Sucrose Phosphate Synthase and Acid Invertase ... - Plant Physiology

Plant Physiol. (1989) 91, 1527-1534
0032-0889/89/91/1 527/08/$01 .00/0
Received for publication April 24, 1989
and in revised form July 24, 1989
Sucrose Phosphate Synthase and Acid Invertase as
Determinants of Sucrose Concentration in Developing
Muskmelon (Cucumis melo L.) Fruits1
Natalie L. Hubbard, Steven C. Huber, and D. Mason Pharr*
Department of Horticultural Science (N.L.H., D.M.P.), U.S. Department of Agriculture, Agricultural Research Service
and Departments of Crop Science and Botany (S.C.H.), North Carolina State University, Raleigh, North Carolina
27695-7609
ABSTRACT
Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis
melo L.) were harvested at different times throughout development
to evaluate changes in metabolism which lead to
sucrose accumulation, and to determine the basis of differences
in fruit sucrose accumulation among genotypes. Concentrations
of sucrose, raffinose saccharides, hexoses and starch, as well
as activities of the sucrose metabolizing enzymes sucrose phosphate
synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC
2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were
measured. Sucrose synthase and neutral invertase activities
were relatively low (1.7 ± 0.3 micromole per hour per gram fresh
weight and 2.2 ± 0.2, respectively) and changed little throughout
fruit development. Acid invertase activity decreased during fruit
development, (from as high as 40 micromoles per hour per gram
fresh weight) in unripe fruit, to undetectable activity in mature,
ripened fruits, while SPS activity in the fruit increased (from 7
micromoles per hour per gram fresh weight) to as high as 32
micromoles per hour per gram fresh weight. Genotypes which
accumulated different amounts of sucrose had similar acid invertase
activity but differed in SPS activity. Our results indicate that
both acid invertase and SPS are determinants of sucrose accumulation
in melon fruit. However, the decline in acid invertase
appears to be a normal function of fruit maturation, and is not the
primary factor which determines sucrose accumulation. Rather,
the capacity for sucrose synthesis, reflected in the activity of
SPS, appears to determine sucrose accumulation, which is an
important component of fruit quality.
Midway through development, muskmelon (Cucumis melo
L.) fruits undergo a metabolic transition marked by both
physical and compositional changes such as netting of the
exocarp, mesocarp softening, and the onset of sucrose accumulation
(1, 7, 10, 12). Attempts to elucidate the changes in
metabolism that lead to accumulation of sucrose have focused
on sucrose metabolizing enzymes during fruit growth and
development (8, 10, 15). McCollum et al. (11) and Shaffer et
' Cooperative investigations of the U.S. Department of Agriculture,
Agricultural Research Service, and the North Carolina Agricultural
Research Service, Raleigh, NC. This work was supported in part by
BARD grant No. 1-1062-86. Paper No. 12165 of the Journal Series
of the North Carolina Agricultural Research Service, Raleigh, NC
27695-7643.
al. (15) suggested that increasing sucrose concentration in
muskmelon fruit may be due to increased sucrose synthase
(EC 2.4.1.13) activity accompanied by a decrease in acid
invertase (EC 3.2.1.26) activity during fruit growth. In both
studies the reported sucrose synthase activities were low (10,
15), and its role, if any, in sucrose accumulation remains
obscure. The inverse relationship between acid invertase activity
and sucrose concentration has been reported in other
fruits (9, 18) as well as in other sink tissues such as roots of
carrot (13) and sugar beet (3). Because sucrose accumulates
in vacuoles, a reduction in acid invertase activity would be a
prerequisite for sucrose accumulation.
Sucrose phosphate synthase (SPS2) (EC 2.4.1.14) is recognized
to participate in sucrose synthesis in source tissues but
has received less attention as an important enzyme in sink
tissues. Substantial SPS activity was recently observed in sugar
beet roots (3), where it was postulated to play a role in sucrose
synthesis. In developmental studies of muskmelon fruit, Lingle
and Dunlap (8) reported that SPS activity increased somewhat
during ripening, and the increase was generally correlated
with sucrose accumulation. However, the measured
enzyme activity was low [less than 3 qmol h-' (g fresh
weight)-'], and in our view, a strong case for involvement of
SPS in sucrose synthesis in melon fruit could not be made.
The purpose of our study was to investigate metabolic
changes associated with the accumulation of sucrose within
the developing muskmelon fruit. Initial studies with orangefleshed
muskmelon fruit indicated that SPS activity was an
order of magnitude greater than previously reported (8), increased
with development, and was associated with sucrose
accumulation. To explore potential biochemical differences
associated with genotypic variation in fruit sweetness, developmental
changes in enzyme activities were compared in one
'nonsweet' and two 'sweet' melon genotypes.
MATERIALS AND METHODS
Plant Material
Orange-fleshed netted muskmelons (Cucumis melo L. cv
Burpee's Hybrid) were field-grown for commercial purposes
in Raleigh, NC. Sampling took place in August 1988.
2 Abbreviations: SPS, sucrose phosphate synthase; daa, days after
anthesis.
1527

1 528 HUBBARD ET AL.
Three green-fleshed muskmelon genotypes (Cucumis melo
L. cv Galia, Noy Yizreel, and Birds Nest) were grown in a
greenhouse from June through September 1988. Plants were
grown in 5.9 L pots in a 1:1:1 (v/v/v) mixture of steamtreated
soil, sand, and peat with regular applications of soluble
fertilizer. Plants were trained to three laterals. Flowers were
hand-pollinated and tagged. Fruit load was limited to one
fruit per plant.
Tissue Sampling
Orange-fleshed muskmelons ranged in maturity from small
with very firm, green, nonaromatic tissue throughout, to large
with softened, orange, aromatic mesocarp. Plugs were removed
from the equatorial section of each fruit with a cork
borer. The netted exocarp, seeds, and internal pulp were
removed and discarded. Each plug was sliced into sections,
approximately 1 to 2 cm wide from just beneath the exocarp
to the innermost tissue. Small fruit were divided into three
sections while larger, more mature fruit with thicker mesocarp
tissue, were sliced into four sections. Immediately after slicing,
fruit sections were frozen in liquid nitrogen. Several corresponding
sections from each individual fruit were pooled and
powdered in liquid nitrogen using a mortar and pestle. Samples
were stored at -80°C.
Greenhouse grown, green-fleshed muskmelons were harvested
during late August and early September. 'Galia' fruit,
which developed at a faster rate than the other two genotypes,
were sampled at 15, 24, and 32 + 4 'Birds Nest' and 'Noy
Yizreel' were sampled at 15, 24, 32, and 37 ± 4 daa. The final
day of fruit sampling for each genotype roughly coincided
with optimal fruit maturity. Three replicates of each were
sampled with the exception of 'Birds Nest' which, due to poor
fruit set and some disease problems, had two replicates on the
final sampling date. As with the field-grown melons, plugs
were removed from the center of the fruit, with exocarp and
endocarp discarded. Plugs from the green-fleshed melons were
sliced into two sections, inner and outer, and immediately
frozen in liquid nitrogen. Several corresponding sections from
each fruit were pooled, powdered in liquid nitrogen using a
mortar and pestle, and stored at -80°C.
Carbohydrate Analysis
Ethanolic extracts from each of the samples were used for
measurements of starch, sucrose, and hexose sugars in melon
tissues. Analyses were conducted using analytical techniques
described by Brown and Huber (2). Ethanolic extracts from
'Noy Yizreel' and 'Birds Nest' mesocarp tissue harvested at
32 daa were used in the determination of raffinose, stachyose,
and galactose concentrations. A Waters Sugar-Pak I3 column
was used on an HPLC system as described by Robbins and
Pharr (14). To confirm the absence of detectable amounts of
starch, mesocarp from orange-fleshed melons, as well as the
internal pulp, were stained with an I2KI solution.
3 Mention of a trademark or proprietary product does not constitute
a guarantee or warranty of the product by the North Carolina
Agricultural Research Service or the U.S. Department of Agriculture
and does not imply its approval to the exclusion of other products
that may also be suitable.
Enzyme Extraction
Plant Physiol. Vol. 89, 1989
Frozen melon tissue was ground in a chilled mortar using
a 1:5 tissue-to-buffer ratio. Buffer contained 50 mm Mops-
NaOH (pH 7.5), 5 mM MgCl2, 1 mm EDTA, 2.5 mM DTT,
0.05% (v/v) Triton X-100, and 0.5 mg mL-' BSA. Homogenates
were centrifuged at 1 0,000g for 30 s. Supernatants were
desalted immediately by centrifugal filtration (5) on Sephadex
G-25 columns equilibrated with 50 mm Mops-NaOH (pH
7.5), 5 mM MgC92, 2.5 mM DTT, and 0.5 mg mL' BSA.
SPS and Sucrose Synthase Assays
Reaction mixtures (70 ,gL) to determine SPS activity contained
50 mM Mops-NaOH (pH 7.5), 15 mM MgC2, 5 mM
fructose 6-P, 15 mm glucose 6-P, 10 mM UDPG, and 45 ,uL
desalted extract. Reaction mixtures were incubated at 25°C
and terminated at 0 and 20 min with 70 ,L of 30% KOH.
Tubes were placed in boiling water for 10 min to destroy any
unreacted fructose or fructose 6-P. After cooling, 1 mL of a
mixture of 0.14% anthrone in 13.8 M H2SO4 was added and
incubated in a 40°C water bath for 20 min. After cooling,
color development was measured at 620 nm.
The procedure for the sucrose synthase assay (measured in
the sucrose synthesis direction) was identical to that of SPS
except the reaction mixtures contained 10 mm fructose and
did not contain fructose 6-P or glucose 6-P.
Acid and Neutral Invertase Assays
Reaction mixtures (100 ,L) to determine acid invertase
activity contained 100 mm citrate-phosphate (pH 5.0), 120
mm sucrose, and 40 ,uL desalted extract. Reaction mixtures
were incubated at 25°C and terminated by placing tubes in
boiling water at 0 and 30 min after initiation with enzyme
extract. Hexose sugar concentration was determined enzymatically
by measuring NAD reduction after incubation with
hexokinase, phosphoglucoisomerase, and glucose 6-P dehydrogenase
(2).
Reaction mixtures (50 ,uL) for determination of neutral
invertase contained 50 mm Mops-NaOH (pH 7.5), 90 mM
sucrose, and 30 ,uL desalted extract. The assay and hexose
determination was otherwise the same as for acid invertase.
RESULTS
Orange-Fleshed Muskmelons
In young, unripe melons (Cucumis melo cv Burpee's Hybrid),
sucrose concentration was low and constant throughout
the mesocarp (Table I). As development proceeded, the melons
accumulated sucrose, with the highest sucrose concentration
in the innermost tissue. Hexose sugars, on the other
hand, remained relatively constant, with roughly equimolar
concentrations of glucose and fructose (data not shown). No
starch was detected at any stage of development using enzymatic
analysis or 12KI staining techniques.
SPS activity in mature, ripe melon tissue was an order of
magnitude greater than previously reported (8) suggesting a
major role for SPS in sucrose synthesis in muskmelon fruit.
SPS activity increased with fruit development over time and

SUCROSE METABOLIZING ENZYMES IN DEVELOPING MUSKMELON FRUIT
Table I. Carbohydrate Concentration and Sucrose Metabolizing Enzyme Activities in Unripe,
Ripenening, and Mature 'Burpee's Hybrid' Muskmelon Mesocarp Segments
Carbohydrate Enzyme Activity
Maturity Segment Concentration
Sucrose Hexose SPS Acid mnva Neut Invb SSC
mg (g fr wtJ' jirnol h-' (g fr wt) '
Young, unripe 1 (outer) 0.7d 34.5 7.4 40.1 6.3 2.4
2 1.2 38.0 8.8 27.7 4.2 1.5
3 (inner) 0.8 41.1 4.4 20.5 4.2 8.1
Ripening 1 2.1 41.2 9.5 20.0 4.1 1.9
2 13.4 39.5 13.5 6.2 1.9 1.2
3 22.0 43.5 20.5 6.6 3.2 2.6
Mature, ripened 1 6.6 41.7 24.1 10.8 3.5 2.5
2 22.1 43.4 23.4 1.0 1.7 3.9
3 44.3 39.8 22.7 0.1 1.3 0.8
4 46.7 39.9 28.3 0.0 1.7 5.6
a Acid invertase. b Neutral invertase. c Sucrose synthase. d Data are the averages of two
melons at each stage of development.
from outer to inner tissues in a manner similar to the increase
in sucrose concentration (Table I). Acid invertase activity was
high throughout the small, unripe fruit and declined to a
negligible level as tissue ripened. Activities of sucrose synthase
and neutral invertase were much lower than acid invertase
and did not fluctuate during fruit development.
Tissues of orange-fleshed muskmelons contained SPS activity
of up to 10 ,mol h-' (g fresh weight)-' with no sucrose
accumulation (Fig. 1A). Conversely, high sucrose concentration
was associated with low acid invertase activity (Fig. 1 B).
Green-Fleshed Muskmelons
To further evaluate the roles of SPS and acid invertase in
sucrose accumulation of muskmelons, three green-fleshed
I,
cw
Lcm
E v
0
L-
U
50
40
30
20
10
0
genotypes, known to differ in sucrose accumulation, were
studied. Two of the genotypes, 'Galia' and 'Noy Yizreel' were
'sweet' melons and the third, 'Birds Nest' was 'nonsweet' ( 15).
Fruit of the genotype 'Galia' reached maturity 32 daa, while
the 'Noy Yizreel' and 'Birds Nest' matured 37 daa.
With all genotypes of green-fleshed muskmelons, the initiation
of sucrose accumulation occurred near 24 daa (Fig. 2).
By maturity, sucrose accumulation in the inner mesocarp
tissue of the two sweet genotypes had reached approximately
40 mg (g fr wt)-'. During the same period of time, inner
mesocarp tissue of the nonsweet genotype accumulated only
10 mg (g fresh weight)-'. Although mesocarp of green-fleshed
melons was divided only into inner and outer tissues, the data
suggested a strong increasing gradient with respect to sucrose
0 10 20 30 0 1 0 20 30 40 50 60
SPS Activity jLmol h '(g fr wt)'l Acid Invertase gimol h (g tr wt)'
Figure 1. Sucrose concentration of orange-fleshed muskmelon fruit in relation to SPS activities (A) and acid invertase activities (B). Data points
represent means of two fruits, sampled at five comparable developmental stages ranging from young, unripe fruit to fully mature, ripenend fruit.
Three to four mesocarp segments were evaluated from each fruit, depending upon fruit size.
1 529

1 530 HUBBARD ET AL.
3:
L-
>
%-
Ec1
0
L- n
50
40
30
20
10
Plant Physiol. Vol. 89, 1989
0 15 20 25 30 35 0 15 20 25 30 35 40
Days Atter Anthesis Days Atter Anthesis
Figure 2. Sucrose concentration in three green-fleshed muskmelon genotypes from 15 daa through maturity. Inner (A) and outer (B) tissues of
two sweet (-, 'Noy Yizreel;' 0, 'Galia') and one nonsweet (5 'Birds Nest') were evaluated. Data points represent the mean sucrose concentration
± 1 SE.
concentration from outer to inner tissue, similar to that
observed in the orange-fleshed muskmelon fruit.
Stachyose and raffinose were present in 'Noy Yizreel' and
'Birds Nest' fruit tissues harvested during the sucrose accumulation
phase in concentrations less than 1 mg (g fresh
weight)' (Table II). Although concentrations were low, stachyose
and raffinose were present in greater concentrations in
the inner tissues than in the outer tissues of both sweet and
nonsweet genotypes evaluated. Raffinose saccharides were
detectable only if extracts were concentrated and detection
levels on the HPLC were very sensitive. HPLC analysis also
indicated that galactose was present, but quantification was
difficult due to overlap of very large glucose and fructose
peaks on the chromatogram.
Hexose sugars increased slightly in concentration during
early fruit development but remained constant during sucrose
accumulation (Table III). As in the case of the orange-fleshed
muskmelons, glucose and fructose were present in roughly
equimolar concentrations. While small amounts of starch
were detectable, the mean throughout development was only
0.15 ± 0.06 mg (g fresh weight)-' with no evidence for starch
accumulation at any time (Table III).
SPS activity increased in all three, green-fleshed melon
Table I. Raffinose Saccharides in 'Noy Yizreel' and 'Birds Nest'
Outer and Inner Mesocarp Tissues at 32 daa
Genotype Tissue Stachyose Raffinose
mg (g fr wt-1
'Noy Yizreel' Outer 0.086 (0.01)8 0.121 (0.03)
Inner 0.233 (0.10) 0.308 (0.12)
'Birds Nest' Outer 0.064 (0.01) 0.051 (0.01)
Inner 0.386 (0.17) 0.160 (0.05)
a Data are the averages of three melons with one standard error
shown in parentheses.
genotypes during development (Fig. 3). SPS activity in the
inner tissue of 'Birds Nest', the nonsweet genotype, increased
from 2 to 15 ,mol h-' (g fresh weight)-', while that of the
sweet genotypes rose from approximately 6 to 24 and 32 Amol
h-' (g fresh weight)-' in 'Noy Yizreel' and 'Galia,' respectively.
The increase in SPS activity during fruit development occurred
in both inner and outer tissues with somewhat lower
activity in outer tissues of each genotype. Genotypic and
tissue differences in SPS activity at maturity appeared to be
reflected in tissue sucrose concentration.
Acid invertase activity decreased with time in all three
green-fleshed melons (Fig. 4). Unlike SPS activity, there were
no substantial genotypic differences with respect to the decline
in acid invertase activity during fruit development. In the
inner tissues of each genotype, acid invertase activity decreased
from 10 ,umol h-' (g fresh weight)-' at 15 daa to less
than 5 ,mol h-' (g fresh weight)-' at 24 daa. Acid invertase
activity in the outer tissue exhibited a steady decline from
approximately 18 to less than 5 ,umol h-' (g fresh weight)-' at
maturity.
Sucrose synthase and neutral invertase activities were low
and changed little during fruit development (Table IV), as in
the case of orange-fleshed muskmelons (Table I). While these
enzymes serve a function in sucrose metabolism, there were
no apparent changes in activities associated with muskmelon
fruit sucrose accumulation.
To evaluate the roles of each sucrose metabolizing enzyme
with respect to sucrose accumulation in developing muskmelon
fruits, the mathematical difference in activity of the
sucrose synthesizing enzyme (SPS) and the potential sucrose
degrading enzymes (sucrose synthase, acid and neutral invertases)
was calculated for each tissue sampled. Figure 5 illustrates
the relationship between tissue sucrose concentration
and the difference between potential sucrose synthesis and
sucrose degradation for the orange-fleshed muskmelons and

40
. -
3-3
T-
. c
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-1
-b
.j I 0
0.
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0
SUCROSE METABOLIZING ENZYMES IN DEVELOPING MUSKMELON FRUIT
Table Ill. Hexose Sugars and Starch Concentrations in Sweet ('Galia', 'Noy Yizreel') and Nonsweet
('Birds Nest') Green-Fleshed Muskmelon Genotypes at 15, 24, 32, and 37 daa
Hexose Concentration Starch Concentration
Genotype daa
Inner Outer Inner Outer
mg (g fr wt)-
'Galia' 15 32.7 (0.7)a 31.4 (1.2) 0.0 (0) 0.0 (0)
24 45.0 (1.9) 38.1 (1.3) 0.1 (0) 0.0 (0)
32 45.5 (1.7) 45.6 (2.2) 0.1 (0) 0.0 (0)
'Noy Yizreel' 15 39.4 (0.8) 35.9 (0.8) 0.2 (0.1) 0.1 (0)
24 46.8 (0.8) 40.7 (2.1) 0.1 (0) 0.0(0)
32 57.8 (0.1) 55.0 (1.6) 0.2 (0.1) 0.1 (0)
37 49.3 (5.7) 43.4 (1.7) 0.0 (0) 0.0 (0)
'Birds Nest' 15 27.5 (1.2) 25.5 (2.5) 1.4 (0.6) 0.3 (0.1)
24 31.1 (0.6) 29.6 (0.5) 0.2 (0) 0.2 (0)
32 45.7 (7.7) 35.3(4.2) 0.2 (0.1) 0.1 (0)
37 38.1 (0.8) 30.5 (0.3) 0.0 (0) 0.0 (0)
a Data are the averages of three melons except 'Birds Nest' at 37 daa when only two fruit were
available for sampling. One standard error is shown in parentheses.
0 15 20 25 30 35 0 15 20 25 30 35 40
Days After Anthesis
Days After AnthesIs
Figure 3. SPS activities in three green-fleshed muskmelon genotypes from 15 daa through maturity. Inner (A) and outer (B) tissues of two sweet
(0, 'Noy Yizreel,' 0, 'Galia') and one nonsweet (El, 'Birds Nest') were evaluated. Data points represent the mean SPS activity ± 1 SE.
each of the three green-fleshed muskmelon genotypes. Sucrose
accumulation occurred only after the enzymatic capacity to
synthesize sucrose exceeded the enzymatic capacity to degrade
sucrose.
DISCUSSION
Muskmelon fruit mesocarp contained a substantial pool of
hexose sugars throughout development, but accumulated sucrose
only during the final stages of development as fruit
ripened. Accumulation was greater in sweet genotypes than
in nonsweet genotypes which verifies that sucrose accumulation
is a major determinant of sweetness among genotypes.
Sucrose accumulation progressed from the innermost to the
outermost portion of mesocarp tissue. Sucrose accumulation
did not occur at the expense of the hexose pool; rather, sucrose
1531
was added to the total sugar pool. The absence of a significant
starch reserve (Table III) and the recognized fact that harvested
muskmelons do not acquire additional soluble sugars
during postharvest ripening (12), provides strong evidence
that sucrose arises from carbohydrates translocated into the
fruit during ripening.
Muskmelons synthesize stachyose and raffinose in their
leaves, and these sugars are translocated to sink tissues (19).
However, previous studies (6, 10) reported these sugars were
absent in developing muskmelon fruit tissues. Gross and
Pharr (4) proposed a metabolic pathway for hydrolysis of
stachyose and raffinose with subsequent conversion of galactose
to sucrose in the fruit pedicel of several stachyose translocating
species, including muskmelon. Sucrose synthase was
implicated in the synthesis of sucrose because high activity
was observed in the pedicel of cucumber, and SPS activity

1 532 HUBBARD ET AL.
a. 20
L-
C7) 15
%-E
< 5
C S
Plant Physiol. Vol. 89, 1989
600 0 1 5 20 25 30 35 0 1 5 20 25 30 35 40
Days After Anthesis
Days After Anthesis
Figure 4. Acid invertase activity in three green-fleshed muskmelon genotypes from 15 daa through maturity. Inner (A) and outer (B) tissues of
two sweet (0, 'Noy Yizreel,' 0 'Galia') and one nonsweet (D, 'Birds Nest') were evaluated. Data points represent the mean acid invertase activity
± 1 SE.
Table IV. Sucrose Synthase and Neutral Invertase Activities in
Sweet ('Galia', 'Noy Yizreel') and Nonsweet ('Birds Nest') Green-
Fleshed Muskmelon Genotypes at 15, 24, 32, and 37 daa
SSa Activity Neut lnvb Activity
Genotype daa
Inner Outer Inner Outer
smoI h-' (g fr wt)-1
'Galia' 15 1.2(1.1)c 0.0(0) 2.1 (0.2) 2.6(0.3)
24 5.2 (1.8) 0.0 (0) 1.0 (0.2) 1.9 (0.1)
32 2.1 (0.3) 1.2 (0.6) 1.6(1.6) 1.8(1.5)
'Noy Yizreel' 15 0.0 (0) 0.0 (0) 3.2 (0.2) 3.7 (0.6)
24 1.5 (1.5) 1.4 (0.9) 0.7 (0.1) 1.2 (0.1)
32 1.4(1.0) 1.0(0.4) 1.2(0) 4.5(2.7)
37 1.5 (0.9) 0.6(0.6) 2.3(0.2) 2.1 (0.6)
'Birds Nest' 15 0.9 (0.6) 1.1 (1.1) 1.3 (0.5) 2.5 (0.4)
24 0.0 (0) 0.0 (0) 0.4 (0.4) 0.9 (0.6)
32 1.2 (0.3) 0.5(0.3) 0.6(0.3) 1.2 (0.7)
37 1.2 (1.2) 0.7(0.7) 1.3(1.3) 1.7(1.7)
a Sucrose synthase. b Neutral invertase. c Data are the averages
of three melons except 'Birds Nest' at 37 daa when only two
fruit were available for sampling. One standard error is shown in
parentheses.
was not detected. Accordingly, it was postulated that sucrose
was the predominant sugar of translocation in these fruits (4).
In the present study, through the use of more sensitive
HPLC sugar analysis, and the use of more concentrated
extracts than used previously, stachyose, raffinose, and galactose
have been identified within muskmelon mesocarp tissue.
Stachyose degradation and sucrose synthesis may occur to
some extent in the fruit pedicel, but the current results suggest
that raffinose saccharides are likely imported into the fruit
mesocarp. The very low concentrations of these sugars (Table
II) suggest that they are rapidly metabolized in the fruit.
5 0 , ,I v
50
,40t _j/O 2
Y0
L30~~~~~~
E 20 00
E0
4) ~~~00
L0 ;o
-50 -25 0 25 50
SPS - (Acid Inv + Neut Inv + 55)
Figure 5. Relationship between sucrose concentration and the difference
between activities of SPS and the potential sucrose degrading
enzymes, acid and neutral invertases, and sucrose synthase. Data
points are from all green-fleshed muskmelon genotypes, outer and
inner tissues, and from unripe, ripening, and mature orange-fleshed
muskmelons. Line for all positive values on x axis fit by linear
regression. Correlation coefficient = 0.85.
Previous studies established the probable metabolism of galactose
via a pyrophosphorylase pathway leading to the formation
of UDP-glucose and hexose-phosphates (16). Galactose
may be metabolized similarly in muskmelon mesocarp
where UDP-glucose and fructose 6-P would be utilized for
sucrose formation and accumulation during ripening.
Even very young, unripe fruit mesocarp contained SPS
activity (Table I). Despite the presence of the enzymatic

SUCROSE METABOLIZING ENZYMES IN DEVELOPING MUSKMELON FRUIT
capacity for sucrose formation, very little sucrose was present
in the tissue, which was apparently due to an excess of
enzymatic capacity for sucrose degradation, with acid invertase
being particularly high. Although sucrose concentration
was low in young fruit, free hexoses, glucose, and fructose,
were high (Table I). Across all fruit and tissues sampled, no
sucrose accumulated in muskmelon mesocarp in circumstances
where the combined activity of sucrose degrading
enzymes exceeded that of SPS as measured in vitro (Fig. 5).
In numerous sink tissues, sucrose concentration has been
shown to be inversely related to acid invertase activity (3, 8,
9, 13). Sucrose may arise in at least two ways in muskmelon
mesocarp. a-Galactosidase attack upon the raffinose sugars
releases galactosyl moieties as well as sucrose. As proposed in
Figure 6, galactose may then be metabolized to sucrose which,
in turn, may be broken down to hexoses by acid invertase
which is presumably in vacuoles. In this way, SPS along with
acid invertase may serve as key enzymes in hexose biosynthesis
in young melon fruits which are not actively accumulating
sucrose.
Later in fruit development, SPS clearly plays a role in fruit
sweetening associated with rapid accumulation of sucrose
during ripening (Table I; Figs. 1, 2, 3). The increased activity
of the enzyme between 15 and 35 daa generally coincided
with the increase in tissue sucrose concentration. The low
concentration of sucrose which accumulated in the nonsweet
genotype, 'Birds Nest,' was clearly associated with lower activity
of SPS than in the sweet genotypes. Sucrose degrading
enzymes, as discussed earlier, also play a developmental role
Glucose + Fructose Stachyose
INVERTASE s
,,, ~~Ratfinose
I---- HP ---- , HP
Sucrose -
SPS
Pi Galactose
Sucrose-6-P lucros
f ATP
ADP
Galactose- -P

1534 HUBBARD ET AL.
6. Hughes DL, Yamaguchi M (1983) Identification and distribution
of some carbohydrates of the muskmelon plant. HortScience
18: 739-740
7. Lester GE, Dunlap JR (1985) Physiological changes during development
and ripening of 'Perlita' muskmelon fruits. Sci
Hortic 26: 323-331
8. Lingle SE, Dunlap JR (1987) Sucrose metabolism in netted
muskmelon fruit during development. Plant Physiol 84: 386-
389
9. Manning K, Maw GA (1975) Distribution of acid invertase in
the tomato plant. Phytochemistry 14: 1965-1969
10. McCollum TG (1987) Metabolism of soluble and structural carbohydrates
during muskmelon fruit development. PhD thesis.
University of Florida, Gainesville
11. McCollum TG, Huber DJ, Cantliffe DJ (1988) Soluble sugar
accumulation and activity of related enzymes during muskmelon
fruit development. J Am Soc Hortic Sci 113: 399-403
12. Pratt HK (1971) Melons. In AC Hulme, ed, The Biochemistry
of Fruits and Their Products, Vol 2. Academic Press, New
York, pp 207-232
Plant Physiol. Vol. 89, 1989
13. Ricardo CPP, ap Rees T (1970) Invertase activity during the
development of carrot roots. Phytochemistry 9: 239-247
14. Robbins NS, Pharr DM (1988) Effect of restricted root growth
on carbohydrate metabolism and whole plant growth of Cucumis
sativus L. Plant Physiol 87: 409-413
15. Schaffer AA, Aloni BA, Fogelman E (1987) Sucrose metabolism
and accumulation in developing fruit of Cucumis. Phytochemistry
26: 1883-1887
16. Smart EL, Pharr DM (1981) Separation and characteristics of
galactose- I - phosphate and glucose- I -phosphate uridyltransferase
from fruit peduncles of cucumber. Planta 153: 370-375
17. Smyth DA, Prescott HE Jr (1989) Sugar content and activity of
sucrose metabolism enzymes in milled rice grain. Plant Physiol
89: 893-896
18. Yamaki S, Ishikawa K (1986) Roles of four sorbitol related
enzymes and invertase in the seasonal alteration of sugar
metabolism in apple tissue. J Am Soc Hortic Sci 111: 134-137
19. Zimmerman MH, Ziegler H (1975) List of sugars and sugar
alcohols in sieve tube exudates. Appendix III. Encyclopedia of
Plant Physiology (New Series), Vol 1. Springer-Verlag, Berlin,
pp 480-503