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RESEARCH ARTICLE
Desiccation-induced ROS accumulation and lipid catabolism
in recalcitrant Madhuca latifolia seeds
Jipsi Chandra
1
•S. Keshavkant
1
Received: 9 December 2016 / Revised: 23 September 2017 / Accepted: 9 November 2017
ÓProf. H.S. Srivastava Foundation for Science and Society 2017
Abstract Loss of viability in desiccation-sensitive seeds
of Madhuca latifolia (Roxb.) J. F. Macbr., an important
multipurpose tropical tree, was correlated with seed water
content (WC). WC declined from 0.59 to 0.19 g g
-1
fresh
mass, 35 days after harvest from mother plant, at ambient
conditions (temperature 25 ±2°C, relative humidity
50 ±2%). The desiccation-induced reduction in viability
was related with an accumulation of reactive oxygen spe-
cies (ROS) that promoted lipid peroxidation associated loss
of membrane integrity. Conducted study revealed 1.6–19
folds rise in lipid peroxidized products in desiccated M.
latifolia seeds, and was found to be linked inversely with
WC and germination percentage. Additionally, increased
activities (7 and 13 folds) of lipid hydrolyzing enzymes;
lipase (EC 3.1.1.3) and lipoxygenase (EC 1.13.11.12)
respectively, were discernible in desiccating M. latifolia
seeds. In summary, increased ROS, lipid oxidation, lipase
and lipoxygenase were strongly correlated with viability
loss in desiccating M. latifolia seeds.
Keywords Desiccation Madhuca latifolia Lipase Lipid
peroxidation Lipoxygenase Reactive oxygen species
Water content
Introduction
Plants have been an indispensable part of human life for
ages. Ever since ancient times, their fruits, seeds even roots
and branches have been used to meet personal and social
needs such as food, curing diseases and beautifying the
planet (Canan et al. 2016; Sorkheh and Khaleghi 2016;
Yazici and Sahin 2016). Madhuca latifolia (Roxb.) J.
F. Macbr., a commercially important tropical tree, is
mostly propagated through seeds (Orwa et al. 2009; Royal
Botanic Gardens Kew 2016). Recalcitrant, desiccation-
sensitive seeds are widespread in nature and their loss of
viability has been ascribed to a variety of factors, including
oxidative stress via reactive oxygen species (ROS) and
resulting physical damage to cell membranes and other
organelles (Berjak and Pammenter 2008). However, the
desiccation sensitivity and associated mechanisms of seed
death in M. latifolia have not been reported.
Recalcitrant seeds possess relatively high
[0.4–4.4 g g
-1
dry mass (DM)] water content (WC) at the
time of shedding from their mother plant, hence are
metabolically highly active, and quite sensitive towards
desiccation and low temperature (Berjak and Pammenter
2004). Such seeds deliberately lose water even under
ambient temperature and optimum relative humidity
(Umarani et al. 2015). Desiccation leads to imbalanced
metabolism, intracellular damage and death of embryos
even at higher WCs within weeks or months time,
depending on the species (Pammenter and Berjak 1999).
This metabolism related seed deteriorations are intimately
related with the over production of ROS due to inefficient
functioning or failure of ROS scavenging system (Anjum
et al. 2015). Accelerated production of ROS causes oxi-
dation of membrane lipids, proteins and nucleic acids
(Berjak and Pammenter 2008). These types of desiccation-
&S. Keshavkant
skeshavkant@gmail.com
1
School of Studies in Biotechnology, Pt. Ravishankar Shukla
University, Raipur 492 010, India
123
Physiol Mol Biol Plants
https://doi.org/10.1007/s12298-017-0487-y
induced ROS mediated damage in membranes and accu-
mulation of toxic by-products of oxidative metabolism may
be the basic cause for viability loss in recalcitrant seeds
under ambient storage (Umarani et al. 2015).
A number of ROS are known, among which superoxide
anion (O
2), hydroxyl radical (
.
OH) and hydrogen peroxide
(H
2
O
2
) are most potent in causing damage to macro-
molecules (Gill and Tuteja 2010). Among the macro-
molecules, cellular lipids, particularly poly unsaturated
fatty acids (PUFAs), are quite sensitive to ROS, and its
peroxidation is the major cause of quality, vigour and
viability loss of seeds, under ambient storage (Shaban et al.
2013). In addition to ROS, lipids are also catabolised by
lipoxygenase (LOX), particularly by hydroperoxidation of
the cis–cis-1,4-pentadiene structures of fatty acids, and
releases lipid hydroperoxide (LOOH), a more reactive form
of fatty acid. The LOOH thus formed is further degraded
into several other cytotoxic reactive products viz.; malon-
dialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE) and
conjugated dienes (CD), which can reduce seed viability
(Anjum et al. 2015). Among these products, the 4-HNE is
highly toxic, and even trace of it is sufficient to attack
protein, nucleic acids and mitochondrial respiration leading
to loss of viability. Additionally, due to high instability/
reactivity, it readily makes conjugates with proteins and
nucleic acids, which are again toxic to the cell (Parkhey
et al. 2012).
Further, ROS are also shown to cause de-esterification
of seed phospholipids, therefore, leading to accumulation
of free fatty acids (FFAs) in desiccating recalcitrant seeds
(Ratajczak and Pukacka 2005). However, in addition to
ROS, activity of lipase (EC 3.1.1.3) also adds FFA by
hydrolysing the ester-carboxylate bonds of lipids, particu-
larly at the organic-aqueous interface, thus playing critical
role in loss of viability (Anjum et al. 2015). Free fatty acid
also serves as membrane destabilizing agent, and substrate
for the LOX, hence may be the initiator of degenerative
reactions. Moreover, due to the detergent like chemistry,
FFAs are found to damage mitochondrial membranes
resulting in reduced energy production (Mosavi Nik et al.
2011). Accumulation of FFA also cause reduced cellular
pH which is highly detrimental to normal metabolism and
functioning of enzymes leading to loss of viability (Xia
et al. 2015). Considering the above discussed facts, the
present study was aimed to monitor changes in the mem-
brane integrity (by measuring electrolyte leakage), tissue
viability status, ROS (O
2,H
2
O
2
and.OH), total lipid
content and its oxidized products (FFA, CD, LOOH, MDA
and 4-HNE), and activities of lipase and LOX enzymes in
tissues of desiccating M. latifolia seeds.
Materials and methods
Seed collection
Madhuca latifolia (Roxb.) J. F. Macbr. seeds were har-
vested by manual plucking off the ripened greenish-yellow
drupes (66 days after flowering), which differentiate them
from unripe green drupes during their development/matu-
ration (Chandra and Keshavkant 2016), from the randomly
selected healthy trees growing at Village Attari, 8 km to
North-West of Pt. Ravishankar Shukla University Campus,
Raipur (22°330Nto21°140N, 82°60to 81°380E, 305 MSL),
India. Within an hour of collection, drupes were brought to
the laboratory and immediately processed to separate
mature seeds. Healthy, uniform sized seeds were sorted out
and kept in perforated plastic trays at ambient conditions
(25 ±2°C temperature, 50 ±2% relative humidity).
These seeds and their parts were assayed for physiological
estimations at every five-day intervals until germination
capacity was lost. For biochemical estimations, embryonic
axes (EA) and cotyledon tissues were separately frozen in
liquid nitrogen, on each of the harvest dates, and were then
stored in sterile vials at -80 °C (U410, Eppendrof, Ger-
many). All the experiments were performed twice and in
five replications, and within 2 months of post harvest
storage of liquid nitrogen frozen tissues at -80 °C.
Germination assessment
To assess germination percentage, 20 seeds each in five
replicates, at five-day intervals, were surface sterilized
(5 min) with 1% (v/v) sodium hypochlorite solution
(50 ml), followed by thorough washing (5 times) with
MilliQ water (MW) (Millipore Gradient A-10, USA).
These seeds were allowed to germinate inside the two
layers of MW saturated filter paper (Whatman No. 1)
towels, rolled inside the plastic sheet, in the dark at
25–27 °C, and till appearance of the radicle (at least 5 mm)
in each of the seeds (Varghese et al. 2002). Germination
test was assessed for 15 days because after this duration the
seeds became black in colour and/or undergo fungal
manifestations.
Determination of WC
Water content of the whole seed was determined following
the procedure of Parkhey et al. (2014). Five independent
sets containing 10 seeds each were weighed using an
electronic balance (BSA 224S-CW, Sartorius, Sweden),
before and after oven drying at 102 °C for 48 h. Water
content was calculated gravimetrically on the fresh mass
(FM) basis and expressed as g g
-1
FM.
Physiol Mol Biol Plants
123
Monitoring of electrolyte leakage
Leakage of electrolyte was measured in five replicates,
following the protocol of Blum and Ebercon (1981). For
this, two seeds in each replicate were dipped in 20 ml of
MW and kept on shaker for 24 h at 50 rpm and at ambient
temperature. Thereafter, electrical conductivity of the
solution was measured (C
0
) using EC-TDS analyzer (CM-
183, Elico, India). Then, samples were boiled (100 °C) for
20 min and electrolyte content was determined (C
1
).
Results were expressed as percentage of electrolyte leakage
Seed
-1
=C
0
/C
1
9100.
Assessment of tissue viability
The tetrazolium test (TZ test) was performed following
Chandra et al. (2015), and in five replicates. Ten de-coated
M. latifolia seeds were immersed in 1% (w/v) solution of
2,3,5-triphenyl tetrazolium chloride and incubated in the
dark at ambient condition for 12 h. Red coloured formazon
thus formed were screened visually and then extracted
separately for EA and cotyledons, with 100% ethanol.
Absorbance of extracted formazon was read at 520 nm
using a UV–Vis spectrophotometer (Lambda 25, Perkin
Elmer, USA) and expressed as A
520
g
-1
FM.
Release of ROS
Weighed amounts (0.2 g) of EA and cotyledons, in five
replicates, were extracted in cold (4 °C) sodium phosphate
buffer (0.2 M, pH 7.2) comprising diethyl dithiocarbamate
(DDC, 10
-3
M) to inhibit SOD activity (Sangeetha et al.
1990). The homogenate was immediately centrifuged for
5 min at 10,000 g. In the supernatant, O
2was measured
by its capacity to reduce nitro blue tetrazolium
(2.5 910
-4
M). The absorbance of the end product was
measured at 540 nm. Amount of the O
2was calculated
following the extinction coefficient of
2.16 910
4
M
-1
cm
-1
and values were expressed as
lmol g
-1
FM.
Amount of H
2
O
2
was assessed in five replicates fol-
lowing the method of Velikova et al. (2000). Seed tissues
(0.2 g) were homogenized with 2 ml of 0.1% (w/v) tri-
chloroacetic acid (TCA) followed by centrifugation at
12,000gfor 15 min at 25 °C. Absorbance was noted at
390 nm by adding 1 ml of supernatant into 1 ml of 10 mM
sodium phosphate buffer (pH 7) and 2 ml of 1 M potas-
sium iodide. Content of H
2
O
2
was calculated following an
extinction coefficient of 0.28 lM
-1
cm
-1
and expressed as
lmol g
-1
FM.
Hydroxyl radicals were estimated following the protocol
of Halliwell et al. (1987). For this, seed tissues (0.2 g) were
incubated in sodium phosphate buffer (10 mM, pH 7.4)
comprising 15 mM 2-deoxyribose, at 37 °C for 2 h. The
0.7 ml of this solution was further incubated with 3 ml of
0.5% (w/v) thiobarbituric acid (TBA) prepared in 5 mM
NaOH and 1 ml glacial acetic acid, at 100 °C for 30 min
and then cooled at 4 °C for 10 min. After centrifugation,
absorbance of the supernatant was recorded at 600 and
532 nm. Its amount was estimated using the extinction
coefficient 155 mM
-1
cm
-1
and mean of five replicates
was expressed as nmol g
-1
FM.
Total lipid: extraction and estimation
Five replicates having 4 g each of liquid nitrogen frozen
EA or cotyledons were ground into fine powder and
extracted in petroleum ether (40–60 °C) using soxhlet
apparatus for 8 h (Raheja et al. 1973). Content of total lipid
was determined gravimetrically and expressed as
gg
-1
FM.
Assessment of lipase activity
The enzyme source was extracted in five replicates.
Weighed amounts (0.2 g) of liquid nitrogen frozen fine
tissue powders were homogenized with 2 ml of borate
buffer (0.2 M, pH 7.4) consisting 20% polyvinyl pyrroli-
done, and centrifuged at 12,000gfor 15 min (Keshavkant
and Naithani 2001). An aliquot (100 ll) of extracted
enzyme was mixed properly with 1 ml of substrate (0.98%
(w/v) sodium chloride, 5 g gum acacia and 5 ml olive oil),
and allowed to stand at 37 °C for 1 h. The tube was then
kept in a water bath (90 °C) for 2 min to stop the reaction.
Thereafter, 6 ml chloroform and 2 ml sodium phosphate
buffer (0.66 mM, pH 6.2) were added to it and kept for
30 min at ambient condition. Then, in the bottom layer,
3 ml of copper triethanolamine reagent (1 M tri-
ethanolamine, 1 N acetic acid, 6.45% (w/v) copper nitrate)
was added and once again incubated for 30 min. Now, in
the bottom layer, 11 mM DDC (100 ll) was mixed and
absorbance was read at 440 nm (Itaya and Ui 1965). A
standard curve of stearic acid was prepared and lipase (EC
3.1.1.3) activity was expressed in terms of lmol min
-1
g
-1
FM.
Determination of FFA
Estimation of FFA was performed in five replicates fol-
lowing the method of Itaya and Ui (1965). The 4 ml of
chloroform and 1 ml of 0.66 mM sodium phosphate buffer
(pH 6.2) were added to 0.1 ml of extracted lipid (Raheja
et al. 1973), and were allowed to stand for 30 min at
Physiol Mol Biol Plants
123
ambient condition. The 3 ml of copper triethanolamine
reagent (as prepared previously) was added to the chloro-
form layer of it and then once again incubated for 30 min.
Now, 20 ll of 11 mM DDC was added and absorbance of
the sample was recorded at 440 nm. Content of FFA was
expressed as mg g
-1
Lipid.
Assay of LOX
Activity of LOX (EC 1.13.11.12) was assayed following
the method of Grossman and Zakut (1979). A mixture of
0.25 ml of Tween-20 and 7 ml of linoleic acid, dissolved in
ethanol, was used as substrate. For LOX assay, borate
buffer (0.2 M, pH 7.4) extracted enzyme (10 ll) was added
to 1990 ll of substrate, in five separate replicates. Initial
and final absorbance of the complex was recorded after 0
and 5 min respectively, at 234 nm. Activity of LOX was
calculated using extinction coefficient 25 mM
-1
cm
-1
and
expressed as lmol min
-1
g
-1
FM.
Estimation of CD
Weighed amounts (0.2 g) of liquid nitrogen crushed sam-
ples, in each five replicates, were homogenized with
methanol containing 0.02% (w/v) ethylenediaminete-
traacetic acid, 1% (w/v) sodium chloride and 2 ml of
chloroform. Homogenate was centrifuged at 12,000 gfor
15 min at 4 °C. An aliquot of supernatant was taken out
and dried under liquid nitrogen. The residue was dissolved
in ethanol and absorbance was recorded at 234 nm (Gidrol
et al. 1989). An extinction coefficient of 25 mM
-1
cm
-1
was used to deduce the amount of CD and values were
expressed as lmol g
-1
FM.
Monitoring of LOOH
To measure LOOH, procedure of Delong et al. (2002) was
followed, and assessed in five replicates. Liquid nitrogen
crushed (0.2 g) EA and cotyledon tissues were homoge-
nized in 2 ml of ice-cold ethanol: MW (80:20, v/v) mixture
and centrifuged at 4 °C for 10 min at 11,000g. Supernatant
(500 ll) thus obtained was mixed with 500 llof10mM
triphenyl phosphine, prepared in methanol, and allowed to
stand at 25 °C for 30 min. Thereafter, 2 ml of the ferrous
xylenol orange reagent (4 mM butylated hydroxyltoluene,
250 lM ferrous ammonium sulphate hexahydrate, 100 lM
xylenol orange, 250 mM H
2
SO
4
and 90% methanol) was
added in it, incubated at room temperature for 60 min, and
absorbance was measured at 560 nm. Content of LOOH
was calculated using extinction coefficient
60,000 M
-1
cm
-1
and were expressed as mmol g
-1
FM.
Monitoring of lipid peroxidation
To measure lipid peroxidized products, weighed amounts
(0.2 g) of liquid nitrogen crushed samples, in five repli-
cates, were homogenized with 0.5% (w/v) TBA dissolved
in 20% (w/v) TCA (Hodges et al. 1999). The homogenate
was incubated for 30 min in a water bath at 100 °C and
then shifted to 0 °C for 15 min, so as to stop the reaction.
Thereafter, centrifuged the sample at 11,000gfor 10 min,
and absorbance of the supernatant was recorded at 532 and
600 nm. The quantity of MDA present was calculated from
the extinction coefficient of 157 mM
-1
cm
-1
and expres-
sed as mmol g
-1
FM.
Assessment of 4-HNE
The 4-HNE contents were estimated in five replicates fol-
lowing the method of Ray et al. (2007). Weighed amounts
(0.2 g) of liquid nitrogen grinded seed tissues were
homogenized with 0.2 M chilled borate buffer (pH 7.4) and
1.5 ml of 10% (w/v) TCA, centrifuged at 11,000gfor
15 min. Collected supernatant was incubated for 2 h at
ambient condition after addition of 1 ml 2,4-dinitro-
phenylhydrazine (1%, w/v), prepared in 0.5 M HCl. Sam-
ples were now extracted with hexane and then evaporated
under liquid nitrogen. Absorbance of the product was
measured at 350 nm by addition of 2 ml of methanol to it.
Amount of the 4-HNE was calculated using an extinction
coefficient of 13,750 M
-1
cm
-1
and expressed in terms of
mmol g
-1
FM.
Statistical analyses
One-way ANOVA was employed to assess the influence of
desiccation on different parameters. Moreover, associations
between various parameters were tested using Pearson’s
correlation coefficient test. All the statistical analyses were
performed using SPSS software (Version 16.0). Values
presented are mean ±standard deviation of five separate
replicates, and all the experiments were performed twice.
Results
Freshly harvested 0 day seeds of M. latifolia with
0.59 g g
-1
FM WC showed 100% germination, which was
maintained up to 10 days after harvest (DAH, 0.48 g g
-1
FM WC). Later, germination percentage declined gradu-
ally, and was lost completely at 35 DAH (0.19 g g
-1
FM
WC) (Table 1). The decline in seed WC was strongly
correlated with loss of germination percentage (Table 2).
The critical water content (CWC) for M. latifolia seed was
0.48 g g
-1
FM.
Physiol Mol Biol Plants
123
Table 1 Changes in water content, germination percentage, electrolyte leakage, viability and reactive oxygen species (superoxide, hydrogen peroxide and hydroxyl radical) levels in
desiccating (temperature 25 ±2°C, relative humidity 50 ±2%) Madhuca latifolia seeds
Days after harvest
0 5 1015 20 253035
WC (g g
-1
FM) 0.59 ±0.010
A
0.59 ±0.010
A
0.48 ±0.027
B
0.43 ±0.029
C
0.40 ±0.012
D
0.28 ±0.019
E
0.25 ±0.020
F
0.19 ±0.015
G
GP (%) 100 ±0
A
100 ±0
A
100 ±0
A
95 ±4
B
70 ±6
C
55 ±8
D
20 ±4
E
0±0
F
EL (%) 0.130 ±0.005
F
0.147 ±0.006
E
0.146 ±0.005
E
0.158 ±0.007
D
0.168 ±0.006
DC
0.176 ±0.007
C
0.217 ±0.010
B
0.285 ±0.010
A
TZ staining
Viability (A
520
g
-1
FM)
Cotyledons 14.51 ±0.69
A
13.62 ±1.01
B
10.97 ±0.65
C
6.12 ±0.45
D
2.56 ±0.27
E
1.84 ±0.012
F
0.58 ±0.04
G
0.21 ±0.02
G
Embryonic axes 41.82 ±0.95
a
40.39 ±0.64
b
39.63 ±0.70
b
27.64 ±0.90
c
13.81 ±0.89
d
11.58 ±0.90
e
5.23 ±0.28
f
3.11 ±0.20
g
O
2(lmol g
-1
FM)
Cotyledons 1.06 ±0.19
H
1.54 ±0.03
G
1.81 ±0.05
F
2.02 ±0.04
E
2.39 ±0.02
D
2.6 ±0.05
C
3.08 ±0.08
B
4.66 ±0.08
A
Embryonic axes 1.13 ±0.01
h
2.32 ±0.01
g
2.88 ±0.01
f
4.57 ±0.07
e
5.06 ±0.05
d
5.36 ±0.05
c
6.02 ±0.07
b
7.58 ±0.4
a
H
2
O
2
(lmol g
-1
FM)
Cotyledons 0.86 ±0.02
G
0.99 ±0.06
G
1.27 ±0.06
F
1.7 ±0.04
E
2.21 ±0.17
D
2.93 ±0.06
C
3.94 ±0.28
B
6.39 ±0.46
A
Embryonic axes 7.82 ±0.40
h
9.41 ±0.15
g
11.32 ±0.33
f
13.3 ±0.17
e
15.25 ±0.36
d
28.52 ±1.12
c
32.73 ±0.62
b
37.8 ±1.37
a
.
OH (nmol g
-1
FM)
Cotyledons 0.72 ±0.03
G
0.76 ±0.01
G
0.91 ±0.02
F
1.01 ±0.01
E
1.16 ±0.02
D
1.27 ±0.01
C
1.4 ±0.04
B
1.56 ±0.03
A
Embryonic axes 3.25 ±0.14
h
4.24 ±0.11
g
5.14 ±0.12
f
5.84 ±0.09
e
6.15 ±0.06
d
6.71 ±0.17
c
7.42 ±0.30
b
9.28 ±0.32
a
Values are means of five replicates ±SD. Data point showing the same letter are insignificant at p\0.05 level
WC water content, GP germination percentage, EL electrolyte leakage, TZ tetrazolium, O
2superoxide anion, H
2
O
2
hydrogen peroxide,
.
OH hydroxyl radical
Physiol Mol Biol Plants
123
Table 2 Pearson’s correlation coefficients of the studied parameters in desiccated (25 ±2°C temperature, 50 ±2% relative humidity) Madhuca latifolia seeds
DAH WC GP EL TZ O
2H
2
O
2
.
OH TL
Cot EA Cot EA Cot EA Cot EA Cot EA
WC -0.98
GP -0.93 0.92
EL 0.88 -0.86 -0.95
TZ
Cot -0.96 0.95 0.84 -0.76
EA -0.96 0.95 0.91 -0.82 0.97
O
2
Cot 0.93 -0.91 -0.94 0.98 -0.83 -0.87
EA 0.98 -0.96 -0.88 0.88 -0.96 -0.95 0.93
H
2
O
2
Cot 0.91 -0.91 -0.96 0.99 -0.80 -0.86 0.98 0.90
EA 0.94 -0.96 -0.97 0.91 -0.86 -0.91 0.92 0.90 0.94
.
OH
Cot 0.99 -0.99 -0.95 0.90 -0.95 -0.97 0.94 0.97 0.93 0.95
EA 0.97 -0.96 -0.91 0.93 -0.91 -0.91 0.97 0.98 0.94 0.92 0.97
TL
Cot -0.89 0.92 0.90 -0.76 0.89 0.93 -0.79 -0.83 -0.83 -0.92 -0.91 -0.82
EA -0.96 0.98 0.94 -0.84 0.94 0.97 -0.87 -0.92 -0.89 -0.96 -0.97 -0.91 0.97
Lipase
Cot 0.95 -0.94 -0.95 0.97 -0.87 -0.90 0.99 0.95 0.98 0.94 0.96 0.98 -0.83 -0.91
EA 0.93 -0.93 -0.97 0.98 -0.83 -0.88 0.98 0.92 0.99 0.95 0.95 0.96 -0.84 -0.91
FFA
Cot 0.99 -0.98 -0.95 0.92 -0.94 -0.95 0.95 0.97 0.94 0.95 0.99 0.98 -0.88 -0.96
EA 0.97 -0.96 -0.98 0.95 -0.89 -0.94 0.97 0.94 0.97 0.97 0.98 0.96 -0.90 -0.95
LOX
Cot 0.90 -0.89 -0.97 0.98 -0.78 -0.85 0.97 0.87 0.99 0.95 0.92 0.92 -0.84 -0.89
EA 0.90 -0.89 -0.95 0.99 -0.77 -0.83 0.98 0.89 0.99 0.93 0.92 0.94 -0.79 -0.86
CD
Cot 0.99 -0.97 -0.91 0.89 -0.94 -0.94 0.94 0.99 0.91 0.93 0.98 0.98 -0.84 -0.93
EA 0.96 -0.95 -0.98 0.96 -0.87 -0.93 0.97 0.93 0.98 0.97 0.97 0.96 -0.89 -0.94
LOOH
Cot 0.99 -0.97 -0.92 0.90 -0.94 -0.95 0.95 0.99 0.93 0.93 0.99 0.98 -0.87 -0.94
EA 0.98 -0.95 -0.85 0.83 -0.95 -0.93 0.90 0.98 0.85 0.88 0.96 0.97 -0.80 -0.90
MDA
Cot 0.99 -0.97 -0.95 0.91 -0.94 -0.96 0.95 0.97 0.93 0.96 0.99 0.97 -0.89 -0.96
EA 0.99 -0.98 -0.93 0.90 -0.95 -0.96 0.95 0.98 0.93 0.95 0.99 0.98 -0.89 -0.96
4-HNE
Cot 20.16 0.18 0.49 20.42 0.02 0.21 20.31 20.07 20.43 20.41 20.22 20.15 20.09 20.11
EA 0.06 20.06 0.29 20.27 20.21 20.01 20.13 0.13 20.24 20.18 0.00 0.05 0.14 0.12
Physiol Mol Biol Plants
123
Table 2 continued
Lipase FFA LOX CD LOOH MDA
Cot EA Cot EA Cot EA Cot EA Cot EA Cot EA
WC
GP
EL
TZ
Cot
EA
O
2
Cot
EA
H
2
O
2
Cot
EA
.
OH
Cot
EA
TL
Cot
EA
Lipase
Cot
EA 0.99
FFA
Cot 0.97 0.96
EA 0.98 0.98 0.97
LOX
Cot 0.97 0.99 0.92 0.97
EA 0.98 0.99 0.93 0.96 0.99
CD
Cot 0.96 0.94 0.98 0.96 0.89 0.90
EA 0.98 0.99 0.97 0.99 0.98 0.97 0.95
LOOH
Cot 0.97 0.95 0.98 0.97 0.91 0.91 0.98 0.96
EA 0.92 0.88 0.96 0.92 0.83 0.84 0.98 0.90 0.98
MDA
Cot 0.96 0.95 0.98 0.98 0.92 0.92 0.98 0.97 0.99 0.96
EA 0.97 0.95 0.98 0.97 0.91 0.91 0.99 0.96 0.96 0.97 0.99
4-HNE
Physiol Mol Biol Plants
123
Leakage of electrolyte increased as seeds desiccated.
With increased desiccation, a significant rise in leachate
conductivity was observed (Table 1). Rate of electrolyte
leakage was negatively correlated with seed WC and ger-
mination percentage, but positively with levels of MDA
and ROS (Table 2).
The results of the TZ staining test revealed gradual loss
of viability of M. latifolia seeds with desiccation and DAH
(Table 1). With increased desiccation, a precipitous fall in
TZ colouration ability of M. latifolia seed was discernible
(Table 1). Loss of viability was negatively correlated with
DAH and level of ROS while positively with seed WC
(Table 2).
All the three ROS (Table 1) gradually increased in M.
latifolia seeds during desiccation. Least levels of all the
three ROS were observed in 0 day seeds, which shoots up
to 2.1–7.4 folds in 35 DAH seeds. A close dependency of
ROS generation was discernible over seed WC and DAH
(Table 2).
In response to desiccation, a gradual decline in total
lipid content was discernible in both EA and cotyledons of
M. latifolia seeds (Fig. 1). In this study, cotyledons con-
tained more total lipids than the EA. Declining lipid con-
tent was positively associated with tissue WC and
germination percentage, but negatively with ROS, lipase
and LOX (Table 2).
Consistent with this, the lipase activity (Fig. 2) and FFA
content (Fig. 3) increased with DAH and desiccation.
Higher lipase activity and FFA content were measured in
the EA compared to the cotyledons. Content of FFA and
activity of lipase were related positively with DAH and
ROS, while negatively with seed WC and germination
percentage (Table 2).
Table 2 continued
Lipase FFA LOX CD LOOH MDA
Cot EA Cot EA Cot EA Cot EA Cot EA Cot EA
Cot 20.29 20.39 20.20 0.36 20.50 20.42 20.12 0.39 20.17 0.00 20.24 20.18
EA 20.10 20.20 20.01 20.14 20.31 20.24 20.08 20.18 20.04 0.20 20.02 0.04
GP germination percentage, EA embryonic axes, EL electrolyte leakage, TL total lipid, Cot cotyledons
Values are significantly different at p\0.05 level. Negative symbol (-) shows negative correlation between two variables, while no symbol meant positive correlation between two variables.
Bold values denotes no significant relationship between two variables at p\0.05 level
Fig. 1 Variation in total lipid content in embryonic axes and
cotyledons of desiccating (temperature 25 ±2°C, relative humidity
50 ±2%) Madhuca latifolia seeds. Values are means of five
replicates ±SD. Data point showing the same letter are not
significantly different at p\0.05 level
Physiol Mol Biol Plants
123
Activity of LOX increased in response to desiccation
and along with DAH in M. latifolia seeds (Fig. 4) with
higher activity in the EA particularly in low viability seeds.
Accumulated data exhibited its positive relationship with
DAH and ROS, but negative with seed WC, germination
percentage and total lipid content (Table 2).
Like LOX, CD too exhibited increasing trend with DAH
and loss of tissue WC (Fig. 5). The level of CD was higher
in the EA compared to the cotyledons throughout. Content
of CD revealed negative association with seed WC, ger-
mination percentage and total lipid content, but positive
with DAH and ROS (Table 2).
Accumulation of LOOH increased along with desicca-
tion and DAH in M. latifolia seeds (Fig. 6). Its levels
measured in the EA were higher than in the cotyledons, all
through the investigation. LOOH exhibited positive asso-
ciation with DAH and ROS, but negative with seed WC,
and germination percentage (Table 2).
Alike LOOH, MDA too was increased in M. latifolia
seeds with extent of desiccation (Fig. 7), approving posi-
tive relationship with DAH, leakage of electrolyte, and
ROS accumulation, but negative with seed WC and tissue
viability (Table 2). Its levels were higher in the EA than
the cotyledons, in general.
In desiccating M. latifolia seeds, an initial gradual rise in
4-HNE was discernible until 15 DAH (0.43 g g
-1
FM WC)
(Fig. 8), thereafter it declined gradually. Higher amount of
4-HNE was measured in the EA, than the cotyledons,
throughout.
Fig. 2 Alteration in lipase activity in embryonic axes and cotyledons
of desiccating (temperature 25 ±2°C, relative humidity 50 ±2%)
Madhuca latifolia seeds. Values are means of five replicates ±SD.
Data point showing the same letter are not significantly different at
p\0.05 level
Fig. 3 Deviation in free fatty acids in embryonic axes and cotyledons
of Madhuca latifolia seeds during desiccation (temperature
25 ±2°C, relative humidity 50 ±2). Values are means of five
replicates ±SD. Data point showing the same letter are not
significantly different at p\0.05 level
Fig. 4 Changes in lipoxygenase activity in Madhuca latifolia seed
tissues with increased rate of desiccation (temperature 25 ±2°C,
relative humidity 50 ±2%). Values are means of five repli-
cates ±SD. Data point showing the same letter are not significantly
different at p\0.05 level
Fig. 5 Contents of conjugated dienes in embryonic axes and
cotyledons of desiccating (temperature 25 ±2°C, relative humidity
50 ±2%) Madhuca latifolia seeds. Values are means of five
replicates ±SD. Data point showing the same letter are not
significantly different at p\0.05 level
Physiol Mol Biol Plants
123
Discussion
Water content is an important factor influencing vigour and
viability of any seed under long term storage (Pammenter
and Berjak 1999; Xia et al. 2015). Our results have con-
firmed recalcitrant nature of M. latifolia seeds that lost their
viability rapidly when desiccated below the CWC
(Table 1). Our findings are in congruence with the reports
of Varghese et al. (2002) who showed a dependence of
viability over the WC of Madhuca indica seeds. M. lati-
folia seed became completely non-viable as the WC
reached to 0.19 g g
-1
FM on 35 DAH (Table 1). Similar
observations of desiccation promoted viability loss was
also reported for several recalcitrant seeds like M. indica,
Knema attenuata,Shorea robusta, etc. (Varghese et al.
2002; Vinayachandra and Chandrashekar 2012; Parkhey
et al. 2014). In addition, results of TZ staining test also
exhibited a steep decline in the tissue coloration ability
suggesting rapid loss of viability in M. latifolia seeds with
DAH and reduced seed WC (Table 1), as was also pub-
lished for desiccating S. robusta seeds (Parkhey 2013).
Increased rate of electrolyte leakage, an indicator of
enhanced permeability of cell membranes (Shaban 2013),
was discernible during loss of viability in M. latifolia
seeds, which showed close relationship with seed WC,
MDA and ROS levels (Table 2). Involvement of the lipid
peroxidation reaction in membrane deterioration and
associated loss of viability has already been documented
for recalcitrant seeds by Berjak et al. (1992) and Parkhey
et al. (2012), among many others. In general, over pro-
duction of ROS due to desiccation-induced disruption in
the metabolism, are responsible for peroxidation of lipid
moieties, particularly PUFAs (Berjak and Pammenter
2008; Roach et al. 2010). Similar was the situation in M.
latifolia seeds, in which all the three ROS significantly
increased in desiccated seeds during 35 DAH, revealing
negative correlation with loss of viability (Table 2). Just
like Quercus robur (Pukacka et al. 2011) and S. robusta
(Parkhey et al. 2012), in M. latifolia ROS seem to have
imposed damage to membrane lipids (PUFA fraction)
associated with the loss of viability during desiccation of
seeds. Peroxidation of lipids also results in the production
of free radical intermediates such as LOOH and an array of
secondary products, which have potential to cause seed
deterioration and reduced germination (Gill and Tuteja
2010; Umarani et al. 2015). In the EA and cotyledons of
desiccating M. latifolia seeds, a gradual loss in total lipid
content (3.8 fold) was discernible with simultaneous rise in
Fig. 6 Changes in lipid hydroperoxide in embryonic axes and
cotyledons of desiccating (temperature 25 ±2°C, relative humidity
50 ±2%) Madhuca latifolia seeds. Values are means of five
replicates ±SD. Data point showing the same letter are not
significantly different at p\0.05 level
Fig. 7 Deviation in lipid peroxidized product malondialdehyde in
embryonic axes and cotyledons of dehydrating Madhuca latifolia
seeds at temperature 25 ±2°C and relative humidity 50 ±2%.
Values are means of five replicates ±SD. Data point showing the
same letter are not significantly different at p\0.05 level
Fig. 8 Pattern of 4-HNE content in embryonic axes and cotyledons
of Madhuca latifolia seeds with extended dehydration at temperature
25 ±2°C and relative humidity 50 ±2%. Values are means of five
replicates ±SD. Data point showing the same letter are not
significantly different at p\0.05 level
Physiol Mol Biol Plants
123
LOOH (3.6 fold) and MDA (2.5 fold) contents (Figs. 6and
7). A good correlation of DAH was calculated for both
LOOH and MDA, which were related negatively with seed
WC, germination percentage and total lipid content
(Table 2). Our findings are in congruence with the obser-
vations reported for desiccating Quercus robur and Knema
attenuata seeds by Pukacka et al. (2011) and Vinay-
achandra and Chandrashekar (2012) respectively.
In the present study, increased lipase was found to be
associated with loss of total lipid by enhanced production
of FFA and LOOH in the EA and cotyledons of desiccating
M. latifolia seeds (Figs. 1,2,3,6). The FFA was a key
source of LOOH and free radicals in the presence of LOX
(Zacheo et al. 2000). Being a membrane destabilizing
agent, accumulation of FFA has largely been shown to be
related with the loss of viability in desiccating seeds (Xia
et al. 2015). Correlation of FFA in deterioration/loss of
viability in M. latifolia seeds have been established with
DAH and germination percentage (Table 2). Similar to our
data, lipase promoted a rise in FFA accumulation and loss
of viability in oil rich seeds of S. robusta (Parkhey et al.
2012). A positive correlation between lipase and FFA
accumulation (Table 2), thereby suggesting the involve-
ment of lipase in de-esterification of lipid moieties, was
found in desiccating M. latifolia seeds.
LOX also exhibited up-regulation (13–69 folds) in the
activity, in both EA and cotyledons of M. latifolia seeds, in
response to desiccation and with extended DAH (Fig. 4).
Our findings suggest that LOX enhanced enzymatic oxi-
dation of lipid fractions by degrading FFA and releasing
toxic aldehydic products such as LOOH, CD and MDA
(Table 2), in the EA and cotyledons respectively. In the
species like Prunus dulcis and S. robusta, existence of
active LOX has been detected even in the low water con-
taining (less than 0.4 g
-1
WC) seed tissues (Zacheo et al.
2000; Parkhey et al. 2012). In the EA and cotyledons of S.
robusta seeds, Parkhey et al. (2012) estimated LOX regu-
lated enhancement in the CD levels by 5.2 and 2.7 folds,
respectively. In addition to CD content, accumulation of
MDA was also related with loss of viability in Avicennia
marina and Antiaris toxicaria seeds during desiccation
(Greggains et al. 2001; Xin et al. 2010). The data obtained
here suggested that desiccation promoted LOX-regulated
lipid peroxidation, resulting in the accumulation of LOOH,
CD and MDA, is involved in the loss of viability in M.
latifolia seeds. Zacheo et al. (2000) stated that both CD and
LOOH may be later broken down or enter into the phe-
nomenon of chemical re-arrangement, thereby forming
secondary cytotoxic products. Similar change in the mag-
nitude of MDA and many other lipid oxidized products
were also recorded for desiccating Theobroma cacao and S.
robusta seeds by Li and Sun (1999) and Parkhey et al.
(2012).
Under oxidative conditions, the lipid peroxidation
reaction also produces highly toxic 4-HNE from its chief
precursor LOOH inside the mitochondria with subsequent
apoptotic cell death (Yin et al. 2015). A significant upsurge
in the 4-HNE content occurred in pathogen-infected
Phaseolus vulgaris and desiccating S. robusta seeds, which
was found to be linked closely with their physiological
quality or viability status (Muckenschnabel et al. 2001;
Parkhey et al. 2012). In contrast to the other products of
lipid peroxidation reaction, an initial, gradual rise in
4-HNE content was noticed until 15 DAH, which thereafter
declined abruptly to their initial levels in both EA and
cotyledons of M. latifolia seeds on 35 DAH (Fig. 8). In
this regard, it could be proposed that most of the 4-HNE
formed during the later stages of desiccation of M. latifolia
seed could not be measured following the protocol
employed, due to their conjugation with proteins formed
during oxidative stress. Further, our spectrophotometric
procedure was able to detect only free forms of 4-HNE;
therefore, conjugated forms remained undetermined in this
study. Additionally, it could also be argued that the high
reactivity and unstable nature of 4-HNE are also possible
reasons for its decline after 15 DAH in desiccating M.
latifolia seeds. A similar decline of 4-HNE was recently
reported in the EA and cotyledons of desiccating S. robusta
seeds after 5 DAH by Parkhey et al. (2012).
Conclusions
Desiccation promoted loss of viability in M. latifolia seeds
was correlated with accumulation of ROS and its lipid
peroxidation products resulting in loss of membrane
integrity. Extended desiccation contributed peroxidation of
PUFAs, correlating with the loss of viability in M. latifolia
seeds. In addition, activity of both lipase and LOX also
linked with the loss of viability through production of FFA,
CD, LOOH, MDA and 4-HNE, in desiccating M. latifolia
seeds. In summary, our findings suggested that several
mechanisms (ROS, lipid oxidation, and lipase and lipoxy-
genase) were simultaneously operative in desiccating M.
latifolia seeds finally contributing to loss of viability.
Acknowledgements The authors would like to thank Pt. Ravishankar
Shukla University, Raipur, and University Grants Commission, New
Delhi, for awarding fellowship to Jipsi Chandra under Research
Fellowship (No. 79/8/Fin.Sch/2014, dated 16.04.14) and National
Fellowship for students of Other Backward Classes (F./2016-17/NFO-
2015-17-OBC-CHH-27902) respectively. Authors are also grateful to
Department of Science & Technology, New Delhi, for financial
support through DST-FIST scheme (Sanction No. 2384/IFD/2014-15,
dated 31.07.2014) sanctioned to the School of Studies in
Biotechnology.
Physiol Mol Biol Plants
123
Authors’ contributions JC performed the experiments, generated
and analyzed the data, and drafted the manuscript. SK conceptualized
the project, designed the experimental protocols, and finalized the
manuscript draft. Both the authors read and approved the final
manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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