Proc. Nati Acad. Sci. USAVol. 79, pp. 7185-7189, December 1982Biochemistry

Isolation of cloned cDNAs to auxin-responsive poly(A)+RNAs ofelongating soybean hypocotyl

(hormone action/cell elongation/gene expression)

JOHN C. WALKER AND JOE L. KEYDepartments of Botany and Biochemistry, University of Georgia, Athens, Georgia 30602

Communicated by Folke Skoog, August 30, 1982

ABSTRACT Auxin-responsive cDNA clones have been iso-lated from a cDNA library prepared from elongating soybean hy-pocotyl poly(A)+RNA. The expression of two such sequences hasbeen assessed by RNA blot hybridization analyses during normaldevelopmental transitions in the soybean hypocotyl and duringincubation of sections excised from the region of cell elongation.The concentrations of these poly(A)+RNAs are higher in the elon-gating zone than in the apical and mature zones of the hypocotyl.Both poly(A)+RNAs are depleted during incubation of the sectionsin the absence ofauxin. The loss ofone ofthese sequences (pJCWI)is prevented by the addition of auxin to the incubation mediumwhile the other sequence (pJCW2) increases above the initial levelin the presence of auxin. The addition of auxin to auxin-depletedtissue in which the sequences are depleted results in rapid accu-mulation of these poly(A)+RNAs; pJCW1 accumulates to the con-trol level while pJCW2 increases well above the control level.These data alongwith others [Baulcombe, D. C. & Key, J. L. (1980)J. BSOL Chem. 255, 8907-8913] demonstrate directly a highly se-lective effect of auxin on the expression of a small number ofmRNAs in tissues undergoing both cell elongation and cell divisionin response to auxin. Although the data are suggestive of a closeassociation betwen auxin action and altered gene expression, acausal relationship is not established. It seems highly unlikely,however, that such specific effects ofauxin on gene expression areunimportant in auxin physiology.

Auxins are a class of plant hormones implicated in the controlof both cell elongation and cell division (1). Based on an analysisofgrowth characteristics and nucleic acid content ofcallus tissueunder different hormone regimes, Skoog (2) suggested that theaction of auxin is closely coupled to altered nucleic acid metab-olism. Subsequently, results from a number of investigationsshowed higher nucleic acid contents of tissue in response toauxin (3, 4). With the availability of relatively specific inhibitorsofRNA and protein synthesis, a requirement for RNA and pro-tein synthesis in auxin-induced cell elongation was demon-strated (5, 6). A generally positive correlation between the abil-ity of auxin to enhance RNA synthesis and the enhancement ofcell elongation by auxin has been shown in a number of studies(7, 8). The parallel incremental inhibition of (mRNA-like) RNAsynthesis by actinomycin D or protein synthesis by cyclohexi-mide and auxin-induced cell elongation also provided supportfor the view of a close association between nucleic acid metab-olism and auxin action (9, 10).The possibility of a causal relationship between the enhance-

ment by auxin of cell elongation and altered RNA synthesis wasgenerally thought to be excluded by the theoretical consider-ations of Evans and Ray (11) relating the kinetics of auxin-in-duced growth to kinetic parameters of RNA synthesis or half-

life ofthose RNAs (or both). Consequently, there was a dramaticshift in research activity relating to auxin action from consid-eration ofRNA and protein synthesis to studies on acidificationof the cell wall by auxin-mediated hydrogen ion extrusion (12,13). Subsequent studies showing a dual response to auxin in thecell elongation process (14)-i.e., one response occurring witha lag of about 15 min and a second with a lag of some 45 minled to increased consideration of the possibility of a causal as-sociation between auxin-regulated RNA and protein synthesisand auxin-mediated growth processes (15).

Hybridization analyses (16, 17) showed that the expressionof some 40,000 different poly(A)+RNAs was not significantlyaltered in tissue induced to proliferate by auxin. However,auxin treatment of soybean hypocotyl did result in a markeddifferential expression of a few highly abundant poly(A)+RNAs,based on both in vitro-translation two-dimensional gel analyses(16, 17) and RNA blot hybridization to cloned cDNAs (18). Soy-bean cells in culture also showed changes in selected mRNAlevels in response to auxin based on in vitro translation data(19). Earlier, Verma et al. (20) showed increased levels of trans-latable cellulase mRNA in pea stem tissue undergoing long-term auxin-induced growth aberrations. In recent studies,Zurfluh and Guilfoyle (21, 22) showed that auxin affects selec-tively the in vivo synthesis of a few proteins in excised soybeanhypocotyl and demonstrated by in vitro-translation two-dimen-sional gel analysis (23, 24) a rapid increase in the concentrationof translatable mRNAs for a few proteins in response to auxinin excised elongating and mature tissue of soybean hypocotyl.Similar observations were made by Theologis and Ray (25) usingpoly(A)+RNAs isolated from excised pea epicotyl tissue.

In the experiments reported here, cloned cDNAs were usedto determine whether auxin induces changes in certain mRNAsin tissue undergoing an enhanced rate of cell elongation in re-ponse to auxin, a quantitative (change in rate) response in con-trast to the induction of cell division. The rate of elongation ofsoybean hypocotyl decreases from about 0.6 mm/hr to a baselevel of about 0.2 mm/hr when excised sections are incubatedin the absence of auxin (15); addition of auxin to the incubationmedium restores the elongation rate to about 0.6 mm/hr. Thissystem thus offers the opportunity to investigate the influenceof auxin on the production ofmRNAs that may be important tothe cell elongation process. The data presented here usingcloned cDNA RNA blot analyses provide a direct demonstrationofan auxin-mediated selective increase in mRNA concentrationin tissue undergoing enhanced rates of cell elongation. Theseobservations along with those of Baulcombe and Key (18) showdirectly that auxin selectively and dramatically alters the con-centration of a few poly(A)+RNA sequences in tissues undergo-ing both cell elongation and cell division in response to the hor-mone.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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METHODS AND MATERIALSPlant Material. Soybean seeds (Glycine max var. Wayne)

were allowed to germinate in moist vermiculite in the dark at28-30'C for 96 hr; 10-mm-long elongating sections (5-15 mmbelow the cotyledons) were harvested into medium at 40C andeither placed in incubation medium or extracted for RNA. Allexperiments were with 10 g of tissue in 40 ml of incubationmedium [10 mM K phosphate, pH 6.0/2% (wt/vol) sucrose]with or without 23 A.M 2,4-dichlorophenoxyacetic acid (2,4-D,a synthetic auxin) for the times indicated in a 250-ml flask at 28-30°C with continuous shaking. Apical (0-5 mm below the coty-ledon) and mature (from 15mm below the cotyledon to the base)zones were harvested and immediately extracted, for RNA.

Isolation of RNA. Tissue samples were extracted with 50mMTris-HCI, pH 8.8/2% NaDodSO4 and phenol/chloroform/isoamyl alcohol (24:24:1) by homogenization with a Polytron.Total nucleic acid was ethanol precipitated, extracted with TEbuffer (10 mM Tris HCI, pH 7.5/1 mM EDTA)/1% Sarkosyland phenol/chloroform/isoamyl alcohol (24:24:1) and repre-cipitated with ethanol. RNA greater than about 5S was thenselectively precipitated with 3 M NaCl, collected by centrifu-gation, and dissolved in TE buffer/0. 1% NaDodSO4.Poly(A)+RNA was isolated by oligo(dT)-cellulose chromatogra-phy as described by Silflow et al. (26).

Quantitation of Poly(A)+RNA. Poly(A)+RNA content wasmeasured by [3H]poly(U) hybridization (27). In a standard 50-,l assay mixture, 0.01-0. 03 pug ofpoly(A)+RNA was hybridizedwith excess [3H]poly(U) (200 p.Ci/pAmol of P; 1 Ci = 3.7 X 1010becquerels; Miles) in 10 mM Tris HCl, pH 7.6/200 mM NaCl/5 mM MgC12 containing yeast RNA at 50 pug/ml for 30 min at250C. Nonhybridized [3H]poly(U) was digested at 250C for 30min by the addition of 10 ,ul of RNase A (100 ,ug/ml).in 150 mMNaCl. The entire reaction mixture was spotted onto WhatmanDE-81 filters, and the filters were washed for 30 min with threechanges of 0.5 M Na phosphate (pH 6.5), rinsed with water andthen with ethanol, dried, and assayed with 5 ml of a toluene-based scintillation fluid. All samples were assayed in triplicate.

Construction of the cDNA Library. The conditions for syn-thesis of double-stranded cDNA molecules were those de-scribed by Wickens et aL (28) with modifications of Baulcombeand Key (18). Poly(A)+RNA from unincubated elongating sec-

tions of soybean hypocotyl was used as template for oligo(dT)-primed synthesis of double-stranded cDNA by reverse tran-scriptase and DNA polymerase. Hairpin loops and single-stranded regions were removed by nuclease S1 digestion, andthe large cDNA molecules were selected on sucrose gradients.Terminal deoxyribonucleotidyltransferase was used to addpoly(dC) tails to the 3' ends of the double-stranded cDNA mol-ecules and poly(dG).tails to Pst I-cut pBR322. The tailedplasmidand cDNA molecules were circularized slowly in dilute solutionto form molecules that were directly transformed into Esche-richia coli SK 1590 (29). Recombinant clones were selected bygrowth on Luria agar plates containing tetracycline at 10 pug/ml. The recombinant cloningwas accomplished in accordancewith the 1980 National Institutes of Health guidelines for re-

combinant DNA research.Selection of Auxin-Responsive Recombinant Plasmids. Col-

onies were screened for auxin-regulated recombinant plasmidsby using the in situ colony hybridization method of Grunsteinand Hogness (30) with the modifications of Chang et al. (31) andSchoffl and Key (32). Colonies were picked and patched (100per 15 x 100 mm Petri dish) onto Luria agar containing tet-racycline at 10 pug/ml and grown for 24 hr at 37°C. Two replicaswere made onto nitrocellulose discs, the colonies were grownand lysed, and the DNA was fixed to the nitrocellulose. One

set of replica filters was hybridized with [a-32P]dCTP-labeledcDNA synthesized from the poly(A)+RNA ofelongating sectionsincubated for 4 hr in the absence of auxin. The second set offilters was hybridized to similarly labeled cDNA made from thepoly(A)+RNA of elongating sections previously incubated for 2hr without auxin and then for 2 hr in the presence of auxin.Colonies showing differential hybridization were transferred tofresh selective nutrient agar plates, grown, and replicated ontonitrocellulose. After growth and fixation ofcolonies, the replicafilters were hybridized with the two cDNA populations as de-scribed above. Conditions for single-strand cDNA synthesis andfilter hybridization, washing, and autoradiography were thosedescribed by Baulcombe and Key (18).

Plasmid Preparation and Insert Isolation. Cells were grownin Luria broth containing tetracycline at 20 /ig/ml at 370C.Plasmid copy number was amplified by treating cells overnightwith chloramphenicol at 150 tug/ml (33, 34). Plasmid DNA wasisolated by the Sarkosyl lysis method (35) with the omission ofRNase and purification of closed-circular plasmid DNA by tworounds of CsCl/ethidium bromide centrifugation (36). cDNAinserts were isolated by Pst I digestion followed by separationand recovery by electrophoresis on agarose gels (37).

In Vitro Labeling of Plasmids. Plasmids were labeled bynick-translation using [a-32P]dCTP (400 Ci/mmol; Amersham)to specific activities of 0.5-1 x 108 cpm/,ug of DNA. Unincor-porated nucleotide was separated from labeled DNA by chro-matography on Sephadex G-50.RNA Blot Hybridization. RNA was electrophoresed in 2%

agarose/6% formaldehyde (38) and transferred to nitrocelluloseaccording to Thomas (39). Baking, prehybridization, hybridiza-tion, washing, and autoradiography were as described by Baul-combe and Key (18). Glyoxylation of RNA and subsequent elec-trophoresis were as described by McMaster and Carmichael(40).

RESULTSSelection and Isolation ofRecombinant Plasmids Containing

cDNA Copies of Auxin-Responsive Poly(A)+RNA. Recombi-nant DNA technology was used to isolate cDNA probes suitablefor studies of individual auxin-reponsive poly(A)+RNAs. cDNAcloning was accomplished in the plasmid pBR322, which nor-mally confers ampicillin and tetracycline resistance to the hostcell; insertion ofDNA into the Pst I site destroys the ampicillinresistance, allowing recombinant clones to be recognized as tet-racycline resistant, ampicillin sensitive. From transformationof 0.14 ,g of double-stranded cDNA, approximately 14,000tetracycline-resistant colonies were obtained; of these, 95%were. ampicillin sensitive.

Plasmid DNA from 19 clones was isolated, labeled by nick-translation, and used individually to probe RNA blots of equiv-alent amounts ofpoly(A)+RNA isolated from auxin-depleted andauxin-induced tissue. RNA blot profiles of two of the cloneschosen for further study are shown in Fig. 1. Both cDNA clonesshow stronger hybridization with RNA from auxin-induced tis-sue than with RNA from auxin-depleted tissue, indicating ahigher relative concentration of these RNAs in the RNA pop-ulation isolated from auxin-treated tissue. The hybridizationdata further indicate that RNA corresponding to pJCW1 is moreabundant than that complementary to pJCW2. The molecularweight of these RNAs is slightly different (Table 1) indicatingthat each clone hybridizes to an independent RNA species.Some properties of the cloned sequences and relative abun-dance of the corresponding RNAs are given in Table 1.

Analysis of Poly(A)+RNA Corresponding to Auxin-Respon-sive cDNA Clones by RNA Blot Hybridization. The selectedclones hybridize to RNAs that are present at relatively higher

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Proc. Natd Acad. Sci. USA 79 (1982) 7187

A A B A B

.d w #

a b c d e fFIG. 1. RNA blot hybridization analysis of poly(A)+RNA from

elongating soybean hypocotyl. Poly(A)+RNA was separated by elec-trophoresis on 6% formaldehyde/2% agarose gels, transferred to ni-trocellulose, and hybridized to 32P-labeled pJCW1 (A and A') orpJCW2 (B). One microgram of poly(A)+RNA was separated by elec-trophoresis from tissue incubated without auxin for 2 hr and then withauxin for 2 hr (lanes b, d, and f) or from tissue incubated without auxinfor 4 hr (lanes a, c, and e). Autoradiography was for 3 hr (A) or for 18hr (A' and B); A is a shorter exposure ofA' and gives a more accuraterepresentation of the difference in levels of RNA hybridizing topJCW1.

concentration in auxin-induced tissue than in auxin-depletedtissue (Fig. 1). If these auxin-responsive RNAs are involved incell elongation in the intact hypocotyl, a higher concentrationmight be expected in the elongating zone than in apical (divid-ing) or mature (growth-quiescent) zones of the soybean hypo-cotyl. RNA blots of poly(A)+RNA obtained from these regions(Fig. 2) show that bothcDNA clones hybridize to RNAs that arepresent in greater relative abundance in actively elongating tis-sue than in apical or mature tissues.

Fig. 3 shows the level of pJCW1 and pJCW2 RNAs fromelongating tissue incubated in the presence (lanes b-d) or ab-sence (lanes f-h) of auxin for 2, 4, and 6 hr after excision.Poly(A)+RNA corresponding to clone pJCW1 (Fig. 3A) is main-tained at control (unincubated) levels (lanes a and e) in tissueincubated with auxin but shows a rapid decrease in tissue in-cubated without auxin. The level of pJCW2 RNA increases inexcised sections incubated with auxin but decreases in auxin-depleted tissue. These changes occur in tissue in which a con-stant growth rate is maintained in the presence ofauxin (6) whilethe elongation rate declines to a low base level after excision inthe absence of auxin (15).

Kinetics ofRNA Induction by Auxin in Auxin-Depleted Tis-sue. RNA blot hybridization analyses show a rapid accumulationof the auxin-responsive poly(A)+RNAs noted above on additionofauxin to auxin-depleted tissue. Hybridization intensity shows

Table 1. Characteristics of auxin-responsive cDNA clonespJCW1 pJCW2

DNA insert size, base pairs 580 + 160* 720Poly(A)+RNA size, bases 1,100 1,050Relative level of RNAUnincubated elongating tissue + + + + + + + +

Auxin-depleted elongating tissue + + + +

Auxin-induced elongating tissue +++++ ++++

DNA insert size was determined by comparison of Pst I-digestedfragments with markers of Alu I-digested DNA of pBR322.Poly(A)+RNA size was determined by comparison of poly(A)+RNAwith Alu I-digested and Sal I/Pst I-digested pBR3Z2 DNA run onglyoxal gels, transferred to nitrocellulose, and hybridized separatelyto32P-labeled plasmid DNA. Relative level ofRNAwasestimated fromRNA and dot blot hybridization analyses.* This insert contains an internal Pst I site.

a b c d e f

FIG. 2. Developmental regulation of poly(A)+RNA hybridizationto 32P-labeled cloned cDNAs. (A) pJCW1. (B) pJCW2. Poly(A)+RNA(A, 0.25 Mg, B, 0.5 ,ug) from apical (lanes a and d), elongating (lanesb and e), and mature (lanes c and f) zones was electrophoresed, trans-ferred, and hybridized as described in Fig. 1.

an increase in pJCW2 RNA within 15 min (Fig. 4B, lane c) whilean increase in pJCW1 RNA level is apparent within 30 min (Fig.4A, lane d). RNA hybridization to pJCWl reaches the controltissue level by 90 min of auxin treatment (lane f). pJCW2 RNAlevels rise above control levels by 30 min (lane d) and continueto increase during longer auxin-induced incubation periods.This auxin-induced increase in pJCW2 RNA levels in excisedsections above control levels was also seen when auxin wasadded immediately after excision (Fig. 3B). The upper weakerband in Fig. 4B is seen in occasional preparations ofpoly(A)+RNA,possibly as a result of nonspecific hybridization to significantlevels of rRNA that are present in some of the poly(A)+RNApreparations.

DISCUSSIONThe data presented here show by direct analysis the capacityof the plant hormone, auxin, to selectively increase the level

A

*WWW jb6 1of

B

wUisI.0w4ww

a b c d e f 9 h

FIG. 3. Depletion of cloned poly(A)+RNA sequences during incu-bation of excised isueinthe absence of auxin. Poly(A)+RNAwas prepared from unincubated tissue (lanes a and e), tissue incubatedwith auxin for 2 (lane b), 4 (lane c), or 6 (lane d) hr, and tissue incubatedwithout auxin for 2 (lane f), 4 (lane g) or 6 (lane h) hr. Lanes contained0.25 Mg (pJCW1; A) or 0.50 ,g (pJCW2; B) of poly(A)+RNA separatedby electrophoresis and transferred to nitrocellulose as described. Au-toradiography was for 4 hr (A) or for 36 hr (B).

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7188 Biochemistry: Walker and Key

A

B

a b c d e fFIG. 4. RNA blot hybridization analysis showing kinetics of in-

duction by auxin of auxin-responsive poly(A)+RNAs in excised elon-gating tissue. Each lane contains 0.25 ,ig (A, pJCW1) or 0.50 ,g (B,pJCW2) of poly(A)+RNA prepared from unincubated tissue (lane a);4-hr auxin-depleted tissue (lane b); or 4-hr auxin-depleted tissue afteraddition of auxin for 15, 30, 60, or 90 min (lanes c-f, respectively).Electrophoresis of RNA and transfer and hybridization were as de-scribed in Fig. 1. Autoradiography was for 25 hr (A) or for 96 hr (B).

of certain poly(A)+RNAs in elongating soybean hypocotyl. Byusing hybridization of cloned cDNA probes in RNA blot anal-yses, we found several lines of evidence consistent with a pos-sible involvement of these RNAs in the cell elongation process:(i) the two poly(A)+RNAs ofnote are developmentally regulatedin the soybean hypocotyl, occurring at relatively higher con-centrations in tissue at maximum rates ofelongation (elongatingzone) compared with apical and mature tissues; (ii) these se-

quences are depleted in excised tissue that has been incubatedin the absence ofauxin and are maintained at or near the controllevel (pJCW1) or increased (pJCW2) if auxin is included in theincubation medium at the outset; (iii) auxin induces accumu-lation of these sequences when added to auxin-deficient tissue(i. e., excised tissue incubated for 4-6 hr in the absence ofauxin)in which the level of these sequences is depleted; and (iv) theresponse ofchanges in concentration ofthese sequences duringauxin-depleted or auxin-induced growth is rapid. The changesin level of these poly(A)+RNAs correspond generally to the al-tered rates ofcell elongation that occur under these growth con-ditions (14, 15).These general correlations in no way establish a causal re-

lationship between the concentration of these poly(A)+RNAsand the rate of cell elongation or between the action of auxinon cell elongation and the effects of auxin on the expression ofthese RNAs. These observations relate more directly to the factthat auxin has the capacity to rapidly and selectively increase(or decrease) the level of some poly(A)+RNAs in auxin-respon-sive tissues. These data, along with those of Baulcombe and Key(16, 18), show by direct analysis that the level of a few mRNAsmay be increased or decreased selectively in response to auxin.The concentrations of pJCW1 and pJCW2 RNAs appear to in-crease some 3- to 5-fold and 5- to 8-fold, respectively, in excisedauxin-depleted elongating tissues in response to auxin. Thethree sequences studied by Baulcombe and Key (18) in prolif-erating tissue decreased by factors of 1/100 to 1/20, dependingon the RNA, in response to auxin. Also, the data presented hereprovide direct evidence supporting the general conclusionsreached from in vitro-translation data of Zurfluh and Guilfoyle(23) and Theologis and Ray (25) and confirmed by us (unpub-

lished observations) that auxin causes a selective increase insome translatable mRNAs during auxin-enhanced elongation ofexcised tissues. One limitation in these studies is the fact thatthe functional identity of the auxin-regulated sequences is notknown.

Having identified a group of poly(A)+RNAs that undergorapid and significant changes in concentration in response toauxin by using cloned cDNA probes, we can now investigatethe mechanism(s) by which auxin alters the expression of thesesequences. Several possibilities must be considered includingenhanced rates ofsynthesis, altered turnover rates, altered pro-cessing, and combinations thereof. The results presented hereand those ofBaulcombe and Key (18), Baulcombe et al. (16, 17),Zurfluh and Guilfoyle (21-24), and Theologis and Ray (25) willprovide the basis for investigating causal relationships betweenauxin-regulated gene expression and auxin-induced physiolog-ical processes (e.g., cell division and cell elongation).We thank Drs. F. Sch6ffl, R. Nagao, and D. C. Baulcombe for helpful

discussions. This research was supported by National Institutes ofHealth Grant GM 30317.

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