Plant Physiol.

(1970) 46, 531-534

Inhibitors from Carob (Ceratonia siliqua L.)

II. EFFECT ON GROWTH INDUCED BY INDOLEACETIC ACID OR GIBBERELLINS A1, A4, A5, AND A7 t

Received for publication March 20, 1970

MARY RITZEL CORCORAN

Department of Biology, San Fernando Valley State College, Northridge, California 91324

ABSTRACT by charcoal adsorption. Inhibitor C was then removed by differ-

ential extraction from aqueous solution into ether, and inhibitor
Two inhibitory fractions (B1 and C) from extracts of im- B1 was removed by subsequent extraction from the aqueous solu-
mature fruit of carob were tested for their ability to inhibit tion into ethyl acetate. Inhibitors B1 and C are extractable to
the action of indoleacetic acid (IAA) in three bioassays. various degrees into diethyl ether from aqueous solutions at
There was no reduction of IAA-induced reactions in the acid pH values, indicating that they may be weak acids (6).
Avena curvature test, abscission of debladed coleus petioles, Concentrations of inhibitory whole extract and fractions B1
or growth of cucumber hypocotyls. The highest ratio of or C are given as the amount of extract obtained from a specified
inhibitor to IAA was 10,000 times greater than the ratio fresh weight of carob fruit. This figure is determined either for a
necessary to inhibit by 50% the growth caused by an equiva- total volume or for the volume given to each assay plant.
lent amount of gibberellin A3 in pea seedlings. At the high- The amount of fraction B1 or C used with a given amount of
est concentration used, fraction C alone caused curvature IAA is related to the activity of these inhibitors with gibberellic
of Avena coleoptiles. The inhibitory fractions appeared acid. In the dwarf pea bioassay, fraction B1 or C extracted from
to enhance the effect of IAA in the cucumber test. 5 mg fresh weight of carob fruit will inhibit by 50% the growth
Concentrated whole extract and fractions B, and C were induced by 0.05 ,ug of GA3 (5). The ratio of extract to IAA is
tested for reduction of growth caused by gibberellins A1, compared to this ratio in peas.
A4, As, A7, and a neutral gibberellin-like substance from Avena Curvature. The assay was used essentially as described
beans in the dwarf-5 maize bioassay. Each gibberellin was by Went and Thimann (23). Curvature was allowed to develop
inhibited and required the same amount of inhibitor for for 90 min after application of the agar block. Phosphate buffer
a 50% reduction of the induced growth. The inhibiting at a pH of 5.9 was used in preparing the blocks. IAA was used
effect could be completely overcome by increasing the at a concentration of 50 or 100 ,Ag/liter with or without the addi-
amount of gibberellin while maintaining the same con- tion of fractions B1 or C.
centration of inhibitor. Fractions B1 and C were also tested Coleus Petiole Abscission. The assay is similar to that used
with gibberellins As and A4 in the cucumber hypocotyl earlier (15, 24). Eight-week-old coleus plants, Coleus blumei
test. Both inhibitory fractions reduced growth but were Benth., which had been derived from cuttings were used. Each
more effective against gibberellin A3 than gibberellin A4 plant had from two to four branches, and the first full size leaf
in the assay. The ability to reduce gibberellin-induced below the apex of each branch was used. Lanolin (0.2 ml) was
growth and not reduce IAA-induced growth indicates that added into half of a gelatin capsule. The leaf was debladed, and
the inhibitors from carob have a greater specificity of action the half-capsule was introduced so that the petiole passed through
than that previously reported for any inhibitor. the open end and the cut petiole surface was imbedded in the
lanolin. Indoleacetic acid (0.1 Mug per plant) or inhibitory frac-
tion B1 or C was added to the lanolin either separately or in
combinations of IAA and inhibitor.
Cucumber Hypocotyl Elongation. The assay was adopted from
Katsumi et al. (11). Seedlings of cucumber, Cucumis sativus L.
cv. National Pickling, were used. About 100 seeds were soaked
Growth inhibitors are usually nonspecific in action and will 2 to 3 hr in distilled water and then planted in a flat, containing a
block responses induced by any of several growth promotors. 1:1 mixture of soil and vermiculite. The flats were placed in a
Coumarin, naringenin, and abscisic acid have been shown to growth chamber which provided a 12-hr light period at 28 C
inhibit responses due to indoleacetic acid, gibberellic acid (GA3), and a 12-hr dark period at 22 C. The seedlings were used 6 days
and in some cases cytokinins (2, 7, 10, 17, 25). Extracts from after planting when the hypocotyls were 25 to 30 mm long.
carob will inhibit the growth induced by GA3 in pea and maize Uniform seedlings were marked with India ink 20 mm below the
seedlings (5, 6). The present paper reports on tests with IAA and cotyledonary node. The test solution (0.01 ml) in 95% alcohol
with gibberellins other than GA3. was added to the apical bud of each seedling with a 0.01-ml
pipette. The length of the marked hypocotyl unit was measured 3
MATERIALS AND METHODS days later.
Carob Extract. Concentrated whole extract and the partially Iaize Assay. Seedlings of maize, Zea mays L., dwarf-5 mutant
purified fractions B1 and C were purified as described previously (d5), were used as previously described (19, 20).
(4, 5). These fractions were separated from the other components Gibberellins. Gibberellins Al, A4, A5, and A7 were supplied by
P. W. Brian of Cambridge University. The "neutral gibberellin-
1 This work was supported in part by National Science Foundation like substance" was provided by L. Rappaport of the University
Grant GB-971. of California, Davis. It was obtained from the Kentucky Wonder
531
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Copyright © 1970 American Society of Plant Biologists. All rights reserved.
532 CORCORAN Plant Physiol. Vol. 46, 1970
Table I. Effect of Fractionzs B, and C on Avena Curvature COLEUS PETIOLE ABSCISSION
Two concentrations of fraction B, and three concentrations of
fraction C were each mixed with IAA in phosphate buffer, pH 5.9.
Curvature was developed for 90 min.

IAA Fraction Bi Fraction C Curvature'

ug Iliter extract from g fresh wI/liter deg z

0
O 0 0 0 Uf)
U)
50 0 0 14.0 1.3 o- lanolin
100 0 0 25.1 1.7 m *- IAA(O.Ipg/plant)
x- fraction BI (extract
50
50
1,000
10,000
0
0
15.7
16.6
1.4
1.2
r~ from lOg FW/plant)
6- IAA + B1
0 0 0 0
50 0 0 14.5 1.5
100 0 0 27.3 2.2
50 0 1,000 12.4 1.7 2 3 4 5 6
50 0 10,000 15.9 1.0
50 0 100,000 26.1 -- 2.5 DAYS AFTER TREATMENT
0 0 1,000 O FIG. 1. Abscission of debladed coleus petioles. Half a gelatin capsule
0 0 100,000 13.6 + 1.1 containing 0.2 ml of lanolin was applied to each petiole stump. The
lanolin was used alone or with the addition of IAA, fraction B,, or a
1 Average and standard error of 12 plants. combination of IAA and fraction B,. Each point represents the average
from 10 plants.
variety of bean seed by the method used to obtain a neutral
fraction from potato tubers (8). CUCUMBER HYPOCOTYL

RESULTS
Effect of Inhibitors on Avena Curvature. Two concentrations of
fraction B1 and three concentrations of fraction C were each E
mixed with 50 ,ug of IAA and assayed in the Avena curvature test.
Fraction B1 did not reduce the curvature induced by IAA alone 41
a
(Table I). At the highest concentration the ratio of B1 to IAA was z
over 1000 times greater than the ratio needed to reduce by 50%
the growth caused by an equivalent amount of GA3 in pea seed- x
fr
lings. Fraction C also did not reduce the curvature induced by
IAA (Table I). The combination of IAA and the highest concen- 0
tration of fraction C resulted in a curvature that was greater than
that induced by IAA alone. The highest concentration of frac-
tion C alone gave a curvature equivalent to that caused by 50 IAA(pg/plant) 0 I I I I I l 0 1

jig of IAA. Whether this curvature was due to the inhibitory INHIBITOR o 0.1 D01 .001
I 0 0 1 .1 .01 .001
component or to an auxin contaminant has not been resolved. (extract from
g FW/plant) FRACTION SI FRACTION C
At the highest concentration the ratio of fraction C to IAA was
over 10,000 times greater than the ratio needed to reduce by 50 %0 FIG. 2. Interaction of fractions B, or C with IAA in hypocotyl
the growth induced by an equivalent amount of GA3 in pea growth of cucumber. The growing tip of each seedling received 0.01 ml
seedlings. of ethanol alone or combined with IAA or with mixtures of IAA and
Effect of Inhibitors on Coleus Petiole Abscission. Fractions B1 different concentrations of fractions B, or C. Each point represents the
and C were applied both separately and in combination with average and standard error from 10 plants.
IAA to debladed coleus petioles. The addition of IAA alone re-
tarded abscission by 2 days (Fig. 1). Fractions B1 or C added amounts of whole extract and assayed. The extract clearly in-
with IAA had no effect on this retardation. Fractions B1 or C hibited the growth induced by each of the gibberellins (Table
alone had no effect on abscission. The ratio of inhibitor to IAA II). With each gibberellin the extract from 5 mg (fresh weight)
was 1000 times more than the ratio necessary to reduce by 50% of carob fruit was able to reduce this growth by 50%. The neu-
the growth induced by an equivalent amount of GA3 in peas. tral gibberellin-like substance was also used as a growth pro-
Effect of Inhibitors on IAA-induced Growth of Cucumber. moter in combination with the whole extract from carob. The
Four concentrations each of fractions B, and C were applied to growth induced by this gibberellin-like substance was also in-
cucumber seedlings. None of them affected hypocotyl growth. hibited by the carob extract (Table II).
The same concentrations were mixed with IAA and assayed. Similar experiments were performed in which gibberellins
Fraction B1 enhanced the IAA response at all concentrations A1, A4, A5, and A7 (0.1 ,ug per plant) were mixed with decreasing
used (Fig. 2). Fraction C also showed indications of enhancing amounts of fractions B1 or C. These fractions also reduced the
the IAA response. There was no evidence of inhibition of IAA- growth caused by each of the gibberellins. With both fractions
induced growth. At the highest concentration the ratio of frac- B1 and C, the extract from 50 mg (fresh weight) of carob fruit was
tions B1 or C to IAA was 10 times greater than the amount the lowest amount able to reduce the gibberellin-induced growth
needed to reduce by 50% the growth induced by GA3 in the same by about 50%.
system. Reversibility of Inhibition. Increasing amounts of gibberellins
Effect of Inhibitors on Growth of Maize. Constant amounts of were added to constant amounts of inhibitor in order to test the
gibberellins A1, A4, A5, and A7 were mixed with decreasing reversibility of the inhibitor effect. The whole extract was used

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Copyright © 1970 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 46, 1970 INHIBIrORS FROM CAROB. II 533
Table II. Inhibition of Gibberellin-induced Growth 80r
by Whole Extract
E
Seedlings treated with gibberellins or a combination of gibberel- E
lin and whole extract from carob. CONTROL
60
CD
hJ
J
z
Gibberellin w
Kind____________ __
C Whole Extract
Concn
Leaf Sheath
Length'
Inhibition -J
Kind Concn z
40
extract from % reduction of w
jug Iplant mg fresh mm gibberellin- in-
wt /plant duced growth
A1 0 0 23.1 i0.8 w WHOLE EXTRACT
0.1 0 42.3 :2.2 -J 20
0.1 50 27.5 ± 1.5 77
0.1 5 29.9 ± 1.4 65
0.1 0.5 40.9 :4 2.0 8
A4 0 0 24.4 ± 0.9 0 0.05 0.5 5 50
0.1 0 55.7 ± 2.7 GIBBERELLIN Al (pg/plant)
0.1 50 34.1 i 1.5 69
0.1 5 36.6 1.8 61 FIG. 3. The effect of gibberellin A1 on the growth of maize seedlings
± 2.4 in the presence and absence of whole extract. Each seedling treated with
0.1 0.5 55.0 2 inhibitor received the extract from 5 mg fresh weight of carob fruit.
Each point represents the average and standard error of 10 plants.
A5 0 0 19.6 0.7
0.1 0 32.4 ± 1.2
0.1 50 20.7 1.0 99 50s
0.1 5 23.8 ± 0.9 69 CUCUMBER HYPOCOTYL
0.1 0.5 31.1 i 1.4 11 IE 40

nn l p
A7 0 0 21.1 ± 0.9 0
4 30
0.1 0 54.3 3.7 a
iii
34.1 ± 1.2
0.1
0.1
50
5 39.7 ± 3.9
61
45 z
20 i'
;i
z
on
0.1 0.5 50.8 2.6 11 H
Ii

0
'I
l0o
Neutral gibberellin 0 0 21.1 ± 0.5 0:
±
from Kentucky
Wonder beans2
10
10
0
5
29.3
23.3 ±
1.0
0.8 73 I I I I _1
I
__
I
Ii0 .01
GA (pg/plant) 0 1 I .01 .01 .01 .01 .01
10 0.5 27.6 ± 1.3 21 INHIBITOR 0 0 I .1 .01 .001 0 0 I .1 .01 .001 .0001
(extract from
l Average and standard error of 10 plants. Assays on dwarf-5 g F W/plant) GIBBERELLIN A3 GIBBERELLIN A4
maize seedlings. Each seedling received a single application of 0.1
ml. Measurements were made 7 days after treatment. FIG. 4. Interaction of fraction C with GA3 and GA4 in hypocotyl
2 Concentration given as extract from g fresh wt/plant. growth of cucumber. The growing tip of each seedling received 0.01 ml
of ethanol alone or combined with either gibberellin or a mixture of
with gibberellin A1 (Fig. 3). The amount of inhibitor was suffi- gibberellin and fraction C. Each point represents the average and stand-
ard error of 10 plants.
cient to reduce strongly the growth induced by the lower concen-
trations of gibberellin. The inhibition was completely reversed
at the highest concentration of gibberellin. Similar results were and maize, which have been the most frequently used for the
obtained using whole extract with gibberellin A5 and fraction carob inhibitors. The fact that the inhibitors are so very different
B1 with gibberellin A4 or A5. Other combinations were not tested. in their effects against IAA and gibberellins in tests which have
Effect of Inhibitors on Gibberellin-induced Growth of Cucum- some similarity and which in the case of cucumber hypocotyl
ber. Two gibberellins, A3 and A4, were used alone and with de- growth are identical indicates that the substances probably act
creasing amounts of fraction C. In this assay GA3 was about 1 % quite differently with the two kinds of growth promotors.
as active as GA4 (Fig. 4). Fraction C inhibited both gibberellins Most plant growth inhibitors have been considered only in
but appeared to be relatively more effective with GA3 than with relation to auxin-induced phenomena (9). Those few which have
GA4. A test in which gibberellins A3 and A4 were assayed with been tested with other promotors have been found to be inhibi-
decreasing amounts of fraction B1 gave similar results. tory. These are discussed by Leopold (12) and by Addicott and
Lyon (1). The inhibitory system from carob extract appears to
DISCUSSION differ from other reported inhibitory substances in its specificity.
The enhancement of IAA-induced growth of cucumber by the
The auxin tests used here show a range in specificity. Avena carob inhibitors, especially fraction B1, is reminiscent of the
curvature is sensitive only to translocatable auxins such as IAA promotive effects of low concentrations of phenolic inhibitors
while the cucumber hypocotyl test gives a similar response to with IAA (16, 22). The phenolic substances become inhibitory
both IAA and gibberellins. These assays were selected because in at higher concentrations. Specific activities cannot be compared
each case a whole seedling or plant was involved, thus making between carob inhibitors and the phenolics because of the lack of
the assays more comparable to the shoot growth assays of peas information on the identity of the carob inhibitors.

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Copyright © 1970 American Society of Plant Biologists. All rights reserved.
534 CORCORAN Plant Physiol. Vol. 46, 1970
The inhibitors from carob prevented growth induced by all 2. ASPINALL, D., L. G. PALEG, AND F. T. ADDIco-r. 1967. Abscisin II and some hor-
four of the gibberellins tested and by the one neutral gibberellin- mone-regulated plant responses. Aust. J. Biol. Sci. 20: 869-882.
3. BUKOVAC, M. J. AND S. H. WIrTWER. 1961. Biological evaluation of gibberellins Al,
like substance. These results indicate that the inhibitors may have A2, A3, and A4 and some of their derivatives. In: R. M. Klein, ed., Plant Growth
a broad spectrum of activity among the gibberellins. The in- Regulation. Iowa State University Press, Ames. pp. 505-520.
hibition of gibberellin-induced growth in maize is shown here 4 CORCORAN, M. R. 1966. Reduction of a-amylase by an inhibitor from carob. Plant
to be overcome by adding more gibberellin. Similar results have Physiol. 41: 1265-1267.
S CORCORAN, M. R. AND C. A. WEST. 1968. Inhibitors from carob (Ceratonia siliqua
also been reported with peas (5). Such results are consistent with L.) I. Nature of the interaction with gibberellic acid on shoot growth. Plant
the interpretation that the inhibitors are involved in a gibberellin Physiol. 43: 859-864.
mechanism. 6. CORCORAN, M. R., C. A. WEST, AND B. 0. PHINNEY. 1961. Natural inhibitors of
There are marked differences in the activities of various gib- gibberellin-induced growth. In: R. F. Gould, ed., Gibberellins. Advan. Chem.
Ser. 28: 152-158.
berellins in bioassays. Gibberellins A4, A7, and Ag are especially 7. GANTZER, E. 1960. Wirkungen von Cumarin auf Wachstum und Entwicklung-
active in stimulating growth of cucurbits (3, 13). Gibberellin svorgange und seine Wanderungs fahigkeit im Pflanzengewebe. Panta 55: 235-
A5 is only 15% as active as A3 in the dwarf-I maize assay (19). 253.
Just as different gibberellins cause different amounts of response, 8. HAYASHI, F. AND L. RAPPAPORT. 1966. Isolation, crystallization, and nartial identifi-
cation of potato factor II from potato tubers. Plant Physiol. 41: 53-58.
it might be expected that inhibitors of gibberellin-induced growth 9. HEMBERG, T. 1961. Biogenous inhibitors. In: W. Ruhland, ed., Encyclopedia of
might vary in their activity with different gibberellins, and this Plant Physiology, Vol. 14. Springer, Berlin. pp. 1162-1183.
seems to be the case here. Thus, it is interesting that the inhibitors 10. KATO, J. 1958. Studies on the physiological effect of gibberellin II. On the interaction
are much more active against GA3 than GA4 in the cucumber of gibberellin with auxin and growth inhibitors. Physiol. Plant. 11: 10-15.
11. KATSUMI, M., B. 0. PHINNEY, AND W. K. PURVES. 1965. The roles of gibberellin and
hypocotyl assay. auxin in cucumber hypocotyl growth. Physiol. Plant. 18: 462-473.
In general, stem growth in intact plants is much more responsive 12. LEOPOLD, A. C. 1964. Plant Growth and Development. McGraw Hill Book Co.,
to gibberellins than to auxins (21). Mature leaves and stems do New York.
not respond to gibberellins; however, they still respond to tro- 13. LOCKHART, J. A. AND P. H. DEAL. 1960. Prevention of red light inhibition of stem
growth in the Cucurbitaceae by gibberellin A4. Naturwissenschaften 47: 141-142.
pisms mediated by endogenous auxin. The same type of differen- 14. MAYER, A. M. AND A. POuAKOFF-MAYBER. 1963. The Germination of Seeds.
tial response has been shown in serial sections of the first leaf of Pergamon Press, New York.
wheat (25). The basal meristematic and next higher sections re- 15. MYERS, R. M. 1940. Effect of growth substances on the abscission layer in leaves of
sponded to GA3 whereas the uppermost sections containing more Coleus. Bot. Gaz. 102: 323-338.
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Acknowledgments-Some of this work was done at the Research Institute for Plant monin and coumarin and its prevention by BAL. Proc. Nat. Acad. Sci. U. S. A.
Pests and Diseases in Teheran, Iran. The author would like to thank the Institute for its 35: 272-276.
generous provision of facilities and equipment and also thank Mrs. Ardehaly, who 23. WENT, F. W. AND K. V. THIMANN. 1937. Phytohormones. The Macmillan Co., New
assisted in the work. York.
24. WETMORE, R. H. AND W. P. JACOBS. 1953. Studies on abscission: The inhibiting
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stances. Annu. Rev. Plant Physiol. 20: 139-164. Growth Substances. Runge Press, Ottawa. pp. 521-542.

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