Polyadenylated RNA Sequences Which Are Reduced in ...

THE JOURNAL

OF

BIOLOGICAL CHEMIBTRY

Vol. 255. No. 18,lasue of September 25. pp. 8907-8913, 1980 Printed in U.S. A.

Polyadenylated RNA Sequences Which Are Reducedin Concentration Following Auxin Treatment of Soybean Hypocotyls* (Received for publication, January 24, 1980)

David C. Baulcombe and Joe L. Key$ From the Departments of Botany and $Biochemistry, University of Georgia, Athens, Georgia 30602

Previous work has shown that any effect of exogenous auxin on gene expression in soybean hypocotyl tissue must be restricted to a relatively small fraction of the polyadenylated RNA. However, kinetic hybridization analysis with cDNA probes revealed that a minor abundant class of sequences is markedly reduced in concentration in the auxin-treated polyadenylated RNA. Recombinant plasmids containing copies of polyadenylated RNA species were constructed using the GC tailing procedure and clones of auxin-regulated sequences were detected by differential in situ hybridization with cDNA of polyadenylated RNA from auxintreated or untreated hypocotyls. Although the 12 clones which were selected all contained different size inserts, and were therefore independent, 11 of these apparently hybridized to just two different RNA species. The rate constant of the auxin-sensitive abundant component of the untreatedpolyadenylated RNA/DNA hybridization was similar to that of the reaction between the two major groups of clones and untreated polyadenylated RNA. This indicates that these cloned sequences are homologous with that cDNA fraction. The twelfth clone is thought to be representative of a group of less abundant auxin-regulated polyadenylated mRNA species which had been detected in an earlier analysis of the in u i t m translation products of soybean hypocotyl RNA. Both the timing and the extent of the influence of auxin on the relative concentration of these cloned sequences are quite consistent with a close relationship between growth regulation by auxin and its effects on gene expression.

where cell division or cell differentiation is the response. On this basis, we are investigating the possibility that other mechanisms, including gene regulation, may mediate auxinregulated plant growth. A large amount of experimental data, largely overlookedin the light of the strong attractions of the wall acidification model, exists to support the idea that auxin action involves an effect on geneaction. Briefly summarized, this evidence is as follows: RNA synthesis is enhanced by auxin in responsive tissues (6); continued RNA synthesis, including presumptive mRNA synthesis, is required for continued elongation of piant tissues, including soybean hypocotyl (6); auxin does not enhance the rate of cell elongation when RNA synthesis is inhibited by a level of actinomycin D which does not impair the endogenous elongation rate for about 2 h ( 7 ) ; there is a rapid change in mRNA utilization, evidenced by increased polysome formation, subsequent to treatment of soybean hypocotyls with auxin (8); also, in soybean hypocotyls, auxin induces a massive increase in RNA polymerase I activity and rRNA synthesis (9) and a general enhancement of synthesis of all classes of RNA including presumptive mRNA (10); in pea epicotyls, auxin treatment enhances the level of cellulasemRNA (11); the in vitro translation products of polyadenylated RNA of auxin-treated soybean hypocotyls are different from the products of untreated polyadenylated RNA (12), indicating that approximately 40 moderately abundant mRNA sequences undergo either upward or downward shifts in concentration following auxin treatment. In soybean hypocotyl, where the response to exogenous auxin is a massive tissue proliferation, we have shown previously, using nucleic acid hybridization, that auxin does not affect the concentration of the large majority of the 40,000 different polyadenylated RNA sequences expressed (12). There is, however, a downward shift in the concentration of the most abundant polyadenylated sequences following auxin application (12). In order to extend these studies and to investigate the influence of auxin on expression of specific genes, we have constructed recombinant plasmids containing auxin-regulated polyadenylated RNA sequences. This paper describes the isolation and characterization of these plasmids.

Auxin is a class of naturally occurring plant growth regulators which is required for growth of plants both by cell expansion and cell division (1).While several mechanisms of auxin action have received attention, including regulation of gene expression, a mechanism involving a loosening of rigidifying links in the cell wall, mediated by extrusion of protons across the plasmalemma (2),is now widelyaccepted. However, as has been explained chiefly by Vanderhoef ( 3 4 , such a mechanism, which mayaccount for a rapid transient response EXPERIMENTALPROCEDURES to auxin, may not be adequate to explain the prolonged effects Plants-Soybean seeds (Glycine max), Wayne variety, were gerof auxin on cell growth. He and others have demonstrated minated in moist vermiculite in the dark at 30°C for 3 days. After this that auxin-enhanced cell elongation may be divided into two time, plants were sprayed with 2,4-dichlorophenoxyaceticacid, a phases which exhibit differential sensitivity to cytokinins (3), synthetic auxin. Hypocotyls were harvested 40 h later. Untreated auxin analogs (4), and cycloheximide (5). Of course, the wall hypocotyls were harvested after 5 days total germination. acidification model cannot explain auxin action in situations Preparation and Analysis of Polyadenylated RNA * This work was supported by Grant CA11624 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Isolation of RNA-Hypocotyls were homogenized with a Polytron homogenizer (Brinkmann) in detergent buffer and extracted with phenol/chloroform/isoamyl alcohol (24:24: 1) as described by Silfow et aZ. (13).Total nucleic acid was ethanol-precipitated and redissolved at more than 1 mg rn” in 50 mM Tris-HC1 (pH 9). 1 mM EDTA, and

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1%sarkosyl (14). After a further extraction with phenol/chloroform/ isoamyl alcohol, RNA of a size greater than 5 S was selectively precipitated away from DNA, 5 S RNA, and tRNAby the addition of NaOAc (pH 6) to 0.1 M and solid NaCl to 3.0 M final concentration. RNA was collected after incubation for 24 h a t 0°C by centrifugation (10,000 rpm, 2"C, Somall SS-34 rotor). Polyadenylated RNA was prepared by oligo(dT) cellulose chromatography (Collaborative Research type T2) as described by Silflow et al. (13). This RNA was essentially free of rRNA (54 >41 >49

172

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0.05

Q 0.2

0.10

9.8

0.01

15.6

15.0

"The rate constant of the hybridization against polyadenylated RNA of either untreated or auxin-treated tissue. Calculated based on the rate constant of 1700-nucleotide ovalbumin mHNA-cDNA hvbridimtion. No account has been made for a size difference between the ovalbumin cDNA and the nick-translated clone inserts, so these values are underestimates. Thus, percentage of RNA = (K/Kf,v.thum,n) X (length of driver HNA/length of ovalbumin mHNA). ' Not determined directly. Semiquantitativeanalysis using the Northern blot technique showed that clones 1 and 5 are copies of sequences which were reduced in relative concentration, following auxin treatment, to a similar extent as other sequencesin this group. " Determined using a double-stranded nick-translated cloned insert probein 80'1 formamide. 0.4 M NaCI, 10 mM Pipes, pH 6.9, 1 mM EDTA. The rate constant values have been corrected by a factor of 12 to account for the decreased rate of RNA/DNA hybridization in this buffer relative to the rate in aqueous buffer with the same salt concentration ( 3 1 ) .

a

b

0 4 8 2049

0 4 8 2040

C

0 4 8 2040

HOURS OF AUXIN TREATMENT FIG. 6. Time course of the effect of auxin on RNA encoded by cDNA containing recombinant plasmids. Aliquots ( 1 pg) of polyadenylated RNA isolated from soybean hypocotyls, which had been treated for different times with auxin, were electrophoresed in 21. agarose gels containing 6% formaldehyde. The gels were then blotted, hybridized to nick-translated plasmid DNA and autoradiographed as described under "Experimental Procedures." a, clone 9; h, clone 3; c, clone 1 1 .

detected by this technique to the 20-h auxin-treated RNA. Examples of data from each of the two major groups andfrom clone 11 are shown (Fig. 6). The cloned sequences therefore are representative of the abundant cDNA class, the concentration of which is reduced following auxin treatment. DISCUSSION

The data in Fig. 1 with unfractionated cDNA and in Fig. 5 and 6 using cloned probes of purified polyadenylated RNA sequences clearly demonstratethatsome polyadenylated RNA sequences arereduced in relative concentration following auxin treatment. It is likely that the two major groups of clones, which are internally homogeneous, are homologous with the highly abundant auxin-sensitive classof cDNA in the untreated cDNA reaction (Fig. l ) , evidenced by repeated isolation of clones of these sequences and the similarity of the rate constant of the cloned-probe reactions (5 and 12.5, Table 111) with that of the cDNA reactions (14, Table I). Precise equivalence of these uncorrected parametersfrom the clonedprobe reactions and the total cDNA reaction is not expected since the nick translated DNA would have been shorter than the reverse transcribed probe. Furthermore, if the length of the RNA driving the abundant class reaction was relatively short, as apparently is the case in untreated soybean hypocotyl, the abundant class seqeunces would have been overrepresented in a cDNA preparation which was primed at the 3' end of the RNA andwhich has a final length substantially less than the template RNA. Hence,the abundantclass cDNA (Fig. 1) was estimated to comprise 4%of the cDNA, whereas the majorgroups of clones which are homologous to relatively short RNA only account for approximately 2% of the untreated polyadenylated RNA based on kinetic analysis of the hybridization of these clones. It is striking thatall three types of clone are homologous to abundant RNAswhich are markedly smaller than the number average lengthof polyadenylated RNA in the soybean hypocotyl. This is reminiscent of the situation in mouse L-cells where there is a general correlationbetweensmall size, increased stability,andgreater abundance amongpolyadenylated mRNA species (32). Clone 11 probably represents a group of less abundant auxin-regulated sequences which are not resolved by cDNA hybridization, but which aredetected in a 2D gel analysis of the translationproducts of polyadenylatedRNAfromauxintreated and untreated tissues (12). Approximately 40 auxinregulated sequences were resolved by translation analysis, divided equally between sequences increased and sequences decreased in relative concentration following auxin treatment. Clearly, a much more extensive colony hybridization screen than was carried outin this studywould be needed in order to isolate more cloned sequences from this less abundant group of auxin-regulated genes. The magnitude of the difference between the untreated and auxin-treated RNA populationsobserved in this and the earlier study (12) is comparable to the difference between RNA populations of quiescent and growth-stimulatedcells of other organisms. In soybean hypocotyl, the effect of auxin on RNA is mainly limitedto an altered concentration of some abundant sequences as indicated by single copy DNA hybridizations and cDNA hybridizations (12). Similarly transformed mouse fibroblasts share virtually all the polyadenylatedRNAsequences of nontransformed cells and vise versa (33), and stimulation of rat liver cell proliferation also causes a reduced concentration of someabundant polyadenylated RNA sequences (34). In contrast, the development of differentiated cell types in tobacco is associated with relatively large differences in the single copy DNAtranscript fraction of polysomal RNA (35). Since auxin-mediated growth must be an early

Regulation Auxin

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stage in all organ differentiation, some other factors must induce this more extensive regulation of gene expression. Of course, part of the extensive developmental regulation of gene expression (35) mayinvolve nonpolyadenylated sequences which were not studied here. It is not clear whether the shift in abundant polyadenylated RNA sequence concentration reported here is mediated via general stimulation of the levels of all other RNA sequences or viaa specific reduction of the levels of the cloned sequences (or both). Messenger RNA levels in this tissue do increase following auxin treatment (8),indicating that theformer possibility may account in part for an effecton the relative concentration of the sequences encoded by clones 8 through 12, which are reduced 10- to 15-fold in relative concentration (Table 111). The sequence of clones 1 through 7, however, is reduced 100-fold in relative concentration, indicating that specific regulation processes must occur. Comparison of the synthesis, processing, and turnover of the regulated and nonregulated sequences will be needed for a precise assessment of these alternatives. The demonstration of quantitative changes in the polyadenylated RNA following auxin treatment does not establish a direct association between auxin/cell interaction and gene regulation. However, hybridization data from Fig.6 and translation product analysis (Ref. 12 and Footnote 2) show that the auxin-associated effect on polyadenylated RNA is initiated during the initial 4 haftertreatment, before detectable changes in the growth pattern occur (36). Thus, the timing of this auxin effect is similar to the timing of the influence of auxin on RNA polymerase I activity (9). In the chick oviduct, the induction of increased levels of egg-white protein mRNA levels occurs over a similar interval (37). The time scale of the effect of auxin on polyadenylated RNAis therefore quite consistent with a direct specific effect of auxin on gene expression. Use of the recombinant DNA clones described in this paper will allow detailed analysis of this possibility. The G-C tailing procedure of cDNA cloningis now standard procedure and the technique of differential colony hybridization has been used previously for isolation of development stage specificmRNA probes (38) for sequences which are abundant but not predominant in the RNA population. A useful improvement in the methods of this paper is in the use of [32P]cDNA probes and small buffervolume for colony hybridizations. This modification allows the detection of recombinant colonies complementary to -0.2% of the template RNA in 1 to 2 days of autoradiography, rather than 2 weeks as in the earlier work (38). Acknowledgments-We thank Dr. Richard Meagher for the use of biocontainment facilities and for provision of materials, Dr. Robert Goldberg for communicating the modification of the Northern blot procedure, Dr. W. Salser for terminal transferase, Dr. W. Dower for ovalbumin mRNA, and Ms. Coco Whelchel for technical assistance. REFERENCES 1. Thimann, K. V. (1969) in The Physiology of Plant Growth and

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