Sci. USA 56. 662 I-6625. Van Dcr Straetcn, D., Van Wicmccrsch. L.. Goodman, H.M. and Van Montagu. M. (1990) Proc. Natl. Acad. Sci. USA 87.48594863. 1161 Sangcr. F.. Nicklcn. S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74. 5463-5467. 117 Dcllaporta. S.L.. Wood, J. and Hicks, J.B. (1983) Plant Mol. Biol. Reporter I. 19-1. I. 1181Church, G.M. atld Gilbert. W. (1984) Proc. Natl. Acad. Sci. USA 81. 1991-1995. R.. \‘;III !-iontagu, M. and Van Dcr II91 Rodripucs-Pousadil, Srractcn. D. (1990) Tcchniqttc 2. 292-294. WI Sambrook, J.. Fritsch, E.F. and Maniatis. T. (1989) Molecular Cloning. B Laboratory Manu:~l. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. XY. Solomos. T. and Latics. G.G. (1971) Plant Physiol. 54, 506-51 I. Stitt. M.. Cseke. C. and Bucl~anaa. B, (1986) Plant Physiol. 80. 246-148. Bennett. A.B.. Smith. G.M. and Nichols, 13.G: (1987) Plant Physiol. 83. 973-976 Ball. K.L..Green. J.H. and Rw.T. (1991) Eur. J. Biochcm. 197, 265-269. Kancllis. A.K.. So!otnos. T. and Roubclakis-Anyelakis, K.A. (1991) Plar11 Physiol. 96. 269-274.
[lj]
Tomato
aicohoi
December
1991
dehyckogenase
Expression during fruit ripening and under hypoxic conditions Dominique
Van Der Straeten, Lrrborn/oriu~~
Renato
A. Rodrigues
voor Gorericu.
Pousada,
Jan Gielen and Marc Van Montagu
GCIII, B-9000
ti~~irrrsi~eit
Grttr,
Bclgirm
Received I :! July 1991: revised version received 35 October I991 WC have isolated and scqucnccd a partial tomato alcohol dehydrogcnasc (/It/h) cDNA clone. Expression of tomato clc/h was studied at the messenger RNA lcvcl in seedlings. roots. and fruit. High induction was obscrvcd under hypoxic conditions. both in tomato seedlings and in roots. In addition. the Al//t mRNA was present a~ the mature green and pink stage ofthc tomato fruit. and was highly induced in late ripening. Morcowzr. an artiftcial ripening trcatmcnt rcsultcd in at IC;ISI50.fold induction compared to the mature green mRNA Icvcl. Gcnomic DNA gel bloomingsugestcd the preswcc of a multigcnc family for ,Il/lf in tomato. Alcoho! dchydrogcnasc;
Anacrobiosis:
Glycolysis:
1. INTRODUCTION
Alcohol dehydrogenase (ADH: alcohol:NAD+ oxidoreductase: EC 1. I, I. 1) is a terminal step in glycolysis. leading to ethanolic fermentation under anaerobic conditions, The enzyme from horse liver has been structurally characterized at the three-dimensional level [I]. Plant ADH has been studied in quite some detail at the molecular level. Genes encoding alcohol dchydrogenasc have been cloned and sequenced both from monocotyledonous (maize [I?]; barley [3]; rice [4.5]) and dicotyledonous species (A~~~bi~o/~sisthliutut [G]; pea [7]; potato [8]). Under hypoxic conditions, plants shift their metabolism from aerobic to fermentative pathways. Alcohol dehydrogcnase has been identified as one of the major anaerobic polypeptides (ANPs; [9]), and the cisacting controlling elements responsible for high anaerobic Ad/t mRNA induction have been extensively characterized [IO.1 I]. Recently it has become clear that Ad/r is not only responsive to anaerobic stress, but to a wide range of stresses, including elicitors, salicylic acid, UV light [8]. and loin temperature treatment [I 21. An interesting and
so far almost unexplored research area concerns the molecular aspects of glycolysis in general and ADH in particular, during the ripening process of fruits and vegetables. This could be important in storage under controlled al:d modified atmospheres, where hypoxia could lead to dramatic increases of mRNA and/or protein levels. thus having an impact on the flavour Corrc.~llo,nlr~,irr otlrlrcss: M. Van hlontagu. Laborutorium voor Gcnctica. Uniwrsiteit Gent, K.L. Lcdcgnnckstraat 35, U-9000 Gent. Eel-
giunl. Fax: (32) (91) 64 5349.
Ripening:
L~wpersicorl
rscdtwfrurr
of the fruit. In this report. we present cloning of a partial tomato Ah cDNA and characterize its expression under anaerobiosis in tomato seedlings and roots as well as in ripening tomato fruit.
quality
2. MATERIALS 2.I. Phi rnarwirrl.
AND METHODS
growlr codi~iot~s. mul urnrwohic itrhcriotrs (L~wpmico~r m-u/mr~rrrr cv. Supersonic)
Tomato plants were grown a~ 24’C and 80% humidity. Hypoxic induction of I3-day-old. dark-grown tomal secdlinps was by complete submergcncc for 20 h in IO mM Tris-HCI. pH 7.0, containing 75 pgfml cltloramphcnicol. as described by Springer CI al. 1131. When applied IO l.5-month-old tomato plants. waterlogging conditions were simulated by placing them in il IOO-liter tank filled with water to 3 cm above the soil surface for 40 h at ll°C. Tomato bruits (green. pink. or red; cv. Orlando) were obtained from a local market. Artilicial ripening was by a combination of I6 h incubation in 50 mM LiCI. 0.5 mM indolcacctic acid. 0. I mM bcnzyladeninc, and 0.6 mM aminooxyaccticacid. followed by 2 h of wounding (as modilicd from [14]). 1.1. Currsrrrtcriurr of N IOIIIOIO cD!VA lirinrr.v The construction of a tomato cDNA library lioni RNA induced .^., . I'.!__._ __ ..,_,I CI.", l...hr:A(;~~,;m. unucr arumal rlprlwll; cljhwubm. 03 HS~, ;; llllcl ,n,Y.aUI.-U..U.. conditions have been described previously [IS].
The tomato ADH cDNA insert was subcloned as en EcoRI fragmcnt in pUCI8. and named TADH I. Scqucnces were obtained by the didcoxy chain termination method [ IG]. 2.4. Mrcleic ~cltl gd blorliug urirl I~ylwidiWio~r Gcnomic DNA was essentially prepared as described [ 171. followed by a CsCl gradient. DNA gels wrc blotted onto Hybond N’ (Amcrsham) and the probe labelled by using a Mcgaprimc kit according IO lhc tnanubcturcr’s specificulions (Amcrshnm). Hybridization was carried out at 60°C mainly as described [ Ia]. Tolal RNA was purified as reported [l9]. and the poly(A)’ fraclion prepared on oligo-dT ccllulosc [SO]. Six pcrccnt rormaldehydc gels
39
Volume
295,
number 1.2.3
FEBSLETTERS
December 1991
1234567 --
were blottedon Hybond N (Amtrsham).RNX gel blothybridization was at PC in jx SSPEiO.S% SDS!50%formnmidcl% Denhardt's solutioniO.l mg'ml ofdcnaturcd herring sperm DNA [IS].For RNA slotblotnnnlysis. samples wcrc prcparcd and fixed01110 I-lybotid N' as recommended by Amersham.
--m-_. ._
__,“%--.,*
-.,_.
.l,.. ....
kb
L
3. RESULTS
IO
3.1. Screenittg of a ?.gtli tottwto cDNA IiDrary ami seqtrettcittg of rhc Adh clone Upon screening a tomato cDNA library for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase [15]. one positive clone was found which contained an extra fragment. identified as AC/IIafter sequence analysis and comparison with sequences in the database. The fragment was subcloned in pUCI8 and named TADH 1. The sequence is presented in Fig. I. The cDNA of 594 bp includes part of the coding region (i-390 bp. containing the last 130 amino acids) and possesses an untranslated sequence of 204 bp. Both the catalytic domain and coenzyme binding domain of the ADH protein reside, at least in part. in the peptide encoded by TADHI. This explains the high degree of similarity with other AP!I species. On the amino acid level, TADH! is 96% homologous to potato ADH, and 79-
8.0
1lC
-1.0
-a ,’
. ..
.
.;._
45 54 21 36 9 18 CCT AM CCA ClT.CM GAG GTA ATTCCT GAG ATCAC7 GAT GGC GGA GTC GAT AGG
--0.5
:,
RKPVQEVIAEHTDGGVDR 99 108 12 81 90 63 ACTGTGGM TGtACT ‘XT CAC All CAT GCl'A;GAll'TCA GCA TTT GM TGT GTC s v E c P GE 1 D A HI S A F E C V
Fig. 7.Gcnoniic DNA gelblol analysisof tomato. Ten pg oTlo:al DNA twSdigjtcdwith &rrrrHl (lane I).EcoRI(lanc2). I-li,rdIll (Ian:
153 162 135 144 117 126 CAT GAT KC TGG GGA GI'G Ccc GTT CTT GTT CU GTA CCC CAT AAA GM GCTGTG
3).;md ~/~~~l(Iitne 1).One, Iwo. and livecopy cquivalcnts oTTADHl arc in laws 5, 6 and 7, rcspcctively. The EcoRI-;Vcol fragment of TADHl(380bp)was nicgaprimclabclicdand uscdasa probe. Exposure was for 36 h on flash-sensitized film.
liDGWGVAVLVGVPBKEAV 207 216 171 189 198 180 Tl'C MC ACACATCCl'CIG AAClWl'lB AATGEACGG ACTCl'C AAAGGAACClX PKTUPLNFLNERTLKGTF 243 261 270 225 234 252 lTTCGAMC TACAAACCTCGTT'XGATATTCCTTGTGTPGTTGAGAAATACATG FGNYRPRSDIPCVVEKYH 297 279 288 306 315 324 MC AAAGM CITGM TIGGAGAAA'IWATCACTCATACAClTCCATITGCTGM !! K s L e L E R F I T A T L P F A E 369 3'18 342 351 333 360 ATCMT MC CCTRC CAT TCA ATGCl'GMC CGAGM GGCClTCGTTGCATCATC INKAPDLHLKGEGLRCII 433 443 403 413 423 381 ACC ATG Ccc GACTM ACETCKl%ThGAAMCGACACl'G~AGAAAAAAGACCMT T H A D.
84% homologous to maize ADH2 and ADH I, respcctively. On the DNA level these homologies are 96% between tomato and potato and 71 c/c between tomato and maize. 3.2. Detectiott of Adh sc~tret~cs it? totttato gctlotuic D&i
Genomic DNA gel blot analysis with the coding part of TADHI as a probe revealed a fairly complex hybridization pattern (Fig. 2). Restriction nf tomato gcnomic DNA with BatttI-II, EcoRI, Nittdlll, and Xhnl showed 3-G hybridizing bands, all of which had intensities of one or two copy equivalents. This complexity indicated the presence of a multigene family in tomato, consistent with the three M/I genes found in potato, another genus of the Solatraceue family [S].
493 503 513 473 483 453 463 AAAlTGTCACi%TCTIAlTlWClTKGTUTlW'l7G~~TTGTAACATTCCATCCATGTC~~TCTTT 563 573 583 543 553 523 533 3.2. Atralysis of ~GCTCITITCCPTAGAn;m;TCA~AT~~~A~~~T~~~TT~~CM tttRNA he/ 593 AAAMAMW The anaerobic Fig. I. Nuclcolidctenddcduccd aminoncid scqucnccs of the tomato ADH cDNA TADHI (EMBL accessionnumber XGOGOO).
40
the espessiot~
of tomato A DH at the
induction of A& messenger was tested in tomato seedlings and roots. As shown in Fig. 3A, a l.4-kb mRNA was detected at low levels in uninduced
Solulne
295. number
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December
I991
resuits of the DNA gel blot. it is very likely that the probe revealed all classes of Arlll mRNA.
kb 4. DISCUSSION 1
Fig. 3. Expression of tomato ADH
under anaerobic conditions (A).
during ripcuing (B and C). and after wounding (C). A J’P-labcllcd EwRI-Ncol fraatnent ol’TADH I was used as a probe. Hvbridization was at 42OC in ibuffer comaining 50% formamibc. (A) KNA gel blot of submcrgcd etiolatcd tomato seedling and waterlogged tomato rools. Each lanr contains 100 fig of total RNA. (Lane I) Etiolaled seedlings: (lime 2) after 20 h submcrgcncc: (Ianc 3) root tissue; (lane 4) root tissc:: alicr 40 h walerlo_rzin_r. Esposurc was 24 h on llashscnsilizcd film. (B) RNA blol analysis or IOIIMIO fruit ;II diffcrcnl siagcs. Each lane conrains IO pugor poly(A)’ RNA. (Lane I) Green tissue: (IiInc 2) pink tissue; (law 3) artiliciully ripcncd pink [issue. Esposuru WiIs2-t h on flash-scnsilizcd film. (C) RXA SIOI blot analysis of naturally and arLificially ripcncd tomato fruit. or afrcr wounding. Each Ianc contains 25 pugor total RNA. (I) Green lissuc; (2) pink tissue: (3) red ripe tissue; (4) arlifici;tlly ripened pink tissue: (5j pink tissue aficr 8 h wounding wcatmcm. Esposurc was 60 II on flashscnsilizcd film.
tissues. After 20 h of submergence of tomato seedlings. the Ad/? mRNA level was at least IO-fold increased, whereas ttt least 30-fold induction was observed in waterlogged tomato roots after 40 h. Fig. 38 presents an RNA gel blot of tomato fruit at different ripening stages. Very little if any difference was seen between mRNA levels in mature green and pink tomatoes, By contrast, there was a dramatic increase of Ad messenger when fruits were artificially ripened. At least 50-fold induction compared to the level in green tissue was observed. This induction range is comparable to that demonstrated for ACC synthasr under the same conditions [IS]. From Fig. 3C it was concluded that approximately the same induction of M/r messenger was reached upon natural ripening. The highest induction was observed after 8 h of wounding of pink bruit tissue. In all cases, the RNA blots were probed with a coding region fragment of TADH I (IZcoRM’coI fragment, 380 bp). Because _#ir/lrgents were found to be highly conserved ttmong different species [4,8], and given the
In this paper we report cDNA cloning and expression analysis of tomato Ad/t. The partial cDNA covers the last third of the ADH protein and is highly conserved with both monocotyledonous and dicotyledonous Adz genes (79-96%). RNA gel blot analysis indicated the existence of a l.4-kb messenger, the same size as was found for the Arubidopsis rhlimcr AC//I mRNA [6]. High induction of tomato Ad mRNA was observed under anaerobiosis, as expected for this terminal step of glycolysis [lo]. Comparable induction levels have been reported for Adit from several plants [2,4,6-81. Hypoxia is not only created under conditions of waterlogging, but also occurs when fruits and vegetables are stored under controlled or modified atmosphere. Research concerning glycolytic activities during fruit ripening and storage has so Tar been limited to the analysis of enzymatic activities [21-251. Glycolytic carbon flux was reported to increase 2-3-fold during ripening [21]. Enhancement of gycolytic activity was detected both in ethylene-treated [32] and in naturally ripened fruit [23,2-1]. A recent paper [25] described the enhancement of ADH activity in avocado fruit ripened in air and under hypoxia. Our results on accumulation of Ad/r mRNA during tomato fruit ripening are consistent with those data. There is a low constitutivc expression of Ad/t mRNA in mature green tomato fruit, and in the pink stage. The fact that the hybridization occurred to a broad rather than a discrete band may indicate the presence of mRNAs from several genes. At the protein level. new ADH isozymes have also been found in avocado late in ripening [35]. Under both natural and artificial ripening conditions, tomato Ad mRNA accumulated to a level at least 50-fold over green fruit. High induction under artificial ripening conditions is probably the result of the combined effects of anoxia, with auxin [4] and wounding. An 8-h wounding treatment of pink tomato fruit was also shown to result in A& messenger ieveis compiirabie io i!iose iii rii;cnifig fruit. Recent!y, potato Ad/? was shown to be responsive to different stresses [8]. Further research will be needed to investigate possible effects of difl’erent inducing factors of AtIll during the ripening process. This is of particular importance with respect to the maintenance of Ravour of fruits during post-harvest periods. ,lrk,~on,/~~/~~~rr~crrrs: WC thank hlartinc De Cock for linal lay-am or ihc manuscripl and our arl team ror the prcparalion of rhc ligurcs. D.V.D.S. is a Senior Rcscarch Assistant of the Nalional Fund l’or Fcicnlilic Rcscarch (Belgium). R.A.R.P. is indebted IO 111~Programa Cicncin of Juma Nacionul dc lnvcsligacao Cicmifica c Tccnok&ca (Portugal) for a rcllowship.
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295. number
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FEBS LETTERS
REFERENCES ii] Ekiund.
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