Industrial Crops and Products 76 (2015) 1106–1113

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Industrial Crops and Products

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Pivotal role of sugar fluxes between the inner integument and

endosperm in lipid synthesis during seed ontogeny in
Jatropha curcas L.
Bharatula Sri Krishna Chaitanya a , Sumit Kumar a , Enti Anjaneyulu b ,
Rachapudi Badari Narayana Prasad b , Pidaparty Seshadri Sastry a ,
Attipalli Ramachandra Reddy a,∗
a
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India
b
Council of Scientific and Industrial Research, Indian Institute of Chemical Technology, Hyderabad 500007, India

a r t i c l e i n f o a b s t r a c t

Article history: Jatropha curcas seed matures in 42 days after anthesis (DAA) with ∼40% oil on dry weight basis. In this
Received 29 April 2015 study, we report significant alterations in sugar fluxes in the inner integument, which might play a signif-
Received in revised form 12 August 2015 icant role in endosperm development during course of seed ontogeny in Jatropha. The inner integument
Accepted 15 August 2015
significantly contributed to seed weight up to 32 DAA which was followed by endosperm development
Available online 27 August 2015
and maturity by 40 DAA. The inner integument rapidly lost its weight from 34 to 40 DAA, accompanied
by a simultaneous gain in the weight of endosperm. Incorporation studies with 14 C-label showed that the
Keywords:
inner integument has the ability to take up both [14 C] sucrose or [14 C] glucose whereas the endosperm
Inner integument
Endosperm
incorporated only [14 C] glucose. The inner integument incorporated most of the 14 C label into esters (80%),
Starch while the endosperm effectively incorporated [14 C] glucose into glycerolipids and no label was found in
Sucrose the ester component. Starch and soluble sugar analysis of inner integument at 34 DAA revealed that ∼54%
Glucose of the dry weight of the tissue was starch while soluble sugars amounted to 10% of which sucrose content
Jatropha curcas ranged from 20 to 30%. It can be inferred that the inner integument was specialised in sucrose uptake
for its conversion mostly into starch and the remaining sugar was utilised for synthesis of esters. A rapid
uptake of glucose by the developing endosperm fueled glycerolipid synthesis. Our data on sugar fluxes
elucidate that the inner integument acts as a transient storage tissue and later the developing endosperm
was responsible for synthesis and accumulation of lipids in Jatropha seed.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Seed development is a key process in plants in which efficient
packaging and processing of genetic material and nutrients ensure
successful propagation of the species. Efficient seed development
relies on a coordinated interaction between various tissue com-
partments of the seed, both genetically and metabolically (Radchuk
and Borisjuk, 2014). Developing seeds utilize sucrose, a major com-
ponent of photosynthate, as a carbon source for the synthesis of
storage nutrients like starch, oil or protein (Bewley and Black,
1994). Sucrose import and its cleavage play a major role in trig-
gering seed development and determining the strength of the sink
Abbreviations: DAA, days after anthesis; TAG, triacylglycerols.
∗ Corresponding author. Fax: +91 4023010120.
E-mail addresses: arrsl@uohyd.ernet.in, arreddy@yahoo.com (A.R. Reddy).
tissue in several plant species (Haughn and Chaudhury, 2005). This
transition stage is of special interest during seed growth which is
characterized by major transcriptional and metabolic reprogram-
ming (Baud et al., 2002). Although the sites and pathways of sucrose
cleavage have been described in legume seeds, knowledge of mech-
anisms controlling sugar unloading and cleavage in oil seeds is
poorly known (Borisjuk et al., 2004). This limited information is
compounded further by differential pattern of development of
seed compartments in oilseeds (Baud et al., 2005). Understanding
the mechanisms underlying the regulation of seed development
in relation to its morphology and the corresponding metabolite
cross-talk between seed compartments of certain economically
important oil seeds should be a preliminary step before accom-
plishing their metabolic engineering.
Triacylglycerols (TAG), produced by domesticated oilseed crops,
are used primarily for nutritional applications. The gravity of food
to fuel ratio has shifted the focus to non-edible oil yielding plants

http://dx.doi.org/10.1016/j.indcrop.2015.08.035
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113
species including Jatropha curcas, Pongamia pinnata and Simarouba
glauca which are being domesticated for biodiesel production. Of
these, J. curcas has shown a great promise as a potential biodiesel
feed stock (Kumar and Sharma, 2008; Kumar et al., 2014). J. cur-
cas L. (Family – Euphorbiaceae) is a perennial, semi-evergreen
plant that can be grown in varied soil conditions with minimal
irrigation inputs. The seeds contain 30–45% of oil on dry weight
basis and would be an economically viable substitute for diesel
(Forson et al., 2004). The molecular basis of J. curcas seed devel-
opment in relation to TAG synthesis has been recently reported
(Xu et al., 2011; Jiang et al., 2012). These molecular studies assisted
by detailed proteomic analysis of individual tissues integrated with
anatomical studies has highlighted some of the regulatory aspects
on cellular events associated with oil biosynthesis during seed dif-
ferentiation (Liu et al., 2009; Rocha et al., 2013; Shah et al., 2015).
However, in order to have a complete understanding on the reg-
ulatory insights of TAG biosynthesis, it is necessary to discern the
operational metabolic transitioning occurring between individual
tissues during seed development which is limited in J. curcas seeds.
One such study examined the histological development of seed
coat of J. curcas and subsequent proteomic analysis within the lay-
ers of the inner integument during its development highlights the
role of certain proteins involved in developmental programmed
cell death in seeds of J. curcas (Soares et al., 2014). The same study
also reported that mature seeds of J. curcas contained the highest
concentrations of phorbol esters in the inner layer of the seed coat,
called the tegmen, from where they diffuse in to the endosperm. It is
therefore imperative to understand the metabolic fluxes occurring
between the integuments and endosperm during seed develop-
ment to fill the gaps in understanding the regulatory mechanisms
associated with oil biosynthesis in Jatropha.
In this study, we complemented the morphological observations
with biochemical characteristics in different seed compartments
to provide an evidence for the role of inner integument in
endosperm development in Jatropha seed. The metabolomics, in
relation to developmental morphology, of the inner integument
and endosperm has also been investigated in order to delineate the
mechanisms of sugar uptake and its metabolic fate in these tissues
during progressive stages of Jatropha seed development.
2. Material and methods
2.1. Plant material and growth conditions
Seeds were collected from J. curcas (cv – Chhattisgarh; saplings
planted in the year 2008; n = 10) plants grown in botanical gardens
of University of Hyderabad (17.3◦ 10 N and 78◦ 23 E at an altitude of
542.6 m above MSL), located 20 km north of Hyderabad metropoli-
tan in Telangana state, India. The plants were grown under natural
photoperiod with the spacing 2 m between the rows and 2 m within
the row. The regional climate was hot-steppe (Köppen climate clas-
sification) with the hot summer months (March–June) followed by
rains in south-west monsoon during July–October and winter dur-
ing November–January. The plants were regularly watered during
hot summer months and alternate watering during winter months
with no watering during monsoon season. Nitrogen was provided
by applying farmyard manure at the rate of 12 kg year−1 twice
in equal splits during growth. Flowering occurred frequently dur-
ing the months of July till December when the day temperatures
ranged between 20 and 34 ◦ C with little or no flowering during
the summer months. Hence, fruits and seeds of various stages for
morphological, biochemical and metabolic studies were collected
during this period (July–December) which comprised two growth
seasons. J. curcas is a monoecious plant as the inflorescence con-
tained both male and female flowers. Natural pollination resulted
1107
in transformation of the female flowers to fruits. In this study, we
have tagged the tertiary branches bearing flowers from the onset of
flowering and monitored seed development upto the harvest phase.
The first visible appearance of open flower in each inflorescence
bunch in each tree was considered as zero days after anthesis (DAA).
The fruits at different stages of development were collected from
mature Jatropha trees for four seasons during 5th and 6th year of
growth (number of fruits collected for each stage n = 60; minimum
10 fruits of each stage per tree). Fruits of 14–42 DAA were collected
and stored in −80 ◦ C for different analyses. Annual weather data
and important soil characteristics recorded at the experimental site
during the course of our study were presented in supplementary
Table S1.
2.2. Gross morphology of seed development
Jatropha fruit and seed development were investigated by fol-
lowing the morphology during four developmental stages (32, 34,
37 and 40 DAA). Transverse and longitudinal sections of fruit and
seed from these stages were taken, fixed in 10% formalin for 12 h
and dehydrated in ethanol series. These sections were observed
under microscope. Individual tissues of seeds at 34, 37 and 40 DAA
were separated and photographed. Further, free hand sections of
endosperm and distal region of inner integument with respect to
endosperm from 34, 37 and 40 DAA seeds were taken and fixed in
10% formalin for 12 h. These tissues were dehydrated in ethanol
series. The inner integument sections were stained with iodine
stain (50% v/v, Lugol’s solution) while the endosperm sections were
stained with oil red-O stain (0.5 g in 100 mL of isopropanol, stock
solution) for 5 min. Excess stain from both inner integument and
endosperm sections was washed off using water and isopropanol
respectively. These sections were placed on slides and observed
under light microscope.
2.3. Analysis of total lipids and starch
Inner integument and endosperm of 34, 37 and 40 DAA stages
were carefully isolated from their respective seeds and were mac-
erated using liquid nitrogen in a mortar and pestle. Total lipids
were extracted by homogenizing the tissue powder with a mix-
ture of chloroform and methanol in such proportions that a miscible
system was formed with the water in the tissue. Dilution with chlo-
roform and water separated the homogenate into two layers, the
chloroform layer containing all the lipids and the methanolic layer
containing all the non-lipids. A purified lipid extract was obtained
by separating the chloroform layer (Bligh and Dyer, 1959). After
extraction, total lipids were gravimetrically measured, separated
on thin layer chromatography plates and visualized by exposure to
iodine vapor. Inner integument and endosperm were freshly col-
lected and air-dried to obtain constant weight of dry tissues. These
tissues were ground with hot ethanol and incubated at room tem-
perature for 15 min. The contents were repeatedly washed with
ethanol, centrifuged and the supernatant was used to estimate sol-
uble sugars. The pellet was percolated with perchloric acid (1 mL of
35% perchloric acid) for starch analysis. An aliquot of 2 mL of sample
was added to 10 mL of anthrone reagent (pre-chilled). The reaction
mixture was shaken thoroughly and heated for 11 min at 100 ◦ C.
The tube was rapidly cooled to 0 ◦ C and absorbance at 630 nm was
measured within 1 h (Hansen and Møller, 1975).
2.4. Uptake of 14 C labelled sucrose, glucose into total lipids by
inner integument and endosperm during seed development
Seeds from 34, 37 and 40 DAA were cut to isolate the inner
integument and endosperm. To obtain 1 g of inner integument,
approximately 5, 10 and 30 seeds from 34, 37 and 40 DAA
1108 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113

Fig. 1. Gross morphology of developing Jatropha curcas fruit, seed and embryo. (A) Size, colour and texture of Jatropha fruits along development; (B) longitudinal section
of fruit (scale, 5 mm); (C) transverse section of lower region of fruit at micropylar region (scale, 1.5 mm); (D) transverse section of middle region 30 DAA stage fruit with
a locule at chalazal region (scale, 1.5 mm); (E) transverse section of 32 DAA stage seed at micropylar region (scale, 1.5 mm); (F) longitudinal section of 32 DAA seed at
micropylar region (scale, 1.5 mm); (G) embryo at 40 DAA (scale, 600 ␮m). [Note: p, placenta; mc, mesocarp; c, caruncle; f, funiculus; s, seed; oi, outer integument; Ii, inner
integument; en, endosperm; e, embryo; h, hypocotyl; cd, cotyledons]. (For interpretation of the references to colour in the text, the reader is referred to the web version of
this article.)

stages respectively were collected whereas 1 g of endosperm was
obtained from 30, 10 and 3 seeds from 34, 37 and 40 DAA stages
respectively. The entire inner integument and endosperm were
sliced into small pieces using a surgical blade under 0.1 M MES
buffer (pH 6.0) to prevent tissue oxidation. These slices were
transferred to 25 mL conical flasks containing 2 mL MES buffer,
10 mg mL−1 ampicillin and 140 ␮moles of 14 C sucrose or glucose.
Incubation was carried out in a shaker incubator at 30 ◦ C for 6 h.
At the end of incubation period, the medium was aspirated, and
the slices were washed thoroughly with cold MES wash buffer to
remove adhering radioactivity, if any. The slices were later ground
to fine powder with liquid nitrogen. Total lipids were extracted
as described above in the Section 2.3 (Bligh and Dyer, 1959). An
aliquot of 10 ␮L of total lipid extract was spotted on a piece of What-
man No. 3 filter paper and washed thoroughly with milliQ water.
The paper was air-dried, suspended in 3 mL of liquid scintillat-
ing fluid [0.5% PPO, 2,5-diphenyloxazole and 0.35% POPOP, 1,4-bis
(5-phenyloxazol-2-yl) benzene in toluene (w/v)] and radioactivity
was determined using a liquid scintillation counter (PerkinElmer,
Waltham, Massachusetts, USA). For autoradiography, 5–8 mg total
lipid was applied as a streak at the origin of the thin layer chro-
matography plate and neutral lipid separation was carried out
using hexane:diethyl ether:acetic acid (55:45:1) as a solvent. After
45 min, the thin layer chromatography plates were exposed to X-
ray films in a cassette with intensifiers for a period of 35 days
to develop autoradiograms. After the autoradiograms were devel-
oped radioactivity in individual lipid components was measured.
Thin layer chromatography plates were exposed to iodine vapor
and the lipid components were marked. These components were
scraped off and radioactivity was measured in them using liquid
scintillation counter.
2.5. Soluble sugar profile of inner integument and endosperm
using high performance liquid chromatography (HPLC)
lized in 50 ␮L MilliQ water. An aliquot (25 ␮L) of this mixture
was injected into HPLC (Agilent 1100 series, Palo Alto, CA, USA)
to analyze soluble sugars. The HPLC was equipped with a Refrac-
tive Index Detector (RID) (Agilent Technologies 1100 series, NY,
USA). The column used was Rezex RPM-Monosaccharide Pb col-
umn (300 cm × 7.8 mm) purchased from Phenomenex, USA. The
oven temperature was set at 65 ◦ C and optical unit temperature was
set at 35 ◦ C. The sugars were eluted within 10 min using a mobile
phase of MilliQ water at a flow rate of 0.8 mL min−1 .
2.6. Chemicals
[U-14 C]sucrose (435 mCi mmole−1 ) and [U-14 C]glucose
(90 mCi mmole−1 ) were obtained from BRIT (Board of Radia-
tion and Isotope Technology, Mumbai, India). Ampicillin was
procured from Sigma–Aldrich (St. Louis, MO, USA). POPOP [1,4-bis
(5-phenyloxazol-2-yl) benzene], PPO [2,5-diphenyloxazole] and
MES buffer was procured from HIMEDIA (Mumbai, India). All
solvents used were of AR grade. Thin layer chromatography plates
were procured from MERCK (White House Station, NJ, USA). X-ray
films and cassettes were procured from KODAK (Rochester, NY,
USA).
2.7. Statistical analysis
All biochemical, metabolic and chromatographic experiments
using seeds at various stages of development were performed
in triplicates and the results were presented as mean of four
seasons ± SD. Individual seeds at various stages were collected ran-
domly from the mature trees and were considered as experimental
units (n = 100). The mean values were compared using ANOVA in
SIGMA PLOT 11.0. The significant differences in the mean values
of the tested variables (P value) were Tukey corrected to reduce
errors.

The inner integuments and endosperm of 34, 37 and 40 DAA 3. Results

seeds were isolated and air-dried to obtain constant weight. Sol-
uble sugars were extracted from 1 g of the above tissues at each
stage according to Hansen and Møller, 1975. Ethanol supernatant
(100 ␮L) from each tissue extract was transferred to 1.5 mL eppen-
dorf tube and subjected to evaporation using a concentrator. After
the ethanol was completely evaporated, the sugars were solubi-
3.1. Morphological changes during seed development
The development of Jatropha seed was complete by approxi-
mately 42 days from the day after anthesis (DAA). The fruit was
tri-locular and housed three seeds. The fruit was green initially
 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113
Fig. 2. Morphological changes in seed and its components during development.
(A–C) Seed at 34, 37 and 40 DAA stages; (D–F) inner integument at 34, 37 and 40
DAA stages; (G–I) endosperm at 34, 37 and 40 DAA stages; (J–L) embryo at 34, 37
and 40 DAA stages. [Note: Scale for all images – 1.5 cm].
and increased in size up to 34 DAA. The fruit undergoes desicca-
tion from 37 DAA with a change in color from green to yellow
(Fig. 1A). The developing seeds receive photosynthates through
placenta (Fig. 1B). The seed was surrounded by a fleshy mesocarp
and the caruncle connected to the placenta through the funiculus
(Fig. 1B and C). The transverse section of the seed at 30 DAA showed
a well-developed inner integument (Fig. 1D). It was noticed that the
endosperm expansion was initiated at 32 DAA right at the centre
of the inner integument (Fig. 1E). Further, the lateral section of the
endosperm at 32 DAA showed a developing embryo (Fig. 1F). A fully
developed embryo with conspicuous cotyledons and hypocotyl was
observed at 40 DAA (Fig. 1G).
The outer integument, which was initially soft (Fig. 2A), sub-
sequently desiccated (Fig. 2B) with further hardening (Fig. 2C)
associated with complete seed development. In contrast, the
inner integument which appeared as a bulk tissue at 34 DAA
(Fig. 2D) diminished in size progressively and finally developed
into a shrunken layer like tissue (Fig. 2E and F). Interestingly,
the endosperm increased in its thickness as the seed developed
towards maturity (Fig. 2G–I). However, the nascent embryo in the
endosperm (Fig. 2J) was progressively conspicuous during 37 and
40 DAA (Fig. 2K and L). The sequence of morphological and histo-
logical changes during different stages of seed development were
diagrammatically represented in Fig. 3.
Table 1 shows the quantitative changes in fresh and dry weights
of inner integument and endosperm from 34, 37 and 40 DAA seeds.
The dry weight of inner integument diminished considerably from
20 mg to 4 mg during 34, 37 and 40 DAA (Table 1; P < 0.01). How-
ever, no significant variation in the moisture content of the inner
integument during seed development was recorded. Dry weight of
the endosperm increased by ∼3 fold (P < 0.001), with a significant
decrease in moisture content (∼90 to ∼25 %; P < 0.05) from 37 to
40 DAA seeds (Table 1). Accumulation of starch and total lipids in
the inner integument and endosperm of seeds from 34, 37 and 40
DAA were depicted in Fig. 4. Sequential decrease in starch reserves
were observed in the inner integument (Fig. 4A–C) while significant
increase in total lipids was noticed in the endosperm (Fig. 4D–F)
from 34, 37 and 40 DAA.
1109
3.2. Starch and lipid fluxes in the developing Jatropha seed
Total lipid content in the inner integument remained constant
(∼4%) during seed development, whereas starch content decreased
from 54% at 34 DAA to 3% at 40 DAA (Fig. 5A and Table 1; P < 0.001).
Total lipid content in endosperm at 32 DAA was minimal, which
could not be determined gravimetrically. However, between 34 and
40 DAA, the endosperm showed a significant increase in total lipids
ranging from 5.4% to 42.7% on dry weight basis, an increase of ∼8
fold (Fig. 5B and Table 1; P < 0.001).
3.3. Incorporation of [U-14 C]sucrose, [U-14 C]glucose by inner
integument and endosperm into total lipids during seed
development
The inner integument from 34, 37 and 40 DAA seeds effec-
tively incorporated both sucrose and glucose into total lipids and
their incorporation was reduced with seed development (Fig. 6A;
P < 0.01). The endosperm, on the other hand, did not incorporate
sucrose, while significant incorporation of glucose was recorded
from 34, 37 and 40 DAA (Fig. 6B; P < 0.001). Since 37 DAA showed
conspicuous inner integument and endosperm, this stage was
selected for incorporation studies through autoradiography in
order to determine the fate of sugars during storage lipid formation
(Fig. 7). Both sucrose (∼81% into esters, ∼20% into phospholipids)
and glucose (∼83% into esters, ∼17% into phospholipids) were
effectively incorporated by the inner integument (Fig. 7A and B).
However, the endosperm did not incorporate labelled sucrose into
total lipids (Fig. 7C) while 14 C glucose was effectively incorporated
into total lipids (glycerolipids) by the endosperm (Fig. 7D).
3.4. Soluble sugar profile in the inner integument and endosperm
during the phase of endosperm expansion
Sucrose, glucose and fructose contents gradually declined in
the inner integument which were completely depleted by 40 DAA
(Fig. 8A). Sucrose content was minimal in the endosperm from 34,
37 and 40 DAA seed, whereas glucose does not seem to increase
but only fructose increased (Fig. 8B; P < 0.001). Sugars other than
sucrose, glucose and fructose were not assessed during our analysis.
4. Discussion
Carbon and nitrogen sources for embryo and endosperm dur-
ing seed development are usually provided by the long distance
transport of photoassimilates including sucrose, amino acids and
amides through the maternal vascular tissue (Patrick and Offler,
2001). The embryo and endosperm development are facilitated
further by the coordinated interaction as well as exchange of sig-
nals and nutrients between the seed compartments (Baud et al.,
2002). Hence, it is necessary to elucidate the regulatory mecha-
nisms involved in the metabolic transitioning between seed tissues
during seed development in relation to its morphology and bio-
chemistry. In this study, significant alterations in sugar fluxes in
the inner integument, which play a significant role in endosperm
development during the course of seed development in Jatropha
were reported.
Seed is an essential sink tissue in flowering plants which houses
the embryo and endosperm. It is well known that embryo devel-
opment depends on endosperm development which in turn is
dependent on the efficient supply and unloading of photosynthates
from source leaves (Zhang et al., 2007). We observed a developing
endosperm and embryo at 32 DAA with a significant expansion
of both the tissues from 37 to 40 DAA followed by a concomitant
reduction in the size of inner integument (Figs. 1 and 2). Prior to
1110 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113

Fig. 3. Morphological changes of Jatropha seed components during development. (A) 34 DAA – soft outer integument, well developed inner integument and endosperm
housing a very small embryo; (B) 37 DAA – hardening outer integument, reducing inner integument and expanding endosperm; (C) 40 DAA – hardened outer integument,
diminished inner integument and fully developed endosperm along with mature embryo. [Note: Oi, outer integument; Ii, inner integument; En, endosperm; E, embryo; Scale
for all images – 1.5 mm].

Table 1
Dry weight, total lipid, starch and moisture content of inner integument and endosperm during Jatropha seed development. The values are mean ± SD (n = 50) and significant
differences was analyzed by one-way ANOVA among the variables between the stages. [Note: Moisture (%) = (fresh weight − dry weight)/fresh weight × 100; ns, not significant;
*P < 0.05; **P < 0.01; ***P < 0.001; ND, not detected].

Inner integument Endosperm

34 DAA 37 DAA 40 DAA 34 DAA 37 DAA 40 DAA

Fresh weight (mg) 102.3 ± 1.22 77.8 ± 0.76 *

39.8 ± 0.23**
234.2 ± 1.89 465.4 ± 5.31***
637.2 ± 9.1***
Dry weight (mg) 20.3 ± 1.22 12.4 ± 0.91* 4.6 ± 0.23** 21.3 ± 1.32 112.5 ± 7.84*** 476.9 ± 9.66***
Moisture (%) 82 ± 4.21 65.4 ± 3.96ns 35.2 ± 1.99** 90.9 ± 3.46 75.8 ± 6.45* 25.1 ± 4.98**
Total lipids (mg) 1.01 ± 0.17 0.62 ± 0.06ns 0.23 ± 0.08ns 1.1 ± 0.86 13.5 ± 1.23* 200.29 ± 7.31***
Starch (mg) 10.96 ± 1.33 2.85 ± 0.57** 0.13 ± 0.02*** ND ND ND

Fig. 4. Histochemical changes during Jatropha seed development. Distal region of inner integument (A–C) and endosperm (D–F) from 34 DAA, 37 DAA and 40 DAA seed
stages stained with iodine and oil red O respectively. [Note: Scale is 1.5 mm for all images; En, endosperm, Ii, inner integument].
 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113 1111

Composition of starch and total lipids 60

A B
on dry weight basis (in %) ***
Starch Total lipid Starch Total lipid
40

**
20
*
ns *** ns
0
34 DAA 37 DAA 40 DAA 34 DAA 37 DAA 40 DAA
Inner Integument Endosperm
Fig. 5. Starch and total lipid content in inner integument (A) and endosperm (B) from 34, 37 and 40 DAA seeds. The values are mean ± SD (n = 50) and significant differences
was analyzed by one-way ANOVA among the variables between the stages. [Note: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001].

40000 160000
Counts min-1 g tissue-1

Counts min-1 g tissue-1

A B ***
Sucrose Glucose
30000 * 120000 Sucrose Glucose
*
20000 80000
**
10000 ** 40000
ns
0 0
34 DAA 37 DAA 40 DAA 34 DAA 37 DAA 40 DAA
Inner Integument Endosperm

Fig. 6. Incorporation of [U-14 C]sucrose and [U-14 C]glucose by inner integument (A) and endosperm (B) into total lipids during seed development. The values are mean ± SD
(n = 80) and expressed as counts min−1 g tissue−1 . Significant differences was analyzed by one-way ANOVA among the variables between the stages. [Note: ns, not significant;
*P < 0.05; **P < 0.01; ***P < 0.001].

Fig. 7. Autoradiograms of [U-14 C]sucrose and [U-14 C]glucose incorporations into total lipids extracted from inner integument (A and B) and endosperm (C and D) from 37
DAA seeds (n = 80) respectively. [Note: E, esters; PL, phospholipids; TAG, triacylglycerol; DAG, diacylglycerol].

32 DAA, inner integument was the principal tissue in the develop- utilization by other components during seed development. Fur-
ing seed (Fig. 1D). The data demonstrate that the inner integument thermore, the reduction in size and desiccation of inner integument
acts as a transient storage tissue in the seed where photosyn- in subsequent days and formation of endosperm just beneath the
thates from source leaves are translocated and stored for further inner integument suggested a possible role of inner integument’s
1112 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113

25 60
A Sucrose Glucose Fructose B Sucrose Glucose Fructose

Soluble sugars (in %)

Soluble sugars (in %)
ns ***
20 50 ***
ns
ns 40 ns
15 ns
30
10
20
5 10 ns
0 0
34 DAA 37 DAA 40 DAA 34 DAA 37 DAA 40 DAA

Fig. 8. Soluble sugar content during Jatropha seed development in inner integument (A) and endosperm (B) from 34, 37 and 40 DAA seeds. The values are mean ± SD (n = 50)
and significant differences was analyzed by one-way ANOVA among the variables between the stages. [Note: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001].

T
Sucrose

Starch

Ii
Glucose
En
Phorbol
Ii ester
En
Glycerol

Fatty
acids
En
TAG

F F F
34 DAA 37 DAA 40 DAA

Fig. 9. Model depicting sugar fluxes between inner integument and endosperm highlighting their role in storage lipid synthesis and accumulation during the ontogeny of
Jatropha seed. [Note: Ii, inner integument; En, endosperm; F, funiculus; T, tegmen].

stored photosynthate in endosperm development. Earlier stud-
ies indicate that accumulation of reserves in endosperm triggers
embryo development which marks the beginning of seed develop-
ment where storage reserves in the seed nourishes the developing
embryo (Raz et al., 2001). This study complements the morpho-
logical observations with biochemical characteristics in different
seed compartments to provide an evidence for the role of inner
integument in endosperm development in Jatropha seed.
Since lipids are the major components of the mature Jatropha
seeds, we analysed the lipid content in the inner integument and
endosperm as these two tissue components are responsible for
accumulation of total lipids. The data on gravimetric estimation of
total lipids also revealed that the endosperm was the major storage
tissue for glycerolipids in the mature seed. Significant increment
in total lipid content in the endosperm was recorded from 34 to
40 DAA, while total lipid content in inner integument remained
almost the same until seed maturity (Fig. 5). It can be inferred that
endosperm is the only tissue within the seed which synthesizes
storage lipids that are later utilized for embryo nourishment dur-
ing germination (Hills, 2004). Also, the data show that the inner
integument had low lipid content (∼5%) all through seed devel-
opment suggesting that majority of available photosynthates were
chanalised for reserves other than lipids in the early stages of devel-
opment before endosperm expansion. Metabolic profile of the inner
integument has clearly shown significant accumulation of sucrose
and starch in the early stages of seed development, which were
possibly utilized by the endosperm for its development at later
stages (Figs. 5 and 8). Further confirmation on this utilization of
stored carbohydrates in the inner integument by the endosperm
into total lipids was achieved through 14 C incorporation studies.
The data on biochemical analysis and 14 C tracer studies confirmed
that the inner integument is highly specialised for sucrose uptake
which was mostly converted into starch (Fig. 5A) while the remain-
ing sugar was utilised for synthesis of esters (Fig. 7A and B). The
synthesis and further diffusion of the esters from inner integu-
ment to endosperm upon seed development has been previously
reported (King et al., 2013). The data on 14 C in this study suggest
that endosperm synthesizes glycerolipids exclusively from glucose
(Fig. 7D). The lipid synthesis in endosperm was probably aided
by sugars released by assimilation of stored starch in the inner
integument. This highlights a possible role of inner integument
in endosperm development during seed ontogeny. Metabolomics
data further confirmed the presence of sucrose in the inner integu-
ment and its absence in endosperm during different stages of seed
development (Fig. 8).
Based on the data, a comprehensive model was proposed depict-
ing sugar fluxes involved in lipid synthesis during Jatropha seed
development and maturation (Fig. 9). The inner integument is a
transient storage tissue which accumulates starch. Later, the fruit
undergoes desiccation and the connection between the seed and
the fruit is disrupted. With the passage of time, the inner integu-
ment acts as a source of glucose (obtained from reserve starch
 B.S.K. Chaitanya et al. / Industrial Crops and Products 76 (2015) 1106–1113
breakdown) for endosperm development and glycerolipid synthe-
sis. Finally, the endosperm becomes the sink and utilizes carbon for
further lipid production and storage, which was evidenced by the
decline in dry weight of inner integument with the simultaneous
expansion of endosperm.
5. Conclusions
In the present study, the relevance of sugar fluxes between
the inner integument and endosperm during seed development
was clearly demonstrated. Studies on seed morphology and
14 C-labelled sucrose and glucose incorporation at different devel-
opmental stages of Jatropha seed indicate that the inner integument
has the ability to utilize both sucrose and glucose as substrates for
metabolism, whereas the endosperm selectively utilizes glucose for
lipid biosynthesis.
Acknowledgements
This work was supported by Department of Science and
Technology (DST), Government of India (Project No. DST/IS-
STAC/CO2 -SR-68/09) to Attipalli R. Reddy. Facilities through FIST
grant to the department from DST, New Delhi, India is gratefully
acknowledged. B.S.K.C. and S.K. acknowledges the fellowship from
DST and UGC, New Delhi, India respectively. We also thank Tree
Oils India Ltd., Zaheerabad, India for providing Jatropha seeds.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.08.
035.
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