UNIVERSITI PUTRA MALAYSIA

MORPHOLOGICAL, PHYTOCHEMICAL AND PROTEOMIC RESPONSES OF ANDROGRAPHIS PANICULATA NEES. TO SALT STRESS

DARYUSH TALEI

FBSB 2012 7

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MORPHOLOGICAL, PHYTOCHEMICAL AND PROTEOMIC RESPONSES OF ANDROGRAPHIS PANICULATA NEES. TO SALT STRESS

By

DARYUSH TALEI

Thesis submitted to the School of Graduate Students, Universiti Putra Malaysia in

Fulfillment of the Requirement for the Degree of Doctor of Philosophy

August 2012

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DEDICATIONS

This Thesis is Special Dedicated to

My Wife, Fatemeh Zaeromali and

My children Sarina and Karina

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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

MORPHOLOGICAL, PHYTOCHEMICAL AND PROTEOMIC RESPONSES OF

ANDROGRAPHIS PANICULATA NEES. TO SALT STRESS

By

DARYUSH TALEI

August 2012

Chairman: Associate Professor Mohd Puad Abdullah, PhD

Faculty: Biotechnology and Biomolecular Sciences

Andrographis paniculata Nees. is a medicinal plant belonging to the family Acanthaceae.

The plant is important due to its pharmaceutical curative compound called diterpenoids. The

compound is extracted from vegetative parts of the plant which was cultivated in the field

from seeds. However, growing the plant is challenging especially in arid and semi-arid

regions where salt concentration is relative high. In different parts of the world, the plant

exists in various accessions but none of them was properly characterized for its level of salt

tolerance. Therefore, the present study was conducted to determine the salt tolerance

thresholds of various accessions of the plant, and finally, to characterize the accessions for

their physiological, phytochemical and proteomic responses to elevated levels of salt. Salt

tolerance thresholds of the various accessions of the plant were determined by exposing 6 of

the accessions to different salt concentrations and exposure times at seedling. The

experiments were carried out in a split split plot arrangement based on the RCBD. Based on

salt tolerance threshold results various dependent parameters related to physiological,

biochemical and proteomic responses of the 32 different accessions of the plant were

evaluated at seedling and mature stages. Changes in metabolites and proteomes were

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monitored by using HPLC and 2D-polyacrylamide gel electrophoresis, respectively. It was

found that the tolerance threshold of the plant was 12 dSm-1 for 15-day of exposure time.

Based on the salt tolerance index of individual accession, the plant could be grouped into salt-

tolerant, moderately salt-tolerant and salt-sensitive accessions. When exposed to a high salt

concentration, the tolerant accessions accumulated higher concentrations of proline, K+, Ca2+

and andrographolide, and lower concentration of Na+ than the sensitive ones, suggesting a

salt tolerance response dictated by metabolite and ion adjustments. However, the levels of

photosynthetic parameters were decreased in all of the accessions, which indicate that the salt

stress directly reduced photosynthesis in the plant. Andrographolide content was positively

correlated with salt concentration; the higher concentration of the salt the larger increase in

andrographolide was observed. The concentration of three main phytochemicals

(andrographolide, neoandrographolide and 14-deoxy-11, 12-didehydroandrographolide)

evaluated were also positively correlated with salt tolerance index and were negatively

correlated with proline content. Overall, these patterns resemble the plant’s salt tolerance

responses based on physiological and phytochemical performances. At proteomic level, the

tolerant accessions showed dissimilar SDS-PAGE banding patterns of soluble leaf protein of

the plant exposed to high salt concentrations. These proteins were further analyzed by 2D gel

electrophoresis in combination with a spot protein analysis by using the PDQuest software.

The analysis led to the detection of 32 induced proteins and 12 up-regulated proteins in the

leaf and root of the salt-treated plants. Of the 44 detected proteins, 12 were sequenced with

three of them matched superoxide dismutase, ascorbate peroxidase and ribulose-1, 5-

bisphosphate oxygenase whereas the rest were unknown. The three known proteins are

generally associated with plant response to environmental stresses and could represent

general stress proteins in the present study. Together, the proteomic data and the diverse

physiological and phytochemical reactions demonstrated by the different accessions of the

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plant to salt stress would potentially be used as salt tolerance indicators in the plant’s

breeding program for salt tolerance improvement.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazzah Doktor Falsafah

RESPONS MORFOLOGI, FITOKIMIA DAN PROTEOMIK DALAM

ANDROGRAPHIS PANICULATA NEES. TERHADAP TEKANAN GARAM

Oleh

DARYUSH TALEI

Ogos 2012

Pengerusi: Prof. Madya Mohd Puad Abdullah, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Andrographis paniculata Nees. adalah tumbuhan ubatan dari famili Acanthaceae.Tumbuhan

itu adalah penting disebabkan sebatian kuratif farmaseutikal yang dipanggil diterpenoid.

Sebatian ini diekstrak dari bahagian-bahagian vegetatif tumbuhan tersebut yang lazimnya

ditanam di ladang melalui bijibenih. Walau bagaimanapun, menanam tumbuhan tersebut

terutamanya di kawasan gersang dan separa-gersang adalah mencabar disebabkan kepekatan

garam yang tinggi di kawasan tersebut. Di berbagai bahagian dunia, tumbuhan ini wujud

dalam pelbagai aksesi tetapi tiada satupun daripada aksesi berkenaan yang telah dicirikan dari

segi ketoleranan terhadap garam. Oleh itu, kajian ini telah dijalankan untuk menentukan

ambang toleransi garam pelbagai aksesi tumbuhan tersebut, dan akhirnya untuk mencirikan

aksesi tersebut dari segi respons fisiologi, fitokimia dan proteomik apabila didedahkan

kepada garam pada kepekatan tinggi. Ambang toleransi garam bagi pelbagai aksesi tumbuhan

tersebut ditentukan dengan mendedahkan 6 aksesi kepada kepekatan garam yang berbeza dan

kali pendedahan pada peringkat anakbenih. Eksperimen ini telah dijalankan dalam

pengaturan plot pecahan pecahan yang berdasarkan rekabentuk blok lengkap (RCBD).

Berdasarkan keputusan ambang toleransi garam pelbagai parameter berkaitan dengan respons

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fisiologi, biokimia dan proteomik tumbuhan tersebut telah dinilai terhadap 32 aksesi berbeza

pada peringkat anak benih dan matang. Perubahan metabolit dan proteom telah dipantau

dengan menggunakan HPLC dan gel elektroforesis dua dimensi, masing-masing. Didapati

ambang toleransi aksesi tumbuhan tersebut adalah pada 12 dSm-1 selama 15 hari masa

pendedahan. Berdasarkan kepada indeks toleransi garam setiap aksesi, tumbuhan tersebut

boleh dikumpulkan ke dalam kumpulan toleran garam, toleran sederhana garam dan sensitif

garam. Apabila terdedah kepada kepekatan garam yang tinggi, aksesi toleran garam

menunjukkan kepekatan prolina, K+, Ca2+ dan andrografolida yang lebih tinggi dan kepekatan

Na+ yang lebih rendah berbanding dengan aksesi sensitif garam, menunjukkan respons

toleransi garam yang didominasi oleh pelarasan pelbagai metabolit dan ion. Walau

bagaimanapun, tahap parameter fotosintesis telah menurun dalam semua aksesi yang diuji,

menunjukkan bahawa tekanan garam memberi kesan buruk kepada proses fotosintesis

tumbuhan berkenaan. Kandungan andrografolida pula berkorelasi secara positif dengan

kepekatan garam, semakin tinggi kepekatan garam yang digunakan semakin besar pula

perubahan penghasilan andrografolida yang diperolehi.Kepekatan tiga fitokimia utama yang

lain (andrografolida, neoandrografolida dan deoksi 14-11, 12-didehidroandrografolida) juga

berkorelasi positif dengan indeks toleransi garam tetapi berkorelasi negatif dengan kepekatan

prolina. Secara keseluruhannya, pola perubahan berkenaan menyerupai respons toleransi

garam bagi tumbuhan tersebut berdasarkan kepada prestasi fisiologi dan fitokimia. Pada

peringkat proteomik pula, aksesi toleran yang didedahkan kepada kepekatan garam yang

tinggi menunjukkan perbezaan dari segi corak SDS-PAGE protein daun. Melalui kombinasi

elektroforesis gel 2D dan analisis spot protein dengan menggunakan perisian PDQuest,

analisis tersebut telah membawa kepada pengesanan 32 protein teraruh dan 12 protein yang

ditingkat-kawal di dalam daun dan akar tumbuhan yang dirawat dengan garam. Daripada 44

protein yang dikesan, 12 daripadanya telah dijujukkan dengan tiga daripada mereka sepadan

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dengan superoksida dismutase, askorbat peroksidase dan ribulose-1, 5-bifosfat oksigenase

manakala selebihnya adalah tidak diketahui. Secara bersama, data proteomik dan pelbagai

respons fisologi dan fitokimia yang ditunjukkan oleh pelbagai aksesi tumbuhan tersebut

terhadap tekanan garam berpotensi untuk digunakan sebagai petunjuk toleransi garam dalam

program pembaik-bakaan tumbuhan berkenaan terutamanya dari segi pembaikan tahap

toleransi garam.

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ACKNOWLEDGEMENTS

First, I must thank to Almighty Allah the Most Gracious and the Most Merciful blessing

enabled me to perceive and for the opportunity given to me to complete the PhD thesis. I

would like to express my sincere appreciation for people who encouraged me and finally

bring me to this sweet moment. I would first like to take this opportunity to express my

deepest gratitude and sincere appreciation to my committee chairman, Associate Professor

Dr. Mohd Puad Abdullah, Department of Cell and Molecular Biology, Faculty of

Biotechnology and Biomolecular Sciences for his professional guidance, constructive

supervision during my study and cooperation to complete the thesis. I am deeply impressed

by his God-given positive energies and the spiritual light whom his carrying on his face. I

especially want to extend my deepest thanks to my dear co-supervisor Professor Dr. Mohd

Khanif Yusop, Department of Land Management; Associate Professor Dr. Mihdzar Abdul

Khadir, Department of Agriculture Technology, Faculty of Agriculture; Associate Professor

Dr. Mohd Said Saad, Department of Crop Science, Faculty of Agriculture, Universiti Putra

Malaysia, 43400 UPM, Serdang, Selangor, Malaysia for providing advice on all aspects of

becoming a scientist. My sincerest gratitude also goes to the secretarial staff for the good-

natured assistance received over the years. Thank you to Mr. Mohd Norhaizan Saliudin,

Department of Crop Science for hosting me in his lab, and for being a constant source of

advice and support over the years. This project would not have been possible without the help

of a handful of excellent field and lab assistants. I have been fortunate to share space with lab

mates that are outstanding both as scientists and as a people. I am certainly grateful and

indebted to my dearest friend Dr. Alireza Valdinai who scarify his times for every of my

problems, thanks from Mr. Mahmoud Danaee and Mr. Ahmad Sharifkhani for his valuable

helps. I also would like to acknowledge Faculty of Agriculture especially Professor Dr.

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Abdul Shukor Juraimi and Associate Professor Dr. Mohd Rafii Yusop, Faculty of

Biotechnology and Biomolecular Sciences especially Professor Dr. Raha Abdul Rahim and

Associate Professor Dr. Rosfarizan Mohamad and School of Graduate Studies, Universiti

Putra (UPM), especially, Professor Dr. Bujang Kim Huat and administrative staff of the

laboratories at the Department of crop science, Department of Land Management,

Department of Agriculture Technology, Faculty of Agriculture, Department of Cell and

Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences for providing the

facilities and their assistance for my study, especially Mrs. Siti Samsiah Yaakup, Ms. Siti

Raziah Rosli, Mr. Abdul Aziz Abdullah, Mrs. Hajah Faridah Aman, Mrs. Mrs. Salmi Yaacob,

Mr. Jamil Omar. I would also like to express my sincerely appreciation to Dr. Hassan Moeini

from Institute of Bioscience, also thank Ms. Siti Raziah Rosli from Department of

Agriculture Technology and Mrs Fatemeh Haddadi and Dr. Vahid Omidvar, Ph.D for their

valuable helps.

Finally, I could not have done any of this without the love and support of my family. I want

to express my deepest gratitude to my parents, my brothers, Almas and Sayyad, my wife,

Fatemeh, my children, Sarina and Karina and my sisters for their moral support and patience

during my study and as well as those from other relatives and friends both in Malaysia.

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I certify that a Thesis Examination Committee has met on 9 August to conduct the final examination of Daryush Talei on his Doctor of Philosophy thesis entitled “Morphological, Phytochemical and Proteomic Responses of Andrographis paniculata Nees. To Salt Stress” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the degree of Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Mohd Arif Syed, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Mohd Yunus bin Abd Shukor, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Mohd Razi bin Ismail, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Mohd Solaiman Ali Fakir, PhD Professor Faculty of Agriculture Bangladesh Agriculture University Bangladesh (External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Mohd Puad Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Mohd Khanif Yusop, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

Mihdzar Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

Mohd Said Saad, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

________________________________

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia 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.

________________

DARYUSH TALEI

Date: 9 August 2012

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TABLE OF CONTENTS

Page

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS ix APPROVAL DECLARATION xiii LIST OF TABLES xviii LIST OF FIGURES xx LIST OF APPENDICES xxiv LIST OF ABBREVIATIONS xxv CHAPTER 1 INTRODUCTION 1

1.1 Research Questions 6

1.2 Research Objectives 6

2 LITERATURE REVIEW 7

2.1 Andrographis paniculata 7

2.1.1 Historical background of A. paniculata 7 2.1.2 Importance of A. paniculata 7 2.1.3 Botany of A. paniculata 8 2.1.4 Taxonomy of A. paniculata 10 2.1.5 Distribution and preferred habitats of A. paniculata 10 2.1.6 Genetic diversity and cytology of A. paniculata 11 2.1.7 Phytochemistry 11 2.1.8 Crude Extract of A. Paniculata and Extraction

Methods 12 2.1.9 Chromatography techniques in herbal medicine 13

2.2 Salinity stress 14

2.2.1 Definition of stress 14 2.2.2 Water and soil salinity 15 2.2.3 General salinity problems on plants 16 2.2.4 Salinity problems associated with cultivation 18 2.2.5 Mechanisms of salt tolerance 19 2.2.6 Salt tolerance 21 2.2.7 Plant response to salinity stress 24 2.2.8 Salinity and yield 25 2.2.9 Growth and morphological responses to salt stress 25 2.2.10 Physiological responses to salt stress 27 2.2.11 Molecular responses to salt stress 29 2.2.12 Strategies and the importance of traits for increasing

salt tolerance 32 2.2.13 Role of proline in salt stress tolerance of plants 33 2.2.14 High Na+ effects on plant cells 35

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2.2.15 Role of potassium in salt tolerance in plants 36 2.2.16 Role of Ca2+ in salt stress 37 2.2.17 Root signaling in response to salinity 38 2.2.18 Salinity effects on secondary metabolites 39 2.2.19 Salinity effects on amino acid status 41 2.2.20 Protein induced salt stress 41

2.3 Proteomics approach 42

2.3.1 Two-dimensional gel electrophoresis 43 2.3.2 Proteome analysis of plant salt stress response 44 2.3.3 Bioinformatics in proteomics 46

3 RESPONSE OF A. PANICULATA SEEDLINGS TO SALINITY

STRESS BEYOND THE SALT TOLERANCE THRESHOLD 48 3.1 Introduction 48

3.2 Materials and Methods 50

3.2.1 Plant materials and chemicals 50 3.2.2 Seed germination conditions 51 3.2.3 Experimental design 53 3.2.4 Determination of proline and chlorophyll content 55 3.2.5 Determination of ion contents of leaf 56 3.2.6 Statistical analysis 57

3.3 Results and Discussion 57

3.3.1 Evaluation of morphological traits of A. paniculata before applying salinity stress 57

3.3.2 Effects of salinity levels and salinity exposure times on growth indices of A. paniculata 58

3.3.3 Changes on dry weight and STI under salinity conditions 70

3.3.4 Salinity effects on chlorophyll and proline content 72 3.3.5 Salinity effects on ion contents 73 3.3.6 Correlations 77 3.3.7 Cluster analysis 80

3.4 Conclusion 82

4 EFFECTS OF SALINITY ON MORPHOLOGICAL AND

PHYSIOLOGICAL TRAITS OF MATURE A. PANICULATA PLANTS 83 4.1 Introduction 83

4.2 Materials and Methods 85

4.2.1 Preparation of A. paniculata seedlings 85 4.2.2 Experimental design 86 4.2.3 Determination of photosynthesis parameters 87

4.3 Results and Discussion 89

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4.3.1 Pre-assessment of morphological traits prior to salinity treatment 89

4.3.2 Salinity effects on morphological traits 89 4.3.3 Salinity effects on dry weight and salt tolerance index 90 4.3.4 Salinity effects on proline content 94 4.3.5 Salinity effects on macro nutrients 95 4.3.6 Salinity effects on micro nutrients 99 4.3.7 Salinity effects on photosynthesis parameters 103 4.3.8 Correlations 109 4.3.9 Broad-sense heritability 110 4.3.10 Principal component analysis (PCA) 116 4.3.11 Cluster analysis 118

4.4 Conclusion 119

5 SALINITY EFFECTS ON SECONDARY METABOLITES IN

A. PANICULATA AT MATURE STAGE 122 5.1 Introduction 122

5.2 Materials and Methods 124

5.2.1 Chemicals 124 5.2.2 Plant material and experimental design 124 5.2.3 Preparation of crude extracts 125 5.2.4 Preparation of samples 126 5.2.5 Preparation of standards 126 5.2.6 Phytochemical analysis 127

5.3 Results and Discussion 127

5.3.1 Effect of different solvents on total crude extract of A. paniculata 127

5.3.2 Analysis of standard samples 128 5.3.3 Effect of salinity levels on the three main metabolites

in A. paniculata 129

5.4 Conclusion 135

6 ANALYSIS OF LEAF AND ROOT PROTEOMES OF SALT-

TREATED A. PANICULATA SEEDLINGS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS 137 6.1 Introduction 137

6.2 Materials and Methods 139

6.2.1 Chemicals and reagents 139 6.2.2 Plant material and germination conditions 139 6.2.3 Growth conditions at seedling stage 140 6.2.4 Growth conditions at mature stage 140 6.2.5 Protein Sample Preparation and Quantification for

SDS-PAGE 141 6.2.6 Protein Separation using SDS-PAGE 141 6.2.7 Staining of Protein Pattern of SDS-PAGE gel 142

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6.2.8 Protein Sample Preparation and Quantification for 2-DE 142

6.2.9 Hurkman and Tanaka method (1986) 143 6.2.10 Damerval et al. method (1986) 144 6.2.11 Saravanan et al. method (2004) 144 6.2.12 HEPES-based buffer system 145 6.2.13 First and Second Dimension Protein Separation 146 6.2.14 Staining and digitization of protein pattern 148

6.3 Results and Discussion 148

6.3.1 SDS-PAGE seed protein profiles 148 6.3.2 Leaf protein profiles of salt-treated A. Paniculataat

seedling stage 151 6.3.3 Leaf protein profiles of salt-treated A. Paniculata at

mature stage 154 6.3.4 Comparison of different protein extraction methods for

two dimensional separation of protein 157 6.3.5 Analysis of Protein spots separated by 2D PAGE by

using the PDQuest software 158 6.3.6 Classification of Functional Identified Proteins 170

7 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH 175 REFERENCES 180 APPENDICES 206 BIODATA OF STUDENT 221 LIST OF PUBLICATIONS 222

MORPHOLOGICAL, PHYTOCHEMICAL AND PROTEOMIC RESPONSES OFANDROGRAPHIS PANICULATA NEES. TO SALT STRESSABSTRACTTABLE OF CONTENTSCHAPTERREFERENCES