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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