Plant Physiol. (1994) 104: 505-513

lnvestigation of Gene Expression, Crowth Kinetics, and Wall Extensibility during Brassinosteroid-Regulated

Stem Elongation'

Daniel M. Zurek, David 1. Rayle, Trevor C. McMorris, and Steven D. Clouse*

Department of Biology, San Diego State University, San Diego, California 921 82 (D.M.Z., D.L.R., S.D.C.); and Department of Chemistry, D-006, University of California, San Diego, La Jolla, California 92093 (T.C.M.)

Brassinosteroids promote stem elongation in a variety of plants but little i s known about the mechanism of action of these plant growth regulators. We investigated a number of physiological and molecular parameters associated with brassinosteroid-enhanced elongation. Continuous growth recordings of soybean (Glycine mar 1. cv Williams 82) epicotyls showed that there was a 45-min lag before 0.1 f i ~ brassinolide (BR) exerted a detectable effect on elongation. BR caused a marked increase in Instron-measured plastic extensibility, suggesting that BR may promote elongation in part by altering mechanical properties of the cell wall (wall loos- ening). Structure-function studies suggested that the dimensions of the brassinosteroid side chain were critical for promotion of elon- gation and expression of BRU1, a gene regulated specifically by active brassinosteroids. Auxin-BR interactions were examined by using small auxin up RNA (SAUR) gene probes and the auxin- insensitive diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.). We have shown that in wild-type tomato, which elongates in response to exogenous auxin, a transcript of identical size to the soybean SAUR 15A is strongly induced within 1 h by 50 f i ~ 2,4-dichlorophenoxyacetic acid or indoleacetic acid, whereas in the dgt mutant, which does not elongate in response to auxin, no transcript i s expressed. Furthermore, BR promotes equal elon- gation of hypocotyls in both wild-type and dgt tomatoes but does not rapidly induce the SAUR 15A homolog in either genotype. BR does not cause rapid induction of SAUR 6B in elongating soybean epicotyls but does lead to increased expression after 18 h. This late BR activation of SAUR 6B is controlled, at least in part, at the transcriptional leve1 and i 5 not accompanied by an increase of free indoleacetic acid in the tissue. We conclude that although both BR and auxin affect wall relaxation processes, BR-promoted elonga- tion in soybean and tomato stems acts via a mechanism that most likely does not proceed through the auxin signal transduction pathway.

Brassinosteroids are growth-promoting natural products with structural similarities to animal steroid hormones (Man- dava, 1988). The wide distribution of brassinosteroids in the plant kingdom, their marked effect on cell proliferation and elongation at nanomolar levels, and their interactions with

'This research was supported in part by U.S. Department of Agriculture/Competitive Research Grants Office grant 90-37261- 5700 (S.D.C., T.C.M.), National Science Foundation grant DCB- 9013409 (S.D.C.), and National Aeronautics and Space Administra- tion grant NAG 1849 (D.L.R.).

* Corresponding author; 1-619-594-5676. 505

other plant hormones suggest that these compounds play a role as endogenous plant-growth regulators (Sasse, 1991). Of the many physiological effects brassinosteroids have on plants, cell elongation is perhaps the best documented. Bras- sinosteroids promote elongation of soybean (Glycine max), mung bean, azuki bean, and pea epicotyls (Yopp et al., 1981; Gregory and Mandava, 1982; Mandava, 1988; Clouse et al., 1992); bean, sunflower, and cucumber hypocotyls (Mandava et al., 1981; Katsumi, 1985); Arabidopsis peduncles (Clouse et al., 1993); and maize mesocotyls and wheat coleoptiles (Yopp et al., 1981; Sasse, 1985). Despite numerous physiological studies, the molecular mechanism of brassinosteroid-induced elongation remains unclear. The aim of the present study was to examine some of the physiological and molecular parameters associated with brassinosteroid-regulated stem elongation.

One parameter of interest with respect to growth regulator action is the time required for induction of the response. Such information is useful in separating primary events leading to the response from secondary events that are caused by or coincidental to the response. For example, auxin-induced elongation proceeds after a lag time of 10 to 15 min, and thus substantial importance is assigned to molecular events preceding the onset of auxin action (Taiz, 1984). Brassinos- teriod-stimulated elongation appears to have a longer latent time (Clouse et al., 1992); however, to date no studies have reported the early kinetics of brassinosteroid action.

Also of interest is whether brassinosteroid-induced growth, like auxin-induced cell elongation, is ultimately mediated by cell wall loosening. The Instron extensometer has been used to follow changes in mechanical (viscoelastic) properties of the cell wall in response to auxins (Cleland, 1984), gibberel- lins (Behringer et al., 1990), and more recently, brassinoster- oids (Wang et al., 1993). Furthermore, severa1 enzymes with suggested or proven wall-loosening capacity have been pu- rified (Fry et al., 1992; McQueen-Mason et al., 1992) and/or cloned (de Silva et al., 1993), thus providing the opportunity to test directly for effects of growth regulators on their activity or synthesis.

A complementary approach to understanding the mecha-

Abbreviations: BR, brassinolide; dgt, diageotropica; KPSC, 10 m potassium phosphate, pH 6.0, 2% SUC, 25 pg/mL chloramphenicol; SAUR, small auxin up RNA; VFN, Verticillium-, Fusarium-, Nema- toda-resistant cultivar of tomato (wild type).

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506 Zurek et al. Plant Physiol. Vol. 104, 1994

nism of hormone-regulated growth is the characterization of mutants that are defective in hormone perception or action, such as the dgt mutant of tomato (Klee and Estelle, 1991). The dgt mutant is nearly insensitive to exogenously applied auxin with respect to hypocotyl elongation (Kelly and Brad- ford, 1986), and Instron analysis has shown that normal auxin-induced increases in plastic extensibility of hypocotyl cell walls that occur in wild-type tomato do not occur in the dgt mutant (Daniels et al., 1989). The dgt mutant provides a useful test of whether the growth-promoting activity of bras- sinosteroids is independent of auxin action (in which case dgt hypocotyls should elongate in response to brassinosteroids) or proceeds by a mechanism requiring auxin activity. In the latter case, one would expect dgt hypocotyls to be insensitive to both auxin and brassinosteroids.

Yet another approach has been the cloning of genes regu- lated by plant hormones in elongating tissue. For example, members of the GH (Hagen et al., 1984), SAUR (McClure and Guilfoyle, 1984), and JCW (Walker and Key, 1982) gene families are rapidly induced at the transcriptional leve1 when auxins are applied to soybean hypocotyls or epicotyls. Al- though the causal role of these genes in elongation has not been established, the availability of these probes has proved useful in monitoring auxin activity in elongating tissue. We previously used these probes to show that BR, a highly active brassinosteroid, can affect elongation and gene expression independently of auxin in soybean epicotyls (Clouse and Zurek, 1991; Clouse et al., 1992). Recently we have cloned pBRU1, a cDNA corresponding to a transcript whose abun- dance is increased specifically by brassinosteroids in elongat- ing soybean epicotyls (Zurek and Clouse, 1993). The derived amino acid sequence of pBRUl shows 89% similarity (77% identity) to the meri-5 gene of Arabidopsis thaliana (Medford et al., 1991) over a contiguous 114-amino acid region and 62% similarity (48% identity) to a nasturtium xyloglucan endo-transglycosylase (NXGl) over the same stretch (de Silva et al., 1993). The NXGl gene product has been localized to the cell wall and has been promoted as a wall-loosening enzyme (de Silva et al., 1993), although recent data may cast doubt on this assertion (Cosgrove, 1993; McQueen-Mason et al., 1993).

In the present paper we study the early kinetics of brassi- nosteroid-enhanced growth and analyze the effects of BR on cell wall extensibility in soybean epicotyls. We also charac- terize the growth-promoting activity of BR in the auxin- insensitive dgt mutant and use BRUl and SAUR gene probes to study the mechanism of brassinosteroid-regulated growth and its relationship to auxin.

MATERIALS AND METHODS

Brassinosteroid Synthesis

Brassinolide [2a,3~~,22(R),23(R)-tetrahydrox~-24(S)-methyl- B-homo-7-oxa-5a-cholestan-6-one] and analogs I through IV were synthesized as previously described (McMorris et al., 1991). Iso-epibrassinolide [2a,3a,22(S),23(S)tetrahydroxy- 24(R) -methyl-B- homo- 7-oxa-5a-cholestan-6-one] and 24-epibrassinolide [2a, 3a, 22(R), 23(R)- tetrahydroxy - 24(R)- methyl-B- homo- 7-oxa-5a-cholestan-6 -one] were synthe-

sized from ergosterol as described by McMoms and Patil (1993). The synthesis of iso-homobrassinolide [2a,3a, 22(S),23(S)-tetrahydroxy-24(S)-ethyl-B-homo-7-oxa-5a-cho- lestan-6-oneI and 28-homobrassinolide [2a,3a,22(R), 23(R)- tetrahydroxy-24(S)-ethyl-B-homo-7-oxa-5~-cholestan-6- one] will be described elsewhere (T.C. McMoms, P.A. Patil, R.G. Chavez, M.E. Baker, and S.D. Clouse, unpublished data). Brassinosteroids were stored as 1 or 10 mM stock solutions in absolute ethanol or DMSO at -2OOC.

Plant Crowth

Soybean seeds (Glycine max L., cv Williams 82, purchased from Wilkens Seed Grains, Pontiac, IL) were allowed to imbibe in water ovemight and sown in 50% vermiculite/50% Perlite. Seedlings were grown 10 to 14 d in a greenhouse (25-27OC) under natural lighting conditions before harvest- ing for the epicotyl assay. Seeds of VFN and dgt tomato (Lycopersicon esculentum Mill.) near isogenic lines were orig- inally obtained from Ken Bradford (University of Califomia, Davis) and thereafter propagated at the Oregon State Uni- versity Botany and Plant Pathology Field Station. Seeds were surface sterilized in a 20% commercial bleach solution for 20 min, followed by extensive washing in distilled water. Seeds were sown on moist Whatman No. 1 filter paper in 150-mm Petri plates and incubated for 6 to 7 d at 27OC in the dark.

Stem Elongation Assays

Epicotyl sections were obtained from the first 1.5 cm im- mediately below the plumule of 10- to 14-d-old soybean seedlings and floated on ice-cold KPSC buffer until required for the assay. Twenty sections were placed in a 50-mL Erlenmeyer flask containing 10 mL of KPSC and rotated at 125 rpm in a 27OC shaking incubator under continuous illumination (25 pE m-* s-l). Sections were preincubated for 2 h as described (Clouse et al., 1992) to deplete endogenous auxin before the addition of fresh KPSC containing brassi- nosteroids or analogs. Controls were incubated in KPSC with appropriate concentrations of ethanol or DMSO. Tomato hypocotyl sections (1 cm) were cut directly below the hypo- cotyl hook of 6- to 7-d-old VFN or dgt seedlings and floated on 1 ITLM potassium phosphate, pH 6.0, containing 2% SUC (without chloramphenicol). Hypocotyl sections were prein- cubated and treated as described for the epicotyl sections. Epicotyl or hypocotyl length was measured to the nearest 1 mm and sections were immediately frozen in liquid nitrogen for RNA isolation or boiled (5 min) in methanol for Instron analysis.

Continuous Crowth Measurements

To determine the detailed kinetics of brassinosteroid-in- duced growth, 1.5-cm soybean epicotyl sections were mounted in a custom growth-recording device as previously described (Rayle and Cleland, 1972) and incubated in KPSC buffer. After a low, relatively steady rate of growth was established (1-2 h), BR or other brassinosteroids were added

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Brassinosteroid-Regulated Stem Elongation 507

to a final concentration of 0.1 or 1.0 p ~ . Growth was recorded continuously for an additional5 to 7 h. To determine the lag time of BR-induced growth, a ruler was placed parallel to the chart tracing just prior to the point at which BR was added and a line was drawn that extended beyond this point. The lag period was defined as the time after BR addition when the chart tracing deviated from this line in the positive direction (showing enhanced elongation). Control tracings (i.e. no BR added) did not show enhanced elongation meas- ured in this way for the duration of the experiment. Rather, such tracings exhibited steady or very slowly declining growth.

Wall Extensibility Measurements

Soybean epicotyl segments (1.5 cm) were incubated for various times in KPSC or KPSC plus various concentrations of BR (see above), after which elongation was recorded and the segments were boiled for 5 min in methanol. One-half of the segments from each treatment were removed at random, dried ovemight at 6OoC, and weighed. The other half of each treatment was stored in fresh methanol at room temperature. Just prior to analysis, methanol-stored segments were rehy- drated (30-60 min) and subjected to Instron analysis as previously described (Cleland, 1967, 1971). From the slopes of the first and second extensions, total elastic and plastic extensibility values were determined. The initial distance between the clamps of the Instron (jaw gap) was 5.0 mm. The rate of extension was 0.5 mm/min and the final load limit was 30 g. Data are expressed as percent plastic exten- sion/100 g load. In some experiments we noticed that BR- treated segments became somewhat thinner than control segments. To make certain that the increased extensibility of BR-treated segments was not due to thinning, which might produce a higher stress (force/area) and thus a geometicall measurement artifact, we normalized each data point to dry weight per unit length to account for differing segment thickness.

RNA lsolation and Analysis

Total RNA was isolated from frozen epicotyl or hypocotyl sections with 4.0 M guanidinium isothiocyanate and acidified phenol by the method of Chomczynski and Sacchi (1987). Polyadenylated RNA was purified from total RNA using biotinylated oligo(dT)- and streptavidin-coated magnetic beads (PolyATtract System, Promega, Madison, WI). For RNA gel-blot analysis, polyadenylated or total RNA was incubated for 15 min at 65OC in 18 pL of formamide, 7 pL of 37% formaldehyde, 3.5 pL of 1OX Mops, and 3.5 pL of ethidium bromide (400 pg/mL). The denatured RNA was electrophoresed on 1.2% agarose gels containing 1.2 M form- aldehyde for 3 h at 140 V. RNA was transferred to Duralon- UV (Stratagene, La Jolla, CA) nylon membranes in 1OX SSC for 30 min under pressure. RNA was cross-linked to the membrane with a Stratalinker apparatus (Stratagene) follow- ing the manufacturer's instructions.

For tomato RNA blots, we synthesized a 59-mer comple- mentary to bases 117 through 163 of the open reading frame of SAUR15A (McClure et al., 1989) and labeled it with T4

polynucleotide kinase and [T-~~PIATP. Blots were prehybrid- ized in 6X SSC, 1.0% SDS, 5X Denhardt's solution, and 200 pg/mL yeast tRNA (partially degraded with NaOH). Hybrid- ization was in the same solution except Denhardt's was reduced to lx. Membranes were hybridized ovemight at 37OC in a rotary hybridization oven (HybAid, Intermountain Scientific, Bountiful, UT) using 1 X 106 cpm/mL of probe. Washes were in 6X SSC, 0.2% SDS once at room tempera- ture, twice at 37OC, and once at 42OC. For soybean RNA blots, the pBRUl (Zurek and Clouse, 1993) cDNA insert was labeled with [32P]dCTP by random priming using the Prime- It I1 Kit (Stratagene). Blots were prehybridized, hybridized, and washed as previously described (Clouse et al., 1992). To confirm equal loading of RNA in each lane, blots were stripped and reprobed with an oligonucleotide complemen- tary to a highly conserved region of plant actin genes (Naim et al., 1988). Each blot was repeated a minimum of two times.

The probe for RNase protection assays was generated by digesting pBRUl with PstI and rendering the resulting 3' overhang blunt-ended with Klenow fragment of DNA po- lymerase I. After proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation, the purified DNA frag- ment was used as a templatefor in vitro RNA transcription. The final reaction contained 1 pg DNA; 1 X transcription buffer (40 m Tris, pH 8.0, 8 mM MgC12, 2 mM spermidine, 50 mM NaCl); 30 mM DTT; 40 units of RNase Block I; 50 pCi of [a-32P]UTP (800 Ci/mmol); 400 p~ each ATP, GTP, and CTP; and 10 units of T7 RNA polymerase. After 30 min at 37OC, 10 units of RNase-free DNase was added and the reaction was incubated for an additional 15 min. Labeled probe was separated from unincorporated nucleotides by passage through a NucTrap column (Stratagene). The result- ing probe represented a 281-bp antisense RNA complemen- tary to the 3' end of the BRUl mRNA. RNase protection was performed by hybridizing 8 X 104 cpm of this probe with 5 pg of total RNA from various treatments using the Ribonu- clease Protection Assay I1 kit (Ambion, Austin, TX) according to the manufacturer's instructions. Gels were exposed to preflashed film with intensifying screens (Laskey and Mills, 1975) and quantitated by scanning densitometry in the linear range of absorbance. The RNase protection was performed in triplicate and the densitometry results were averaged.

Measurement of Free IAA Levels

Soybean epicotyl segments were incubated in buffer plus or minus 1.0 p~ BR for 5 and 21 h as described above. After incubation, 0.5 g of tissue was weighed out and free IAA levels were determined at the U.S. Department of Agriculture Plant Hormone Laboratory (Beltsville, MD) by GC-MS analy- sis (Chen et al., 1988). IAA levels were determined on dupli- cate 0.5-g samples and the values were averaged.

Nuclear lsolation and Run-On Transcription

Epicotyl sections (apical 1.5 cm) were excised from 9- to 11 -d-old soybean seedlings and treated as described above with or without 0.1 /*M BR. Nuclei were isolated at 18 h using the procedure of Luthe and Quatrano (1980) as modified by

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508 Zurek et ai. Plant Physioi. Vol. 104, 1994

0.30 - Lawton and Lamb (1987) with the exceptions that (a) tissue was used immediately without freezing; (b) homogenized tissue was filtered successively through 60-, 52-, and 20-pm meshes; and (c) the Perco11 step gradient was centrifuged for 45 rather than 30 min. Isolated nuclei were examined micro- scopically after staining with 4’,6’-diamidine-2-phenylindole dihydrochloride and quantitated in a hemocytometer. Typical yields were 1.5 X 106 to 2.8 X 106 nuclei per extraction. Nuclei were assayed for transcriptional activity by determin- ing the incorporation of [w3’P]UTP (600 Ci/mmol) in re- sponse to different times of incubation and number of nuclei. Optimal reaction conditions were detennined to be a 30-min incubation with 5 X 105 nuclei at 26OC. Reaction buffers and other details of preparing labeled run-on transcripts were as described by Lawton and Lamb (1987).

Duplicate slot blots were generated by blotting (in tripli- cate) 10 pg of test or control cDNAs on Zeta Probe Nylon membranes (Bio-Rad) following the manufacturer’s instruc- tions. The SAUR 6B and soybean actin cDNAs were obtained from an 18-h BR-treated soybean epicotyl library (Clouse et al., 1992) and the SAUR cDNA was shown by sequence analysis to be identical to the previously published SAUR 6B from auxin-induced soybean hypocotyls (McClure et al., 1989). A partia1 cDNA for tomato hydroxymethyl glutaryl COA reductase cDNA was obtained from Dr. Carole Cramer (Virginia Polytechnic Institute and State University, Blacks- burg, VA). As an additional control, we included 100 ng of a cDNA for 25s ribosomal RNA obtained from a Helianfhus tuberosus cDNA library (M.A. Buchanan and S.D. Clouse, unpublished data).

Duplicate blots were hybridized for 18 h at 42OC with 107 cpm/mL nuclear run-on transcripts from control or BR- treated tissue in 40% formamide, 1.0% SDS, 5X Denhardt’s solution, and 100 pg/mL sheared, denatured, salmon sperm DNA. Blots were washed in 2 X SSC, 0.1% SDS once at room temperature and once at 42OC, and in 0.2X SSC, 0.1% SDS twice at 42OC and twice at 5OOC. Blots were exposed to preflashed film with one intensifying screen (Laskey and Mills, 1975) and developed in the linear range of intensity for scanning densitometry.

RESULTS

Growth Kinetics and Wall Properties during BR-Regulated Elongation

We have previously shown that BR is a potent inducer of elongation in soybean epicotyl sections, with application of 0.1 PM BR resulting in a measurable increase over control after 6 h and up to 200% by 48 h (Clouse et al., 1992). In that study, epicotyl sections were measured to the nearest mm, which is not sensitive enough to determine the earliest response of sections to BR. To examine the detailed growth kinetics produced by 0.1 p~ BR, 1.5-cm soybean epicotyl sections were clamped into a growth-recording device (Rayle and Cleland, 1972) and elongation was monitored continu- ously. A typical recording is shown in Figure 1. Lag times for BR-enhanced elongation ranged from 41 to 51 min. The average value for a11 trials was 46.5 & 4.4 min. Once BR- enhanced growth is initiated there is a gradual acceleration

E E I 1

K z a

TIME (h)

Figure 1. Continuous recording of BR-induced growth in soybean epicotyl sections. Growth was initially recorded in KPSC buffer. After 1 h, BR was added to the incubation medium (final concen- tration, 0.1 p ~ ) and a gradual acceleration of growth began after approximately 45 min. Five or more hours were required to achieve maximum steady-state, BR-induced growth. T h e graph is repre- sentative of numerous experiments, all of which gave similar results.

in rate that occurs over a period of several hours. Application of 1.0 p~ BR did not shorten the latent period or the overall kinetics of the subsequent response, indicating that the rather long period required to initiate elongation and establish a maximum rate is not related to BR uptake but rather to the intrinsic mechanism of BR action.

We also obtained continuous growth recordings for several other brassinosteroids whose structures are shown in Figure 2. The overall pattem of growth and the latent time (40-50 min) for 24-epiBR, iso-epiBR, and 28-homoBR at 1.0 p~ were similar to those of BR (data not shown). However, 1.0 p~ iso-homoBR produced no detectable enhancement of elon- gation over the time interval measured.

Figure 3A shows a time course of the effect of 10-7 M BR on wall extensibility of soybean epicotyl sections using In- stron analysis. BR had a significant effect on plastic extensi- bility (wall loosening) at 2 h. Plastic extensibility continued to increase up to about 6 h and then remained constant. Figure 3B shows that BR also had a significant effect on plastic extensibility at 10-’ M. The 2-h treatment reached near maximum extensibility at 10-7 M, a concentration that is optimal for elongation in the standard epicotyl elongation assay (Clouse et al., 1992). BR had no significant effect on elastic extensibility (data not shown).

Structure-Function Relationships in Crowth and Cene Expression

Numerous studies have examined the structural require- ments of brassinosteroids for biological activity (reviewed by Adam and Marquardt, 1986; Mandava, 1988). We synthe- sized a variety of brassinosteroids and structural analogs (see Fig. 2) and tested their ability to enhance soybean epicotyl

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Brassinosteroid-Regulated Stem Elongation 509

Brasrinolida 24-epibrarsinolide iso-epibrarrinolide

28-homobrarsinolide iso-homobrassinolide Analog I

Anslog I1 Analog 111 Analog IV

Figure 2. Structures of the brassinosteroids used in these experiments

elongation and to regulate the expression of BRUl. We have shown elsewhere that BR causes an increase in BRUl expres- sion in soybean epicotyls concomitant with an increase in epicotyl length versus control (Zurek and Clouse, 1993). Figure 4 shows that 24-epiBR, iso-epiBR, and 28-homoBR a11 promote similar elongation of soybean epicotyls and cause a 2.5-fold or greater increase in BRUZ transcript levels. Sur- prisingly, iso-homoBR, which differs from 28-homoBR only by the configuration of hydroxyls at C-22 and C-23, results in 10-fold less elongation of epicotyls and a reduced leve1 of BRUl transcript. This is in agreement with our continuous growth experiments (see above), in which iso-homoBR failed to produce detectable epicotyl elongation during the first severa1 hours of measurement. A11 four synthetic analogs, which differ in the side chain or oxygen functional groups in the A or B ring, failed to promote any elongation and resulted in either no increase or a decrease in BRUZ transcript levels when compared with the control.

BR lnduces Elongation but Not SAUR Cene Expression in an Auxin-lnsensitive Mutant

The soybean SAURs respond rapidly to exogenous auxin with transcriptional increases observed within 2 to 5 min after the addition of 50 JLM 2,4-D to hypocotyl sections (McClure and Guilfoyle, 1987). Although the function of SAURs and their causal role in elongation remains uncertain, they provide useful molecular markers for auxin activity. To further examine the molecular mechanisms of elongation in tomato, we tested for the presence of SAUR gene homologs

in VFN (wild-type) and dgt (auxin-insensitive) hypocotyls. Figure 5A shows that a transcript of identical size to the soybean SAUR 15A is expressed in VFN hypocotyls treated for 1 h with 50 PM IAA. The dgt mutant shows very low transcript expression under the same conditions, providing further evidence that the dgt mutant is defective in auxin perception or primary action. The strength of hybridization, size, and induction kinetics of this transcript indicate that it is fairly homologous to the soybean SAUR 15A. The presence of SAUR genes in plants other than soybean may point to their general importance. SAUR homologs have also been recently identified in mung bean (Yamamoto et al., 1992) and Arabidopsis (Newman et al., 1993).

It has been shown previously that exogenously applied auxins lead to increased hypocotyl lengths in VFN but not in dgt tomatoes (Kelly and Bradford, 1986) and that BR promotes elongation of wild-type tomato hypocotyls (Takatsuto et al., 1983). Figure 5B shows that BR also promotes elongation in the dgt mutant and that BR does not cause rapid induction of the SAUR homolog in either genotype. With respect to auxin-BR interaction, it is notable that we have shown BR- induced hypocotyl elongation in an auxin-insensitive mutant.

Transcriptional Activation of a SAUR Gene by BR

We have previously shown (Clouse et al., 1992) that BR does not cause rapid induction of members of the SAUR (McClure and Guilfoyle, 1987), JCW (Walker and Key, 1982), or GH (Hagen et al., 1984) gene families during BR-promoted elongation of soybean epicotyls. Nevertheless, with longer

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510 Zurek et al. Plant Physiol. Vol. 104, 1994

6 8 10 12 14 16 18 20

TIME OF INCUBATION (h)

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Figure 3. Effect of BR on plastic wall extensibility. A, Soybeanepicotyl sections (1.5 cm) were incubated with or without 10~7 MBR for the times indicated, after which they were boiled in methanolfor 5 min. Upon rehydration, their plastic extensibility was deter-mined by the Instron technique. Each measurement was multipliedby a coefficient that normalized for differences in segment thickness(see "Materials and Methods"). Data points represent the averageof 10 replicate segments ± SE. B, Soybean epicotyl sections (1.5cm) were treated with the indicated concentrations of BR for 24 hand plastic extensibility was determined as described above.

incubations (18 and 24 h) there is substantial induction ofSAUR 6B in soybean epicotyls treated with BR alone (Clouseet al., 1992). To further investigate the mechanism of thisdelayed increase in SAUR expression in BR-treated tissue, weisolated nuclei from soybean epicotyls treated with or without0.1 IJ.M BR and performed run-on transcription assays. Figure6 clearly shows that BR induces SAUR 6B expression at 18 hat least in part by transcriptional activation. Actin, which wasintended as a control, also showed a slight but reproducibleincrease in transcription rate in BR-treated tissue that mightbe related to increased cytoskeleton synthesis in the enlargingcells. Neither hydroxymethyl glutaryl CoA reductase or the25S ribosomal gene showed any difference in transcriptionrate between control and BR-treated tissue and were thusvalid controls.

To determine if BR causes a slow increase in auxin levels,which would result in a gradual increase in SAUR geneexpression in tissue treated with BR alone, we measured freeIAA levels in soybean epicotyls treated with or without 0.1MM BR. After 5 and 21 h of incubation in buffer, free IAAlevels were 6.8 and 19.9 ng/g, respectively. After 5 and 21 hof BR treatment, free IAA levels of 3.8 and 8.5 ng/g were

found. The fact that BR seems to reduce free IAA levels ratherthan increase them makes it clear that BR does not stimulateSAUR gene transcription (or stem elongation) by a mecha-nism related to enhanced auxin biosynthesis or deconjuga-tion. To determine whether BR affects the activity or numberof auxin receptors will require suitable molecular probes forsuch receptors.

DISCUSSION

In a previous study we found that the kinetics of BR-promoted elongation was slower than that of auxin, but thatBR was more effective than auxin after long incubation(Clouse et al., 1992). In this paper we have identified theearliest time of BR action on soybean epicotyl elongation bymonitoring growth continuously. It takes approximately 45min for 0.1 /IM BR to enhance the rate of elongation ofsoybean epicotyl segments and several hours for a maximum

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Figure 4. Brassinosteroid structure-function relationships in soy-bean epicotyl elongation and BRU1 gene expression. Twenty-fivereplicate soybean epicotyl sections (1.5 cm) for each treatmentwere incubated for 19 h in 1.0 HM of the indicated brassinosteroidor KPSC buffer (control). Total RNA was then isolated from thesegments and 5 ng of RNA from each treatment was subjected toRNase protection analysis using a 281-bp antisense RNA probecomplementary to the 3' end of the 8RU1 transcript. Numbersbelow the autoradiograph represent relative transcript abundanceand are an average of scanning densitometry results from threeseparate experiments. Error bars are ± SE. Control segments elon-gated 0.26 ± 0.02 cm. www.plantphysiol.orgon September 16, 2020 - Published by Downloaded from

Copyright © 1994 American Society of Plant Biologists. All rights reserved.

Brassinosteroid-Regulated Stem Elongation 511

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1 iio2 x8 3

oE 2

s § i s

SAUR15A PROBE

*»•_•» w w ACTIN PROBE

Figure 5. Effect of auxin and BR on SAUR gene expression andelongation in excised VFN and dgt tomato hypocotyls. A, VFN ordgt tomato hypocotyl sections were incubated in buffer or buffer+ 50 fiM IAA for 1 h prior to RNA isolation. Polyadenylated RNA (2Mg) was run on an agarose/formaldehyde gel and blotted to a nylonmembrane. The membrane was hybridized with 1 x 106 cpm/mLof a 32P-labeled 59-mer oligonucleotide complementary to thepublished sequence of soybean SAUR 15A (McClure et al., 1989).The soybean auxin control lane contained 2 Mg of polyadenylatedRNA from 2,4-D-treated soybean epicotyls. B, One-centimetersections from the apical portion of the hypocotyl of VFN and dgttomatoes were excised and pretreated in buffer as described in"Materials and Methods." The buffer was then removed and re-placed with fresh buffer only, buffer + 50 MM 2,4-D, or buffer + 0.1MM BR. Hypocotyl sections were incubated for 1 h and RNA wasisolated and northern blot analysis was performed as describedabove. An additional 20 hypocotyl sections for each treatment wereincubated for 22 h, after which hypocotyl length was determinedto the nearest mm. Error bars are ± SE for 20 replicates. VFN controlsegments elongated 0.048 ± 0.006 cm and dgt control segmentselongated 0.0 ± 0.005 cm.

rate to be established. We believe these kinetics reflect theintrinsic mechanism of BR action for the following reasons:(a) a 10-fold excess of BR (1.0 MM) over optimal levels (0.1MM) produced similar kinetics; (b) all active BR analogs testedhave response times very similar to BR; and (c) similar kineticswere observed with abraded and nonabraded Avena coleop-tile sections (D.L. Rayle and S.D. Clouse, unpublished data).It is interesting to contrast the elongation kinetics of BR withthose of auxin. In a variety of species tested (Taiz, 1984),auxin-induced elongation commences after a lag period of 10to 15 min and a maximum rate is achieved within 30 to 45min. Such different kinetics for BR and auxin seem to suggestthat a rather different set of signaling events occur in responseto these growth regulators.

Recently, Wang et al. (1993) examined some of the param-eters of BR-stimulated elongation in Brassica chinensis hypo-cotyls and found that BR decreased the cellular osmoticpressure during elongation, suggesting that BR does notenhance water uptake via increasing cell solutes. Instead, itwas found using the pressure block technique that BR causedan increase in wall-relaxation properties (wall loosening).This type of wall loosening did not appear to greatly alter themechanical properties of the wall as measured by Instronanalysis. Our results with soybean epicotyl segments alsosuggest that BR stimulates wall loosening. However, in soy-bean this form of loosening appears to alter the mechanicalproperties of the wall, since we observed an increase in plasticextensibility as measured by Instron analysis. At present wedo not know why Instron analysis produces different resultsin Brassica and soybeans.

Numerous studies of structure/activity relationships forbrassinosteroids have shown that minor structural modifica-tions can result in loss of biological activity, although struc-tural requirements vary somewhat with the assay systemused (Thompson et al., 1982; Takatsuto et al., 1983; Artecaet al., 1985). Such strict structural requirements for activityare consistent with the hypothesis that BR effects are me-diated by a protein receptor. In this paper we show thatstructural modifications that reduce or eliminate epicotyl

CONTROL + BR

SAUR 6B

HMG CoA Red.

ACTIN

RIBOSOMAL

18 HR 18 HR

Figure 6. Transcriptional activation of SAUR 68 by BR. Nuclei wereisolated from soybean epicotyls 18 h after treatment with buffer(control) or 0.1 MM BR (+BR). Labeled run-on transcripts wereprepared and hybridized to slot blots of the indicated cDNAs asdescribed in "Materials and Methods."

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512 Zurek et al. Plant Physiol. Vol. 104, 1994

elongation also result in a conesponding reduction of BRUl expression. Altering the configuration at the C-22 and C- 23 hydroxyls or the C-24 methyl had little effect on elonga- tion or expression, 24-epiBR [22(R),23(R),24(R)] and iso- epiBR [22(S),23(S),24(R)] showed similar activity to BR [22(R),23(R),24(S)] at 1.0 p ~ . The replacement of the C-24 methyl with an ethyl group was also tolerated as long as the 22(R),23(R),24(S) configuration of BR was conserved (28- homoBR). However, iso-homoBR [22(S),23(S),24(S)] exhib- ited less than 10% of the elongation activity of BR and a reduction of BRUl gene expression (although not to the same extent). A computer analysis of interatomic distances in en- ergy-minimized structures of the various isomers (T.C. McMorris, P.A. Patil, R.G. Chavez, M.E. Baker, and S.D. Clouse, unpublished data) showed that the optimum respec- tive distances between C-16 on the ring and the C-22, C-23, C-24, and C-28 carbons, as well as the 0-22 and 0-23 oxygens, were similar in a11 of the isomers except iso- homoBR, which differed significantly. This indicates that the overall dimensions of the side chain may be more important than the configuration at the individual chiral carbons.

More pronounced alterations of structure (analogs I through IV) caused a complete loss of activity with respect to both elongation and increases in BRUl gene expression over that in control tissue. The correlation of BRUl expression with elongation observed in the structure/function studies does not prove a causal role for BRUl in BR-enhanced elongation. However, increases in BRUl transcript levels in elongating soybean epicotyls are dependent on BR, since auxins and GA3 both stimulate epicotyl elongation without increasing BRUl expression (Zurek and Clouse, 1994). Fur- thermore, the initial BR-promoted increase in BRUZ transcript abundance observed previously (Zurek and Clouse, 1994) and the initial increase in plastic wall extensibility caused by BR (Fig. 3A) both occur around 2 h after BR addition. The homology of BRUZ with a xyloglucan endotransferase strengthens the argument for a role of BRUl in wall altera- tions during elongation, but a definitive evaluation must await purification of the BRUl gene product, followed by localization and activity studies.

A number of studies have attempted to unravel the com- plex interaction between auxin and brassinosteroids (Man- dava, 1988). Based on early physiological work, it was proposed that responses induced or accelerated by brassino- steroids require the activity of endogenous auxin (Yopp et al., 1981). We have examined this problem in a new way by studying the effects of BR on auxin-insensitive mutants and on auxin-inducible gene expression. The dgt mutant of tomato shows multiple characteristics of auxin insufficiency includ- ing diagravitropic shoot growth, abnormal vascular tissue, lack of lateral root branching, and marked insensitivity to exogenously applied auxin with respect to hypocotyl elon- gation and ethylene production (Daniels et al., 1989). In this paper we show that a SAUR 15A homolog is rapidly induced by exogenous auxin in VFN but not in dgt hypocotyls, pro- viding further molecular evidence that the dgt lesion affects a gene responsible for primary auxin response, possibly an auxin receptor. Furthermore, the fact that BR fails to induce the SAUR 15A transcript supports our previous conclusion that BR-promoted elongation in soybean is not accompanied

by rapid induction of auxin-regulated genes (Clouse et al., 1992). The ability of BR to stimulate hypocotyl elongation equally in the wild-type and the auxin-insensitive tomato argues against the requirement of auxin action for BR activity in this case. This agrees with our previous results demonstrat- ing the effect of brassinosteroids on auxin-resistant mutants of A. thaliana (Clouse et al., 1993). Exogenous auxin causes inhibition of root elongation in wild-type Arabidopsis but not in the auxin-resistant axr-1 mutant (Lincoln et al., 1990). We found that 24-epiBR also inhibited root elongation of wild- type A. thaliana, but that 24-epiBR just as effectively inhibited root elongation in the axr-1 mutant (Clouse et al., 1993).

The transcriptional activation of SAUR 68 by BR in soy- bean epicotyls observed at 18 h might be due to a direct effect of BR on SAUR gene transcription or it may result from a slow BR-promoted increase in endogenous auxin action in the tissue. Severa1 hypotheses have been presented to explain the reported enhancement of auxin activity by BR (Sasse, 1991b), including (a) BR may be involved in sensitizing the tissue to auxin, perhaps by increasing the activity of auxin receptors, or (b) BR may increase active auxin levels in the tissue by increasing synthesis, transport, or deconjugation of auxin. The second hypothesis seems to have been discounted by work of Cohen and Meudt (1983), who demonstrated that BR did not increase IAA synthesis, metabolism, or transport in first intemodes of Phaseolus vulgaris. Our results on IAA levels in control and BR-treated tissue agree with those of Cohen and Meudt (1983), who also reported a decrease in free IAA levels in BR-treated tissue. The possibility remains that hypothesis (a) accounts for BR-mediated SAUR gene transcription. On the other hand, if BR is directly affecting SAUR gene expression it would be of interest because the SAURs were previously thought to be auxin specific (McClure and Guilfoyle, 1987).

In summary, we have combined biophysical, molecular, and genetic approaches to further understand the mechanism of brassinosteroid-promoted stem elongation. Our results with the dgt mutant and SAUR gene expression support the contention that BR can act independently of auxin, although more definitive molecular probes will be required to study subtle interactions between these growth regulators. Whereas the kinetics of BR-enhanced elongation differs from that of auxin in soybean epicotyls, both growth regulators alter the mechanical properties of the wall. Obviously, different bio- chemical events could lead to the same result (wall loosening) as measured by Instron analysis. The cloning of BRUI, a gene whose expression is enhanced by BR but not by auxin, and whose sequence shows homology to wall-loosening and/or wall-metabolizing enzymes, will be the focal point of further studies. Construction of transgenic plants overexpressing BRUl or expressing BRUl antisense RNA will allow critica1 testing of the importance of BRUl in stem elongation.

ACKNOWLEDCMENTS

We thank Robert Cleland and Suzanne Bagshaw for assistance with the Instron measurements. We are also grateful to Jerry Cohen and David Ribnicky for performing the GC-MS analysis of free IAA levels. Finally, we thank Carole Cramer for the tomato hydroxy- methyl glutaryl COA reductase cDNA.

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Brassinosteroid-Regulated Stem Elongation 513

Received July 7, 1993; accepted October 13, 1993. Copyright Clearance Center: 0032-0889/94/104/0505/09.

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