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Endogenous cytokinins from developing ‘Shamouti’ orange fruits derived from leafy and leafless inflorescences
Scientia Horticulturae, 26 (1985) 35--41
35
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ENDOGENOUS CYTOKININS FROM DEVELOPING 'SHAMOUTI' ORANGE FRUITS DERIVED FROM LEAFY AND LEAFLESS INFLORESCENCES
TEKCHAND SAIDHA1, E.E. GOLDSCHMIDT and S.P. MONSELISE Hebrew University o f Jerusalem, Department of Horticulture, Rehovot, 76100 (Israel) (Accepted for publication 19 December 1984)
ABSTRACT
Saidha, T., Goldschmidt, E.E. and Monselise, S_P., 1985. Endogenous cytokinins from developing 'Shamouti' orange fruits derived from leafy and leafless inflorescences. Scientia Hortic., 26: 35--41. Purified extracts of developing fruits of 'Shamouti' orange, derived from leafy and leafless inflorescences, showed two paper chromatographic zones of cytokinin activity by the Amaranthus bioassay. The first zone corresponded to Rf 0.0--0.3 and the second to R~ 0.6--0.9. Sephadex LH-20 fractionation of the cytokinin extracts tentatively showed the occurrence of zeatin and zeatin-riboside. Irrespective of the origin of fruit, highest cytokinin levels were obtained at petal fall (Stage I) and markedly declined thereafter. Levels appeared to be relatively higher in fruit from the leafy than in fruit from the leafless inflorescence type. The involvement of cytokinins in fruit set and growth is discussed. Keywords: Citrus sinensis (L.) Osbeck; cytokinin; fruit set; zeatin; zeatin riboside.
INTRODUCTION
The spring flush of citrus produces a wide range of shoot types. In addition to purely vegetative or purely generative shoots, mixed forms occur. While fruiting is achieved in both leafy and leafless inflorescences, the degree of fruit set and fruit drop appears to vary depending upon the type of inflorescence from which the fruit originates. The element of leafiness plays a vital role in improving set over leafless types (Lenz and Cary, 1969), and in early fruit development by substantially contributing photosynthetic assimilates (Moss et al., 1972) and necessary native regulators (Goldschmidt and Monselise, 1972). Fruit development and drop are considered to be under hormonal control. Relationships between endogenous hormonal levels and different phases of growth and development of citrus fruit have been emphasized (Takahashi et al., 1975; Goldschmidt, 1976; Monselise, 1977). Initial increase in fruit size 1Present address: Institute for Photobiology, Brandeis University, Waltham, MA 02154, U.S.A. 0304-4238/85/$03.30
© 1985 Elsevier Science Publishers B.V.
36 in many species is related to a period of rapid cell division and cytokinin activity has been demonstrated in many fruits at this time (Letham, 1963; Blumenfeld and Gazit, 1970; Gazit and Blumenfeld, 1970; Chacko et al., 1977; Hopping et al., 1979). In citrus, the duration of this cell division period, which is associated with rapid peel growth, is limited to the first few weeks after petal fall (Bain, 1958; Goren and Monselise, 1965). Cytokinin activity has been detected in lemon seeds (Khalifah and Lewis, 1966) and in all flower organs, but petals were found to be a particularly rich source (E.E. Goldschmidt, unpublished data, 1976). Cytokinin occurrence was also reported in relation to the rough fruit condition of the 'Shamouti' orange (Erner et al., 1976). Herein we describe the changes in the cytokinin levels in developing 'Shamouti' fruit derived from leafy and leafless inflorescences. Tentative identification of cytokinins present is also reported. M A T E R I A L S AND M E T H O D S - - Fruits originating from leafy and leafless inflorescences were picked from trees of 'Shamouti' orange ( C i t r u s s i n e n s i s [L.] Osbeck) on four dates, viz. 15 and 27 April and 5 and 24 May 1981. These were designated as Stages I, II, III and IV, respectively. The first two stages correspond to petal fall and stylar abscission, respectively, the latter two relate to somewhat more advanced stages of fruit development. On each occasion, the fruits were taken to the laboratory under refrigerated conditions, and a known weight was fixed in liquid nitrogen and stored at -20°C. Fruits.
E x t r a c t i o n , p u r i f i c a t i o n a n d bioassay. - - Cytokinins in the fruit sample were
extracted and purified according to the procedure outlined by Van Staden (1976) with slight modifications. The ethanolic fruit extract was filtered, adjusted to pH 2.5 and passed through a Dowex 50W-X8 cation exchange resin (H + form, 200--400 mesh, column 1.2X8 cm) at a flow-rate of 15 ml h -~ . After preparatory column washing with 50 ml distilled water followed by 15 ml of 70% ethanol, the cytokinins were eluted from the column with 250 ml of 5N NH4OH. The ammonia was removed in an evaporator and the residue dissolved in 3 ml of 35% ethanol. Extract equivalent to 2 g fresh weight was strip-loaded on Whatman No. 1 chromatography paper and separated with iso-propanol:25% ammonium hydroxide:water (10:1:1 v/v) by descending chromatography. The dried chromatogram was divided into 10 equal Rf strips and these were assayed for cytokinin activity by the A r n a r a n t h u s bioassay (Biddington and Thomas, 1973). In order to obtain more information about the nature of cytokinins, 5 g of fruit material pooled from leafy and leafless inflorescences at Stage I, extracted and purified as above but omitting the paper chromatography step, were further fractionated on a Sephadex LH-20 column (2.1×60 cm) and eluted with 35% ethanol (Armstrong et al., 1969). Forty-ml fractions
37
were collected, dried in an evaporator at 30°C and assayed for cytokinin activity by the Amaranthus bioassay. All bioassays were run in duplicate and averages were calculated. RESULTS
Cytokinin activity was detected in the various developmental stages of fruit from leafy and leafless inflorescences. Fruit extracts (Fig. 1) showed 2 zones of activity; one, more polar, at Rf 0.0--0.3, the other, less polar, at Rf 0.6--0.9. Most of the activity of the fast-running zone cochromatographed with zeatin and zeatin-riboside. The presence and the tentative identification were confirmed by Sephadex LH-20 fractionation, wherein the zone where zeatin and zeatin-riboside overlap (Fig. 1) was separated and two biologically active peaks at the elution volume o f zeatin and its MIXED
GENERATIVE STAGE
0.04
Z._ER
r
z
0.03
ZR Z
10-4 10-5 10-6
0.02
10-7
"•O.O o i
O.OC 0.02
i0-9 N CONTROL
l.J 1-r
IO"7
'~ 0.0
8 r,1
Z
10. 9 CONTROL
~ o.oc
Z
~z 0.02
0.04 IO " r
F, 0 , 0 1
Z
b
0.03
gNgTROL
O.O0 0.02
ZR
N ~)
ITr
tO
z 0.02 "Iv -7
I.,J tJ
z
O.OI
iO-9 ;ONTROL
0.00
t,,J n,t,.I ~" 0
Rf VALUES
O.OI 0
; ELUTION VOLUME (ml)
Fig. 1. (Left). Cytokinin-like activity in developing 'Shamouti' orange fruits from mixed and generative types. The Dowex-purified extract was chromatographed with isopropanol: 25% ammonium hydroxide:water (10:1:1 v/v) and ~_myed for cytokinin activity by the Amaranthus b i o e ~ y . Zfzeatin; ZRfzeatin riboside. Fig. 2. (Right). Sephadex LH-20 fractionation of Dowex-purified fruit extract showing cytokinin-like activity at Stage I. Zfzeatin; ZRfzeatin riboside. Horizontal line denotes control.
38
riboside were observed (Fig. 2). The identity of other peaks, however, remains obscure. In order to express the total cytokinin activity detected from paper chromatograms to best advantage, zeatin equivalent concentrations of both active zones were calculated, pooled and plotted against time, on a semilogarithmic scale. The drop in concentrations is enormous; about 4 and 3.5 logarithmic units for fruits borne on leafy and leafless inflorescences, respectively. Highest concentrations were detected at Stage I, and markedly declined thereafter in both kinds of fruit (Fig. 3). The fresh weight of developing fruit, on the other hand, showed a consistent increase during this period, as expected (Fig. 4), which is depicted on a Cartesian scale; this is the early stage o f the smooth sigmoidal curve of citrus fruit growth (Bain, 1958; Goren and Monselise, 1965). Differences were observed in the cytokinin levels o f fruits from the different inflorescence types. The difference at the first stage is largest (&fold); at other stages it ranges between 2- and 5-fold only (Figs. 1 and 3). The increase in fresh weight of fruits during the different stages was also higher in leafy than in leafless inflorescences; the largest absolute difference measured occurred at Stage IV (Fig. 4). ~ 10 -~ !
I
I
I
T
~ 10-6
i
~ 10-7
LEAFY
~-10-s LEAFLESS
i
'-"
"/ /
I
I
I
•
l
II
Ill
IV
i0-9
ffl LIA
I l
I II
STAGES
I III
I IV
~
u_
S I
STAGES
Fig. 3. (Left). Cytokinin levels in 'Shamouti' orange fruits at different stages, on a semilogarithmic scale. Standard error o f control equals 2.5 x 10 -1° Mol zeatin equivalents. Fig. 4. (Right). Changes in fruit fresh weight o f 'Shamouti' orange fruits at different stages.
39
DISCUSSION Studies with applied cytokinins (Crane, 1965; Weaver et al., 1965; Letham, 1969) have shown that young developing fruits are very responsive to these hormones, which participate in the regulation of fruit set as well as in determining the ultimate fruit size. High cytokinin contents have also been detected in a number of fruits during the period of active cell division. Even in 'Shamouti', cytokinins have been found to be very active, together with gibberellins, at a young fruit stage (Erner et al., 1976). Their values for kinetin equivalents are in the same order of magnitude as ours. In our study, the high values found at the early stages strongly suggest the involvement of cytokinins in cell proliferation of the developing citrus fruit. On the basis of studies with other fruits, it may be predicted that cytokinin activity at early stages might also be associated with fruit set. Higher cytokinins levels found in fruits resulting from leafy rather than leafless inflorescences further suggest an active role for cytokinins in improving fruit set. Differences between fruit borne on different inflorescence types are very similar to those found between the peels of rough and smooth 'Shamouti' fruits (Erner et al., 1976) (about 18-fold). However, the role of cytokinins in the improved persistence of fruitlets borne on leafy inflorescences should not be overestimated. The differences between the two types, apart from Stage I, are not very large, and much more extraction and assaying work is necessary to make sure about the reproducibility of these data. Cytokinins are synthesized in roots, occur in the xylem sap of citrus (Saidha et ah, 1983), are translocated in the transpiration stream to leaves, and partially re~listributed to other organs, including fruit, causing them to function better as sinks for translocated metabolites (Kriedemann, 1968). Young leaves of the leafy inflorescence may be instrumental in supplying more native cytokinins to leafy than to leafless inflorescences, so that fruits borne on them may build up more cytokinins, as found in this study. It should, moreover, be noted that transpiration rates of young immature leaves, found at this stage, is greater than that of mature leaves (Erickson, 1968) having an efficient stomatal apparatus and a thick cuticular protection. In citrus the distribution of labelled assimilates from inflorescences leaves to the developing fruit indicate that these leaves provide sufficient photosynthates to support early fruit development, while fruit on leafless inflorescences must rely on older leaves. It has been concluded that leaves contribute to the better set of fruit on leafy inflorescences with their photosynthetic activity (Moss et al., 1972). On the other hand, high endogenous gibberellin-like substances were found in leafy inflorescences as compared to leafless types (Goldschmidt and Monselise, 1972), indicating the involvement of plant hormones in the improved set of these fruits. A way to conciliate native regulator and carbohydrate effects is to consider the sink capacity of fruitlets. Those which are provided with larger cytokinin and gibberellin activities are also capable of better nutrient mobili-
40 z a t i o n (Erner and Bravdo, 1983). A higher c y t o k i n i n activity o f fruits b o r n e on leafy inflorescences is h i n t e d in this w o r k , and m a y be o n e o f the causes o f b e t t e r persistence o f these fruits. ACKNOWLEDGEMENT
This work was supported by a grant from the United States--Israel, Binational Agriculture and Development Fund {BARD).
REFERENCES Armstrong, D.J., Burrows, W.J., Evans, P.K. and Skoog, F., 1969. Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun., 37: 451--456. Bain, J.M., 1958. Morphological, anatomical and physiological changes in the developing fruit of the 'Valencia' orange. Aust. J. Bot., 6: 1--24. Biddington, N.L. and Thomas, T.H., 1973. A modified A m a r a n t h u s betacyanin bioassay for the rapid determination of cytokinins in plant extracts. Planta, 111: 183--186. Blumenfeld, A. and Gazit, S., 1970. Cytokinin activity in avocado seeds during fruit development. Plant Physiol., 46: 331--333. Chacko, E.K., Saidha, T., Dote Swamy, R., Reddy, Y.N. and Kohli, R.R., 1977. Studies on cytokinins in fruits. I. Occurrence and levels of cytokinin-like substances in grape berries at different developmental stages. Vitis, 15: 221--226. Crane, J.C., 1965. The chemical induction of parthenocarpy in the 'Calymirna' fig and its physiological significance. Plant Physiol., 40: 606--610. Erickson, L.C., 1968. The general physiology of citrus. In: W. Reuther, L.D. Batchelor and Hd. Webber (Editors), The Citrus Industry, Vol. 2. University of California Division of Agricultural Sciences, Berkeley, pp. 86--126. Erner, Y. and Bravdo, B., 1983. The importance of inflorescence leaves in fruit setting of 'Shamouti' orange. Acta Hortic., 139: 107--112. Erner, Y., Goren, R. and Monselise, S-P., 1976. The rough fruit condition of the 'Shamouti' orange -- connection with the internal hormonal balance. J. Hortic. Sci., 51: 367--374. Gazit, Y. and Blumenfeld, A., 1970. Cytokinin and inhibitor activities in the avocado fruit mesocarp. Plant Physiol., 46: 334--336. Goldschmidt, E.E., 1976. Endogenous growth substances of citrus tissues. HortScience, 11: 95--99. Goldschmidt, E.E, and Monselise, S-P., 1972. Hormonal control of flowering in citrus trees and other woody perennials. In: D~I. Cart (Editor), Plant Growth Substances. Springer Verlag, Berlin, pp. 758--766. Goren, R. and Monselise, S.P., 1965. Interrelations of hesperidin, some other natural components and certain enzyme systems in developing 'Shamouti' orange fruits. J. Hortic. Sci., 40: 83--99. Hopping, M.E., Young, H. and Bukovac, M.J., 1979. Endogenous plant growth substances in developing fruit of Prunus cerasus L. VI. Cytokinins in relation to initial fruit development. J. Am. Soc. Hortic. Sci., 104: 47--52. Khalifah, R.A. and Lewis, L.N., 1966. Cytokinins in citrus: isolation of a cell division factor from lemon seeds. Nature (London), 212: 1472--1473. Kriedemann, P.E., 1968. An effect of kinetin on the translocation of labelled carbon photosynthates in citrus. Aust. J. Biol. Sci., 21: 569--571.
41
Lenz, F. and Cary, P., 1969. Relationships between the vegetative and reproductive growth in 'Washington' navel oranges as affected by nutrition. In: H.D. Chapman (Editor), Proc. First Int. Citrus Syrup., University of California, Riverside, 3:1625 --1633. Letham, D.S., 1963. Regulators o f cell division in plant tissues. I. Inhibitors and stimulants of cell division in developing fruits: their properties and activities in relation to the ceil division period. N. Z. J. Bot., 1 : 336--350. Letham, D.S., 1969. Regulators o f cell divisions in plant tissues. IV. The effect o f zeatin and other stimulants o f cell divisions on apple fruit development. N. Z. J. Agric. Res., 12: 1--20. Monselise, S-P., 1977. Citrus fruit development: endogenous systems and external regulation. Proc. Int. Soc. Citriculture, 1977, pp. 664--668. Moss, G.I., Steer, B.T. and Kriedemann, P.E., 1972. The regulatory role of inflorescence leaves in fruit setting by sweet orange (Citrus sinensis). Physiol. Plant., 27: 432--438. Saidha, T., Goldschmidt, E.E. and Monselise, S~P., 1983. Endogenous growth regulators in tracheal sap of citrus. HortScience, 18: 231--232. Takahashi, N., Yamaguchi, I., Kono, T., Igoshi, M., Hirose, K. and Suzuki, K., 1975. Characterization of plant growth substances in Citrus unshiu and their change in fruit development. Plant Cell Physiol., 16: 1101--1111. Van Staden, J., 1976. Seasonal changes in the cytokinin content o f Ginkgo biloba leaves. Physiol. Plant., 38: 1--5. Weaver, R.J., Van Overbeek, J. and Pool, R.M., 1965. Induction o f fruit set in Vitis vinifera L. by a kinin. Nature (London), 206: 952--953.
35
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ENDOGENOUS CYTOKININS FROM DEVELOPING 'SHAMOUTI' ORANGE FRUITS DERIVED FROM LEAFY AND LEAFLESS INFLORESCENCES
TEKCHAND SAIDHA1, E.E. GOLDSCHMIDT and S.P. MONSELISE Hebrew University o f Jerusalem, Department of Horticulture, Rehovot, 76100 (Israel) (Accepted for publication 19 December 1984)
ABSTRACT
Saidha, T., Goldschmidt, E.E. and Monselise, S_P., 1985. Endogenous cytokinins from developing 'Shamouti' orange fruits derived from leafy and leafless inflorescences. Scientia Hortic., 26: 35--41. Purified extracts of developing fruits of 'Shamouti' orange, derived from leafy and leafless inflorescences, showed two paper chromatographic zones of cytokinin activity by the Amaranthus bioassay. The first zone corresponded to Rf 0.0--0.3 and the second to R~ 0.6--0.9. Sephadex LH-20 fractionation of the cytokinin extracts tentatively showed the occurrence of zeatin and zeatin-riboside. Irrespective of the origin of fruit, highest cytokinin levels were obtained at petal fall (Stage I) and markedly declined thereafter. Levels appeared to be relatively higher in fruit from the leafy than in fruit from the leafless inflorescence type. The involvement of cytokinins in fruit set and growth is discussed. Keywords: Citrus sinensis (L.) Osbeck; cytokinin; fruit set; zeatin; zeatin riboside.
INTRODUCTION
The spring flush of citrus produces a wide range of shoot types. In addition to purely vegetative or purely generative shoots, mixed forms occur. While fruiting is achieved in both leafy and leafless inflorescences, the degree of fruit set and fruit drop appears to vary depending upon the type of inflorescence from which the fruit originates. The element of leafiness plays a vital role in improving set over leafless types (Lenz and Cary, 1969), and in early fruit development by substantially contributing photosynthetic assimilates (Moss et al., 1972) and necessary native regulators (Goldschmidt and Monselise, 1972). Fruit development and drop are considered to be under hormonal control. Relationships between endogenous hormonal levels and different phases of growth and development of citrus fruit have been emphasized (Takahashi et al., 1975; Goldschmidt, 1976; Monselise, 1977). Initial increase in fruit size 1Present address: Institute for Photobiology, Brandeis University, Waltham, MA 02154, U.S.A. 0304-4238/85/$03.30
© 1985 Elsevier Science Publishers B.V.
36 in many species is related to a period of rapid cell division and cytokinin activity has been demonstrated in many fruits at this time (Letham, 1963; Blumenfeld and Gazit, 1970; Gazit and Blumenfeld, 1970; Chacko et al., 1977; Hopping et al., 1979). In citrus, the duration of this cell division period, which is associated with rapid peel growth, is limited to the first few weeks after petal fall (Bain, 1958; Goren and Monselise, 1965). Cytokinin activity has been detected in lemon seeds (Khalifah and Lewis, 1966) and in all flower organs, but petals were found to be a particularly rich source (E.E. Goldschmidt, unpublished data, 1976). Cytokinin occurrence was also reported in relation to the rough fruit condition of the 'Shamouti' orange (Erner et al., 1976). Herein we describe the changes in the cytokinin levels in developing 'Shamouti' fruit derived from leafy and leafless inflorescences. Tentative identification of cytokinins present is also reported. M A T E R I A L S AND M E T H O D S - - Fruits originating from leafy and leafless inflorescences were picked from trees of 'Shamouti' orange ( C i t r u s s i n e n s i s [L.] Osbeck) on four dates, viz. 15 and 27 April and 5 and 24 May 1981. These were designated as Stages I, II, III and IV, respectively. The first two stages correspond to petal fall and stylar abscission, respectively, the latter two relate to somewhat more advanced stages of fruit development. On each occasion, the fruits were taken to the laboratory under refrigerated conditions, and a known weight was fixed in liquid nitrogen and stored at -20°C. Fruits.
E x t r a c t i o n , p u r i f i c a t i o n a n d bioassay. - - Cytokinins in the fruit sample were
extracted and purified according to the procedure outlined by Van Staden (1976) with slight modifications. The ethanolic fruit extract was filtered, adjusted to pH 2.5 and passed through a Dowex 50W-X8 cation exchange resin (H + form, 200--400 mesh, column 1.2X8 cm) at a flow-rate of 15 ml h -~ . After preparatory column washing with 50 ml distilled water followed by 15 ml of 70% ethanol, the cytokinins were eluted from the column with 250 ml of 5N NH4OH. The ammonia was removed in an evaporator and the residue dissolved in 3 ml of 35% ethanol. Extract equivalent to 2 g fresh weight was strip-loaded on Whatman No. 1 chromatography paper and separated with iso-propanol:25% ammonium hydroxide:water (10:1:1 v/v) by descending chromatography. The dried chromatogram was divided into 10 equal Rf strips and these were assayed for cytokinin activity by the A r n a r a n t h u s bioassay (Biddington and Thomas, 1973). In order to obtain more information about the nature of cytokinins, 5 g of fruit material pooled from leafy and leafless inflorescences at Stage I, extracted and purified as above but omitting the paper chromatography step, were further fractionated on a Sephadex LH-20 column (2.1×60 cm) and eluted with 35% ethanol (Armstrong et al., 1969). Forty-ml fractions
37
were collected, dried in an evaporator at 30°C and assayed for cytokinin activity by the Amaranthus bioassay. All bioassays were run in duplicate and averages were calculated. RESULTS
Cytokinin activity was detected in the various developmental stages of fruit from leafy and leafless inflorescences. Fruit extracts (Fig. 1) showed 2 zones of activity; one, more polar, at Rf 0.0--0.3, the other, less polar, at Rf 0.6--0.9. Most of the activity of the fast-running zone cochromatographed with zeatin and zeatin-riboside. The presence and the tentative identification were confirmed by Sephadex LH-20 fractionation, wherein the zone where zeatin and zeatin-riboside overlap (Fig. 1) was separated and two biologically active peaks at the elution volume o f zeatin and its MIXED
GENERATIVE STAGE
0.04
Z._ER
r
z
0.03
ZR Z
10-4 10-5 10-6
0.02
10-7
"•O.O o i
O.OC 0.02
i0-9 N CONTROL
l.J 1-r
IO"7
'~ 0.0
8 r,1
Z
10. 9 CONTROL
~ o.oc
Z
~z 0.02
0.04 IO " r
F, 0 , 0 1
Z
b
0.03
gNgTROL
O.O0 0.02
ZR
N ~)
ITr
tO
z 0.02 "Iv -7
I.,J tJ
z
O.OI
iO-9 ;ONTROL
0.00
t,,J n,t,.I ~" 0
Rf VALUES
O.OI 0
; ELUTION VOLUME (ml)
Fig. 1. (Left). Cytokinin-like activity in developing 'Shamouti' orange fruits from mixed and generative types. The Dowex-purified extract was chromatographed with isopropanol: 25% ammonium hydroxide:water (10:1:1 v/v) and ~_myed for cytokinin activity by the Amaranthus b i o e ~ y . Zfzeatin; ZRfzeatin riboside. Fig. 2. (Right). Sephadex LH-20 fractionation of Dowex-purified fruit extract showing cytokinin-like activity at Stage I. Zfzeatin; ZRfzeatin riboside. Horizontal line denotes control.
38
riboside were observed (Fig. 2). The identity of other peaks, however, remains obscure. In order to express the total cytokinin activity detected from paper chromatograms to best advantage, zeatin equivalent concentrations of both active zones were calculated, pooled and plotted against time, on a semilogarithmic scale. The drop in concentrations is enormous; about 4 and 3.5 logarithmic units for fruits borne on leafy and leafless inflorescences, respectively. Highest concentrations were detected at Stage I, and markedly declined thereafter in both kinds of fruit (Fig. 3). The fresh weight of developing fruit, on the other hand, showed a consistent increase during this period, as expected (Fig. 4), which is depicted on a Cartesian scale; this is the early stage o f the smooth sigmoidal curve of citrus fruit growth (Bain, 1958; Goren and Monselise, 1965). Differences were observed in the cytokinin levels o f fruits from the different inflorescence types. The difference at the first stage is largest (&fold); at other stages it ranges between 2- and 5-fold only (Figs. 1 and 3). The increase in fresh weight of fruits during the different stages was also higher in leafy than in leafless inflorescences; the largest absolute difference measured occurred at Stage IV (Fig. 4). ~ 10 -~ !
I
I
I
T
~ 10-6
i
~ 10-7
LEAFY
~-10-s LEAFLESS
i
'-"
"/ /
I
I
I
•
l
II
Ill
IV
i0-9
ffl LIA
I l
I II
STAGES
I III
I IV
~
u_
S I
STAGES
Fig. 3. (Left). Cytokinin levels in 'Shamouti' orange fruits at different stages, on a semilogarithmic scale. Standard error o f control equals 2.5 x 10 -1° Mol zeatin equivalents. Fig. 4. (Right). Changes in fruit fresh weight o f 'Shamouti' orange fruits at different stages.
39
DISCUSSION Studies with applied cytokinins (Crane, 1965; Weaver et al., 1965; Letham, 1969) have shown that young developing fruits are very responsive to these hormones, which participate in the regulation of fruit set as well as in determining the ultimate fruit size. High cytokinin contents have also been detected in a number of fruits during the period of active cell division. Even in 'Shamouti', cytokinins have been found to be very active, together with gibberellins, at a young fruit stage (Erner et al., 1976). Their values for kinetin equivalents are in the same order of magnitude as ours. In our study, the high values found at the early stages strongly suggest the involvement of cytokinins in cell proliferation of the developing citrus fruit. On the basis of studies with other fruits, it may be predicted that cytokinin activity at early stages might also be associated with fruit set. Higher cytokinins levels found in fruits resulting from leafy rather than leafless inflorescences further suggest an active role for cytokinins in improving fruit set. Differences between fruit borne on different inflorescence types are very similar to those found between the peels of rough and smooth 'Shamouti' fruits (Erner et al., 1976) (about 18-fold). However, the role of cytokinins in the improved persistence of fruitlets borne on leafy inflorescences should not be overestimated. The differences between the two types, apart from Stage I, are not very large, and much more extraction and assaying work is necessary to make sure about the reproducibility of these data. Cytokinins are synthesized in roots, occur in the xylem sap of citrus (Saidha et ah, 1983), are translocated in the transpiration stream to leaves, and partially re~listributed to other organs, including fruit, causing them to function better as sinks for translocated metabolites (Kriedemann, 1968). Young leaves of the leafy inflorescence may be instrumental in supplying more native cytokinins to leafy than to leafless inflorescences, so that fruits borne on them may build up more cytokinins, as found in this study. It should, moreover, be noted that transpiration rates of young immature leaves, found at this stage, is greater than that of mature leaves (Erickson, 1968) having an efficient stomatal apparatus and a thick cuticular protection. In citrus the distribution of labelled assimilates from inflorescences leaves to the developing fruit indicate that these leaves provide sufficient photosynthates to support early fruit development, while fruit on leafless inflorescences must rely on older leaves. It has been concluded that leaves contribute to the better set of fruit on leafy inflorescences with their photosynthetic activity (Moss et al., 1972). On the other hand, high endogenous gibberellin-like substances were found in leafy inflorescences as compared to leafless types (Goldschmidt and Monselise, 1972), indicating the involvement of plant hormones in the improved set of these fruits. A way to conciliate native regulator and carbohydrate effects is to consider the sink capacity of fruitlets. Those which are provided with larger cytokinin and gibberellin activities are also capable of better nutrient mobili-
40 z a t i o n (Erner and Bravdo, 1983). A higher c y t o k i n i n activity o f fruits b o r n e on leafy inflorescences is h i n t e d in this w o r k , and m a y be o n e o f the causes o f b e t t e r persistence o f these fruits. ACKNOWLEDGEMENT
This work was supported by a grant from the United States--Israel, Binational Agriculture and Development Fund {BARD).
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