PHYSIOLOGIA PLANTAKtfM 79: 4O4MO6.

Copenhagen l » 0

Hormonal changes associated with fruit set and development in

mandarins differing In their parthenocarpic ability
Manuel Talon, Lorenzo Zacarias and Eduardo Primo-Millo

Talon, M., Zacadas, L. & Primo-Millo, E. 1990. Hormonal changes associated with
fruit set and development in mandarins diffedng in their parthenocarpic ability. —
Physiol. Plant. 79: 400-^06.

Satsuma [Citrus unshiu (Mak) Marc] and Clementine [Citrus reticulata (Hort.) Ex.
Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in
their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocar-
picaUy whereas Clementine mandadns show very low ability to set frait in the
absence of cross-polUnation. The endogenous levels of gibberellins (GAs) and free
and conjugated indole-acetic add (lAA) and abscisic acid (ABA) throughout early
stages of fruit development were investigated in seedless cultivars of both species.
Analyses performed by full-scan combined gas chromatography-mass spectrometry
(GC-MS) of extracts from ovaries at anthesis demonstrated the presence of GA,,,
GA20, GA29, GAi, GA^, GA3 and iso-GA3 in Satsuma mandarin, whereas only GA^^,
GA3 and trace levels oif GAj were detected in Clementine. At this developmental
stage GA-like substances, as estimated by bioassay, reached their highest levels in
Satsuma, while Clementine mandadns contained relatively lower levels. In both
species the highest levels of free IAA were found at petal-fall stage at which time free
A B A levels also peaked. Developing fruits of Clementine had higher amounts of
both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low
throughout reproductive development whereas in Clementine they increased as the
free form declined. In contrast, conjugated ABA was at low levels in Clementine but
reached higher concentrations in Satsuma. These results suggest that in these manda-
rins the potential for setting parthenocarpic fruits is mainly influenced by the hor-
monal status of the fruit dudng the later stages of cell division and early stages of cell
enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to
be associated with lower levels of active GAs, lower capability to catabolize ABA to
conjugated ABA and higher ability to conjugate IAA dudng this period.

Key words - Absdsic add. Citrus reticulata, Citrus unshiu, fruit set and development,
gibberellins, indoleacetic acid, mandarin, parthenocarpy.

M. Talon et al., Dept of Citriculture, Instituto Valenciano de Investigaciones Agrarias,

46113 Moncada, Valencia, Spain. Present address: M. Talon (corresponding author),
MSU-DOE Plant Res. Lab., Mich. State Univ., East Lansing, MI 48824, USA; L.
Zacarias, Environ. Hortic, Univ. of California, Davis, CA 95616, USA.

fruit development in seedless and seeded Clementine

mtroduction
The importance of plant hormones in reproductive de-
veloptnent is now well accepted (Goodwin 1978). At-
tempts have been made to study the relationship be-
tween endogenous plant hortnones and fruit develop-
ment in citrus (see Goldschmidt 1976 and references
therein). Thus, it has been suggested that the control of
mandarins is carried out through the hortnonal balance
between ABA and atixin-like substances and ABA and
GA-like substances, respectively (Garcia-Papi and Gar-
cia-Martinez 1984). Seedless Cletnentine mandarin cul-
tivars are self-incotrtpatible and iti the absence of cross-
pollination show a very low ability to set fruit. In these
varieties it has been showti that exogenous applications

Received 2 October, 1989; revised 23 February, 1990

400 Physiol. Plant. 79. W90

of GA3 increase fruit set (Soost and Burnett 1961).
IdeDtificatian and determination of GAs
Furthermore, localized GA3 treatment of pistils in citrus
flowers resulted in a stronger mobilization of metabo- Ten g of dry weight of ovaries at .anthesis from each
lites to young ovaries, which appears essential for fruit mandarin were homogenized in 100 ml cold 80% aque-
set and development (Powell and Krezdorn 1977). ous methanol (MeOH) and the homogenate was stirred
However, seedless citrus cuttivars which show high male overnight at 4°C. The extract was filtered and the resid-
sterility, such as Satsuma mandarins, also have a high ue re-extracted twice with 100% MeOH for 3 h at room
degree of natural parthenocarpy and a high fruiting temperature. The combined filtrates were reduced to
ratio. Typically, GA applications to these varieties have the aqueous phase, an equal volume of 0.1 M potassium
not been efficacious in increasing fruit set (Coggins and phosphate buffer at pH 8.2 was added and the pH was
Hield 1968). adjusted to 8.2. The aqueous phase was partitioned 3
The objective of the present investigation was to de- times against ethyl acetate (EtOAc) and the EtOAc
termine differences in the hormonal content of Satsuma fractions were discarded. The pH of the extract was
and Clementine mandarins throughout reproductive de- readjusted to 2.5 and the extract were partitioned 3
velopment and to study its relationship to the partheno- times against EtOAc. The EtOAc fractions were com-
carpic ability of these two species. We report the identi- bined, evaporated to dryness and purified on a polyvi-
fication of GAs by GC-MS in developing seedless fruits nylpolypyrrolidone (PVPP) column (Glenn et al. 1972).
of Satsuma and Clementine mandarins and in addition, The eluate was .acidified to pH 2.5 and partitioned 3
the estimation of the GA, IAA .and ABA levels during times against EtOAc. The EtOAc fractions were com-
the early phases of fruit growth. bined, evaporated to dryness and redissolved in 15 ml
of 0.1 M potassium phosphate buffer, pH 8.0. The pH
Abbreviations - ABA, abscisic acid; BHT, butylated hydroxy- was readjusted to 2.5 and the extract was passed
toiuene; DAA, days after anthesis; EtOAc, ethyl acetate; through a C,g Sep-Pak cartridge (Waters Associates,
GA(s), gibberellin(s); GC-MS, combined gas chromatogra-
phy-mass spectrometry; HPLC, high performance liquid chjo- Milford, MA, USA) which was washed with 5 ml of 5%
matography; IAA, indoleacetic acid; KRl, Kovats retention acetic acid in water. The GAs were then eluted with
index; MeOH, methanol; MeTMS, methyl ester trlmethylsilyl 5 ml of 80% MeOHiHjO (v/v). The dried 80% MeOH
ether; MSTFA, N-methyl-N-trimethylsilyltriflaoracetamide; fraction was redissolved in a few drops of MeOH and
PVPP, polyvinylpolypyrrolidone.
the pH was adjusted to 8.0 after addition of 5 ml of
water. The solution was applied to a 1 x 5 cm column of
QAE-Sephadex-25 previously equilibrated with 0.5 M
Materials and methods sodium formate and washed with 10 volumes of water.
Plant material After application of the sample the column was washed
Clementine [Citrus reticutata (Hort) Ex. Tanaka, cv. with 15 ml of pH 8.0 water and the GAs were eluted
Oroval] and Satsuma [Citrus unsttiu (Mak) Marc, j man- with 20 ml of 0.2 M formic acid. The eluate was dried
darins were selected for this study since these two culti- and subjected to reverse phase HPLC on a Kontron
vars yield exclusively seedless fruit (Ortiz et al. 1988). HPLC System 600 (Kontron Instruments Ltd., St. Al-
Reproductive organs from both mandarins were col- bans, UK). The extract was injected onto an ODS Hy-
lected at random at the following reproductive stages, persil (5 \im) column (250 X 4.9 mm i.d.; Ailtech Asso-
described for citrus by Frost and Soost (1968): stage A, ciated Inc., Deerfield, IL, USA). The elution was car-
bud probably containing pollen mother cells before the ried out with a 40 min linear gradient of 20-100%
prophase of the first meiotic division [-21 days after MeOH in aqueous acetic acid (50 [tl T') at a flow rate
anthesis (DAA)]; stage C, ovary at stage of young pol- of 1 ml min"' and 1 ml fractions were collected. In
ien (—12 DAA); stage E, ovary at pollination order to determine elution volumes on HPLC, ca
(0 DAA); Stage F, ovary at petal fall (6 DAA) and 800 Bq [1,2-^HJ-GA,, [1,2-'H2]:-GA4, [l,2,3-'H3]-GA,o
stages H and I, developing fruits (12 and 42 DAA, and [2,3-'H2]-GA5 were added to the extract. Based on
respectively). After determination of fresh weight, sam- the retention volumes of the internal standards, dried
ples were frozen in hquid nitrogen, lyophilized and HPLC fractions were combined and methylated with
stored at -20°C until analysis. Batches of 200 fruits at ethereal diazomethane. Residual sugars were removed
each developmental stage were used for measurements from the methylated samples by adding 0.5 ml of water
of fruit growth. Abscission rates of ovaries and fruits and partitioning against EtOAc (3x0.5 ml). The com-
were determined on 10 populations of 250 tagged fruits bined organic phases were dried, trimethylsilylated with
on 10 different trees. Growth and abscission parameters 10 yd of MSTFA and analyzed using a Kratos MS80RFA
were studied over two consecutive seasons. The results GC-MS system (Kratos Analytical Ltd., Manchester,
presented herein are representative experiments, since UK). The samples were co-injected with Parafilm (Gas-
absolute values, mainly for abscission rates, varied con- kin et al. 1971) into a fused silica wall coated open
siderably. tubular (WCOT) BP-1 capillary column (25 m x
0.32 mm x 0.33 (xm film thickness) at an oven temper-
ature of 50°C in the splitless mode. After 1 min the oven

Physiol. Plant. 79.1990 401

temperature was increased at 20°C min^' to 240°C and
then at 4°C min^' to 295°C. The inlet pressure was 0.04
MPa and the injector, interface and source temper-
atures were 220°C, 250°C and 2OO''C, respectively. Elec-
tron impact mass spectra were obtained at 70 eV, scan-
ning from 700-50 amu at 1 s per decade.
Estimation of the GA levels in the reproductive or-
gans of both varieties was carried out by bioassay. Five
g dry weight of each sample were extracted, partitioned
and purified on a PVPP column as above. The eloate at
pH 2.5 was partitioned 3 times against EtOAc and the
combined organic fractions were reduced to dryness.
The dried extract was dissolved in 1 ml 100% MeOH
and irradiated with UV light (254 nm for 1 h) to mini-
mize the effect of potential inhibitors such as ABA
(Aharoni et al. 1977). Aliquot volumes of this solution
were then loaded onto strips of Whatman No. 3 chroma-
tography paper and separated by ascending chromatog-
raphy with 80% isopropyl alcohol. After drying, the
strips were cut into 10 equal sections and individually
assayed by the barley endosperm bioassay (Jones and
Varoer 1967). Each enzymatic solution was read in
1:600, 1:100, 1:25, and 1:5 dilutions at 620 nm with an
automatic enzyme-immunoassay processor-spectropho-
tometer. The GA content was estimated from a log dose
curve which was fitted for each polystyrene microtest
plate. Authentic GAs, namely, GA,, GAj, GA3, GA,,
GA5, GA,, GA,, GA]3, GAjo and GA55, were chroma-
tographed and bloassayed as described to determine R,
values and relative activities.
Determination of [AA and ABA
Five g dry weight of each sample were extracted with
80% MeOH (20 ml g"") containing butylated hydroxy-
totuene (BHT) at 100 mg T' with continuous stirring
for 16 h in darkness. After filtration, the residue was
re-extracted twice with 80% MeOH for 3 h. The com-
bined methanolic extracts were concentrated and the
aqueous residue centrifuged at 5 000 g for 30 min. The
supernatant was divided in 2 subsamples: 2/3 was used
for free IAA and free and bound ABA determinations
and the remaining 1/3 for bound IAA. The fomaer
subsample was dried, redissoived io 0.1 M potassium
phosphate buffer at pH 8.0 and purified on a PVPP
column (Glenn et al. 1972). The eluate was adjusted to
pH 2.5 and partitioned against diethyl ether containing
BHT (100 mg r ' ; 3 X 1/3 volume). The combined orga-
nic phases were evaporated to dryness, re-dissolved in
MeOH and used for free IAA and ABA estimations.
The remaining aqueous fraction containing bonnd ABA
was hydrolyzed at pH 11 and at 60°C for 1 h. The hy-
drolysate was acidified to pH 2.5 and partitioned
against diethyl ether containing BHT (100 mg T';
3x 1/3 volume). The combined organic phases were
evaporated and used foi bound ABA determination.
Although fram-ABA was also detected in this fraction,
no attempts to study this compound were made.
402
Coejugated forms of IAA were estimated by sub-
jecting the remaining 1/3 aqueous fraction from the
methanolic extract to mild alkaline hydrolysis in 1 M
NaOH at 25°C for 1 h (Bandurski and Schulze 1977).
The hydrolysate was adjusted to pH 2.5 and partitioned
against diethyl ether containing BHT (100 mg 1~';
3x1/3 volume). The combined ether phases were re-
duced to dryness, purified on a PVPP column and parti-
tioned as described above. IAA recovered in this frac-
tion was the free form plus the ester-bound IAA, which
probably contained the IAA-methyl ester (Takahashi
et al. 1975). The ester-bound IAA was calculated by
subtracting the free IAA value from the total amount
estimated after hydrolysis. IAA was determined by flu-
orometric measurement of the indole-a-pyrone (Stoessl
and Venis 1970, Horemans et al. 1984). ABA was quan-
tified after methylation usiog a Per'kin-Elmer Sigma 3-B
gas chromatograph (Perkin-Etoer Ltd., Beaconfield,
UK) fitted with a ''Ni electron capture detector. A
bonded phase SE-54 (5% phenyl silicone) fused silica
capillary column (10 m x 0.53 mm) was used and the
injector, detector and column temperatures were
275°C, 300°C and 210°C, respectively.
The hormone estimations were determined twice in
fruits from 2 consecutive seasons. Qualitatively, similar
data were obtained each time, although estimations of
absolute levels varied.
Results
Growtli and abscission in mandarin fmits
The pattern of fruit growth in Satsuma and Clementine
mandarins through the early phases of development,
measured as the increase in fresh weight, is shown in
Fig. 1. Citrus fruit exhibit a sigmoidal growth curve
which can be divided into three stages (Bain 1958):
a) stage of cell division and slow growth, b) stage of cell
enlargement and rapid fruit growth and c) maturation
period. In Satsuma and Clementine mandarins the pe-
riod of cell division and slow growth lasted about 1
month including flowering. The stage of cell enlarge-
3,0
S" 2.0
B 1.0
0.0
36
Days after anthesis
Fig. 1. Fresh weight of reproductive organs of Satsuma (open
circles) and Clementine (filled circles) mandarins throughout
the early stages of development. The error bars show SD.
Where error bars are omitted the circle is bigger than the SD.
Physiol. Plant. 79. 1990

-2 -1 1 2 3 4 5 6 7 trace levels of GAj, were detected in Clementine. The
Weeks after anthesis
Fig. 2. Abscission rates ot reproductive organs of Satsuma
(open columns) aod Clementine (filled columns) mandarins
throughout the early stages of development. The error bars
show SD.
ment or phase of rapid fruit growth proceeded during
the following 5 months until the beginning of matura-
tioti. Satsunja fruits showed a higher rate of growth and
a 35% difference in fruit weight was observed 42 DAA.
Two waves of abscission were distinguishable io both
mandarins, although these varied io magnitude between
the 2 cultivars (Fig. 2). The first wave occurred at anthe-
sis and petal-fall stages and iticluded the drop of flowers
and developing ovaries. The second corresponded to
the abscission of developing fruits about 1 toonth later.
Clementine, the self-incompatible mandarin, had a
higher percentage of fruit abscission, settitig only 3% of
the ioitial fruits io comparison with 23% in Satsuma.
Gililierellins
For GA identification purposes, extracts of ovaries at
anthesis from Satsuma and Clementine mandarins were
purified and examined by full-scanning capillary
GC-MS. The identity of the GAs as their MeTMS deriv-
atives was established by comparison with reference
spectra (Takahashi et al. 1986, Hedden 1987, Poling
and Maier 1988) and from their relative retention times
on capillary GC. Table 1 lists the GAs identified in both
mandarios. Satsuma ovaries contained tbe following 13-
hydroxylated GAs: GA^, GAjo, GAj,, GAj, GAg, GA3
and iso-GAj. Of these, only GA3 and 2 inactive end
products of the 13-hydroxylalion pathway, GA29 and
characterization of the latter 2 GAs may suggest the
presence of previous 13-hydroxylated precursors at very
low levels. In order to estimate the levels of the active
GAs in both mandarins, the barley endosperm bioassay
was used as a non-specific GA detector to pick up
obvious differences between Satsuma and Clementine
fruits. Reproductive organs from both mandarins
showed 2 zones of GA-like activity at R( 0.4—0.6 and
0.7-0.9 which co-chromatographed with dihydroxy- and
monohydroxy-GAs, respectively. Since GA,,, GA^o,
GAi and GA3 were the active GAs identified by
GC-MS, the chromatographic distribution of the GA-
like activity may suggest the presence of the dihydroxy-
lated OAs GAj and GA3 in the active zone at R,
0.4-0.6, aod the presence of the tnonohydroxylated
GAs GAi, and GA^) at R, 0.7--0.9. The changes io
GA-like activity during ovary and early fruit develop-
ment, as assayed by the barley endosperm bioassay, are
shown in Fig. 3A,B. It should be noted that in this
system 3/J-hydroxy-Cj9 GAs such as GA3 and GAj ex-
hibit higher biological activity (ca 10-fold) than non-3fi-
hydroxy GAs such as GA,, and GAjj. In both chroma-
tographic zones GA activity was significantly higher in
Satsuma than in Clementine ovaries at anthesis.

Tab. 1. Gibberellins of Satsuma and Clementine mandarins identified as their MeTMS derivatives by Ml scan GC-MS from
ovaries at anthesis- Reference spectra and relative retention times are also shown (Hedden 1987, Poling and Maier 1988.
Takahashi et aL 1986), * = Weak spectrum.

GA Source HPLC GCR, KRl Characteristic ions

fractions (min) (relative intensity)

GA,, Satsuma 21-25 20.45 2608 M+ 462(23), 434(100), 375(54), 374(36)

GA,, Reference 2612 M+ 462(10), 434(100 , 375(57), 374(59)
GAjo Satsuma 16-20 19.13 2507 M+ 418(100), 375(35 , 359(10), 301(12)
GAao Reference - 2512 M+ 418(100), 375(45 , 359(12), 301(13)
Satsuma 1H5 21.51 2678 M+ 506(100), 491(17), 448(22), 377(13)
GAI Referetice 2676 M+ 506(100), 491(13), 448(20), 377(12)
GA,, Satsuma 6-10 21.68 2685 M+ 506(100), 491(12), 375(18), 303(13)
GAJ, Clemendne 6-10 21.51 2684 M+ 506(100), 491(10), 375(16), 303(12)
GA25 Reference 2680 M+ 506(100), 491(11), 375(15), 303(17)
GAs Satsuma 6-10 23.58 2822 M+ 594(100), 579(31), 535(03). 504(02)
Clementine* 6-10 23.53 2820 M* 594(100), 579(36), 535(15), 504(17)
GA Reference 2818 M+ 594(100), 579(10), 535(10), 504(05)
GA3 Satsuma 1H5 21.83 2707 M+ 504(100), 489(10), 473(03), 445(04)
GA3 Clementine 11-15 21.70 2704 M+ 504(100), 489(11),, 473(05), 445(08)
GAJ Reference 2714 M+ 504(100), 489(10), 473(02), 445(05)
IS0-GA3 Satsuma 6-10 20.93 2643 M+ 504(100), 489(14), 475(15), 445(lO)
Iso-GA, Reference 2645 M* 504(100), 489(11), 475(19), 445(15)

Physiol. Plasil. 79. 19W 403


-12 0 12 24 36 -1:2
Days after anthesis
Free and bound IAA
In both mandarins free IAA levels showed similar
trends (Fig. 4A). Free IAA was relatively low in repro-
ductive buds, increased during the flowering period and
reached a maximum 1 week after aothesis. On the other
haod, in Satsuma mandario the content of bouod-IAA
increased slightly throughout flowering aod early fruit
growth, whereas in Clementine it increased dramat-
ically (Fig. 4B), coincident with the decline of the free
form. As a result of these changes the ratio of
free/bound IAA in Clementine decreased from 3.7 in
developing ovaries (6 DAA) to 0.7 in fruitless at 12
DAA. In Satsuma this ratio was always higher than
tioity.
Free and bound ABA
The cootent of free aod bouod ABA in Satsuma and
Clementioe tnandarins is shown io Fig. 5A and 5B, re-
spectively. Clementine had higher amounts of free
ABA, which in both species reached its highest level at
petal-fall stage, at which time bound ABA levels io
Satsutna also peaked. In contrast, bouod ABA levels in
Clementine remained low during all stages analyzed.
Thus, io Clementine the ratio of free/bound ABA was
higher than 1, whereas in Satsuma this ratio was less
than 1 during anthesis and subsequeot stages.
-12 0 12 24 36 *12 0 12 24
Days after anthesis
Fig. 4. Free (A) and bound (B) IAA in reproductive organs of
Satsuma (open circles) and Clementine (filled circles) manda-
rins during the early stages of development.
404
Fig. 3. Gibberellin-like activity as detected
by the barley endosperm bioassay at R,
0.4-0.6 (A) and 0.7-0.9 (B) in reproductive
organs of Satsuma (open circles) and Clem-
entine (filled circles) mandarins during the
early stages of development.
0 12 24 36
Discussion
Plant hormones are involved in the regulation of cell
division and enlargement, which are two main processes
determining fruit development. Thus, the influence of
plant hormones on the induction of fruit growth should
take place at the same time or before these processes
occur. Differences in the pattern of growth between
Satsuma and Clementine fruits were mainly observed
soon after anthesis at the beginning of the phase of
rapid fruit growth. Developing fruitlets of Satsuma had
a higher rate of growth (Fig. 1) and higher parthenocar-
pic ability (Fig. 2). The GAs identified in Satsuma,
GA19, GA20, GA29, GA], GAg, GA3 and iso-GA3,
(Tab. 1) are all 13-hydroxylated GAs. GA2, and GAj
are inactive GAs arising from the 2/3-hydroxylatioo of
GA20 aod GAi, respectively. GA,,, the only C-20 com-
pound identified, is the precursor of GA20, the itnmedi-
ate precursor of GAj, which is assumed to be the active
GA of the 13-hydroxyl,ation pathway in vegetative or-
gaos (Phinney 1984). The presence of these GAs in
Satsuma ovaries suggests that this GA biosynthetic
pathway operates io the reproductive tissues of this
variety. Except for GA3, whose metabolic origin is still
uncertain, all these GAs have also been detected in
vegetative shoots of Navel oranges by Poling and Maier
(1988), who in addition identified GA^^. These observa-
tions indicate that the 13-hydroxy pathway is the major
GA biosynthetic pathway in both vegetative and repro-
36 .12 0 12 24 3S .12 C 12 24 36
Days after anthesis
Fig. 5. Free (A) and bound (B) ABA in reproductive organs of
Satsuma (open circles) and Clementine (filled circles) manda-
rins during the early stages of development.
Physiol. Plant. 79, 1990
ductive citrus tissues. This GA sequence appears to
operate also in Clementine ovaries, since the only GAs
identified in these tissues, GA3, GA29 and GAg (Tab. 1)
are also 13-hydroxylated. Furthermore, the identifica-
tion of 2 end inactive products of the 13-hydroxy path-
way may indicate the presence of other active members
of this series, although at lower levels. The results ob-
tained by bioassay also suggest that the reproductive
organs of Clementine have low amounts of monohy-
droxy GAs and that ovaries at the anthesis stage io
particular contain much lower levels of active GAs in
comparison with those in Satsuma (Fig. 3A,B). The
physiologically-active GAs which are present during the
initial stages of fruit growth are probably involved in
cell expansion in the fruit, as suggested for pea (Garcia-
Martioez et al. 1987) and tomato (Bohoer et al. 1988).
Thus, these results suggest that seedless fruits of Clem-
entine do not reach certain GA threshold levels which
are required during anthesis for adequate fruit set aod
development. Supporting this conclusion, exogenous
applications of GAs increase fruit set of seedless Clem-
entine mandarins (Soost and Burnett 1961). Further-
more, these treatments are more effective when GAs
are applied between anthesis and 2 weeks later (Garcia-
Martinez and Garcia-Papi 1979). However, GAs have
not been satisfactory in improving the fruit set of other
seedless citrus cultivars such as Satsuma (Coggios and
Hield 1968). Whether the absence of fertilization proc-
esses in Clementine is responsible for this GA defi-
ciency remains unanswered. In general GAs io seeded
fruits accumulate in the seeds (Goodwin 1978) and
fruits which have been prevented from setting seeds
have lower levels of GAs (Garcia-Martinez et al. 1987).
However, fruits that naturally set parthenocarpically,
for example pear (Gil et al. 1972), contain higher levels
of GAs, which may explain their tendency to set fruit in
the absence of pollination or fertilization.
The endogenous levels of free IAA found in this
study are io reasonable agreement with those reported
previously in citrus (Igoshi et al. 1971, Takahashi et al.
1975, Garcia-Papi and Garcia-Martinez 1984). During
the studied period both species show similar amounts of
free IAA (Fig.4A). Since applications of exogenous
auxins are also ineffective in promoting fruit set of
either Clementine (Garcia-Martinez and Garcia-Papi
1979) or Satsuma (Guardiola and Lazaro 1987) manda-
rins, it seems unlikely that the content of free IAA was
a limiting factor in controlling fruit development in
these species. In Clementine the decline in the levels of
free IAA coincided with the increase in the concentra-
tion of conjugated IAA (Fig.4B), suggesting a pre-
cursor-product relationship between these two com-
pouods. Similar correlations have also beeo fouod in the
development of strawberry (Archbold and Dennis 1984)
and apple fruits (Treharne et al. 1985). In these tissues
conjugates of IAA could act as a temporary storage
form, controlling the concentration of free IAA (Cohen
and Bandurski 1982). From our data, however, this
Physiol. Plant. 79. 1990
orrelation does not seem to occur in Satsuma tnanda-
rins. In this species, Takahashi et al. (1975) showed that
abscission of fruit and levels of free IAA varied over the
years, indicating that both are susceptible to environ-
mental conditions. In contrast, the levels of bound IAA
appear to be rather constant (Fig.4B), indepeodeot of
the levels of free IAA (Takahashi et al. 1975).
In both cultivars, the concentration of free ABA re-
aches its highest level at the petal-fall state (Fig. 5A).
Developiog fruitlets of Clementine cootain higher
amounts of free ABA but very low levels of bound
ABA (Fig.5B). In contrast, larger amouots of bound
ABA than free ABA accumulate in Satsuma fruits, as is
the case in other citrus cultivars (Goldschmidt et al.
1973, Harris and Dugger 1986). Although the physio-
logical role of ABA conjugation is not clear, bound
ABA is assumed to be an inactive storage form by
which free ABA may be sequestered (Milborrow 1983).
Thus, although the total ABA content in these two
kinds of mandario fruits is similar, Clemeotine fruits
have higher amounts of free ABA.
In conclusion, the hormonal status of developing
fruits of Satsuma aod Clementioe maodarins differs
maioly during the later stages of cell division and early
stages of cell enlargement. During this period the low
ability to set parthenocarpic fruits appears to be related
to lower levels of GAs, a low capability to conjugate
ABA and a higher ability to conjugate IAA.
Acknowledgements - The authors wish to thank Dr P. Hedden
for guidance during the GC-MS identification of GAs at Long
Ashton Research Station, Bristol, UK.
References
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