Annals of Botany 87: 805±812, 2001
doi:10.1006/anbo.2001.1415, available online at http://www.idealibrary.com on
Carbohydrate Availability in Relation to Fruitlet Abscission in Citrus
R . R U I Z * , A . G A R C IÂ A - L U I S , C . M O N E R R I and J . L . G U A R D I O L A {
Departamento de BiologõÂa Vegetal, Universidad PoliteÂcnica de Valencia, 46071 Valencia, Spain
Received: 15 December 2000 Returned for revision: 1 February 2001 Accepted: 1 March 2001
Abscission of ¯owers and fruitlets in the Washington navel orange (Citrus sinensis [L.] Osbeck) has been characterized
in relation to carbohydrate availability. A main wave of ¯ower abscission occurs shortly after anthesis while the
carbohydrate reserves in the tree are high. Fruitlet abscission starts approx. 30 d after the commencement of
¯owering, while carbohydrates (mainly starch) are being accumulated in the leaves. Flower and early fruitlet
abscission are not caused by carbohydrate shortage. During late fruitlet abscission sucrose concentration in the leaves
falls to a low value demonstrating a limitation in supply and competition among the developing fruitlets for
carbohydrates. Concentrations of sucrose and reducing sugars in the peel of the fruitlets also fall to low values, and a
relationship could be demonstrated between these free sugar levels and abscission. Ringing increases carbohydrate
supply to fruit and reduces late fruitlet abscission, but only has a marginal e€ect on the growth of the fruitlets, which
seems less sensitive than abscission to carbohydrate shortage. The limitation of carbohydrate supply to the fruitlets
occurs while starch levels in the leaves remain high. Slow mobilization of starch reserves may be one factor limiting set
# 2001 Annals of Botany Company
in Citrus.
Key words: Carbohydrate supply, citrus, fruit growth and abscission, ringing, navel orange, starch, sugar
metabolism.
I N T RO D U C T I O N
Fruit set rather than ¯owering is the step that limits yield in
most Citrus cultivars. The number of fruits ®nally harvested
is usually less than 2 % of the number of ¯owers formed,
and values as low as 0.1 % have been reported (Monselise,
1986). A massive abscission of ¯owers and fruitlets, which
starts at ¯ower opening or earlier and lasts for several weeks
(AgustõÂ et al., 1982), continuously reduces the number of
developing fruitlets. This abscission has been interpreted as
a self-regulatory mechanism which adjusts fruit number to
the capacity of metabolite supply by the tree (Goldschmidt
and Monselise, 1977; Guardiola, 1988). From the changes
in the amount of reserves, it has been postulated that
carbohydrates may be the limiting factor for set (Scha€er
et al., 1985; Sanz et al., 1987; GarcõÂ a-Luis et al., 1988a). A
correlation between abscission and the carbohydrate levels
in leaves and fruitlets at the end of the period of abscission
has been found in trees in which competition was altered by
changing the number of fruitlets (GarcõÂ a-Luis et al., 1988b)
or the number of leaves (Mehouachi et al., 1995; GoÂmezCadenas et al., 2000).
However, little is known about the carbohydrate nutrition of fruitlets in relation to abscission, or the mechanism
that triggers abscission (Goldschmidt, 1999). Ruiz and
Guardiola (1994) found that during the early stages of
fruitlet abscission this process was preceded by a reduction
* Permanent address: EstacioÂn Experimental La Platina, PO Box
439/3, Santiago, Chile.
{ For correspondence. Fax ‡34 963877419, e-mail jlguardiola@
bvg.upv.es
0305-7364/01/060805+08 $35.00/00
in transport to the abscising fruitlets. A low mobilizing
ability of these fruitlets rather than a limitation in supply
was identi®ed as the cause of this reduction in transport. In
this paper we present evidence that late fruitlet abscission in
the Washington navel cultivar of sweet orange is caused by
a limitation in carbohydrate supply, and it is suggested that
free sugar levels in the peel may be the signal triggering
abscission.
M AT E R I A L S A N D M E T H O D S
Plant material and experimental design
The experiments were performed in two plots of 7-year-old
Washington navel orange trees (Citrus sinensis [L.] Osbeck)
grafted onto Troyer citrange (Citrus sinensis [L.]
Osbeck Poncirus trifoliata Raf) rootstock, growing in
the open. In the years in which the experiments were
performed the number of ¯owers formed per tree ranged
from 20 000 to 40 000. This number of ¯owers is adequate
for the production of a maximum yield in this cultivar
(Guardiola et al., 1984). Flowering commenced on
23 March on experimental plot I, and on 27 March on
experimental plot II. These dates were used as a reference
for all measurements, and the time elapsed is referred to as
days from the commencement of ¯owering (abbreviated to
`days' in the text). Usually, the analytical determinations
were carried out on ¯owers that opened at a later date
(indicated in the ®gures when appropriate).
To determine the in¯uence of the type of in¯orescence on
the pattern of abscission and set, 200 in¯orescences of each
of the four in¯orescence types found in Citrus (lea¯ess
# 2001 Annals of Botany Company
806
Ruiz et al.ÐFruitlet Abscission in Citrus
single-¯owered; lea¯ess multiple-¯owered; leafy single¯owered; and leafy multiple-¯owered) were tagged. These
in¯orescences were evenly distributed on 20 trees (ten
in¯orescences of each type on each tree). Flower and fruitlet
abscission were recorded periodically. Anthesis started
earlier (by day 0) in the lea¯ess in¯orescences, and by days
15±25 in the single-¯owered leafy in¯orescences. The ®rst
¯ower opened by day 7 in the multiple-¯owered leafy
in¯orescences.
The relationship between carbohydrate levels in the
leaves and fruitlets and abscission was determined in two
groups of four trees each, which served as sampling units.
Starting at ¯ower opening and ending by day 92, leaves that
formed during the spring ¯ush of the preceding year and
that were 1-year-old at bud break (referred to in the text as
old leaves) and from the single-¯owered leafy in¯orescences
of the current year (referred to in the text as young leaves)
were sampled for analysis at 7 d intervals. Fruitlets formed
in single-¯owered leafy in¯orescences were also sampled.
The fruitlets sampled were the largest present on the tree.
After day 60, fruitlets were separated into peel and juice
vesicles for analysis. To determine the pattern of abscission
and the amount of dry matter accumulated in the fruitlets
( persisting and abscised), ®ve branchlets, each with 55±65
nodes (ˆold leaves) and 150±165 ¯owers, were tagged on
each tree. In all, the tagged branchlets had 6339 ¯owers.
The number of surviving ¯owers and fruitlets was determined at weekly intervals, and the rate of accumulation of
dry matter in the fruitlets calculated from their diameter,
which was measured weekly with calipers. The relationship
between fruitlet diameter and dry weight was determined
each week from a random sample of fruitlets taken from
non-experimental trees on the same experimental plot.
The in¯uence of ringing on carbohydrate contents in the
leaves, the pattern of abscission and the accumulation of
dry matter in the fruitlets was determined in the same
experimental plot (Plot I). Two groups of four trees each
were ringed on day 33 (25 April), making a single cut in the
bark deep at the base of the trunk with a sharp knife.
Samplings and measurements of fruitlet abscission were
performed as described above. Treatments (ringed or nonringed) were allocated at random in the four groups of four
trees used.
The relationship between sugar content in the fruitlets
and abscission was determined in experimental plot II,
comparing fruitlet populations di€ering in their likelihood
of survival. All the trees in this plot were ringed by day 36
(2 May) as described above. The criterion used to
distinguish fruitlets with a high and a low probability of
survival was early fruitlet growth rate (Ruiz and Guardiola,
1994). By day 30 from the commencement of ¯owering, a
sucient number of single-¯owered leafy in¯orescences were
tagged. We selected 300 of these in¯orescences with the
largest fruitlets present on the tree (9.9 + 0.1 mm in
diameter; referred to in the text as large fruitlets). In the
other 300 in¯orescences, the fruitlets had a lower growth
rate and were of an average size (5.7 + 0.13 mm in diameter,
referred to in the text as small fruitlets). The tagged
in¯orescences were evenly distributed among 60 trees, and
were used for samplings (240 in¯orescences of each type)
and to monitor abscission. Fruitlets from the two populations, as well as old and in¯orescence leaves, were sampled at
7 d intervals during the period of fruitlet abscission. Three
separate samples consisting of 30 old leaves or the young
leaves from ten in¯orescences (40 to 60 leaves) were picked at
random from two groups of 30 trees each which were used as
sampling units. Sampling of the old leaves began 50 d before
the commencement of ¯owering to determine the changes in
carbohydrate reserves before bud break.
Determination of carbohydrate and mineral elements
Samples were transported to the laboratory under
refrigeration and dried in a forced air oven at 65 8C to
constant weight. Dry weight was measured and the tissues
were ground. Carbohydrate and mineral analyses were
performed as described earlier (Ruiz and Guardiola, 1994).
Total soluble sugars and starch were extracted and
determined with anthrone. The sum of sugars and starch
is referred to in the text as metabolizable (non-structural)
carbohydrates. Sucrose was determined in the soluble sugar
fraction as described by Cardini et al. (1955). Total nitrogen
was determined by a Kjeldahl method. After digestion of
the samples with an acid mixture, phosphorus was
determined colorimetrically and potassium by ¯ame
photometry (Ruiz and Guardiola, 1994).
Statistical analysis of data
Seasonal changes in composition were subjected to an
analysis of variance using the date of sampling and fruitlet
type as variables, as appropriate. The residual sum of
squares was used to calculate the standard error of the
individual determinations. The lowest signi®cant di€erence
at P 4 0.05 (which equals 2.9-times the standard error) was
used for the comparison of individual determinations. The
percentages of fruit set and abscission were compared using
a w2 test.
R E S U LT S
The pattern of abscission
The pattern of fruitlet abscission was markedly a€ected by
the characteristics of the in¯orescence. Most (87 %) of the
multiple-¯owered lea¯ess in¯orescences had lost all their
fruitlets by day 30 after ¯ower opening (Table 1). In the
13 % of the in¯orescences with fruitlets, their number had
fallen from the initial 4.0 to 1.8 per in¯orescence. In
contrast, most of the multiple-¯owered leafy in¯orescences
(62 %) had at least one fruitlet by day 30. As in the lea¯ess
in¯orescences, the number of fruitlets per in¯orescence had
fallen from an initial value of 4.7 to 1.7 (Table 1). This
initial reduction in fruitlet number resulted from abscission
at the pedicel. Abscission continued up to day 60 after
¯ower opening. This late abscission resulted mainly in a
reduction in the number of in¯orescences with fruitlets, and
occurred at the calyx. These two waves of abscission
occurring at the pedicel and at the calyx overlap in the tree,
Ruiz et al.ÐFruitlet Abscission in Citrus
since anthesis in leafy in¯orescences started 7±15 d later
than in the lea¯ess in¯orescences.
Initial competition among fruitlets within the in¯orescence did not a€ect ®nal fruit set on an in¯orescence basis,
which was similar in single-¯owered and in multiple¯owered in¯orescences (Table 2). Therefore, we determined
the carbohydrate nutrition of the fruitlets during abscission
in single-¯owered leafy in¯orescences, as fruitlet growth was
easier to monitor in them. Abscission in these in¯orescences
occurred mainly between day 40 and day 85 after the
commencement of ¯owering in the tree (30±60 d after
¯ower opening in these in¯orescences). At this time, the
fruitlets had already grown signi®cantly, having a fresh
weight of 9.0 g (2.34 g d.wt) by day 60. Most of this weight
corresponded to the peel (mesocarp plus exocarp), which
represented 86 % of the fresh weight and 88.5 % of the dry
weight of the fruitlets. At this time, the growth rate of the
peel was 690 mg d ÿ1 on a fresh weight basis (146 mg d ÿ1
d.wt), and that of the juice vesicles was 169 mg d ÿ1 f.wt
(22 mg d ÿ1 d.wt). Abscission was usually preceded by
senescence of the fruitlets, which was apparent as a
yellowing at the stylar zone, in contrast to the dark green
colouration of the non-abscising fruitlets.
Carbohydrate levels in leaves during ¯owering and abscission
Metabolizable carbohydrate concentration in the old
leaves decreased steadily during ¯owering reaching its
lowest value by day 22 which coincided with the end of
petal fall in the lea¯ess in¯orescences (Fig. 1). Afterwards,
there was a transient increase to an intermediate value by
day 36, before another decrease from day 64 until the end of
fruit drop. Changes in starch concentration accounted for
most of these changes. Only from day 71 onwards was there
a slight reduction in the soluble sugar concentration in
leaves. Sucrose concentration remained nearly constant
(4.0 + 0.17 %) until day 50, except for a low value on day
22 coinciding with the initial depletion of starch reserves. It
30
In¯orescence type
0
30
60
Lea¯ess in¯orescences
In¯orescences with fruitlets ( %)
Fruitlets per in¯orescence
100
4.0
13
1.8
3 .7
1 .1
Leafy in¯orescences
In¯orescences with fruitlets ( %)
Fruitlets per in¯orescence
100
4.7
62
1.7
35
1 .3
Carbohydrates (% d. wt)
T A B L E 1. Fruitlet retention during post-anthesis in multiple¯owered in¯orescences
Days from ¯ower opening
In¯orescence type ( ¯ower number/
leaf number)
Lea¯ess in¯orescences
Single ¯owered (1.0/0)
Multiple ¯owered (4.0/0)
Leafy in¯orescences
Single ¯owered (1.0/3.4)
Multiple ¯owered (4.7/2.2)
Percentage of set
Per ¯ower
Per in¯orescence
3.4
0.9
3.4
3.7
26.9
6.7
26.9
31.3
The di€erences in the percentage of set per ¯ower between the single¯owered and the multiple-¯owered in¯orescences are statistically
signi®cant at P 4 0.01. w2 values are 7.2 and 75.5 for the leafy and
lea¯ess in¯orescences respectively. The number of ¯owers in the
in¯orescence did not a€ect set on an in¯orescence basis (w2 5 0.78;
NS). Both per ¯ower and per in¯orescence, the percentage of set was
higher (P 4 0.01) in the leafy than in the lea¯ess in¯orescences
(w2 4 15).
Carbohydrates (% d. wt)
T A B L E 2. Final fruit set as related to the characteristics of
the in¯orescences
A
ringing
20
10
0
30
w2
The
test demonstrated statistically signi®cant di€erences
(P 4 0.01) between leafy and lea¯ess in¯orescences in the percentage
of in¯orescences with fruitlets by days 30 (w2 ˆ 102.4) and 60
(w2 ˆ 63.8) after ¯ower opening.
807
C D
E
F
G
F
G
B
ringing
20
10
0
B
0
C D
E
30
60
90
Days from the commencement of flowering
F I G . 1. Changes in sucrose (h, j), total soluble sugars (n, m), starch
(s, d) and total metabolizable carbohydrates (e, r) in the
in¯orescence leaves (A) and the old leaves (B) of control (h, n,
s, e) and ringed trees (j, m, d, r). The trees were ringed on 25 April
(arrow), 33 d after the commencement of ¯owering. Phenological
stages: B, commencement of ¯owering; C, full bloom; D, petal fall in
lea¯ess in¯orescences and full bloom in the leafy in¯orescences; E,
beginning of fruitlet abscission; F, commencement of abscission in the
single-¯owered leafy in¯orescences; G, end of abscission. Standard
errors calculated from the residual sum of squares from the analysis of
variance: sucrose, 0.17; total soluble sugars, 0.6; starch, 0.35; total
carbohydrates, 0.72 (on % d.wt basis).
Ruiz et al.ÐFruitlet Abscission in Citrus
then fell to a value of 3.2 % by day 57, and further to 2.2 %
at the end of fruit drop (day 92).
In¯orescence leaves had expanded fully by day 22,
coinciding with anthesis in the leafy in¯orescences (data
not shown). The transition from sink to source occurred by
day 15, when they had expanded by 80 %, as revealed by
the marked increase in sucrose concentration (Fig. 1) and a
sharp reduction in soluble invertase activity (data not
shown). These in¯orescence leaves accumulated metabolizable carbohydrates (mainly starch) until day 36. As for the
old leaves, concentrations of starch and soluble sugars fell
steadily from day 64 until the end of fruit drop (Fig. 1).
Sucrose concentration in the in¯orescence leaves paralleled
the changes described above for the old leaves, with a fall in
content by day 57 and a further decrease by day 78 (Fig. 1).
Both in the old and in the in¯orescence leaves, the sucrose
concentration by day 78 was 55 % of the mean value
recorded from day 15 to day 50.
80
Weekly abscision rate (%)
808
ringing
60
40
20
B
0
C D
0
E
F
G
30
60
90
Days from the commencement of flowering
F I G . 2. Weekly relative abscission rate in control (d) and ringed trees
(s). Values are percentages of the number of fruitlets present on the
tree at the beginning of each weekly interval (Zucconi et al., 1978).
Di€erences in abscission between control and ringed trees are not
signi®cant (w2 test). Phenological stages (B±G) as in Fig. 1.
The e€ect of ringing on leaf carbohydrates and abscission
Ringing the trees on day 33 resulted in the gradual
accumulation of starch and, to a lesser extent, soluble
sugars, both in the old and in the in¯orescence leaves
(Fig. 1). The accumulation of starch was markedly higher in
the old leaves (9.4 % of leaf d.wt above the unringed control
trees; P 4 0.01) than in the leaves of the in¯orescences
(2.4 % of leaf d.wt; P 4 0.01). In both type of leaf, starch
content decreased from day 64 onwards, coinciding with the
mobilization of starch from the leaves in the control
(unringed) trees. By day 92, at the end of fruitlet abscission,
di€erences in starch concentration between girdled and
control trees had fallen to 1.3 % of d.wt (P 4 0.05) in the
old leaves, and 0.3 % d.wt (not signi®cant) in the
in¯orescence leaves (Fig. 1).
Ringing only had a transient and marginal e€ect on
sucrose concentration in the in¯orescence leaves, and no
di€erences between girdled and control trees were found
Sugar contents (% d. wt)
There were two main waves of fruitlet abscission in the
tree (Fig. 2). During the ®rst of these, which occurred within
15 d of the commencement of ¯owering, 65 % of the ¯owers
formed were shed. The fruitlets arising from single-¯owered
leafy in¯orescences were shed mostly from day 57 onwards
(data not shown), during the latter part of the second peak
of abscission. The growth rate of these fruitlets and the
sugar concentrations in the peel and the juice vesicles are
presented in Fig. 3. Abscission coincided with a marked
increase in the rate of accumulation of dry matter in the
fruitlets. Most of this accumulation occurred in the peel.
The concentrations of sucrose and reducing sugars in the
peel increased with fruitlet age until day 64 (Fig. 3). The
main period of drop in these in¯orescences (days 71±78)
coincided with a transient reduction in reducing sugars both
in the peel and in the juice vesicles, which was followed soon
afterwards by a reduction in the sucrose content of the peel.
During this time, the sucrose concentration in the juice
vesicles increased steadily (Fig. 3).
Fruitlet growth rate (mg d. wt d–1)
Carbohydrate contents in the fruitlets and abscission
400
A
300
200
100
0
14
B
12
10
8
6
4
2
0
ABSCISSION
0
D
E
F
G
30
60
90
Days from the commencement of flowering
F I G . 3. A, Growth rate of the fruitlets (d), the peel (m) and the juice
vesicles (n). Fruitlets from single-¯owered leafy in¯orescences.
Anthesis occurred by day 15. B, Sucrose (s, d) and reducing sugar
(n, m) contents in the peel (s, n) and the juice vesicles (d, m) of
fruitlets from leafy in¯orescences. The mean peak of abscission of the
fruitlets from these in¯orescences is indicated by the horizontal bar.
Phenological stages in the tree (D±G) as in Fig. 1. Standard errors,
calculated from the residual sum of squares in the analysis of variance:
sucrose, 0.27; reducing sugars, 0.4 (on % d.wt).
Ruiz et al.ÐFruitlet Abscission in Citrus
Fruit set and fruitlet parameters
Fruit set ( % of ¯owers)
Fruit set (number per branchlet)
Mean dry weight (g)
Abscised fruitlets
Persisting fruitlets
Girdled
trees
58.0
156
63.0
160
Signi®cance
(t-test)
NS
NS
2.04
3.2
2.54
4.3
NS
P 4 0.01
0.152
3.34
0.161
3.65
NS
NS
Dry matter accumulated (g per branchlet)
In abscising fruitlets
23.3
In persisting fruitlets
10.7
Total in fruitlets
34.0
25.1
15.7
40.8
NS
P 4 0.01
P 4 0.10
Balance set of dry matter in the branchlets calculated by day 92
(23 June). NS, Not signi®cant.
after day 57 (Fig. 1). On the contrary, ringing markedly
increased the sucrose concentration in old leaves (P 4 0.01),
but did not prevent the fall in sucrose concentration which
occurred by day 57. After this date, sucrose concentrations
in old leaves were 10 % higher (0.2 % on a leaf d.wt basis) in
the ringed than in the control trees (P 4 0.05).
The increase in carbohydrate levels in the leaves brought
about by ringing resulted in an increase in the accumulation
of dry matter in the developing fruitlets which ®nally set.
Their number was increased by 35 % by ringing, from 3.2 to
4.3 per branchlet (P 4 0.01, Table 3). Ringing did not
signi®cantly a€ect the pattern of fruitlet abscission (Fig. 2)
or the individual dry weight of the abscised (5.6 % increase)
or the persisting (9.2 % increase) fruitlets (Table 3).
Fruitlet survival and carbohydrate contents
The seasonal trend in carbohydrate concentration in the
leaves in experimental plot II is presented in Fig. 4. The
earlier samplings in this experiment showed that a
signi®cant part of the carbohydrate reserves in the old
leaves accumulated in early spring after bud break. These
reserves were mobilized during anthesis, falling to a
minimum value by day 18 (Fig. 4). Starch accumulated in
the leaves after day 34 but, at variance with the previous
experiment (see Fig. 1), these starch reserves were not
mobilized after day 65 during late fruitlet abscission.
Sucrose concentrations were nearly constant up to day 49,
but fell by about 30 % (P 4 0.01) by day 56 both in the old
and in the in¯orescence leaves.
Carbohydrate concentrations in the in¯orescence leaves
closely paralleled changes in the old leaves. Values shown in
Fig. 4 are means for in¯orescences with large and small
fruitlets. By day 84, at the end of fruit drop, the sucrose
concentration was signi®cantly higher (P 4 0.05) in leaves
of in¯orescences with large fruitlets (3.4 %) than in
in¯orescences with small fruitlets (2.5 %). Apart from
Carbohydrates (% d. wt)
Branchlet parameters
Number of old leaves
Number of ¯owers
Control
trees
30
ringing
20
10
0
–60
A
B
C D E
F
G
–30
0
30
60
90
Days from the commencement of flowering
F I G . 4. Changes in metabolizable carbohydrate contents in the old (h,
s, n, e) and in¯orescence (j, d, m, r) leaves during bud sprouting,
¯owering and fruitlet abscission in expt 2. Phenological stages: A, bud
break; B±G as in Fig. 1. Standard errors calculated from the residual
sum of squares in the analysis of variance: sucrose (h), 0.21; total
soluble sugars (n), 0.38; starch (s), 0.51; total carbohydrates (e), 0.84
(on % d.wt).
15
Carbohydrates (% d. wt)
T A B L E 3. Characteristics of the branchlets used for
monitoring fruit set and dry matter accumulated in the
fruitlets in control and girdled tress
809
10
5
0
30
50
70
90
Days from the commencement of flowering
F I G . 5. Changes in starch (s, d), sucrose (h, j) and reducing sugars
(n, m) in the peel of small (d, j, m) and large (s, h, n) fruitlets. The
F values in the analysis of variance demonstrated statistically
signi®cant di€erences (5 % level or higher) between the fruitlets in
starch, sucrose and reducing sugars concentrations. Standard errors,
calculated from the residual sum of squares in the analysis of variance:
starch, 0.33; sucrose, 0.26; reducing sugars, 0.35 (on % d.wt).
this, no di€erences were found in carbohydrate concentrations between either type of in¯orescence.
Throughout the study period, starch and sugar concentrations in the peel were higher in the large than in the small
fruitlets (Fig. 5). Despite di€erences in concentration, the
patterns of change were similar for the two populations of
fruitlets. Sucrose accumulated in the peel until day 63, and
then fell in 7 d to a very low value (16 % and 19 % of the
value on day 63 for small and large fruitlets, respectively).
The concentration of reducing sugars increased with fruitlet
age, but transient reductions were found on days 56±63,
and by day 77. Starch concentration increased up to day 77.
The pattern of abscission was similar for the two
populations of fruitlets (Fig. 6). Peaks of abscission were
found by day 57, and during days 70±77. At both dates,
810
Ruiz et al.ÐFruitlet Abscission in Citrus
Accumulation of mineral elements in the fruitlets
Remaining fruitlets (%)
and weekly abscissions rate (%)
100
75
50
25
0
30
50
70
90
Days from the commencement of flowering
Phosphorus (% d. wt)
Nitrogen and potassium (% d. wt)
F I G . 6. The pattern of abscission in the two populations of fruitlets
from single-¯owered leafy in¯orescences de®ned in this study. Relative
abscission rates shown for large (n) and small (m) fruitlets. The w2 test
demonstrated statistically signi®cant di€erences (P 4 0.05) between
fruitlets by days 77 and 84. The percentage of persisting fruitlets (d) is
the mean for the two populations. Anthesis in these in¯orescences
occurred by days 10±18 from the commencement of ¯owering.
3.5
3
2.5
2
1.5
1
0.5
0
0.2
Nitrogen
Potassium
A
B C D E
F
0.15
G
Phosphorus
0.1
0.05
0
–60
A
B C D E
F
G
–30
0
30
60
90
Days from the commencement of flowering
F I G . 7. Changes in nitrogen, phosphorus and potassium in the old (d)
and the in¯orescence leaves from large (m) and small fruitlets (m).
Standard errors, calculated from the residual sum of squares in the
analysis of variance: nitrogen, 0.05; phosphorus, 0.07; and potassium,
0.025 (on % d.wt).
relative abscission was higher for the small fruitlets,
particularly during the second peak of abscission
(P 4 0.05). Final set, as predicted, was higher (P 4 0.01)
for large (40 % of the initial fruitlet number) than for small
(21 %) fruitlets.
During the study period, the accumulation of nitrogen,
phosphorus and potassium in the fruitlets was linearly
related to the accumulation of dry matter (r2 5 0.998;
n ˆ 12; P 4 0.01). For each gram of increase in dry weight
the fruitlets accumulated 24.5 + 0.2 mg of nitrogen,
2.05 + 0.14 mg of phosphorus and 13.7 + 0.4 mg of
potassium. These ®gures were identical for large and
small fruitlets. In contrast, the small fruitlets accumulated
more calcium than the large fruitlets. Calcium accumulation in the fruitlets was exponentially, not linearly, related
to the increase in dry matter (data not presented).
The three mineral elements studied were mobilized from
the old leaves during bud break and ¯owering as shown by
the marked reduction in concentration (Fig. 7). There was
some recovery in the leaf contents of nitrogen and
potassium by day 49, coinciding with the accumulation of
starch shown in Fig. 4, to decrease again after day 56. The
concentration of mineral elements in the in¯orescence
leaves decreased steadily during fruit set (Fig. 7). This
decrease in concentration was caused by dilution due to the
increase in dry weight of these leaves. The total amount of
N, P and K per leaf remained constant ( potassium) or
increased slightly (nitrogen and phosphorus; data not
shown). During late fruitlet abscission, nitrogen and
phosphorus concentrations were the same in in¯orescences
with large and small fruitlets. Potassium concentration was
higher (P 4 0.05) during days 70±77 in leaves of in¯orescences with large fruitlets, both in absolute value (mg per
leaf, data not shown) and in concentration (Fig. 7).
DISCUSSION
The two waves of abscission shown in this study di€er in the
characteristics of ¯owers/fruitlets shed and in the carbohydrate status of the aerial part of the tree as indicated by
carbohydrate concentrations in the leaves. This parameter
is a good indicator of carbohydrate availability since
carbohydrate concentrations in the bark of twigs and
branches closely parallels the carbohydrate concentrations
in leaves (GarcõÂ a-Luis et al., 1995). During the ®rst wave of
abscission, which occurs within 30 d of the commencement
of ¯owering, it is mainly the ¯owers and fruitlets borne on
lea¯ess in¯orescences that are shed. These ¯owers are
smaller in size than ¯owers from leafy in¯orescences
(Guardiola et al., 1984). A low sink strength rather than a
limitation in supply seems to be the cause of this abscission,
as carbohydrate reserves are maximal at this time. This
early abscission has been reported to be unrelated to sugar
transport to the ¯owers/fruitlets (Erner, 1989). Fruit set in
these early abscising ¯owers/fruitlets is not increased by
ringing, a technique which increases carbohydrate availability to the fruitlets (Scha€er et al., 1985). This early
abscission also occurs in the multiple-¯owered leafy
in¯orescences, thereby thinning them to a single or at
most two ¯owers/fruitlets. The events leading to the
thinning of ¯owers/fruitlets within an in¯orescence have
not been investigated, but the presence of several ¯owers
does not reduce ®nal set on an in¯orescence basis. For
Ruiz et al.ÐFruitlet Abscission in Citrus
several orange cultivars and grapefruit, fruit set on an
in¯orescence basis is reportedly higher in multiple-¯owered
than in single-¯owered in¯orescences (Jahn, 1973; Primo
et al., 1977; Erner and Shomer, 1996).
The rapid decrease in carbohydrate (mainly starch)
concentrations in the old leaves during ¯owering found in
this study contrasts with the gradual decrease from the
onset of ¯owering until the end of fruitlet growth reported
earlier (GonzaÂlez-Ferrer et al., 1984; Sanz et al., 1987). In
the present experiments carbohydrate concentration in the
leaves fell to a minimum by day 20, so that during the
second wave of abscission (days 30±92) carbohydrate
supply relies mainly on current photosynthesis. The root
system, which may represent an important storage repository for carbohydrates (Goldschmidt and Golomb, 1982),
does not contribute to fruitlet nutrition at this time. Rather,
the marked accumulation of starch in leaves caused by
ringing demonstrates that roots compete with the developing fruitlets for current photosynthate.
During the second wave of abscission ( fruitlet abscission),
the carbohydrate balance of the tree changes as shown by
the accumulation of starch in the leaves, the sucrose
concentration in the leaves (which indicates sugar availability at the source) and the free sugar concentration in the
peel of the fruitlets (which is a measure of the amount of
sugar at the sink). Sucrose is unloaded from the sieve tubes
in the pericarp, and from that point is directed to the
di€erent fruit parts through a non-vascular pathway (Koch,
1984; GarcõÂ a-Luis et al., 1991). Therefore, free sugar
concentrations in the peel of the fruitlets results from the
balance between discharge and utilization (transport and
metabolism). At the beginning of fruitlet abscission starch
accumulates both in the old and in the in¯orescence leaves,
and sucrose concentration in the leaves remains high.
During this period, which lasts from day 30 to day 50
(experiment 2) or 58 (experiment 1), carbohydrate availability at the source may not limit fruitlet growth.
Accordingly, Ruiz and Guardiola (1994) found that fruitlets
abscising at this time had higher free sugar concentrations in
the peel than persisting fruitlets. Hormone-regulated carbohydrate utilization in the fruitlet rather than availability at
the source was identi®ed as the cause of the reduction in
fruitlet growth, leading to abscission.
Later in development the accumulation of dry matter in
the developing fruitlets increases markedly to exceed the
photosynthetic capacity of the leaves of the in¯orescence
(Moss et al., 1972; MartõÂ nez-Cortina and Sanz, 1991). The
carbohydrate reserves previously accumulated in the leaves
may be mobilized, and sucrose levels in the leaves fall to a
low value, which demonstrates a limitation in carbohydrate
supply. The developing fruitlets compete for the available
carbohydrates, and the increase in growth rate is matched
by a reduction in fruitlet number through abscission so that
the rate of use of dry matter in fructi®cation remains nearly
constant (Guardiola, 1988). This late peak of fruitlet
abscission coincides with a reduction in sucrose concentration in the leaves. Slow growing fruitlets, which have a
smaller sink-drawing ability (ˆsink intensity) are shed
preferentially (Zucconi et al., 1978; AgustõÂ et al., 1982). It is
demonstrated here that this shedding is related to free sugar
811
concentrations in the peel of the fruitlets since: (1) the main
peak of abscission coincides with, or shortly follows, a
decrease in sucrose and, transiently, in reducing sugars
(compare Figs 5 and 6); and (2) the free sugar concentration (sucrose and reducing sugars) is lower in the peel of
the population of small fruitlets, which have a higher
cumulative abscission.
Sucrose concentration in the juice vesicles appears to be
unrelated to abscission as it increases at a time when its
concentration is minimal in the peel. Abscission at this time
is caused by ethylene, as it is inhibited by the application of
cobalt (Ortola et al., 1997). The shortage of free sugars may
trigger ethylene synthesis since defoliation, which drastically reduces sucrose transport to the fruitlets, increases
ethylene synthesis (Ortola et al., 1997) and ACC accumulation (GoÂmez-Cadenas et al., 2000) by fruitlets. This
sequence of events seems to operate in several abscission
systems (GonzaÂlez-Carranza et al., 1998). However, the
causal link between sugar levels and ethylene synthesis
remains to be established.
Since this late abscission is correlated to carbohydrate
levels in the fruitlet, fruitlet survival may be determined by
carbohydrate availability and its sink-driving ability relative
to the competing fruitlets. Dominance among sinks appears
to involve both nutritional factors and hormonal signals
(Bangerth, 1989). A main factor is the type of in¯orescence
in which the ¯ower is formed, since both fruit set and size are
bigger in leafy than in lea¯ess in¯orescences (Moss, 1970;
Erner and Bravdo, 1983; Guardiola and LaÂzaro, 1987).
Di€erences among fruitlets formed in leafy in¯orescences
may arise from the time of ¯ower opening, as demonstrated
in tomato (Bangerth and Ho, 1984), the number of leaves
and leaf area in the in¯orescence (Ruiz and Guardiola, 1994)
and the size of the vascular connections (Erner and Shomer,
1996). These di€erences in sink-driving ability are established early in development, although the di€erences in the
rate of abscission mainly occur after day 65 when
competition for carbohydrates begins. Ruiz and Guardiola
(1994) also observed that the sink-driving ability (ˆgrowth
rate) of some fruitlets changed markedly several weeks after
anthesis for no apparent reason. As discussed by Ganeshaia
and Uma Shaanker (1994), factors unrelated to resource
availability and to hormone signals also in¯uence the
dominance hierarchy among fruitlets. On the other hand
ringing, which eliminates competition by the root system for
photosynthates, increases the amount of metabolites
diverted to fructi®cation and increases set without a€ecting
fruitlet growth. Abscission seems more sensitive to carbohydrate shortage than fruitlet growth.
A reduced carbohydrate supply to the fruitlets occurs
during late abscission, while at the same time a signi®cant
amount of carbohydrate reserves is retained in the leaves. A
slow mobilization of leaf starch reserves in Citrus leaves has
previously been reported (Erickson, 1968; Goldschmidt and
Koch, 1996), but its cause has not yet been ascertained. The
di€erent rate of mobilization during late abscission found
in the two experimental plots (compare Figs 1 and 5)
demonstrates the in¯uence of an uncontrolled factor. In
view of the well documented e€ect of potassium on
photosynthate transport from the leaves (Marschner,
812
Ruiz et al.ÐFruitlet Abscission in Citrus
1995), a transient shortage of this mineral element could be
the cause since potassium levels in leaves fall to a very low
value at the end of fruitlet drop and seem to be related to
set. This assumption remains to be tested under a wide
range of potassium levels, since an extreme potassium
de®ciency inhibits, rather than enhances, starch accumulation in Citrus leaves (Lavon et al., 1995). The correction
of this situation, whose rate-limiting step is the transport
from the chloroplast to the cytoplasm, may signi®cantly
increase yield. In our experiments, the amount of carbohydrates stored in the leaves at the end of fruitlet drop
represents about 50 % of the amount diverted to the
fruitlets by ringing.
AC K N OW L E D G E M E N T S
This research was funded by a grant from Ministerio de
EducacioÂn y Ciencia, Spain (Grant PB95-0736). Thanks are
due to Mr J. Llusar and Mr F. Petit for providing orchard
facilities.
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