Chapter 7 Jasmonates in macroalgae: exogenous application of

methyl jasmonate and its physiological effects in Gracilaria dura

 

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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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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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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.