Temporal and spatial expression of mRNAs ... - Wiley Online Library

Plant, Celt and Envlrontnent(^997)2Q, 1517-1524

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra^ S. A. COUPE,'-r ;. E. TAYLOR-& J. A. ROBERTS' 'School of Biolo!>y, University of Nottitigliatii. Stittoti Botutigton Catnptts. Lotighhorotigh, Leicestershire LE12 5RD, UK, and ^Division of Biological Sciences, Institttte of Ettvirotvncntal and Biological Sciences. Lancaster Utiiversity, Laticaster LAI 4YQ, UK

ABSTRACT Differential screening of a cDNA library generated from RNA extracted from etiiylene-treated leallet abscis.sion zones of Sawhucus iiigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesisrelated (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase aiid a metallothionein-like protein (Coupe, Taylor & Roberts 1995. Planta 197, 442-447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to etiiylene at a time when abscission of the leaflet explants bad reached 70%. Expression of the PPO and the Chia4-type ebitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in tbe absence of tbe gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed. Key-words: Satnhttctts nigra; abscission; e(hylene; gene expression; pa(hogetiesis-t-elated proteins; woutiding.

INTRODUCTION Abscission is the physiologieal process by which a plan( sheds an organ s(tch as a leaf, flower or fruit. This event is (he consequence of cell wall dissolution and involves the separation of cells coniprising the abscission zone (Sexton *The tnwleotide sequence data reported will appear in the EMBL, Genhank and DDBJ Nucteotida Sequence Databases wider the accession numbers Z46946 (JETH), Z46947 (JET9), Z46948 (.IETI5). Z46949 (JETII). Z46950 (,IETi9). •\Presetit address: Leviti Research Centre, Crop and Food Research, 4005, Levin, New ZeaUnid. Correspotidetice: Jerettiy A. Roberts. Fa.\: 0115 951 6334: e-tnail: jeremy.roberts@nottinj>ham.ac.uk © 1997 Blaekwell Seienee Ltd

& Roberts 1982). Thete is compelling evidence that the plant gtowth (egulator ethylene p(omotes the process, and that this is a consequence of the synthesis of new RNAs and ptoteins. Sotiie of these niay encode peptides such as cell wall hydrolases (Sexton & Roberts 1982). Abscission of the leallets of the herbaceous plant Sanihtictis nigra has proved to be an effective model system for the study of abscission, as the zone of sepatation is made up of a sufficient number of rows of cells to facilitate the extraction of proteins and nucleic acids. S(udies on this plant have revealed that during ethylene-stitnulated leaflet abscission (he activities of both polygalacturonase and /3-1,4-glucanase increase specifically at the site of separation (Taylor et al. 1993; Webb et al. 1993) and that this is correlated with an inctease in the expression of genes encoding these etizytnes (Taylor et al. 1994; Roberts et al. 1997). Additional ptoteins have been teported to be produced dut-ing leaf abscission in other species (Kelly et al. 1987; Gomez Lim et al. 1987). For instance, del Campillo & Lewis (1992a) showed that abscission of (he pritiiaiy leaves of bean was acconipanied by the accumulation of a nutnber of PR ptoteins. Upon N-tertninal sequencing, the proteins were found to have homology to chitinases, PR-1 type, PR-2 type iP -1,3-glucanase) and PR-5 type (tbaumatin-like) ptoteins. Such ptoteins were also found to accumulate in bean anthet\s and pistils during flower ab.scission (del Campillo & Lewis 1992b). del Catnpillo & Lewis (1992b) pt-oposed that the PR ptoteins were either involved in the cell separation process itself or protected the plant frotn pathogenic attack. Although extensive wotk has been undettaken on the biochemistty and physiology of abscission, little is known about the niolecular events and signalling components that ate involved in regulating cell separation. One apptoach (o studying this has been to isolate the genes encoding certain key enzynies, such as cell wall hydtolases, and analyse (he protTioters regulating their expiession (Koehler et al. 1996). However, the exptession of additional geties tnay conttibute, in a critical way, to the abscission process and their identification will be itiiportant in furthering our understanding of how the ptocess is regulated. With this in tnind we have etnbarked on a diffetential scteening strategy using an ethylene-stimulated leaflet abscission cDNA 1517

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S. A. Coupe etal.

library from S. tiigra. This approach led us to the isolation and characterization of a r-netallothior-rein (MT)-like protein (Coupe et al. 1995) that was found to be highly expressed in ethylene-tr-eated abscission zone tissues at a tir-ne when cell separation was taking place. The peptide was highly homologous with that of another MT-like protein isolated from a Brassica napus leaf senescence cDNA library (Buchanan-Wollaston 1994). The funciion of this group of proteins remains unknown, but they could play a scavenging role in the pr-otection of cells from free radical groups that n-right be generated during the induction of pathogenesis-related pr-oteins (Buchanan-Wollaston 1994; Coupe et al. 1995). In this paper we report the isolation and identification of additional abscission-r-elaled gene pr-oducts whose expr-ession is stimulated during cell separ-alion. These r-riRNAs encode PR proteins and their specific functions are unknown, but they r-nay protect the exposed fractur-e face frorrr pathogenic attack.

MATERIALS AND METHODS Plant material Leaves of S. nigra (elder) wer-e collected fror-n bushes growing near the Sutton Bonington Campus. The leaflets wer-e excised and the rachis severed at sites 2 mm distal and 10 r-nm pr-oxin-ral fVorn the position of leaflet attachments to generate 'explants". For ethylene tr-eatrrrents, the basal end of these explants was embedded in 1 % plain agar in a 90 rrrm Petri dish and then incubated in an environment of ethylene (10 cm^ WT""). The ethylcne-tr-eated explants wer-e then harvested into 'zone' and 'non-zone' tissues at various tir-ne intervals and the per-centage abscission calculated as deser-ibed in Taylor et al. (1993). Explants wer-e also maintained in an atrnospher-e devoid of ethylene by the inclusion of an absorber of the gas (ethysor-b). Ethylene accur-nulation was rrreasured during this tirrre using a Pye Series 104 Gas Chr-omalogr-aphy using the proccdur-c outlined in Meakin & Roberts (1990). All tissues were imrrrediately har-vested into liquid N2 before stor-age at -70 °C for RNA isolations.

Library screening Total RNA was exlracled as described in Taylor et al. (1990) and used to isolate poly(A)^ RNA to construct a cDNA libr-ary (Rober-ts et al. 1993). Differential scr-eening was performed using single-stranded cDNA probes synthesized from 1 /yg poly(A)'' RNA isolated from the zone or non-zone material. The pr-obes wer-e synthesized using the method of Picton et al. (1993) and used lo scr-een 150 000 r-ecombinanl plaques plated on three plates of 50 000 each. Replicate lifts wer-e pr-obed and hybr-idized as described in Coupe et al. (1993). Plaques that differ-entially hybridized to the zone cDNA probe were le-screened at lower densities. Selected positive phage clot-res were conver-ted to pBluescript SK , using in vivo excision following the manufactur-ers' instr-uctions (Str-atagene, USA).

DNA sequencing Plasn-rid DNA was isolated using standard methods (San-rbr-ook, Fritsch & Maniatis 1989). Both str-ands of the appropriate plasmid insert were sequenced using modified primer-s, hon-rologous lo Ihe T3 and T7 bacleriophage pr-omoter sites contained in the pBluescript plasmid, as well as internal oligonucleotide primers (Oswel DNA, Bdinbur-gh). The methodology employed was based on the dideoxynucleolide chain termination n-rethod (Sanger, Nicklen & Coulson 1977) and ulilized a Taq dye deoxy ter-minator cycle sequencing kit and a 373 A DNA sequencer (Applied Biosystems Inc.). The obtained sequence was compared lo EMBL and GenBank DNA databases for homology. Amino acid sequence analyses wer-e performed by translation of the obtained DNA sequence using cither the DNAstar or GCG/Wisconsin prograni package (Devereux, Haeberii & Smithies 1984) and searched against the Swissprot EMBL databank using Ihe BLAST (Altschul et al. 1990) and FASTA algorithm (Pear-son & Lipman 1988).

RNA isolation and northern analysis Total RNA was extr-acted IVom frozen explatit tissue using the method described by Coupe et al. (1993) for northern analysis. Northern analysis was carried out using 10 ^g lolal RNA and the recommended Genescreen protocols (NEN, DuPont, UK). Radio-labelled probes were gener-ated using 100 ng of insert from the various plasn-rid pJET clones. The inser-l was Gelase (Epicerilr-e, c/o Cambio, UK) extracted from low-melting point agar-ose (FMC products) and labelled using the method of Nick Tr-anslation (Rigby el al. 1977). The blots were hybridized and washed as described in Coupe ('/ al. (1995) except that Ihc nor-thern was r-epeatedly stripped and re-pr-obcd.

RESULTS Isolation of abscission-zone-specific clones Differ-ential screening of a cDNA libr-ar-y constructed IVor-n ethylene-tr-eated abscission-zone tissue (Roberts et al. 1993; Coupe et al. 1995) led lo Ihc i.solation of 20 clones whose expression was up-regulated during the process leading to cell separation. Upon partial sequencing and cross-hybridization studies these cDNAs were gr-ouped into seven fan-rilies and fur-ther char-acterized by northern analysis. One abscissiori-r-elaled clone (termed pJET12) was found to be also expr-essed dur-ing leaf senescence, and sequencing of this cDNA revealed Ihal it encoded a nietallothionein-likc ptotein (Coupe et al. 1995). Representative cDNAs which contained the largest inserts IVom the six remaining families wer-e sequenced fully and wer-e found to encode peptidcs that could be classified as pathogenesis-r-elated (PR) pi-oteins. Accor-ding to van Loon et al. (1994) PR proteins can be defined as 'proteins encoded by the hosl plaril but induced only in pathological or related situations'. The

' 1997 Blackwell Scierrce Lrd. Plain. Cell and Environment. 20. 1517-15

Expression of PR proteins during leaflet abscission

following cDNAs were further characterized by northern and sequence analysis: pJET5, pJET8, pJET9, pJETll, pJET15at-tdp.IET19. Expression of JET mRNAs during ethylenepromoted leaf abscission Eigure 1 shows the accumulation pattern of the transcripts encoded by each of the cDNA clones. The transcript sizes of the messages wer-e 2-0 (JET5), 0-8 (JET8), 0-9 (JET9), 0-4 (JETII), 10 (JET15) and 1-0 kb (JET19). In each case the expression of the message was primarily restricted to the abscission zone tissue. Only .IETI5 showed a significant level of expr-ession in the non-zone tissue (Eig. le). Expr-ession of the abscission-related

0

12

18

Ethylene treatment (h) 24 24 0 12 18

Non-zone

24

24

(b) 0.8 kb -•

mRNAs was detectable within 18 h of exposure to ethylene at a time when cell separation had commenced in the majority of explants. By 24 h, further accumulation of the mRNAs encoding JET5 (Eig. la), JET9 (Eig. lc) and JETll (Fig. Id) had taken place, while that of JET8 (Eig. lb) and JET 19 (Fig. 10 had reached a plateau at 18 h. In eonttast, the level of expression of JET15 appeared to decline in both zone and non-zone tissue after 18 h of treatment with ethylene. Expression of .IET5 and JET 19 in cells of the abscission zone was highly dependent upon ethylene, with no detectable accur-nulation of tiiessage in the absence of the gas. A sn-rall level of accumulation of the mRNAs encoded by JET9, JETll and JET 15 took place in the abscission zone tissue of explants not exposed to ethylene, while expr-ession of JET8 took place even without tr-eatment with the gas. To show that equal atnounts of RNA were loaded for each tir-ne point a photograph of a representative ethidium-stained sjel is also included (Eig. lg).

Zone

(a) 2.0 kb -*

1519

-f •••

(c) 0.9 kb ->

(d) 0.4 kb

(e) 1.0kb->

(f) 1.0 kb

(g)

Figure 1. Nor-thern analysis of total RNA (l()/(g) IVom S. nigra leaflet ahscission-zonc (Zone) and non-ahscissiort-zorre (Non-zone) tissues that had been exposed to tlitfercnr durations of cthylene treattnent (10 ctn' tn ) or incuhatcd lor 24 It in the ahscttcc of the gas (-CTHJ). The rior-rhern hlot was hyhr-idizeil ro rhe following radiolabelled inser-ts: (a) JET."); (h) JET8; (c) JET9: (d) JETl 1; (e) JET 15; (f) JET 19. Panel (g) shows ar-epr-eserrtative erliidiuriistained gel to demonsrrate equal loading of the RNA. © 1997 Blackwell Science Ltd. Plain. Celt and Enyironment. 20. I.SI 7-1."524

DNA sequence analysis of the JET Clones JET5 The insert size of this cDNA was 700 bp which hybridized to a tt-anscript 2 kb in length. Sequence analysis r-evealed that the insert had greatest hormology, at a nucleic acid and amino acid level, with polyphenol oxidases (PPO) from a spcctr-urn of plant species. Highest identity, 65% in a 70 amino acid overlap, was with a PPO from Mcdns domestiea (Boss etal. 1995). JETS

,

The nucleic acid and deduced protein sequence of JET8 can be seen in Eig. 2a. When the putative peptide sequence was compared to that of other proteins in the databases, it was found to show extensive identity with a class of PR proteins that include the potato win senes. Upon sequencing, the insert was found to be 674 bp in length and. although not full length, it had 72% identity over 140 amino acids with the pt-oduct of ihe potato win] gene and 67 and 74% identities over 140 amino acids with similar proteins from tobacco (Eriedrich et al. 1991) and tomato (Linthorst er al. 1991), respectively. The deduced ptotein sequence also contained the signature of the Bar-win domain (underlined in Eig. 2a) thorjght to be involved in the binding of chitin (Svensson et al. 1992) and proposed to play a role in protection against pathogens. However, the putative JET8 ptotein lacked the N-tct-rninal chitin-binding domain found in the win genes. The sequence also contained a polyadenylation signal (Joshi 1987).

JET9

----:-,

Sequencing of this cDNA revealed that JBT9 encoded a full-length open reading fr-ame with an insert size of length

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S. A. Coupe etal.

874 bp (Eig. 2b). The deduced pr-otein cor-npt-iscd 167 amino acids coding for a ptotein with a ptedicted tnoleeular tnass of 18-4 kDa and a pi of 7-90. The sequence contained a signal cleavage site (indicated by the arrow in

Fig. 2b) based on the method of von Heijne (1986), a single glycosylalion site denoted by the motif 'N-X-S or T" (Kornfeld & Kor-nfcld 1985) and a polyadenylation signal (Joshi 1987). After cleavage of the putative transit peptide the molecular mass of the protein would be 14 8 kDa. The JET9 peptide sequence shated hornology with several mRNAs encoding PR-1 type proteins (Eig. 3). The most extensive sequence identities are with PR-1 proteins from barley (56%, Mur-adov et al. 1993) and tobacco (57%, Pfitzner-, Pfitzner & Goodman 1988; 56%, Eyal, Sagee & Fluhr 1992). The function of PR-1 lype proteins is unknown, but they have been characterized in a spectrum of plant species. -

(a) CTTGTGTGTGTTGGTGTTTGTGTGCTTGAGCCTGCTGGTGGGTGG 4 5 L C - V L V F V C L S L L - V G G 1 5 TGGCACTGCCCAAAGTGCCTCTAATGTTAGGGCAACCTACCATAT 9 0 G T A Q S A S N V R A T Y H I 3 0 CTACAATCCTCAGCAAATTAACTGGGATTATAACCGCGCTAGCGT

135

TTATTGCGCAACTTGGGACGCTAACAGGCCGCTAGAGTGGCGGCG 1 8 0 Y C A T W D A N R P L E W R R 6 0 TAGGTATGGTTGGACCGCCTTTTGTGGCCCCGTCGGACCTCGTGG 2 2 5 R Y G W T A F C G P V G P R G 7 5 CCGAGACTCTTGCGGGAGGTGCTTAAGGGTAACTAATACAGG2VAC 2 7 0 R D S C G R C L R V T N T G T 90 TGGAACCTCAGAAACGGTGAGAATTGTAGATCAATGTGCCAATGG G T S E T V R I V D Q C A N G

315 105

AGGTCTGGATTTGGAACAAGGCGTTTTCCAAAGACTGGACACAGA G L D I i E Q G V F Q R L D T D

360 120

.lETIl From sequence data and northern analysis, we estimate that Ihc itisett ftom this cDNA is only just short of encoding a full-length message. The 424 bp sequence (Fig. 2c) has a single glycosylation site and two putative polyadenylation signals. A database search revealed that the JETl 1 sequence had substantial .sequence identity at the amino acid level with a class of PR proteins described as proteinase inhibitors. Closest identity (62 and 51%, respectively) was with peptides from bitter gour-d (Momoidia all rant ia) (Ogata et a I. 1991) and pumpkin (Cucurhiia tnaxitna) (Krishnamoorthi, Gong & Richardson 1990).

TGGAAGGGGCTACGCTCGAGGCAACCTCAATGTGAACTACCAATT 4 0 5 G R G Y A R G N L N V N Y Q F 135 TGTCAACTGCAATGATTAAGGGATGTTTGACTACTTATTATGAAA V N C N D -*

450 140

AATTTACGAGATAAATAATGCTATGCATTTTAAAAAATATTATAG

495

AACAATAAAATGTGAGCTCAACTTGTTGAGATGCTTGTGAGATGG

540

AGATCTAAGAATAAAAATTATATAATATAAATGAATAGTCTTCGA

585

CTATTAATCAAAATGTTTTACATTGAATTTGTTTGCTCTACATGA

63 0

AATGATCAAGTCAATCGTCCGATAACAAAAAAAAAAAAAAAAAA

674

(b) ATrTTCGAAT7AGCAATACAAAGAAACCCAAAATGGCGCATAACC M A H N

45 4

ATTGGTGTAACCTCTTTTCAGTAGCCCTAGTCTGTGTGGTAGCGT H W C N L F S V A L V C V V A TAGTTATGGTGCAATATTCTGTTGCCCAAAACTCACCCCAGGACT L V M V Q Y S V A Q N S P Q D ACGTGGATGCTCACAACGCGGCACGAAGTGCTGTGAATGTGGGGC Y V D A H N A A R S A V N V G CGGTGACATGGGACGAGTCAGTGGCAGCCTTTGCGCGACAGTACG P V T W D E S V A A F A R Q Y CGCAATCAAGGGCGGGGGATTGTAGGCTCGTGCATTCGGGCGACC A Q S R A G D C R L V H S G D CGCGATATGGTGAGAACCTGGCTTTTGGGTCCGGCTTTGAATTAA P R Y G E N L A F G S G F E L CGGGGAGAAACGCCGTGGACATGTGGGTGGCAGAGAGGAATGACT T G R N A V D M W V A E R N D ACAACCCCAACACCAACACGTGCGCTCCGGGGAAGGTGTGTGGAC Y N P N T N T C A P G K V C G ACTACACTCAAGTGGTATGGCGAAACTCGGTCCGGATAGGGTGCG H Y T Q V V W R N S V R I G C CTAGGGTTCGGTGTAACAACGGGGCATGGTTCATCACATGCAACT A R V R C N N G A W F I T C J^

90 19 135 34 180 49 225 64 270 79 315 94 360 109 405 124 450 139 495 154

JET15ctndJET19 These two cDNAs have been grouped together as they were found to have similar sequences (Eig. 4), being 66% identical over an overlap of 261 amino acids. Both sequences are just short of full length, with JET 15 being

(c) AAAGATGGAAGCATGTGCAAGGAGAGTAAGTCATTGCAGAGATGT 4 5 K M E A C A R R V S H C R D V 1 5 GGGAAAGAACACATGGCCAGAGCTGTGTGGGGCAAGAGGAGAGGA 9 0 G K N T W P E L C G A R G E E 3 0 AGCTGCAGCCACCGTTGAGACGGAAAACCCTTCTGTCACTGCTGT 13 5 A A A T V E T E N P S V T A V 45 TATTGTGCCAGAAGGATCGATCGTCACAACAGATGAGCGGTGTGA 180 TAGGGTTCGTGTTTGGGTCGATGAAAATGGCATTGTTACCAGGGT 225

ATTCTCCTCCCGGCAACTACGCAGGACAGCGTCCATACTAGTTTT 540

CCCTGTCATTGGTTAGGTTTCCAACAAAATTGCTCXSCTCaAAATG 270 P V I G * 79

ACACAATCCAATAAAAAAAATTATTATTATGAATAATTGCATTGA 585

TGCTATAATTAAATAAATATTATTTTACTTTTGTAAGGTGCATCT 315

GCACTTCTAGTTTTATATTTTATGCAACATAAAAGTTCGGATTGC 63 0

GCCCCCATTACAATTGTAAGACGGGCCATTCTATTGTGCTATATT 3 60

ATACATAATCTGAAACGATCAAGTACTAGTAAGCTGTTAGTCTTG 67 5 CAGCAACTACAGCTAATAAAATGTAATCGAGAAATAAGCTGTATC 7 20 TCGGGTGCATAAATGTAGGGATAATTGGAATATCTAGGGCGGTGA 765 AGTGAGAGATTAAGACATAGATGTAATTATCCCCTAAATGCCATA 810 TTTAAGTCTTTTGTTGAAAACAGACTTTTGTCTATTTGCTTGTGT 855 AAAAAAAAAAAAAAAAAAA

874

CTAAATAAGAGCGGCATAATAAAATAATGTTATTGTTTCCCTGGA 405 AAAAAAAAAAAAAAAAAAA 424 Figure 2, Nucleotide and deduced amino aeid sequences of (a) JETS cDNA, (b) JET9 cDNA and (c) JETl 1 eDNA. The terrriination eodons ar-e indieated by a single asterisk. Putative glycosylation and polyadenylation sites are doubly untlerlined. The Barwiii titotils rite singly trndetlincd in JETX. The putative cleavage site of the signal sequence in JET9 is indieated by the ariow.

© 1997 Blackwell .Scienee Ltd. Plam. Cell and Enyironmenl. 20. I.SI7-1.524

Expression of PR proteins during leaflet abscission

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JET9.PRO PRIATOB.PRO PR1BTOB.PRO PR1HORVU.PRO JET9.PR0 PR1ATOB.PRO PR1BTOB.PRO PR1HORVU.PRO JET9.PR0 PR1ATOB.PRO PR1BTOB.PRO PR1HORVU.PRO JET9.PRO PR1ATOB.PRO PR1BTOB.PRO PR1HORVU.PRO

-

JVCCi

HYTQVVWRNSVRVGC

" V C G H Y T O V V W r ••" '- - •• ' -

156 157 157 E 153

GYVVSCNYD GYVVSCNYD

' V C C? H Y T O V V W n

JET9.PR0 PRIATOB.PRO PR1BTOB.PRO PR1HORVU.PRO

167 168 168 164

Figure 3, Comparison olplanr PR-1 type proteins with the derived prorein sequence ol JET9. The sequences aligned with JET9.PRO are from robacco. PR 1 ATOIB.PRO (Plitzner el al. 19