* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Characterization and expression of an mRNA encoding a wound
RNA silencing wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Point mutation wikipedia , lookup
RNA interference wikipedia , lookup
Non-coding RNA wikipedia , lookup
Gene expression programming wikipedia , lookup
Designer baby wikipedia , lookup
Protein moonlighting wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Messenger RNA wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
History of genetic engineering wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Primary transcript wikipedia , lookup
Gene expression profiling wikipedia , lookup
Epitranscriptome wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Journal of Experimental Botany, Vol. 48, No. 311, pp. 1223-1227, June 1997 Journal of Experimental Botany Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue1 Neil Harris3, Jane E. Taylor4 and Jeremy A. Roberts2'5 2 Department of Physiology and Environmental Science, Faculty of Agricultural and Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK 3 Department of Biology, University of St Andrew's, St Andrew's, Fife KY169TH, UK 4 Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK Received 23 September 1996; Accepted 25 February 1997 of several hydrolytic enzymes which have been proposed to be responsible for bringing about the degradation of A cDNA clone (TAB7) encoding a putative woundthe middle lamella, and hence cell separation. The two induced (Win) protein has been isolated from a tomato most widely documented enzymes falling into this cat(Lycopersicon esculentum Mill. cv. Ailsa Craig) leaf egory are jft-1,4 glucanase (cellulase) (EC 3.1.2.4) and abscission zone cDNA library using a differential polygalacturonase (PG) (EC 3.1.1.5) (Taylor et ai, 1993; screening strategy. The clone has a high degree of Webb et al., 1993). In tomato, mRNAs encoding these homology at the amino acid level to both the potato enzymes have been characterized from both leaf and win1 and 2 genes, Hevea brasiliensis hevein and flower abscission zones, and Northern analysis has shown Nicotiana tabacum PR-4a and PR-4b proteins. The that they are up-regulated prior to the onset of organ mRNA encoded by TAB7 is up-regulated within 12 h of shedding (Lashbrook et al., 1994; Kalaitzis et al., 1995; exposure to ethylene (10/<ir 1 ) and its expression del Campillo and Bennett, 1996). increases steadily within the cells comprising the leaf The activity of a spectrum of other enzymes has been abscission zone and to a lesser extent in the adjacent shown to increase during leaf abscission (Sexton and non-zone tissue. This rise precedes the onset of cell Roberts, 1982). Some of these, such as_$-l,3 glucanase separation. Southern analysis indicates that the mRNA (Abeles and Forrence, 1970), peroxidase, and chitinase is encoded by either a single gene or a small gene (Gomez et al., 1987), have been proposed to play a role family. The role of the protein during abscission is in protecting the fracture surface from pathogenic attack discussed. once cell separation has taken place and, in the early stages of ethylene-promoted abscission of Phaseolus vulKey words: Lycopersicon esculentum, abscission zone, garis leaves, it has been shown that there is a synchronized ethylene, tomato, wound-induced proteins. accumulation of several pathogen-related (PR) peptides (del Campillo and Lewis, 1992). In accord with this hypothesis, work by Eyal et al. (1993) has revealed that Introduction a PR-1 protein promoter is active in tobacco leaf abscission zones and it has been proposed that a pre-emptive Leaf abscission occurs as the result of cell separation at up-regulation of defence proteins takes place prior to the a specific zone comprising several layers of morpholoonset of cell separation. Moreover, in leaflets of Sambucus gically distinct cells (Sexton and Roberts, 1982). Upon nigra an mRNA encoding a metallothionein-like protein exposure to ethylene there is an increase in the activity Abstract 1 The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U89764. 5 To whom correspondence should be addressed. Fax: +44 1159 516334. E-mail: Jeremy.Robertsenottingham.ac.uk 6 Oxford University Press 1997 1224 Harris et al. has been shown to be rapidly be up-regulated in the leaflet abscission zone tissue and it has been proposed that this peptide may play a role in sequestering free radicals associated with the induction of PR-proteins during the cell separation process (Coupe et al., 1995). In this paper a differential screening strategy was employed to identify mRNAs that are specifically expressed during ethylene-promoted leaf abscission in tomato. The identification of an mRNA encoding a peptide which has close similarity to the wound-induced proteins from potato (Stanford et al., 1989) is reported and it is shown that the gene product is up-regulated specifically in the leaf abscission zone after exposure to ethylene. Materials and methods Plant material and growth conditions The growth of Lycopersicon esculentum Mill. cv. Ailsa Craig plants and preparation of leaf abscission zone explains were as described in Taylor et al. (1990). Abscission was determined as described in Roberts et al. (1984). RNA extraction Explants were incubated in ethylene (10 /tl I" 1 ) for 0, 12, 24 or 48 h and then RNA extracted from a piece of tissue approximately 2 mm wide containing the leaf abscission zone (zone tissue) or from an adjacent piece of stem (non zone tissue) using the method of Christoffersen and Laties (1982). Poly (A + ) RNA was separated from total RNA using the method detailed for the Poly A + Quick mRNA Purification kit (Stratagene, USA) by oligo (dT)-cellulose column chromatography. Construction of a cDNA library Double-stranded cDNA was prepared from abscission zone total poly(A + ) RNA isolated from explants exposed to ethylene (10/xl I" 1 ) for 24 or 48 h and a library constructed and packaged using the AZap cDNA synthesis kit and Gigapack Gold II packaging extract (Stratagene, USA). Library screening The cDNA library was screened using random primed probes labelled with 32 P constructed from either abscission zone or non-zone poly(A + ) RNA using the method of Picton et al. (1993). Plated aliquots of the library were transferred to Hybond N + filters (Amersham, UK) and hybridization was carried out as per manufacturers instructions. 0.2 ^g ml * ethidium bromide), heated to 65 °C for 15 min and then 4 JX\ of loading buffer (0.025% (w/v) bromophenol blue, 8% (w/v) sucrose) added. The samples were then electrophoresed in a 1% (w/v) agarose gel (dissolved in 0.02 M Na 2 HPO 4 buffer (pH 6.8), 3% (v/v) formaldehyde) at 75 V, with constant buffer recirculation, for 3 h. Samples were then blotted on to Genescreen membrane (Du-Pont, UK) as per the manufacturer's instructions. Blots were hybridized in 1 M NaCl, 50% (v/v) formamide, 0.1% (w/v) SDS, 20% (w/v) dextran sulphate, and 0.1 ^g ml" 1 denatured salmon sperm DNA at 42 °C overnight. They were then washed sequentially in 2, 1 and 0.1 x SSPE; 0.1% (w/v) SDS for 20 min at 42 °C then again at 65 °C as necessary before exposure to autoradiographic film. Southern blot hybndization Genomic DNA was extracted as described in Coupe et al. (1993). Enzyme digestion and electrophoresis were carried out according to Sambrook et al. (1989). Samples were blotted onto Genescreen Plus membrane (Du-Pont, UK) and hybridized as per manufacturers instructions. Probing of Southern and Northern blots Northern and Southern blots were probed with random primed probes made from the cDNA for TAB7 and labelled with 32 P using the method of Feinberg and Vogelstein (1983). Results Expression of TAB7 in abscission zone and non-zone tissue Differential screening of the abscission zone library using mRNA extracted from ethylene-treated non-zone tissue led to the isolation of a clone (pTAB7) that was identified as being abscission-related. A Northern analysis was carried out on RNA extracted from either abscission zone or non-zone tissue after exposure to ethylene (10 ^xl I" 1 ) for 0, 12, 24 or 48 h and probed with the cDNA for TAB7. Figure 1 shows that within 12—24 h a hybridizing transcript of approximately 0.8 kb was detectable and that the intensity of hybridization increased in both zone and non-zone tissue reaching a peak by 48 h after treatment. Expression of the TAB7 transcript increased to a greater extent in the abscission zone tissue than in the adjacent stem (non zone) tissue with no signal being detectable in fresh tissue from either the zone or non- Zone Oh 12h 24h 48h Non zone Oh 12h 24h 48h DAM sequencing The cDNA encoding TAB7 was sequenced using Sequenase version 2.0 (US Biochemical Corp, USA) by the method of Sanger et al. (1977). Synthetic oligonucleotides were obtained from Genosys, UK. Northern hybridization Northern blotting was performed using 10^g total RNA per lane. Each sample was mixed with an equal volume of 2 x sample buffer (50% (v/v) formamide, 16.5% (v/v) formaldehyde, 0.01 M EDTA (pH7.5), 0.04 M Na 2 HPO 4 (pH 6.8), and 0.8 kb Fig. 1. Northern blot analysis of mRNA isolated from leaf abscission zone or non zone tissue after exposure to ethylene (10 (J I" 1 ) for 0, 12, 24 or 48 h. Total mRNA, 10 ^g per lane, was probed with "P-labelled TAB7 cDNA and exposed to X-ray film for 3d at - 7 0 ° C with intensifying screens. win Expression during tomato leaf abscission zone material. This rise in expression could be detected before the first signs of cell separation were observed. Sequence analysis of TAB7 Both strands of the cDNA for TAB7 were fully sequenced. The encoded mRNA is 694 nucleotides in length with an open reading frame (ORF) of 201 amino acids. The largest ORF does not begin with an initiation codon and is therefore not full length (Fig. 2). Further screening of the cDNA library failed to isolate a full length clone. An analysis of TAB7 sequence has revealed that the putative peptide shares a high degree of homology at the amino acid level (see Fig. 2) with the potato winl and 2 genes (87% and 90%, respectively) (Stanford et al, 1989), the hevein precursor from Hevea brasilensis (77%) (Broekaert et al., 1990) and the PR4A protein from tobacco (76%) (Friedrich et al., 1991). Based on the sequences of the winl and 2 genes from potato which are its closest relatives (Fig. 3), TAB7 would appear to be approximately 10 amino acids short of encoding the full open reading frame. However, it does possess the remaining (15 out of 25) amino acids of the leader TAB7 WINl WIN2 HEVEA sequence, present in winl and 2, as well as its proposed cleavage site. TAB7 also contains the highly conserved cysteine-rich domain (Gln 16 -Gly 58 ) present in class I chitinases and the potato win genes. This region is bound by flanking sequences of 9 bp imperfect direct repeats (CAACGCGCA upstream and TAACCCGCA downstream) consistent with the findings of Shinshi et al. (1990) that the cysteine-rich domain was introduced by transposition from a common ancestral gene. Genomic Southern analysis Genomic DNA from tomato digested with Eco RI and Bam HI was Southern blotted and probed with 32 P radiolabelled TAB7 cDNA. The result shows that with both restriction enzyme digests two fragments hybridized, suggesting TAB7 is encoded by either a single gene or possibly a small gene family (results not shown). Discussion During cell separation at tomato leaf abscission zones there is an associated increase in the activity of several ^^^^^ H|^^| I S N S T ^ ^ H H S C G P • N H F I V V L PR4AT0B | E R V N N Y K 1225 A '0/Q C G R A "Q Q C I I A. ' Q S M V M c TAB7 WINl WIN2 HEVEA PR4ATOB TAB7 WINl WIN2 HEVEA PR4ATOB G;.W..T A F G;W T A F G*G;L D L-D N V F . Q i '&' G"'X" D L D V N V F P.'*Q V V D R - - - S S V I N j t> n JJ i v « i Q 2 G'H L I..V»VY.Q ; 2 G H L I V N Y Q D.T D G V 3 G K V 'N Y Q D T D G - G T ' G V G Q Q G H L F V N: j (^^P F'.-V C ; G - D F V N c'G D F V, C. G D 188 99 98 39 N 201 200 211 204 147 Fig. 2. Multiple sequence alignment of TAB7 to potato Winl and Win2 proteins, accession numbers PO9761 and PO9762 (Stanford et al., 1989); hevein precursor (hevea), accession number PO2877 (Broekaert et al., 1990) and PR4A protein from tobacco (PR4ATOB), accession number P29062 (Friedrich et al., 1991). The alignment was generated using the Clustal method with PAM250 residue weight table on DNAStar program. Dark shading indicates identical residues. 1226 TAB7 WIN2 WIN1 HEVEA PR4ATOB 16 3 . 16 14 12 10 FIE 3 PhvloKenet.c tree of TAB7 to Win I and Win2, hevein and PR4A protein from tobacco. Generated using DNAStar Oustal method (PAM250 S d u e w e , S m b ! e ) ^ e scale beneath the tree measures the distance between sequences. Units ind.cate the number of subst.tut.on events. hydrolytic enzymes including>l ,4 glucanase and polygalacturonase (PG) (Taylor et al, 1990). Preceding the rises in the activity of these enzymes in leaves and flowers is a specific increase in expression of genes encoding these cell wall hydrolases (Lashbrook et al, 1994; Kalaitzis et al, 1995; del Campillo and Bennett, 1996). Several other proteins have been demonstrated to accumulate in the area adjacent to cell separation and some of these have been identified as peptides that may play a role in preventing pathogenic attack such as chitinases (Gomez et al, 1987) and PR-proteins (del Campillo and Lewis, 1992). In this paper, it is shown that an mRNA, encoding a putative win protein, primarily accumulates in tomato leaf abscission zones exposed to ethylene. As this protein shares significant homology with several PR proteins it is possible that, as proposed by Stanford et al. (1989), the peptide protects the exposed fracture surface from pathogenic invasion. Some expression is also apparent in nonzone cells and, whilst no cell separation takes place in this tissue, the process of explant generation may provide a sufficient wound signal to promote transcription of the TAB7 gene in the presence of ethylene (Weiss and Bevan, 1991). The amino acid sequence for TAB7 contains the conserved cysteine-rich residues present in several PR proteins and consistent with highly stable protease resistant structures (Stanford et al, 1989). Little is known about the role of Win proteins although work with the two potato genes, winl and 2, has demonstrated that ethylene plays a role in regulating transcription of a Win2-GUS fusion in a systemic response to either biological or mechanical stress at the site of wounding (Weiss and Bevan, 1991). These workers demonstrated that the promoter was active in the leaf abscission zone of potato although they concluded that expression was probably a consequence of a wound response associated with cell separation. However, the results indicate that tomato win expression is initiated within 12 h of exposure to ethylene and as cell separation within the leaf abscission zone is not detectable until 24 h after ethylene exposure (Taylor et al, 1990) it is likely that expression of TAB7 is the consequence of a pre-emptive rather than a wound-induced signal. This hypothesis is supported by the observations of del Campillo and Lewis (1992) who showed the ethylenepromoted accumulation of PR proteins in bean abscission zones prior to that of cellulase, an enzyme thought to be involved in cell separation. Furthermore, in the publication by Eyal et al (1993) an abscission zone-specific ethylene-inducible promotor element has been identified for the PR-1 protein from Nicotiana tabacum. These workers found that GUS expression driven by the PR-1 promoter appeared in the abscission zone and proposed that the activation of this gene represented a pre-emptive induction of the plant defence system against possible infection. Recently, a spectrum of pathogenesis-related gene products have been found to be up-regulated in the leaflet abscission zone of Sambucus nigra after ethylene exposure and that these also appear immediately prior to cell separation (Roberts et al, 1997). Morover, the upregulation of the genes encoding these PR-proteins is associated with the expression of an mRNA encoding an MT-like protein that may play a role in protecting the separating cells from free radicals generated during the process of protection against pathogenic invasion (Coupe et al, 1995). The results indicate that the rapid expression of the Win peptide in tomato leaf abscission tissue may contribute to the protection of the exposed fracture surface from fungal and bacterial attack. Further studies are now needed to isolate the gene encoding TAB7 and identify the promotor elements responsible for enhancing the ethylene effect within the abscission zone tissues. Such work may also highlight whether certain promoter domains play a critical role in the regulation of gene expression within tomato leaf abscission zone cells in general and also at other sites where cell separation takes place. Acknowledgement NH gratefully acknowledges receipt of a BBSRC research studentship (P239RS). References Abeles FB, Forreoce LE. 1970. Temporal and hormonal control of>l,3-glucanase in Phaseolus vulgaris L. Plant Physiology Broekaert W, Lee HI, Kush A, Chua NH, Raikhel N. 1990. Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree. Proceedings of the National Academy of Science, USA 87, 7633-7. win Expression during tomato leaf abscission del Campillo E, Lewis LN. 1992. Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones. Plant Physiology 98, 955—61. del Campillo E, Bennett AB. 1996. Pedicel breakstrength and cellulase gene expression during tomato flower abscission. Plant Physiology 111, 813-20. Christoffersen ER, Laties GC. 1982. Ethylene regulation of gene expression in carrots. Proceedings of the National Academy of Science, USA 79, 4060-3. Coupe SA, Taylor JE, Isaac PG, Roberts JA. 1993. Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.). Plant Molecular Biology 23, 1223-32. Coupe SA, Taylor JE, Roberts, JA. 1995. Characterization of an mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets. Planta 197, 442-7. Eyal Y, Meller Y, Lev-Yadun S, Fluhr R. 1993. A basic type PR-1 promotor directs ethylene responsiveness, vascular and abscission zone specific expression. The Plant Journal 4, 225-34. Feinberg AP, Vogelstein B. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13. Friedrich L, Moyer M, Ward E, Ryals J. 1991. Pathogenesisrelated protein 4 is structurally homologous to the carboxyterminal domains of hevein, Win-1 and Win-2. Molecular General Genetics 230, 113-19. Gomez LM, Kelly P, Sexton R, Trewavas AJ. 1987. Identification of chitinase mRNA in abscission zones from bean. Plant, Cell and Environment 10, 741-6. Kalaitzis P, Koehler SM, Tucker ML. 1995. Cloning of a tomato polygalacturonase expressed in abscission. Plant Molecular Biology 28, 647-56. Lashbrook CC, Gonzalez-Bosch C, Bennett AB. 1994. Two divergent endo-j8-l,4-glucanase genes exhibit overlapping expression in ripening fruit and abscizing flowers. The Plant Cell 6, 1485-93. Picton S, Gray J, Barton S, Abu Baker U, Lowe A, Grierson D. 1993. cDNA cloning and characterization of novel ripening related mRNAs with altered patterns of accumulation in the 1227 ripening inhibitor (rin) tomato ripening mutant. Plant Molecular Biology 23, 193-207. Roberts JA, Scbindler CB, Tucker GA. 1984. Ethylene promoted flower abscission and the possible involvement of an inhibitor. Planta 160, 159-63. Roberts JA, Coupe SA, Taylor JE, Whitelaw CA. 1997. Spatial and temporal expression of abscission-related genes during ethylene-promoted organ shedding. In: Kancllis AK, ed. Biology and biotechnology of the plant hormone ethylene. NATO series, 185-90. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning. A laboratory manual. New York: Cold Spring Harbour Laboratory Press. Sanger F, Nkklen S, Coulsen AR. 1977. DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Science, USA 74, 5463. Sexton R, Roberts JA. 1982. Cell biology of abscission. Annual Review of Plant Physiology 33, 133—62. Shinshi H, Neuhaus JM, Ryals J, Meins Jr F. 1990. Structure of a tobacco endochitinase gene: Evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine rich domain. Plant Molecular Biology 14, 357-68. Stanford A, Bevan M, Nortbcote D. 1989. Differential expression within a family of novel wound-induced genes in potato. Molecular and General Genetics 215, 200-8. Taylor JE, Tucker GA, Laslett Y, Smith CJS, Arnold CM, Watson CF, Schuch W, Grierson D, Roberts JA. 1990. Polygalacturonase expression during leaf abscission of transgenic and normal tomato plants. Planta 183, 133-8. Taylor JE, Webb STJ, Coupe SA, Tucker GA, Roberts JA. 1993. Changes in polygalacturonase activity and solubility of polyuronides during ethylene stimulated leaf abscission in Sambucus nigra. Journal of Experimental Botany 44, 93-8. Webb STJ, Taylor JE, Coupe SA, Ferrarese L, Roberts JA. 1993. Purification of ,£-1,4 glucanase from ethylene-treated abscission zones of Sambucus nigra. Plant, Cell and Environment 16, 329-333. Weiss C, Bevan M. 1991. Ethylene and a wound signal modulate local and systemic transcription of win2 genes in transgenic potato plants. Plant Physiology 96, 943-51.