Membranes"2 - NCBI

Plant Physiol. (1981) 68, 1289-1293 0032-0889/81/68/1289/05/$00.50/0

Demonstration of Auxin Binding to Strawberry Fruit Membranes"2 Received for publication August 1, 1980 and in revised form April 9, 1981

KOMARATCHI R. NARAYANAN, KENNETH W. MUDGE3, AND B. W. POOVAIAH4 Department of Horticulture and Landscape Architecture, Washington State University, Pullman, Washington 99164 ABSTRACT Presence of specific auxin-binding sites in strawberry fruit (Fragaria cv. Ozark Beauty) membranes has been demonstrated, These 1-naphthaleneacetic acid (NAA)-binding sites in the 80,000g to 120,000g fraction of the strawberry fruit membrane were pronase sensitive with an estimated equilibrium dissociation constant for NAA of 1.1 x 10' molar. The minimum concentration of NAA required to stimulate strawberry fruit growth was at least one order of magnitude higher than the minimum concentration of NAA required to stimulate corn coleoptile elongation. This was consistent with the higher equilibrium dissociation constant (lower affinity) for auxin binding to strawberry fruit membranes than to corn coleoptiles. Twelve auxin analogs, ranging from very strong to weak auxins, were tested for abilities to stimulate in situ strawberry fruit growth and to bind (displace or compete with NAA) to strawberry fruit membranes. The observed positive correlation (r = 0.74) between the in vitro binding to the 80,000 to 120,000 membrane fraction and the in situ biological activity of these analogs indicated that the NAA-binding sites in strawberry fruit membranes may represent physiologically relevant auxin receptors.

aanassa Duch.

parthenocarpic fruit set and subsequent receptacle growth. In the strict anatomical sense, the fleshy portion of the strawberry is not a true fruit, i.e. it is not a matured ovary, but rather the flower receptacle bearing true fruits or achenes. But, in the physiological sense, it is a fruit in that it is the flower-associated tissue which, in response to fertilization, undergoes enlargement, accumulation of sugars, and ripening. The strawberry receptacle represents an excellent system in which to investigate and compare the mechanism of auxin action in fruit with that in vegetative shoots. One aspect of auxin action studied in the last decade has been auxin binding to receptor macromolecules which presumably mediate the primary cellular response to the hormone (14). Several reports indicate that auxin binding sites located on microsomal membranes from corn coleoptiles (24, 31) and other auxin-responsive tissue have the characteristics expected of physiologically relevant auxin receptors. Inasmuch as little is known regarding auxin binding to fruit membranes, the object of this study has been to demonstrate and characterize auxin binding to membranes of strawberry receptacles and to determine the possible physiological significance of this phenomenon. MATERIALS AND METHODS

Plant Material. Everbearing strawberries (Fragaria ananassa The role of auxin in plant growth and development has been studied most extensively in etiolated vegetative tissues such as grass coleoptiles and to a limited extent in other plant organs such as tubers, bulbs, leaves, roots, and fruits. A probable role of auxin in fruit growth has been implied in several cases, but there are very few fruits which have an unambiguous auxin response. The strawberry is a fruit which not only responds to exogenous auxin, but also one in which endogenous auxin probably plays a pivotal role in normal growth and development. This was demonstrated by Nitsch (19) who showed that the growth of the strawberry receptacle could be stopped at any time after pollination by removal of the achenes (true fruit) and that the receptacle resumed its growth after treatment with auxin in lanolin paste. The removal of achenes deprived the receptacle of endogenous auxin. This was deduced from the finding that the achenes, but not the receptacle, were a rich source of native auxin, IAA (19, 20). Subsequently, Thompson (29) and others (17, 28) showed that auxin in aqueous sprays or lanolin paste applied to unpollinated flowers resulted in

Duch. cv. Ozark Beauty) were grown in the field for use from May through October, and in the greenhouse and growth chamber for use during the winter months. In the greenhouse and growth chamber, plants were grown in 15-cm pots with a 16 h/day photoperiod to maximize flowering. Corn (Zea mays cv. Patriot) was germinated in moist vermiculite and grown at room temperature in the dark, except for 2 h of dim red light per day. Chemicals. [1-14C]NAA,5 55.4 mCi/mmol, was purchased from Amersham and stored in acetonitrile at -20°C. [12C]NAA and other auxin analogs were obtained from Sigma, recrystallized from 95% ethanol, and stored in acetonitrile at -20°C. Acetonitrile was obtained from Mallinckrodt and redistilled prior to use. All other reagents were obtained from Sigma. Fines were removed from PVPP and wetted with extraction buffer prior to use. Membrane Isolation. Strawberry fruits were harvested for binding experiments at "berry swelling" stage of development as defined by Darrow (6) which is 8 to 12 days after pollination for Ozark Beauty. This stage is characterized by maximum endogenous auxin level (20) and presumably, high auxin responsiveness. After the pedicel and calyx were removed and achenes scraped

'Supported by National Science Foundation Grant PCM 78-05292. 2 Scientific Paper 5556, College of Agriculture Research Center, Wash5Abbreviations: NAA, 1-napthaleneacetic acid; PVPP, polyvinylpolyington State University, Pullman, Project 0321. 3Present address: Department of Floriculture apd Ornamental Horti- pyrrolidone; gfw, gram fresh weight; CM, crude membrane; 120 K, 80,000 to 120,000g membrane fraction; DMSO, dimethylsulfoxide; Kd, equilibculture, Cornell University, Ithaca, NY. rium dissociation constant. 4 To whom reprint requests should be addressed. 1289

1290

NARAYANAN ET AL.

off with a spatula, receptacles were put into 5.8 ml/gfw of ice-cold extraction buffer (medium I) consisting of (unless otherwise specified) 100 mm Mes, 250 mm sucrose, 1 mm EDTA, and 0.1 mM MgCl2 and with pH adjusted to 7 with HCI. The pH of buffer solutions was adjusted at room temperature. The large volume and high concentration of Mes in the extraction medium were necessary in order to adequately buffer the relatively acidic strawberry receptacle homogenate. Because of the abundance of phenolic compounds in strawberry receptacles, 0.4 g/gfw of wetted insoluble PVPP was added to the extraction buffer along with the tissue. PVPP has been reported to prevent browning and associated inactivation of proteins by absorbing phenolics (15). Preliminary experiments revealed that the addition of PVPP resulted in a small increase in specific auxin binding. Receptacles, medium I, and insoluble PVPP were ground in a Waring blender and further homogenized with a Polytron homogenizer at maximum speed. The homogenate was filtered through one layer each of Miracloth (Chicopee Mills, Inc.) and cheesecloth to remove PVPP and larger tissue fragments. The filtrate was centrifuged at 8,000g for 10 min and the pellet containing all debris, including cell walls, nuclei, etc., was discarded. For CM preparations, the supernatant was centrifuged at 250,000g for 20 min and the supernatant was discarded. For differential centrifugation, the supernatant from the 8,000g centrifugation was first centrifuged at 60,000g for 20 min and the pellet designated as 60 K fraction. The supernatant was then centrifuged at 80,000g for 20 min and the pellet designated as 80 K fraction. The supernatant from this centrifugation was centrifuged again at 120,000g for 20 min and the pellet labeled as 120 K fraction. The 60 K, 80 K, 120 K, and CM pellets were washed by resuspending the pellets with a glass rod in medium II which consisted of 10 mm citric acid, 250 mm sucrose, and 0.5 mM MgC12, adjusted to pH 6 with NaOH. The resuspended membrane pellets were again centrifuged at 250,000g for 20 min. In the case of the experiments shown in Table II, the resuspended membranes were treated with combinations of phospholipase and pronase prior to recentrifugation. After appropriate incubation periods, the membrane suspensions were repelleted at 250,000g for 20 min. The resulting washed membrane pellet was resuspended with a tight-fitting Potter-Elvehjem type homogenizer in 1 to 2 ml per initial gfw of binding assay buffer (medium III) consisting of 10 mm citric acid, 250 mm sucrose, and 5 mM MgC12 (pH 4.0, except for crude membranes, where the pH was 3.5). The pH of medium III ranged from 3.0 to 6.0 in the pH series experiments. In Vitro Auxin-Binding Assay. The theoretical rationale for the competitive hormone binding assay used in these experiments has been discussed at length elsewhere (11, 23). To an appropriate amount of membrane suspension, radioactive NAA in acetonitrile was added to a final concentration of about 5 x l0-7 M (about 70,000 dpm/ml). The membrane suspension was then divided into two or more aliquots. To one was added [12C]NAA to a final concentration of 3.16 x l0-4 M (Fig. 2, Tables I and II). In auxin analog binding experiments [12C]NAA or other auxin analog was added to a concentration of l0-4 M. The second aliquot in each of these experiments received an equal volume of acetonitrile. In the case of displacement experiments (Fig. 3), the membrane suspension containing [14C]NAA was divided into 14 aliquots to which a range of concentrations from 5.6 x 10-8 to l0-3 M of [12C]NAA was added, as well as one aliquot with acetonitrile only. The final concentration of acetonitrile in all cases was 0.75% (v/v). Membrane suspensions were then incubated on ice for 10 min after which time triplicate l-ml samples were centrifuged at 120,000g for 15 min. After centrifugation (to separate membrane-bound NAA from free NAA) the supernatant was poured off and 1 ml of methanol was added to each centrifuge tube. Tubes were sealed with Parafilm and left at room temperature overnight to extract radioactivity from the membrane pellets. The methanol, along with the pellet, was then transferred by Pasteur pipetts to a

Plant Physiol. Vol. 68, 1981

scintillation vial containing 10 ml of Scintiverse scintillation cocktail. Each tube was rinsed with an additional 1 ml methanol which was immediately transferred to the same scintillation vial. Samples were counted to 1% error at about 70% counting efficiency in a Packard Tricarb Liquid Scintillation Spectrometer. Radioactivity in the pellet from the assay mixture containing only ["4C]NAA was taken as a measure of total NAA binding, while radioactivity in the ?ellet from the assay mixture containing [14C]NAA plus excess [ 2C]NAA (or other auxin analogs) was taken as a measure of nonspecifically bound NAA. The difference in radioactivity between the two was taken as a measure of specifically bound NAA. Results expressed as percentage specific binding were calculated by dividing the specifically bound radioactivity in the pellet by the total radioactivity in 1 ml of assay mixture. Total radioactivity was determined by counting a sample of the assay mixture before centrifugation. NAA was reextracted from membrane preparations that had been incubated with [14C]NAA. Both the original [14C]NAA and reextracted [14CJNAA eluted at the same time from a ,uBondapak C18 column in HPLC indicating that the bound ligand was NAA

(data not shown). Bioassays. Two methods were used for testing the effects of various auxin analogs on strawberry fruit growth in situ. The test used in the analog specificity experiments (Fig. 5) was adapted from Nitsch (19), and involved applying the test compounds in lanolin paste to receptacles of previously pollinated flowers at the berry swelling stage of development (10 ± 2 days after pollination) after scraping the achenes. The other method, used for the doseresponse curve in Figure 1, involved application of NAA in aqueous solution containing 2% DMSO to newly opened, unpol-

linated, emasculated flowers. In the former case, the auxin response was restricted to enlargement of a partially developed receptacle, while in the latter both parthenocarpic fruit set and

receptacle enlargement occurred. Lanolin pastes were prepared by first dissolving the test compounds in 95% ethanol and then adding 100 ,l1 of the ethanolic solution to 10 g molten anhydrous lanolin at 45 ± 2°C and mixing thoroughly. It was found that the variability of the assay was minimized by using berries at the same developmental stage rather than at exactly the same chronological age. The calyx and anthers were removed to ensure complete and uniform coverage of the receptacle with lanolin paste. After we had measured the receptacle diameter at its widest point, we gently scraped offthe achenes with a spatula and applied the lanolin paste to the surface of the receptacle as uniformly as possible with gloved fingers. One week following treatment, receptacle diameter measurements were begun and were continued every other day until the receptacle had reached its maximum diameter (about 10 days after treatment20 days after pollination). Results were expressed as percentage increase in receptacle diameter (Fig. IA) or increase in receptacle diameter expressed as percentage of NAA (Fig. 5). The doseresponse curve (Fig. IA) was obtained from newly opened, emasculated flowers which were dipped for 10 s in 2% DMSO solution containing NAA at the concentrations indicated, and the growth response measured as described above. Corn coleoptile bioassays (Fig. 1B) were performed with 6-dayold, dark-grown coleoptile segments, cut to 10 mm, starting 3 mm below the tip. Segments were floated on solution (pH 6.3) containing 2% DMSO and 10 mm KH2PO4, 10 iM Na3 citrate, and 1.5% sucrose, plus NAA at the concentrations indicated in Figure lB. The segments were incubated in the dark at 25°C for 18 h and their length measured. RESULTS Figure 1, A and B show the growth responses of strawberry receptacles and of corn coleoptile segments respectively to NAA. The minimum concentration of NAA required to stimulate straw-


Table I. NAA Binding to

0

A. Strawberry

200

0

c.E

0

.0

0150

CLO 50

0,

0 E

B. Corn

14[

-CL

131 o 0

C

J -j

12

I,

.

.

.

.

D8

7

6

5

4

12

3

-log[NAA] ,M

4

4t

FIG. 1. Effect of NAA concentration on strawberry receptacle enlarge-

Z

ment (A) or on corn coleoptile elongation (B). Newly opened strawberry flowers were emasculated and dipped in treatment solutions. Fruit growth

tli 0.

was measured were cut from

after approximately 2 weeks. Ten-mm coleoptile segments 6-day-old, dark-grown corn seedlings and placed in treatment solutions containing 2% DMSO. Each datum is the mean + SEM for 10 to 30 strawberry fruits or 15 coleoptile segments.

CO

o 0Cl,, 25

0.V

6

35

5

4

[NAA],M

FIG. 3. Effect of [12 CNAA on binding of ['4C]NAA to 120 K fraction from strawberry fruit. Concentration of ['2C]NAA indicated on the abscissa were added to membrane suspensions containing 5 x 10-' M ['4C] NAA and then centrifuged at 120,000g for 15 min.

.o c

7

-Log

20

cr

Different Membrane Fractions of Strawberry

Fruit After 8,000g centrifugation of the fruit homogenate to remove the cell debris, the homogenate was centrifuged sequentially at 60,000g, 80,000g, and 120,000g for 20 min. The resultant membrane pellets were washed and assayed for NAA binding as described in the text. Specifically Bound Membrane NAA Fraction dpm/mg protein 8,000g to 120,000g (crude membrane) 1,686 ± 101 8,000g to 60,000g (60 K) 1,469 ± 87 60,000g to 80,000g (80 K) 3,256 ± 164 80,000g to 120,000g (120 K) 20,682 ± 397

0

-W

1291

AUXIN BINDING TO STRAWBERRY FRUIT MEMBRANES

3

~,10

0.I

CP

Kd,= 1.I x I0-M

4I

z

X0

e

4)

0 0

pH of Assay Buffer FIG.

2.

Effect of pH of assay buffer on specific binding of ['4C]NAA to

80,O00g

120 K fraction (membranes that pelleted between

to

z

.~0.05

120,O00g) 0

from strawberry fruit. Specific binding was the difference in the radioactivity between membranes incubated in 5 x without 3.2 x

10-4

10-7

M ['4C]NAA with or

M ['2C]NAA and pelleted at 120,000g for 15 min.

berry receptacle growth (5 x

10-5

M) iS about an order of magni-

tude higher than for coleoptile elongation (5 X

lo-6

M).

0

The binding assay used in this study is based on the assumption that

equilibrium

had

been

achieved

before

the

separation

membrane-bound auxin from the free auxin. No significant

of

dif'-

ference was found in the level of specific NAA binding between

samples incubated from 10 to as long as 90 min. Thus, samples were routinely incubated for 10

min

membranes, specific binding of NAA was determined in the pH range between 3.0 and 6.0. Figure 2 shows that the maximum

specific binding to 120 K fraction occurred at pH 4.0 with very little specific binding at pH 6.0. On the other hand, when the CM was used, the optimal pH for the specific NAA binding appeared

3.5

(data not shown).

shows specific NAA binding to 60 K, 80 K, 120 K, and fractions expressed as dpm bound/mg protein. It is apparent that, per unit protein, the 120 K fraction has the highest NAA binding. Table

CM

I

40

80

120

Specifically Bound NAA (pmol/gfw) FIG. 4. Scatchard analysis of NAA binding to 120 K fraction from strawberry fruit. Data in this figure were calculated from the displacement curve shown in Figure 3.

prior to centrifugation.

To determine the optimal pH for NAA binding to strawberry

to be

0

In order to quantitatively analyze the binding sites of the strawberry membrane, displacement experiments were conducted and data analyzed according to Scatchard (26). Binding assays were performed with a constant concentration of [14C]NAA (5 x 10-7 M) and a range of concentrations of [12C]NAA from 5 x 10-8 to 10-3 M. Figure 3 shows the displacement curve for the 120 K membrane fraction. As expected, increasing concentrations of [12C]NAA resulted in progressively greater displacement of [14C]NAA from the membrane pellet. Data in Figure 3 were transposed into a Scatchard plot (Fig. 4) after correcting for nonspecific binding by the formula described by Chamness and

1292

NARAYANAN ET AL.


McGuire (5).

B5P = BT -F(lim BT/F) Z

where BT and B8p refer to the pmol of ["4CJNAA + [12CJNAA bound, i.e. total bound and specifically bound NAA, respectively. F refers to the pmol of [14C]NAA + [12C]NAA which remain free. The limit of B/F as B -X oo was determined from a Scatchard plot of BT/F versus BT. This limit was the minimum value of BT/F which occurred as the curve plateaued at high values of BT. The shape of the Scatchard plot (Fig. 4) and the displacement curve (Fig. 3) suggest positive cooperativity. From the negative reciprocal of the slope, obtained by dividing the ordinate intercept by the abscissa intercept (expressed as molar concentration), the Kd was estimated to be 1.1 x 1o-6 M with 100 pmol of binding sites/ gfw. If the entity responsible for auxin binding in this system is a protein, treatment of the membrane with proteases should destroy or reduce specific binding. Table II shows the effects of phospholipase and pronase treatment to 120 K membrane fraction on NAA binding. Wtih CM, phospholipase + pronase treatment was ineffective in reducing the NAA binding (data not shown). In contrast, treatment of the 120 K membrane fraction with phospholipase + pronase resulted in virtual elimination of specific binding. The specificity of hormone binding for biologically active hormone analogs is considered an important criterion for establishing the physiological relevance of in vitro hormone binding in any system (4, 25). Twelve compounds including strong, weak, and structurally related nonauxins were tested for their effect on strawberry receptacle growth (lanolin test) as well as for their ability to displace [14CJNAA from (affinity for) binding sites in the 120 K membrane fraction (Fig. 5). There was a general tendency for the most effective growth stimulators to be very active in the binding assay while less effective growth stimulators tended to be less active in the binding assay, with notable exceptions being benzoic acid and 2-naphthaleneacetic acid. The correlation coefficient (r) calculated from a linear regression of the data is 0.74 for the 120 K fraction. Table II.

Effects of Phospholipase and Pronase on Specfifc Binding of [14CJNAA to Strawberry Fruit Membranes

After the first 250,000g centrifugation, 120 K pellet was resuspended in medium II and divided into 5 aliquots. Phospholipase C (100 mg/ml) was added to two aliquots, pronase (5 mg/ml) was added to two aliquots and all four treatments were incubated at 37°C. One aliquot was held on ice as control. After I h, crystalline pronase was added to one of the aliquots containing phospholipase, and phospholipase was added to one of the aliquots containing pronase and all five treatments were incubated for an additional h. Then, all the aliquots were centrifuged at 250,000g for 20 min and assayed for NAA binding in the usual manner. Change 1'4CJNAA Bound Treatment

Total

Nonspecific

S

ciiec

cpm/gfw Control Pronase

Lipase Pronase + lipase

Lipase + pronase

2,069 ± 23 2,294 ± 21 2,675 ± 34 1,997 + 32 2,577 + 90

1,119 ± 14 1,987 ± 41 2,037 ±: 18 1,639 + 30 2,603 ± 77

-/NAA

100

o BX-

Due to Treatment %

950 307 638 358

-68 -33 -62 -100

80

o 2.4-D

-/

X P02-NOA

0R

o IAA

o

PCPA

40

o 2-NAA

OBA

Z

oOH -IAA 20

-

/IA

OMI

0

20

40

60

80

100

In Vitro Binding Ability (% of NAA) FIG. 5. Correlations between the ability of auxin analogs to displace 1'4C]NAA from 120 K fraction of strawberry fruit and the ability of auxin analogs to stimulate strawberry receptacle growth. Membranes were incubated in 5 x 10-7 M ['4C]NAA with or without I x 10-4 M test compound and pelleted at 120,000g for 15 min. Receptacle growth was assayed by applying 1 x 10-4 M test compound dissolved in lanolin to the surface of 10-day old fruits from which achenes had been removed prior to the treatment. Growth was measured approximately 10 days after treatment. Abbreviations: IBA, 3-indolebutyric acid; 2-NOA, 2-naphthoxyacetic acid; PCPA, p-chlorophenoxyacetic acid; PAA, phenylacetic acid; 2-NAA, 2naphthaleneacetic acid; BA, benzoic acid; OH-IAA, 4-hydroxyindoleacetic acid; IPA, 3-indolepropionic acid; MI, methylindole.

DISCUSSION

Since strawberry fruit is auxin-responsive (19) and under the control of auxin for its growth and development to a large extent (17, 20), it is logical to look for specific auxin-binding sites (receptors) in the fruit tissue. Strawberry fruit is an ideal system to compare auxin binding with the in vivo biological response because of the ability to nondestructively remove the endogenous hromonal source, the achenes. One of the main differences between auxin binding to vegetative (7-10, 12, 13, 21-24, 30, 32, 33) and fruit tissue appears to be the low pH optimum. The pH optimum for the 120 K membrane fraction is 4.0 (Fig. 2), while the pH optima for vegetative tissues range from 5.0 in zucchini (12) and tobacco pith callus (32), 5.5 in com (1, 23), to 8.0 in mung bean (13, 33). The physiological relevance of this low pH optimum is unknown, but fruits in general appear to have a low pH optimum for auxin binding as evidenced by pH optima of 3.75 for cucumber fruit and 3.5 for bean and tomato fruit CM fraction (16, 18). The estimated Kd for NAA binding to strawberry fruit membranes is higher (lower affinity) than the minimum Kd reported for NAA binding to com coleoptile membranes (1, 2, 23). One criterion for the physiological relevance of in vitro hormone binding is that the Kd should be similar to the Km of the biological response (25), even though in an in situ biological response, transport and penetration of the compounds would tend to raise the Km (concentration at which the response is half saturated). Comparison of the Kd for NAA binding to com coleoptile and strawberry fruit membranes with their respective dose-response curves (Fig. 1, A and B) do indeed suggest that the kinetics of binding in these two systems is compatible with the physiological relevance of in vitro auxin binding. Of course, caution must be exercised when comparing the dose-response curves for two entirely different bioassay systems. To minimize the difference between the two systems and make them as comparable as possible,


AUXIN BINDING TO STRAWBERRY FRUIT MEMBRANES

DMSO (2% in aqueous solution) was used as a solvent in both bioassays. DMSO had the desired effect in the strawberry bioassay since flowers treated in this manner (Fig. IA) respond to NAA more rapidly (about 4 days after treatment) than flowers treated with the same concentration of NAA in lanolin paste (about 2 weeks after treatment-data not shown). Nevertheless, the minimum concentration of NAA required to stimulate fruit set and receptacle enlargement was the same irrespective of the method of application. This suggests that the uptake was not the limiting factor in the strawberry bioassay and that the differences between the minimum effective NAA concentrations for strawberry (5 x 10-5 M) and for corn (5 X 10-6 M) (Fig. 1, A and B) represent real differences in the Km (sensitivity) between the two systems. This lower sensitivity of strawberry fruits to auxin is entirely consistent with the lower affinity (higher Kd) of NAA for strawberry fruit membrane binding sites. The displacement curve (Fig. 3) and Scatchard plots (Fig. 4) suggest positive cooperativity, although not to the extent found in cucumber fruits (18). Positive cooperativity narrows the range of ligand concentration to which the receptor is receptive, can produce a kind of threshold effect at low ligand concentrations, and even allows an on/off type of regulation (3). IAA was less effective than either NAA, 3-indolebutyric acid, 2,4-D, or 2-naphthoxyacetic acid in the in situ strawberry fruit growth bioassay, but it was the most effective auxin tested in the com coleoptile bioassay (24). This relative difference in activity may be due to photolability of IAA since the strawberry test was conducted in a brightly lit growth chamber over a 10-day period, whereas the corn coleoptile elongation test was performed in darkness over a 24-h period. Other differences between analogs in the strawberry and corn coleoptile systems include the strong and weak inhibitory action of 2-NAA and phenylacetic acid, respectively in coleoptile elongation (24), but both were weakly stimulatory in strawberry receptacle growth. If auxin binding to strawberry membranes does represent the formation of a physiologically relevant hormone-receptor complex, then the auxins which are most effective in stimulating receptacle growth should be most effective in binding to the receptacle membranes in vitro. Similarly, structurally similar but biologically inactive analogs should be relatively ineffective in binding to the fruit membranes. A weak positive correlation (r = 0.36 for corn coleoptiles [24]; r = 0.37 for zucchini hypocotyls [12]) between in vitro binding and in vivo biological activity was still considered relevant because of the different factors such as differing rates of diffusion or transport to the active sites as well as differing rates of degradation which might be expected to minimize correlation. Even in strawberry, when CM are used for in vitro binding, the positive correlation is only r = 0.43 (16). On the other hand, when the 120 K membrane fraction is used for in vitro binding, the positive correlation is 0.74 (Fig. 5), the highest value reported so far in any plant auxin study. This would seem to indicate that the auxin-binding sites in the 120 K membrane fraction of strawberry fruit may be physiologically relevant. Acknowledgments-The authors wish to thank Dr. M. Griswold for his advice and assistance during the course of this investigation, and Mr. R. R. Baddam for technical assistance.

1293

LITERATURE CITED

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