DEPARTMENT OF BIOTECHNOLOGY

SEMINAR

ON

Sucrose accumulation in watermelon fruits: Genetic

variation and biochemical analysis

Merav Yativ, Idan Harary, Shmuel Wolf
Journal of Plant Physiology (2010) 167: 589-596
PRESENTED BY GUIDED BY
Amrutha B Dr. Vinita Balasubramanya
1st M.Sc Biotechnology Guest faculty

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 CONTENTS
 INTRODUCTION

 MATERIALS AND METHODS

 RESULTS AND DISCUSSION

 CONCLUSION

 REFERENCES

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3
 INTRODUCTION

 Watermelon (Citrullus lanatus) is a widely cultivated member of

the Cucurbitaceae family.
 Systematic improvement of watermelon over millenia has
resulted in the present day cultivars which are of large size and
considerable sweetness.
 Watermelon sweetness is determined by the total sugar content
and the ratios of accumulated sugars mainly, glucose ,fructose
and sucrose.
 Glucose and fructose are found in early stages of fruit
development, while sucrose accumulation is detected 3-4 weeks
post anthesis*.
Source: https://myplantin.com/blog/how-to-
 Rise in sucrose levels may or may not correlate with an grow-watermelon-from-seed
accompanying decrease in glucose and fructose, depending
upon the cultivar (variety).

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* anthesis: full opening of the flower bud
 SUCROSE- THE PRIMARY SWEETENER

 Sucrose is derived from the raffinose family

oligosaccharides (RFOs)
 RFOs in melon include stachyose and
raffinose, the main photo-assimilates
translocated in the phloem of cucurbit crops.
 Stachyose and raffinose move up the
phloem and are hydrolysed by α-
galactosidase
 Hydrolysis of RFOs is crucial for sugar
accumulation and transportation in the fruit.

Source: Tester and Karkalas (2003)

ENZYMES INVOLVED IN SUGAR METABOLISM
IN MELON
 Invertases: Sucrose  Glucose + Fructose

Type of pH Cellular
Invertase optimum localisation
Soluble acid 4.5-5.5 Vacuole
invertase
Insoluble 3.5-5.5 Cell wall
acid
invertase
Neutral 7.0-7.8 Cytosol
invertase

 Soluble acid invertase activity decreases

with sucrose accumulation
 Insoluble invertase activity is constant
even though sucrose concentration
gradually increases in the fruit
Source: Wang et al., (2013) 5
SuSy (Sucrose synthase) and SPS (Sucrose phosphate synthase)

 SuSy (Sucrose synthase) catalyzes both: sucrose synthesis and hydrolysis.

pH 7.0-9.0
Glucose + Fructose Sucrose
pH 6.2- 7.0

 Km values for fructose are in the range of 30–150 for sucrose synthesis and 1–5 mM for
hydrolysis

 SPS (Sucrose phosphate synthase) catalyzes the transfer of a glycosyl group from an activated
donor sugar,(uridine diphosphate glucose or UDP-Glc), to a saccharide acceptor like fructose 6-
phosphate (F6P) to give sucrose-6′-phosphate (S6P).
 Localized in cytosol of photosynthetically active tissue
 Increase in SPS activity is seen at late stages of melon fruit development
 This, along with a decrease in invertase activity, results in accumulation of sucrose in watermelon.
Source: Sinha et al., (1997)
In this paper, the authors have analysed the activity of enzymes
involved in sugar metabolism in different varieties of watermelon to
study:
- the variation in the proportions of sugar
- the mechanism of sucrose accumulation
With this they attempt to correlate the sugar metabolism to the high
genetic variation in watermelon fruits.
 MATERIALS AND METHODS

PLANT MATERIAL AND SUGAR DETERMINATION

GROWTH CONDITIONS
 Four Citrullus species: C.lanatus, C.colocynthis, Fruit samples (5-10) g from the center of fleshy
C.ecirrhosus, C.rhemii fruit into test tubes & keep on ice
 Two cultivars of C.lanatus: Sugar baby and Mallali
 F7-F9 lines (stemming from a cross between
Sugar baby and Mallali of C.colocynthis)were Store sample at -80°C for the further analysis of sugar
analysed for sugar content in the mature fruit content and enzyme activity
 Seven were selected for detailed analysis
 An additional three commercial varieties served
as controls in the various experiments: Hybrid Squeezed fruit juice was centrifuged at 9000 rpm for 10 mins at 4°C
313, Odem, Crimson sweet
 Developing fruits were collected at 12, 19, 27, 35,
42 days after pollination for sugar analyses and Dilute juice with ddH₂O & filter through a nylon filter
determination of invertase activity.
 Fruits were collected at four stages for the
determination of SuSy and SPS activities at 20, Analyse separate sugars on HPLC system (SugarPak column,
30, 40, 50 after pollination. refractive index detector)
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 INVERTASE EXTRACTION AND ASSAY

200mg of lyophilized melon flesh

The supernatant was dialyzed in a
samples were homogenized in 3
membrane & stirred gently for
ml extraction buffer
16hrs at 4°C against 25mM NaOH
Each sample is centrifuged at
& 0.25mM Na - EDTA
18000 rpm for 30mins at 4°C

The solution remaining in the

membrane was collected for
analysis of soluable invertase
activity
While the pellet were
homogenized with 3ml extraction
buffer and collected for analysis of
insoluble invertase activity

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 EXTRACTION OF SuSy AND SPS

Proteins were
5ml of separated on
extraction Samples a 5ml
buffer & 20µl were Sephadex gel
of PMSF were centrifuged filtration
added to 2g at 3600rpm column &
frozen for 5 mins at washed with
watermelon 4°C 3 volumes of
flesh samples Column was then Eluted extractextraction
buffer
centrifuged at solution was taken
100rpm for 1min at for analysis of SuSy
4°C and SPS activities.

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 SPS ACTIVITY
SuSy ACTIVITY
Addition of 40 µl of
extract to 30µl Hepes
buffer ( it includes SuSy assay is identical to that of SPS activity .
fructose 6-P and UDP
glucose) Except that the reaction mixture contained
25mM fructose and did not contain fructose
Samples were incubated
for 30 mins at 37°C and 6-p & glucose-6-P
reaction was stopped by
the addition of 70µl of 30
% KOH
Tubes were placed in STATISTICAL ANALYSIS
boiling water for 10min
to destroy any non
reacted fructose and
fructose-6-P Data were tested by one way
ANOVA or simple regression
After cooling, 3 ml of a for analysis of multiple
mixture of 0.14%
anthrone in 13.8M H₂SO₄ variant regression.
was added

Incubated in a water
bath at 40°C for 20 mins
Colour developed
measured at 620nm 9
RESULTS AND DISCUSSION:
GENETIC VARIABILITY IN THE
PROPORTIONS OF THE VARIOUS
SUGAR IN WATERMELON

 Commercial varieties of C.lanatus (Sugar baby &

Mallali) were crossed with wild type of Citrullus

 Sucrose concentration of 20-40 mg/ml similar in 3

varieties (sugar baby, mallali, hybrid 313)

 Sucrose and glucose concentration ranged from

10-100mg/ml and fructose – 120mg/ml

 Percentage of sugar , fructose, glucose were

20:45:35 (sugar baby) , 30:40:30 (Mallali),
Fig. 1. Sucrose (A), fructose (B) and glucose (C) concentrations in mature 20:50:30 (hybrid313)
watermelon fruits of the commercial varieties Mallali, Sugar Baby and hybrid
313 and over 150 F2 and backcrosses among the wild species Citrullus
colocynthis, C. ecirrhosus and C. rhemii and the commercial C. lanatus
cultivars (Sugar Baby and Mallali).
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 SUGAR ACCUMULATION IN
SEVEN F8 LINES OF
DIFFERENT GENOTYPES
 Sugar accumulation was analysed
from a cross between C. lanatus &
C.colocynthis characterized by low %
of sucrose and high % of fructose in
mature fruit ( plant line – 7, 13 & 19)

 F8 lines characterized by high % of

sucrose (plant line – 30, 35, 46, 52)

 The commercial C.lanatus variety

odem served as control

 The control fruits were characterized

by early accumulation of fructose and
glucose where sucrose began to
Fig. 2. Sugar accumulation in seven F8 lines of genotypes differing in level of sucrose accumulation accumulate after 3 weeks of
and in the control variety Odem. Plant lines 7, 13 and 19 accumulate a low level of sucrose while
plant lines 30, 35, 46 and 52 accumulate a high level of sucrose. Fruits were collected at 7- to 10-
pollination.
day intervals from field-grown plants under a 40-mesh net.
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 In vitro ACTIVITY OF INSOLUBLE
INVERTASE IN ACIDIC AND
BASIC pH

 Various plant lines were tested for the

activities of the insoluble invertases in
both acidic and basic buffers

 A sharp decline in enzyme activity was

measured 4 weeks after pollination in
the four lines that were characterized by
very high sucrose content

 Insoluble invertase activity in the control

variety remained high and similar to that
of three lines accumulating a low level of
sucrose (7, 13, 19)
Fig. 3. In-vitro activity of insoluble invertase under acidic (pH 5.0) and basic (pH 7.5)
conditions and in watermelon fruits collected at various developmental stages from field-
grown plants. Full symbols, low-sucrose-accumulating genotypes; empty symbols, high-
sucrose-accumulating genotypes.
 RELATIONSHIP BETWEEN ACID
INSOLUBLE INVERTASE AND ACID
SOLUBLE INVERTASE
Regression analyses were performed to
examine the relationship between insoluble
invertase activity and sucrose concentration
in the fruit.

A significant negative correlation between

insoluble invertase activity and fruit sucrose
concentration was obtained (r=0.91)

In all lines, this enzymes activity was close to zero

during the last stage of fruit development.

The correlation coefficient between soluble invertase

activity and sucrose levels was also found to be (r=0.57)
Fig. 4. Relationship between sucrose concentration in watermelon fruits and: (A)
acid insoluble invertase activity and (B) acid soluble invertase activity. Fruit
samples were collected from the control variety Odem and seven genotypes
differing in their fruit sucrose content at various stages of fruit development. Full
symbols, low-sucrose accumulating genotypes; empty symbols, high-sucrose
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 SOLUBLE INVERTASE
ACTIVITY UNDER ACIDIC
AND BASIC pH
 Values of soluble invertase activity
were much lower than those of
insoluble invertase.

 Peak activity values for soluble

invertase in basic buffer is lower than
that of measured insoluble invertase
similar in all plant line

 During first 4 weeks after pollination ,

activities of soluble invertase in acidic
buffer were higher in the low sucrose
accumulating lines (7, 13, 19) than in
Fig. 5. In-vitro activity of soluble invertase under acidic (pH 5.0) and basic high sucrose accumulating lines
(pH 7.5) conditions and in watermelon fruits collected at various
developmental stages from field-grown plants. Full symbols, low-sucrose-
accumulating genotypes; empty symbols, accumulating genotypes.

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IN VITRO ACTIVITY OF SuSy
 An additional set of experiments was
performed with progeny of lines 7( low
sucrose) 30 ( high sucrose ) and the
commercial variety crimson sweet as a
control .

 Activity levels of SuSy were low and

similar for all lines during the first
month of fruit growth.

 SuSy activity was much lower than the

SPS activity .

 As a result reduction in soluble acid

invertase and an increase in SPS
activity.

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 IN VITRO ACTIVITY OF SPS

 Difference between the low sucrose

accumulating line 7 and the other two
examined lines are (30 and crimson sweet)
were also found for SPS activity.

 SPS activity almost constant throughout the

fruit growth, Fruit of plant line 7 were
significantly lower than in fruit of plant line 30
and crimson sweet.

 An increase in SPS activity following a

decrease in soluble acid invertase has been
observed.

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 RELATIONSHIP BETWEEN SUCROSE
CONCENTRATION IN WATERMELON

 Regression analyses revealed a higher correlation

between SuSy activity and sucrose concentration
between SPS activity and sucrose concentration.

 These results indicate that SuSy activity in the

direction of sucrose synthesis also play important
role in determining the composition in
watermelon fruits.

Fig. 8. Relationship between sucrose concentration in watermelon fruits

and: (A) sucrose synthase (SuSy) activity and (B) sucrose phosphate
synthase (SPS) activity. Fruit samples were collected from the control
variety Crimson Sweet (CS), low sucrose-accumulating plant line 7 and high-
sucrose-accumulating plant line 30 at various stages of fruit development .
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 CONCLUSION
 Characterization of high genetic variation in the proportions of sugars in watermelon fruits was done.

 Within the genus Citrullus, some genotypes that accumulate a high percentage of sucrose, while others
accumulate high percentage of glucose and fructose.

 Analyses of the activity of enzymes involved in sugar metabolism showed a significant negative correlation
between insoluble invertase activity and fruit sucrose level.

 This suggests that sucrose accumulation is determined both by its synthesis as well as breakdown.

 The mechanism of sucrose accumulation in the fruit is the major factor contributing to the genetic
variation in the different cultivars.

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 REFERENCES
 Brown AC, Summers WL. Carbohydrate accumulation and color development in watermelon. J
Am Soc Hortic Sci 1985;110:683–6.
 Chrost B, Schmitz K. Changes in soluble sugar and activity of a-galatosidases and acid invertase
during muskmelon (Cucumis melo L.) fruit development. J Plant Physiol 1996;151:41–50.
 Dali N, Michaud D, Yelle S. Evidence for the involvement of sucrose phosphate synthase in the
pathway of sugar accumulation in sucrose accumulating tomato fruits. Plant Physiol
1992;99:434–43.
 Elmstrom GW, Davis PL. Sugars in developing and mature fruits of several watermelon cultivars. J
Am Soc Hortic Sci 1981;106:330–3.
 Gao Z, Schaffer AA. Novel alkaline a-galactosidase from melon fruit with a substrate preference
for raffinose. Plant Physiol 1999;119:979–88.
 Godt DE, Roitsch T. Regulation and tissue-specific distribution of mRNAs for three extracellular
invertase isoenzymes of tomato suggests an important function in establishing and maintaining
sink metabolism. Plant Physiol 1997;115: 273–82.

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 Hubbard NL, Huber SC, Pharr DM. Sucrose phosphate synthase and acid invertase as
determinants of sucrose concentration in developing muskmelon (Cucumis melo L.) fruits. Plant
Physiol 1989;91:1527–34.
 Huber SC, Huber LH. Role and regulation of sucrose phosphate synthase in higher plants. Annu
Rev Plant Physiol Plant Mol Biol 1996;47:431–44.
 Sinha AK, Pathre U and Sane PV. (1997). Purification and characterization of sucrose-
phosphate synthase from Prosopis juliflora. Phytochemistry 46 (3): 441-447.
 Tester RF and Karkalas J (2003). Carbohydrates: classification and properties In Encyclopedia of
Foos Sciences and Nutrition (2nd edition), Benjamin Caballero (Ed.), Academic Press.
 Wang, Jianping & Nayak, Spurthi & Koch, Karen & Ming, Ray. (2013). Carbon partitioning in
sugarcane (Saccharum species). Frontiers in plant science. 4. 201. 10.3389/fpls.2013.00201.