Planta (1984)162:353 360

pl~HA[~ 9 Springer-Verlag 1984

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue A.D. Shirras* and D.H. Northcote Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 IQW, UK

Abstract. Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage MI3 rap8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of R N A complementary to two of the clones show a marked increase during the 24 h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated R N A per microgram of total R N A which increases 2.5-fold during the same period. The wound-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants. Key words: Bacteriophage (DNA cloning) - Complementary DNA (clones) - Gene expression - Solanum (cDNAs) Wound response.

Introduction The slicing (wounding) and subsequent aerobic incubation (ageing) of previously quiescent plant storage organs leads to a wide variety of metabolic and morphological changes (van Stevininck 1975; Kahl 1978). For instance, there is an increased synthesis of R N A (Kahl 1971; Byrne and Setterfield 1977; Sato et al. 1978) and protein (Sato et al. 1978). Increased synthesis of m R N A is apparent immediately following wounding whereas a 2 to cDNA=complementary DNA; poly(A)=polyadenosine; poly(A) + RNA=polyadenylated RNA; poly(U) = polyuridine Abbreviations.

3-h lag precedes synthesis of ribosomal R N A (Byrne and Setterfield 1977; Sato et al. 1978). Concomitant with the increase in protein synthesis is a rapid recruitment of ribosomes into polysomes (Leaver and Key 1967; Kahl 1971). Since ribosomes from unscliced tissue are incapable of incorporating labelled amino acids into protein in vitro unless supplied with exogenous m R N A (Kahl 1971) and newly synthesised m R N A is rapidly incorporated into polysomes (Byrne and Setterfield 1977; Sato et al. 1978), newly synthesised m R N A may be necessary for this process. However, unsliced Jerusalem-artichoke tubers contain polyadenylated R N A (poly(A)+RNA) and polysome formation is not inhibited by c~-amanitin (Byrne and Setterfield 1978). These data indicate that stored, inactive m R N A is important for polysome formation, at least during the early stages of ageing. The cell-wall glycoprotein of carrots has been shown to accumulate following wounding (Chrispeels et al. 1974) and a number of enzymes also show increased activity (Sacher et al. 1972). The most dramatic increases are seen in phenylalanine ammonia-lyase (Zucker 1965, 1968; Sacher et al. 1972) and other enzymes concerned with phenylpropanoid metabolism (Rhodes and Wooltorton 1975; Lamb and Rubery 1976; Smith and Rubery 1981), peroxidase (Borchert 1978) and fatty-acid synthase (Willemot and Stumpf 1967a). These increased enzyme activities may be correlated with some of the characteristic features of wound metabolism: the accumulation of phenolic compounds (Rhodes and Wooltorton 1978), the synthesis of substances such as the suberin complex at the wound surface (Kolattukudy and Dean 1974; Rhodes and Wooltorton 1978; Gould and Northcote 1984) and the synthesis of fatty acids (Mazliak and Kader 1978). From the use ofinhibi-

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A.D. Shirras and D.H. Northcote: cDNAs complementary to mRNAs from wounded potato tissue

tors of RNA synthesis, the synthesis of at least some of these proteins has been shown to be regulated at the transcriptional level (Rhodes et al. 1976; Lamb 1977; Willemot and Stumpf 1967b; Chrispeels et al. 1974). More direct evidence for transcriptional control of specific genes in wounded plant storage tissue has come from the analysis of the in-vitro translation products of polysomal RNA from potato slices (Ishizuka et al. 1981) or of total cellular RNA from carrot discs (Smith 1981). Here we report the isolation of cDNA clones complementary to a number of mRNAs from aged potato (Solanum tuberosum cv. King Edward) tuber discs and characterise the levels of these mRNAs during the initial 24 h of ageing. Material and methods Preparation of aged discs. Discs (24 mm in diameter, 1 mm thick) of potato tuber tissue (variety King Edward) were aged as described by Ishizuka et al. (1981) in the dark at 26 ~ C between sheets of filter paper soaked in 20 mM sodium-phosphate buffer (pH 6.8) containing 50 gg ml 1 chloramphenicol.

Potato tissue culture. Suspension cultures of potato callus were grown in PRL 4 medium supplemented with 2,4-dichlorophenoxyacetie acid (6 m g l 1) (Gamborg 1966; Owens and Northcote 1981).

(pH 8.3), 140 mM KC1, 4 I~g m l - 1 oligo(dT)12_ls, 740 kBq [all] deoxyguanosine triphosphate (dGTP) (351.5 GBq mmol-1), 0.25 mM dGTP, 2 m M each of dATP, deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), 13 gg poly(A)+RNA and 128 units AMV reverse transcriptase (Life Sciences, St. Petersburg, Fla., USA) in a final volume of 100 gl. The reaction mix was incubated at 40 ~ C for 60 min. The reaction was stopped, the R N A - D N A duplexes denatured by boiling for 3 rain and the tube was placed on ice for 5 rain. The mixture was centrifuged in a micro-centrifuge for 3 min at 4 ~ C and the supernatant transferred to a fresh tube. An equal volume of 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid (Hepes) buffer, pH 7.0 was added to a final concentration of I00 m M before synthesis of the second strand using 25 units of Klenow fragment (Amersham International, Amersham, Bucks., UK) of D N A polymerase I at 15 ~ C for 5 h. The double-stranded c D N A was then phenol-extracted and ethanol-precipitated overnight.

Preparation of eDNA clones. Approximately 50 ng of doublestranded c D N A in 10 gl were digested for 30 rain with 1 unit Sau 3A (Bethesda Research Laboratoiries, Cambridge, UK) according to the manufacturer's instructions. To the mixture were added 40 gl of 10 mM Tris-HCl (pH 8.0), 0.1 m M E D T A and the mixture was extracted with an equal volume of buffersaturated phenol. Residual phenol was removed by ether extraction. Sau-3A-digested c D N A (2-ng lots) was ligated to 20 ng of Bam-Hl (Bethesda)-digested, alkaline-phosphatase-treated M13 rap8 (Bethesda) (Messing 1981). Escherichia coli strain JM 103 cells (Bethesda) were made competent by CaC12 treatment and transformed with the ligation mix. The transformed cells were plated on lawns containing isopropyl thiogalactoside and 5-bromo-4-chloro-3-indolyl-fl-l>galactoside. Colourless plaques were picked onto fresh lawns without indicator.

Purification of RNA. Total cellular R N A was isolated by a modification of the method of Martin and Northcote (1981). Tissue was frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. The tissue was allowed to thaw in R N A extraction buffer [100 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol(Tris)-HC1 (pH 9.0), 150 mM LiCI, 5 mM ethylenediaminetetraacetic acid (EDTA), 5% sodium dodecyl sulfate (SDS)], 3 ml per g of tissue. This was further ground with acid-washed sand to a fine slurry, strained through four layers of muslin and extracted three times with an equal volume of water-saturated phenol: chloroform (1:1, v/v). The final aqueous phase was made 0.2 M with solid LiC1 and the nucleic acids plus contaminating polysaccharide precipitated with 2.5 vol. ethanol at - 2 0 ~ C for 16 h. The precipitate was collected by centrifugation and washed three times with 3 M sodium acetate (pH 5.5), and once with 70% ethanol. The pellet was dried under vacuum, dissolved in distilled water and reprecipitated with 2 vol. 3 M LiC1 at 4 ~ C for 16 h. The precipitate was collected, washed with 70% ethanol, dried, dissolved in distilled water and ethanol-precipitated. The final R N A pellet was dissolved in distilled water to a concentration of 10 m g m l -x and stored at - 2 0 ~ C. Polyadenylated R N A was prepared by two cycles of oligo(dT) cellulose chromatography according to Martin and Northcote (1981). The poly(A) + R N A was precipitated with 0.1 vol. 3 M sodium acetate (pH 5.5) and 2.5vol. ethanol at - - 2 0 ~ for 16 h. The pellet was dissolved in distilled water to a concentration of I mg m l - 1.

Synthesis of complementary DNA (cDNA). Single-stranded c D N A was synthesised from 13 gg poly(A)+RNA prepared from tuber discs aged for 20 h. The reaction mixture contained 10raM MgC12, I mM dithiothreitol, 100raM Tris-HC1

Plaque hybridisation. Plaques were transferred to nitrocellulose filters according to Benton and Davies (1977) and hybridised with poly(A)+RNA, from 20-h-aged tuber discs, end-labelled with y-[32p]ATP according to Maizels (1976) to a specific activity of 2.106 cpm ~g- 1. Conditions for hybridisation and washing of the filters were according to Maniatis et al. (1982). Filters were autoradiographed with intensifying screens at - 7 0 ~ (Laskey and Mills 1977). Preparation of single-stranded DNA. Single-stranded M13DNA-containing e D N A inserts were prepared according to Brown et al. (1982). Cloned D N A was stored in I0 m M TrisHCI(pH 8.0), 0.1 m M E D T A at 25 g g m l 1.

Hybrid-release translation. Diazobenzyloxymethyl (DBM) paper discs, a c m diameter were prepared by the method of Alwine et al. (1977). Hybrid-release translation was carried out essentially according to Smith et al. (1979). Single-stranded clone D N A (5 gg) was bound to each DBM disc in 80% dimethyl sulphoxide, 10 mM sodium acetate (pH 5.5) for 16 h at 20 ~ C. Discs were washed three times with water, four times with 0.4 M N a O H and a further four times with water. Discs were then stored in 50% formamide, 100 mM sodium phosphate (pH 7.0), 0.6 M NaC1, 0.1% SDS, 0.2 mgm1-1 polyadenosine [poly(A)], 0.2 mg m l - 1 calf liver transfer R N A (tRNA) at 4 ~ C for 16 h. The storage buffer was removed and hybridisation solution containing 50% formamide, 40 mM 1,4-piperazinediethanesulfonic acid (Pipes; pH 6.4), 0.9 M NaC1, 1 mM EDTA, 0.4% SDS, 50 ~tg ml 1 poly(A), 0.2 mg m l - 1 calf liver t R N A and 500 I~g m l - 1 total aged-tuber R N A added. Hybridisation was carried out at 41 ~ C for 16 h. The discs were washed four times with 50% formamide, 8 m M sodium citrate, 20 mM

A.D. Shirras and D.H. Northcote: cDNAs complementary to m R N A s from wounded potato tissue

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NaC1, 1 m M EDTA, 0.2% SDS at 32 ~ C, 20 rain each wash. The R N A was eluted from the discs twice with 150 ~1 90% formamide, 20 m M Pipes (pH 6.4), 1 mM EDTA, 0.5% SDS at 60 ~ C for 20 min. Released R N A was ethanol-precipitated with 20 gg calf liver t R N A as carrier at - 20 ~ C for 16 h. The R N A pellet was washed with 70% ethanol to remove residual formamide, dried and dissolved in 5 gl distilled water.

In-vitro translation, In-vitro translation was carried out using the message-dependent reticulocyte-iysate system of Pelham and Jackson (1976). Each 10 txl reaction contained 222 kBq [35S]methionine and either 10 ~tg of totaI R N A or 2 gt of hybrid-released R N A solution. Translation products were separated on 15% polyacrylamide gels according to Laemmli (1970). Dried gels were fluorographed according to Laskey and Mills (1975).

Probe preparation. Probes were prepared from single-standed D N A by the primer-extension method of Hu and Messing (:1982) as modified by Brown et al. (1982) to a specific activity of 108cpmpg 1 Hybridisation to immobilised RNA. Northern transfers of poly(A) + R N A (1 gg per track) and dot blots of total R N A (10 pg in 2 gl) were carried out according to Thomas (1980). Hybridisation conditions were according to Maniatis et al. (1982). After washing, blots were dried and autoradiographed. Additionally, dot blots were cut at individual time points and counted in triton/toluene-based scintillant.

Polyuridine [poty (U)] hybridisation. Polyadenylated R N A was determined by hybridisation to excess [3H]poly(U) according to Byrne and Setterfield (1978). [3H]poly(U) (1.85 kBq) was hybridised to I gg of total RNA.

Results

Preparation and selection of cDNA clones. Doublestranded cDNA was prepared from poly(A) + R N A from 20-h-aged discs by a rapid method whereby the first-strand reaction is terminated by boiling, diluted with buffer at pH 7.0 and the secondstrand reaction carried out immediately by addition of Klenow fragment. This eliminates the need for ethanol precipitation between the two reactions. Removal of the terminal hairpin bends and size selection were unneccessary since the cloning procedure used internal digestion by Sau 3A before ligating into Bam-Hl-digested M13 rap8. Recombinant clones were detected by the blue-white plaque assay afforded by this vector. The efficiency of cloning was such that approximately 200 recombinants could be obtained per 1.0 ng of Sau-3Adigested cDNA with a control transformation rate o f l 0 6 plaques per 1.0 txg of vector. Recombinant plaques were screened by hybridisation to poly(A)+RNA from aged discs. Singlestranded DNA was prepared from 24 hybridising plaques and used for hybrid-release translation and hybridisation to dot blots of RNA from fresh

Fig. 1. Fluorograph of [35S]methionine-labelled polypeptides translated in vitro from R N A selected by c D N A clones. Track A, M13 rap8 control; tracks B-F, clones 5, 14, 17, 19 and 22, respectively; track G, translation products of R N A from fresh potato tubers; track H, translation products of R N A from tuber discs aged for 20 h; track L result of separate experiment using clone 14 showing the translated polypeptide of Mr 17k. The prominent band at 42k is an endogenous product of the reticulocyte system

and aged tubers. Figure 1 shows the translation products of the RNAs selected by five clones chosen for further study. Clones 14, 17, 19 and 22 are complementary to RNAs encoding polypeptides of Mr 17k, 87k, 40k and 59k, respectively. Clone 5 gave no detectable polypeptide product. Carrying out the hybridisation and washing at lower stringency merely increased the background. Labelling with [3H]lysine instead of [3~S]methionine also did not reveal a polypeptide product. Figure 2 shows the results of hybridisation of clones 5, 14, 17, 19 and 22 to poly(A)+RNA from 20-h-aged tuber discs and from fresh tubers. The RNA complementary to clones 5 and 17 shows a marked induction during ageing. Clone-14 RNA and clone-22 R N A show a slight induction and RNA complementary to clone 19 is induced little, if at all. The possibility that clone 5 contains an insert complementary to ribosomal RNA was tested by hybridising to non-polyadenylated RNA; no hybridisation was detected. Clone 5 hybridised to an R N A species of approx. 1.2 kilobases following transfer of total aged

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A.D. Shirras and D.H. Northcote: cDNAs complementary to mRNAs from wounded potato tissue 14

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Changes in mRNA levels after wounding. The amount of m R N A complementary to each of the clones during the first 24 h of ageing was determined by hybridisation to dot blots of total RNA. Figure 4 shows the amount of radioactive probe hybridising to dot blots of equal amounts of total R N A from discs taken at 4 h-intervals following tissue slicing. Ctone-5 R N A , after a lag of between 4 and 8 h, increases 12-fold to a maximum at 20 h before decreasing. Clone-14 R N A initially decreases slightly followed by a lag up to 12 h before increasing and reaches a 3.5-fold increase at 24 h. Clone17 R N A starts to increase during the first 4 h of

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Fig. 4A-E. Amount of radioactive probe prepared from each of the e D N A clones hybridising to dot blots of 10 gg of total R N A extracted from potato tuber discs at intervals after slicing. A, B, C, D, E Clones 5, 14, 17, 19 and 22, respectively

ageing, then there is a lag between 8 and 12 h before it increases again at a higher rate to an overall 71-fold increase at 24 h. Clone-19 R N A shows a small decrease during the first 4 h and then maintains a low steady level, approximately one third

A.D. Shirras and D.H. Northcote: cDNAs complementary to mRNAs from wounded potato tissue

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Fig. 5. Amount of poly(A)-containing RNA per gg of total RNA as determined by hybridisation of [3H]poly(U) to RNA extracted from potato tuber discs at intervals after slicing

of the initial amount. Clone-22 RNA increases slightly during the first 4 h and stays at about the same level for the rest of the time course. There was some slight variability in the quantitative dotblot assays between individual experiments, especially when low concentrations of the mRNA were present. This was particularly true for the dot blots of clones 19 and 22. Byrne and Setterfield (1977, 1978) showed that the amount of poly(A)+RNA in Jerusalem-artichoke tuber slices changes during the initial phase of ageing. In order to differentiate between a change in the amount of a particular mRNA caused by the underlying general change in the proportion of total poly(A) § RNA and one caused by a specific change in the amount of the particular mRNA, the amount of poly(A) § RNA per gg of total RNA was determined by [3H]poly(U)hybridisation for each point in the time course (Fig. 5). The amount of poly(A)+RNA increases approx. 2.5-fold after 16 h. This agrees well with the amount of poly(A) § RNA recovered by oligo(dT) cellulose chromatography. Taking this into account, the RNA complementary to clone 19 must account for proportionally less of the poly(A)+RNA population following wounding, whereas the three clones 5, 14 and 17 are complementary to RNAs which specifically increase during ageing. The slight rise in mRNA complementary to clone 22 probably represents the increase in total mRNA over the first 4h. Figure 6 shows the in-vitro translation prod-

Fig. 6. Fluorograph of [3SS]methionine-labelled polypeptides translated in vitro from total RNA extracted from potato tuber discs at intervals after slicing (0-24 h). The bands ~ d are discussed in the text

ucts of total cellular RNA during the ageing time course. The most prominent induced band has an Mr of about 24k (band d) and appears to reach a maximum intensity at 12-16 h after wounding. Band a probably corresponds to the 87k protein coded for by clone-17 RNA. A number of other induced bands can be seen (e.g. bands b, c).

Levels of mRNA in diluted suspension cultures. Similar metabolic events to ageing (e.g. increase in the activity of phenyalanine ammonia-lyase and other enzymes of phenylpropanoid synthesis and polysome formation) occur when stationary plant cell suspension cultures are diluted into fresh medium (Hahlbrock and Schr6der 1975; Ebel et al. 1974; Bevan and Northcote 1981). To determine whether the mRNAs studied during ageing of tuber slices would show similar changes after dilution of potato suspension culture cells, RNA was extracted from stationary cells (10 d after subculture) 5 h and 24 h after dilution (1:20) in fresh medium. This RNA was blotted onto nitrocellulose and hybridised to probes prepared from the clones. Figure 7 shows the results of these hybridisations. The total translation products are shown in Fig. 8.

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A.D. Shirras and D.H. Northcote: cDNAs complementary to mRNAs from wounded potato tissue

Fig. 7. A Autoradiograph of dot-blots of 10 gg total RNA extracted from fresh potato tubers (0), tuber discs aged for 8 h (8) and discs aged for 20 h (20), hybridised with probes prepared from the cDNA clones (5, 14, 17). B Autoradiograph of dots blots of 10 gg of RNA extracted from stationary potato suspension-culture cells (0), cells 5 h after dilution into fresh medium (5) and 24 h after dilution (24), hybridised with probes prepared from cDNA clones (5, 14, 17)

Fig. 9. Autoradiograph of dot-blots of 10 gg total RNA extracted from leaves (L), stems (S) and roots (R) of potato plants, hybridised with probes prepared from each of the cDNA clones

(5, 14, 17, 19, 22)

14 shows very little change in level after dilution. There was insufficient radioactivity hybridising to the m R N A s of clones 19 and 22 to show any small variations in their amounts, although it was obvious that there was no major increase in these mRNAs. Comparison of the pattern of dilutioninduced polypeptides with wound-induced polypeptides (Fig. 8) indicates that few of the major induced bands are common to both stimuli.

Fig. 8. Fluorograph of [35S]methionine-labelled polypeptides translated in vitro from total RNA extracted from stationary potato suspension-culture cells (A), cells 5 h after dilution into fresh medium (B), and cells 24 h after dilution (C). Tracks D and E show the translation products of RNA from fresh tubers and tuber discs aged for 20 h, respectively

Organ specificity. The R N A extracted from leaves, stems and roots of potato plants, was spotted onto nitrocellulose and hybridised with probes prepared from each of the five clones. The results are shown in Fig. 9. The RNAs of clones 14, 19 and 22 are present in very small amounts in all three organs. Clone-17 R N A has an abundance in roots comparable to that in fresh tubers but is present in very small amounts in leaves and stems. Clone-5 R N A is present in about equal amounts in stem and root - comparable to the abundance in tuber discs 8 h after wounding - and in slightly higher amounts in leaves. Discussion

While the pattern of polypeptide products changes markedly following dilution, the wound-induced RNAs complementary to clones 5 and 17 decrease. In stationary cells, clone- 5 R N A is present in levels comparable to, or slightly higher than the maximum value following wounding, whereas clone-17 R N A is at a similar level to that in unsliced tubers and decreases to a low level in the first 5 h following dilution. The m R N A complementary to clone

A rapid cDNA-cloning technique was used to generate, in the single-stranded bacteriophage M13 rap8, a small library of Sau 3A fragments of c D N A prepared from poly(A)§ from potato tuber discs aged for 20 h. The main advantage of this method, apart from its speed, is that cloned, singlestranded c D N A can be prepared easily and used for hybrid-release translation, preparation of sensitive probes (Hu and Messing 1982; Brown et al.

A.D. Shirras and D.H. Northcote: cDNAs complementaryto mRNAs from woundedpotato tissue 1982) or D N A sequencing by the dideoxy-nucleotide method (Sanger et al. 1980). Against this must be set the disadvantages of small insert size and incompleteness of the library (since the ability to clone a given sequence will depend on a favourable distribution of restriction sites). The latter may be overcome by using other restriction enzymes with a four-base recognition sequence and ligating into alternative sites in the vector. The wounding of potato tuber tissue has previously been shown to induce the synthesis of a variety of translatable m R N A s (Ishizuka et al. 1981). The objective of this study was to clone cDNAs for a number of m R N A s from wounded tuber tissue (both induced and non-induced) and to use these clones as probes to measure the amount of their complementary RNAs following tissue slicing. Five clones were chosen to represent a range of abundance and response to wounding. These were characterised by hybrid-release translation, N o r t h e r n blotting and hybridisation to dot blots of R N A from fresh and aged tubers. The reasons that clone 5 did not give a detectable product in the hybrid-release translation are unclear. Sequence analysis showed that the insert in clone 5 was very adenosine-thymidine rich, so it is possible that the hybridisation and washing procedures were too stringent. However, when these were altered a specific m R N A species was again not selected. Another possibility is that clone-5 R N A translates very inefficiently in the reticulocyte cell-free system. The relative intensities of the polypeptide bands in the hybrid-release translation were not comparable to the relative amounts of the R N A detected by the dot blots, so that the translation efficiency of individual m R N A s could possibly vary. The results of the hybridisation to dot blots indicate that wounding induces a series of events at the level of transcription or post-transcriptional processing, acting on specific genes or their transcripts. As well as these specific effects there is an increase in the total amount of poly(A)+RNA. It is likely that most of the genes respond only to the general stimulus while some respond in a specific manner. Genes such as the clone-19 gene may respond to neither stimulus. In addition the turnover rate of individual m R N A s may change during ageing. Ishizuka et al. (1981) reached similar conclusions from in-vitro translation of polysomal R N A from wounded potato tuber tissue. Two RNAs which are induced by wounding of potato tuber tissue decrease in suspension-culture cells following dilution into fresh medium, indeed the pattern of induced bands in the total in-

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vitro translation products is different in the two cases. This is not what one would expect if these major bands correspond to enzymes of general phenylpropanoid metabolism or proteins important for preparation for cell division. The m R N A complementary to clone 5 is abundant in both stationary cell suspension cultures and in wounded tuber tissue. It is possible that some secondary metabolic pathways may be common to wounded tissue and stationary suspension cultures. It is known that secondary metabolites accumulate towards the stationary phase of cell growth in culture (Yeoman etal. 1982). Clone-5 R N A is reasonably abundant in leaves, stems and roots of intact plants, all of which may be active in secondary metabolism. The RNAs of clones 19 and 22 may code for "housekeeping" enzymes since they seem to be equally abundant in all the tissues tested and are little affected by wounding or by dilution shock of suspension culture cells. Clone-17 R N A seems to show some tissue specificity as it is more abundant in roots than in stems or leaves. The level of this R N A in fresh tubers decreases with time of storage which may indicate that it is present in higher levels in developing tubers. The use of these cDNA clones and the isolation of other clones for other wound-induced m R N A s will allow the wound response of plant tissue to be studied in detail. Run-off assays using isolated nuclei will allow the importance of transcriptional versus post-transcriptional control to be determined. These clones should also allow the isolation of their corresponding genomic sequences. We would like to thank Dr. C. Martin and Dr. J. Beeching for helpful discussionand comment. A.D.S. was supported by a Science and Engineering Research Council research studentship. References Alwine, J.C., Kemp, D.J., Stark, G.R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl paper and hybridisation with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350-5354 Benton, W.D., Davies, R.W. (1977) Screening2 gt recombinant clones by hybridisation to single plaques in situ. Science 196, 180-192 Bevan, M., Northcote, D.tt. (1981) Subculture-inducedprotein synthesis in tissue cultures of Glycine max and Phaseolus vulgaris. Planta 152, 24-31 Borchert, R. (1978) Time course and spatial distribution of phenylalanine ammonia-lyase and peroxidase activity in wounded potato tuber tissue. Plant Physiol.62, 789-793 Brown, D.M., Frampton, J., Goelet, P., Karn, J. (1982) Sensitive detection of RNA using strand-specific MI3 probes. Gene 20, 139-144 Byrne, H., Setterfield, G. (1977) Activation of ribosomal and

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messenger RNA synthesis in Jerusalem artichoke tuber slices. Planta 136, 203-210 Byrne, H., Setterfield, G. (1978) Utilization of stored messenger RNA during early ageing of Jerusalem artichoke tuber slices. Planta 143, 75-83 Chrispeels, M., Sadava, D., Cho, Y.P. (1974) Enhancement of extensin biosynthesis in ageing discs of carrot storage tissue. J. Exp. Bot. 25, 1157-1166 Ebel, J., Schaller-Hekeler, B., Knobloch, K.H., Wellmann, E., Grisebach, H., Hahlbrock, K. (1974) Coordinated changes in enzyme activities of phenyl-propanoid metabolism during the growth of soybean cell suspension cultures. Biochim. Biophys. Acta 362, 417-424 Gamborg, O.L. (1966) Aromatic metabolism in plants. II. Enzymes of the shikimate pathway in suspension cultures of plant cells. Can. J. Biochem. 44, 791-799 Gould, J., Northcote, D.H. (1984) Characteristics of plant surfaces. In: Bacterial adhesion: mechanisms and physiological significance, Fletcher, M.M., Savage, D.C., eds. Plenum Press, New York (in press) Hahlbrock, K., Schr6der, J. (1975) Specific effects of enzyme activities upon dilution of Petroselinum hortense cell cultures into water. Arch. Biochem. Biophys. 171, 500-506 Hu, N.T., Messing, J. (1982) The making of strand-specific M13 probes. Gene 17, 271-277 Ishizuka, M., Sato, T., Watanabe, A., Imaseki, H. (1981) Alteration of coding properties of polysome-associated messenger RNA in potato tuber slices during ageing. Plant Physiol. 68, 154-157 Kahl, G. (1971) Activation of protein synthesis in ageing potato tuber slices. Z. Naturforsch. Teil B 26, 1058-1064 Kahl, G. (ed) (1978) Biochemistry of wounded plant tissue. de Gruyter, Berlin Kolattukudy, P.E., Dean, B.B. (1974) Structure, gas chromatographic measurement, and function of suberin synthesised by potato tuber tissue slices. Plant Physiol. 54, 116-121 Laemmli, U.K. (1970) Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 Lamb, C.J. (1977) Phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase: characterisation of the concomitant changes in enzyme activities in illuminated potato tuber discs. Planta 135, 169-175 Lamb, C.J., Rubery, P.H. (1976) Photocontrol of chlorogenic acid biosynthesis in potato tuber discs. Phytochemistry 15, 665-668 Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of aH and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 Laskey, R.A., Mills, A.D. (1977) Enhanced autoradiographic detection of 32p and 125I using intensifying screens and hypersensitized film. FEBS Lett. 82, 314-316 Leaver, C.J., Key, J.L. (1967) Polysome formation and RNA synthesis during early ageing of carrot-root tissue. Proc. Natl. Acad. Sci. USA 57, 1338-1344 Maizels, N. (1976) Dictyostelium 17S, 25S and 5S rDNAs lie within a 38000 base pair repeated unit. Cell 9, 431-438 Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning - a laboratory manual, pp. 326-327. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Martin, C., Northcote, D.H. (1981) Qualitative and quantitative changes in mRNA in castor beans during the initial stages of germination. Planta 151, 18%197 Mazliak, P,, Kader, J.C. (1978) Lipid metabolism in ageing plant storage tissues. In: Biochemistry of wounded plant tissue, pp. 123-154, Kahl, G., ed. de Gruyter, Berlin Messing, J. (1981) M13mp2 and derivatives: a molecular clon-

ing system for DNA sequencing, strand specific hybridisation, and in-vitro mutagenesis. In: 3rd Cleveland Symposium on Macromolecules: Recombinant DNA, pp. 143153, Walton, A., ed. Elsevier, Amsterdam Owens, R.J., Northcote, D.H. (1981) The location of arabinosyl:hydroxyproline transferase in the membrane system of potato tissue culture cells. Biochem. J. 195, 661-667 Pelham, H.R.B., Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256 Rhodes, M.J.C., Hill, A.C.R., Wooltorton, L.S.C. (1976) Activity of enzymes involved in the lignin biosynthesis in swede root disks. Phytochemistry 15, 707-716 Rhodes, M.J.C., Wooltorton, L.S.C. (1975) The p-coumarate CoA ligase of potato tubers. Phytochemistry 14, 2161-2164 Rhodes, MJ.C., Wooltorton, L.S.C. (1978) The biosynthesis of phenolic compounds in wounded plant storage tissues. In: Biochemistry of wounded plant tissue, pp. 243-286, Kahl, G., ed. de Gruyter, Berlin Sacher, J.A., Towers, G.H.N., Davies, D.D. (1972) Effect of light and ageing on enzymes, particularly phenylalanaine ammonia-lyase, in discs of storage tissue. Phytochemistry 11, 2383-2391 Sanger, F., Coulson, A.R., Barrell, B.G., Smith, A.J.H., Roe, B.A. (1980) Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143, 161-178 Sato, T., Watanabe, A., Imaseki, H. (1978) RNA synthesis in the early stage of aerobic incubation of potato tuber discs. Plant Cell Physiol. 19, 609-616 Smith, B.G., Rubery, P.H. (1981) The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue. Planta 151, 535-540 Smith, D.F., Searle, P.F., Williams, J.G. (1979) Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA. Nucleic Acids Res. 6, 487-506 Smith, M.A. (1981) Characterisation of carrot cell wall protein. I. Effect of c~,~'-dipyridyl on cell wall protein synthesis and secretion in incubated carrot discs. Plant Physiol. 68, 956-963 Thomas, P.S. (1980) Hybridisation of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205 van Stevenick, R.F.M.(1975) The washing or ageing phenomenon in plant tissues. Annu. Rev. Plant Physiol. 26, 237-258 Wiltemot, C., Stumpf, P.K. (1967a) Fat metabolism in higher plants. XXXIII. Development of fatty acid synthase in "ageing" storage tissue slices. Can. J. Bot. 45, 579 584 Willemot, C., Stumpf, P.K. (1967b) Fat metabolism in higher plants. XXXIV. Development of fatty acid synthetase as a function of protein synthesis in ageing potato tuber slices. Plant Physiol. 42, 391-397 Yeoman, M.M., Lindsey, K., Miedzybrodzka, M.B., McLauchlan, W.R. (1982) Accumulation of secondary products as a facet of differentiation in plant cell and tissue cultures. In: Differentiation in vitro (The Fourth Symposium of the British Society for Cell Biology), pp. 65-82, Yeoman, M.M., Truman, D.E.S., eds. Cambridge University Press, Cambridge, UK Zucker, M. (1965) Induction of phenylalanine deaminase by light and its relation to chlorogenic acid synthesis in potato tuber tissue. Plant Physiol. 40, 77%784 Zucker, M. (1968) Sequential induction of phenylalanine ammonia-lyase and a lyase-inactivating system in potato tuber disks. Plant Physiol. 43, 365-374 Received 26 March; accepted 1 May 1984