universiti putra malaysia - psasir.upm.edu.mypsasir.upm.edu.my/id/eprint/39934/1/fbsb 2013...

UNIVERSITI PUTRA MALAYSIA

REVANDY ISKANDAR MUDA DAMANIK

FBSB 2013 22

CHANGES IN ANTIOXIDANT ENZYMES AND GROWTH PARAMETERS IN RICE (Oryza sativa L.) GENOTYPES UNDER SUBMERGENCE

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CHANGES IN ANTIOXIDANT ENZYMES AND GROWTH PARAMETERS

IN RICE (Oryza sativa L.) GENOTYPES UNDER SUBMERGENCE


DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2013

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By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirement for the Degree of Doctor of Philosophy

June 2013

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COPYRIGHT

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photographs and all other artwork, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use may be made of any material contained within the thesis

for non-commercial purposes from the copyright holder. Commercial use of material

may only be made with the express prior, written permission of Universiti Putra

Malaysia.

Copyright © Universiti Putra Malaysia

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DEDICATED TO:

My MOTHER, BROTHERS, SISTER, BROTHER IN LAW, SISTER IN LAWS,

NEPHEWS (Nathan, Frans, Frederik) and

NIECES (Rinda, Felicia, Cornelia, Lika, Lina)

WHO ALWAYS HAVE SUPPORTING AND SHOWERING ME WITH THEIR

SPIRIT, LOVE AND PRAYER.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Doctor of Philosophy



By


June 2013

Chairperson : Professor Maziah Mahmood, PhD

Faculty : Biotechnology and Biomolecular Sciences

Submergence is one of the main abiotic stresses limitations for yield and quality

production of many important crops. In order to better understand the physiological

and biochemical responses by submergence stress in rice (Oryza sativa L.), this thesis

was mainly examined to clarify the biochemical markers of contrasting rice

genotypes/cultivar during cell level, germination and seedling stages, and discusses

the basis of tolerance related genotypes/cultivar. For that reason, in order to recognize

and also understand better for the biochemical mechanism of submergence tolerance,

the objective of this study was to find out the activities of antioxidant enzymes

(superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione

reductase, GR), as well as for lipid peroxidation in rice plant subjected to different

periods of submergence stress. The isoenzymic profile analysis of the antioxidant

enzymes also examined in anoxia-stressed suspension culture to get further

understanding of the mobilization of protein reserves as well as apperearance of new

isoenzymes in submergence stress.

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Until now, there have been some researchs and reports related to the influence of

submergence stress on the growth of rice plants at seedling growth (Colmer and

Pedersen, 2008; Das et al., 2005; Ella et al., 2003; Sarkar et al., 2001), but only a

small amount of research has been focused on the influence of submergence stress at

early stage (a week after germination) of seedling. Plant growth rate and antioxidative

defense responses of early seedlings in six selected Malaysian rice genotypes, MR84,

MR185, MR219, MR219-4, MR220, and MR219-9, and FR13A cultivar (known as

cultivated plant for submergence tolerant) at different submergence periods (4, 8, and

12 days) were examined. Eleven day old seedlings of hypoxia resistant Malaysian rice

genotypes and tolerant rice cultivar (FR13A) were hydroponically grown in normoxic

or hypoxic conditions inside the laboratorium. The result on chlorophyll content in

rice seedlings suggested that photosynthesis was affected beginning as early as 4 d

under submerged condition, except for FR13A cultivar. Activities of superoxide

dismutase (SOD) increased linearly with the longer periods of submergence stress

until up to 8 d submerged for FR13A cultivar, and MR84, MR185, MR219-4, and

MR219-9 genotypes when compared with the control. In contrast, the result showed

that catalase (CAT) activity was activated while ascorbate peroxidase (APX) activity

was deactivated, and vice versa, in response for submergence conditions for FR13A

cultivar and MR219-9 genotype. Additionally, submergence stress led to a significant

increase in glutathione reductase (GR) activity for all genotypes started at 4 d to 8 d

after being submerged, except for MR219 genotype. From the results obtained in this

study, give strength to our hypothesis that detoxification of O2_

to H2O2 was

maintained at a stable level throughout the submergence stress until up to 8 days

(specifically for MR219-4 and MR219-9 genotypes) and the role of GR might be

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more efficient in cytotoxic H2O2 necessary for the observed protection from

submergence stress.

The potential involvement of activated oxygen species by submergence stress was

studied also focusing on two Malaysian rice genotypes, MR219-4 and MR219-9, and

cultivar FR13A at the seedling stages. Fourteen-day-old seedlings of three rice

cultivars were subjected to different submergence periods (4, 8 and 12 days) in

greenhouse. Under 8 days of complete submergence, FR13A cultivar showed higher

lipid peroxidation in terms of malondialdehyde (MDA) level and activities of

antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), ascorbate

peroxidase (APX), and glutathione reductase (GR) when compared to the MR219-4

and MR219-9 genotypes. MR219-9 genotype showed higher SOD, APX and GR

activities after 12 days of submergence. The levels of SOD activity indicated that

detoxification of O2.- to H2O2 was maintained at a stable level throughout the

submergence stress until up to 8 days and increased rapidly at 12 days of

submergence. The results showed that tolerance to submergence is linked until 8 days

submergence for MR219-4 genotype and FR13A cultivar. In addition, CAT activity

has much higher affinity for scavenges H2O2 than APX, therefore ascorbate

glutathione cycle might be more efficient to scavenge H2O2.

The consequence of oxygen deprivation (anoxia) on the antioxidant system in

suspension culture of anoxia intolerant Malaysian rice genotypes and FR13A

genotype cells was examined. The antioxidative enzymes were decreased for MR219-

4 and MR219-9 genotypes for CAT and APX activities, and increased in FR13A

cultivar starting at 20 days in suspension culture compared to that of control. CAT

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and APX activities were maintained higher in oxygen depriviation conditions for all

genotypes and cultivar suggested that anoxia stress in suspension cultures induced the

level of H2O2 to toxic levels.

The relationship between the enzyme activity and isoenzyme of the SOD and

peroxidase (POD) in cell suspension rice submitted to anoxia stress (oxygen

depriviation) has been evaluated. Cell suspension culture of rice exhibited three kinds

of SOD bands, identified as MnSOD, FeSOD and Cu/ZnSOD. A substantial

improvement was identified in the activity of MnSOD isozymes since it has been

prominent appeared for all genotypes and cultivar at different periods of stress. It is

practical to take on that mitochondrial partition senses oxygen depletion condition

faster than the rest of the cell (peroxisome, chloroplast, etc) partition because it is the

most important consume of oxygen. The higher activity of POD and its isoenzymes

activities for MR219-4 genotype and FR13A cultivar, maybe compensate for the

decline in CAT activity. These results recommend that POD, SOD and CAT activities

play necessary and synergy protection responsibility aligned with H2O2 toxic levels in

rice suspension culture in anoxia condition. Induction of their isoenzymes/isoforms

and activities of POD, CAT and SOD provide protection from oxidative damage,

hence these parameters might be taken as selection criteria for study of submergent

tolerance in rice.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PERUBAHAN DALAM ENZIM ANTIOKSIDAN DAN PARAMETER

PERTUMBUHAN PADI (Oryza sativa L.) DALAM TEKANAN

PERENDAMAN

Oleh


Jun 2013

Pengerusi: Professor Maziah Mahmood, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Perendaman merupakan salah satu tegasan utama abiotik untuk menghadkan hasil

dan kualiti tanaman yang penting. Dalam usaha untuk lebih memahami tindak balas

fisiologi dan biokimia dalam tekanan perendaman pada padi (Oryza sativa L.), tesis

ini terutamanya diperiksa untuk menjelaskan penanda biokimia genotip padi berbeza

pada tahap sel, percambahan dan peringkat anak benih dan membincangkan asas

toleransi genotip yang berkaitan. Oleh itu, dalam usaha untuk lebih memahami

mekanisme biokimia toleransi perendaman, matlamat kajian ini adalah untuk

menentukan aktiviti enzim antioksidan (superoxide dismutase, SOD; catalase, CAT;

askorbat peroksidase, APX; glutathione reductase, GR), serta sebagai peroksidaan

lipid dalam tanaman padi diserahkan kepada tekanan sederhana atau teruk semasa

tekanan perendaman pendek atau jangka panjang. Analisis profil isoenzymic enzim

antioksidan juga diperiksa dalam tekanan anoxia dalam suspensi kultur untuk

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mendapatkan pemahaman lanjut dan mobilisasi rizab protein serta apperearance

daripada isoenzim baru dalam tekanan perendaman.

Sehingga kini, telah ada beberapa laporan mengenai pengaruh tekanan perendaman

pada pertumbuhan tanaman padi pada kadar pertumbuhan anak benih (Colmer dan

Pedersen, 2008; Das et al, 2005; Ella et al, 2003;. Sarkar et al, 2001), tetapi sedikit

penyelidikan tumpuan kepada pengaruh tekanan perendaman pada peringkat awal

anak benih. Laju kadar pertumbuhan dan tindak balas pertahanan antioksida benih

awal dalam pilihan enam beras genotip Malaysia, MR84, MR185, MR219, MR219-4,

MR220 dan MR219-9, dan FR13A (kultivar toleran perendaman) pada tempoh

perendaman yang berbeza (4, 8, dan 12 hari) telah diperiksa. Sebelas hari anak benih

genotip padi Malaysia dan padi kultivar toleran (FR13A) telah secara hidroponik

berkembang dalam keadaan normoxic atau hipoksia di dalam makmal. Hasil kajian

terhadap kandungan klorofil dalam anak benih padi mencadangkan bahawa

fotosintesis terjejas bermula seawal sebagai 4 d bawah keadaan tenggelam, kecuali

bagi FR13A kultivar. Aktiviti superoxide dismutase (SOD) meningkat secara linear

dengan tempoh perendaman sehingga 8 hari bagi kultivar FR13A, dan MR84,

MR185, MR219-4, dan MR219-9 genotip berbanding dengan kawalan. Sebaliknya,

hasilnya menunjukkan bahawa katalase (CAT) aktiviti telah diaktifkan manakala

askorbat peroksidase (APX) aktiviti telah dinyahaktifkan, dan sebaliknya, sebagai

tindak balas untuk keadaan terendam, untuk kultivar FR13A dab MR219-9 genotip..

Selain itu, tekanan perendaman membawa kepada peningkatan yang ketara dalam

aktiviti glutation reductase (GR) bagi semua kultivar atau genotip pada 4 dan 8 hari

perendaman, kecuali bagi MR219 genotip. Daripada keputusan yang diperolehi dalam

kajian ini, memberi kekuatan kepada hipotesis kami bahawa detoksifikasi O2_ untuk

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H2O2 dikekalkan pada tahap yang stabil sepanjang tekanan perendaman sehingga 8

hari (MR219-4 dan MR219-9 genotip) dan peranan GR mungkin menjadi lebih cekap

dalam H2O2 perlindungan sitotoksik perlu untuk diperhatikan daripada tekanan

perendaman.

Penglibatan potensi spesies oksigen yang diaktifkan oleh tekanan perendaman telah

dikaji juga memberi tumpuan kepada dua beras genotip Malaysia, MR219-4 dan

MR219-9, dan FR13A kultivar di peringkat anak benih. Benih tiga kultivar beras yang

berusia empat belas hari telah tertakluk kepada tempoh perendaman yang berbeza (4,

8 dan 12 hari) di dalam rumah kaca. Di bawah 8 hari tenggelam, kultivar FR13A

menunjukkan peroksidaan lipid yang lebih tinggi dari segi tahap malondialdehyde

(MDA) dan aktiviti-aktiviti enzim antioksida, superoxide dismutase (SOD), katalase

(CAT), peroxidase ascorbate (APX), dan glutation reductase (GR) apabila

dibandingkan kepada genotip MR219-4 dan MR219-9. MR219-9 menunjukkan

genotip SOD yang lebih tinggi, APX dan aktiviti GR selepas 12 hari tenggelam.

Tahap aktiviti SOD menunjukkan bahawa detoksifikasi O2 menjadi H2O2 telah

dikekalkan pada tahap yang stabil sepanjang tekanan perendaman sehingga 8 hari dan

meningkat pesat pada 12 hari perendaman. Keputusan menunjukkan bahawa toleransi

untuk tenggelam dalam beras dikaitkan sehingga 8 hari tenggelam pada genotip

MR219-4 dan kultivar FR13A. Di samping itu, aktiviti CAT mempunyai pertalian

yang lebih tinggi untuk mengurangkan H2O2 daripada APX, itu askorbat kitaran

glutation mungkin lebih berkesan untuk mengurangkan H2O2.

Kesan kekurangan oksigen (anoxia) pada sistem antioksidan dalam kultur suspensi

anoxia pada beras genotip Malaysia dan sel-sel genotip FR13A diperiksa. Enzim

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antioksidan telah menurun pada MR219-4 dan MR219-9 genotip untuk CAT dan

aktiviti APX, dan meningkat pada kultivar FR13A bermula pada 20 hari dalam

suspensi kultur berbanding dengan kultur kawalan. Aktiviti CAT dan APX dikekalkan

lebih tinggi dalam keadaan kekurangan oksigen untuk semua genotip dan kultivar

mencadangkan bahawa tekanan anoxia dalam suspensi kultur mendorong tahap H2O2

ke tahap toksik.

Hubungan antara aktiviti enzim dan isoenzim superoxide dismutase (SOD) dan

peroxidase (POD) dalam suspensi kultur sel diserahkan kepada tekanan anoxia

(oksigen depriviation) telah dinilai. Suspensi kultur beras memberikan tiga jenis band

SOD, dikenalpasti sebagai MnSOD, FeSOD dan Cu / ZnSOD. Satu peningkatan yang

ketara telah dikesan dalam aktiviti isozymes MnSOD sejak ia telah terkemuka muncul

untuk semua genotip dan kultivar pada tempoh yang berbeza tekanan. Ia adalah

munasabah untuk menganggap bahawa mitokondria kekurangan oksigen lebih cepat

daripada seluruh sel kerana ia adalah mengambil yang paling penting oksigen.

Aktiviti yang lebih tinggi POD dan isoenzim aktiviti untuk genotip MR219-4 dan

kultivar FR13A, mungkin mengimbangi penurunan dalam aktiviti CAT. Keputusan

ini menunjukkan bahawa aktiviti POD, CAT dan SOD memainkan peranan penting

perlindungan dan sinergi terhadap H2O2 tahap toksik dalam suspensi kultur padi

dalam keadaan anoxia. Induksi isoenzim/isoforms dan aktiviti POD, CAT dan SOD

menyediakan perlindungan daripada kerosakan oksidatif, maka parameter ini

mungkin diambil sebagai kriteria pemilihan untuk kajian toleransi perendaman dalam

beras.

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ACKNOWLEDGEMENTS

First of all, I would like to thank to the chairman of my supervisory committee, Prof.

Dr. Maziah Mahmood for her professionalism, her high quality, and furthermore for

her patience by giving the excellence guidance and allowing me to join and work in

the group of laboratory 235 in order to conduct my research. Special thanks also to

my co-supervisors of supervisory committee, Prof. Dr. Mohd. Razi Ismail and Dr.

Syahida Ahmad for their supports and assistances throughout this research.

I would like to thank to Rector University of North Sumatera (USU) Medan, North

Sumatera, Indonesia, and the staff who gave me permission doing my further study

and help me with their cooperation. Besides, I want to say thank you to Prof. Madya.

Dr. Abd. Mohd. Zain, who help provided rice seeds of some cultivars/genotypes I

need to conduct my research from Malaysian Agricultural Research and Development

Institute (MARDI) Seberang Perai, Malaysia. Moreover, I also would like to thank to

Graduate Research Fund (GRF) of Universiti Putra Malaysia (UPM) who was

supported this research. I cannot forget towards my laboratory mates from lab.235 for

their knowledge sharing, ideas and friendship during the course of this project.

Finally, I would like to express my gratitude to my mother, brothers, sister, and all of

family for supporting and showering me with their love and prayer.

May GOD bless all of you

Revandy Iskandar Muda Damanik, June 2013.

Trust in the LORD with all thine heart;

and lean not unto thine own understanding. Proverbs 3:5

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

member of Supervisory Committee were as follows:

Maziah Mahmood, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Mohammad Razi Ismail, PhD

Professor

Institute of Tropical Agriculture


(Member)

Syahida Ahmad, PhD

Lecturer

Faculty of Biotechnology and Biomolecular Sciences


(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any

other institution.


Date: 14 June 2013

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vii

ACKNOWLEDGEMENTS xi

APPROVAL xii

DECLARATION xiv

LIST OF TABLES xviii

LIST OF FIGURES xx

LIST OF ABBREVIATIONS xxiii

CHAPTER

I INTRODUCTION 1

II LITERATURE REVIEW 8

2.1 Rice cultivation 8

2.2 The general effect of submergence conditions on the growth of rice 11

Seedlings

2.2.1 Floodwater condition 14

2.2.2 Morphological and Anatomical responses 15

2.2.3 Aerenchyma formation 17

2.2.3.1. Lysigenous aerenchyma 18

2.2.3.2. Schizogenous aerenchyma 18

2.2.4 Photosynthetical performance 19

2.2.5 Biochemical response 20

2.3 Reactive oxygen species (ROS) roles under abiotic stress 22

2.3.1 Superoxide radical (O2•) 24

2.3.2 Singlet oxygen (1O2) 25

2.3.3 Hydroxyl radicals (OH•) 26

2.3.4 Hydrogen peroxide (H2O2) 27

2.4 Physiological responses towards on submergent conditions 29

2.4.1 Anaerobic induction of sugar supply and energy metabolism 30

2.4.2 Alcohol dehydrogenase (ADH) and pyruvate decarboxylase 32

(PDC)

2.4.3 Hormonal regulation 33

2.5 Reactive oxygen species (ROS) in plant cells 35

2.5.1 Non-enzymatic antioxidant 37

2.5.1.1 α-Tocopherol 37

2.5.1.2 Ascorbic acid 38

2.5.1.3 Glutathione 39

2.5.1.4 Phenolics 40

2.5.2 Antioxidant enxymes 41

2.6 Aeration stress induced physiology changes in plant tissue 44

culture

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III EFFECT OF SUBMERGENCE DURING EARLY SEEDLING 47

GROWTH OF SELECTED MALAYSIAN RICE GENOTYPES

(Oryza sativa L.) 3.1 Introduction 47

3.2 Materials and methods 49

3.2.1 Plant materials and stress condition 49

3.2.2 Determination of seed germination 50

3.2.3 Determination of plant biomass and chlorophyll analysis 51

3.2.4 Preparation of crude enzyme extracts 51

3.2.5 Total soluble protein assay 52

3.2.6 ROS scavenging enzyme assays 53

3.2.7 Statistical analysis 54

3.3 Results and discussions 55

3.3.1 Seed germination and growth of the seedlings 55

3.3.2 Effect of submergence on chlorophyll analysis 60

3.3.3 Effect of submergence on protein concentration 63

3.3.4 Effect of submergence on antioxidant enzymes activities 64

3.4 Conclusions 78

IV RESPONSES OF THE ANTIOXIDATIVE ENZYMES IN 80

MALAYSIAN RICE (Oryza sativa L.) CULTIVARS

UNDER SUBMERGENT CONDITION 4.1 Introduction 80


4.2.1 Plant materials and stress conditions 82

4.2.2 Chlorophyll analysis 84



4.2.5 Lipid peroxidation 85


4.3 Results and discussions 86

4.3.1 Effect of submergence condition on total chlorophyll 86

4.3.2 Effect of submergence condition on antioxidant enzymes 90

activities

4.3.3 Effect of submergence condition on lipid peroxidation 99

4.4 Conclusions 101

V RESPONSE OF ANTIOXIDANT SYSTEM IN OXYGEN 103

DEPRIVED SUSPENSION CULTURES OF RICE (Oryza sativa L.)

5.1 Introduction 103


5.2.1 Plant materials 107

5.2.2 The effect of different types of auxin on callus induction 107

5.2.3 Callus maintenance medium 108

5.2.4 Determination callus growth pattern 108

5.2.5 Establishment of rice cell suspension cultures 109

5.2.6 Treatment for oxygen deprivation (anoxic) condition 109

5.2.7 Determination of growth of rice suspension cultures 110



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5.2.10 Lipid peroxidation 111


5.3 Results and discussion 112

5.3.1 The effect of different types of auxin on callus induction 112

5.3.2 Callus maintenance medium 114

5.3.3 Determination callus growth pattern 115

5.3.4 Growth of rice suspension cultures 116

5.3.5 Effect of anoxia condition on antioxidant enzymes 118

activities

5.3.6 Effect of anoxia condition on lipid peroxidation 126

5.4 Conclusions 128

VI DETECTION OF ENZYMES ACTIVITY ON 129

ELECTROPHORETIC GELS FROM RICE GENOTYPES

INVOLVED IN ANOXIA CONDITION 6.1 Introduction 129


6.2.1 Plant materials 132


6.2.3 Identification of isoenzymes 133


6.3 Results and discussion 135

6.3.1 Response of SOD isoenzymes to anoxia condition 135

6.3.2 Response of POD isoenzymes to anoxia condition 142

6.4 Conclusions 147

VII GENERAL CONCLUSIONS AND SUGGESTIONS 149

7.1 General conclusions 149

7.2 Suggestions 151

BIBLOGRAPHY 152

APPENDICES 175

BIODATA OF STUDENT 181

LIST OF PUBLICATIONS 182

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