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Characterization of a cytokinininduced cDNA showingsequence homologies tohybrid proline (or glycine) richproteins in Catharanthus roseuscell suspension culturesKarim Chahed a , Ferid Limam a , Ali Gargouri b ,Rachid Ghrir a & Lazhar Ouelhazi aa Laboratoire de Biochimie Végétale , InstitutNational de la Recherche Scientifique et Technique ,BP 95, 2050 , Hammam-Lif , Tunisieb Centre de Biotechnologie de Sfax , BP “W”, 3038 ,Sfax , TunisiePublished online: 26 Apr 2013.

To cite this article: Karim Chahed , Ferid Limam , Ali Gargouri , Rachid Ghrir &Lazhar Ouelhazi (2000) Characterization of a cytokinin induced cDNA showingsequence homologies to hybrid proline (or glycine) rich proteins in Catharanthusroseus cell suspension cultures, Acta Botanica Gallica, 147:4, 311-321, DOI:10.1080/12538078.2000.10515863

To link to this article: http://dx.doi.org/10.1080/12538078.2000.10515863

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Acta Bot. Gallica, 2000, 147 (4), 311-32/.

Characterization of a cytokinin induced eDNA showing sequence homologies to hybrid proline (or glycine) rich proteins in Catharanthus roseus cell suspension cultures

by Karim Chahed( 1), Ferid Limam( 1), Ali Gargourie). Rachid Ghrir( 1) and Lazhar Ouelhazi( 1)

(I) Laboratoire de Biochimie Vegetale, lnstitut National de Ia Recherche Scientifique et Technique,

BP 95, 2050 Hammam-Lif, Tunisie

(2) Centre de Biotechnologie de Sfax, BP "W", 3038 Sfax, Tunisie

arrive le 22 novembre 1999, accepte le 10 mai 2000

Ahstract.-ln a preliminary screening of a eDNA library we isolated a eDNA that accumulates to high levels in cytokinin treated cells. The expression of the rela­ted transcript was induced alter treatment with benzylaminopurine (BAP), zeati­ne and kinetine. Sequence analysis showed the presence of multiple initiation and stop codons and the absence of a long open reading frame (OAF). However, classic polyadenylation signals and poly A tail were found. Searching for nucleo­tide and amino acid similarities revealed a strong homology (up to 90%) with cDNAs corresponding to hybrid proline (or glycine) - rich proteins (HyPAP/ HyGAP). A particular homology was found with a recently reported tobacco eDNA which encodes a novel glycine rich hydrophobic protein.

Key-words : Catharanthus roseus- cytokinins- HyPAP- HyGAP.

Resume.- Un ADNc dontl'expression est fortement stimulee par les cytokinines a ete isola a Ia suite d'un criblage d'une banque ADNc de C. roseus. L'analyse de Ia sequence nucleotidique correspondante a montre Ia presence de plusieurs phases de lecture courtes et !'absence d'une longue OAF. La recherche des homologies de sequences a revele de fortes homologies avec des transcrits induits lors de Ia suppression du 2,4-D, a Ia suite de !'addition des cytokinines ainsi que lors de !'induction des tumeurs. Les polypeptides homologues presen­tent une region C-terminale hydrophobe fortement conservee et une region N­terminale variable. D'apres ces travaux ces polypeptides auraient un domaine trans-membranaire C-terminal hautement conserve traversant Ia membrane plasmique, oriente du cote du cytoplasme, et une region N- terminale orientee du cote de Ia paroi cellulaire.

Mots-des : Catharanthus roseus- cytokinines - HyPAP - HyGAP.

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I. INTRODUCTION

Phytohormones have pleiotropic effects on plant growth and developmental processes. During the last decade studies at the biochemical and molecular levels revealed that phy­tohormones regulate gene expression at the transcriptional, post-transcriptional, translatio­nal and post-translational levels (Hagen, 1987; Cotton et a/., 1990; Jacobsen & Gubler, 1992). Cytokinins were less studied than the other phytohormones and their molecular mechanism of action remains unknown. Recently, studies at the biochemical and molecu­lar levels suggested that these phytohormones modify the abundance of specific mRNAs and therefore the corresponding polypeptides ( Dominov et a/., 1992; Chen et a/., 1993; Sano & Youssefian, 1994; Teramoto eta/., 1995). These effects seem to occur through the regulation of gene expression at the transcriptional and/ or post-transcriptional levels (Schmulling eta/., 1989; Lu eta/., 1992; Schmulling eta/., 1997). To determine the effects of cytokinins on gene expression, a multitude of biological models were used. The most frequently experimental system that have been chosen to investigate the influence of these phytohormones are cell suspension cultures.

Catharanthus roseus (C20) cell cultures have been widely used as a model system to identify molecular markers of the promotive action of cytokinins on indole alkaloid accu­mulation. Merillon eta/. (1991) found that transferring C20 cells from a medium contai­ning 2,4-D to a 2,4-D depleted one induced the alkaloid synthesis. Adding cytokinins to induced cells (cultured on a 2,4-D free medium) highly stimulated the alkaloid amounts (Merillon et a/., 1991; Decendit et a/., 1992; Ouelhazi eta/., 1993 ). Recently, the analysis of cytokinin effects on gene expression in C. roseus C20 cells showed a correlation bet­ween the abundance of specific cytosolic proteins in response to cytokinins and alkaloid production (Ouelhazi et a/., 1994; Carp in et a/., 1998; Limam et a/., 1998 ). It was shown particularly that exogeneously added cytokinins induce the accumulation of stress related proteins such as pathogenesis related proteins (Carpin eta/., 1998). In a preceeding study we investigated the effects of cytokinins (BAP) on gene expression in C. mseus cells through the analysis of microsomal polypeptide patterns by 2D-PAGE. This approach sho­wed that cytokinins enhance specifically, in a C. roseus plasmalemma enriched fraction, the accumulation of a 33 kDa membrane polypeptide. Time course studies under different hormonal conditions. revealed that this enhancement occurs after 24 hours of cytokinin treatment and persists for at least 96 hours (Chahed eta/., 1999). In the litterature, it was shown that high concentrations of cytokinins (1-10 11M) strongly amplify the expression of stress induced genes corresponding to structural proteins such as proline or glycine rich proteins (Sheng eta/., 1991; Watillon eta/., 1991; Harding & Smigocki, 1994; Cheng et a/., 1996; Goodwin eta/., 1996; Marty et a/., 1996 ).

It is well known that several cytokinin-regulated genes were identified by northern blot analysis when the reactions of already isolated genes towards cytokinins were tested. In the present work we report the characterization of one eDNA whose corresponding mRNA increases in abundance during cytokinin treatment. Sequence analysis revealed however, the presence of several stop codons throughout the sequence and the absence of a long open reading frame. Searching for nucleotide and amino acid similarities revealed great homologies with cDNAs induced after the suppression of 2,4-D or after cytokinin treat­ment. A striking homology was found with cDNAs corresponding to hybrid proline rich proteins and particularly with a recently reported tobacco eDNA which encodes a novel glycine rich hydrophobic protein.

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II. MATERIAL AND METHODS

A. Cell culture and hormonal treatments The 2,4-D-dependent, cytokinin independent C20 line of periwinkle (Catharanthus

roseus ( L.) G. Don, Apocynaceae) was cultured on a rotary shaker ( 100 rpm) at 25 oc in the dark (Ouelhazi et a/., 1993 ). Cells were subcultured weekly (dilution 1: 10) in B5 medium (Gamborg eta/., 1968) additionned with 58 mM sucrose and 4.5 J.!M 2,4-D. For experimental purposes cells were first subcultured in a 2,4-D-free medium then 8J.!M BAP were added to the culture at day 3 as described previously (Ouelhazi et a/., 1993 ).

B. Isolation of a cytokinin induced eDNA and northern blot experiments A cytokinin induced eDNA showing sequence homologies to hybrid glycine rich-pro­

teins was isolated after screening a C. roseus eDNA library with oligonucleotides corres­ponding to the N-terminal aminoacid sequence (E A E V K K E E P) of a previously characterized cytokinin enhanced membrane polypeptide (Chahed eta/., 1999). After two rounds of screening, eight clones were isolated. In a preliminary approach we investigated the responses of already isolated genes towards cytokinins by northern blot. For this pur­pose, cells corresponding to control and cytokinin treated cultures were harvested at days 4, 5 and 7 (respectively 24, 48 and 96 h after addition of BAP) and homogenized in extra­ction buffer: 50 mM Tris-HCI pH 8.0, 5 M guanidine thiocyanate, 2% N-lauroylsarcosine, I% 2-mercaptoethanol. Total RNAs were then isolated according to the procedure descri­bed by Chomszynski and Sacchi ( 1987). RNA quality was checked through visualisation of ribosomal RNA bands (25S and 18S). For northern analysis equal amounts of RNAs were loaded onto I% formam ide/ formaldehyde denaturant agarose gel using a standard protocol (Sambrook eta/., 1989). Inserts corresponding to the different clones were isola­ted and labeled with a digoxygenin-DNA labeling Kit (Boehringer Mannheim). Hybridization was carried at 42 oc during 16 h in a solution consisting of 50 mM Tris-HCI pH 8.0, 5 X sse, 50% formamide, 0.02% SDS, 0.1% N-lauroylsarcosine and 2% blocking reagent. Washes were realized at high stringency : 0.1 X sse at 68 oc for 2 X 15 min. Blocked sheets were then incubated with an alcaline phosphatase labeled anti-digoxigenin antibody and revealed with NBT and X-Phosphate according to the manufacturer's ins­tructions (Boehringer Mannheim).

C. Genomic DNA extraction and southern blot experiments Due to the high polysaccharide content of C. roseus cells, genomic DNA extractions

were carried as described by Rogers and Bendich ( 1994 ). DNA was quantified by spectro­photometry and quality was checked by electrophoresis on I% agarose gel. DNA was sepa­rately digested with five restriction enzymes that do not cut within the CR2.15 eDNA (EcoRl, Hindlll, BamHl, Xhol or Sail). The southern steps were carried out according to Sambrook eta/. ( 1989). Washes were realized at high stringency: 0.1 X sse at 68 oc for 2 x 15 min.

D. Sequencing Sequencing was carried out using the Thermosequenase and the sequenase version 2.0

kits (Amersham). Both forward and reverse primers were used in sequence reactions. Subclones were generated after digestion with appropriate restriction enzymes and ligation into pUC 19 or pBluescript KS vectors. Sequencing was performed using a combination of forward and reverse primers and internal oligonucleotide priming.

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III. RESULTS AND DISCUSSION

Catharanthus roseus cells were maintained in 85 medium containing 4,5 11M 2,4-D (+2,4D). For experimental purposes cells were first subcultured in a 2,4-D free medium(-2,4-D) then 8 11M BAP was added to the culture at day 3. For northern blot experiments, cells corresponding to control and cytokinin treated cultures were harvested at days 4, 5, and 7 (respectively 24, 48 and 96 hours after addition of BAP).

A. eDNA library screening and northern blot After screening the eDNA libra­

ry, the expression of the isolated cDNAs towards cytokinins was determined by northern blotting. Visualization of ethidium bromide stained gels confirms equal loading of RNA in all lanes (not shown). Northern analysis revealed that the steady state levels of only one eDNA referred to as CR2.15 (I ,6 Kb in length) were highly increased in cytokinin treated cultures. Using a digoxygenin labeled probe the hybridization signal of the corres­ponding transcript was clearly amplified after 24, 48 and 96 hours of BAP treatment (Fig. I). Three other cytokinins ( iade, zeatine, kinetine) as well as abscisic acid stimulated the expression of this mRNA.

Based on RNA markers, the length of the CR2.15 transcript was estimated to be I ,2 Kb. Therefore, the CR2.15 eDNA sequence seems to cover more than the full length of it's corresponding mRNA.

B. Southern blot As shown in Figure 2, southern

analysis predicts a small multigene family or a single gene containing at least one intron. In fact, based on the restriction endonuclease reco­gnition sites within the cytokinin induced eDNA a single band was observed in digests from BamHI and two bands in EcoRI and

Fig.1.- Changes in the levels of CR2.15 transcripts after cytokinin treatment. Hormones were added at day 3 and total RNAs (corresponding to control and cytokinin treated cells) were extracted after 24 (A), 48 (B) or 96 h (C) of cytokinin treatment. Equal amounts of RNAs were loaded. For A and 8 (res­pectively 24 and 48 hours of cytokinin treatment): tracks 1,2: 10 IJg; tracks 3,4: 20 IJg,; tracks 5,6: 30 IJg. For C: 30 1-1g of total RNAs were loaded for each treatment. A 1, 3, 5, B 1, 3, 5 and C 6: control cells. A 2, 4, 6, B 2, 4, 6 and C 1: cells treated at day 3 with 8 iJM BAP. C 2: cells treated with 10 iJM abscissic acid. C 3: cells treated with 5 IJM zeatine. C 4: cells treated with 10 IJM kinetine. C 5: cells treated with 10 iJM isopentenyladenine (iade).

Fig.1.- Analyse par northern de l'effet des cytokinines sur !'expression du gene correspondant au transcrit CR2.15.

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Fig. 2.- Southern analysis of C. roseus genomic DNA probed with the CR2.15 eDNA. DNA was digested separately with EcoRI (E), Hindlll (H), BamHI (B) or Xhol (X). DNA was also double digested: H+B, E+H, E+B. Position of molecular markers is indicated on the left.

Fig. 2.- Analyse par southern de !'organisation genomique du gene correspondant a I'ADNc CR2.15.

Hindlll digests (Fig. 2). This result suggest that the encoding region of the related trans­cript corresponds to more than two genes or is separated by a few introns.

C. Sequence analysis The nucleotide sequence of the cytokinin induced eDNA was determined (Fig. 3 ). It

involves many termination codons and no long open reading frame. However, classic poly­adenylation signals and poly A tail were found. Positions of potential initiation and termi­nation codons in the three possible reading frames are shown (Fig. 4 ). A cytokinin repressed eDNA with similar characteristics was described by Teramoto et a/. ( 1995, 1996). The amino acid sequences deduced in the three possible reading frames revealed many termination codons throughout each sequence and no long ORF was found. These authors suggested in the absence of a significant homology with data bases that the cor­responding RNA could not be translated and may function as a regulatory factor.

599 1090

Fig. 4.- Search for OAFs (in the three reading frames) using the DNA Strider program. Short vertical bars indicate position of initiation codons (ATG ) and long ones indicate position of termination codons (TAA, TAG and TGA).

Fig. 4.- Recherche des phases de lecture ouvertes le long de I'ADNc CR2.15.

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1 l',6GCGTITCTCC'ITACCAGA1IiTAIIiC:.V.GCG.1IiAA'ITATCAAMATIGCTGGTGTATTiuillll 66

6? CCAAGGGT'ITCTI'GTGCTGCAGC'ITCTGCAGAmGTGc:::I:GACC.IACOATCCTGCTGTTGc:;:r:GAACCT 13 2

13 3 CCT~CCT'ITGCTGGAGAGGC'ITGCAAAGGAGCTCCGAGTCCAATIGGGCCTGCAGCT'ITTC 19 8

199 MTA'l'lU\c:A.l'AA'ITCGAGAGTAIIiGGAC:.V.GAGATCTATA'IT.A.I!irGA'l'lU\CATC:.V.CTAT'ITC 264

2 6 5 CCTGGATAIIiG.v.J>.AAmCCGGM TACGAGC'.AJIZT A T'IT ACAGA 'IT'ITC'ITCTCAGCCT.I:GAAC:.V. 3 3 0

3 31 AGCAAAA TACAAGMGCCT AC'I'G'l'liiAGJ:AGCC.AIIiGAGTCCTCCAAMAGTGGMGIAAAGIAAA 3 9 6

529 T'ITATI'GTATA'ITCC'ITGTGGACATCATATCTACTCA.:I'AAT.IACZCAMTCTIGI IGGMGM'IT'IT 594

59 5 TCA 'ITC'ITACCCAGCCAGCCTC:.V.GCTC:.V.GGAGTGTTCT'ITC'ITCjCTTG'l'lU\ TCTCCCAGMG 6 6 0

6 61 TGCAGCCCCG'ITCTCAGATGACA 'ITTCTCGACA~GCA'l'lU\~GGGTTCTTGGAT:I:A.AAA 7 26

7 2 7 GMGA~GM'I'JliAATGAGGCT'ITA.'IGTACCA:I:GATGA'ITTGG~M~GA'IT 7 9 2

793 Il"IGIIIIGTGIIICTA'ITT'ITAc:;:r:GACAAAT'ITAT'ITAT'ITA T'IT TCA AAA 'ITA GCT 850 F S K L A

851 CCA 'ITA TCA ATC 'ITC CTA 'ITA GTG AAC T'IT CTC 'ITC T'IT GCT CTA ATC 898 P L S IFLLVNFLFFALI

899 MT GCC TCA TCT TCT TGT GTT ACC TGC CCC AAT AAC CCC CCT TCA A'IT . 946 N A S S S C V T C P N N P P S I

9 4 7 CCA GGA ACC GGA GGC GGC AGT GGC GGT GGC GGT GGC AGT GGC GGT GGC 9 9 4 P G T GGGSGGGGGSGGG

995 AAT GGA GGA GGA GGT GGA GGT GGA GGA GGA AGG TGC CCT AGA GAT GCA 1042 N G G GGGGGGGRCPRDA

1043 TIG AAA TIG GGA GTA TGT GCC AAT 'ITA C'IT GGA GGA 'ITA GTT GGG GTG 1090 L K L GVCANLLGGLVGV

1091 AGA A'IT GGG AGT CCA CCA ACA 'ITA CCA TGC TGC AGT TTG C'IT AGT GGA 1138 RIG SPPTLP CCSLLSG

1139 'ITA GCA GAT C'IT GAA GCA GCA GCT TGC C'IT TGC ACA GCC ATA AGA GCT 1186 L A D L E A A A C L C T A I R A

1187 MC A'IT TTG GGT ATA AAT CTA AAT GTC CCA A'IT TCC CTC AGT TTG A'IT 1234 N I L G I N L N V P I S L S L I

1235 C'IT MC AAT TGC GGA AGG AAT CCT CCT ACT GGC 'ITC A'IT TGT TGA 'ITC 1282 L N N C G R N P P T G F I C

1283 ACCAAACAATACIIIGIIATA'ITCGGGGCTCGAGTC'ITCTGTATCACIIIIIGTACCA'ITCTCTAT 1348

13 4 9 ATCACTTTCTAATCTGATATATAGATAAATACACAAAATGATACAGMGATCCGGATACAGAGAAC 1414

1415 GTCCTAATAA'ITA'ITCCCATGIIIIGIIIIGGGTCATGGGAIIIIGIGIGTGGTT'ITCCGTTGG'IT 1480

1481 'ITCTTCMGTTGTAA'ITM'ITA'?.GG'ITTGGACTAAAAGG'ITC'ITAA'IT~ 15 46

1546 1611

Fig. 3.- Nucleotide sequence of the CR 2.15 eDNA. Putative initiation and termination codons (from nt 1 to nt 850) are underlined. Significant nucleotide and amino acid homo­logy starts from the underlined GGA (glycine) to the termination codon TGA (nt 1279). The putative amino acid sequence showing sequence homologies to hybrid proline (or glycine) rich-proteins is indicated.

Fig. 3.- Sequence nucleotidique de I'ADNc CR2.15.

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Searching for CR2.15 nucleotide and amino acid similarities revealed, in contrast with the previous study, a great homology from nt 950 to nt 1279 (Figs. 5, 6) with cDNAs indu­ced after removal of auxin from the cell cultures (Aieith & Richter, 1990; Hotze et a/., 1994) or after cytokinin treatment (Subramaniam eta/., 1994). This homology (up to 90%) covers only a highly hydrophobic C-terminal domain that contains membrane spanning regions (Goodwin eta/., 1996; Yasuda eta/., 1997); the cDNAs CR.l4 (562 pb) and DC2.15 ( 600 pb) issued respectively from Catharanthus roseus (Hotze et a/., 1994) and Daucus carota (Aleith & Richter, 1990) saw their expression enhanced after removal of 2,4-D. The hydropathy plot of the deduced polypeptides indicates a predominance of hydrophobic amino acids and the presence of putative membrane associated helices within the C-terminal domain. The cuscuta HyPRP eDNA (1,6 Kb) correspond to an mRNA the levels of which increased following cytokinin treatment. This increase was prevented by auxin (Subramaniam eta/., 1994). As shown in figure 6, the deduced polypeptide corres­pond to an hybrid prot in rich-protein of 329 amino acids containing a partially hydropho­bic N-terminal region and an hydrophobic C-terminal domain of I 09 amino acids (Subramaniam et a/., 1994 ).

A similar homology was also found with cDNAs that were preferentially expressed after a stress (maize HyPRP) or during the development of tumorous tissues (Fujita & Ichikawa, 1994; Yasuda et a/., 1997). The protein corresponding to the maize HyPRP eDNA (MM 31,6 kDa) presents a proline rich region characteristic of cell wall associated proline rich proteins (Ye eta/., 1991) followed by the C-terminal hydrophobic domain (Estanyol eta/., 1992, Fig. 6). Transcripts corresponding to cDNAs NTI6 (900 pb) and TID23 were res­pectively induced by the T-DNA genes of Agrobacterium tumefaciens (Yasuda eta/., 1997) and during the development of tobacco genetic tumors (Fujita & Ichikawa, 1994). The accumulation of NT 16 transcripts was also induced by mechanical wounding (Yasuda et a/., 1997).

The comparison of the domain configurations overall these homologeous polypeptides (Fig. 5, 6) revealed the presence of three different domains (Yasuda eta/., 1997):

- the domain I, a non conserved amino terminal hydrophobic region containing putative signal peptides responsible for membrane insertion of proteins (Wickner & Lodish, 1985); -the domain II (Fig. 5, 6) is proline rich (CR.I4, DC2.15, cuscuta HyPRP, maize HYPRP and BNPRP) or glycine rich (NTI6, CR.l5); - domain III is a highly conserved carboxy-terminal hydrophobic region (Fig. 5, 6).

Overall these polypeptides, eight cysteine residues are well conserved (Fig. 5, 6).

Yasuda eta/. ( 1997) suggested that these proteins are anchored in the plasma membra­ne via the carboxyl hydrophobic domain and may act as connectors between the cytoske­leton and the cell wall. Similarly, Goodwin et a/. ( 1996) suggested that the BNPRP encoded protein is secreted from the cell wall and is anchored in the plasma membrane via the C-terminal domain (Fig. 6) that contains three putative membrane spanning regions (Goodwin et a/., 1996). They suggested that BNPRP maintains cellular integrity in cold stressed plants by forming strong linkages between the cell wall and the plasma membra­ne.

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a) CR2.15

b)NT16

a) CR2.15 b)NT16

a)CR2.15 b) NT16

a)CR2.15 b) NTI6 c) pZRP3 d)DC2.15 e)CR.l4 I) TrmRNA

a) CR2.15 b)NT16 c) pZRP3 d)DC215 e)CR.l4 t)TrmRNA

al CR2.15 b)NTI6 c)pZRP3 d)DC215 e)CR. 14 f)TrmRNA

a) <...""R2.15 b)NTI6

c) pZRP3 d)DC215 e)CR.l4 t)TrmRNA

836 [FisK L!AlP L 5 l [Fi). V f-Y} ~Ll N AS ss!C]v r[ClPN!Nl

l.£1KT R~ 5 V TL[_0; L ~~IV 5 G T D -E.F 5 E_j-1 Hl.:::J

PPSTGNGGGNGSNTGGSG NGGG SG

[Llfil~~ R liJ G 5 P P fTl fLl P C C 5 L [g 5 L!:Jtjl£.] I V G 5 P P W I!J P C C 5 L l A --- V KVGL prQly E [§Jcc[BL L E VHN V VriJGS r'-J!'[!j[!JP CC 5 L L E II!ISAVl!JG~PPKT PCC SLI E L!JI]@J A I V G 5 P P [!j [g P C C 5 L I A

::~~s ~ ~~c : r 871 c) pZRP3 F T C P N X [631 d)DC215 F E C T X [i11 elCR.l4 F l!J C A X [691 t)Tr mRNA F T C X [831

Domain III

Fig. 5.- Comparison of the putative CR2.15 amino acid sequence with the homologous poly­peptides. Identical amino acids are boxed. (X): indicates the stop codon. The polypep­tides reported are: NT16 (Yasuda et at., 1997); pZRP3 (John et a/.,1992); DC2.15 (Aieith & Richter, 1990); CR14 (Hotze eta/., 1994) and TID23 (Fujita & Ichikawa, 1994). The percentage of homology is indicated.

Fig. 5.- Comparaison de Ia sequence en acides amines deduite a partir de I'ADNc CR2.15 avec les polypeptides homologues: NT16, pZRP3, DC2.15, CR.14 et TID23.

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a)CR2.15 b) RCc2

a)CR215 b) RCc2

c) BNPRP d)SACSI

~lADRI1.2

t) Cuscuta HvPRP g)TPRP-Fl

hlPVRS i) Matze H,·PRP

a)CR2.15 b)BNPRP c) RCc2 d)SACSI

e)ADR11.2 t) CuscutLl f---.,·PRP

g)TPRP-Fl h)PVRS t) Ma1ze H,·PRP

tl Matze H\"PRP - K H A P

X X I 48[ X I 71[

319

Dom.~in 111

Fig. 6.- Comparison of the putative CR2.15 amino acid sequence with the homologous polypeptides. Identical amino acids are boxed. (X): indicates the stop codon. The poly­peptides reported are: BNPRP (Goodwin eta/., 1996); RCc2 (Fang eta/., 1991); SAC51 (Coupe eta/., 1993); ADR11.2 (Datta eta/., 1993); Cuscuta HyPRP (Subramaniam eta/., 1994); TPRP-F1 (Salts et a/., 1992); PVR5 (Choi et a/., 1996) and Maize HyPRP (Estanyol eta/., 1992). The percentage of homology is indicated.

Fig. 6.- Comparaison de Ia sequence en acides amines deduite a partir de I'ADNc CR2.15 avec les polypeptides homologues: BNPRP, RCc2, SAC51,ADR11.2, cuscuta HyPRP, TPRP-F1, PVR5 et maize HyPRP.

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IV. CONCLUSION

It is well known that cytokinins are involved during systemic responses to stress. It was shown particularly that these phytohormones enhance the levels of stress related cytosolic proteins such as pathogenesis related proteins. Also, elevated levels of cytokinins strongly amplify the expression of genes corresponding to structural proteins such as proline (or glycine) rich-proteins. The tatters as those described by Goodwin et a/. ( 1996) and Yasuda eta!. ( 1997) are known to be induced by stress. They are postulated to act as connectors between the cytoskeleton and cell wall and are membrane or periplasmic associated.

In spite of the absence of a long open reading frame, the CR2.15 eDNA shows strong similarities with a highly conserved domain issued from cDNAs that are preferentially induced after suppression of 2,4-D or after cytokinin treatment. Further investigations may help in determining the role of the corresponding transcript in alkaloid production.

Acknowledgements - This work was supported by a grant from the Tunisian government (Secretariat d'Etat a Ia Recherche Scientifique et a Ia Technologie. Project code number: P93/ IEP 09). We wish to thank Pr M. Rideau and Pr S. Hamdi for the gift of the eDNA library.

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