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Chapter 7 Jasmonates in macroalgae: exogenous application of
methyl jasmonate and its physiological effects in Gracilaria dura
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150
7.1. Introduction
Jasmonates are ubiquitously occurring lipid derived signals that mediate a plethora of
essential biological processes from stress and defense responses (Browse and Howe, 2008;
Howe and Jander, 2008) to reproductive development (Browse, 2005; Mandaokar et al.
2006; Wasternack et al. 2012), secondary metabolism and senescence (Browse, 2009) in the
plant kingdom. They are generated via the AOS branch of LOX pathway of lipid oxidation.
The stress responses that depend on jasmonate signaling include defense responses against
insects and pathogens (biotic stress and herbivory), responses to ozone, UV light, wounding,
and other abiotic stresses (Wasternack, 2007; Browse and Howe, 2008; Browse, 2009) while
developmental process include the modulation of root growth, flower development, tendril
coiling, senescence, and carbon portioning in healthy plants (Mandaokar et al. 2006;
Wasternack, 2007; Yan et al. 2007; Browse, 2009). Further, jasmonates exert their effects by
orchestrating large-scale reprogramming of gene expression as revealed by transcriptional
profiling and hundreds of downstream JA-regulated and JA-co-regulated genes
(Wasternack, 2007; Yan, 2007; Kombrink, 2012).
Methyl jasmonate (MeJA) or jasmonic acid methyl ester (JAME) is one of the most
active form of jasmonic acid in plants that is formed by methylation of C1 of jasmonic acid
by jasmonic acid-specific methyl transferase (JMT) (Seo et al., 2001). It was first isolated
from the essential oil of Jasminum grandiflorum in 1962 (Demole et al., 1962) and
thereafter this area of research has spun the plant biology with focus on its physiological
roles to the identification of its binding sites, respective enzymes, their cloning and
characterization to crystallization using the molecular tools of genomics, transcriptome
profiling, metabolomics and proteomics. Now the researchers are extending their expertise
to other kingdoms outside the plant such as fungus and algae to unravel its role and
metabolism.
The presence of JA and MeJA has been reported in several lineages of nonvascular
plants (Hamberg and Gardner, 1992), including unicellular green algae (Fujii et al., 1997),
Euglenophytes (Ueda et al., 1991), and the Rhodophyte Gelidium latifolium (Krupina and
Dathe, 1991). Even the entire set of enzymes necessary for the biosynthesis of JA from
linolenic acid have also been identified in the marine red algae Gracilariopsis sp. (Hamberg
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and Gerwick, 1993) and Lithothamnion corallioides (Hamberg, 1992). But their presence in
other red, green or brown macroalgae is still an enigma. However, researchers in the last
decade have succeeded a step in establishing their roles in a few algae such as Chondrus
crispus (Bouarab et al. 2004; Collén et al. 2006; Gaquerel et al. 2007), Fucus vesiculosus
(Arnold et al. 2011) and Laminaria digitata (Küpper et al. 2009) Chlorella vulgaris
(Czerpak et al. 2006) and Scendesmus spp. (Fedina and Benderliev, 2000; Christov et al.
2001; Kováčik et al. 2011). Among microalgae, MeJA regulates growth of algal cells in
Chlorella and Scenedesmus, (Pouneva et al. 1994; Czerpak et al. 2006). Application of
MeJA into algal cell suspension also inhibited the development of bacterial pathogens and
had a positive effect on the Scenedesmus incrassulatus tolerance to temperature and salinity
stress (Fedina and Benderliev, 2000; Christov et al. 2001). However, the occurrence of
MeJA/JA in both these microalgae Chlorella and Scenedesmus have not been verified.
Similarly, the treatment with MeJA conferred an induced resistance to the C. crispus against
an endophytic pathogen, Acrochaete operculata and upregulated defense enzyme activities,
such as fatty acid oxygenases, phenyl ammonia-lyase (PAL) and shikimate dehydrogenase
(SD) involved in the secondary metabolism leading to an induced resistance to the pathogen
attack. Collén et al. (2006) reported that the transcription of defense-related genes is also up
regulated after MeJA addition, including glutathione-S-transferase (GST) in C. crispus.
MeJA was also found to be strong triggers of oxidative stress in kelp Laminaria digitata
(Küpper et al., 2009). Arnold et al. (2001) reported that the exposure of F. vesiculosus to
MeJA during periods of tidal emergence causes induction of polyphenolic chemical defense,
identical to that caused by herbivory, suggesting a probable role of jasmonates as natural
elements of antiherbivore responses in Fucus. Despite the physiological relevance of MeJA
in these three macroalgae, C. crispus, F. vesiculosus and L. digitata, it is not clear, whether
MeJA is an endogenous compound in these macroalgae. Although MeJA has been detected
in cell-free extracts of C. crispus after the addition of linolenic acid (Bouarab et al. 2004),
the attempts to identify JA in C. crispus cell homogenates have remained unsuccessful.
Wiesemeier et al. (2008) were also unable to detect JA/MeJA and even their biosynthetic
precursor12-oxophytodienoic acid (12-OPDA) in seven brown macroalgal species of
Dictyota, Colpomenia, Ectocarpus, Fucus, Himanthalia, Saccharina and Sargassum.
Moreover, treatment with ecologically relevant concentrations of JA and MeJA did not lead
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to a significant change in the profile of medium- and non-polar metabolites of the tested
algae. Only after the application of higher concentrations of ≥ 500 μg ml-1 medium of the
phytohormones a metabolic response of unspecific stress was observed (Wiesemeier et al.
2008). In contrast, Ritter et al. (2008) detected 12-OPDA in response to copper stress in
Laminaria digitata, indicating that Laminaria sp. do employ the plant-like octadecanoid
metabolites to regulate protective mechanisms at least towards copper stress.
Our understanding of jasmonates in macroalgae is limited to these few reports and
the role of jasmonates in other macroalgae has not been undertaken. Thus, we attempted to
study the effect of MeJA on the lipids, fatty acids (FA) and oxylipin profiles of a
commercially important red macroalgae Gracilaria dura. We addressed the key issues like:
do MeJA triggers lipid peroxidation and oxidative stress response in G. dura like Laminaria
sp.? What are the lipidomics and hydroxy-oxylipins changes in response to MeJA in G.
dura? Does this red alga also show induction of phenolic compounds and PPO/PAL/SD
activities involved in secondary metabolism?
7.2. Materials and Methods
7.2.1. Algal culture and methyl jasmonate treatment
Gracilaria dura was collected from Adri coast (N 20º 57.58'; E 70º 16.76'), Gujarat,
India in the month of March 2010. The selected healthy thalli were carried in a cool pack to
the laboratory. They were cleaned with autoclaved seawater to remove epiphytic foreign
matters and the rhizoidal portions were removed to eliminate further contaminants. The
cleaned algal thalli were maintained under laboratory conditions in aerated flat-bottom
round flasks in Provasoli enrichment seawater (PES) medium (Provasoli, 1968) at 25 ± 1°C
temperature under daylight white fluorescent lamps at 15 μmol photon m–2 s–1 irradiance
with a 12:12 h light: dark photoperiod. The culture medium was renewed weekly.
For methyl jasmonate (MeJA) treatment, healthy algal thalli maintained under
laboratory conditions for almost six months were taken. G. dura thalli were treated by
increasing concentration of MeJA (1 μM, 10 μM and 100 μM) in ethanol in autoclaved
seawater for variable time periods (6 h, 12 h, 24 h and 48 h). In addition untreated algal
thalli were incubated with the same amount of ethanol for control for the same time periods.
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7.2.2. Determination of Lipid peroxidation (TBARS), ROS and in situ localization of ROS
The level of lipid peroxidation was determined as described by Heath and Packer
(1968). Algal tissue (0.2 g) was extracted with 2 ml of 0.5% thiobarbituric acid (TBA)
prepared in 20% trichloroacetic acid (TCA), heated at 95 °C for 30 min, cooled in ice and
centrifuged at 10,000×g for 10 min. The absorbance of the supernatant was measured at 532
nm and corrected for non-specific absorbance by subtracting the value recorded for 600 nm.
The level of lipid peroxidation was expressed as nmols of malondialdehyde (MDA) formed
using the extinction coefficient of 155 mM cm-1.
The O2•− production rate was measured according to Liu et al. (2010b). The algal
samples were homogenized in 65 mM potassium phosphate buffer (pH 7.8) (1:4, w/v) and
centrifuged at 5,000×g for 10 min. The incubation mixture contained 0.9 ml of 65 mM
potassium phosphate buffer (pH 7.8), 0.1 ml of 10 mM hydroxylaminoniumchloride and 1
ml of the supernatant. After incubation at 25 °C for 20 min, 17 mM sulphanilic acid and 7
mM α-naphthylamine were added to the incubation mixture. After reacting at 25°C for a
further 20 min, the absorbance was read at 530 nm. A standard curve, with NaNO2 was used
to calculate the production rate of O2•−. For the estimation of H2O2, the samples (100 mg
FW) were extracted in 200 μl of Na-acetate buffer (50 mM, pH 6.5) and incubated in the
reaction mixture containing 50 mM Na-acetate buffer, 1 mM 4-aminoantipyrine, 1 mM 2,4-
dichlorophenol, 50 mM MnCl2 and 0.2 mM NADH for 24 h. The oxidation of
aminoantipyrine was recorded at 510 nm and the absorbance was compared to the standard
curve prepared with H2O2 in the same reaction mixture.
Determination of HO• production was performed based on the degradation of 2-
deoxyribose by HO• (Halliwell, 2006). The samples (250 mg FW) were homogenized with
1.2 ml of 50 mM potassium phosphate buffer (pH 7.0) and centrifuged at 10,000×g for 15
min. Thereafter, 0.5 ml of supernatant was mixed with 0.5 ml of 50 mM potassium
phosphate buffer (pH 7.0) containing 2.5 mM of 2-deoxyribose. The reaction was developed
at 35°C in dark for 1 h. After adding 1 ml of 1% TBA in 0.05 M NaOH and 1 ml of acetic
acid, the mixture was boiled for 30 min and immediately cooled for 10 min on ice. The
production of HO• was followed by measuring of absorbance at 532 nm and the HO• content
was expressed as absorbance units per gram FW.
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The in situ localization of O2•− and H2O2 production was performed according to the
method of Castro-Mercado et al. (2009). For the detection of O2•− radicals, the hand cut
sections of control and MeJA-treated thalli (25 in numbers each) were immersed in 5 ml of
detection solution containing 0.05% nitroblue tetrazolium (NBT) in 50 mM potassium
phosphate buffer (pH 6.4) and 10 mM sodium azide (NaN3). The sections were infiltrated
under vacuum for 3 min in the same solution and illuminated for 2 h until the appearance of
dark spots, characteristic of blue formazan precipitates. Stained sections were cleared by
boiling in acetic acid/glycerol/ethanol (1:1:3, v/v/v) solution before photographs were taken.
H2O2 production was visually detected by an endogenous peroxidase-dependent staining
procedure using 3, 3′-diaminobenzidine (DAB). Hand cut sections of control and MeJA-
treated thalli (25 in numbers each) were immersed in DAB solution 1 mg ml-1 (pH 5.0),
vacuum-infiltrated for 5 min and then incubated at room temperature for 4 h in the presence
of light till brown spots appeared. Sectioned were bleached by immersing in boiling ethanol
to visualize the brown spots and photographs were taken.
7.2.3. Pigments analyses
The photosynthetic pigments were estimated by following the method of Dawes et
al. (1999). Chlorophyll a (80% acetone) and phycobiliproteins (100 mM phosphate buffer,
pH 6.5) were extracted by grinding the sample in their respective extraction solutions (1:4
w/v) in the dark and cold conditions followed by centrifugation at 3,000 g at 4°C for 10 min.
Absorbances were recorded at 665 nm for chlorophyll a and 620, 650, and 565 nm for
phycobiliproteins. Extinction coefficient of 11.9 was used for calculating chlorophyll a
content. Phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE) contents were
estimated using the equations mentioned below as described by Tandeau and Houmard
(1988).
PC (mg ml-1) = (OD620nm − 0.7OD650nm)/7.38
APC (mg ml-1) = (OD650nm − 0.19OD620nm)/5.65
PE (mg ml-1) = (OD565nm – 2.8 [PC] – 1.34 [APC])/12.7
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7.2.4. Determination of total lipids, ESI-MS polar lipid profiling and fatty acids
Lipids were extracted by modified Bligh and Dyer method using
chloroform/methanol/phosphate buffer (pH-7.5) (1/2/0.9, v/v/v) as described in chapter 2
and total lipid content was determined. Further, the lipid extracts were completely dried
filled with nitrogen in 2 ml glass vials and samples were sent to Kansas State Lipidomics
Research Center (KLRC), USA for the quantitative analysis of polar lipid profile of MeJA-
treated and control samples. An automated electrospray ionization-tandem mass
spectrometry approach was used, and data acquisition and analysis and acyl group
identification were carried out as described previously (Devaiah et al. 2006) with
modifications. The samples were dissolved in 1 ml chloroform. An aliquot of 500 µl of
extract in chloroform was used. Precise amounts of internal standards, obtained and
quantified as previously described (Welti et al. 2002), were added in the following quantities
(with some small variation in amounts in different batches of internal standards): 0.6 nmol
di12:0-PC, 0.6 nmol di24:1-PC, 0.6 nmol 13:0-LPC, 0.6 nmol 19:0-LPC, 0.3 nmol di12:0-
PE, 0.3 nmol di23:0-PE, 0.3 nmol 14:0-LPE, 0.3 nmol 18:0-LPE, 0.3 nmol di14:0-PG, 0.3
nmol di20:0 (phytanoyl)-PG, 0.3 nmol 14:0-LPG, 0.3 nmol 18:0-LPG, 0.23 nmol 16:0-
18:0-PI, 0.16 nmol di18:0-PI, 0.2 nmol di14:0-PS, 0.2 nmol di20:0 (phytanoyl)-PS, 0.3
nmol di14:0-PA, 0.3 nmol di20:0 (phytanoyl)-PA, 0.49 nmol 16:0-18:0-DGDG, 0.71 nmol
di18:0-DGDG, 2.01 nmol 16:0-18:0-MGDG, and 0.39 nmol di18:0-MGDG. The sample and
internal standard mixture was combined with solvents, such that the ratio of
chloroform/methanol/300 mM ammonium acetate in water was 300/665/35, and the final
volume was 1.4 ml.
Unfractionated lipid extracts were introduced by continuous infusion into the ESI
source on a triple quadrupole MS/MS (4000 QTrap, Applied Biosystems, Foster City, CA).
Samples were introduced using an autosampler (LC Mini PAL, CTC Analytics AG,
Zwingen, Switzerland) fitted with the required injection loop for the acquisition time and
presented to the ESI needle at 30 l min-1.
Sequential precursor and neutral loss scans of the extracts produce a series of spectra
with each spectrum revealing a set of lipid species containing a common head group
fragment. Lipid species were detected with the following scans: PC and LPC, [M + H]+ ions
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156
in positive ion mode with Precursor of 184.1 (Pre 184.1); PE and LPE, [M + H]+ ions in
positive ion mode with Neutral Loss of 141.0 (NL 141.0); PG, [M + NH4]+ in positive ion
mode with NL 189.0 for PG; LPG, [M – H]- in negative mode with Pre 152.9; PI, [M +
NH4]+ in positive ion mode with NL 277.0; PS, [M + H]+ in positive ion mode with NL
185.0; PA, [M + NH4]+ in positive ion mode with NL 115.0; DGDG, [M + NH4]
+ in positive
ion mode with NL 341.1; and MGDG, [M + NH4]+ in positive ion mode with NL 179.1.
The scan speed was 50 or 100 u per sec. The collision gas pressure was set at 2 (arbitrary
units). The collision energies, with nitrogen in the collision cell, were +40 V for PC, +28 V
for PE, +20 V and PG, +25 V for PI, PS and PA, +24 V for DGDG, and +21 V for MGDG.
Declustering potentials were +100 V for PE, PC, PA, PG, PI, and PS, and +90 V for DGDG
and MGDG. Entrance potentials were +15 V for PE, +14 V for PC, PG, PI, PS, and PA, and
+10 V for DGDG and MGDG. Exit potentials were +11 V for PE, +14 V for PC, PG, PI,
PS, and PA, and +23 V for DGDG and MGDG. The mass analyzers were adjusted to a
resolution of 0.7 u full width at half height. For each spectrum, 9 to 150 continuum scans
were averaged in multiple channel analyzer (MCA) mode. The source temperature (heated
nebulizer) was 100C, the interface heater was on, +5.5 kV or -4.5 kV were applied to the
electrospray capillary, the curtain gas was set at 20 (arbitrary units), and the two ion source
gases were set at 45 (arbitrary units).
The background of each spectrum was subtracted, the data were smoothed, and peak
areas integrated using a custom script and Applied Biosystems Analyst software. After
isotopic deconvolution, the lipids in each class were quantified in comparison to the two
internal standards of that class (Brügger et al. 1997; Welti et al. 2002). The first and
typically every 11th set of mass spectra were acquired on the internal standard mixture only.
Peaks corresponding to the target lipids in these spectra were identified and molar amounts
calculated in comparison to the internal standards on the same lipid class. To correct for
chemical or instrumental noise in the samples, the molar amount of each lipid metabolite
detected in the “internal standards only” spectra was subtracted from the molar amount of
each metabolite calculated in each set of sample spectra. The data from each “internal
standards only” set of spectra was used to correct the data from the following 10 samples.
Finally, the data were corrected for the fraction of the sample analyzed and normalized to
the sample “dry weights” to produce data in the units nmol mg-1.
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For fatty acid analysis lipids were extracted from algal samples and transmethylated
with 1ml of 1% NaOH in methanol, followed by heating for 15 min at 55°C, adding 2 ml of
5% methanolic HCl and again heated for 15 min at 55°C then adding 1ml milli-Q water
(Carreau and Dubacq, 1978). Nonadecanoic acid was used as an internal standard. FAMEs
were extracted in hexane and separated on RTX-5 fused silica capillary column, 30 m x 0.25
mm x 0.25 µm (Rastek) by GC-MS with helium (99.9% purity) as the carrier gas. The GC-
MS conditions were the same as described in chapter 2.
7.2.5. Determination of oxylipins and lipoxygenase (LOX) enzyme
Oxylipins were extracted by modified method of Küpper et al. (2006) and analyzed
by reverse phase HPLC (RP-HPLC) on Waters alliance model (2695 separation module with
autosampler) equipped with photodiode array detector (Waters 2996) using Luna-C18
reversed-phase column (5.0 µm, 4.6 × 150 mm, Phenomenex, USA) using an isocratic
mobile phase of acetonitrile/water/acetic acid (55:45:0.1, v/v/v) as described in chapter 5.
Lipoxygenase (LOX) enzyme was extracted according to modified Tsai et al. (2008)
method. The algal samples were homogenized in 50 mM potassium phosphate buffer (pH
7.5) containing 0.2 mM CaCl2, 1 mM glutathione reduced, 1 mM phenylmethylsulphonyl
fluoride (PMSF), 0.3 mM dithiothreitol (DTT), 0.2 mM EDTA, 1% polyvinyl
polypyrrolidone (PVPP) and 0.1% Triton X-100 and centrifuged at 15, 000 g at 4 °C for 30
min. The supernatant was assayed for LOX activity by measuring the increase in absorbance
at 234 nm with 100 μM substrate solutions of linoleic acid (LA), α-linolenic acid (ALA) and
arachidonic acid (AA) prepared in ethanol. LOX activity was determined using extinction
coefficient 25,000 L mol-1 cm-1. The protein content of the three enzyme extracts was
estimated by Bradford method (Bradford, 1976) using bovine albumin serum (BSA) as a
standard.
7.2.6. Determination of total phenolic compounds, polyphenol oxidase, phenyl ammonia-
lyase and shikimic dehydrogenase activities
Total phenolic compounds (TPC) were extracted by following the method of Folin
and Ciocalteu (1927). Algal samples were homogenized with 80% methanol (1:4, w/v) and
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158
incubated at 70 °C for 1 h. Samples were centrifuged at 13,000 g for 10 min. The reaction
assay consisted of 200 μl sample extract, 50 μl Folin-Ciocalteu reagent (3 min incubation),
and 750 μl of 7.5% (w/v) Na2CO3. The reaction assay was incubated at room temperature in
dark for 2 h and the absorbance was recorded at 765 nm. The concentration of phenolic
compounds in the tissue was determined against a standard curve of catechol.
Polyphenol oxidase (PPO) was extracted according to the method of Chen et al.
(2000). Algal samples were homogenized with 0.1 M sodium phosphate buffer (pH 6.4)
containing 0.5 g of PVP (1:4, w/v) at 4 °C. The homogenate was centrifuged at 15,000 g for
30 min at 4 °C, and the supernatant was used for enzyme assays. The PPO activity was
determined by adding 1 ml of enzyme preparation to 2 ml of catechol as a substrate, and the
change was measured immediately in absorbance at 398 nm (A398). The activity was
expressed as A398 per minute per milligram of protein.
Phenylalanine ammonia-lyase (PAL) was extracted from algal samples by
homogenizing with 50 mM sodium borate buffer (pH 8.8) containing 5 mM β-
mercaptoethanol and 0.5 g of polyvinyl pyrrolidone (PVP) at 4°C (Qin and Tian, 2005). The
mixture was centrifuged at 15,000 × g for 30 min at 4°C, and the supernatant was collected
for enzyme analysis. For assay, 1 ml of enzyme extract was incubated with 2 ml of borate
buffer (50 mM, pH 8.8) and 0.5 ml of L-phenylalanine (20 mM) for 60 min at 37 °C. The
reaction was stopped with 0.1 ml of 6 N HCl. PAL activity was determined by the
production of cinnamate, measured by the absorbance change at 290 nm. The blank was the
crude enzyme preparation mixed with L-phenylalanine with zero time incubation.
For shikimate dehydrogenase (SD), algal samples were homogenized with 100 mM
Tris-HCl, (pH 7.8) at 4 °C (Magalhães et al. 2002). SD catalyzes the NADPH-dependent
reduction of 3-dehydroshikimate to form shikimate and ADP+. The enzyme activity was
assayed in the reverse direction by continuously monitoring the increase in NADPH
absorbance at 340 nm (ε NADPH = 6.22 x 103 M-1 cm-1). The assay mixture contained 100
mM Tris–HCl, pH 9.0, 4 mM shikimic acid, and 2 mM NADP+.
The protein content of all the three enzyme extracts was estimated by Bradford
method (Bradford, 1976) using bovine albumin serum (BSA) as a standard.
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7.2.7. Statistical analysis
All the analyses were performed in triplicates except quantitative lipid profiling
which had 4 replicates and mean values are reported. Data analysis was carried out by one-
way analysis of variance (one-way ANOVA), and a significant difference was considered at
the level of p < 0.05. Further, two-way analysis of variance (two-way ANOVA) was also
performed with concentration and time as the two factors for all the parameters to identify
the significant distinct responses of MeJA in G. dura.
7.3. Results
7.3.1. Lipid peroxidation, ROS production and in situ localization
MeJA treated G. dura thalli showed a dose-dependent increase in TBARS-MDA
level by 1.1-2.0-fold as compared to control (p < 0.05). In addition, the increase in lipid
peroxidation was also observed with the increase in time, with treated thalli showing a 1.03-
1.4-fold increase as compared to control thalli (1.01-1.04-fold) (Fig. 7.1). This increase in
lipid peroxidation was probably due to the increased ROS production resulting into
oxidative stress. Subsequently, treated thalli also showed an increase of 1.1-1.5-fold in H2O2
content with the increase in exogenous MeJA concentration and an increase of 1.1-1.3-fold
with time (Fig. 7.1). Similarly, HO· and O2•− also showed both the dose (1.1-1.9-fold and
1.1-1.7-fold respectively) and time dependent (1.2-1.4-fold and 1.5-3.2-fold respectively)
increase in treated thalli as compared to control which showed a change of 1.01-1.05-fold in
HO· and 1.09-fold in O2•− contents (p < 0.05) (Fig. 7.1). The highest rate of increase in H2O2
and HO· was observed at 12 h, then the rate of increase in their content slowed down and
again increased after 24 h while the highest rate of increase in O2•− was observed at 24 h
indicating that generation O2•− radicals precedes H2O2 and HO· burst.
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Table 7.1 Details of two-way ANOVA for different biochemical parameters of Gracilaria dura treated with methyl jasmonate.
Concentration Time Interaction Model SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value
ROS, pigments, phenolic compounds and enzymes MDA 156.7 52.25 65.02 1.04x10-13 10.57 3.52 4.38 0.01 5.68 0.63 0.78 0.63 173.03 11.53 14.3 3.64x10-10 H2O2 1.14 0.38 10.57 5.47x10-5 0.36 0.12 3.42 0.02 0.16 0.018 0.51 0.85 1.68 0.11 3.10 0.0034 OH− 7689.6 2563.2 164.7 0 2627.8 875.9 56.2 7.3x10-13 761.1 84.5 5.43 0.00015 11078.5 738.5 47.4 0 O2
·− 15.3 5.13 125.0 0 5.71 1.9 46.3 9.3x10-12 1.49 0.16 4.05 0.0015 22.6 1.5 36.7 6.6x10-16 PC 0.056 0.018 16.27 5.5x10-6 0.0029 0.0010 0.89 0.4 0.0025 0.0004 0.36 0.89 0.06 0.0056 4.8 0.00064
APC 0.012 0.004 4.85 0.0088 0.0011 0.0005 0.65 0.53 0.0006 0.0001 0.13 0.99 0.014 0.0013 1.51 0.19 PE 0.24 0.08 93.6 2.1x10-13 0.11 0.05 65.3 1.9x10-10 0.04 0.007 7.9 8.7x10-5 0.4 0.03 41.7 6.2x10-13
Chl a 5113.5 1704.5 40.8 1.3x10-9 146.8 73.4 1.76 0.19 525.6 87.6 2.1 0.09 5786.1 526.0 12.6 1.8x10-7 TPC 18881.0 6293.6 83.12 8.0x10-13 14242.1 7121.0 94.05 4.4x10-12 4137.1 689.5 9.1 3.0x10-5 37260.4 3387.3 44.7 2.8x10-13 PPO 5218.4 1739.4 36.5 4.1x10-9 2084.4 1042.2 21. 3.8x10-6 2724.7 454.1 9.5 2.1x10-5 10027.6 911.6 19.14 2.8x10-9 PAL 0.16 0.05 74.8 2.5x10-12 0.05 0.02 38.2 3.4x10-8 0.025 0.004 5.7 0.00082 0.25 0.023 30.48 2.1x10-11 SD 14617.8 4872.6 152.8 8.8x10-16 2600.0 1300.0 40.7 1.9x10-8 1116.0 186.0 5.8 0.00072 18333.9 1666.7 52.2 4.9x10-14
LA-LOX 640.05 213.35 21.95 4.5x10-7 402.8 201.4 20.7 5.8x10-6 24.4 4.07 0.41 0.85 1067.3 97.03 9.98 1.67x10-6 ALA-LOX 7457.2 2485.7 64.6 1.2x10-11 32372.7 16186.3 421.1 0 7895.6 1315.9 34.2 1.2x10-10 47725.7 4338.7 112.8 0 AA-LOX 823.6 274.5 23.5 2.5x10-7 73.1 36.5 3.13 0.06 22.5 3.76 0.32 0.91 919.3 83.5 7.16 3x10-5
Lipids TL 1.02 0.34 5.22 0.006 0.028 0.014 0.22 0.8 0.1 0.016 0.25 0.95 1.15 0.105 1.6 0.16
DGDG-32C 0.06 0.023 4.13 0.012 0.012 0.006 1.09 0.34 0.034 0.0058 1.01 0.43 0.11 0.0105 1.88 0.07 DGDG-34C 4.26 1.42 6.89 0.0008 1.19 0.59 2.9 0.067 2.83 0.47 2.28 0.056 8.29 0.75 3.65 0.001 DGDG-36C 7.57 2.52 2.68 0.06 21.8 10.9 11.6 0.0001 37.04 6.17 6.55 9.5x10-5 66.4 6.04 6.41 9.2x10-6 DGDG-38C 0.053 0.017 1.27 0.29 0.17 0.089 6.44 0.004 0.133 0.022 1.59 0.17 0.36 0.03 2.39 0.024 DGDG-40C 0.23 0.079 0.6 0.61 0.23 0.11 0.90 0.41 1.25 0.2 1.6 0.17 1.73 0.15 1.2 0.31 Total DGDG 15.05 5.01 2.8 0.0504 28.9 14.4 8.25 0.0011 62.3 10.3 5.9 0.0002 106.4 9.6 5.5 4.3x10-5 MGDG-30C 0.019 0.006 0.87 0.46 0.022 0.011 1.5 0.23 0.022 0.011 1.5 0.23 0.06 0.01 1.3 0.26 MGDG-32C 0.12 0.04 2.22 0.10 0.55 0.27 14.4 2.4x10-5 0.60 0.10 5.29 0.0005 1.28 0.11 6.13 1.4x10-5 MGDG-34C 10.13 3.3 14.3 2.5x10-6 1.43 0.71 3.06 0.059 6.04 1.007 4.29 0.002 17.6 1.6 6.8 4.8x10-6 MGDG-36C 58.1 19.3 26.4 3.2x10-9 231.4 115.7 157.9 0 27.25 4.5 6.2 0.0001 316.8 28.8 39.3 1.1x10-16 MGDG-37C 0.003 0.001 2.32 0.09 0.005 0.002 5.8 0.006 0.005 0.0008 1.8 0.11 0.013 0.0012 2.7 0.011 MGDG-38C 0.07 0.024 0.36 0.78 1.37 0.68 10.1 0.0003 0.50 0.08 1.24 0.30 1.95 0.177 2.62 0.014 MGDG-40C 40.5 13.5 10.7 3.4x10-5 47.5 23.7 18.8 2.4x10-6 42.7 7.12 5.65 0.0003 130.8 11.8 9.44 1.1x10-7 Total MGDG 50.8 16.9 11.65 1.7x10-5 381.3 190.6 131.0 0 14.7 2.45 1.68 0.15 446.9 40.6 27.9 2.3x10-14 Values in bold are significant at p < 0.05. SS-sum of square; MSS-Mean sum of square; PC-Phycocyanin; APC-Allophycocyanin; PE-Phycoerythrin; Chl a-Chlorophyll a; TPC-Total phenolic
compounds; PPO-Polyphenol oxidase; PAL-Phenyl ammonia-lyase; SD-Shikimic dehydrogenase; LA-LOX-linoleate lipoxygenase; ALA-LOX- linolenate lipoxygenase; AA-LOX-aracidonate
lipoxygenase; TL-Total lipid; DGDG-Digalactosyldiacylglycerol; MGDG-Monogalactosyldiacylglycerol
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Concentration Time Interaction Model SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value
PG-26C 0.028 0.009 0.35 0.78 0.068 0.03 1.29 0.28 0.37 0.06 2.35 0.05 0.47 0.04 1.61 0.13 PG-27C 3.6x10-5 1.2x10-5 1.01 0.39 4.5x10-5 2.2x10-5 1.9 0.16 0.0001 1.7x10-5 1.42 0.23 0.0001 1.6x10-5 1.39 0.21 PG-29C 2.79 9.3x10-6 0.76 0.52 0.00014 7.0x10-5 5.8 0.0065 6.9x10-5 1.1x10-5 0.95 0.4 0.0002 2.1x10-5 1.78 0.09 PG-30C 0.007 0.0024 1.42 0.25 0.026 0.013 7.6 0.0016 0.04 0.0066 3.92 0.004 0.073 0.0067 3.92 0.0008 PG-31C 0.0001 6.4x10-5 1.29 0.29 0.0018 0.0009 18.8 2.4x10-6 0.0008 1.4x10-4 2.9 0.02 0.0029 0.0002 5.36 5.6x10-5 PG-32C 0.011 0.003 2.22 0.102 0.14 0.007 40.9 5.4x10-10 0.052 0.008 4.95 0.0008 0.20 0.019 10.74 2.2x10-8 PG-33C 0.0002 6.7x10-5 0.64 0.59 0.0027 0.0018 10.3 0.0002 0.0006 1.1x10-4 1.1 0.38 0.003 0.0002 2.65 0.013 PG-34C 0.088 0.029 2.31 0.09 0.41 0.209 16.4 8.5x10-6 0.18 0.03 2.38 0.04 0.69 0.062 4.91 0.00012 PG-35C 0.0017 5.7x10-4 8.96 0.0001 0.0086 0.0043 66.7 7.8x10-13 0.0016 0.0002 4.34 0.002 0.012 0.001 16.94 4.4x10-11 PG-36C 1.66 0.55 23.5 1.2x10-8 1.32 0.66 28.0 4.5x10-8 0.97 0.16 6.92 5.9x10-5 3.96 0.36 15.3 1.8x10-10 PG-37C 0.0001 6.6x10-5 6.7 0.0009 0.0007 0.0003 38.0 1.3x10-9 0.0002 4.7x10-5 4.83 0.001 0.0012 0.0001 11.3 1.03x10-8 PG-38C 3x10-5 1.2x10-5 3.25 0.032 1.5x10-4 7.8x10-5 20.5 1.1x10-6 0.0001 2.0x10-5 5.32 0.0005 0.0003 2.8x10-5 7.52 1.6x10-6 PG-40C 0.0001 3.7x10-5 4.34 0.010 8.7x10-5 4.3x10-5 4.99 0.012 9.4x10-5 1.5x10-5 1.81 0.12 0.0002 2.6x10-5 3.08 0.005 Total PG 2.9 0.99 12.9 6.7x10-6 7.8 3.9 51.1 3.0x10-11 4.9 0.83 10.7 7.6x10-7 15.8 1.44 18.7 1.0x10-11 PC-28C 9x10-6 3.0x10-6 0.40 0.75 4.6x10-5 2.3x10-5 3.11 0.056 5.9x10-5 9.9x10-6 1.32 0.27 0.0001 1.0x10-5 1.4 0.21 PC-30C 3.6x10-4 1.2x10-4 2.89 0.04 0.0005 0.0002 7.03 0.002 0.0002 3.7x10-5 0.87 0.52 0.0011 0.0001 2.54 0.017 PC-32C 0.0006 0.0002 0.83 0.48 0.007 0.003 15.9 1.1x10-5 0.0014 0.0002 1.01 0.43 0.0098 0.0008 3.67 0.0014 PC-34C 0.048 0.016 7.25 0.0006 0.16 0.08 37.0 1.8x10-9 0.021 0.003 1.57 0.18 0.23 0.021 9.57 9.4x10-8 PC-36C 0.08 0.36 9.44 9.6x10-5 2.5 1.2 32.7 8x10-9 0.69 0.11 3.0 0.017 4.28 0.38 10.1 4.4x10-8 PC-38C 6.49 2.1 41.4 9x10-12 14.07 7.03 134.8 0 5.82 0.97 18.6 1x10-9 26.3 2.39 45.9 0 PC-40C 60.0 20.0 29.6 7.6x10-10 276.8 138.4 205.2 0 79.3 13.2 19.6 5x10-10 416.3 37.8 56.1 0 PC-42C 0.40 0.13 29.9 6.7x10-10 0.32 0.16 35.3 3.2x10-9 0.10 0.017 3.81 0.004 0.83 0.076 16.6 5.5x10-11 PC-44C 0.04 0.015 15.2 1.4x10-6 0.01 0.005 4.9 0.012 0.008 0.0014 1.4 0.23 0.066 0.006 5.85 2.4x10-5 Total PC 147.6 49.2 45.7 2.3x10-12 514.3 257.1 238.8 0 135.2 22.5 20.9 2.1x10-10 797.2 72.4 67.3 0 PE-28C 0.002 0.0008 33.9 1.3x10-10 0.001 0.0009 38.1 1.2x10-9 0.002 0.0004 17.6 2x10-9 0.007 0.0006 25.8 8x10-14 PE-30C 0.014 0.004 16.2 7.6x10-7 0.015 0.007 26.4 8.5x10-8 0.014 0.0024 8.37 1.0x10-5 0.043 0.004 13.8 7.8x10-10 PE-32C 0.035 0.011 7.31 0.0005 0.056 0.028 17.6 4.4x10-6 0.04 0.007 4.45 0.0018 0.13 0.012 7.64 1.3x10-6 PE-34C 0.052 0.017 11.6 1.7x10-5 0.13 0.06 43.8 2.2x10-10 0.069 0.011 7.58 2.6x10-5 0.25 0.023 15.2 1.9x10-10 PE-36C 0.41 0.13 11.6 1.7x10-5 0.85 0.42 35.4 3.1x10-9 0.48 0.08 6.74 7.5x10-5 1.75 0.15 13.2 1.3x10-9 PE-38C 0.079 0.026 11.9 1.3x10-5 0.18 0.09 40.7 5.6x10-10 0.10 0.017 7.8 1.8x10-5 0.36 0.033 14.9 2.5x10-10 PE-40C 1.55 0.51 13.8 3.6x10-6 2.5 1.25 33.4 6.2x10-9 1.62 0.27 7.2 4.1x10-5 5.6 0.51 13.7 8x10-10 PE-42C 0.0002 9.9x10-5 11.9 1.3x10-5 0.0005 0.0002 30.3 1.8x10-8 2.41 4.02 4.8 0.001 0.001 9.4x10-5 11.4 9.9x10-9 PE-44C 1.0x10-5 3.6x10-6 3.35 0.029 8.5x10-6 4.2x10-6 4.0 0.026 5x10-6 9x10-7 0.84 0.54 2.4x10-5 2.2x10-6 2.1 0.04
Total PE 7.61 2.53 15.2 1.4x10-6 13.6 6.8 41.03 5.1x10-10 8.35 1.39 8.36 1.04x10-5 29.6 2.69 16.1 8.5x10-11 PI-30C 0.00017 5.8x10-5 1.02 0.39 1.7x10-4 8.9x10-5 1.57 0.22 0.0007 0.00012 2.17 0.06 0.001 9.9x10-5 1.74 0.105
Values in bold are significant at p < 0.05. PG-Phosphatidylglycerol; PC-Phosphatidylcholine; PE-Phosphatidylethanolamine
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Concentration Time Interaction Model SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value
PI-31C 7.3x10-5 2.4x10-5 0.55 0.65 0.0003 1.6x10-4 3.68 0.034 0.0005 8x10-5 1.98 0.09 9.2x10-4 8.4x10-5 1.9 0.07 PI-32C 0.003 0.0010 3.1 0.037 0.0015 0.0007 2.24 0.12 0.006 0.0010 3.11 0.012 0.011 0.001 2.9 0.006 PI-33C 1.1x10-5 3.8x10-6 0.85 0.47 4x10-5 2x10-5 4.47 0.018 9x10-5 1.5x10-5 3.43 0.008 1.4x10-4 1.3x10-5 2.91 0.007 PI-34C 0.003 0.0012 4.02 0.014 0.0027 0.0013 4.26 0.021 0.003 0.0006 2.0 0.09 0.01 0.0009 2.9 0.006 PI-35C 2.7x10-5 9x10-6 2.55 0.07 4x10-5 2x10-5 5.66 0.007 2.6x10-7 4.4x10-6 1.25 0.30 9.4x10-5 8.5x10-6 2.41 0.023 PI-36C 0.0027 0.0009 3.98 0.015 0.004 0.002 9.96 0.0003 0.028 0.0004 2.02 0.08 0.01 0.0009 4.0 0.0007 PI-38C 0.0002 8.2x10-5 1.84 0.15 0.0004 0.0002 4.67 0.015 0.0005 9.9x10-5 2.21 0.06 0.0012 0.0001 2.55 0.016 PI-40C 0.024 0.082 2.94 0.045 0.026 0.013 4.71 0.015 0.03 0.005 1.82 0.12 0.08 0.007 2.6 0.01 PI-42C 0.016 0.005 1.99 0.13 0.002 0.001 0.36 0.69 0.022 0.003 1.32 0.27 0.041 0.003 1.32 0.24 PI-44C 0.0004 0.00013 2.06 0.12 0.0004 0.0002 3.46 0.04 0.0007 0.0001 1.9 0.1 0.001 0.00014 2.24 0.03 Total PI 0.045 0.015 1.16 0.33 0.097 0.048 3.7 0.03 0.14 0.024 1.93 0.1 0.29 0.026 2.06 0.0502 PS-32C 3.7x10-6 1.2x10-6 1.31 0.28 1.9x10-6 9x10-7 1.02 0.37 9x10-6 1.5x10-6 1.66 0.15 1.5x10-5 1.3x10-6 1.45 0.19 PS-34C 1.1x10-5 3.7x10-6 3.0 0.04 6.2x10-6 3.1x10-6 2.5 0.09 5x10-6 8.4x10-7 0.67 0.67 2.2x10-5 2x10-6 1.64 0.12 PS-36C 5.22 1.7x10-5 4.5 0.008 1.9x10-5 9.6x10-6 2.48 0.09 6.8x10-5 1.1x10-5 2.96 0.018 0.0001 1.2x10-5 3.29 0.003 PS-38C 3.7x10-6 1.2x10-6 0.25 0.85 1.6x10-5 8x10-6 1.64 0.20 3.5x10-5 5.9x10-6 1.2 0.32 5.5x10-5 5x10-6 1.02 0.44 PS-40C 0.0001 4.9x10-5 5.45 0.0034 6x10-6 3.3x10-5 3.68 0.03 0.0001 2x10-5 2.29 0.055 0.0003 3x10-5 3.41 0.002 PS-42C 3.2x10-6 1x10-6 1.72 0.18 2x10-7 1x10-7 0.16 0.85 6.8x10-6 1.1x10-6 1.8 0.12 1x10-5 9x10-7 1.48 0.18 PS-44C 4.4x10-5 1.4x10-5 8.49 0.0002 1x10-5 5.4x10-6 3.14 0.055 1x10-5 1.8x10-6 1.05 0.4 6.5x10-5 5.9x10-6 3.46 0.002
Total PS 0.0008 0.0002 6.8 0.0009 0.0002 0.0001 2.8 0.07 0.0004 7.9x10-5 1.81 0.12 0.0016 1.4x10-4 3.36 0.002 PA-32C 0.003 0.001 2.32 0.09 0.005 0.002 6.11 0.005 0.003 0.0006 1.27 0.29 0.01 0.011 2.44 0.02 PA-34C 0.0016 0.0005 4.3 0.01 0.009 0.004 34.9 3.7x10-9 0.004 0.0007 5.64 0.0003 0.015 0.0018 10.6 2.6x10-8 PA-36C 0.017 0.005 10.2 5.2x10-5 0.08 0.04 76.0 1.1x10-13 0.037 0.006 11.1 5.3x10-7 0.13 0.012 22.6 5.8x10-13 PA-38C 0.025 0.008 24.5 8x10-9 0.15 0.076 224.7 0 0.04 0.006 19.9 4x10-10 0.21 0.019 58.4 0 PA-40C 0.33 0.11 39.9 1.5x10-11 1.7 0.85 302.8 0 0.46 0.077 27.5 4.5x10-12 2.51 0.22 81.0 0
Total PA 0.7 0.23 28.9 1.0x10-9 3.57 1.78 220.6 0 1.28 0.21 26.4 8.3x10-12 5.56 0.5 62.4 0 LPG-16C 0.0002 8x10-5 0.71 0.55 0.0001 5.8x10-5 0.47 0.62 0.0005 9.2x10-5 0.74 0.61 0.0009 8.4x10-5 0.68 0.73 LPG-18C 0.0001 5.5x10-5 1.57 0.21 0.0001 9x10-5 2.63 0.085 0.0004 7x10-5 1.98 0.09 7x10-4 7x10-5 1.98 0.059 Total LPG 0.00078 0.0002 1.31 0.28 0.0005 0.0002 1.5 0.23 0.0011 0.0001 0.93 0.47 0.0025 0.0002 1.14 0.35 LPC-16C 0.008 0.002 0.92 0.44 0.009 0.004 1.5 0.22 0.04 0.007 2. 0.03 0.06 0.005 1.9 0.067 LPC-18C 0.038 0.012 2.1 0.10 0.027 0.013 2.28 0.11 0.12 0.02 3.48 0.008 0.18 0.017 2.91 0.007 LPC-20C 0.72 0.24 3.15 0.03 1.7 0.85 11.1 0.0001 2.2 0.36 4.8 0.001 4.62 0.42 5.5 4.3x10-5 LPC-22C 6.3x10-7 2x10-7 0.13 0.93 4.6x10-6 2.3x10-6 01.46 0.24 1.3x10-5 2.2x10-6 1.4 0.23 1.8x10-5 1.6x10-6 1.07 0.4 Total LPC 1.25 0.41 2.58 0.06 2.19 1.09 6.78 0.003 4.16 0.69 4.27 0.0023 7.62 0.69 4.27 0.0004 LPE-16C 0.0004 0.0001 3.95 0.015 0.0013 0.0006 16.02 1x10-5 0.0005 8.4x10-5 2.05 0.08 0.0023 0.0002 5.11 8.9x10-5 LPE-18C 4.3x10-5 1.4x10-5 3.11 0.037 0.0001 5.4x10-5 11.7 0.0001 6.3x10-5 1x10-5 2.28 0.056 0.0002 1.9x10-5 4.23 0.0004
Values in bold are significant at p < 0.05. PI-Phosphatidylinositol; PS-Phosphatidylserine; PA-Phosphatidic acid; LPG-Lyso-phosphatidylglycerol; LPC- Lyso-phosphatidylcholine; LPE-
Lysophosphatidylethanolamine
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Concentration Time Interaction Model SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value SS MSS F-value P-value
LPE-20C 0.001 0.0003 6.22 0.0016 0.003 0.0016 28.9 3.2x10-8 0.002 0.0003 6.08 0.0001 0.006 0.0006 10.2 3.9x10-8 Total LPE 0.0037 0.0012 6.61 0.0011 0.0108 0.0054 28.7 3.4x10-8 0.005 8.9x10-4 4.74 0.0011 0.019 0.0018 9.6 8.8x10-8
Fatty acids C14:0 1.33 0.44 0.29 0.82 1.82 0.91 0.61 0.54 6.02 1.004 0.67 0.66 9.1 0.83 0.56 0.84 C15:0 0.45 0.15 1.67 0.19 0.35 0.17 1.96 0.16 0.76 0.12 1.39 0.25 1.57 0.14 1.57 0.17 C16:0 2.74 0.91 0.065 0.97 113.3 56.6 4.07 0.02 23.09 3.4 0.27 0.94 139.1 12.65 0.91 0.54 C17:0 0.22 0.07 0.83 0.48 0.0037 0.0018 0.02 0.97 0.77 0.12 1.43 0.24 1.008 0.091 1.01 0.46 C18:0 2.49 0.83 0.71 0.55 4.2 2.1 1.79 0.18 8.4 1.4 1.19 0.34 15.1 1.37 1.17 0.35 C20:0 0.22 0.07 2.81 0.06 0.14 0.07 2.64 0.091 0.44 0.073 2.78 0.033 0.8 0.07 2.77 0.017 C22:0 0.55 0.18 0.92 0.44 1.2 0.6 2.98 0.06 1.33 0.22 1.1 0.38 3.09 0.28 1.39 0.23 C24:0 0.11 0.037 0.42 0.73 0.5 0.25 2.83 0.078 0.66 0.11 1.23 0.32 1.28 0.11 1.30 0.28
C16:1n7 5.27 1.75 1.06 0.38 0.75 0.37 0.22 0.79 12.9 2.15 1.3 0.29 18.9 1.72 1.04 0.44 C16:1n9 4.28 1.42 1.12 0.35 11.85 5.92 4.67 0.019 8.25 1.37 1.08 0.39 24.38 2.21 1.75 0.12 C18:1n9 12.77 4.25 1.96 0.14 1.84 0.92 0.42 0.65 29.0 4.8 2.22 0.075 43.6 3.9 1.8 0.104 C18:1 n9t 3.54 1.18 2.64 0.07 0.35 0.17 0.402 0.67 2.85 0.47 1.06 0.409 6.76 0.61 1.37 0.24 C18:2 n6 0.78 0.26 0.4 0.75 0.87 0.43 0.67 0.52 3.48 0.58 0.89 0.51 5.14 0.46 0.72 0.70 C20:3 n6 2.2 0.73 3.41 0.03 4.95 2.47 11.5 0.0003 1.5 0.25 1.16 0.35 8.6 0.78 3.6 0.003 C20:4 n6 29.7 9.91 0.85 0.47 67.2 33.6 2.9 0.07 205.9 34.3 2.9 0.02 302.8 27.5 2.38 0.03
SFA 20.96 6.9 0.41 0.74 169.5 84.75 4.9 0.015 66.7 11.13 0.65 0.68 257.2 23.3 1.37 0.24 MUFA 26.65 8.88 1.4 0.26 31.6 15.8 2.5 0.1 37.8 6.3 0.99 0.44 96.07 8.73 1.38 0.24 PUFA 11.24 3.74 0.26 0.84 67.3 33.6 2.4 0.11 193.02 32.1 2.29 0.06 271.65 24.6 1.76 0.11
Oxylipins Toxl 219388.8 73129.6 83.06 8.1x10-13 3254.0 16270.1 18.4 1.3x10-5 12281.2 2046.8 2.32 0.065 264210.2 24019.1 27.2 6.7x10-11
THETE 113802.0 37934.0 49.8 1.8x10-10 9490.4 4745.2 6.23 0.006 8262.1 1377.0 1.8 0.13 131554.6 11959.5 15.7 2.1x10-8 THODE 1022.6 340.8 22.1 4.1x10-7 1489.0 744.5 48.4 3.7x10-9 348.5 58.08 3.78 0.008 2860.2 260.02 16.9 1.01x10-8 THOTrE 7322.8 2440.9 114.8 2.2x10-14 4043.5 2021.7 95.1 3.9x10-12 1443.8 240.6 11.3 5.2x10-6 121810.2 1164.5 54.7 2.8x10-14 15-HETE 5949.5 1983.2 32.9 1.1x10-8 2506.5 1253.2 20.83 5.7x10-6 1583.4 263.9 4.38 0.0039 110039.4 912.6 15.17 3.05x10-8 12-HETE 11988.3 3996.1 10.8 0.0001 6280.4 3140.2 8.5 0.0016 2874.1 479.02 1.29 0.29 21142.9 1922.0 5.21 0.0003 8-HETE 1547.2 515.7 8.05 0.0006 65.74 32.8 0.51 0.6 73.57 12.26 0.19 0.97 1686.5 153.3 2.39 0.035 5-HETE 1317.6 4379.2 44.6 5.6x10-10 326.9 163.4 1.65 0.2 546.2 91.03 0.92 0.49 14040.8 1273.7 12.9 1.4x10-7 9-HOTrE 238.02 79.34 21.1 6.3x10-7 466.07 233.03 62.1 3.2x10-10 103.3 17.2 4.5 0.003 807.4 73.4 19.5 2.2x10-9
13-HOTrE 4995.7 1665.2 81.9 9.4x10-13 1766.3 883.1 43.47 1.05x10-8 801.5 133.5 6.57 0.00033 7563.6 687.6 33.8 6.4x10-12 9-HODE 124.4 41.4 14.5 1.2x10-5 332.4 166.2 58.4 5.9x10-10 9.68 1.61 0.56 0.75 466.5 42.4 14.9 3.6x10-8
13-HODE 463.4 154.4 12.2 4.7x10-5 413.6 206.8 16.3 3.3x10-15 267.2 44.5 3.5 0.012 1144.3 104.02 8.21 9.4x10-6 Values in bold are significant at p < 0.05. LPE-Lysophosphatidylethnolamine; SFA-Saturated fatty acid; MUFA-Monounsaturated fatty acid; PUFA-Polyunsaturated fatty acid; Toxl-Total
oxylipins; THETE-Total hydroxyeicosatetraenoic acids; THODEs-Total hydroxyoctadecadienoic acids; THOTrEs-Total hydroxyoctadecatrienoic acids; HETE-Hydroxyeicosatetraenoic acid;
HOTrE-Hydroxyoctadecatrienoic cid; HODE-Hydroxyoctadecadienoic acid.
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Fig. 7.1 TBARS-MDA level and reactive oxygen species contents Gracilaria dura thalli treated with methyl
jasmonate (A) TBARS-MDA, (B) H2O2, (C) HO· and (D) O2•−. Different letters on same shade of
columns indicate that mean values for the particular incubation time were significantly different at p <
0.05.
Further, histochemical staining for the detection of in situ accumulation of H2O2 and
O2•− radicals using NBT and DAB respectively confirmed the MeJA induced ROS
production (Fig. 7.2). A blue formazone formed by the reduction of NBT by O2•− clearly
showed the generation of superoxide radical that appeared first in the epidermal cells, then
gradually progressed to cortical cells and later distributed all over the tissue. Similarly, the
formation of H2O2 dependent brown precipitates was contingent with the exposure duration
and the concentration of exogenous MeJA applied. Moreover, two-way ANOVA revealed
that though the ROS production and lipid peroxidation observed in MeJA thalli significantly
increased with the increase in exogenous concentration of MeJA and with time, this increase
was independent of each other and the interaction of concentration and time was significant
only for HO· and O2·− (Table 7.1). The increase in content of HO· and O2
·− was higher as
compared to the increase observed in H2O2 indicating the larger contribution of hydroxyl
and superoxide radicals in lipid peroxidation and ROS mediated oxidative stress in MeJA
treated thalli. Further, the G. dura thalli at 48 h also showed the signs of bleaching due to
increased oxidative stress and thus the experimental period was limited to 24 h for the study
of other biochemical and lipidomics responses.
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Fig. 7.2 ROS generation in Gracilaria dura thalli treated with methyl jasmonate (I) H2O2 by 3, 3-
diaminobenzidine (DAB) staining and (II) O2·− by nitroblue tetrazolium (NBT) staining.
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Fig. 7.3 Effect of methyl jasmonate on (A) chlorophyll a and phycobiliptroteins (B) phycoerythrin, (C)
phycocyanin and (D) allophycocyanin in Gracilaria dura. Different letters on same shade of
columns indicate that mean values for the particular incubation time were significantly different at p
< 0.05.
7.3.2. Pigments
Chlorophyll a (Chl a) content decreased in MeJA treated thalli as compared to
control as a result of increased ROS generation by 1.03-2.0-fold with the increase in MeJA
concentration and by 1.02-1.4-fold with treatment time (Fig. 7.3). The decrease in
chlorophyll content was accompanied by the increase in the contents of phycobiliptroteins.
The highest increase was observed in phycoerythrin content, which showed 1.2-2.1-fold
increase in treated thalli with the increase in exogenous MeJA concentration applied as well
as 1.3-1.7-fold with time, as compared to control (p < 0.05). Phycocyanin (PC) showed only
dose dependent increase of 1.2-1.8-fold in the treated thalli (Fig. 7.3, Table 7.1) Further,
1μM MeJA treated thalli showed a constant marginal increase in PC content (1.02-1.1-fold)
while 10 μM and 100 μM MeJA treated thalli showed an initial increase of 1.2-fold till 12 h
and then slightly decreased, may be due to increased oxidative damage. Allophycocyanin
(APC) also showed only dose-dependent increase in treated thalli (1.1-1.4-fold) as compared
to control while the change in APC content was non-significant with time duration.
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7.3.3. Lipids and Fatty acids
Total lipid content increased by 1.2-1.5-fold in the treated thalli as compared to
control (p < 0.05). Further, with the increase in time period, only 1 μM MeJA treated thalli
showed a constant increase till 24 h while 10 μM and 100 μM MeJA treated thalli showed a
slight decrease in TL content after 12 h (Fig. 7.4).
Fig. 7.4 Effect of methyl jasmonate on total lipid content of Gracilaria dura. Different letters on same shade of
columns indicate that mean values for the particular incubation time were significantly different at p <
0.05.
ESI-MS lipidomic profiling is shown in Fig. 7.5 and total contents of different lipid
classes in Fig. 7.6. Monogalactosyldiacylglycerol (MGDG) was the dominant lipid class
followed by digalactosyldiacylglycerol (DGDG), phosphatidylcholine (PC), and
phosphatidylglycerol (PG) in both the control and treated thalli. Phosphatidylinositol (PI),
phosphatidylethanolamine (PE) and phosphatidylserine (PS) were present as minor lipids
while phosphatidic acid (PA) was present in appreciable amounts contributing upto 0.4-
1.23% of polar lipids in treated thalli as compared to 0.3-1.1% of polar lipids in control. The
lysolipids, lyso-phosphatidylglycerol (LPG), lyso-phosphatidylethanolamine (LPE) and
lyso-phosphatidylcholine (LPC) together accounted to 0.17-0.6% of polar lipids in control
and 0.18-1.5% of polar lipids in treated thalli. Further, there was a predominance of C36
(36:4) and C34 (34:1) acyl carbons in chloroplastic lipids DGDG, MGDG and PG indicating
the presence of 18:2/18:2 or 18:1/18:0 acyl chains (Table 7.2, Fig. 7.7). In addition MGDG
was also characterized by the presence of C40 carbons (40:8 and 40:9) which together
represented 13.8-27.8% of total MGDGs and indicated the presence of 20:4/20:4 or
20:4/20:5 acyl chains.
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Fig. 7.5 Polar lipid profile of Gracilaria dura thalli treated with methyl jasmonate.
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Fig. 7.6 Lipid class composition of Gracilaria dura thalli treated with methyl jasmonate. Different letters on
same shade of columns indicate that mean values for the particular incubation time were significantly different
at p < 0.05.
The presence of C20:4 and C20:5 in MGDGs reveal that these fatty acids (FAs) are
imported from the endoplasmic reticulum and thereafter incorporated into the chloroplastic
galactolipids. Apart from long chain PUFAs, G. dura thalli also showed the presence of PG
26:0 as one of the prominent lipid contributing to 10.5-19.7% of total PGs, which could be
comprised of either 12:0/14:0 or 13:0/13:0 (Fig. 7.8). The extraplastidic phospholipids PC,
PE, PI and PA were dominated by C40 acyl carbons (mainly 40:8 or 40:7) and represented
60-68% of total PC, 36.6-43.7% of total PE, 35.4-45.4% of total PI and 23.2-58% of total
PA except PS, where C36 acyl carbons were dominant (28.0-43.6% of total PS) followed by
C40 acyl carbons (15.4-39.8% of total PS) (Fig. 7.8, 7.9, 7.10, 7.11). Furthermore, 2-way
ANOVA revealed that MGDG, PG, PC, PE and PA showed the significant dose- and time-
dependent changes with respect to control and the interaction effect of exogenous MeJA
concentration and time were also significant for these lipids except MGDG, in which the
interaction effect was not significant (p < 0.05) (Table 7.1). The lipid species that exhibited
the highest treatment-specific responses were all of high abundance in each lipid class.
MGDG showed a dose- and time-dependent decrease of 1.01-1.1-fold in MGDG in the
treated thalli due to 1.1-1.6-fold decrease in the contents of C36 (36:4, 36:5, and 36:6), C38
(38:5, 38:4, and 39:7) and C40 (40:8) lipid molecular species (Fig. 7.7).
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Table 7.2 Polar lipid chain lengths in Gracilaria dura treated with methyl jasmonate (mol% of class).
Control 1MeJA 10MeJA 100MeJA 6H 12H 24H 6H 12H 24H 6H 12H 24H 6H 12H 24H DGDG (% of DGDG) With 32 acyl carbons 1.0 ±0.2 1.2±0.1 1.3±0.1 1.1±0.2 1.5±0.2 1.6±0.7 1.4±0.2 1.7±0.2 1.3±0.2 1.5±0.2 1.6±0.5 1.4±0.1 With 34 acyl carbons 14.9±1.2 15.0±1.6 16.3±1.5 15.4±0.7 16.3±0.8 19.9±5.4 16.5±1.8 18.7±1.9 17.2±1.1 16.2±1.1 19.1±1.6 18.3±1.2 With 36 acyl carbons 76.9±0.6 78.2±1.6 76.4±1.8 76.8±1.2 74.7±2.2 84.9±18.5 75.9±1.0 74.0±0.9 74.1±3.0 75.6±1.0 73.4±1.1 73.3±1.0 With 38 acyl carbons 4.0±0.7 3.6±0.6 3.1±0.1 3.7±0.2 3.2±0.3 3.8±1.0 3.5±0.3 3.4±0.4 3.2±0.5 3.8±0.6 3.6±0.6 4.1±0.5 With 40 acyl carbons 3.2±0.8 2.0±0.5 2.8±1.6 2.9±0.6 4.4±1.8 2.9±0.8 2.6±2.1 2.3±1.2 4.1±2.4 2.9±1.4 2.2±0.6 3.0±1.0 MGDG (% of MGDG) With 30 acyl carbons 0.3±0.1 0.2±0.1 0.5±0.3 0.4±0.1 0.4±0.2 0.3±0.1 0.5±0.2 0.4±0.1 0.3± 0.1 0.4±0.1 0.4±0.1 0.5±0.1 With 32 acyl carbons 0.9±0.1 1.9±0.5 1.8±0.3 1.0±0.01 1.4±0.2 1.6±0.3 1.2±0.3 2.1±0.3 1.0±0.7 1.2±0.2 1.4±0.3 1.7±0.2 With 34 acyl carbons 9.1±0.8 10.3±0.8 9.3±1.4 8.8±0.6 8.5±0.2 10.8±1.3 9.5±1.1 12.8±1.2 9.0±1.9 9.8±0.7 13.7±2.3 12.6±0.8 With 36 acyl carbons 56.5±0.4 54.1±2.0 54.5±0.6 56.5±0.6 52.8±0.9 57.0±2.6 56.6±0.8 51.9±1.0 46.3±3.2 55.5±1.3 51.5±1.9 50.7±1.8 With 37 acyl carbons 0.2±0.01 0.2±0.01 0.2±0.01 0.2±0.01 0.2±0.1 0.3±0.01 0.3±0.01 0.2±0.01 0.2±0.1 0.3±0.1 0.3±0.1 0.2±0.01 With 38 acyl carbons 6.0±0.5 6.0±0.3 5.7±0.2 6.2±0.5 5.9±0.2 6.1±0.8 6.3±0.1 6.4±0.2 4.7±1.3 6.1±0.3 6.3±0.6 6.8±0.5 With 40 acyl carbons 26.6±0.8 27.1±2.6 27.8±2.0 26.7±1.0 30.6±1.2 23.8±4.0 25.4±1.1 26.0±1.2 13.8±3.1 26.6±2.2 26.4±2.2 27.1±2.0 PG (% of PG) With 26 acyl carbons 10.5±4.5 11.6±3.0 14.1±5.0 10.9±5.4 14.0±3.2 10.7±5.3 15.7±3.2 12.0±2.8 13.1±3.7 19.7±4.8 14.4±4.9 12.8±4.0 With 27 acyl carbons 0.2±0.1 0.3±0.03 0.3±0.1 0.3±0.1 0.3±0.1 0.2±0.1 0.4±0.1 0.3±0.1 0.2±0.1 0.4±0.1 0.4±0.1 0.4±0.1 With 29 acyl carbons 0.2±0.1 0.3±0.1 0.3±0.1 0.4±0.2 0.4±0.2 0.3±0.1 0.4±0.1 0.4±0.1 0.3±0.1 0.4±0.1 0.4±0.1 0.5±0.1 With 30 acyl carbons 3.5±1.0 4.5±0.9 5.9±1.2 3.6±1.2 4.6±0.5 4.2±1.5 5.5±0.5 5.1±1.6 4.4±0.8 6.0±1.0 5.1±1.0 5.6±1.5 With 31 acyl carbons 0.5±0.1 0.7±0.1 1.0±0.3 0.5±0.01 0.7±0.2 0.7±0.1 0.6±0.2 0.8±0.3 0.7±0.2 0.7±0.2 1.0±0.2 1.1±0.1 With 32 acyl carbons 3.8±0.3 5.5±0.6 8.1±1.8 4.3±0.3 5.9±1.0 6.6±0.6 4.4±0.6 7.7±1.5 5.7±0.5 4.8±0.7 6.2±1.5 7.5±0.5 With 33 acyl carbons 0.4±0.01 0.5±0.1 0.8±0.2 0.4±0.1 0.5±0.1 0.7±0.01 0.5±0.1 0.7±0.1 0.9±0.7 0.6±0.2 0.8±0.3 0.8±0.1 With 34 acyl carbons 13.9±4.9 10.9±0.8 13.4±3.1 10.8±2.3 9.5±0.5 15.1±2.3 10.1±2.3 11.6±2.8 15.1±3.5 9.3±0.6 13.2±3.0 12.4±0.9 With 35 acyl carbons 0.7±0.1 2.0±0.2 1.5±0.2 0.7±0.2 1.3±0.3 1.2±0.2 0.7±0.1 1.8±0.3 1.2±0.2 0.9±0.1 1.3±0.5 1.5±0.3 With 36 acyl carbons 64.9±8.2 62.4±3.7 53.2±9.2 66.7±6.2 61.8±4.6 58.7±4.9 60.4±2.5 58.2±6.1 57.0±8.4 55.9±5.9 55.9±6.0 55.9±5.4 With 37 acyl carbons 0.3±0.1 0.4±0.1 0.6±0.2 0.3±0.1 0.4±0.1 0.5±0.03 0.3±0.1 0.4±0.1 0.5±0.1 0.4±0.1 0.3±0.1 0.4±0.1 With 38 acyl carbons 0.3±0.1 0.4±0.05 0.3±0.01 0.4±0.1 0.3±0.01 0.4±0.1 0.4±0.03 0.5±0.1 0.4±0.03 0.4±0.1 0.5±0.1 0.5±0.1 With 40 acyl carbons 0.6±0.1 0.6±0.1 0.5±0.1 0.6±0.1 0.4±0.1 0.5±0.1 0.6±0.2 0.5±0.1 0.5±0.1 0.5±0.1 0.4±0.1 0.6±0.1 PC (% of PC) With 28 acyl carbons 0.01±0.001 0.01±0.01 0.01±0.01 0.01±0.01 0.02±0.01 0.01±0.01 0.02±0.01 0.03±0.01 0.01±0.01 0.01±0.01 0.01±0.01 0.03±0.01 With 30 acyl carbons 0.1±0.01 0.1±0.02 0.1±0.04 0.1±0.03 0.1±0.1 0.1±0.03 0.1±0.02 0.1±0.05 0.1±0.04 0.1±0.03 0.1±0.01 0.1±0.02 With 32 acyl carbons 0.1±0.01 0.2±0.1 0.3±0.1 0.1±0.1 0.2±0.2 0.4±0.1 0.2±0.1 0.2±0.1 0.3±0.1 0.2±0.1 0.3±0.1 0.2±0.1 With 34 acyl carbons 1.9±0.1 1.7±0.2 2.5±0.4 2.0±0.2 1.8±0.2 2.9±0.2 2.2±0.2 2.0±0.3 2.6±0.2 2.1±0.1 2.1±0.6 1.9±0.1 With 36 acyl carbons 14.2±1.3 10.1±0.4 16.0±0.8 14.7±0.8 11.7±0.9 16.9±0.6 14.5±0.4 11.5±0.9 16.2±0.9 14.3±0.6 12.4±2.1 11.2±1.1 With 38 acyl carbons 16.6±1.0 17.2±0.3 15.9±0.6 17.4±1.3 15.2±0.4 16.2±0.6 17.4±0.4 17.4±1.4 17.9±1.3 16.3±0.7 17.3±0.2 18.2±1.2 With 40 acyl carbons 64.8±1.1 67.7±0.5 62.9±0.8 63.4±1.5 68.5±1.9 60.9±1.8 62.2±0.6 66.0±1.4 60.4±1.1 63.2±1.0 64.5±2.2 65.1±1.2
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Control 1MeJA 10MeJA 100MeJA 6H 12H 24H 6H 12H 24H 6H 12H 24H 6H 12H 24H With 42 acyl carbons 1.9±0.1 2.6±0.2 2.0±0.4 2.0±0.3 2.1±0.4 2.1±0.4 2.8±0.2 2.3±0.4 2.1±0.1 3.0±0.3 2.7±0.5 2.7±0.3 With 44 acyl carbons 0.4±0.0 0.5±0.1 0.3±0.1 0.3±0.1 0.4±0.3 0.4±0.2 0.6±0.1 0.4±0.2 0.4±0.1 0.7±0.1 0.7±0.2 0.6±0.2 PE (% of PE) With 28 acyl carbons 1.0±0.6 2.7±0.6 0.9±0.4 1.1±0.7 3.0±0.1 0.6±0.2 1.3±0.6 0.9±0.3 0.6±0.2 2.1±1.1 0.3±0.03 0.4±0.3 With 30 acyl carbons 2.6±1.2 4.8±0.6 4.2±1.9 3.1±1.2 4.0±0.7 2.8±0.5 3.7±1.0 3.2±0.4 2.8±0.5 3.8±1.1 3.9±1.6 2.2±1.6 With 32 acyl carbons 2.3±0.7 6.1±1.0 7.5±1.3 3.4±1.7 5.5±1.8 4.7±0.8 3.2±1.0 3.7±1.0 5.7±0.9 4.5±1.2 6.8±1.6 4.3±1.6 With 34 acyl carbons 7.0±0.7 10.3±0.7 9.7±2.4 6.1±1.1 8.2±1.1 8.7±0.6 7.9±1.2 9.3±0.3 9.4±0.6 9.4±0.8 12.1±1.9 10.7±1.9 With 36 acyl carbons 34.4±1.7 25.7±1.2 24.8±2.1 34.4±1.4 25.3±1.4 26.5±2.2 33.7±1.0 30.1±2.0 29.9±1.0 30.5±1.8 28.5±3.5 30.9±3.5 With 38 acyl carbons 12.3±1.6 9.8±0.1 10.4±1.3 10.6±1.0 9.8±0.5 12.5±0.8 11.4±0.9 11.4±0.6 11.7±0.7 12.8±0.6 12.6±1.4 12.8±1.4 With 40 acyl carbons 39.4±2.2 40.0±6.0 41.6±6.3 40.2±3.0 43.7±1.9 43.4±2.1 38.3±3.5 40.7±1.9 38.8±1.1 36.6±4.2 34.7±4.3 38.1±4.3 With 42 acyl carbons 0.7±0.2 0.5±0.2 0.6±0.2 0.9±0.5 0.6±0.2 0.7±0.2 0.5±0.2 0.5±0.2 1.1±03 0.4±0.1 0.9±0.1 0.7±0.1 With 44 acyl carbons 0.1±0.1 0.1±0.03 0.1±0.1 0.1±0.1 0.1±0.1 0.1±0.1 0.1±0.01 0.02±0.01 0.1±0.01 - - - PI (% of PI) With 30 acyl carbons 5.1±0.6 4.6±0.6 4.2±0.7 4.7±0.9 6.2±2.5 4.9±0.7 6.0±1.1 5.2±1.3 5.4±0.8 6.6±1.4 5.7±0.2 6.0±0.8 With 31 acyl carbons 2.5±0.4 2.5±1.2 2.3±0.4 1.4±0.8 3.8±1.6 2.9±0.2 3.2±0.2 2.6±0.9 3.4±0.8 3.5±0.8 2.6±1.3 3.2±0.6 With 32 acyl carbons 6.8±1.0 7.3±0.8 7.1±1.8 6.6±0.6 9.0±2.6 7.3±1.7 8.5±1.5 10.5±2.8 6.4±1.2 10.6±1.4 9.9±3.0 12.9±3.6 With 33 acyl carbons 0.8±0.2 0.8±0.05 1.0±0.2 0.6±0.1 0.9±0.3 1.2±0.3 0.9±0.3 1.0±0.3 0.8±0.4 1.2±0.3 1.2±0.2 1.0±0.2 With 34 acyl carbons 11.6±1.4 13.1±1.8 10.3±1.2 8.3±1.8 12.2±3.2 10.5±2.4 11.4±1.3 11.5±1.7 9.6±1.0 12.3±1.9 12.5±0.8 13.1±0.9 With 35 acyl carbons 0.9±0.2 0.9±0.2 0.9±3.0 1.0±0.3 0.8±0.3 1.0±0.6 1.1±0.2 0.8±0.2 1.4±0.4 0.9±0.4 0.9±0.1 0.9±0.2 With 36 acyl carbons 15.5±1.4 17.0±0.4 13.9±4.8 14.6±2.9 13.9±2.6 15.7±1.4 15.4±0.8 15.0±3.1 17.1±1.6 15.1±1.1 16.1±2.3 15.4±0.6 With 38 acyl carbons 4.5±0.3 5.5±0.6 4.3±1.2 4.6±0.3 4.2±0.1 5.2±1.3 5.4±0.7 5.3±0.8 4.8±0.6 5.4±1.0 6.3±0.4 6.0±0.6 With 40 acyl carbons 41.3±3.1 45.4±3.2 35.5±4.1 39.8±3.9 33.8±8.1 45.3±6.1 41.5±2.0 38.6±4.2 43.0±2.6 35.4±1.3 41.2±4.3 36.6±2.6 With 42 acyl carbons 9.1±4.2 2.6±0.7 7.4±1.6 15.1±7.9 13.8±1.3 5.6±2.5 5.7±2.7 8.3±1.7 7.3±3.7 7.6±2.3 3.6±1.0 4.4±2.9 With 44 acyl carbons 1.8±0.6 0.4±0.3 0.9±0.1 3.3±1.5 1.3±0.1 0.6±0.3 0.7±0.3 1.1±0.2 0.9±0.5 1.4±0.4 0.3±0.1 0.5±0.4 PS (% of PS) With 32 acyl carbons 4.3±2.6 6.1±3.9 9.0±7.4 5.0±1.7 11.5±1.8 10.7±5.4 8.9±4.5 5.6±2.3 3.9±2.2 7.9±2.8 7.7±3.5 5.2±2.6 With 34 acyl carbons 12.8±2.2 13.2±4.6 7.0±6.8 8.3±3.5 12.4±1.6 7.2±2.9 9.8±3.3 12.7±6.1 10.6±1.9 13.1±1.6 9.7±2.7 7.3±3.6 With 36 acyl carbons 33.1±4.1 29.7±5.8 32.9±11.5 43.6±13 32.4±15.0 39.0±6.4 30.1±3.8 29.7±8.3 28.0±4.2 28.1±7.7 26.9±6.5 36.6±8.2 With 38 acyl carbons 12.2±4.5 20.4±5.9 21.4±11.4 14.0±8.9 13.1±4.7 16.9±5.5 14.2±3.0 16.4±2.5 14.3±5.3 11.1±1.9 13.1±7.0 7.9±5.3 With 40 acyl carbons 21.8±6.6 15.4±5.5 22.7±4.1 22.4±9.5 15.7±8.6 17.1±6.2 16.2±5.6 28.4±9.6 39.8±6.8 17.3±1.5 27.4±10 29.7±9.7 With 42 acyl carbons 8.5±4.3 8.1±4.9 2.6±2.2 3.9±1.0 2.4±1.4 8.1±1.6 3.9±1.7 2.2±2.5 1.4±1.8 5.1±1.5 3.8±3.5 3.3±4.4 With 44 acyl carbons 7.3±3.8 7.4±4.9 4.4±1.5 3.0±1.9 12.7±1.1 1.6±0.9 17.4±5.9 5.8±4.3 2.2±1.6 17.3±5.7 11.6±4.5 10.1±5.0 PA (% of PA) With 32 acyl carbons 2.9±1.7 9.5±4.7 3.5±1.5 2.2±0.3 17.2±7.6 4.6±2.2 1.9±0.6 9.6±5.5 5.3±2.5 2.5±1.0 6.7±6.2 7.0±2.5 With 34 acyl carbons 4.6±0.3 11.1±1.6 7.4±2.1 4.6±0.3 9.4±1.5 6.0±0.3 5.0±0.5 14.7±2.1 7.5±1.3 5.7±0.6 18.3±4.0 16.7±2.0 With 36 acyl carbons 17.7±1.3 35.3±3.4 20.3±0.7 17.7±1.4 35.9±4.4 23.7±4.0 18.9±0.9 40.0±2.5 25.1±0.9 19.8±1.2 39.9±4.3 41.0±2.3 With 38 acyl carbons 16.9±0.8 11.8±0.5 15.5±0.8 18.0±1.8 10.4±1.6 15.8±1.3 18.2±0.6 11.4±1.6 18.0±1.1 17.4±1.1 11.9±1.7 13.9±0.8 With 40 acyl carbons 57.9±1.8 58.0±2.1 55.6±1.1 48.3±12.5 31.9±3.5 25.6±3.0 24.0±2.3 23.2±2.9 52.2±1.3 47.3±4.6 39.2±10.6 20.8±0.6
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Control 1MeJA 10MeJA 100MeJA 6H 12H 24H 6H 12H 24H 6H 12H 24H 6H 12H 24H LPG (% of LPG) With 16 acyl carbons 69.9±8.7 47.6±4.7 70.8±19.9 33.3±2.9 63.3±6.6 41.5±4.8 66.4±3.6 81.4±9.1 40.4±16.8 36.7±4.8 72.3±13.9 76.8±19.8 With 18 acyl carbons 5.9±1.2 51.6±9.2 43.9±9.6 65±10.9 36.8±6.8 32.9±4.7 33.8±3.2 16.6±4.5 60.1±19.2 56.7±6.5 27.9±12.7 22.5±8.1 LPC (% of LPC)) With 16 acyl carbons 3.5±0.4 8.3±0.4 5.4±1.5 3.6±1.0 9.8±0.6 7.6±0.2 3.7±3.1 8.7±0.6 7.9±1.8 3.1±0.5 9.9±3.2 6.7±3.0 With 18 acyl carbons 11.4±1.1 12.2±1.3 10.0±1.4 12.2±1.2 14.2±1.8 13.4±6.3 12.8±1.3 14.1±0.5 15.7±4.4 12.6±1.6 15.1±3.5 11.1±2.3 With 20 acyl carbons 85.0±1.3 78.2±1.2 84.1±2.4 84.0±2.0 76.0±1.5 78.8±5.4 83.3±3.9 76.7±0.7 76.2±5.9 84.1±1.9 74.4±5.8 81.5±4.3 With 22 acyl carbons 0.2± 0.1 1.3±0.2 0.4±0.9 0.2±0.5 0.1±0.1 0.3±0.1 0.2±0.1 0.3±0.1 0.1±0.1 0.2±0.1 0.6±0.4 0.7±0.7 LPE (% of LPE) With 16 acyl carbons 33.2±4.1 22.8±9.0 38.4±3.4 26.3±2.1 47.3±14 30.3±9.9 28.8±5.9 49.0±11.8 30.3±4.5 11.3±1.4 37.8±3.3 37.6±2.8 With 18 acyl carbons 11.4±1.6 25.6±8.0 9.2±5.0 22.1±6.0 7.7±5.0 11.1±2.5 21.9±7.9 16.0±4.5 14.9±3.5 11.9±1.1 14.4±1.8 29.8±2.6 With 20 acyl carbons 55.5±5.6 51.7±12.0 52.0±7.9 51.8±12.5 45.3±12 58.7±9.7 49.4±12.3 35.1±8.7 54.9±11.2 76.8±6.8 22.7±2.8 33.2±3.2
Fig. 7.7 Molecular species of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) (mol% of total polar lipids analyzed)
Gracilaria dura thalli treated with methyl jasmonate.
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Fig. 7.8 Molecular species of phosphatidylcholine (PC) (mol% of total polar lipids analyzed) in Gracilaria
dura thalli treated with methyl jasmonate.
Fig. 7.9 Molecular species of phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) (mol% of total
polar lipids analyzed) in Gracilaria dura thalli treated with methyl jasmonate.
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Fig. 7.10 Molecular species of phosphatidylinositol (PI) and phosphatidylserine (PS) (mol% of total polar
lipids analyzed) in Gracilaria dura thalli treated with methyl jasmonate
Fig. 7.11 Molecular species of phosphatidic acid (PA), lyso-phosphatidylglycerol (LPG), lyso-
phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) (mol% of total polar lipids
analyzed) in Gracilaria dura thalli treated with methyl jasmonate.
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The content of PG decreased with the increase in MeJA concentration in treated
thalli by 1.1-1.6- fold due to decrease in C34 (34:3, 34:2, 34:1), C36 (36:4, 36:2), C37 (37:2,
37:1), and C40 (40:8, 40:7) lipid molecular species (Fig. 7.8). However, an increase in PG
content (1.1-1.7-fold) was observed with time except in 100 μM MeJA that showed lower
PG content at 24 h. The contents of phospholipids PC, PE and PA increased by 1.03-1.4-fold
with the increase in MeJA concentration (except 100 μM MeJA treated thalli that showed
1.4-3.3-fold lower PE and PA contents). This increase in PC, PE and PA could be attributed
to the increase in C30 (32:2, 32:1, 30:0), C32 (32:2, 32:1), C34 (34:4, 34:3, 34:2), C38
(38:7, 38:6, 38:4, 38:3, 38:2, 38:1, 38:0), C40 (40:8, 40:7, 40:6, 40:5, 40:4), C42 (42:11,
42:8, 42:6, 42:5, 42:4) and C44 (44:12, 44:11, 44:10, 44:9, 44:4, 44:3) for PC, C36 (36:4,
36:3) and C40 (40:8, 40:7) for PE and C32 (32:4, 32:2, 32:1), C34 (34:2, 34:1) and C36
(36:4, 36:3, 36:2) lipid molecular species for PA (Fig. 7.8, 7.9, 7.11). However, the response
of these phospholipids PC, PE and PA varied with time. The treated thalli showed 1.1-1.7-
fold increase in PC with time but its content at 24 h was 1.2-1.3-fold lower as compared to
12 h while an initial decrease followed by 2.4-3.0-fold increase at 24 h in PA and a constant
increase of 1.4-8.7-fold in PE contents. Further, DGDG showed a time-dependent increase
of 1.1-1.2-fold changes at 24 h (p < 0.05) except 100 μM MeJA. However, there was a
decrease in lipid species containing two 18:3 acyl carbons in both MGDG and DGDG
(especially at longer duration), indicating these chloroplastic galactolipids would have been
hydrolyzed for 18:3. This 18:3 could have been utilized as a substrate of LOX for the
biosynthesis of 13-hydroperoxylinolenic acid and further channeled downstream either into
the jasmonate pathway or other alternate pathways of fatty acid oxidation cascade,
analogous to the higher plants. No such reduction in 18:3 containing phospholipids was
observed except in PA (36:6). Among minor lipids, PS showed a time dependent increase of
1.1-1.8-fold in treated thalli except 100 μM while PI showed a time dependent decrease of
1.1-1.2 fold in MeJA treated thalli (Fig. 7.10). Among lysolipids, LPE and LPC showed an
increase of 1.1-2.1-fold and 1.2-7.9-fold respectively in treated thalli with the increase in
concentration of exogenous MeJA and 3.8-5.4-fold and 1.4-1.5-fold with time except 100
μM MeJA treated thalli (especially 16:0, 18:1, 18:2, 20:3 and 20:4) while LPG showed non-
significant changes as compared to control (Fig. 7.11).
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Table 7.3 Fatty acid composition (% of fatty acid methyl ester, FAME) in Gracilaria dura treated with methyl
jasmonate (expressed as means ± S.D.; n=3).
Fatty acids Treatment 6 h 12 h 24 h
C14:0 Control 5.2 ± 0.9 5.4 ± 0.7 5.3 ± 0.6
1μM MeJA 4.4 ± 0.8 4.0 ± 0.7 4.6 ± 0.6
10μM MeJA 4.0± 1.7 4.9 ± 1.2 4.7 ± 1.5
100μM MeJA 4.8 ± 1.4 5.3 ± 1.2 4.4 ± 0.7
C15:0 Control 0.8 ± 0.4c 0.9 ± 0.2 0.9 ± 0.3
1μM MeJA 0.9 ± 0.2b 0.7 ± 0.1 0.9 ± 0.1
10μM MeJA 0.8 ± 0.4a 1.2 ± 0.1 1.1 ± 0.3
100μM MeJA 1.1 ± 0.3b 0.7 ± 0.1 0.9 ± 0.4
C16:0 Control 37.4 ± 4.8 38.2 ± 4.3 37.2 ± 3.8
1μM MeJA 39.3 ± 5.3 32.6 ± 3.8 34.8 ± 2.5
10μM MeJA 35.7 ± 2.8 33.5 ± 2.5 36.0 ± 2.5
100μM MeJA 38.2 ± 6.3 33.8 ± 3.5 35.5 ± 2.4
C17:0 Control 0.5 ± 0.1 0.5 ± 0.1b 0.5 ± 0.1
1μM MeJA 0.6 ± 0.2 0.3 ± 0.1c 0.4 ± 0.1
10μM MeJA 0.6 ± 0.4 0.4 ± 0.1bc 0.4 ± 0.1
100μM MeJA 0.4 ± 0.1 0.8 ± 0.2a 0.4 ± 0.1
C18:0 Control 5.0 ± 0.5 4.8 ± 0.9 5.2 ± 0.7
1μM MeJA 5.7 ± 0.4 4.1 ± 0.4 4.9 ± 0.6
10μM MeJA 4.1 ± 0.4 4.7 ± 1.0 4.2 ± 0.7
100μM MeJA 6.1 ± 2.1 4.4 ± 1.1 3.9 ± 0.9
C20:0 Control 0.5 ± 0.2 0.5 ± 0.1 0.5 ± 0.3a
1μM MeJA 0.5 ± 0.2 0.4 ± 0.1 0.8 ± 0.3a
10μM MeJA 0.5 ± 0.1 0.4 ± 0.1 0.3 ± 0.2b
100μM MeJA 0.4 ± 0.1 0.6 ± 0.1 0.5 ± 0.2ab
C22:0 Control 0.9 ± 0.5 1.0 ± 0.2 0.9 ± 0.4
1μM MeJA 0.8 ± 0.3 0.7 ± 0.1 1.1 ± 0.2
10μM MeJA 0.7 ± 0.1 0.7 ± 0.3 0.5 ± 0.2
100μM MeJA 0.7 ± 0.2 0.6 ± 0.1 1.6 ± 0.5
C24:0 Control 1.0 ± 0.2 0.9 ± 0.2 1.0 ± 0.5
1μM MeJA 0.7 ± 0.2 0.4 ± 0.1 0.6 ± 0.2
10μM MeJA 0.9 ± 0.4 0.5 ± 0.2 0.4 ± 0.2
100μM MeJA 0.8 ± 0.2 0.5 ± 0.1 0.9 ± 0.6
C16:1(n-7) Control 2.3 ± 0.7 2.0 ± 0.3 2.2 ± 0.9
1μM MeJA 2.3 ± 1.5 2.2 ± 1.0 3.7 ± 1.4
10μM MeJA 1.9 ± 0.8 3.7 ± 1.3 2.8 ± 1.5
100μM MeJA 3.4 ± 1.4 2.3 ± 1.2 1.8 ± 1.1 a-c: Values in a column for each fatty acids are significantly different at p≤ 0.05.
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Fatty acids Treatment 0 h 6 h 12 h 24 h
C16:1(n-9) Control 2.1 ± 0.6 2.0 ± 1.6 2.0 ± 1.1 2.2 ± 1.2
1μM MeJA 1.9 ± 1.1 3.4 ± 1.3 1.4 ± 0.9
10μM MeJA 2.1 ± 0.6 4.1 ± 1.4 2.1 ± 1.3
100μM MeJA 2.2 ± 1.3 3.9 ± 1.6 2.7 ± 0.9
C18:1(n-9) Control 3.1 ± 1.8 2.7 ± 0.9b 2.7 ± 0.7 2.9 ± 0.9
1μM MeJA 5.4 ± 1.4a 3.8 ± 0.8 3.8 ± 1.0
10μM MeJA 3.4 ± 0.6b 3.4 ± 0.5 3.4 ± 1.1
100μM MeJA 2.3 ± 0.9b 2.3 ± 0.5 3.6 ± 1.2
C18:1(n-9) t Control 2.1± 0.9 2.2 ± 1.1 2.1 ± 0.5b 2.0 ± 0.5
1μM MeJA 2.5 ± 1.0 2.3 ± 0.8b 2.4 ± 0.9
10μM MeJA 2.1 ± 0.6 3.1 ± 0.4a 2.5 ± 0.5
100μM MeJA 2.5 ± 0.5 1.9 ± 0.3b 2.4 ± 0.9
C18:2(n-6) Control 1.5 ± 0.8 1.8 ± 0.2 1.5 ± 0.7 1.6 ± 0.5
1μM MeJA 2.6 ± 0.8 2.0 ± 0.4 3.3 ± 0.5
10μM MeJA 2.6 ± 0.9 2.4 ± 0.9 2.7 ± 1.0
100μM MeJA 2.7 ± 0.4 2.3 ± 0.8 2.4 ± 0.7
C20:3(n-6) Control 2.2 ± 0.4 1.9 ± 0.5 2.1 ± 0.7 2.0 ± 0.5
1μM MeJA 2.2 ± 0.2 2.7 ± 0.3 2.0 ± 0.3
10μM MeJA 2.7 ± 0.9 2.5 ± 0.3 1.9 ± 0.1
100μM MeJA 2.4 ± 0.6 2.8 ± 0.4 2.0 ± 0.2
C20:4(n-6) Control 37.0 ± 4.7 38.2 ± 4.8 37.2 ± 3.7 37.9 ± 3.8
1μM MeJA 30.3 ± 5.2 41.4 ± 2.7 35.2 ± 2.1
10μM MeJA 38.0 ± 3.7 34.6 ± 1.5 37.1 ± 2.7
100μM MeJA 31.6 ± 4.6 37.9 ± 2.7 36.9 ± 3.3
SFA Control 51.9 ± 5.3 51.2 ± 4.5 52.1 ± 4.1 51.5 ± 4.8
1μM MeJA 53.0 ± 7.5 43.3 ± 2.2 48.1 ± 3.3
10μM MeJA 47.3 ± 2.3 46.3 ± 1.9 47.6 ± 1.8
100μM MeJA 52.4 ± 8.8 46.6 ± 3.1 48.1 ± 2.8
MUFA Control 9.6 ± 1.9 9.2 ± 2.9 8.8 ± 4.2 9.3 ± 2.1
1μM MeJA 12.2 ± 1.1 11.7 ± 1.5 11.4 ± 1.6
10μM MeJA 9.5 ± 1.3 14.3 ± 1.4 10.7 ± 1.5
100μM MeJA 10.3 ± 2.7 10.4 ± 1.3 10.6 ± 3.2
PUFA Control 40.7 ± 3.9 41.9 ± 5.4 40.8 ± 2.9 41.5 ± 2.1
1μM MeJA 35.0 ± 3.6 46.1 ± 3.3 40.5 ± 2.8
10μM MeJA 43.3 ± 3.6 39.5 ± 1.6 41.7 ± 2.0
100μM MeJA 37.3 ± 4.3 43.0 ± 2.9 41.4 ± 2.5
PUFA/SFA Control 0.8 ± 0.1 0.8 ± 0.2 0.8 ± 0.1 0.8 ± 0.1
1μM MeJA 0.7 ± 0.2 1.1 ± 0.1 0.8 ± 0.1
10μM MeJA 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1
100μM MeJA 0.7 ± 0.2 0.9 ± 0.1 0.9 ± 0.1 a-c: Values in a column for each fatty acids are significantly different at p≤ 0.05; SFA-Saturated fatty acid; MUFA-Monounsaturated fatty
acid; PUFA-Polyunsaturated fatty acid
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Further, FA profiles of treated and control thalli showed non-significant changes for
almost all the fatty acids quantified except C16:0, C16:1 (n-9), C20:3 (n-6), C20:4 (n-6) and
total saturated fatty acids (SFA) (Table 7.1). The treated thalli showed a time dependent
decrease in SFAs (Table 7. 3). MUFAs contents increased with the increase in exogenous
MeJA concentration mainly due to increase in the contents of C16:1 (n-7), C16:1 (n-9),
C18:1 (n-9t) and C18:1 (n-9). Although polyunsaturated fatty acids (PUFAs) did not showed
any significant changes, C18:2 (n-6) and C20:4 (n-6) showed 1.1-1.6-fold increase in treated
thalli except at 12 h while C20:4 (n-6) showed 1.2-1.4-fold increase in 1 and 100 μM MeJA
treated thalli with time. The non-significant changes in FA profile indicated continued
cycling of FAs, desaturation of SFAs/MUFAs to PUFAs and their incorporation into
different lipid molecular species.
7.3.4. Oxylipin contents and lipoxygenase activity
The contents of all the oxylipins significantly increased with the exogenous
application of MeJA in both the dose and time dependent manner, except 8- and 5-HETE (p
<0.05) (Table 7.4 and Fig. 7.12).
Table 7.4 Oxylipin groups’ contents (ng g-1 FW) in Gracilaria dura treated with methyl jasmonate.
Control 1 μM MeJA 10 μM MeJA 100 μM MeJA
Total oxylipin contents (Toxl)
6 h 187.6 ± 9.6c 192.8 ± 26.4c 297.2 ± 36.2b 380.7 ± 34.0a
12 h 191.1 ± 8.2c 228.2 ± 16.2c 302.9 ± 10.5b 396.3 ± 28.5a
24 h 206.8 ± 6.2d 328.8 ± 14.8c 372.1 ± 32.3b 430.4 ± 22.8a
Total hydroxyeicosatetraenoic acid (THETE)
6 h 165.2 ± 10.2c 164.6 ± 21.2c 255.9 ± 30.5b 308.2 ± 25.8a
12 h 166.0 ± 15.1b 177.0 ± 26.4b 259.5 ± 12.3a 312.7 ± 24.1a
24 h 177.6 ± 7.4c 259.0 ± 21.6b 284.5 ± 28.0ab 320.0 ± 13.4a
Total hydroxyoctadecatrienoic acid (THOTrE)
6 h 8.4 ± 0.4c 14.3 ± 1.7b 16.5 ± 3.1b 35.1 ± 3.0a
12 h 8.0 ± 2.4c 22.5 ± 3.4b 25.1 ± 1.3b 38.4 ± 3.7a
24 h 11.2 ± 0.5d 35.7 ± 4.3c 52.3 ± 9.6b 73.1± 9.4a
Total hydroxyoctadecadienoic acid (THODE)
6 h 8.6 ± 0.2c 7.9 ± 0.9c 10.8 ± 0.5b 20.7 ± 2.1a
12 h 9.3 ± 0.8c 13.0 ± 3.6bc 16.5 ± 0.7b 22.9 ± 2.6a
24 h 13.4 ± 0.7c 32.0 ± 6.5b 30.1 ± 5.1ab 32.7 ± 4.6a a-c: Values in a row are significantly different at p < 0.05.
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Fig. 7.12 Effect of methyl jasmonate on oxylipin contents in Gracilaria dura. Different letters on same shade
of columns indicate that mean values for the particular incubation time were significantly different at
p < 0.05.
The total oxylipin contents (Toxl) increased by 1.03-2.1-fold, total
hydroxyeicosatetraenoic acids (THETEs) by 1.1-1.9-fold, hydroxyoctadecadienoic acids
(HODEs) by 1.3-2.5-fold and hydroxyoctadecatrienoic acids by 1.7-6.7-fold with the
increase in exogenous MeJA concentration applied except 1 μM MeJA treated thalli that
showed lower THETEs at 6 h. Similarly, Toxl increased by 1.02-1.7-fold, THETEs by 1.01-
1.6-fold, THODEs by 1.1-4.0-fold and THOTrEs by 1.1-3.2-fold with the increase in
incubation period (Table 7.4, Fig. 7.12). These oxylipin contents showed that the effect of
exogenous concentration of MeJA applied had more profound effect than time duration, as
apparent from greater increase observed in the content of different oxylipins with the
exogenous concentration of MeJA applied as compared with time. Moreover, the highest
increase observed in HOTrEs, especially 13-HOTrE (2.6-13.5-fold increase) showed the
upregulation of 13-LOX metabolism, as 13-HOTrE is produced by reduction of 13-LOX
product, 13-hydroperoxyoctadectrienoic acid (by peroxidases), one of the key substrate of
jasmonate biosynthesis. Similarly, the contents of 9- and 13- HODE also increased in dose
dependent manner (1.2-4.0-fold and 1.2-3.3-fold respectively) and with time (1.4-4.8-fold
and 1.1-3.8-fold respectively). The ratio of 13/9-HODE was higher in all the treated samples
as well as control but the ratio of 13/9-HODE decreased in treated samples as compared to
control at 6 h and 24 h and showed increase only at 24 h (2.0-2.8-fold). Further, HETEs
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were the predominant oxylipins in control as well as MeJA treated thalli followed by
HODEs and HOTrEs. Among HETEs (15-, 12-, 8- and 5-HETEs), 12-HETE was the
dominant fraction that showed an increase of 1.1-1.5-fold in a dose dependent and 1.3-2.2-
fold in time dependent manner except 1 μM MeJA treated thalli that showed lower content
at 6 h and 12 h as compared to control (Fig. 7.12). However, the highest increase among
different HETEs was shown by 5-HETE that showed an increase of 2.2-6.9-fold with the
increase in exogenous MeJA concentration applied followed by 15-HETE (1.1-1.9-fold) and
8-HETE (1.3-1.8-fold).
The LOX activities for the three substrate FAs (LA, ALA and AA) also increased
significantly (p < 0.05) with the increase in the concentration of exogenous MeJA and with
time in agreement with the increase in oxylipins contents (Fig. 7.13). The highest increase
was shown by linolenate-LOX (ALA-LOX) which showed an increase of 1.6-3.5-fold with
the increase in exogenous MeJA concentration and 1.1-6.3-fold with time. Similarly, the
activities of linoleate-LOX (LA-LOX) and arachidonate-LOX (AA-LOX) also increased by
1.1-1.5-fold and 1.01-1.3-fold respectively with the increase in the concentration of
exogenous MeJA and by 1.3-2.1-fold and 1.1-1.9-fold respectively with time.
Fig. 7.13 Effect of methyl jasmonate on lipoxygenase (LOX) activity in Gracilaria dura. Different letters on
same shade of columns indicate that mean values for the particular incubation time were
significantly different at p < 0.05.
7.3.4. Total phenolic compounds, polyphenol oxidase, phenyl ammonia-lyase and shikimate
dehydrogenase activities
The MeJA treated thalli showed a significant increase in the content of total phenolic
compounds (TPC) both with the increase in the concentration of exogenous MeJA and with
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time by 1.2-1.7-fold and 1.1-1.4-fold respectively as compared to control (p <0.05) (Fig.
7.14). However, the content of TPC in treated G. dura thalli was 1.3-fold lower at 24 h than
those of 12 h. The increase in TPC was in agreement with the increase in the activity of
polyphenol oxidase (PPO) which increased by 1.5-3.0-fold with the increase in the
concentration of exogenous MeJA applied (Fig. 7.14). However, PPO activity increased
with the increase in time period till 12 h by 1.1-2.0-fold but decreased at 24 h followed by a
concomitant decrease in polyphenolic content at 24 h. The activities of phenyl ammonia-
lyase (PAL) also increased significantly in treated thalli with the increase in exogenous
MeJA concentration (1.5-13.2-fold) as well as time (1.7-7.0-fold increase) as compared to
control (p <0.05) (Fig. 7.14). Further, the activity of shikimate dehydrogenate (SD) also
increased by 2.4-7.2-fold in dose-dependent manner in MeJA treated thalli as compared to
control. However, SD activity increased till 12 h with the increase in time (1.1-1.3-fold) and
then decreased at 24 h like PPO activity by 1.4-1.7-fold. Moreover, two-way ANOVA also
confirmed that the changes observed in TPC, PPO, PAL and SD were not only dose and
time dependent but the cumulative effect of concentration and time was also significant and
thus played an additive role in MeJA treated G. dura thalli.
Fig. 7.14 Effect of methyl jasmonate on phenolic compounds and the activities of polyphenol oxidase (PPO),
phenyl-ammonia lyase (PAL) and shikimate dehydrogenase (SD) in Gracilaria dura. Different
letters on same shade of columns indicate that mean values for the particular incubation time were
significantly different at p < 0.05.
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7.4. Discussion
Methyl jasmonate is an important signaling molecule that affects various growth and
developmental responses in higher plants as well as non-vascular plants including fungi and
algae (Bouarab et al. 2004; Collén et al. 2006; Gaquerel et al. 2007; Yan et al. 2007; Küpper
et al. 2009; Wasternack et al. 2012). MeJA upregulates the genes involved in jasmonate
biosynthesis, secondary metabolism, cell wall formation, stress and defense proteins in
higher plants and algae (Cheong and Choi, 2003; Bouarab et al. 2004; Collén et al. 2006;
Zulak et al. 2009; Geyter et al. 2012). In the present study, MeJA induced a state of
oxidative stress in G. dura thalli as found earlier in red alga C. crispus (Collén et al. 2006)
and brown alga L. digitata (Küpper et al. 2009) due to induced ROS production (H2O2, HO·
and O2•−) (Fig.7.1 and 7.2). Similar enhanced ROS production due to MeJA treatment
especially at higher concentrations of 10 and 100 μM, has also been observed in higher
plants (Jung, 2004; Maksymiec and Krupa, 2006; Xue et al. 2008) and microalgae such as
Scendesmus spp. (Fedina and Benderliev, 2000; Kováčik et al. 2011). Further, Collén et al.
(2006) attributed the production of ROS to NADPH oxidase as apparent from its increased
transcripts on C. crispus array while Küpper et al. (2009) found that the source of ROS was
only partially inhibited by diphenylene iodonium (a suicide substrate inhibitor of NAD(P)H
oxidases). This MeJA induced oxidative stress plays a crucial role in conferring resistance
against algal endophytes such as Acrochete operculta in C. crispus and Laminariocolax
tomentosoides in L. digitata (Bouarab et al. 2004; Küpper et al. 2009). There is a specific
cross-talk between MeJA and ROS levels (H2O2) that act as second messengers and aid in
upregulation of various stress genes such as glutathione S-transferase, heat shock protein 20,
xenobiotic reductase and genes involved in phenylpropanoid pathway (Orozco-Cárdenas et
al. 2001; Collén et al. 2006; Hung and Kao, 2007; Liu et al. 2008).
Further, this increased ROS production resulted in lipid peroxidation that further
damaged photosynthetic pigments especially Chl a (Fig. 7.3). In microalgae and freshwater
algae, many researchers have demonstrated that JA/MeJA at lower concentrations (10-6 to
10-8 M) increases the cell vialbility, cell number, photosynthetic pigments, polysaccharides
and soluble proteins while at higher concentrations (10-4 to 10-5 M or higher), MeJA acts as
a stress substance and promotes typical senescence symptoms, decreases cell viability and
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pigments (Fedina and Benderiev, 2000; Czerpak et al. 2006; Piotrowska et al. 2010). In
macroalgae, Gaquerel et al. (2007) reported that 100 μM MeJA was not detrimental to the
thalli of C. crispus and observed strong necrosis only at concentrations >100 μM. However,
G. dura in the present study showed the symptoms of depigmentation and bleaching in 100
μM MeJA treated thalli at 48 h, which may be possibly due to higher levels of ROS
accumulation. Moreover, the degradation of Chl a has also been observed in higher plants in
response to MeJA induced oxidative stress (Ananieva et al. 2007; Gómez et al. 2010; Chen
et al. 2011). Several studies have reported that MeJA treatment induced ROS production not
only degrades Rubisco but also causes down-regulation of photosynthesis-related genes,
thereby decreasing the photosynthetic rate (Popova and Vaklinova, 1988; Ananieva et al.,
2007; Zhai et al., 2007; Matsuda et al., 2009; Chen et al. 2011). The decrease in Chl a
content was accompanied by the increase in phycobiliptroteins (especially PE) as found in
Gracilaria thalli exposed to abiotic stresses (Kumar et al. 2010a, b, 2011b). However, there
was a decrease in PC content at 24 h after an initial increase till 12 h (Fig. 7.3), in
congruence with the increased transcripts of phycocyanin lyase found in C. crispus
microarray (Collén et al. 2006) while APC did not showed any significant changes. This
indicated that PC is more vulnerable to oxidative damage among phycobiliptroteins in G.
dura. Similarly, exogenous MeJA at lower concentration (1 μM) increased the total lipid
content throughout the studied period (Fig. 7.4) while at higher doses (10 and 100 μM)
showed a decrease on longer incubation period (24 h), in agreement with the dose and time
dependent increase in lipid peroxidation (Fedina and Benderiev, 2000; Piotrowska et al.
2010).
The quantitative ESI-MS profiling revealed that G. dura lipids were highly
unsaturated and exhibited a large amount of long chain PUFAs (C18:2, 20:3 and 20:4) in
their polar lipids (Fig. 7.7, 7.8, 7.9, 7.10 and 7.11). The lipids species containing C40 acyl
chains contributed to 20-30% of total polar lipids, of which, most of them were localized in
MGDG in congruence to the earlier reports (Khotimchenko, 2002). Further, two-way
ANOVA analyses helped in deciphering MeJA induced significant changes in such a huge
polar lipid repertoire of G. dura. The detailed analysis revealed that MGDG, PC, PE and PA
showed significant dose time dependent changes in response to MeJA treatment (Fig. 7.7,
7.8, 7.9 and 7.11). Among these lipid classes, MGDG and PC were the most affected lipid
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classes may be due to high flux of these lipid classes during lipid metabolism as they are the
primary sites for de novo fatty acid allocation (Ohlrogge and Browse, 1995). As the flux
through these lipid classes is greater than through other classes, it is more likely that they are
more sensitive to changes in precursor supply. Moreover, the lipid species with higher
abundances exhibited the highest treatment specific responses in each lipid class. Further,
there was a 1.1-1.6-fold decrease in MGDG in response to MeJA (1-100 μM), while the
contents of PG decreased only at higher MeJA concentration (100 μM). DGDG contents
were almost constant at 6 and 12 h, and showed 1.1- and 1.2-fold increase in 1 and 10 μM
treated thalli while a decrease of 1.1-fold in 100 μM treated thalli at 24 h (Fig. 7.7). In
contrast Cacho et al. (2012) reported that that MeJA did not alter the overall lipid content in
Silybum marinum cells treated with 100 μM MeJA up to 48 h, except a small progressive
increase in DGDG and MGDG. Further, the contents of phospholipids, PC, PE and PA
increased except at the higher MeJA doses, a decrease in PE and PA were observed. The
contents of minor lipids PS and PI were not significantly altered. This indicated that most of
the fatty acid acyl chains were degraded form MGDG and included 18:2, 18:3, 20:3 and
20:4 that were channeled downstream the fatty acid oxidation pathway as evident from
higher LOX activities for LA, ALA and AA (Fig. 7.13). It is noteworthy to note that the
decrease in lipid molecules containing 18:3 (36:6 and 36:5) was found mainly in
galactolipids MGDG and DGDG (which showed decrease only at 24 h) while most of the
phospholipids showed an increase in 36:6 acyl lipid chain except PI. This indicated that
MGDG could play a crucial role in JA/MeJA biosynthesis (if occurs in Gracilaria spp., as
endogenous JA/MeJA has not been detected till yet in Gracilaria spp.) and JA/MeJA-
mediated defense responses analogous to those reported in higher plants (Hyun et al. 2008;
Wang et al. 2009). Transgenic plants in which MGDG synthase activity was down-regulated
by using RNA interference technology produced lower levels of JA than wild-type plants in
response to wounding. Moreover, the expression of genes involved in jasmonate
biosynthesis such as encoding lipoxygenase (LOX1), allene oxide cyclase (AOC) as well as
hydroperoxide lyase (HPL) and proteinase inhibitor (PI-I and PI-II) was strongly activated
by mechanical wounding in wild-type plants but was diminished in transgenic plants (Wang,
2009). Further, Hyun et al. (2008) also reported that chloroplast lipid hydrolysis is a critical
step for JA biosynthesis. They illustrated the role of DONGLE and a homolog of
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DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) that encodes a chloroplast targeted
lipase with strong galactolipase and a weak phospholipase A1 activity. This DGL plays an
important role in maintaining basal levels of JA under normal conditions in higher plants
and its expression regulates vegetative growth and is required for a rapid JA burst after
wounding while DAD1 plays an important role in the late phase of JA production especially
under stress (wounding). However, phospholipases (PLA1 and PLD) are also demonstrated
to contribute to JA biosynthesis in plants and are upregulated by MeJA treatment (Profotova
et al. 2005; Salzman et al. 2005; Yang et al. 2007b; Cacho et al. 2012). It is believed that
phospholipase activity results in modification of lipid constituents of membrane and
generation of one or more products that are able to recruit or modulate specific target
proteins (Meijer and Munnik, 2003). Hyun et al. (2008) also reported that while DGL and
DAD1 are necessary and sufficient for JA production while phospholipase D appears to
modulate wound response by stimulating DGL and DAD1 expression in Arabidopsis. The
high levels of PA (40:8, 40:7, 38:5, 38:4, 36:4 and 36:3) and lyso-lipids LPC (20:4, 20:3,
18:3, 18:2) and LPE (20:4) probably generated from PC and PE in treated thalli (Fig. 7.10)
showed higher phospholipase activity and phospholipid turnover in congruence to the earlier
reports of Profotova et al. (2005), Salzman et al. (2005) and Cacho et al. (2012). Despite the
significant changes in polar lipid composition of G. dura under MeJA response, the FA
composition was not altered significantly (Table 7.3), except C16:0, C16:1 (n-9), C20:3 (n-
6) and C20:4 (n-6), similar to the earlier reports in several brown macroalgae (Dictyota
dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia
elongata, Saccharina latissima, Sargassum muticum and Laminaria digitata) and Silybum
marianum (Wiesemeier et al. 2008; Küpper et al. 2009; Cacho et al. 2012). Wiesemeier et
al. 2008 also demonstrated that no changes were found in brown macroalgal samples on
elicitation with JA/MeJA at ecologically relevant conditions (0.1 mg ml-1 to 0.5 mg ml-1)
and significant metabolic changes were observed only at concentrations >0.5 mg ml-1 which
was much higher than the concentrations applied in the present study. At such higher
exogenous application of JA, they found upregulation of 16:0, 16:1 and 18:1 in all the brown
macroalgae investigated.
Further, as Gracilaria spp. exhibit both the C18 and C20 oxidative pathways
wherein, the role of these C18 PUFAs leading to JA/MeJA biosynthesis may not be defined,
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but the formation of hydroperoxy- and hydroxy-FAs form both the C18 and C20 PUFAs are
well documented both under normal and stress conditions (Lion et al. 2006; Nylund et al.
2011; Weinberger et al. 2011; Rempt, 2012). In the present study, MeJA induced a cascade
of oxygenation of C18 and C20 PUFAs (C18:2, C18:3 and C20:4) leading to a dose and
time dependent accumulation of hydroxy-oxylipins (HETEs, HODEs and HOTrEs) (Fig.
7.12). Similar upregulation in the content of hydroxy-oxylipins such as13-HODE, 13-oxo-
ODE, 15-HETE, 12-HETE as well as ketols derived from C18:2 and C20:4 was reported in
C. crispus and L. digitata after treatment with MeJA (Bouarab et al. 2004; Gaquerel et al.
2007; Küpper et al. 2009). This upregulation was more pronounced at higher concentrations
(10 and 100 μM) as reported earlier (Bouarab et al. 2004; Gaquerel et al. 2007; Küpper et al.
2009). Further, Gaquerel et al. (2007) also discovered MeJA induced activation of a new
enzyme, bisallylic hydroxylase (BAH) that oxidizes the ω-7 carbon position of PUFAs and
generates the stereoselective (R)-hydroxylated metabolites with a large enantiomeric excess.
The higher content of 13/9-HOTrE (during entire 24 h duration) and 13/9-HODE (at 24 h)
indicated the upregulation of 13-LOX pathway. Similarly, a greater increase in the activities
of 5-LOX, 15-LOX and 8-LOX were observed as apparent from higher increase in 5-HETE
and 8-HETE. Moreover, the accumulation of these hydroxy-oxylipins occurred
concomitantly with the oxidative burst as found in L. digitata (Küpper et al. 2009). These
authors further illustrated that although it is not known that the oxidation of PUFAs occurs
after or before their release from membranes, the latter possibility is more pronounced as
LOX isoforms that oxidizes PUFAs attached to lipids such as MGDG, DGDG, PG and PC
are already reported in higher plants (Fuller et al. 2001; Buseman et al. 2006; Vu et al.
2011). The untargeted profiling of oxidized lipids may help in gaining insight in this regard.
Further, MeJA was found to be a potent enhancer of LOX (linoleate-, linolenate- and
arachidonate-LOX) in agreement with the increase in HODEs, HOTrEs and HETEs in
treated thalli (Bouarab et al. 2004; Gaquerel et al. 2007; Küpper et al. 2009). The highest
increase was observed in linolenate- followed by linoleate- and arachidonate-LOX (Fig.
7.13). Gaquerel et al. (2007) also reported that LOX has relatively less specificity for
arachidonate as compared to linoleate/linolenate in red macroalgae and this gives the scope
to the bisallylic hydroxylation of PUFAs.
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In addition to the activation of fatty acid oxidation cascade in G. dura treated thalli,
MeJA also significantly increased the content of phenolic compounds and enzymes involved
in secondary metabolism such as PPO, PAL and SD (Fig. 7.14). Similar accumulation of
phenolic compounds and upregulation of PPO has been found in higher plants after MeJA
treatment (Koussevitzky et al. 2004; Kim et al. 2006; Cacho et al. 2012; Nafie et al. 2011).
The phenylpropanoid pathway originating from shikimate pathway is one of the important
pathways of secondary metabolism. SD catalyzes the reduction of shikimate to 3-
dehydroshikimate and participates in the formation of aromatic amino acids such as
phenylalanine, tyrosine and tryptophan while PAL catalyzes the formation of trans-cinnamic
acid by L-deamination of phenylalanine. Both the enzymes are strongly upregulated by
MeJA, as observed in the present study in G. dura (Sharan et al. 1998; Bouarab et al. 2004;
Kim et al. 2006; Liu et al. 2008; Nafie et al. 2011). Bouarab et al. (2004) also showed the
upregulation SD and PAL after MeJA treatment in C. crispus. In contrast, Collén et al.
(2006) only found the gene for DHAP synthase on C. crispus microarray that was
overexpressed at 6 h and no other transcripts involved in shikimate pathway was identified.
Further, the maximum accumulation of TPC was found at 12 h and its content decreased at
24 h concomitatnt with the decrease in PPO activity 24 h as compared to control. Similarly,
the activity of SD also decreased at 24 h as compared to control while maximum PAL
activity was found at 24 h. This indicated that PAL activity lagged behind those of PPO and
SD, or conversely, PAL activity is induced in the late phase of MeJA induced oxidative
stress as compared to PPO/SD. Liu et al. (2008) also reported that no significant change was
observed in PAL activity in Pea leaves in initial 12-14 h of JA treatment and maximum
activity was observed at 36-48 h of JA application. Moreover, the induction of PAL activity
in response to JA/MeJA has been directly linked to H2O2 burst and it was found that PAL
activity can be completely blocked by pretreatments with H2O2 scavengers (superoxide
dismutase and catalase) and quenchers (DMTU) (Liu et al. 2008).
In conclusion, the present study revealed that MeJA (1-100 μM) is a strong trigger of
ROS production (H2O2, HO· and O2•−) in G. dura thalli and causes oxidative stress as
observed in Laminaria sp. (Küpper et al. 2009). This further leads to lipid peroxidation and
degradation of photosynthetic pigments (Chl a and PC) with a concomitant increase in PE
that protects the photosynthetic apparatus from damage by neutralizing reactive oxidants and
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helps in combating oxidative stress (Cano-Europa et al. 2010). Further, MeJA induced
oxidative burst also induced the fatty acid oxidation cascade, resulting in the synthesis of
hydroxy-oxylipins and upregulation of 13-LOX (one of the key enzyme of jasmonate
biosynthesis). Most of these FAs were obtained from the degradation of MGDG. In addition,
G. dura thalli modulated the lipid acyl chains in such a way that no significant change was
observed in the FA profile of treated thalli as compared to control except for C16:0, C16:1,
C20:3 and C20:4. This may be a strategy to maintain the membrane fluidity and integrity of
membrane to combat oxidative stress. Further, MeJA caused a redirection from primary to
secondary metabolism as a defense strategy in treated G. dura and caused the accumulation
of phenolic compounds as well as the upregulation of enzymes involved in secondary
metabolism, PPO, SD and PAL. However, Collén et al. (2006) failed to identify cDNA spots
involved in jasmonate biosynthesis on C. crispus microarray while genes involved in
secondary metabolism were poorly represented (except for DHAP synthase). They stated
that the genes for jasmonate biosynthesis and secondary metabolism are likely to be present
on the array but could not be identified due to limited knowledge of red algal genes. It is
clear from this study that MeJA does induces oxidative burst that significantly affect the
lipid metabolism, fatty acid oxidation cascade and upregulates the secondary metabolism in
G. dura. Further, transcriptional analysis of MeJA treated G. dura will surely confirm the
present findings.