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Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plantlets and tissue cultures. Rudy Dolferus 1, G~rard Marbaix 2, and Michel ...
Mol Gen Genet (1985) 199:256-264 © Springer-Verlag1985

Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plantlets and tissue cultures Rudy Dolferus 1, G~rard Marbaix 2, and Michel Jaeobs 1 1 Laboratorium voor Plantengenetica, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium 2 D6partment de Biologic Mol6culaire, Universit~ Libre de Bruxelles, B-t640 Sint-Genesius-Rode, Belgium

Summary. Alcohol dehydrogenase (ADH) activity is expressed in Arabidopsis seeds and tissue cultures. During the germination process, ADH activity declines rapidly and is no longer detectable in 9- to 10-day-old seedlings. The synthesis of ADH could be demonstrated in seedlings submitted to anaerobiosis by 35S-methionine incorporation studies. Callus, induced from seeds or leaves on a 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, and cell suspension cultures are characterized by a high level of ADH activity. The incorporation of aSS-methionine and two-dimensional electrophoresis indicated that ADH induction was due to de novo synthesis of the polypeptides. In vitro translation of total poly (A)+-RNA from seedlings and callus showed that only callus mRNA was able to direct the synthesis of ADH polypeptides. This demonstrates the de novo synthesis of ADH mRNA during callus induction. Northern blot hybridization, using in vitro labelled ADH1-F DNA from maize as a probe, revealed sequence homology at the mRNA level between Arabidopsis and maize.

Introduction The small autogamous plant Arabidopsis thaliana (L) Heynh. from the Crueiferae family is considered as a model system for the genetics of higher plants. This is because of its short life cycle, its small size and consequent ability to be propagated in large quantities under laboratory conditions, the availability of numerous geographical races and the development of in vitro culture techniques (Redei 1974; R6bbelen 1965). An even more interesting property of Arabidopsis for molecular geneticists is the extremely low DNA content of the Arabidopsis genome which is the smallest known among higher plants: it contains only 2 x 108 base pairs per haploid genome (Bennet and Smith 1976). More recently, Leutwiler et al. (1984) reported that the haploid genome from Arabidopsis (n = 5 chromosomes) contains only 7 x l 0 V b a s e pairs, which is 20times larger than the Escherichia coli genome and 5 times the size of the Saccharomyces cerevisiae genome. The advantages of such a small

Offprint requests to: M. Jacobs Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal

genome for gene isolation and for the study of genome organization and gene expression are obvious. In this respect Arabidopsis could serve the role of the plant counterpart of Drosophila. Attempts have already been made to isolate the Arabidopsis genes coding for arginine and leucine biosynthesis by transforming auxotrophic mutants from yeast with DNA froln an Arabidopsis eosmid genomic library (Werner et al. 1982). More recently Mesnard and Lebeurier (1983) have tried to isolate genes involved in pyrimidine metabolism, by complementation of analogous E. cob mutants, using Arabidopsis cDNA. The goal of our work is to develop the alcohol dehydrogenase enzyme (E.C. 1.1.1.1.) of Arabidopsis as a biochemical marker system for transformation studies and for analysing the molecular organization and function of a plant gene. Alcohol dehydrogenase could be an excellent marker for this purpose: 1) it is quite a stable enzyme and easily detectable by histochemical staining, spectrophotometry and activity staining on acrylamide gels. 2) ADHdeficient mutants are already available for maize (Schwartz and Osterman 1976; Freeling and Cheng 1978), yeast (Lutstorf and Megnet 1968; Ciriacy 1975) and also for Arabidopsis (Jacobs and Dolferus, in preparation). 3) Selection for ADH + cells in an A D H - background is also possible, by using acetaldehyde detoxification, anaerobic stress resistance (Jacobs and Dolferus, 1983) and antimycine A resistance (Shimamoto and King 1983). Amongst higher plants, the ADH system from maize is the best characterized (see review by Freeling and Birchler 1981). It has been demonstrated that ADH in maize is one of the "stress proteins" produced during anaerobic treatment (Ferl et al. 1979; Sachs and Freeling 1978; Sachs et al. 1980). Furthermore Ferl et al. (1980) have shown that in response to anaerobiosis mRNAs for maize ADH are produced among a small class of messengers, which are specifically induced by such treatment. This observation has led to the cloning of the maize ADH cDNA (Gerlach et al. 1982) and the maize ADH gene (Dennis et al. 1984). The polymorphism of Arabidopsis ADH has already been characterized at the genetic and biochemical level. The Arabidopsis ADH enzyme behaves as a dimer and is under the control of a single genetic locus ADHI, with three codominant alleles, called S (slow), F (Fast) and A (Superfast), following their migration rate with respect to the anode (Dolferus and Jacobs 1984). This paper deals with the induction of ADH activity in plantlets and tissue cultures. Evidence is also presented

257 for homology between the Arabidopsis and maize A D H nucleotide sequences, using Northern blot analysis and a maize A D H I - F cDNA as the radioactive probe.

Materials and methods

Plant and tissue cultures. The Arabidopsis races and methodologies used for growing plants and tissue cultures were as described previously (Dolferus and Jacobs 1984). Callus cultures were induced from germinating seeds on a PG 2 medium, and from leaves on a PG 1 medium (Negrutiu et al. 1975). Hormone concentrations in PG 1 are 0.05 rag/1 kinetin and 2 mg/1 2,4-dichlorophenoxyacetic acid; PG 2 has basically the same composition, except for a lower 2,4-D concentration (1 rag/l). Callus material was inoculated in liquid PG 2 medium in order to establish suspension cultures (Negrutiu and Jacobs 1977). Plantlets were grown on a perlite solid substrate, using hormone-free PG o medium. Anaerobic induction and labelling conditions. The Arabidopsis plantlets were collected from the perlite substrate and immersed in induction buffer (10 mM sodium phosphate buffer, pH 6.0; 1% sucrose; 50 gg/ml chloramphenicol) in a closed chamber, through which water-saturated argon gas flowed continuously (Sachs et al. 1980). During anaerobic induction, about 0.125 mCi 35S-methionine (Amersham, 1,000 Ci/mmol) was added to each 0.5 ml of induction buffer. For aerobic control samples, which were allowed to grow further on the perlite substrate, the radioactive precursor was injected into the PG o medium. Callus material was labelled in liquid, hormone-containing medium (PG 1 or PG2) in conical centrifuge tubes, using the same concentrations of 35S-methionine as for the anaerobically induced plants. The tubes were then incubated on a rotary shaker at an angle of about 20 30 degrees. After the specified labelling period the plant material was washed several times in cold incubation solution and immediately frozen in liquid nitrogen. The samples were then homogenized in electrophoresis sample buffer (62.5 mM Tris-HC1 pH 6.8, 10% glycerol and 5% fl-mercaptoethanol) in a 1/2 (w/v) ratio. ADH activity assay and electrophoretic separation methods The activity of A D H was measured in the ethanol to acetaldehyde direction by following spectrophotometrically the increase in OD at 340 nm, caused by N A D H production (Dolferus and Jacobs 1984). Protein determination was accomplished according to Bradford (1976). Methods for one-dimensional native or sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and for two-dimensional native-SDS and SDS-IEF (isoelectric focusing) electrophoresis have been described in detail previously (Dolferus and Jacobs 1984). For non-denaturing native polyacrylamide gels and acrylamide concentration of 7.5% (w/v) was used: for SDS gels we used a separation gel of 10-12% w/v acrylamide. The SDS-1EF two-dimensional electrophoresis is a reversed O'Farrell (1975) technique, consisting of classical SDS electrophoresis in the first dimension and denaturing IEF in the presence of 8 M urea in the second dimension, using the LKB 2117 multiphor system.

Autoradiography andfluorography. After fixation, staining and destaining, the gels were dried under vacuum, then placed on a Fuji RX X-ray film, covered with an intensifying screen and autoradiographed at - 7 0 ° C. Fluorography was accomplished according to Chamberlain (1979). The gels were soaked in water after destaining to remove acetic acid, then shaken slowly in scintillation fluid (1 M sodium salicylate) for 20 min. Gels were then dried and treated as for classical autoradiography. RNA isolation procedure. Plant material was frozen in liquid nitrogen and ground to a fine powder in a mortar. This powder was allowed to thaw in 10 vol extraction buffer (10 mM Tris-HC1, pH 8.3; 150 mM NaC1; I mM EDTA; 0.1% SDS; Nokin et al. 1975). After addition of an identical volume of a phenol: chloroform: isoamylalcohol mixture (50:50:1) the slurry was extracted vigorously with a Warren Blendor. The suspension was centrifuged at 10,000 rpm for 30 min (10 ° C) in a Sorvall HB-4 rotor. Phenol extraction was repeated with the aqueous phase until no protein interphase was observed. The R N A was precipitated with 0.35 M NaC1 and two volumes freezer-cold ethanol. Poly(A+)-RNA was isolated by oligo-dT cellulose chromatography (Aviv and Leder 1972). Total R N A was first dissolved in elution buffer (10 mM Tris-HC1, pH 7.5 ; 1 mM EDTA; 0.5% SDS) and precipitated by addition of the same volume of 4 M LiCk After overnight precipitation at - 2 0 ° C the R N A pellet was dissolved in oligo-dT hybridization buffer (10 mM Tris-HC1, pH 7.5; 1 mM EDTA; 0.35 M NaC1 and 0.5% SDS). This solution was applied to an oligo-dT cellulose column and the poly(A+)-RNA fraction was eluted with elution buffer. This m R N A was precipitated, redissolved in sterile water at a concentration of 1 g/1 and stored at - 70 ° C. In vitro translation. The cell-free rabbit reticulocyte system was used to translate poly(A+)-RNA. The rabbit reticulocyte lysate was prepared and treated with micrococcal nuclease as described by Pelham and Jackson (1976). Typically 1 gg of poly(A+)-RNA was added to a 25 ktl reticulocyte translation mixture containing aSS-methionine. The lysates were incubated for 80 rain at 37 ° C. Radioactive translation products were monitored by electrophoresis and autoradiography. Electrophoresis of RNA and Northern blotting. Electrophoresis of R N A was carried out on denaturing 1.5% agarose gels containing 2.2 M formaldehyde (Lehrach et al. 1977; Goldberg et al. 1980). P o l y ( A + ) - R N A samples were first denatured by heating for 5 rain at 55 ° C in gel buffer containing 2.2 M formaldehyde, 50% formamide, before they were loaded on the gel. After the run the gel was first washed in several changes of water and then in 20 X SSC (3 M NaC1; 0.3 M trisodium citrate) for 40 min. The RNA was blotted onto nitrocellulose filters using 20X SSC as transfer solution, with the same technique as described for D N A blottings (Southern 1975). After a transfer of 24 h the filter was air dried and baked for 2 h in a vacuum OVeD.

Nick translation and hybridization conditions. Plasmids PZML793, containing a nearly full-length cDNA insert from maize A D H I - F (1,587 bp) cloned in the PstI site of pBR322, and PZML841 containing the maize ADH2

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cDNA ( _ 700 bp) were kindly supplied by Dr. W.J. Peacock. The complete plasmids were radioactively labelled with 32P-dATP by nick translation (Rigby et al. 1977). Northern blot nitrocellulose filters were prehybridized in a mixture containing 6X SSPE (3 M NaC1; 0.2 M sodium phosphate, pH 7.0; 20 mM EDTA) with 0.02% Ficoll 400, 0.02% polyvinyl pyrrolidine 360, 0.1% SDS and 50 ~tg/ml denatured salmon sperm DNA, for 4 h at 55 ° C. Hybridization was carried out overnight in the same solution containing 4 X SSPE. The filters were then washed twice in 2 X SSPE, 0.1% SDS, for 15 rain at room temperature and twice at 55 ° C in 0.1 X SSPE, 0. J % SDS, for 15 min.

Results

The activity of ADH rapidly declines during seed germination Seeds of Arabidopsis were sown in a petris dish on filter paper, moistened with PG o medium. After a swelling period of one night in the cold room, the seeds were allowed to germinate in the light in a growth chamber (24 ° C). At different time intervals, samples were withdrawn, extracted and A D H activity was determined spectrophotometrically. Figure 1 shows that A D H activity declines rapidly during the germination process and is no longer detectable in 9- to 10-day-old seedlings. The specific activity of A D H on the other hand increases during the first days of germination, reaching a maximum value after 3~4 days. During the germination process the protein content of the tissue also decreases rapidly.

Anaerobic induction of Arabidopsis ADH in plantlets A complete change in the protein synthesis pattern of maize roots exposed to anaerobic treatment has been observed; maize A D H is one of the major polypeptides synthesized under these circumstances (Ferl etal. 1979; Sachs and Freeling 1978 ; Sachs et al. 1980). Repeated attempts to demonstrate spectrophotometrically an increase in A D H specific activity after anaerobic induction of Arabidopsis plantlets did not give reliable and reproducible results because of the very low amount of

A D H activity and of protein of the plant tissue. Therefore we used a more sensitive pulse-labelling technique to study changes in the protein synthesis pattern of anaerobically treated plantlets on two-dimensional gels. Five-hour pulses of 35S-methionine were given to the plantlets at 5 h time intervals and samples were withdrawn when the next pulse was given. The first series of experiments was carried out with 9 to 10-day-old plantlets in which A D H activity is still detectable. Figure 2A shows that the intensity of protein synthesis is markedly affected by the anaerobic treatment. The A D H polypeptides however are synthesized very actively during up to 15 h of treatment and appear together as one of the most abundant proteins produced under these circumstances. The increase of the relative abundance of the A D H protein is shown in Fig. 2B, C, giving a total survey of the protein pattern after 5 and 24 h of anaerobiosis. Although the importance of the A D H enzyme under conditions of anaerobic stress is illustrated very clearly by this experiment, the induction phenomenon itself could not be visualized by this technique, because control, aerobically grown plantlets already showed a strongly labelled A D H spot. Therefore, another attempt was made using older plantlets (15 days), which had already developed their first leaves and which were completely devoid of A D H activity. The entire plantlets were treated as before and a first sample was taken after 2 h of anaerobiosis. Figure 3 A, B shows that A D H is very faintly visible on the fluorograph after 2 h treatment and slightly more so after 5 h treatment; the rest of the results were analogous to those obtained for the younger plantlets, although the labelling intensity obtained in this experiment was considerably lower. Under anaerobic circumstances only the small root sections incorporated radioactive tracer. The results obtained with these experiments indicate that A D H is synthesized very actively under anaerobic stress periods and that the A D H gene is induced under these circumstances. The study of the kinetics of A D H gene induction however needs a faster and more sensitive screening technique than pulse labelling.

Induction of ADH activity during callus induction In tissue cultures a very high A D H specific activity could be demonstrated. During callus induction from seeds on PG z medium containing the plant hormones 2,4-D (1 rag/l) and kinetin (0.05 mg/1) A D H specific activity increased progressively (Fig. 4A). The same event could be demonstrated when leaf, root or stem pieces were used as the inoculum for callus induction. It was observed that, after a short adaptation period, A D H specific activity increases to a level of more than ten times the initial activity. Maximal activity is reached in the stationary phase, 27 30 days after sowing, in the mature callus stage. From then on activity starts to decrease and calluses start to turn brown. A high level of A D H specific activity can however be maintained by subculturing callus material, grown on solid agar medium, in suspension cultures (Fig. 4B). During the exponential growth phase of the cell suspension A D H specific activity increases again until the cells stop dividing and turn brown. These results indicate that callus material from Arabidopsis is a very good source of ADH. The A D H enzyme activity (units/ml, 1:2 extract) in one month old calluses can be 10~15 times higher than in plantlets. In plant materi-

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Fig. 2A-C. Anaerobic treatment of 9- to 10-day-old Arabidopsis plantlets. A Effect of anaerobiosis on 35S-methionine incorporation in the ADH spot. Only the region containing the ADH protein was used for second-dimension SDS electrophoresis. B, C Demonstration of the increase in relative abundance of ADH between 5 h (B) and 24 h (C) of anaerobiosis. The position of the ADH spot was ascertained using a set of marker proteins ml, m2, and m 3 (Dolferus and Jacobs 1984). The position of the ADH spot is indicated by an arrow, marked with an asterisk al A D H remains nearly measureable spectrophotometrically. Induction of A D H at the protein level At the plant level A D H activity cannot be demonstrated except for some specific cell types in the neighbourhood of the vascular bundles (Dolferus and Jacobs 1984). In seeds activity declines rapidly during the germination process. When plant material without any measurable A D H activity such as leaf, root or stem pieces is used to initiate cultures, A D H is induced to a very high level. Comparison of twodimensional protein patterns of a leaf or seedling extract and a callus extract, shows clearly the appearance of a par-

ticular spot, identified as A D H using electrophoretic variants. This induction phenomenon was studied using the pulse labelling technique. The appearance of the A D H spot can be visualized directly on the autoradiographs by following the increase in labelling intensity of the A D H spot (Fig. 5A-C). We observed that the A D H spot becomes visible as soon as 9 days after sowing but that the relative labelling intensity is highest in mature callus (29 days old), as already expected from specific activity measurements. At the beginning of callus induction total protein synthesis seems to be activated considerably. This is possibly due to the action of the synthetic auxin 2,4-D in the medium. These results indicate that the A D H polypeptide is synthesized very actively de novo during callus induction.

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Fig. 3A, B. Anaerobic induction of ADH in 14-day-old Arabidopsis plantlets, A after 2 h and B after 5 h of labelling with 35S-methionine. Symbols as in Fig. 2

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The pulse labelling experiments dearly demonstrate that there is no activation of preexisting inactive A D H proteins.

De novo synthesis of A D H mRNA during callus formation In order to determine whether the A D H mRNAs are also synthesized de novo during callus growth or preexist as stored or masked mRNAs (Jenkins et al. 1978; Giles et al. 1977), total cellular R N A obtained from plantlets and callus was used in an in vitro translation system. Poly(A+)-RNAs were isolated from plantlets and from callus material, and tested for their ability to stimulate the synthesis of A D H polypeptides in a rabbit reticulocyte lysate system. To be able to identify the ADH protein spot on the two-dimensional gel, mRNAs from two A D H electrophoretic variants, S and A, were used. Translation products were first separated by a nativeSDS two-dimensional gel electrophoresis system (Fig. 6A). A clear difference between the two variants could not be obtained with this separation technique, probably due to an abnormal configuration of the proteins synthesized in such an in vitro translation system. However, the position of the ADH spot could be ascertained by co-migration of translation products with a callus extract. Figure 6A shows the identification of the A D H spots, the A D H polypeptides are detectable as translation products of RNA extracted from callus material. No A D H spot was observed in the case of translation products obtained from plantlet RNA. Such seedling m R N A was not able to stimulate the synthesis of A D H polypeptides in the in vitro translation system. The identification of the ADH polypeptides was confirmed by using an SDS-IEF two-dimensional electrophoresis system (Dolferus and Jacobs 1984). Translation products are first denatured with SDS and separated according to their molecular weight in the first dimension. In the second dimension they are separated according to their charge in an 8 M urea IEF gel.

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Fig. 8A, B. Induction of ADH during callus development. Autoradiographs of two-dimensional native-SDS polyacrylamide gels after incorporation of aSS-methionine into protein of A 9-day-old, B It-day-old and C 30-day-old callus. Total protein content increases markedly during early the days of callus induction until 12 to 14 days after sowing. Symbols as in Fig. 2

Figure 6B shows, respectively, translation products from poly(A+)-RNA, obtained from ADH1-A callus, a mixture of these two callus lysates and from ADH1-A plantlet mRNA. Messenger R N A from A D H I - S callus clearly gives a slower migrating translation product than the A D H I - A m R N A product. The plantlet R N A did not stimulate the translation of an A D H protein, indicating again that A D H proteins are absent in seedling plants and that they are induced de novo during callus induction.

Screening for sequence homology between Arabidopsis RNA and the maize ADH cDNA In order to estimate the homology at the R N A sequence level between maize and Arabidopsis A D H we carried out a Northern blot hybridization experiment, using cDNA clones from both maize A D H genes (Gerlach et al. 1982) as radioactive probes. The result of this hybridization is shown in Fig. 7A. Both the ADH1 and A D H 2 maize probes clearly gave a hybridization response with Arabidop-

sis' poly(A+)-RNA, extracted from one-month-old callus material. The R N A was obtained from callus tissue from two A D H charge variants, S and A, showing the same molecular weight on SDS gels but a different mobility on nondenaturing acrylamide gels (Dolferus and Jacobs 1984). As a negative control for hybridization we used m R N A from 14-day-old, aerobically grown plantlets where A D H activity is no longer detectable spectrophotometrically. An analogous experiment is shown in Fig. 7B where poly (A+)-RNA extracted from mature Arabidopsis plants and from callus cultures obtained from an A D H null mutant (R002) were used as negative controls. With the null mutant we did not find an ADH-like protein spot on twodimensional gels, nor A D H Cross-reacting material ( C R M - ) on immunodiffusion gels using antibodies raised against purified Arabidopsis A D H (Jacobs and Dolferus, in preparation). In neither did we observe a hybridization band with the maize A D H probes. These results show that sequence homology at m R N A level is present between Arabidopsis and maize ADH. Both the ADH1 and ADH2

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Fig. 6A, B. Autoradiographs from A native - SDS and B SDS-IEF two-dimensional separations from in vitro translation products, showing the de novo induction of ADH mRNA synthesis during callus induction from Arabidopsis plantlets. In both cases the position of the ADH spot was ascertained by comigration of a normal callus extract with the reticulocyte lysate, and the use of charge variants (S, A). In (B) renaturation of the ADH polypeptides in the comigrated callus extract allows the determination of the position of ADH on the autoradiographs by activity staining. Symbols as in Fig. 2

Fig. 7A, B. Hybridization of maize ADH 32p-labelled cDNA probes to Northern blot filters, carrying ArabidopsismRNA. A Hybridization of Arabidopsis poly (A+)-RNA, isolated from ADH A-type (lanes 1 and 2) and S-type (lane 3) callus, and plantlet poly (A+)-RNA (14-day-old, lane 4), with maize ADHt (PZML 793) and ADH2 (PZML 841) probes. B Hybridization of poly (A+)-RNA from Aand S-type callus, poly (A÷)-RNA from R002 null mutant callus and poly (A+)-RNA from mature Arabidopsis plants (PL) to the maize ADHI eDNA probe. No hybridization was observed for R002 and mature plant mRNA, which are completely devoid of any measurable ADH enzyme activity. The hybridization band with S- and A-type callus mRNA is about 1,500-1,600 base pairs which is in close agreement with the expected size for the ArabidopsisADH messengers probes from maize seem to hybridize to the same extent with Arabidopsis A D H m R N A . Discussion The induction o f A D H activity in Arabidopsis was investigated in seedlings and in tissue cultures. U n d e r n o r m a l de-

velopmental conditions, enzyme activity declines during the germination process. This situation is c o m p a r a b l e to the maize system: A D H activity seems to be required only during the germination process when metabolic activity is shifted from an inactive to a very active stage. W e also investigated whether the inducibility o f the enzyme by anaerobic stress circumstances could be c o m p a r e d

263 to the maize system (Sachs et al. 1980). The availability of a set of inducible anaerobic stress proteins was important as an m R N A enrichment step leading to the isolation of the maize cDNA (Gerlach et al. 1982). Results presented in this paper show that A D H proteins are synthesized very actively during the anaerobic treatment of plantlets in an argon atmosphere and that A D H is one of the most abundant proteins synthesized under these conditions. Only after 15 h of anaerobiosis does activity slowly decrease. However, a drastic change in the protein pattern on two-dimensional gels to a new stress protein pattern was not apparent. The changes observed were merely quantitative in that the labelling intensities of most proteins changed markedly during anaerobiosis: many proteins disappeared from the gel pattern, while only some of them (about ten were not affected much during up to 24 h of anaerobiosis. By using older plantlets (14 days old), in which A D H activity had never been demonstrated, neither spectrophotometrically nor with Northern blotting using the maize A D H cDNA as a probe, we could observe a very faintly labelled A D H spot after only 2 h of treatment. The rest of the experiment showed a course like that of the younger plantlets. This should indicate that the enzyme is inducible under anaerobic stress periods. However, for a more quantitative survey of the onset of A D H gene activity, a more sensitive and faster screening method was needed. In vivo labelling techniques with 35Smethionine allow only the screening of proteins which have accumulated or been repressed during the labelling period; the amount of time required to obtain enough label for gel analysis and autoradiography is a limiting factor for studying quantitatively the induction of A D H under anaerobiosis. A faster and more reliable technique for studying gene activity or enzyme induction is accomplished at the m R N A level by Northern blot hybridization, using a homologous c D N A probe to measure the concentration of m R N A accumulated after certain induction periods (Hake et al. 1984; Gerlach et al. 1982). When plant material (seeds or leaves) is used for callus induction on a 2,4-D-containing medium, A D I t was induced during the dedifferentiation process. This induction is the consequence of the de novo synthesis of A D H polypeptides as could be ascertained by pulse-labelling experiments with 3SS-methionine. The presence of ADH-like inactive polypeptides in the inoculum was investigated first by the protein pattern from a two-dimensional gel. Then the relative labelling intensity of the A D H spot was followed during callus induction and the highest specific activity was found in mature, one-month-old callus material. Using in vitro translation we found that only poly (A+) R N A from callus material was able to stimulate the synthesis of ADH-like polypeptides, while poly (A+)-RNA from plantlets, cultured on hormone-free medium, did not. These results indicate that the A D H messengers are also synthesized de novo during callus induction and show that there are no preexisting A D H messengers in the starting material for callus induction. It has been demonstrated previously that post-transcriptional control of gene expression can occur through sequestering of m R N A by compartmentalization (Taneja and Sachar 1976; Brooker et al. 1978; Martin and Northcote 1981), or by an inactivation or masking of the sequestered messengers by association with proteins (Jenkins et al. 1978). In both cases the stored messengers

are still in an active form, and the occurrence of their protein products should be demonstrable after in vitro translation. The trigger of this high rate of A D H synthesis in Arabidopsis tissue cultures is not known. Thomas and Murashige (1979 a, b) have observed that most tissue cultures of plants accumulate large amounts of CO 2 (carbon dioxide), ethylene, ethane and the two A D H substrates, ethanol and acetaldehyde. The dedifferentiation process and the proliferation of cells in tissue cultures is characterized by very high metabolic activity in these cells. The synthetic auxin 2,4-D might therefore be responsible for the induction of A D H in tissue cultures. Auxins are known to stimulate the transcriptional mechanism in plant cells (Verma et al. 1975; Bevan and Northcote 1981). They are also able to induce the synthesis of new messengers or to amplify the synthesis of some specific messengers in plant tissues (Zurfluh and Guilfoyle 1982). Evidence for sequence homology between the maize ADH1 and ADH2 cDNAs and Arabidopsis A D H m R N A has also been demonstrated by Northern blot hybridization. Formerly it has been argued that considerable homology existed between the maize and the Arabidopsis A D H protein (Dolferus and Jacobs 1984). It was even possible to make hybrid enzymes in vitro between maize and Arabidopsis A D H which still showed activity, and cross reaction between anti-maize A D H antibodies and Arabidopsis A D H was also observed. Maize A D H seems to be quite a conserved enzyme among higher plants. This sequence homology will be used to find the Arabidopsis A D H cDNA in a cDNA bank. Since Arabidopsis has been shown to be susceptible to infection by Agrobacterium tumefaciens (Aerts et al. 1979), the A D H system could serve the role of a biochemical marker for DNA-mediated transformation using Ti plasmids. The susceptibility of Arabidopsis to Cauliflower Mosaic Virus infection (Balazs and Lebeurier 1981) also creates possibilities for plant transformation. Selection techniques for A D H as a biochemical marker in tissue cultures which are A D H deficient, are also currently under investigation.

Acknowledgements. This research was supported by the "Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" (IWONL 4027A) and the Services of the Prime Minister (00A 80/85-8). R.D. is a research fellow of the IWONL. References Aerts M, Jacobs M, Hernalsteens J-P, Van Montagu M, Schell J (1979) Induction and in vitro culture of Arabidopsis thaliana crown gall tumours. Plant Sci Lett 17:43-50 Aviv H, Leder P (1972) Purification of biologically active globin mRNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69:1408-1412 Balazs E, Lebeurier G (1981) Arabidopsis is a host of Cauliflower mosaic virus. Arab Inf Serv 18 : 130-134 Bennet MD, Smith JB (1976) Nuclear DNA amounts in angiosperms Proc R Soc Lond [Biol] 274:227-274 Bevan M, Northcote DH (1981) Subculture-induced protein synthesis in tissue cultures of Glycine max and Phaseolus vulgaris. Planta t 52:24-35 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bidning. Anal Biochem 72:248-254 Brooker JD, Tomaszewski M, Marcus A (1978) Preformed messenger RNAs and early wheat embryo germination. Plant Physiol 61 : 145-149

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Communicated by J. Schell

Received September 6 / December 17, 1984