Plant Physiol. (1987) 84, 1068-10730032-0889/87/84/1068/06/$01 .00/0

Physiological Studies of a Synthetic Gibberellin-LikeBioregulatorII. EFFECT OF SITE OF APPLICATION ON BIOLOGICAL ACTIVITY

Received for publication December 8, 1986 and in revised form March 30, 1987

JEFFREY C. SUTTLE* AND JULIE F. HULTSTRANDUnited States Department ofAgriculture, Agricultural Research Service, Metabolism and RadiationResearch Laboratory, State University Station, Fargo, North Dakota 58105

ABSTRACT

The biological activity of the synthetic gibberellin agonist AC-94,377(143-chlorophthalimidoFcyclohexanecarboxamide) in certain plants isstrictly dependent on the site of application. Root application of AC-94,377 at concentrations greater than or equal to 1 micromolar toseedlings of dwarf corn (Zea mays L. var d), dwarf rice (Oryza sativa L.cv Tan-inbozu), and sunflower (Helianthus annuus L. cv NK265) seed-lings resulted in readily measurable gibberellin-like biological activity.Application of up to 10 micrograms of AC-94,377 to the shoots of thesesame species had no effect. AC-94,377 was metabolized to more polarproducts in both dwarf corn and sunflower seedlings. After 4 days ofcontinuous root treatment with 1'4qAC-94,377, greater than 70% of therecovered 14C was found in the form of unmetabolized AC-94,377. Incontrast, only 30 to 40% of the recovered 14C was unmetabolized 4 daysafter shoot treatment. Translocation studies demonstrated that the move-ment of 14"CAC-94,377 was limited and occurred almost exclusively inan apoplastic fashion. Four days after leaf treatment, less than 1.5%(corn) or 4% (sunflower) of the recovered radioactivity had moved awayfrom the treated area. It was concluded that the lack of biological activityof AC-94,377 following shoot treatment resulted principally from limitedphloem mobility and to a lesser extent from accelerated metabolic break-down.

The observed biological activity of any externally appliedcompound ultimately depends on the ability of that compoundto reach the appropriate target site (i.e. enzyme or receptor) insufficient quantity. Many interrelated biological and physicalfactors, including absorption (uptake), translocation, and metab-olism may intercede and thereby alter the activity of any xeno-biotic (5). These considerations apply to both naturally occurringand synthetic compounds including metabolic intermediates andgrowth regulators. With agricultural chemicals (notably herbi-cides), the alteration in biological activity can be both beneficial(i.e. differential toxicity) or detrimental (loss of efficacy).

Despite encouraging projections, plant growth regulator usagein agricultural situations has lagged considerably behind that ofother types of agents (i.e. herbicides, fungicides, and so forth).Inconsistent response is often cited as a principal factor in thissituation. In spite of this, there have been very few systematicstudies examining the behavior and fate of growth regulatorsunder conditions of variable response.The substituted phthalimide AC-94,377' (henceforth AC-94;

' Abbreviation: AC-94, AC-94,377 (1-[3-chlorophthalimido]cyclo-hexanecarboxamide).

Fig. 1) has been shown to elicit GA-like activity in a number ofspecific bioassay systems (12). Besides having value in agricul-tural situations, this family of synthetic bioregulators could be ofuse to physiologists exploring the molecular bases of hormoneaction (for review see Ref. 1 1). Although generally active in GAbioassays, AC-94 elicited no response in the standard dwarfmaize (d5) bioassay. Subsequent studies found that AC-94 elicitedconsiderable GA-like activity when supplied to the roots of thed5 seedling via the nutrient solution.

This type of differential biological activity afforded a uniqueopportunity to examine the relationship between biological ac-tivity and the factors listed above versus site of application.Portions of these studies have been presented in abstract form(13).

MATERIALS AND METHODS

Plant Material and Experimental Protocol. Seeds of dwarfcorn (Zea mays L., d5 variety); dwarf rice (Oryza sativa L. cvTan-ginbozu); and sunflower (Helianthus annuus L. cv NK265)were germinated in vertically oriented cylinders of moist papertoweling and were subsequently transferred to foil-wrapped glassjars (about 500-ml volume) that contained nutrient solution (1).Seedlings were raised in a growth chamber under a 14-h photo-period provided by a mixture of cool-white fluorescent andincandescent bulbs (light intensity at plant height: 300 AE m-2s-', PAR). Day/night temperatures were 25°C/23°C, respectively,and a relative humidity of roughly 50% was maintained. Allexperiments described in this paper were conducted a minimumof three times and, where possible, each treatment within anexperiment was replicated. Data from typical experiments arepresented.Growth Response Studies. For shoot treatment studies, the

test compounds were dissolved in 30% (v/v) aqueous acetonecontaining 0.05% (v/v) Tween 20. In the case of root treatment,the test compounds were dissolved in the nutrient solution.Dwarf corn seedlings were transferred to the hydroponic system7 d after sowing and were treated the same day. For shoottreatment, 100 ,gl of solution was applied directly to the innerleaf whorl. Leaf sheath elongation was measured after 7 d.

0

¢N

Cl 0 CONH2FIG. 1. Chemical structure of AC-94,377 (1-[3-chlorophthalimido]-

cyclohexanecarboxamide).1068 www.plantphysiol.orgon October 17, 2020 - Published by Downloaded from

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TREATMENT SITE VERSUS BIOLOGICAL ACTIVITY

Sunflower growth studies were conducted similarly except thatonly 75 gl of treatment solution were applied to the apex ofeachseedling. Dwarf rice seedlings were transferred and treated 12 to14 d after sowing. For shoot treatment, a 2 MAl droplet of acetonecontaining the test compounds was placed between the inside ofthe first leaf sheath and the emerging second leaf sheath. Leafsheath elongation was measured 7 d after treatment.Metabolism Studies. Dwarf corn seedlings were transferred

and treated as described above. For shoot treatment, a total of 5seedlings were each treated with 10 Mg AC-94 containing 0.23MCi of [14C]AC-94. Root treatment was conducted using a totalof 10 seedlings that were exposed to a 10 Mm solution of AC-94(500 ml) containing 21 MCi of [14C]AC-94. Shoot-treated seed-lings were harvested 4 d after treatment and, initially, the treatedarea was rinsed alternately with 30% (v/v) acetone and water (3times). Root-treated seedlings were also harvested after 4 d. Inthis case, roots ofthe treated seedlings were immersed in distilledwater containing 100 uM unlabeled AC-94 for 2 h. The seedlingswere frozen in liquid N2 and were homogenized in 80% (v/v)aqueous acetone. The extracts were clarified by filtration and theresultant filtrate was slurried with a 1:1 (w/w) mixture ofcharcoaland celite in order to remove pigments. Preliminary experimentsdemonstrated that this step did not influence the quantitativepattern of metabolites recovered. The slurry was stirred for 1 h(4C) and was then filtered. This filtrate was reduced in volumeby rotary film evaporation. Where necessary, these extracts werefurther concentrated by evaporation (40C) under a stream ofN2. The extracts were then fractionated on 250 ,um silica plates(Silica Gel HF; Analtech, Inc.)2 using 1:1 (v/v) acetone-dichloro-methane as the developing solvent. After drying, the plates werescanned for radioactivity, the silica was removed from the plates,placed in scintillation vials, and counted. Sunflower metabolismstudies were conducted similarly except that for shoot treatmentstudies a total of 3 seedlings were each treated with 1 Mg AC-94containing 0.23 MCi ['4C]AC-94 and root treatment (3 seedlings)used 10 ,M AC-94 containing 2.7 MCi [14C]AC-94 (500 ml totalvolume).

Translocation Studies. Dwarf corn seedlings were transferredand treated 7 d after sowing. In the case of leaf treatment, 1 jigAC-94 containing 0.05 MACi [14C]AC-94 or 100 ng GA3 containing0.05 MCi [14C]GA3 was applied in a 50 Ml droplet (30% [v/v]aqueous acetone plus 0.05% Tween 20) to the distal portion ofthe first leaf. A lanolin barrier was placed between the treatmentzone and the remainder of the leaf to prevent movement of thetreatment solution. Root treatment studies were conducted using6-d-old seedlings. The test compounds (10 Mm AC-94 containing0.5 MCi ['4C]AC-94 or 0.1 MuM GA3 containing 0.5 ,Ci ['4C]GA3)were dissolved in the nutrient solution (500 ml). Seedlings wereexposed to this solution for 24 h and were then transferred tofresh nutrient solution lacking the 14C. In both cases, the seedlingswere harvested 4 d after the start of treatment. After dissectioninto respective tissues, the plant material was lyophilized and the'4C content was determined after combustion by scintillationspectroscopy. Sunflower translocation studies were conducted ina similar fashion using 9-d-old seedlings. In the case of roottreatment, sunflower seedlings were treated with ['4C]AC-94 for2 d. Plants were harvested 4 d after treatment.

Exudation Studies. Methodologies used here are essentiallythose originally described by King and Zeevaart (6). Two smallareas on fully expanded leaves from 16-d-old sunflower seedlingswere lightly abraded using jewelers abrasive. The test compounds(0.24 MACi [14C]AC-94 or 0.26 MACi ['4C]GA3; both carrier-free)

2 Mention of trademark or proprietary product does not constitute aguarantee or warranty of the product by the U.S. Department of Agri-culture and does not imply its approval to the exclusion ofother productsthat may also be suitable.

were applied to the abraded areas in 10 Ml droplets (30% [v/v]aqueous acetone + 0.05% Tween 20). One h later, the treatedleaves were excised, the petioles recut under distilled water, andplaced in beakers (25 ml) containing 2 ml glass-distilled water ±20 mm EDTA (pH 7.0). At the indicated times, the leaves weretransferred to fresh solutions. The EDTA (where used) was onlypresent during the first collection period (1 h total). The radio-activity in the exudation solutions was determined by scintilla-tion spectroscopy.

Partitioning Behavior. The test compounds (0.02 uCi) weredissolved in 5 ml of buffer solution (universal buffer: 1 mm eachof phosphoric, boric and phenylacetic acids titrated with NaOH).To this, 5 ml of octanol were added. These mixtures were shakenin closed vials for 0.5 h. After standing for an additional h, the'4C in each phase was determined.Chemicals. AC-94,377 (1-[3-chlorophthalimido]-cyclohexa-

necarboxamide) was kindly provided by American CyanamidCo. I-Amino-[carbonyl-'4C]- 1-(3-chlorophthalimido)cyclohex-ane (58 mCi/mmol) and [1,7,12,18-'4C] GA3 (10 mCi/mmol)were obtained from Amersham Corp. These compounds werepurified by HPLC prior to use. All other chemicals used in thisstudy were obtained from commercial supply houses.

RESULTS

Application of up to 10 Mg of AC-94 to the leaf whorl (shoottreatment) ofd5 corn seedlings resulted in no measurable increasein subsequent leaf sheath elongation (Figure 2, upper left panel).In contrast, applications of AC-94 to the root system via thenutrient solution at concentrations in excess of 1 AM elicitedmeasurable increases in subsequent leaf sheath elongation (Fig.2, upper right panel). GA3 effectively stimulated growth in eithersituation. A similar phenomenon with respect to site of applica-tion was observed using sunflower seedlings (Fig. 2, lower panels).We have previously reported (12) that the dwarf rice cultivarTan-ginbozu responded to AC-94 if the entire seedling wastreated (i.e. the immersion assay). When care was taken to treatthe root and shoot systems separately, only root treatment withAC-94 was effective (Table I). In both sunflower and rice seed-lings, root-applied AC-94 was nearly as active as GA3 (on aconcentration basis). In contrast, AC-94 exhibited a 100-foldhigher threshold concentration in d5 seedlings. At a concentrationof 100 FM, both compounds elicited comparable effects.

In attempting to identify the physiological bases for the diver-gent behavior of this compound with respect to treatment site,our attention was directed to differences in metabolism andtranslocation of AC-94. These two processes have been shownto influence the biological activity of a variety of xenobiotics.Due to the relative ease of manipulation, the remainder of thisstudy will focus on dwarf corn and sunflower seedlings.

['4C]AC-94 was readily metabolized by dwarf corn seedlings(Fig. 3). As judged by TLC, the metabolism of AC-94 by thedwarf corn seedlings resulted in the formation of more polarmaterials. When root versus shoot treatments were compared,nearly twice as much metabolism of AC-94 occurred when thismaterial was applied to the shoot. In the case of root treatment,80% of the applied 14C was found associated with unmetabolizedAC-94 4 d after application.The situation was much the same when ['4C]AC-94 was ap-

plied to sunflower seedlings (Fig. 4). As with corn seedlings,metabolism ofshoot-applied AC-94 was nearly twice that ofroot-applied material. Again, metabolism of AC-94 resulted in thegeneration of more polar compounds.

In addition to metabolism, the degree of translocation ofexternally applied compounds may have a profound impact ontheir resultant biological activity (5, 7). For this reason, themovement of ['4C]AC-94 was followed in both corn and sun-flower seedlings. In these studies the movement of ['4C]GA3 was

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SUTTLE AND HULTSTRAND Plant Physiol. Vol. 84, 1987

SHOOT-APPLIED

XI- AC-94

*-fF---4 -_i___{-.

15-

i 13--

(a 11-

-

e 7-so

S 5-

0

' 3-O-

1 10

SHOOT-APPLIED

C

AC-94/

__--

ROOT-APPLIED

00 9 8 7 6 5

Concentration in Nutrient Solution (-og M)

O 0.001 0.01 0.1 1 10 9 8 7 6 4ag/Seedling Concentration In Nutrient Solution (-Iog M)

FIG. 2. Effect of shoot or root treatment with AC-94 or GA3 on subsequent leaf sheath elongation in dwarf corn seedlings (upper panels) andinternode elongation in sunflower seedlings (lower panels). Solid lines, GA3-treated seedlings; dashed lines, AC-94-treated seedlings. Bars indicateSE.

Table I. Effect ofShoot versus Root Application ofGA3 or AC-94 on

Subsequent LeafSheath Elongation in Tan-ginbozu DwarfRiceSeedlings

Thirteen-d-old seedlings were treated with the test compounds as

described in the text. After 7 d, the combined lengths of the second andthird sheaths were determined.

Leaf Sheath Length (cm)Dose/Concentration

GA3 AC-94

Shoot treatment0 7.0 +0.3a0.00Ib 6.7 ±0.10.01 9.0 ± 0.3 7.0 ± 0.40.1 13.7 ± 0.5 7.0 ± 0.11.0 14.7 ± 0.6 6.7 ± 0.2

10.0 13.9 ± 0.8 6.9 ± 0.3Root treatment

0 7.7 0.30.0lc 7.1 ± 0.2 7.6 ± 0.40.1 9.3 ± 0.5 7.7 ± 0.31.0 14.1±1.4 11.0±0.5

10.0 17.6 ± 0.6 16.0 ± 0.6100.0 19.1 ± 1.5 17.5 ± 0.4

aMean±sE(n=6).in nutrient solution (.M).

b Dosages in ptg/seedling. Concentrations

Rf

FIG. 3. Radiochromatogram scans of extracts prePared from dwarfcorn seedlings treated with ['4CJAC-94 and frationated by TLC. Upperpanels, extracts from shoot-treated seedlings; lower panel, extracts fromroot-treated seedlings. Percentage figures indicate fractions of total '4Cfound associated with various regions on each chromatogram. Bar indi-cates position of AC-94 standard.

1070

17 -

-; 15-

13-

3 11-

0

0

9-um

Go

3-

0-

0 0.001 0.01 0.1

ag/Seedling4

7-

E 6-

5-

i 4-

.5 3-

d: 2-

ROOT-APPUED

-I

1 I Ao I I

ilo

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TREATMENT SITE VERSUS BIOLOGICAL ACTIVITY

1.U-

0.5'

t10-

K 1.0-. SUNFLOWER

FIG. 4. Radiochromatogram scan ofextracts prepared from sunflowerseedlings treated with ["C]AC-94 and fractionated by TLC. Upper panel,extracts from shoot-treated seedlings; lower panel, extracts prepared fromroot-treated seedlings. All else as in Figure 3.

100- LEAF-APPLIED

JE AC-9498- Total 14C Recovered (dpm) =3GA396- AC-94: 123,970 5,559

w¢ 92- GA3: 113,422 7,764

Ej ,94 m h j203--

uJ

_L

applied ['4C]GA3 exhibited greater mobility. Greater than 5% ofthe '4C was recovered in tissues distant from the treatment zone.Of this, some 3% was found in the younger leaf tissues.A different pattern emerged from the studies on root supplied

material. In this case, only 20% of the recovered '4C fromAC-94 treatment was found associated with the root tissues 4 dafter pulse treatment (Fig. 5, lower). The bulk of the 14C wasfound in the growing regions. In contrast, nearly half of the 14Cfrom GA3 treatment was still found in the root tissues. Theremaining half was distributed throughout the growing regions.The situation was much the same in sunflower seedlings (Fig.

6). When AC-94 was applied to a leaf, greater than 96% of the14C was still associated with the treated zone 4 d after treatment.The remaining 4% of the '4C was equally distributed throughoutthe seedling. As with corn, GA3 was more mobile in theseseedlings. Greater than 18% of the applied 14C was found in sitesdistant from the treatment zone. The bulk of this radioactivitywas found in the growing region (internode and second leaves).Once again, root treatment resulted in a completely different

pattern of movement (Fig. 6, lower). Greater than 95% of therecovered 14C from AC-94 was found outside of the root tissues.The majority of the translocated 14C was found in the cotyledonsand first true leaves. In the case of GA3, approximately 60% ofthe recovered '4C was found in the root system 4 d after treat-ment. The remaining radioactivity was distributed throughoutthe other seedling tissues.These results suggest that while AC-94 readily translocates via

the apoplast, little symplastic movement occurs. As symplasticmovement occurs primarily in the phloem tissues, these resultscan be interpreted as demonstrating limited phloem mobility. In

100-

90-

o

iz

0

Ul

I~-

zw

ROOTS SEED 1at LEAF 2nd LEAF 3rd & 4th 1st LEAF(UNTREATED) LEAVES (TREATED)

ROOT-APPLIEDTotal 14C Recovered

AC-94: 2074 dpm;! GA3 2874 dpm

80-

12-

11-

10-

9-8-

7-6-~

5-

4-

3-2-

1,0-

r AC-94"Az

I-

;I.-z

a:

0.

I.

SHEATH JR BLADE

FiG. 5. Recovery of 14C from various portions ofdwarfcorn seedlings4 d after application of either ['4CJAC-94 or ['4C]GA3. Upper panel,distribution of '4C in leaf-treated seedlings; lower panel, distribution of'4C in root-treated seedlings.

also monitored for comparative purposes.Following application of ['4CJAC-94 to the leaf-whorl of corn

seedlings, less than 1.5% of the '4C was recovered at sites distantfrom the treatment zone (Fig. 5). The translocated 14C was foundto be equally distributed throughout the seedling. In contrast,

60-

SO-

IL

z 40.

o 30-

z

20-

10-

LEAF-APPLIED

TOTAL 14C RECOVERED:AC-94 :139,972 dpm

GA3 :114,778 dpm

Roots

Fl

AC-94U] GA3

Hypocotyl Cotyledons UntreatedFirstLeaf

ROOT-APPLIEDTOTAL 14C RECOVERED:AC-94 : 8,800 dpm

GA3 : 4,800 dpm n

Intenode andSecond Leavs

TreatedFirstLaf

AC-94

Eza GA,

Roots Hypocotyl Cotyledons First First Internode &Leaves Second Leaves

FIG. 6. Recovery of "4C from various portions of sunflower seedlings4 d after application of either ["C]AC-94 or ["4C]GA3. Upper panel,distribution of 14C in leaf-treated seedlings; lower panel, distribution of'C in root-treated seedlings.

Shoot-Applied.AC-94

I 50,% § % %01

1071

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-I ff %W - - - --

-. . .1 1. . . .1. - 1. I I

0 , , , " - . . .1 . . . - 1. . .1 1. .1 1.

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VAr-i VA F] VA F-1 n.

L\."Ij GA

r-i

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SUTTLE AND HULTSTRAND

an attempt to more closely examine phloem mobility per se, theefflux of ['4C]AC-94 and ['4C]GA3 was studied in sunflowerleaves using the EDTA-enhanced phloem exudation techniqueoriginally described by King and Zeevaart (6).

Recovery of 14C in the exudation media following leaf treat-ment with '4C-GA3 was greatest during the first two collectionperiods and was strictly dependent ofEDTA pretreatment (TableII). A similar situation was found when the movement of 3H-sucrose was monitored (data not presented). In contrast, exuda-tion of '4C from leaves treated with ['4C]AC-94 and EDTA wasminimal.The observed differences in phloem mobility of AC-94 versus

GA3 could be the result of many factors. However, in studieswith other xenobiotics the lipophilicity of the test compoundshas been shown to be a significant determinant ofoverall phloemmobility (7). This characteristic is usually determined by meas-uring the octanol/water partitioning behavior as a function ofpH. The partitioning behavior ofGA3 was found to be typical ofthat of a weak acid (Fig. 7). As the pH was increased, anincreasing amount of GA3 was found in the aqueous phase. Incontrast, at pH values between 2 and 8, essentially all of theAC-94 remained in the organic (octanol) phase. In the case ofAC-94, pH values in excess of 8 were not studied as we havefound rapid decomposition of this material in mildly alkalinesolutions (JC Suttle, JF Hultstrand, unpublished data).

DISCUSSION

The results presented in this manuscript demonstrate that, ifa suitable application method is used, the N-substituted phthal-imide AC-94 elicits GA-like agonist activity in sunflower, dwarfrice, and dwarf corn seedlings (Fig. 2; Table I). The ability to

Table II. Exudation of'4Cfrom Sunflower Leaves Treated with J'4C]GA3 or f4C]AC-94 in the Presence or Absence ofEDTA

The test compounds were applied to lightly abraded areas on fullyexpanded sunflower leaves in 10-Mgl droplets of 30% (v/v) acetone con-taining 0.05% (v/v) Tween 20. After 1 h, the treated leaves were excisedand were placed in vials containing 1.5 ml of glass-distilled water (pH7.0) ± 20 mM EDTA. After 1 h, the EDTA solution was removed andwas replaced with distilled water for the remainder of the experiment.

Collection Period'Compound EDTA'

0-1 1-3 3-6 6-12

14C recovered (dpm/1eaJ)GA3 Yes 405 ± 56 192 ± 27 51 ± 15 45 ± 11GA3 No 6 ± 3 7 ± 3 7±1 10±AC-94 Yes 13 ± 2 7 ± 2 5 ± 1 7 ± 2

a Present only during the first collection period.after leaf excision (in hours).

100-

8o-

60-

40-

20-

0-

- GA3

_ 4AC-94

2 3 4 5 6 7 8pH

I Time elapsed

0

9 10 11

FIG. 7. Octanol to water partitioning behavior of ['4C]AC-94 versus

['4CJGA3 as a function of pH.

elicit GA-like activity in the GA-deficient dwarf rice and cornseedlings supports the earlier contention (1 1, 12) that this classof bioregulators possesses intrinsic GA-like activity and does notsimply stimulate endogenous gibberellin biosynthesis.Not all species examined exhibit this type of differential re-

sponse. Treatment of intact seeds with AC-94 elicits GA-likeactivity (14, 16). In some species such as cucumber, tomato,chrysanthemum, and strawberry, foliar application of AC-94elicits biological activity (3, 8, 9, 15). At present, it is not clearhow these species differ from those in the present study. It ispossible that the spray treatments used resulted in direct contactof the phthalimide with the growing zone thereby circumventingthe limited phloem mobility of this compound (see below).While not excluding other possible factors, the data presented

in this study suggest two underlying physiological mechanismsfor the differential biological activity as affected by treatmentsite. When AC-94 is applied to the shoot or leaf, little biologicalactivity is observed (Fig. 2; Table I). This is associated with agreater degree of metabolic conversion (Figs. 3 and 4) and withlimited movement away from the treatment zone (Figs. 5 and6). In contrast, application of AC-94 to the root system (via thehydroponic medium) elicits considerable biological activity thatis associated with a greater percentage of unmetabolized bio-regulator and a higher degree of translocation. Of these twoprocesses, we feel that the limited phloem mobility of AC-94 isof greater importance in determining the final level of biologicalactivity. Our reasoning is as follows: (a) when applied to theshoot, there is still a fair percentage (34-47%) of unalteredAC-94 present after 4 d, and (b) in contrast, there appears to beessentially no phloem movement of this compound.With respect to metabolism, the picture presented in Figures

3 and 4 is oversimplified. When extracts from treated plants arefractionated by reverse-phase HPLC a more complex pattern canbe demonstrated (JC Suttle, JF Hultstrand, unpublished data).The low RF peak observed following TLC separation (Figs. 3 and4) readily resolves into at least five distinct peaks. The pattern ofmetabolites is qualitatively similar in the three species examined.Due to the chemical instability of these metabolites, their ident-ities and biological activities are presently unknown.The phloem mobility of many xenobiotics is thought to be

inversely related to their hydrophobicity (5, 7). One possibleexception concerns those compounds that are weakly acidic (7).It is generally accepted that weak acids readily enter the phloemin an undissociated state and, once in contact with the mildlyalkaline phloem symplast, they dissociate and are therebytrapped. AC-94 readily partitions into octanol at all physiologicalpH levels (Fig. 7). This chemical property may explain the neartotal lack ofphloem (basipetal) mobility of this compound (Figs.5 and 6; Table II). GA3 is a weakly acidic molecule that exhibitsboth acropetal and basipetal movement (for review see Ref. 4).This observation has been verified in our studies with dwarfcornand sunflower (Figs. 5 and 6). The chemical nature of the 14Ctranslocated after the application of the test compounds was notinvestigated.AC-94 is labile in mildly akaline solution (pH 2 8) and the

breakdown products lack biological activity (JC Suttle, JF Hult-strand, unpublished data). Given the alkaline nature of thephloem symplast (roughly 8), any AC-94 that enters this com-partment would be subject to rapid decomposition and loss ofactivity.

Aside from its effects on phloem mobility, the apolar natureofAC-94 may favor immobilization of the compound within thecuticle (10). Due to the lack ofconsensus concerning the validityof previously used methodologies to study cuticular absorption(2), this possibility was not explored further.

Presumably, the physiological impediments that preclude theexpression of biological activity of this bioregulator will only

1072 Plant Physiol. Vol. 84, 1987

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TREATMENT SITE VERSUS BIOLOGICAL ACTIVITY

manifest themselves during in vivo studies. Therefore, we wishto reemphasize that AC-94 and related chemistries should bevaluable probes ofGA action at the molecular level (i.e. in vitrostudies).

Acknowledgments-The authors gratefully acknowledge Mrs. Jeri Schmidt andMr. Tom Hlavaty for their assistance in preparing this manuscript.

LITERATURE CITED

1. BLANKENDAAL M, RH HODGSON, DG DAVIS, RA HOERAUF, RH SHIMABU-KURO 1972 Growing plants without soil for experimental use. USDA-ARSMisc Publ No 1251. Washington, DC, 17 pp

2. CHAMEL A 1986 Foliar absorption of herbicides: study of the cuticular pene-tration using isolated cuticles. Physiol Veg 24: 491-508

3. CHOMA ME, DG HIMELRICK 1984 Responses of day-neutral, June-bearing andeverbearing strawberry cultivars to gibberellic acid and phthalimide treat-ments. Sc Hortic 22: 257-264

4. GRAEBE JE, HJ ROPERS 1978 Gibberellins. In DS Letham, PB Goodwin, TJVHiggins, eds, Phytohormones and Related Compounds: A ComprehensiveTreatise, Vol. I. Elsevier/North Holland, Amsterdam, pp 107-204

5. HEss FD 1985 Herbicide absorption and translocation and their relationshipto plant tolerances and susceptibility. In SO Duke, ed, Weed Physiology, VolII. CRC Press, Boca Raton, FL, pp 191-211

6. KING RW, JAD ZEEVAART 1974 Enhancement of phloem exudation from cutpetioles by chelating agents. Plant Physiol 53: 96-103

7. LICHTNER FT 1986 Phloem transport of agricultural chemicals. In J Cronshaw,WJ Lucas, RT Glanquinta, eds, Phloem Transport. AR Liss, New York, pp601-608

8. Los M, CA KuST, G LAMB, RH DIEHL 1980 Phthalimides as plant growthregulators. HortScience 15: 22

9. McDANIEL GL 1984 Peduncle elongation of pompom chrysanthemums withsubstituted phthalimides. HortScience 19: 434-436

10. SCHONHERR J, M RIEDERER 1986 Plant cuticles sorb lipophilic compoundsduring enzymatic isolation. Plant Cell Environ 9: 459-466

11. SUTrLE JC 1983 N-substituted phthalimides as plant bioregulants. News Bull,Br Plant Growth Regul Grp 6: 11-19

12. SUTTLE JC, DR SCHREINER 1982 The biological activity of AC-94, 377 [1-(3-chlorophthalimido)-cyclohexane-arboxamide]. J Plant Growth Regul 1:139-146

13. SUTTLE JC, JF HULTSTRAND 1986 Physiological studies of a synthetic gibber-elin-like bioregulator. Plant Physiol 80: S-1 15

14. THOMAS TH 1984 Gibberellin-like stimulation of celery (Apium graveolens L.)seed germination by N-substituted phthalimides. Sci Hortic 23: 113-117

15. Xu SY, MJ BUKOVAC 1983 Phthalimide-modification of sex expression ingynoecious and monoecious cucumbers. J Am Soc Hortic Sci 108: 278-282

16. ZENG GW, AA KHAN 1984 Alleviation of high temperature stress by preplantpermeation of phthalimide and other growth regulators into lettuce seeds viaacetone. J Am Soc Hortic Sci 109: 782-785

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